Elephant
Bibliographic
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Elephant Bibliographic Database
www.elephantcare.org

References updated October 2009 by date of publication, most recent first.

Bartlett, S.L., Abou-Madi, N., Kraus, M.S., Wiedner, E.B., Starkey, S.R., Kollias, G.V., 2009. Electrocardiography of the Asian elephant (Elephas maximus). J. Zoo. Wildl. Med. 40, 466-473.
Abstract: Electrocardiograms (ECGs) are infrequently performed on Asian elephants (Elephas maximus), and few studies have been reported in the literature. The aim of this study was to determine reference ranges of ECG parameters in Asian elephants and to ascertain if age, body weight, and position of the elephant significantly affected the ECG. Electrocardiograms were obtained from 27 captive, nonsedated apparently healthy Asian elephants while they were standing (ST), in right lateral recumbency (RL), and/or in left lateral recumbency (LL). Six-lead ECGs were obtained using novel clamps and long ECG cables (71 cm). From lead I, standard waveforms and intervals were analyzed, including PR interval, QT interval, ST segment, P, QRS, T, and U waves if they were present. One animal was determined to have a previously undiagnosed conduction abnormality and was not included in the study. Most elephants had a sinus arrhythmia in at least one position. With increasing age, there was a trend toward a slower heart rate and significantly longer P waves. Increasing body weight was significantly correlated with longer QT intervals and T waves with lower amplitude. Compared with measurements in ST, LL resulted in P waves and QRS complexes with shorter amplitude, U waves with greater amplitude, PR intervals with shorter duration, and an increased heart rate. Compared with measurements in LL, RL resulted in larger QRS complexes. U waves were most commonly detected in RL and LL. Mean electrical axis calculated in the frontal plane were as follows: standing range -125 to +141 degrees, mean -5 degrees; left lateral range -15 to +104 degrees, mean 27 degrees; right lateral range -16 to +78 degrees, mean 9 degrees. Position-specific reference ranges should be used when interpreting ECGs, and clinicians must be aware of how age and body weight may affect the ECG

Behr, B., Rath, D., Hildebrandt, T.B., Goeritz, F., Blottner, S., Portas, T.J., Bryant, B.R., Sieg, B., Knieriem, A., de Graaf, S.P., Maxwell, W.M., Hermes, R., 2009. Germany/Australia index of sperm sex sortability in elephants and rhinoceros. Reprod. Domest. Anim 44, 273-277.
Abstract: Flow cytometric sexing of spermatozoa followed by application in artificial insemination or in vitro fertilization provides a unique opportunity to predetermine the sex of offspring and might enhance the conservation management of endangered species in captivity such as the elephant and rhinoceros. To obtain an indication of the sortability of spermatozoa from these species, the relative DNA differences between X and Y chromosome bearing spermatozoa (fresh, frozen thawed, epididymal) from three rhinoceros species [white (Ceratotherium simum), black (Diceros bicornis), Indian (Rhinoceros unicornis)] and both elephant species, the Asian and the African elephant (Elephas maximus, Loxodonta Africana), were determined through separation of spermatozoa into X and Y chromosome bearing populations, using a modified high speed flow cytometer. The head profile areas of spermatozoa from all five species were measured using light microscopy. By multiplying the relative DNA differences and the head profile areas, the sperm sorting indices were calculated to be 47, 48 and 51 for white, black and Indian rhinoceros respectively. The calculated sorting index for the Asian elephant was 66. In the African elephant, we determined the highest sorting index of 76. These results indicate the practicability of flow cytometric sex sorting of spermatozoa from the tested rhinoceros species and both elephant species. The lower sorting indices in rhinos indicate that sex sorting of spermatozoa from the rhinoceros will be more challenging than in elephants

Chandrajith, R., Kudavidanage, E., Tobschall, H.J., Dissanayake, C.B., 2009. Geochemical and mineralogical characteristics of elephant geophagic soils in Udawalawe National Park, Sri Lanka. Environ. Geochem. Health 31, 391-400.
Abstract: Geophagy or deliberate ingestion of soils was observed among Asian elephants (Elephas maximus) in the Udawalwe National Park, Sri Lanka, for several years. The geochemical and mineralogical composition of the clayey soil layers which are purposefully selected and eaten by elephants in the park were studied, in order to identify the possible reasons for elephant geophagy. The concentrations of major and trace elements were determined by means of X-ray fluorescence spectrometry in 21 soil samples from eight geophagic sites and six soil samples collected from four non-geophagic sites. The mineralogical composition of selected soil samples was investigated using X-ray diffractometry (XRD). These geochemical analyses revealed that geophagic soils in the study areas are deeply weathered and that most of the elements are leached from the soil layers under extreme weathering conditions. The XRD data showed that the soils of the area consisted mainly quartz, feldspar, and the clay minerals kaolinite, Fe-rich illite, and smectite. Although no significant geochemical differences were identified between geophagic and non-geophagic soils, a clear difference was observed in their clay mineralogical content. Soils eaten by elephants are richer in kaolinite and illite than non-geophagic soils, which contain a higher amount of smectite. It is suggested that elephants in Udawalawe National Park ingest soils mainly not to supplement the mineral contents of their forage but to detoxify unpalatable compounds in their diet

Greenwald, R., Lyashchenko, O., Esfandiari, J., Miller, M., Mikota, S., Olsen, J.H., Ball, R., Dumonceaux, G., Schmitt, D., Moller, T., Payeur, J.B., Harris, B., Sofranko, D., Waters, W.R., Lyashchenko, K.P., 2009. Highly accurate antibody assays for early and rapid detection of tuberculosis in African and Asian elephants. Clin. Vaccine Immunol. 16, 605-612.
Abstract: Tuberculosis (TB) in elephants is a reemerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current methods for screening and diagnosis rely on trunk wash culture, which has serious limitations due to low test sensitivity, slow turnaround time, and variable sample quality. Innovative and more efficient diagnostic tools are urgently needed. We describe three novel serologic techniques, the ElephantTB Stat-Pak kit, multiantigen print immunoassay, and dual-path platform VetTB test, for rapid antibody detection in elephants. The study was performed with serum samples from 236 captive African and Asian elephants from 53 different locations in the United States and Europe. The elephants were divided into three groups based on disease status and history of exposure: (i) 26 animals with culture-confirmed TB due to M. tuberculosis or Mycobacterium bovis, (ii) 63 exposed elephants from known-infected herds that had never produced a culture-positive result from trunk wash samples, and (iii) 147 elephants without clinical symptoms suggestive of TB, with consistently negative trunk wash culture results, and with no history of potential exposure to TB in the past 5 years. Elephants with culture-confirmed TB and a proportion of exposed but trunk wash culture-negative elephants produced robust antibody responses to multiple antigens of M. tuberculosis, with seroconversions detectable years before TB-positive cultures were obtained from trunk wash specimens. ESAT-6 and CFP10 proteins were immunodominant antigens recognized by elephant antibodies during disease. The serologic assays demonstrated 100% sensitivity and 95 to 100% specificity. Rapid and accurate antibody tests to identify infected elephants will likely allow earlier and more efficient treatment, thus limiting transmission of infection to other susceptible animals and to humans

Greenwald, R., Lyashchenko, O., Esfandiari, J., Miller, M., Mikota, S., Olsen, J.H., Ball, R., Dumonceaux, G., Schmitt, D., Moller, T., Payeur, J.B., Harris, B., Sofranko, D., Waters, W.R., Lyaschenko, K.P., 2009. Highly accurate antibody assays for early and rapid detection of tuberculosis in African and Asian elephants. Clinical and Vaccine Immunology 16, 605-612.
Abstract: Tuberculosis (TB) in elephants is a reemerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current methods for screening and diagnosis rely on trunk wash culture, which has serious limitations due to low test sensitivity, slow turnaround time, and variable sample quality. Innovative and more efficient diagnostic tools are urgently needed. We describe three novel serologic techniques, the ElephantTB Stat-Pak kit, multiantigen print immunoassay, and dual-path platform VetTB test, for rapid antibody detection in elephants. The study was performed with serum samples from 236 captive African and Asian elephants from 53 different locations in the United States and Europe. The elephants were divided into three groups based on disease status and history of exposure: (i) 26 animals with culture-confirmed TB due to M. tuberculosis or Mycobacterium bovis, (ii) 63 exposed elephants from known-infected herds that had never produced a culture-positive result from trunk wash samples, and (iii) 147 elephants without clinical symptoms suggestive of TB, with consistently negative trunk wash culture results, and with no history of potential exposure to TB in the past 5 years. Elephants with culture-confirmed TB and a proportion of exposed but trunk wash culture-negative elephants produced robust antibody responses to multiple antigens of M. tuberculosis, with seroconversions detectable years before TB-positive cultures were obtained from trunk wash specimens. ESAT-6 and CFP10 proteins were immunodominant antigens recognized by elephant antibodies during disease. The serologic assays demonstrated 100% sensitivity and 95 to 100% specificity. Rapid and accurate antibody tests to identify infected elephants will likely allow earlier and more efficient treatment, thus limiting transmission of infection to other susceptible animals and to humans.

Hakeem, A.Y., Sherwood, C.C., Bonar, C.J., Butti, C., Hof, P.R., Allman, J.M., 2009. Von Economo neurons in the elephant brain. Anat. Rec. (Hoboken. ) 292, 242-248.
Abstract: Von Economo neurons (VENs), previously found in humans, all of the great ape species, and four cetacean species, are also present in African and Indian elephants. The VENs in the elephant are primarily found in similar locations to those in the other species. They are most abundant in the frontoinsular cortex (area FI) and are also present at lower density in the anterior cingulate cortex. Additionally, they are found in a dorsolateral prefrontal area and less abundantly in the region of the frontal pole. The VEN morphology appears to have arisen independently in hominids, cetaceans, and elephants, and may reflect a specialization for the rapid transmission of crucial social information in very large brains

Hermes, R., Behr, B., Hildebrandt, T.B., Blottner, S., Sieg, B., Frenzel, A., Knieriem, A., Saragusty, J., Rath, D., 2009. Sperm sex-sorting in the Asian elephant (Elephas maximus). Anim Reprod. Sci. 112, 390-396.
Abstract: In captive Asian elephants, there is a strong need for production of female offspring to enhance reproduction, counter premature aging processes in female animals and reduce challenging management situations derived from husbandry of several bulls in one institution. Artificial insemination of flow cytometrically sex-sorted spermatozoa offers the possibility to predetermine the sex of offspring with high accuracy. The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometrical sex-sorting of Asian elephant spermatozoa. In total 18 semen samples were collected by manual rectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders (MES-HEPES-skim milk, MES-HEPES, TRIS-citric acid) and DNA staining. MES-HEPES-skim milk was the only semen extender found suitable to sex Asian elephant spermatozoa. From 18 ejaculates collected, 12 were successfully sorted with a purity of 94.5+/-0.7% at an average sort rate of 1945.5+/-187.5 spermatozoa per second. Sperm integrity, progressive and total motility were 42.6+/-3.9%, 48.1+/-3.3%, 59.4+/-3.8% after DNA labelling, and 64.8+/-3.2%, 58.0+/-5.0%, 70.8+/-4.4% after sorting, respectively. After liquid storage of sorted spermatozoa for 12h at 4 degrees C, sperm integrity, progressive and total motility were 46.4+/-5.2%, 32.2+/-4.2% and 58.2+/-3.9%, respectively. The obtained results provide a promising base to inseminate Asian elephants with sexed semen

Mason, G.J., Veasey, J.S., 2009. How should the psychological well-being of zoo elephants be objectively investigated?
47. Zoo. Biol.
Abstract: Animal welfare (sometimes termed "well-being") is about feelings - states such as "suffering" or "contentment" that we can infer but cannot measure directly. Welfare indices have been developed from two main sources: studies of suffering humans, and of research animals deliberately subjected to challenges known to affect emotional state. We briefly review the resulting indices here, and discuss how well they are understood for elephants, since objective welfare assessment should play a central role in evidence-based elephant management. We cover behavioral and cognitive responses (approach/avoidance; intention, redirected and displacement activities; vigilance/startle; warning signals; cognitive biases, apathy and depression-like changes; stereotypic behavior); physiological responses (sympathetic responses; corticosteroid output - often assayed non-invasively via urine, feces or even hair; other aspects of HPA function, e.g. adrenal hypertrophy); and the potential negative effects of prolonged stress on reproduction (e.g. reduced gametogenesis; low libido; elevated still-birth rates; poor maternal care) and health (e.g. poor wound-healing; enhanced disease rates; shortened lifespans). The best validated, most used welfare indices for elephants are corticosteroid outputs and stereotypic behavior. Indices suggested as valid, partially validated, and/or validated but not yet applied within zoos include: measures of preference/avoidance; displacement movements; vocal/postural signals of affective (emotional) state; startle/vigilance; apathy; salivary and urinary epinephrine; female acyclity; infant mortality rates; skin/foot infections; cardio-vascular disease; and premature adult death. Potentially useful indices that have not yet attracted any validation work in elephants include: operant responding and place preference tests; intention and vacuum movements; fear/stress pheromone release; cognitive biases; heart rate, pupil dilation and blood pressure; corticosteroid assay from hair, especially tail-hairs (to access endocrine events up to a year ago); adrenal hypertrophy; male infertility; prolactinemia; and immunological changes. Zoo Biol 28:1-19, 2009. (c) 2009 Wiley-Liss, Inc

Sherwood, C.C., Stimpson, C.D., Butti, C., Bonar, C.J., Newton, A.L., Allman, J.M., Hof, P.R., 2009. Neocortical neuron types in Xenarthra and Afrotheria: implications for brain evolution in mammals. Brain Struct. Funct. 213, 301-328.
Abstract: Interpreting the evolution of neuronal types in the cerebral cortex of mammals requires information from a diversity of species. However, there is currently a paucity of data from the Xenarthra and Afrotheria, two major phylogenetic groups that diverged close to the base of the eutherian mammal adaptive radiation. In this study, we used immunohistochemistry to examine the distribution and morphology of neocortical neurons stained for nonphosphorylated neurofilament protein, calbindin, calretinin, parvalbumin, and neuropeptide Y in three xenarthran species-the giant anteater (Myrmecophaga tridactyla), the lesser anteater (Tamandua tetradactyla), and the two-toed sloth (Choloepus didactylus)-and two afrotherian species-the rock hyrax (Procavia capensis) and the black and rufous giant elephant shrew (Rhynchocyon petersi). We also studied the distribution and morphology of astrocytes using glial fibrillary acidic protein as a marker. In all of these species, nonphosphorylated neurofilament protein-immunoreactive neurons predominated in layer V. These neurons exhibited diverse morphologies with regional variation. Specifically, high proportions of atypical neurofilament-enriched neuron classes were observed, including extraverted neurons, inverted pyramidal neurons, fusiform neurons, and other multipolar types. In addition, many projection neurons in layers II-III were found to contain calbindin. Among interneurons, parvalbumin- and calbindin-expressing cells were generally denser compared to calretinin-immunoreactive cells. We traced the evolution of certain cortical architectural traits using phylogenetic analysis. Based on our reconstruction of character evolution, we found that the living xenarthrans and afrotherians show many similarities to the stem eutherian mammal, whereas other eutherian lineages display a greater number of derived traits

Thitaram, C., Chansitthiwet, S., Pongsopawijit, P., Brown, J.L., Wongkalasin, W., Daram, P., Roongsri, R., Kalmapijit, A., Mahasawangkul, S., Rojanasthien, S., Colenbrander, B., van der Weijden, G.C., van Eerdenburg, F.J., 2009. Use of genital inspection and female urine tests to detect oestrus in captive Asian elephants
116. Anim Reprod. Sci. 115, 267-278.
Abstract: Captive Asian elephant (Elephas maximus) populations are decreasing due to low birth rates compared to wild elephants. Improving oestrous detection in female elephants is required to ensure successful mating in captive and semi-captive herds. Responsive behaviours of eight semi-captive bull elephants to the uro-genital area (genital inspection test) or urinary pheromones (urine test) of 14 female elephants throughout the oestrous cycle were evaluated. Weekly blood samples were collected for 27 consecutive months (14 months for the genital inspection test and 13 months for the urine test) from female elephants to characterize the patterns of circulating progestagen. Responsive behaviours of bulls were compared between females in the follicular versus the luteal phase of the cycle. The sensitivity and specificity of the genital inspection test were 65% and 68%, while those of the urine test were 52% and 61%, respectively. The bulls showed significantly higher "genital inspection", "flehmen from genital area" and "trunk on back" behaviours during the genital inspection test, and "flehmen" behaviours during the urine test in oestrous than in non-oestrous females. In sum, this study showed that monitoring sexual behaviours of Asian elephant bulls towards females or their urine can be used to detect the oestrous period. Although the sensitivity and specificity of both tests were not as high as expected, still, these methods appear to be more efficient at detecting oestrous than traditional methods based on mahout estimations of female receptivity. The use of genital inspection and urine tests may lead to more successful matings and thus to creating self-sustaining populations of captive elephants in range countries

Aupperle, H., Reischauer, A., Bach, F., Hildebrandt, T., Goritz, F., Jager, K., Scheller, R., Klaue, H.J., Schoon, H.A., 2008. Chronic endometritis in an Asian elephant (Elephas maximus). J. Zoo. Wildl. Med. 39, 107-110.
Abstract: A 48-yr-old female Asian elephant with a history of pododermatitis developed recurrent hematuria beginning in 2002. Transrectal ultrasonography and endoscopic examination in 2004 identified the uterus as the source of hematuria and excluded hemorrhagic cystitis. Treatment with Desloreline implants, antibiotics, and homeopathic drugs led to an improved general condition of the elephant. In July 2005, the elephant was suddenly found dead. During necropsy, the severely enlarged uterus contained about 250 L of purulent fluid, and histopathology revealed ulcerative suppurative endometritis with high numbers of Streptococcus equi ssp. zooepidemicus and Escherichia coli identified on aerobic culture. Additional findings at necropsy included: multifocal severe pododermatitis, uterine leiomyoma, and numerous large calcified areas of abdominal fat necrosis. Microbiologic culture of the pododermatitis lesion revealed the presence of Streptococcus agalactiae, Streptococcus equi ssp. zooepidemicus, Staphylococcus sp., Corynebacterium sp., and Entercoccus sp

Bechert, U., Southern, S., Chase, M. Minimally invasive molecular health analysis in elephants. Proc American Associaton of Zoo Veterinarians and Assoc of Reptile and Amphibian Veterinarians.  88. 2008. 11-10-2008.
Ref Type: Conference Proceeding
Abstract: This paper describes the application of a new assay platform called Stress Response Profiling (SRP) to the analysis of health status in elephants. SRP assays use a large biomarker panel as an indicator of chronically perturbed physiologic homeostasis ("chronic stress"),1,2 which is a known predictor of increased morbidity, infertility and mortality rates.3-8 SRP assays have a broad-based sensitivity to diverse types of stressors in multiple species of vertebrates.2 A minimally invasive SRP assay is based on skin microsamples obtained using routine biopsy procedures.9 The skin SRP assay was applied to captive African elephants with clinically diagnosed gastrointestinal infections and to healthy wild elephants.10 The elephant health status was classified using a reference database of SR biomarker profiles corresponding to eight species of normal and stressed animals. The biomarker profiles were converted into pathway profiles indicating that the molecular mechanism of the elephant gastrointestinal infections preferentially involved responses to misfolded proteins and DNA lesions. To rapidly and economically screen samples from 70 free-ranging African elephants sampled in Northern Botswana, we used a multiplexed SRP assay called multi-SRP.1,2 Statistical analysis of the multi-SRP scores showed correlations with population density, movements, and human-elephant conflict reports. In
summary, this paper documents that SRP and multi-SRP assays are suitable for the elephant skin and relevant to both symptomatic diseases and asymptomatic effects of environmental and anthropogenic stressors. We anticipate that the SRP technology might have a wide range of potential applications in veterinary medicine and ecosystem conservation.
LITERATURE CITED
1. Southern, S.O., A.C. Allen, and N. Kellar. 2002. Molecular signature of physiological stress in dolphins based on protein expression profiling of skin. Administrative Report LJ-02-27, National Marine Fisheries Service, SW Fisheries Science Center, La Jolla, California.
2. Southern, S.O., and G.W. Lilienthal. 2008. New technology for early detection of health threats. Proc. SPIE 69450F.
3. Camougrand, N., and M. Rigoulet. 2001. Aging and oxidative stress: studies of some genes involved both in aging and in response to oxidative stress. Respir. Physiol. 128:393-40.
4. Epel, E.S., J. Lin, F.H. Wilhelm, O.M. Wolkowitz, R. Cawthon, N.E. Adler, C. Dolbier, W.B. Mendes, and E.H. Blackburn. 2006. Cell aging in relation to stress arousal and cardiovascular disease risk factors. Psychoneuroendocrinology. 31:277-87.
5. Feder, M.E., and G.E. Hofmann. 1999. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Ann. Rev. Physiol. 61:243-82.
6. Kapahi, P., M.E. Boulton, and T.B.L. Kirkwood. 1999. Positive correlation between mammalian life span and cellular resistance to stress. Free Radical Biol. Med. 26:495-500.
7. Selye, H.A. 1936. Syndrome produced by diverse nocuous agents. Nature 138:32.
8. Wilson, J.F., and E.J. Kopitzke 2002. Stress and infertility Curr. Womens Health Rep. 2: 194

Drews, B., Hermes, R., Goritz, F., Gray, C., Kurz, J., Lueders, I., Hildebrandt, T.B., 2008. Early embryo development in the elephant assessed by serial ultrasound examinations. Theriogenology 69, 1120-1128.
Abstract: The elephant has an extraordinary long pregnancy, lasting 21 months. However, knowledge on embryo development is limited. To date, only single morphological observations of elephant embryo development associated with placentation are available, all lacking correlation to gestational age. The present study describes morphological characteristics of early embryo development in the elephant with exact biometric staging. Six pregnancies in five Asian and one African elephants with known conception dates were followed by 2D and 3D ultrasound, covering the embryonic period from ovulation to day 116 post-ovulation. The embryonic vesicle was earliest observed was on day 50 p.o. The proper embryo was not detected until day 62 p.o. Embryonic heartbeat was first observed on day 71 p.o. The allantois, which became visible as a single sacculation on day 71 p.o. was subdivided in four compartments on day 76 p.o. By day 95 p.o., head, rump, front and hind legs were clearly distinguished. Between days 95 and 103 p.o. the choriovitelline placenta was replaced by the chorioallantoic placenta. A physiological midgut herniation was transiently present between days 95 and 116 p.o. On the basis of the late appearance of the embryonic vesicle, delayed implantation in the elephant is discussed. The study provides a coherent description of elephant embryonic development, formation of the extraembryonic organs and their role in placenta formation, all of which are of interest for both comparative evolutionary studies and the improvement of assisted reproduction techniques

Kilgallon, C., Flach, E., Boardman, W., Routh, A., Strike, T., Jackson, B., 2008. Analysis of biochemical markers of bone metabolism in Asian elephants (Elephas maximus). J. Zoo. Wildl. Med. 39, 527-536.
Abstract: Two human enzyme immunoassays (EIA) and one radioimmunoassay (RIA) were validated and used to measure osteocalcin (OC), bone alkaline phosphatase (BAP), and the cross-linked telopeptide domain of type I collagen (ICTP), in serum from Asian elephants (Elephas maximus). Sera from four adult females sampled on 7 consecutive days were also analyzed to assess the existence and magnitude of intraindividual day-to-day variability of the serum concentration of these markers. Sample dilution curves were parallel with assay standard curves, which demonstrated that excellent cross reactivity existed between assay antibodies and elephants marker antigens. Statistically significant inverse correlations were found between age and concentrations of all three markers: BAP, r = -0.862 (P < 0.01); OC, r = -0.788 (P < 0.002); and ICTP, r = -0.848 (P < 0.01). Strong positive correlations were found between BAP and OC (r = 0.797, P < 0.01), OC and ICTP (r = 0.860, P < 0.01), and between BAP and ICTP (r = 0.958, P < 0.01). No statistically significant intraindividual variability was found over 7 days in the four adult females for any of the markers assessed (OC: P = 0.089; ICTP: P = 0.642; BAP: P = 0.146; n=4 in each case). The overall coefficient of variability observed in this group of animals was 10.3%, 7.4%, and 5.5% for OC, BAP, and ICTP, respectively. These results suggest a potential role for biochemical markers of bone turnover in monitoring skeletal health and bone disease in Asian elephants

Thongtip, N., Saikhun, J., Mahasawangkul, S., Kornkaewrat, K., Pongsopavijitr, P., Songsasen, N., Pinyopummin, A., 2008. Potential factors affecting semen quality in the Asian elephant (Elephas maximus). Reprod. Biol. Endocrinol. 6, 9.
Abstract: BACKGROUND: One of the major obstacles in using artificial insemination to manage genetics of elephant population in captivity is the large variations in semen quality among ejaculates within the same and among individuals. The objectives of this study were to determine the influences of (1) age (2) seasonality (3) and circulating testosterone (SrTest), triiodothyronine (SrT3) and tetraiodothyronine (SrT4), as well as seminal (4) testosterone (SpTest), zinc (SpZn) and protein (SpTP) on semen quality in the Asian elephant METHODS: Analyses, including motility, viability and morphology were performed in semen samples collected twice monthly from 13 elephant bulls (age range, 10-to 72-years) by manual stimulation between July 2004 and June 2005. Serum samples obtained monthly were assessed for SrTest, SrT3, SrT4, and seminal plasma samples were evaluated for, SpTest, SpZn and SpTP. RESULTS: The highest semen quality was observed at age 23 to 43 years. Percentages of progressive motility and viable sperm were lowest at age 51 to 70 years (P < 0.05); on the other hand, sperm concentration was lowest at age 10 to 19 years (P < 0.05). Percentage of sperm with normal morphology was highest at age 23 to 43 years. The levels of SrT3, SrTest, SpTest and SpZn were lowest at age 51 to 70 years, whereas SrT4 was lowest at age 23 to 43 years. Seasonality significantly affected semen characteristics in which percentage of viable sperm and cell concentration were highest during rainy season and lowest during summer months (P < 0.05). However, percentage of sperm with normal morphology was highest in summer and lowest in rainy season (P < 0.05). Seasonality significantly influenced SrTest with elevated concentrations observed in rainy season and winter (P < 0.05). CONCLUSION: This study indicates that age and seasonality had influence on semen characteristics in the Asian elephant. The knowledge obtained in this study will improve our understanding of the reproductive biology of this species

Viijoen, J.J., Ganswindt, A., du Toit, J.T., Langbauer, W.R., 2008. Translocation stress and faecal glucocorticoid metabolite levels in free-ranging African savanna elephants. South African Journal of Wildlife Research 38, 146-152.
Abstract: There are local populations of African elephants (Loxodonta africana) which have increased to levels where they are implicated in altering vegetation types. The local reduction of elephant numbers for wildlife management objectives can involve contraception, killing excess animals, or translocation to alternative habitats. The effects these management decisions can have on the physiological stress response of free-ranging African savanna elephants are still not fully understood. We examined the effect of translocation on faecal glucocorticoid metabolite levels of an African elephant family group, which was translocated within the Kruger National Park, South Africa. We found that translocation resulted in a significant increase in faecal glucocorticoid metabolite levels (up to 646 ng/g wet weight) compared to (1) pre-translocation levels in this group, (2) post-translocation levels in this group, and (3) levels measured in undisturbed 'control' groups in the area. However, the faecal glucocorticoid metabolite levels had returned to <100 ng/g by the time the translocated animals had navigated their way back to their previous home range, covering 300 km in 23 days.

Hildebrandt, T., Drews, B., Gaeth, A.P., Goeritz, F., Hermes, R., Schmitt, D., Gray, C., Rich, P., Streich, W.J., Short, R.V., Renfree, M.B., 2007. Foetal age determination and development in elephants. Proc. Biol. Sci. 274, 323-331.
Abstract: Elephants have the longest pregnancy of all mammals, with an average gestation of around 660 days, so their embryonic and foetal development have always been of special interest. Hitherto, it has only been possible to estimate foetal ages from theoretical calculations based on foetal mass. The recent development of sophisticated ultrasound procedures for elephants has now made it possible to monitor the growth and development of foetuses of known gestational age conceived in captivity from natural matings or artificial insemination. We have studied the early stages of pregnancy in 10 captive Asian and 9 African elephants by transrectal ultrasound. Measurements of foetal crown-rump lengths have provided the first accurate growth curves, which differ significantly from the previous theoretical estimates based on the cube root of foetal mass. We have used these to age 22 African elephant foetuses collected during culling operations. Pregnancy can be first recognized ultrasonographically by day 50, the presumptive yolk sac by about day 75 and the zonary placenta by about day 85. The trunk is first recognizable by days 85-90 and is distinct by day 104, while the first heartbeats are evident from around day 80. By combining ultrasonography and morphology, we have been able to produce the first reliable criteria for estimating gestational age and ontological development of Asian and African elephant foetuses during the first third of gestation.

Lacasse, C., Terio, K., Kinsel, M.J., Farina, L.L., Travis, D.A., Greenwald, R., Lyashchenko, K.P., Miller, M., Gamble, K.C., 2007. Two cases of atypical mycobacteriosis caused by Mycobacterium szulgai associated with mortality in captive African elephants (Loxodonta africana). J. Zoo. Wildl. Med. 38, 101-107.
Abstract: Mycobacterium szulgai was associated with mortality in two captive African elephants (Loxodonta africana) housed at Lincoln Park Zoo. The first elephant presented with severe, acute lameness of the left rear limb. Despite extensive treatments, the animal collapsed and died 13 mo after initial presentation. Necropsy revealed osteomyelitis with loss of the femoral head and acetabulum and pulmonary granulomas with intralesional M. szulgai. The second elephant collapsed during transport to another institution with no premonitory clinical signs. This animal was euthanized because of prolonged recumbency. Granulomatous pneumonia with intralesional M. szulgai was found at necropsy. Two novel immunoassays performed on banked serum samples detected antibody responses to mycobacterial antigens in both infected elephants. It was not possible to determine when the infection was established or how the elephants were infected. When reviewing the epidemiology of this organism in humans, however, transmission between elephants seemed unlikely because human-to-human transmission of this organism has never been reported and a third elephant in the herd was not affected. In addition to Mycobacterium bovis and Mycobacterium tuberculosis, atypical mycobacterial organisms need to be considered potentially pathogenic in elephants

Morley, R.C., van Aarde, R.J., 2007. Estimating abundance for a savanna elephant population using mark-resight methods: a case study for the Tembe Elephant Park, South Africa. Journal of Zoology 271, 418-427.
Abstract:  Elephants living in dense woodlands are difficult to count. Many elephant populations in Africa occur in such conditions. Estimates of these populations based on total counts, aerial counts and dung counts often lack information on precision and accuracy. We use standard mark-recapture field methods to obtain estimates of population size with associated confidence limits. We apply this approach to a closed elephant population in the Tembe Elephant Park (300 km(2)), South Africa. A registration count completed in 4 months gives a known population size. We evaluate mark-recapture models against the known population size. Individual identification profiles obtained for elephants during the registration count and mark-recapture events indicate that at least 167 elephants live in the park. We consider this value as an estimate of the minimum number alive. We include 189 sightings of bulls and 37 sightings of breeding herds in the mark-recapture modelling. Of the models we test (Petersen, Schnabel, Schumacher, Jolly-Seber, Bowden's, Poisson and negative binomial), Bowden's gives an estimate closest to the registration count. Assumptions of the model are not violated. For all models except one (negative binomial), our estimates improve with increased sampling intensity. Confidence intervals do not improve with increased effort except for the Schnabel model. Mark-recapture methods should be considered as reliable estimators of population size for elephants occurring in dense woodlands and forests when other methods cannot be relied on.

Wittemyer, G., Ganswindt, A., Hodges, K., 2007. The impact of ecological variability on the reproductive endocrinology of wild female African elephants. Hormones and Behavior 51, 346-354.
Abstract: Non-invasive endocrine methods enable investigation of the relationship between ecological variation and ovarian activity and how this impacts on demographic processes. The underlying physiological factors driving high variation in inter-calving intervals among multi-parous African elephants offer an interesting system for such an investigation. This study investigates the relationship between Normalized Differential Vegetation Index (NDVI), an ecosystem surrogate measure of primary productivity, and fecal progestin concentrations among wild female elephants. Matched fecal samples and behavioral data on reproductive activity were collected from 37 focal individuals during the two-year study. Linear mixed models were used to explore the relationship between fecal 5 alpha-pregnane-3-ol-20-one concentrations and the independent variables of NDVI, calf sex, female age, gestation day, and time since last parturition. Among both non-pregnant and pregnant females, fecal 5 alpha-pregnane-3-ol-20-one concentrations were significantly correlated with time-specific NDVI indicating a strong relationship between ecological conditions and endocrine activity regulating reproduction. In addition, the age of a female and time since her last parturition impacted hormone concentrations. These results indicate that the identification of an individual's reproductive status from a single hormone sample is possible, but difficult to achieve in practice since numerous independent factors, particularly season, impact fecal hormone concentrations. Regardless of season, however, fecal 5 alpha-pregnane-3-ol-20-one concentrations below 1 mu g/g were exclusively collected from non-pregnant females, which could be used as a threshold value to identify non-pregnant individuals. Collectively the information generated contributes to a better understanding of environmental regulation of reproductive endocrinology in wild elephant populations, information salient to the management and manipulation of population dynamics in this species.

Witter, K., Egger, G.F., Boeck, P., 2007. Renaut bodies in nerves of the trunk of the African elephant, Loxodonta africana. J. Morphol. 268, 414-422.
Abstract: Renaut bodies are loosely textured, cell-sparse structures in the subperineurial space of peripheral nerves, frequently found at sites of nerve entrapment. The trunk of the elephant is a mobile, richly innervated organ, which serves for food gathering, object grasping and as a tactile organ. These functions of the trunk lead to distortion and mechanical compression of its nerves, which can therefore be expected to contain numerous Renaut bodies. Samples of the trunk wall of an adult African elephant (Loxodonta africana) were examined histologically using conventional staining methods, immunohistochemistry, and lectin histochemistry. Architecture of nerve plexuses and occurrence of Renaut bodies in the elephant trunk were compared with those in tissues surrounding the nasal vestibule of the pig. Prominent nerve plexuses were found in all layers of the elephant trunk. Almost all (81%) nerve profiles contained Renaut bodies, a basophilic, discrete subperineurial layer resembling cushions around the nerve core. In contrast, Renaut bodies were seen in only 15% of nerve profiles in the porcine nasal vestibule. Within Renaut bodies, fusiform fibroblasts and round, ruff-like cells were placed into a matrix of acidic glycosaminoglycans with delicate collagen and very few reticular fibers. The turgor of this matrix is thought to protect nerves against compression and shearing strain. Renaut bodies are readily stained with alcian blue (pH 2.5) favorably in combination with immunohistochemical markers of nerve fibers. They should be regarded as a physiological response to repeated mechanical insults and are distinct from pathological alterations. alterations

Agnew, D.W. Brain removal in charismatic mega-vertebrates:  A not-so-charismatic chore.  2006 Proceedings American Association of Zoo Veterinarians.  2006.
Ref Type: Conference Proceeding
Abstract: Examination of the central nervous system, both grossly and histologically, is an important component of a complete necropsy.  Cerebral nematodiasis, West Nile Virus infection, rabies, distemper, and organophosphate toxicity are just a few of the possible diseases with serious herd and public health significance which may only be diagnosed by careful analysis of the brain and/or spinal cord.  Removal of the brain is strongly suggested for a complete necropsy, and though it may appear a daunting task, a few guidelines and power tools will allow efficient removal of the brain and a complete necropsy.
It is usually preferred that the brain be removed whole by removal of the skull cap.  This technique has been well documented in necropsy texts and is commonly taught in veterinary schools.  Briefly, after skinning the skull, a saw or ax may be used to cut on either side from the foramen magnum and the occipital condyles cranially and dorsally in a circular pattern (Fig. 1). This technique is useful to examine the brain in situ and remove it whole, but unfortunately requires skinning of the head, can be time-consuming, and is almost impossible to complete in rhinoceros and elephants. There are many alternative approaches to brain removal, but the author has found the following methods using commonly available tools are quick, leave a relatively intact skull, and the brain itself is removed in two parts.  Certainly, the techniques presented here can be adapted to the individual preferences of the prosector and to other similar species.  If nothing else, a discussion of brain removal techniques will reinforce the importance of collecting a complete set of tissues during a post-mortem examination.

Ball, R., Fad, O. Serum cortisols in captive Asian elephants (Elephas maximus) in different management systems at Busch Gardens Tampa Bay.  2006 Proceedings American Association of Zoo Veterinarians.  177-180. 2006.
Ref Type: Conference Proceeding
Abstract: Introduction:Cortisol is a widely accepted measure of stress in wild and captive animals.  In the past, captive elephant management systems have been criticized as potential stress inducers. The analysis of fecal cortisols is non-invasive and has been used to give long term evalutions of social and ecologic pressures in elephants and other species.  Salivary cortisols have also been used as a minimally invasive technique to measure social stress in captive elephants. The herd of Asian elephants at Busch Gardens Tampa Bay (BGT) changed from a traditional contact managemnt (free ccontact, FC) to a protected contact (PC) system utilizing positive-reinforcement based operant conditioning in 2004.  Serum cortisols were measured after the change and evaluated along wth banked samples from before. Long term sampling will be utilized to measure this transition but evaluating a single process will hopefully reflect the overall changes that can be expected with this change in management. While the individual variations are notable and other issues potentially confound the issue, it appears that this transition has lowered the serum cortisols in this herd.  In addition to serum cortisol measurements, the actual process of collecting the samples appears to be less stressful behaviorally. Pathologic processes should not be discounted when considering cortisol levels in evalauting stress in captive elephants.

Methods and Materials: Six female Asian elephants (Studbook numbers 30, 32, 304, 34, 35, 3) had been managed in a free contact system for many years.  Studbook number 304 was captive born and the others were wild born. Serum was collected intermittently during this management system to bank and for reproductive hormone analysis.  The elephants were placed in lateral recumbency by the handlers and blood collected from the ear vein on the caudal aspect of the down ear.  Reproductively sound animals were bled more frequently than the others.  Serum was frozen at -80°C until analyzed.  In August 2004, the first group of three animals was moved to the new barn and started the new positive-reinforcement, PC management system.  Within 5 wk, all animals had been moved over. All animals had been trunkwashed and were culture negative for Mycobacterium tuberculosis and negative on the newly developed MultiAntigen Print ImmunoAssay (MAPIA) and lateral-flow technology (Rapid Test) developed to detected antigen to M. tuberculosis.  As the caudal aspect of the ear was used for sampling, each elephant was asked to station in a static chute designed to allow training of voluntary ear-presentation for manipulation and blood collection. Handler safety and creating an effective learning environment for the elephants required training each to proceed to the chute solo and station there calmly. General desensitization techniques were applied as session durations were increased. Within the chute,individual elephants had significant room to maneuver. Since no physical restraint or sedation was utilized,animals were trained to cooperate fully and voluntarily allowing for blood sampling and other husbandry procedures. By May 2005, training for voluntary bloods draws was firmly established on all six animals.  The first approximately 20 samples collected under this new system were matched against the samples collected in the previous system.  Samples were selected against if the animal had an active problem or was on therapy for any reason.  Several animals had undergone a drug trial and these samples were selected against as well.  Serum was again stored in -80°C freezer until analyzed at Conservation and Research Center (CRC) Endocrine Research Laboratory, Smithsonian Institution, National Zoological Park, Front Royal, VA.  T-tests were utilized to discern any statistically significant results in the mean serum cortisols collected from animals before and after the implementation of the new husbandry systems.  Results were considered significant at alpha levels <0.05.

Results: The results and simple means of serum cortisols are listed in Table 1. Elephant No. 34 had essentially the same level of cortisol in both systems.  Elephant No. 32 had a reduction in the mean cortisol level of approximately 32% (20.84 versus 14.28 ng/ml) from the FC to the PC system.  Elephant No. 304 had a similar reduction of 37% in the mean cortisol (22.59 versus 14.29 ng/ml).  Statistical analyses results are reported here (means, standard deviations, t-test results).

Discussion: Serum was chosen over salivary and fecal sampling as a means to measure cortisol for several reasons. While fecal and salivary cortisol changes can reflect stresses within a reasonable period after the stressor (approximately 24 hr), serum cortisols is more likely to be reflective of the stressors closer to the moment of sampling.  The methodology is straightforward and less subject to the hazards for sample storage.  Timeliness of the sample result is also a benefit to serum sampling.  Blood sampling is a required husbandry practice in all elephant holding facilities belonging to the American Zoo and Aquarium Association (AZA).  While fecal cortisol samples may be useful to look at over a long term period to evaluate the transition from FC to PC, we choose to additionally look at how one specific task, blood collection, was affected by making this transition.  Fecal cortisols have been used to measure stress in transportation and environmental stress in some species, but are not thought to be reflective of the stress in a diagnostic procedure itself.  For this evaluation, the lag time period between the potential stressor (blood collection) and the means to measure the stressor are same.  Elephants No. 304 and 32 both had significant reductions in the mean serum cortisol levels.  Both are in good health and had no apparent inflammatory problems.  The logical deduction here is that the sampling process itself is less stressful in the PC management than the FC management.  Elephant 34 and 30 had essentially the same level of serum cortisol as measured by the mean in the different management systems.  Elephant 34 has developed significant uterine leiomyomas during the time period measured.  Elephant 30 has recently had clinical bouts of anterior enteritis and is suspected of having a dietary hypersensitivity to wheat.  Even with these two pathologic processes, the serum cortisol did not rise.  Elevations in cortisol are quite often explained as resulting from social, behavioral, or environmental causes and little attention is paid to inflammatory causes.  Associations between infections and elevated cortisols  have been noted in wild animals.  It is reasonable to assume that if these two processes did not exist, these levels would indeed be lower. Based on the other two elephants, a reduction of approximately 30% could be expected. Overall it appears that collecting blood from the elephants at BGT in the PC system is less stressful that the FC system.  As this is an example of how the routine husbandry and medical husbandry is now conducted, it can be expected that the overall net effect is going to be lowered stress in the elephants at BGT. ……………………………………………………………………………

Ball, R., Dumonceaux, G., Olsen, J., Burton, M.S., 2006. Comparison of trunk wash results matched to Multiantigen Print Immunoassay (MAPIA) in a group of captive Asian elephants (Elephas maximus). Proceedings International Elephant Conservation and Research Symposium 242-243.

Ball, R.L., Dumonceaux, G., Olsen, J.H., Burton, M.S., Lyashchenko, K. Comparison of trunk wash results matched to multiantigen print immunoassay (MAPIA) in a group of captive Asian elephants (Elephas maximus). 2006 Proceedings American Association of Zoo Veterinarians.  303-304. 2006.
Ref Type: Conference Proceeding
Abstract: Introduction: Between 1994 and June 2005, there were 34 confirmed cases of tuberculosis in elephants in the U.S. population. Thirty-one Asian (Elephas maximus) and three African (Loxodonta africana) elephants were affected. Mycobacterium tuberculosis was the etiologic agent in 33 cases and M. bovis in one case. Cases of tuberculosis caused by an unusual nontuberculous mycobacteria, M. szulgai have recently occurred as well.  Currently, TB in elephants remains a diagnostic dilemma. The sensitivity of trunk wash culture, the currently recommended test for diagnosis, is unknown. False negatives have been documented (trunk wash negative elephants that were subsequently found to be culture positive at necropsy).  Other non-culture techniques for TB diagnosis include ELISA, and PCR. A novel technology, MultiAntigen Print ImmunoAssay (MAPIA) and lateral-flow technology (Rapid Test)  has been evaluated and used to diagnose tuberculosis in captive elephants with encouraging results.  One concern with this serologic testing is the possibility of Mycobacterium other than tuberculosis (MOTT) cross-reacting with the antigen used in the Rapid Test or the MAPIA and leading to a false positive.  With numerous MOTT routinely cultured from trunk washes, this is a valid concern. Methods and Materials: A retrospective analysis was done at Busch Gardens Tampa Bay and Chembio, Inc. that matched trunk wash results to serum samples.  All serum was collected within 7 days of the trunk wash and analyzed with the Rapid Test and MAPIA. Four Asian elephants with a total of 18 samples met this criteria and had serum submitted for testing. Results and Discussion: Table 1 lists the results and the organisms cultured. While the sampling is limited in this pilot project, it appears that MOTT does not evoke a response when assayed with the Rapid Test or MAPIA. The recent cases of M. szulgai do demonstrate the potential usefulness for this test when a disease develops from MOTT.  The usefulness of this new technology, taken in conjunction with other clinical data including trunk washes when indicated, is a valuable tool in the healthcare of captive elephants.

LITERATURE CITED
1 Lacasse, C., K.C. Gamble, K. Terio, L.L. Farina, D.A. Travis, and M.Miller. 2005. Mycobacterium szulgai osteroarthritis and pneumonia in an African elephant (Loxdonta africana). Proc. Am. Assoc. Zoo Vet. Ann. Meet. Pp. 170-172.
2 Larsen, R.S., M.D. Salman, S.K. Mikota, R. Isaza, R.J. Montali, and J. Triantis.  2000.  Evaluation of a multiple-antigen enzyme-linked immunosorbent assay for detection of Mycobacterium tuberculosis infection in captive elephants.  J. Zoo Wildl. Med. 31:291-302.
3 Lyashchenko, K., et al.  2000. A multiantigen print immunoassay for the serological diagnosis of infectious diseases.  J. Immunol. Methods  242:91-100
4 Lyashchenko, K., M. Miller, and  W.R. Waters. 2005. Application of multiple antigen print immunoassay and rapid lateral flow technology for tuberculosis testing of elephants. Proc. Am. Assoc. Zoo Vet. Ann. Meet.  Pp. 64-65

Drews, B., Göritz, F., Hermes, R., Streich, J.W., Rich, P., Schmitt, D., Lung, N., Renfree, M.B., Gaeth, A.P., Short, R.V., Hildebrandt, T.B. Morphological and ultrasonographic characterization of the embryonic development in elephants. Proceedings International Elephant Conservation & Research Symposium.  82-83. 2006. 2006.
Ref Type: Conference Proceeding

Fidgett, A.L., Newman, E.C., Sanderson, S. Using faecal analysis as an indicator of dental condition: A case study at Chester Zoo. Proceedings International Elephant Conservation & Research Symposium.  250. 2006. 2006.
Ref Type: Conference Proceeding

Hildebrandt, T.B., Hermes, R., Ratanakorn, P., Rietschel, W., Fickel, J., RetNat, D., Frey, R., Reid, C., Goeritz, F. Ultrasonographic assessment and ultrasound-guided biopsy of the retropharyngeal lymph nodes in elephants.  2006 Proceedings American Association of Zoo Veterinarians.  117-118. 2006.
Ref Type: Conference Proceeding
Abstract: So far there are no valid diagnostic tools available for identifying latent carriers of endotheliotropic elephant herpes virus (EEHV).  For this reason, the lateral retropharyngeal lymph node complex (LARELYNOC) of elephants, identified during postmortem studies as target organ for EEHV and suitable for transcutaneous biopsy, was grossly described. Transcutaneous ultrasound (3.5 MHz) was applied behind the ear region to identify the LARELYNOC containing up to four single lymph nodes on each side. The lymph node tissue is situated 20-50 mm below the skin surface. An ultrasonographic assessment of the LARELYNOC and two biopsies were performed on 39 healthy Asian elephants (Elephas maximus). Samples were tested for EEHV via PCR. Whole blood samples were also collected and tested for active EEHV infection. Lymph nodes were ultrasonographically classified as active (calculated mean volume=17.4 ± 6.9 cm3, P>0.001), inactive (calculated mean volume=3.1 ± 0.6 cm3, P<0.001), or chronic active (calculated mean volume=10.6 ± 1.0 cm3, P<0.05). Histology confirmed not only the presence of lymph tissue but also the ultrasonographically diagnosed reactivity status of the lymph node biospies.  Although all samples including whole blood were found to be negative for the EEHV DNA particles, the successful development of this procedure in elephants could prove beneficial for the screening of not only latent EEHV infections but might also be a less dangerous alternative method for the diagnosis of zoonotic infections such as tuberculosis.

Kilgallon, C., Flach, E., Boardman, W., Routh, A., Strike, T., Jackson, B. Biochemical markers of bone in Asian elephants (Elephas maximus): a cross sectional analysis of two serum markers of bone formation and one serum marker of bone resorption.
2006 Proceedings American Association of Zoo Veterinarians.  183-184. 2006.
Ref Type: Conference Proceeding
Abstract: Conventional radiography has traditionally been employed for investigations of skeletal disease of captive elephants.  However, it is predominantly cortical bone which is assessed by standard radiography, and quantitative assessment of bone is only possible when pathology is advanced. A precise and relatively non-invasive method of quantitatively assessing bone, in isolation, or as a compliment to standard radiography would have positive health and welfare implications for elephants, because skeletal disease is prevalent in both extant species in captivity. The advent of biochemical markers of bone metabolism represents a watershed in non-invasive diagnostics of normal bone homeostasis and pathology in humans and animals alike. These markers are classified as markers of formation and resorbtion and are comprising of enzymes expressed by osteoblasts or osteoclasts, or organic compounds released during the synthesis or resorption of bone matrix. In this study, two human enzyme immunoassays (METRA™ Osteocalcin EIA kit, METRA™ BAP EIA kit, Quidel Corporation, San Diego, California 92121 USA) and one radioimmunoassay (UniQ™ ICTP RIA, Orion Diagnostica, Espoo, Finland) were validated and used to measure osteocalcin (OC), bone alkaline phosphatase (BAP), and the C-terminal telopeptide domain of type I collagen (ICTP) respectively, three biochemical markers of bone, in serum procured from a small sample population (n=12) of captive Asian elephants (Elephas maximus) of various ages, from three European zoos. Serum from four adult females sampled on 7 days consecutively were as also analyzed to assess the existence and magnitude of the intra-individual, day-to-day variability of these markers. Excellent cross reactivity was found to exist between assay antibodies and elephants marker antigens. Significant inverse correlations were found between the age of the animals and concentrations of all three markers. Strong significant positive correlations were also noted between serum concentrations of all three markers. No statistically significant intra-individual variability was found over 7 days in the population of adult females for any of the markers assessed. The results suggest a promising role for biochemical markers of bone turnover in monitoring skeletal growth and bone disease in captive Asian elephants.

Lyashchenko, K.P., Greenwald, R., Esfandiari, J., Olsen, J.H., Ball, R., Dumonceaux, G., Dunker, F., Buckley, C., Richard, M., Murray, S., Payeur, J.B., Andersen, P., Pollock, J.M., Mikota, S., Miller, M., Sofranko, D., Waters, W.R., 2006. Tuberculosis in elephants: antibody responses to defined antigens of Mycobacterium tuberculosis, potential for early diagnosis, and monitoring of treatment
438. Clin. Vaccine Immunol. 13, 722-732.
Abstract: Tuberculosis (TB) in elephants is a re-emerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current diagnosis relies on trunk wash culture, the only officially recognized test, which has serious limitations. Innovative and efficient diagnostic methods are urgently needed. Rapid identification of infected animals is a crucial prerequisite for more effective control of TB, as early diagnosis allows timely initiation of chemotherapy. Serology has diagnostic potential, although key antigens have not been identified and optimal immunoassay formats are not established. To characterize the humoral responses in elephant TB, we tested 143 serum samples collected from 15 elephants over time. These included 48 samples from five culture-confirmed TB cases, of which four were in Asian elephants infected with M. tuberculosis and one was in an African elephant with Mycobacterium bovis. Multiantigen print immunoassay (MAPIA) employing a panel of 12 defined antigens was used to identify serologic correlates of active disease. ESAT-6 was the immunodominant antigen recognized in elephant TB. Serum immunoglobulin G antibodies to ESAT-6 and other proteins were detected up to 3.5 years prior to culture of M. tuberculosis from trunk washes. Antibody levels to certain antigens gradually decreased in response to antitubercular therapy, suggesting the possibility of treatment monitoring. In addition to MAPIA, serum samples were evaluated with a recently developed rapid test (RT) based on lateral flow technology (ElephantTB STAT-PAK). Similarly to MAPIA, infected elephants were identified using the RT up to 4 years prior to positive culture. These findings demonstrate the potential for TB surveillance and treatment monitoring using the RT and MAPIA, respectively

Mikota, S.K., Dumonceaux, G., Miller, M., Gairhe, K., Giri, K., Cheeran, J.V., Abraham, D., Lyashchenko, K., Larsen, S., Payeur, J., Waters, R., Kaufman, G., \, 2006. Tuberculosis in elephants: An update on diagnosis and treatment; implications for control in range countries. Proceedings International Elephant Conservation and Research Symposium 109-118.

Mikota, S.K., Miller, M., Dumonceaux, G., Giri, K., Gairhe, K., Hamilton, K., Paudel, S., Vincent, B. Elephant tuberculosis diagnosis: implications for elephant management in Asian range countries. 2006 Proceedings American Association of Zoo Veterinarians.  142-143. 2006.
Ref Type: Conference Proceeding
Abstract: Serologic tests including the ELISA, MAPIA (Multi-Antigen Print Immunoassay), and a rapid test, VetTB StatPak® (Chembio Diagnostic Systems, Inc., Medford, New York 11763 USA) have recently been developed and show great promise for the diagnosis of tuberculosis (TB) in elephants. These serologic tests detect antibodies to antigens of  Mycobacterium tuberculosis complex organisms and in some cases have detected infection years in advance of active disease and mycobacterial shedding. The diagnosis of active TB (by culture) or serologic conversion presents management challenges for captive elephants in Asian range countries.  Of the 2 billion humans world-wide infected with TB, fewer than 10% will develop active disease. This figure is unknown for elephants. The identification and management of infected elephants has ramifications for elephants and humans alike and issues such as public health and tourism may be impacted. TB is endemic among humans in Asia and where there is intermingling of elephants and humans, both species may act as reservoirs for disease transmission.  The various situations in which elephants are kept in Asia (government-owned, privately-owned, festivals, temples, zoos, etc.) make it difficult to develop a management strategy that will address all circumstances.  Other concerns are the cost of treatment for an elephant (~ $50,000 USD) and appropriate monitoring in resource-poor countries. The authors have recently undertaken the screening of 120 elephants in Nepal to further evaluate the above-mentioned (and other) diagnostic tests.  To our knowledge, this is the first organized, large-scale initiative to screen Asian elephants within a range country.  Preliminary discussions regarding the management of both culture and serologically positive government-owned and privately-owned elephants in Nepal have been initiated and may serve as a starting point for other countries as more elephants are screened within Asia.  Basic options for active (culturepositive) cases include (1) treatment, (2) segregation or (3) euthanasia.  Options for latent disease (culture-negative, serologically positive) cases include (1) treatment, (2) segregation and monitoring for active disease and (3) euthanasia.  The particular ownership/husbandry system, available resources and cultural constraints may dictate final management choices in range countries.

Moller, T., Roken, B.O., Lewerin, S.S., Lyashchenko, K., 2006. The elephant Rapid Test (RT) the future diagnostic test for TB (M. tuberculosis) in elephants? Call for a validation study in Europe. Proceedings International Elephant Conservation and Research Symposium 119-124.

Riley, L.W., 2006. Of mice, men, and elephants: Mycobacterium tuberculosis cell envelope lipids and pathogenesis
454. J. Clin. Invest 116, 1475-1478.
Abstract: Mycolic acids and structures attached to them constitute a major part of the protective envelope of Mycobacterium tuberculosis, and for this reason, their role in tuberculosis pathogenesis has been extensively studied. In this issue of the JCI, Rao et al. examine the effect of trans-cyclopropanation of oxygenated mycolic acids attached to trehalose dimycolate (TDM) on the murine immune response to infection (see the related article beginning on page 1660). Surprisingly, they found that an M. tuberculosis mutant lacking trans-cyclopropane rings was hypervirulent in mice. The recent recognition of a hypervirulence phenotype in mice associated with laboratory and clinical M. tuberculosis strains with altered cell wall components has provided new insights into how M. tuberculosis may establish persistent infection. However, to date, characterization of these bioactive products in pathogenesis has been largely reductionistic; the relationship of their effects observed in mice to the persistent infection and tuberculosis caused by M. tuberculosis observed in humans remains obscure

Siegal-Willott, J., Isaza, R., Johnson, R., Blaik, M. Clinical evaluation of distal limb radiography and growth plate closure in the juvenile Asian elephant  (Elephas maximus).
2006 Proceedings American Association of Zoo Veterinarians.  181-182. 2006.
Ref Type: Conference Proceeding
Abstract: The thoracic limb digits of 11 healthy juvenile Asian elephants (Elephas maximus) were evaluated radiographically to assess normal developmental anatomy.  Parameters evaluated included: the location(s) of centers of ossification, relative age at time of phalangeal ossification, and relative times of growth plate closure in the bones of the distal forelimb.  Specifically, the third phalanx (P3) of each digit was evaluated, as well as the first (P1) and second (P2) phalanges of the third digit (D3). A retrospective evaluation of radiographs from juvenile elephants was also done to augment the data set.  This study reports the methods used to obtain high-quality radiographs of the elephant foot, the locations of centers of ossification based on radiographic evaluation, and the relative times of growth plate closure within the digital bones. The settings used to obtain the radiographs used in this study for P3 are presented in Table 1. Radiographs of D3, P1, and P2 were obtained in a similar manner, using a 45° angle for focal spot positioning.  The kilovoltage power and milliampere seconds were adjusted as needed. Radiographic evaluation of the juvenile Asian elephants revealed variability in the shape of P3 based on age of the animal and degree of ossification of P3.  The relative times of growth plate closure and number of ossifications were also determined.  The information presented will help clinicians in radiographing elephants, interpreting foot radiographs, and recognizing normal versus abnormal anatomy.  It will also help in aging juvenile elephants, investigating diseases and deaths, and recognizing normal patterns of toe and foot development.

Suedmeyer, W.K., Fine, D. Indirect oscillometric blood pressure measurement in four African elephants (Loxodonta africana).
2006 Proceedings American Association of Zoo Veterinarians.  170-172. 2006.
Ref Type: Conference Proceeding
Abstract: The elephant is the largest living land mammal and in danger of extinction. The few literature citations involving blood pressure (BP) measurements have utilized direct arterial measurement of immobilized or stationary conditioned elephants. These investigations determined that BP's in the healthy elephant are generally higher than most other clinically normal mammals studied but similar to unsedated domestic cattle and horses, and increased in laterally recumbent elephants. This project was undertaken to compare cited direct arterial measurements to indirect oscillometric BP measurement of systolic, diastolic, and mean arterial pressure (MAP), and heart rate (HR) in four stationary, non-sedated African elephants. Four female African elephants ranging in age from 28-38 yr of age were used in this study. One elephant (E3) had a history of fetal retention of 5 yr and bilateral scleral injection but was clinically normal in all other regards. The three remaining elephants had no significant clinical histories. All four elephants were conditioned to present the tail for placement of a standard occlusive BP cuff (Cardell™, CAS Medical Systems, Inc. Branford, Connecticut 06405 USA). Use of this indirect oscillometric unit has been compared with simultaneous direct arterial measurement in anesthetized African lions (Panthera leo), and an immobilized African elephant at the Kansas City Zoo. Blood pressure results in each animal studied were virtually identical in both techniques. The width of the cuff was approximately 40% the circumference of the tail (12 cm cuff on an average 27.5 cm tail circumference) of the elephant, in accordance with general recommendations for obtaining BP measurements in domestic animals. Cuff placement was at the distal extent of the caudal tail fold. Three sets of BP's, heart rates, and respiratory rates were obtained on three different occasions in each elephant (Table 1). Each elephant was sampled at the same time of day and had not been exercised. Blood pressure measurements obtained in three of the four elephants in this population compared favorably with reference ranges obtained invasively (direct arterial) in unsedated African elephants. In the elephant with scleral injection and retained fetal mummy (E3), overall BP measurements were higher, on average, than the other three elephants and ranges reported in a previous study of direct arterial pressures in unsedated African elephants. This may reflect a hypertensive state related to increased systemic vascular resistance associated with a retained calf. However, this elephant is the oldest of the four animals studied, and blood pressure parameters generally increase with age in humans and this may be the case with this elephant. Further investigation into the potential causes for a clinical hypertensive state in this elephant is being pursued. The advantages of this technique are the non-invasive application, portability, and comparable results to direct arterial measurement. Disadvantages are that BP measurement can be altered by cuff size, placement, and movement. In this study, cuff placement and size was identical in all elephants, and the only movement was associated with masticatory efforts involved with positive food enrichment, eliminating two of the three variables. Additional elephants are being evaluated and refinement of BP measurement techniques is being completed to help define normal indirect oscillometric BP values in the African elephant. Use of an indirect oscillometric measuring device for obtaining BP measurements in African elephants may prove to be an easily applied valuable ancillary diagnostic tool when evaluating cardiovascular parameters without the need for sedation or immobilization.

Tremblay, Y., Shaffer, S.A., Fowler, S.L., Kuhn, C.E., McDonald, B.I., Weise, M.J., Bost, C.A., Weimerskirch, H., Crocker, D.E., Goebel, M.E., Costa, D.P., 2006. Interpolation of animal tracking data in a fluid environment. J Exp Biol. 209 (Pt 1), 128-140.
Abstract: Interpolation of geolocation or Argos tracking data is a necessity for habitat use analyses of marine vertebrates. In a fluid marine environment, characterized by curvilinear structures, linearly interpolated track data are not realistic. Based on these two facts, we interpolated tracking data from albatrosses, penguins, boobies, sea lions, fur seals and elephant seals using six mathematical algorithms. Given their popularity in mathematical computing, we chose Bézier, hermite and cubic splines, in addition to a commonly used linear algorithm to interpolate data. Performance of interpolation methods was compared with different temporal resolutions representative of the less-precise geolocation and the more-precise Argos tracking techniques. Parameters from interpolated sub-sampled tracks were compared with those obtained from intact tracks. Average accuracy of the interpolated location was not affected by the interpolation method and was always within the precision of the tracking technique used. However, depending on the species tested, some curvilinear interpolation algorithms produced greater occurrences of more accurate locations, compared with the linear interpolation method. Total track lengths were consistently underestimated but were always more accurate using curvilinear interpolation than linear interpolation. Curvilinear algorithms are safe to use because accuracy, shape and length of the tracks are either not different or are slightly enhanced and because analyses always remain conservative. The choice of the curvilinear algorithm does not affect the resulting track dramatically so it should not preclude their use. We thus recommend using curvilinear interpolation techniques because of the more realistic fluid movements of animals. We also provide some guidelines for choosing an algorithm that is most likely to maximize track quality for different types of marine vertebrates. University of California, Santa Cruz, Long Marine Laboratory, Center for Ocean Health, 100 Shaffer Road, Santa Cruz, CA 95060, USA. tremblay@biology.ucsc.edu

Varma, S., Pittet, A., Jamadagni, H.S., 2006. Experimenting usage of camera-traps-for population dynamics study of the Asian elephant Elephas maximus in southern India. Current Science 91, 324-331.
Abstract: To evaluate the application of camera-trap technology in population dynamics studies of the Asian elephant, indigenously designed, cost-effective infrared-triggered camera-traps were used. Usability of pictures was defined based on quality, clarity and positioning of the subject. With 99 pictures of 330 elephants, 20 sequence's were obtained and 44 distinct individuals were identified. It was found that 38.6% were adult females, 4.5% adult males 13.6% sub-adult females, 6.8% sub-adult males, 20.4% juvenile females, while juvenile males were poorly represented (2%), and 13.6% were calves. These results were surprisingly identical with those of other systematic and long-term studies.

Weissengruber, G.E., Egger, G.F., Hutchinson, J.R., Groenewald, H.B., Elsasser, L., Famini, D., Forstenpointner, G., 2006. The structure of the cushions in the feet of African elephants (Loxodonta africana)
380. Journal of Anatomy 209, 781-792.
Abstract: The uniquely designed limbs of the African elephant, Loxodonta africana, support the weight of the largest terrestrial animal. Besides other morphological peculiarities, the feet are equipped with large subcutaneous cushions which play an important role in distributing forces during weight bearing and in storing or absorbing mechanical forces. Although the cushions have been discussed in the literature and captive elephants, in particular, are frequently affected by foot disorders, precise morphological data are sparse. The cushions in the feet of African elephants were examined by means of standard anatomical and histological techniques, computed tomography (CT) and magnetic resonance imaging (MRI). In both the forelimb and the hindlimb a 6th ray, the prepollex or prehallux, is present. These cartilaginous rods support the metacarpal or metatarsal compartment of the cushions. None of the rays touches the ground directly. The cushions consist of sheets or strands of fibrous connective tissue forming larger metacarpal/metatarsal and digital compartments and smaller chambers which were filled with adipose tissue. The compartments are situated between tarsal, metatarsal, metacarpal bones, proximal phalanges or other structures of the locomotor apparatus covering the bones palmarly/plantarly and the thick sole skin. Within the cushions, collagen, reticulin and elastic fibres are found. In the main parts, vascular supply is good and numerous nerves course within the entire cushion. Vater-Pacinian corpuscles are embedded within the collagenous tissue of the cushions and within the dermis. Meissner corpuscles are found in the dermal papillae of the foot skin. The micromorphology of elephant feet cushions resembles that of digital cushions in cattle or of the foot pads in humans but not that of digital cushions in horses. Besides their important mechanical properties, foot cushions in elephants seem to be very sensitive structures

Weissengruber, G.E., Fuss, F.K., Egger, G., Stanek, G., Hittmair, K.M., Forstenpointner, G., 2006. The elephant knee joint: morphological and biomechanical considerations
513. Journal of Anatomy 208, 59-72.
Abstract: Elephant limbs display unique morphological features which are related mainly to supporting the enormous body weight of the animal. In elephants, the knee joint plays important roles in weight bearing and locomotion, but anatomical data are sparse and lacking in functional analyses. In addition, the knee joint is affected frequently by arthrosis. Here we examined structures of the knee joint by means of standard anatomical techniques in eight African (Loxodonta africana) and three Asian elephants (Elephas maximus). Furthermore, we performed radiography in five African and two Asian elephants and magnetic resonance imaging (MRI) in one African elephant. Macerated bones of 11 individuals (four African, seven Asian elephants) were measured with a pair of callipers to give standardized measurements of the articular parts. In one Asian and three African elephants, kinematic and functional analyses were carried out using a digitizer and according to the helical axis concept. Some peculiarities of healthy and arthrotic knee joints of elephants were compared with human knees. In contrast to those of other quadruped mammals, the knee joint of elephants displays an extended resting position. The femorotibial joint of elephants shows a high grade of congruency and the menisci are extremely narrow and thin. The four-bar mechanism of the cruciate ligaments exists also in the elephant. The main motion of the knee joint is extension-flexion with a range of motion of 142 degrees . In elephants, arthrotic alterations of the knee joint can lead to injury or loss of the cranial (anterior) cruciate ligament

Wiedner, E., Alleman, R., Isaza, R. Urinalysis in Asian elephants (Elephas maximus).
2006 Proceedings American Association of Zoo Veterinarians.  265. 2006.
Ref Type: Conference Proceeding
Abstract: Elephants have been reported with renal disease, and arthritic elephants are often maintained on long-term administration of non-steroidal anti-inflammatory drugs which have been associated with renal injury in other species.  Therefore, establishment of the reference values of normal urine would be a useful tool to elephant health care. Routine urinalysis was performed on urine collected from healthy female adult Asian elephants (Elephas maximus, n=30). Elephants were included in the study if they had normal serum urea nitrogen and creatinine serum concentrations, no history of urinary tract disease, and had not received any medication within the past 3 mo.  Urinalysis included gross description of urine color and clarity, measurement of pH and specific gravity, biochemical analysis and sediment evaluation.   Presence of protein was assessed by the sulfosalicylic acid tubidimetric test.  All urine samples were submitted for aerobic bacterial culture. Initial findings included identification of calcium carbonate crystals in the urine of most of the animals in this study; some (n=5) contained small numbers of struvite crystals (magnesium ammonium phosphates). Most samples were alkaline and clarity ranged from clear to flocculent. Specific gravity tended to be fairly low, and isosthenuria was frequent. Trace bilirubin was detected in some samples.  No ketonuria or proteinuria was found.  Glucosuria, which has been reported as a transient and normal finding in captive elephants, was not detected in any samples.

Wynne, J., Greer, L. Management of digital osteomyelitis in an Asian elephant (Elephas maximus).
2006 Proceedings American Association of Zoo Veterinarians.  185-186. 2006.
Ref Type: Conference Proceeding
Abstract: A 47-yr-old female Asian elephant was diagnosed with osteomyelitis of the left front digit 5, involving phalynges 1 and 2. Based on culture results of Pseudomonas and Bacteroides, enrofloxacin and metronidazole rectal suppository treatment was started. Serum levels were measured and different formulations were developed to attempt to deliver appropriate drug levels. The osteomyelitis progressed over the next 55 days. Enrofloxacin was discontinued based on culture and sensitivities (C&S) and regional limb perfusion (RLP) using amikacin started. From this point on, daily treatments with RLP have been performed. The 3-g amikacin dose was based on 5% of the elephant's systemic dose.  Two weeks later, RLP with 6 g of ampicillin was started on alternate days based on C&S, and the following week, 400 mg fluconazole was added on a third day in response to C&S and tissue biopsies indicating invasive Candida. Despite aggressive medical therapy, radiographs and bone biopsy indicated the osteomyelitis continued. Surgery was performed 3 mo after systemic antibiotics were initiated.  All infected bone and tissue was identified with methylene blue, and removed.  Only the most proximal third of P1 remained post surgery.  Post surgery, daily sterile bandage changes were performed and rotational RLP treatment was continued with amikacin (8 g), ampicillin (15 g), and fluconazole (800 mg).  This daily treatment regime, with some drug adjustments, has been continued for 6 mo. One month after surgery P1 was radiolucent at the distal margin, and was progressing to a fragmented appearance, indicating the osteomyelitis may still be present.  Amikacin serum levels were collected post RLP, before the tourniquet was removed.  Systemic theraputic levels were reached, but not the recommended 10 times MIC. Amikacin was replaced with 12 g of ceftazidime in the RLP rotation. Two months post surgery a fragment of the remaining P1 was easily biopsied from the healing surgical tract with culture results indicating Enterococcus, but not Pseudomonas. Three months post surgery we reinstituted enrofloxacin suppositories at a higher dose. At 5 mo post surgery, cultures indicated that we had successfully eliminated Pseudomonas and anaerobic growth; however, the healing site continued to yield various gram-negative bacteria, including a Klebsiella resistant to ceftazidine.  We replaced ceftazidine with 12 g of ceftriaxone and continued ampicillin and fluconazole in the 3-day RLP rotation. Since this last medical alteration the remaining P1 fragments have been radiographically unchanged for 3 mo and the surgical wound has been reduced to a tract that is <2 mm in diameter and 4 cm deep. The current success of this treatment is attributed to a very tractable patient that has allowed daily medical care for over 8 mo. We are continuing her daily treatments and I will give an update on the progression of the case.

Zuba, J.R., Oosterhuis, J.E., Pessier, A.P. The toenail "abscess" in elephants: treatment options including cryotherapy and pathologic similarities with equine proliferative pododermatitis (canker).  2006 Proceedings American Association of Zoo Veterinarians.  187-190. 2006.
Ref Type: Conference Proceeding
Abstract: Foot problems potentially represent the single most important clinical disease of captive elephants.  Predisposing factors include obesity, lack of exercise, nail or sole overgrowth, improper foot care, poor hygiene, inappropriate enclosure surfaces, poor conformation, malnutrition and secondary skeletal disorders such as degenerative joint disease.  Furthermore, factors such as elephant management philosophy, disposition of elephants, facilities and competency of staff in caring for elephant feet will contribute significantly to the foot health of captive animals.  It is important to note that these conditions are rarely reported in free-ranging elephants. The elephant toenail abscess is characterized grossly by proliferative outgrowth of "crab meat-like" tissue that may acutely rupture through the surface of the nail wall and/or adjacent cuticle or sole. True abscess formation with localized collections of suppurative material is not a consistent clinical feature.  In most cases, the inciting cause of these lesions are typically not found and are likely due to one or more of the predisposing factors listed above.  Once established, these frustrating lesions require extensive, intensive and prolonged medical attention.  If not cared for properly, these wounds may progress to phalangeal osteomyelitis and the need for surgical intervention.  Sole abscesses are equally frustrating and difficult to manage with proposed etiologies similar to toenail lesions. There are no reports in the literature describing the pathology of the classic proliferative abscess tissue of the elephant nail abscess.  Although variously interpreted as fibrous or granulation tissue, the authors are unaware of previous histologic descriptions of this tissue.  Biopsy samples of toenail abscess tissue from two Asian elephants (Elephas maximus) at the San Diego Wild Animal Park (SDWAP) consisted of stratified squamous epithelium arranged in columns resembling horn tubules.  The predominant histologic finding was marked, near diffuse, hydropic degeneration of keratinocytes.  There were multifocal areas of suppurative inflammation with admixed bacterial colonies.  Inflammatory foci comprised only a small portion of the lesion and were interpreted as the external surfaces of the biopsy with likely secondary bacterial colonization. Because descriptions of the normal histology of the elephant toenail could not be located, a grossly normal toenail from a different Asian elephant was obtained to compare histologic features with those of the toenail abscesses.  Sections demonstrated formation of the toenail in a manner similar to that of the hoof of the horse and cattle with tubular, intertubular and laminar horn.  Primary and secondary epidermal laminae were identified.  Proliferative lesions of horn-producing epithelium associated with ballooning degeneration and inadequate keratinization of keratinocytes, have been described in horses as equine "canker" and coronary band dystrophy.  Equine canker is most commonly observed in the hind feet of draft horses and begins in the frog sometimes with extension to the sole and hoof wall.  Grossly, lesions are characterized by soft white papillary to "cauliflower-like" tissue associated with a foul odor. Similar to what is noted in elephant foot problems, predisposing factors for the development of equine canker include poor hygiene or wet environmental conditions. There is a lack of gross and histologic description of the normal nail and sole tissue of the elephant and further investigations are warranted.  A review of the anatomy and histology of the normal equine hoof may provide a basic understanding of the elephant nail until more specific and detailed elephant information is available.  From our investigation, the authors offer that a more accurate description of the elephant toenail abscess would be proliferative pododermatitis, the term synonymous with equine canker.  A more colloquial term such as "elephant canker" may be appropriate, as well. Canker in the horse is an uncommon but difficult to treat disease of the hoof.  Historically, treatment options for elephant toenail abscesses include corrective trimming, superficial debridement and application of topical disinfectants or antibiotics. Others have constructed innovative sandals to treat and protect the affected sole or nail with success. The use of regional intravenous perfusion of the affected limb with antibiotics has also been successful. Since the elephant nail abscess now appears to be histologically and clinically comparable to equine canker, this novel characterization of an old disease may offer unique insight for treatment.  In the least, it has provided our practice with a new list of treatment options and experienced equine clinicians for consultation who have been managing patients with a similar disease for many years. One of the Asian elephants at the SDWAP has had chronic toenail abscesses for over 2 yr. Radiographs of the affected digits, as reported by others to assess degree of involvement, have fortunately been negative for evidence of osteomyelitis.  Several bacterial and fungal cultures of deep tissue biopsies and swabs of affected lesions have resulted in a mixture of organisms with no consistent single etiologic agent.  Biopsies were found negative for presence of viral DNA (elephant papillomavirus and herpesvirus) by PCR.  Typical elephant foot care at the SDWAP includes trimming and debriding with hoof knives, foot soaks and topical antibiotics.  Although difficult, attempts are made in keeping the affected foot clean and dry.  Following recommendations for the treatment of equine canker, we recently implemented the routine use of cryotherapy in all elephants with proliferative pododermatitis with improved success in the control and recession of exuberant nail lesions. The proliferative tissue of the nail is first cleaned then disinfected, debrided, trimmed with hoof knives and allowed to dry. Modified brass branding tools with contact surfaces of variable size (2-5 cm diameter) and shape (round or ovoid) are placed into liquid nitrogen (-196 C) for several minutes and then placed directly on the cankerous tissue for 30-60 sec.  This process is then repeated 4-5 min later, following a complete thaw of tissue.  Within 24 hr, the cryoburned tissue becomes macerated and necrotic and is readily removed with gentle scrubbing.  Cryotherapy offers the advantage of destroying tissue to a deeper level than trimming alone and provides hemostasis, as well.  Because of decreased sensation at the cryotherapy treatment site, a memorable painful event is avoided and the elephant patient is more routinely accepting of this technique. With the use of hoof knives, we typically remove 2-3 mm of proliferative tissue before the patient refuses further treatment, presumably due to discomfort.  With cryotherapy, we are able to remove an additional 3-5 mm of tissue by cell freezing and necrosis.  The result is quicker resolution of cankerous lesions without the need for aggressive, and potentially painful, interventions. In conclusion, it appears that elephant nail abscesses can best be described as proliferative pododermatitis, or canker, as is seen in other species.  Further gross and microscopic descriptions of normal and pathologic nail or sole lesions are necessary.  Routine cryotherapy has shown promise in the treatment of these chronic, frustrating and potentially devastating lesions of our captive elephants.

Agnew, D.W., Hagey, L., Shoshani, J., 2005. The elephants of Zoba Gash Barka, Eritrea: part 4. Cholelithiasis in a wild African elephant (Loxodonta africana). J. Zoo. Wildl. Med. 36, 677-683.
Abstract: A 4.0-kg cholelith was found within the abdominal cavity of a dead wild African elephant (Loxodonta africana) in Eritrea. Analysis of this cholelith by histochemistry, electron microscopy, electrospray mass spectroscopy, and energy-dispersive x-ray spectroscopy revealed it was composed of bile alcohols but no calcium, bilirubin, or cholesterol. Bacteria were also found in the cholelith. Similar, but smaller, bile stones have been identified previously in other wild African elephants and an excavated mammoth (Mammuthus columbi). Choleliths have been reported only once in a captive Asian elephant (Elephas maximus). Elephants, along with hyraxes (Procavia capensis) and manatees (Trichechus manatus), are unique among mammals in producing only bile alcohols and no bile acids, which may predispose them to cholelithiasis, particularly in association with bacterial infection. Dietary factors may also play an important role in cholelith formation.

Andrews, J., Mecklenborg, A., Bercovitch, F.B., 2005. Milk intake and development in a newborn captive African elephant  (Loxodonta africana). Zoo Biology 24, 275-281.
Abstract: In August 2003 the San Diego Zoo's Wild Animal Park (WAP) and the Lowry Park Zoo, under the auspices of the AZA's Elephant SSP and a USFWS permit, imported 3.8 African elephants (Loxodonta africana ) from the Kingdom of Swaziland. When they were captured, transrectal ultrasound examinations revealed that one nulliparous cow was approximately 10 months pregnant.  At the time of their arrival (August 2003), all of the animals were estimated to be approximately 13 years old and were thought to be nulliparous.  Based on the ultrasound examination results and the average African elephant gestation period, parturition was predicted to be 20 February 2004. In this report, we provide the first detailed data about nursing activity around the clock and newborn calf development, describe maternal and neonatal nighttime activity budgets, and explore maternal weight changes during suckling and lactation. The newborn calf suckled significantly more at night than during the day, but suckled for only about 2 hr per 24-hr period. Regression analysis revealed that through the first 3 months of life the calf gained 0.385 kg/day while it suckled on a regular basis. We compare our findings with published information on wild elephants, and conclude that although the growth rate is reduced compared to hand-reared elephant calves, the suckling patterns are almost identical to those reported for wild calves.

Bonar, C.J., Lewandowski, A.H., Arafah, B., Capen, C.C., 2005. Pheochromocytoma in an aged female African elephant (Loxodonta africana). J. Zoo. Wildl. Med. 36, 719-723.
Abstract: A 43 yr-old female African elephant (Loxodonta africana) collapsed acutely and died. Necropsy revealed an enlarged right adrenal medulla. Histologic appearance was typical of pheochromocytoma. Special stains and electron microscopy demonstrated chromaffin granules, suggesting that the tumor was derived from catecholamine secreting cells of the adrenal medulla, and may have been functionally secretory. Serum levels of both norepinephrine and epinephrine were elevated at time of death, supporting the functional nature of the tumor. Histologic findings of arteriolar sclerosis and smooth muscle hyperplasia suggested that the animal may have suffered from chronic systemic hypertension. Pheochromocytoma should be considered as a differential diagnosis in cases of suspected hypertension and acute death in elephants

Cousins, D.V., Florisson, N., 2005. A review of tests available for use in the diagnosis of tuberculosis in non-bovine species. Rev. sci. tech. Off. int. Epiz. 24, 1039-1059.
Abstract: Bovine tuberculosis is an important disease that has impacts on regional and international trade. The disease can affect both social and economic stability and have a deleterious affect on species diversity. The intradermal tuberculin test has been in use for almost a century and, despite the technological advances of the last two decades, is still the only prescribed test for the
diagnosis of tuberculosis in cattle. Many other species of animal, including humans, can be infected with Mycobacterium bovis. This paper reviews the various tests that have been used by researchers for detecting infection with M. bovis in a variety of animal species, and attempts to prioritise or comment on the importance of having appropriately validated diagnostics for the different species. The difficulties of test validation using small numbers of animals, especially when tuberculosis occurs in only a few instances or the species of animal affected is rare and/or valuable, are discussed.

Hildebrandt, T.B., Hermes, R., Ratanakorn, P., Rietschel, W., Fickel, J., Frey, R., Wibbelt, G., Reid, C., Goritz, F., 2005. Ultrasonographic assessment and ultrasound-guided biopsy of the retropharyngeal lymph nodes in Asian elephants (Elephas maximus)
552. Veterinary Record 157, 544-548.
Abstract: Endotheliotropic herpesvirus causes a fatal disease in young Asian elephants, but there are no methods for identifying latent carriers of the virus. During the postmortem study of one female African elephant and three male and two female Asian elephants, a lymph node located bilaterally caudoventral to the parotid gland, approximately 1.5 to 5 cm below the skin, was identified as suitable for transcutaneous ultrasound-guided biopsy. An ultrasonographic assessment and two biopsies were performed on 39 Asian elephants, and these lymph nodes were classified ultrasonographically as active, inactive or chronically active. The calculated mean (se) volume of 10 active lymph nodes was 17.4 (6.9) cm(3), and that of three chronically active lymph nodes was 10.6 (1.0) cm(3), whereas the mean volume of 17 inactive lymph nodes was 3.1 (0.6) cm(3). The presence of lymph node tissue in samples obtained by ultrasound-guided biopsy from three animals that were maintained under conditions that allowed for additional sampling was confirmed histologically. The dna extracted from the lymphoid tissue and the whole blood of all the elephants was negative for endotheliotropic herpesvirus by PCR.

Lacasse, C., Gamble, K.C., Terio, K., Farina, L.L., Travis, D.A., Miller, M. Mycobacterium szulgai osteoarthritis and pneumonia in an African elephant (Loxodonta Africana). 2005 Proceedings AAZV, AAWV, AZA Nutrition Advisory Group.  170-172. 2005.
Ref Type: Conference Proceeding
Abstract: Tuberculosis, particularly Mycobacterium bovis and M. tuberculosis, is an important health issue in zoological collections.  Zoos are a particular public health concern because of the close contact between tuberculosis-susceptible animals and humans, specifically animal handlers and visitors.¹⁶ Evidence of M. tuberculosis transmission between humans and elephants, confirmed by DNA fingerprinting, has been reported.¹³ Between 1994 and 2001, M. tuberculosis was isolated from trunk washes of captive elephants from 11 herds in the United States.¹⁷  To date, most reported cases of tuberculosis have occurred in captive Asian elephants (Elephas maximus).¹⁴ In 1997, the National Tuberculosis Working Group for Zoo and Wildlife Species partnered with the USDA to formulate the "Guidelines for the Control of Tuberculosis in Elephants." ¹⁵ This document outlines criteria for the testing, surveillance, and treatment of tuberculosis in elephants. The guidelines recommend annual monitoring of elephants by mycobacterial culture of three direct trunk washes collected over 1 wk.  Isolation of Mycobacterium avium and non-tuberculous mycobacteria from elephant trunk wash samples is common, but these organisms have not been associated with clinical disease.^14,18 This case report details clinical disease with fatal complications of an atypical mycobacterial infection in an African elephant (Loxodonta africana). In September 2003, an African elephant presented with acute, severe lameness of the left rear limb with subsequent swelling of the stifle.  Diagnostic procedures included aspiration cytology of the swelling, radiographs, and thermographic imaging.  The exact location of the injury could not be detected, but a lesion to the stifle or coxofemoral articulation was suspected.  After 13 mo of treatment, including pulse therapy with a variety of nonsteroidal anti-inflammatory drugs (NSAIDs), weekly to biweekly injections of polysulfated glycosaminoglycan, and intensive foot care efforts to treat secondary pedal lesions of both rearlimbs, the animal died acutely.  Gross necropsy revealed granulomatous osteomyelitis with necrosis/loss of the femoral head and acetabulum and pulmonary granulomas.  Both of these lesions contained acid-fast bacteria on cytology. While awaiting confirmatory culture results, quarantine procedures were established for the elephant facility and a program was established to screen all zoo personnel in close contact with the elephant or who participated in the necropsy.  All personnel were tested by the Chicago Department of Public Health without documented conversion. Mycobacterium szulgai was ultimately cultured from both coxofemoral and pulmonary lesions. Mycobacterium szulgai is an uncommon nontuberculous mycobacterium that is usually isolated from pathologic lesions in humans.²¹ This bacterial species was first identified in 1972.¹¹ The lungs are the main locality for pathologic manifestation in humans and several cases have been in patients with acquired immunodeficiency syndrome.^9,20,21 Infection due to M. szulgai most frequently produces thin-walled cavities in lungs resembling tuberculosis.⁴ Other documented sites of infection include the skin, bone, and tendon sheath (causing a carpal tunnel syndrome).^{2,9,10,12,19,20}  Intra-operative contamination from ice water has led to M. szulgai keratitis after laser-assisted ophthalmic surgeries^.6 A case of disseminated disease in a previously healthy young human has been reported.⁵  No evidence of human-to-human transmission of this organism has been documented and human cases are believed to originate from environmental sources.¹²  The natural habitat of the organism is unknown, but previous reports suggest an association of the bacteria with water of swimming pools and fish tanks.^1,21 The organism has been cultured from a snail and tropical fish.^1,3 No standard recommendation for the treatment of M. szulgai infection currently exists.  In general, triple antibiotic therapies used in standard mycobacterial treatments are reported with a low rate of relapses and sterilization of sputum cultures within a mean of 3 mo.³ Pulmonary lesions in this elephant were chronic; it was not possible to determine when initial infection occurred. Infection could have occurred in captivity or in the wild prior to captivity. Three trunk washes over the past year had been negative for mycobacterial culture. Osteomyelitis in the hip may have developed secondary to hematogenous spread from the lungs with the acute lameness resulting from a pathologic fracture associated with this infection. Alternatively, though considered less likely, a traumatic fracture of the hip could have occurred, with bacterial inoculation and secondary osteomyelitis as a result of increased blood flow to the site. The source of infection for this elephant remains unknown.  Prevalence of this organism in the natural habitat or captive environment of the elephants has not been previously documented.
LITERATURE CITED
1 Abalain-Colloc, M.L., D. Guillerm, M. Salaun, S. Gouriou, V. Vincent, and B. Picard.  2003.  Mycobacterium szulgai isolated from a patient, a tropical fish, and aquarium water.  Eur. J. Clin. Microbiol. Infect. Dis.  22: 768-769.
2.Cross, G.M., M. Guill, and J.K. Aton.  1985.  Cutaneous Mycobacterium szulgai infection. Arch. Dermatol. 121: 247-249.
3. Davidson, P.T. 1976. Mycobacterium szulgai: a new pathogen causing infection of the lung.  Chest 69: 799- 801.
4. Dylewski, J.S., H.M. Zackon, A.H. Latour, and G.R. Berry.  1987.  Mycobacterium szulgai: an unusual pathogen.  Rev. Infect. Dis.  9: 578-580.
5. Gur, H., S. Porat, H. Haas, Y. Naparstek, and M. Eliakim.  1984.  Disseminated mycobacterial disease caused by Mycobacterium szulgai. Arch. Intern. Med. 144: 1861-1863.
6.Holmes, G.P., G. Bond, R.C. Fader, and S.F. Fulcher.  2002. A cluster of cases of Mycobacterium szulgai keratitis that occurred after laser-assisted in situ keratomileusis.  Clin. Infect. Dis. 34: 1039-1046.
7.Horusitzky, A., X. Puechal, D. Dumont, T. Begue, M. Robineau, and M. Boissier.  2000.  Carpal tunnel syndrome caused by Mycobacterium szulgai. J. Rheumatol 27: 1299-1302.
8.Hurr, H., and T. Sorg.  1998.  Mycobacterium szulgai osteomyelitis.  J. Infect.  37: 191-192.
9.Luque, A.E., D. Kaminski, R. Reichman, and D. Hardy. 1998.  Mycobacterium szulgai osteomyelitis in an AIDS patient. Scand. J. Infect. Dis. 30: 88-91.
10.Maloney, J.M., C.R. Gregg, D.S. Stephens, F.A. Manian, and D. Rimland.  1987.  Infections caused by Mycobacterium szulgai in humans.  Rev. Infect. Dis.  9: 1120-1126.
11.Marks, J., P.A. Jenkins, and M. Tsukamura.  1972.  Mycobacterium szulgai: a new pathogen.  Tubercle 53: 210.
12.Merlet, C., S. Aberrane, F. Chilot, and J. Laroche.  2000.  Carpal tunnel syndrome complicating hand flexor tenosynovitis due to Mycobacterium szulgai. Joint Bone Spine 67: 247-248.
13.Michalak, K., C. Austin, S. Diesel, J.M. Bacon, P. Zimmerman, and J. N. Maslow.  1998. Mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants. Emerg. Infect. Dis. 4: 283-287.
14.Mikota, S.K., R.S. Larsen, and R.J. Montali.  2000.  Tuberculosis in elephants in North America.  Zoo Biol. 19: 393-403.
15.National Tuberculosis Working Group for Zoo and Wildlife Species. 2000. Guidelines for the control of tuberculosis in elephants.  USDA Animal and Plant Health Inspection Services.
16.Oh, P., R. Granich, J. Scott, B. Sun, M. Joseph, C. Stringfield, S. Thisdell, J. Staley, D. Workman-Malcolm, L. Borenstein, E. Lehnkering, P. Ryan, J. Soukup, A. Nitta, and J. Flood.  2002.  Human exposure following  Mycobacterium tuberculosis infection of multiple animal species in a metropolitan zoo.  Emerg. Infect. Dis. 8: 1290-1293.
17.Payeur, J.B., J.L. Jarnagin, J.G. Marquardt, and D.L. Whipple.  2002.  Mycobacterial isolations in captive elephants in the United States.  Ann. N.Y. Acad. Sci. 969: 256-258.
18.Shojaei, H., J.G. Magee, R. Freeman, M. Yates, N.U. Horadagoda, and M. Goodfellow.  2000. Mycobacterium elephantis sp. nov., a rapidly growing non-chromogenic Mycobacterium isolated from an elephant.  Int. J. Syst. Evol. Microbiol.  50: 1817-1820.
19.Stratton, C.W., D.B. Phelps, and L.B. Reller.  1978.  Tuberculoid tenosynovitis and carpal tunnel syndrome caused by Mycobacterium szulgai.  Am. J. Med.  65: 349-351.
20.Tappe, D., P. Langmann, M. Zilly, H. Klinker, B. Schmausser, and M. Frosch.  2004.  Osteomyelitis and skin ulcers caused by Mycobacterium szulgai in an AIDS patient.  Scand. J. Infect. Dis. 36: 883-885.
21.Tortoli, E., G. Besozzi, C. Lacchini, V. Penati, M.T. Simonetti, and S. Emler.  1998.  Pulmonary infection due to Mycobacterium szulgai, case report and review of the literature.  Eur. Respir. J.  11: 975-977.

Larsen, R.S., Kay, M., Triantis, J., Salman, M.D. Update on serological detection of Mycobacterium tuberculosis infection in Asian elephants. 2005 Proceedings AAZV, AAWV, AZA Nutrition Advisory Group.  62-63. 2005.
Ref Type: Conference Proceeding
Abstract: Tuberculosis has become an important disease in captive elephants, particularly Asian elephants (Elephas maximus). Diagnosing tuberculosis in elephants has been problematic as many tests have inadequate sensitivity or specificity.2-4 A multiple-antigen enzyme-linked immunosorbent assay (ELISA) was previously investigated for detecting infection in Asian elephants and African elephants (Loxodonta africana); this test had excellent sensitivity and specificity, but needed further evaluation.1 Modifications to the multiple-antigen ELISA panel have since been made. Valuable antigens were retained, other antigens were removed, and new ones were added.  This modified ELISA was re-evaluated, using serum from 68 Asian elephants. Sixteen had M. tuberculosis -positive trunk cultures, while 52 were either culture negative at necropsy or had a history of negative trunk cultures and no contact with infected elephants. Seven elephants were evaluated over time. The test was 100% (95% CI; 95-100%) specific and 94% (95% CI; 79-100%) sensitive using two of the six antigens (M. bovis strain AN5 culture filtrate and M. tuberculosis early secretory antigenic target 6). "Effectively-treated" elephants had decreasing seroreactivity, but those that were culture-positive post-treatment were more consistently seroreactive.  Although "effectivelytreated" elephants had declining seroreactivity, they still usually had higher values than animals that had never been infected. Serology continues to show great promise in detecting tuberculosis in elephants, often detecting infection months-to-years sooner than trunk wash culture.  Advances in techniques may soon make serology even more practical.  While serology should not replace trunk-wash culture, it is a useful adjunct for early detection of infection in elephants and for monitoring treatment.
ACKNOLWEDGMENTS We thank the many veterinarians, owners, caretakers, and managers of elephant-owning institutions that participated in this investigation, as well as Drs. Michele Miller and Susan Mikota for helping to coordinate sample collection. We also thank Kimberly Deines and other laboratory personnel who processed ELISA samples.  The study was partially funded by a grant from USDA, CSREES to Colorado State University Program of Economically Important Infectious Animal Diseases.
LITERATURE CITED
1.Larsen, R.S., M.D. Salman, S.K. Mikota, R. Isaza, R.J. Montali, and J. Triantis. 2000.  Evaluation of a multiple-antigen enzyme-linked immunosorbent assay for detection of Mycobacterium tuberculosis in captive elephants.  J. Zoo Wildl. Med. 31: 291-302.
2. Mikota, S.K., L. Peddie, J. Peddie, R. Isaza, F. Dunker, G. West, W. Lindsay, R.S. Larsen, M.D. Salman, D. Chatterjee, J. Payeur, D. Whipple, C. Thoen, D.S. Davis, R.J. Montali and J. Maslow.  2001. Epidemiology and diagnosis of Mycobacterium tuberculosis in six groups of elephants.  J. Zoo Wildl. Med. 32: 1-16.
3. Mikota, S.K., R.S. Larsen, and R.J. Montali.  2000.  Tuberculosis in elephants in North America.  Zoo Biol. 19: 393-403.
4. U.S. Department of Agriculture.  2003.  Guidelines for the control of tuberculosis in elephants.  Animal and Plant Health Inspection Service; Animal Care. Washington, D.C. http://www.aphis.usda.gov/ac/TBGuidelines2003.pdf.

Luikart, K.A., Stover, S.M., 2005. Chronic sole ulcerations associated with degenerative bone disease in two Asian elephants (Elephas maximus). J. Zoo. Wildl. Med. 36, 684-688.
Abstract: Chronic foot lesions and degenerative joint disease are common causes of morbidity in elephants. Lesions may become intractable and progressive despite intensive treatment regimens. The forelimbs of two Asian elephants (Elephas maximus) with chronic nonhealing sole ulcerations were examined using manual dissection and computed tomography. Both elephants had abnormal limb conformation that preceded the development of sole ulcerations. In both cases, sole ulcers were associated with remodeling and degeneration of underlying bones of the digits. Conformational abnormalities and altered weight distribution in these individuals may have induced compensatory bony degeneration and secondary ulcer formation. Sole ulcerations associated with digital abnormalities may have a guarded prognosis for resolution, even with aggressive treatment. Because limb conformational abnormalities could predispose to or result from chronic digital lesions, elephants with conformational abnormalities may have increased likelihood of having chronic sole ulcerations

Lyashchenko, K., Miller, M., Waters, W.R. Application of MAPIA (Multiple antigen print immunoassay) and rapid lateral flow technology for tuberculosis testing of elephants. 2005 Proceedings AAZV, AAWV, AZA Nutrition Advisory Group.  64-65. 2005.
Ref Type: Conference Proceeding
Abstract: Tuberculosis (TB) remains a serious re-emerging disease in wildlife and zoo animals. Mycobacterium tuberculosis has been isolated from 30 captive Asian elephant (Elephas maximus within 14 herds in the United States (1994-2004) and Mycobacterium bovis has been isolated from one African elephant (Loxodonta africana) (Mikota, pers. comm.).3 There are several challenges with elephant TB diagnosis. Culture of trunk wash has relatively poor sensitivity and is subject to contamination.  Skin test is not validated in elephants and there is little reliability in these results.4   Serologic tests are appealing because  samples can be stored for future analysis, archived samples can be analyzed, various assay platforms can be directly compared, and these assays are amenable to serial analysis (e.g., to monitor therapy).  There is currently a multiple antigen ELISA test available for experimental use in elephants.1

To improve tuberculosis control, new diagnostic tools should be rapid, accurate, and host species-independent. Two novel serologic methods, MultiAntigen Print ImmunoAssay (MAPIA) and lateral-flow technology (Rapid Test), have been adapted for use in white-tailed deer, European badger, cattle, and Asian and African elephants for the detection of TB-specific antibody. Serologic markers of diagnostic importance have been identified for each host tested so far. With MAPIA, a machine prints specific antigens horizontally on a nitrocellulose membrane which can be cut into strips and used in Western blot.2   Strips are incubated with test serum samples, then an anti-Ig conjugate and color developer.  Using this assay, an antibody response to multiple mycobacterial antigens has been observed in sera from M. tb-infected elephants. No antibody response was detected to any antigens in non-infected elephant sera.  Additionally, the kinetics of antibody responses by elephants undergoing antibiotic therapy indicates that the MAPIA could be used for monitoring treatment and to determine recrudescence of infection. 

Using selected antigens, a lateral-flow test was developed for rapid antibody detection that can be used in multiple species. The Rapid Test can use serum, plasma, or whole blood and provides results within 15 min.  These tests are similar to in-clinic tests for FIV/FeLV detection (snap test, IDDEX). If a band is present in the test strip, it indicates a positive reaction (antibody present).
A panel of sera from healthy and TB infected elephants showed good correlation between the MAPIA and the rapid test (Table 1).

In summary, it appears that TB-infected elephants produce a robust antibody response that can be detected in serologic assays.  Of special significance is the kinetics of the response, which may permit earlier detection of infection than current diagnostic methods.  While initial results are promising, additional studies are required to validate these two assays.  A relatively small set of serum samples from documented infected and non-infected elephants was used, and more samples are needed to further validate the tests. MAPIA has been used to optimize antigen selection in order to make the most sensitive and specific Rapid Test. This strategy may also allow for identification of "treatment-sensitive" antigens that could be used in the MAPIA format to monitor TB therapy.  While elephants will be used as an initial "proof of concept" species for test development, additional samples from other species will also be evaluated to determine applicability to other species (i.e., a host species-independent test), thus benefiting other groups such as primates, rhinos, cervids, etc.

ACKNOWLEDGMENTS
The authors thank the zoos and individuals that have provided samples and assistance with this research, including Ray Ball, Carol Buckley, Jenifer Chatfield, Genny Dumonceaux, Javan Esfandiary, Rena Greenwald, Scott Larsen, Susan Mikota, Torsten Moller, Dick Montali, Mike Richards, Heidi Riddle, Mo Salman, Scott Terrell, and many others.  This research was supported by Chembio Diagnostics, Inc.
LITERATURE CITED
1 Larsen, R.S., M.D. Salman, S.K. Mikota, R. Isaza, R.J. Montali, and J. Triantis. 2000.  Evaluation of a multiple-antigen enzyme-linked immunosorbent assay for detection of Mycobacterium tuberculosis  
  infection in captive elephants.  J. Zoo Wildl. Med. 31:291-302.
2 Lyashchenko, K., et al.  2000. A multiantigen print immunoassay for the serological diagnosis of infectious diseases.  J. Immunol. Methods  242:91-100. 
3 Mikota, S.K., and J. Maslow.  2002.  Epidemiology and treatment of tuberculosis in elephants:  2002.  Proc. Am. Assoc. Zoo Vet. Annu. Meet.  Pp. 384-387.

Mobasheri, A., Gent, T.C., Womack, M.D., Carter, S.D., Clegg, P.D., Barrett-Jolley, R., 2005. Quantitative analysis of voltage-gated potassium currents from primary equine (Equus caballus) and elephant (Loxodonta africana) articular chondrocytes
618. Am. J. Physiol Regul. Integr. Comp Physiol 289, R172-R180.
Abstract: In this comparative study, we have established in vitro models of equine and elephant articular chondrocytes, examined their basic morphology, and characterized the biophysical properties of their primary voltage-gated potassium channel (Kv) currents. Using whole cell patch-clamp electrophysiological recording from first-expansion and first-passage cells, we measured a maximum Kv conductance of 0.15 +/- 0.04 pS/pF (n = 10) in equine chondrocytes, whereas that in elephant chondrocytes was significantly larger (0.8 +/- 0.4 pS/pF, n = 4, P </= 0.05). Steady-state activation parameters of elephant chondrocytes (V = -22 +/- 6 mV, k = 11.8 +/- 3 mV, n = 4) were not significantly different from those of horse chondrocytes (V = -12.5 +/- 4.3 mV, k = 12 +/- 2, n = 10). This suggests that there would be slightly more resting Kv activation in elephant chondrocytes than in their equine counterparts. Kinetic analysis revealed that both horse and elephant chondrocyte Kv currents had similar activation and inactivation parameters. Pharmacological investigation of equine chondrocyte Kv currents showed them to be powerfully inhibited by the potassium channel blockers tetraethylammonium and 4-aminopyridine but not by dendrotoxin-I. Immunohistochemical studies using polyclonal antibodies to Kv1.1-Kv1.5 provided evidence for expression of Kv1.4 in equine chondrocytes. This is the first electrophysiological study of equine or elephant chondrocytes. The data support the notion that voltage-gated potassium channels play an important role in regulating the membrane potential of articular chondrocytes and will prove useful in future modeling of electromechanotransduction of fully differentiated articular chondrocytes in these and other species

Moller, T., Roken, B., Petersson, L., Vitaud, C., Lyashchenko, K. Preliminary results of a new serological test for detection of TB-infection (Mycobacterium tuberculosis) in elephants (Elephas maximus and Loxodonta africanum) - Swedish Case studies. Verh.ber.Erkrg.Zootiere. 42, 173-181. 2005.
Ref Type: Conference Proceeding

Raubenheimer, E.J., Ngwenya, S.P., 2005. The role of ivory in the survival of the African elephant
510. SADJ. 60, 426, 430.
Abstract: The unique chequered pattern of polished ivory has created a perverted commercial demand for elephant tusks. The morphologic basis of the pattern, which makes ivory a sought after product for the manufacturing of works of art, is discussed. Chemical analyses of ivory holds great potential in tracing the source of illegally harvested tusks and exposing poorly managed elephant sanctuaries. The impact of uncontrolled ivory hunting on the population genetics of the African elephant is briefly reviewed

Rush, E.M., Brawner, W.R., Ogburn, A.L., Marshall, A., Hathcock, J.T. Comparison of radiographs versus computed tomography evaluation of the distal limb in an Asian elephant. 2005 Proceedings AAZV, AAWV, AZA Nutrition Advisory Group.  68-69. 2005.
Ref Type: Conference Proceeding
Abstract: Feet problems are the most commonly seen ailment in captive elephants.  In the field of zoo and wildlife medicine, radiographs are the accepted standard of skeletal evaluation of the distal limb of elephants, to show changes in bone density and conformation .¹ Although radiographs are considered reliable to show severe degenerative change in the distal phalanges, it is difficult to assess detail of the carpus and tarsus due to the anatomy and superimposition of the large carpal and tarsal bones. Radiographic images of the distal limbs of a geriatric, female Asian elephant, were compared with postmortem computed tomography (CT) images.  This animal had a long history of clinical nail disease treated for many years with diligent foot care and aggressive paring of multiple nails. Arthritis of the carpi, tarsi and/or digits was suspected and had been treated with non-steroidal anti-inflammatory medications. Serial radiographs from several years showed obvious degenerative change in multiple digits, especially those most severely affected clinically at the nail. Osseous detail in the carpi and tarsi was suboptimal on radiographs even when postmortem specimens were radiographed with a stationary, high capacity radiograph machine designed for large animal radiology.  CT images of the distal limbs revealed degenerative skeletal changes that were not readily apparent on radiographs.  Most degenerative change was noted in the periosteal areas of the carpal and tarsal bones, particularly at articular surfaces. Realizing that CT of feet and distal limbs of live elephants is impractical, if not impossible, this comparison of radiographs and CT demonstrates that radiographs may not reveal all abnormalities present in joints of the distal extremities. Comparative CT images of younger or clinically normal animals were not available, so it has not been possible to determine the clinical significance of the apparent degenerative changes noted on these radiographs and CT images at the time of this publication.  Nonetheless, consideration should be given for the lack of detail when evaluating radiographs of elephant feet. When radiographic changes are noted in the distal limbs of elephants suffering from arthritis with a history of nail disease, the attending veterinarian may consider prophylactic antibiotic therapy to treat possible osteomyelitis in the bones of the distal limb.  Also, in animals with arthritic change on radiographs and no nail disease, implementation of appropriate antiinflammatory drugs and/or joint supplements should be considered.  Hydrotherapy, acupuncture, limb exercise and other topical therapies may be warranted, depending on each individual case and the clinical signs exhibited. Routine and diagnostic radiographs should be taken from several angles, including oblique views, to assure the most accurate assessment of bony change in the distal limb and to give the best overall images for retrospective comparison. Radiographs should include the carpus and tarsus if the radiograph machine has the capacity for the bone density of that region.
ACKNOWLEDGMENTS
Thanks to the State Veterinary Diagnostic Laboratory, Auburn University College of Veterinary Medicine Department of Radiology for all of their time, expertise and contributions to this study.  Also, thanks to Marcia Riedmiller and the pachyderm care staff at the Birmingham Zoo.
LITERATURE CITED
1.Fowler, M.E., and R.E. Miller. 2003. Zoo and Wild Animal Medicine, 5th Edition. St. Louis: Elsevier Science. Pp 547-548.

Suedmeyer, W.K., Oosterhuis, J., Kollias, G., Fagan, D., Hornoff, B., Dodam, J., Shafford, H. Elephant restraint device assisted anesthesia in an African elephant (Loxodonta africana). 2005 Proceedings AAZV, AAWV, AZA Nutrition Advisory Group.  189-191. 2005.
Ref Type: Conference Proceeding
Abstract: Modern elephant management programs often include the use of protected contact. This allows improved safety for the elephant staff but may limit access to medical conditions occurring in elephants.
A 27-yr-old female African elephant (Loxodonta africana) weighing an estimated 3,700 kg was anesthetized for evaluation of a chronic, progressive, fistulous tract of the left ventral mandible. The mandible was routinely cultured, flushed with diluted peroxide, chlorhexidine, betadine solution, or alternating antibiotics, based on microbial sensitivities. To properly assess the left mandible, the elephant had to be placed in right lateral recumbency, which was accomplished with the use of a commercially available rotational elephant restraint device (ERD). Because of the protected contact management program, right lateral recumbency could not be guaranteed at the time of immobilization. Malpositioning, tusk fracture and/or related injury could occur upon recumbency without the additional control afforded by the ERD. The ERD is a hydraulically operated unit that comfortably restrains an elephant, minimizing safety risks to the animal and staff. The ERD consists of one solid wall, three side panels, and hinged floor. The ends of the restraint are closed with moveable shift doors. The three side panels can be moved independently depending upon the size of the animal and are further subdivided with moveable "subpanels" to allow direct access to various areas of the animal. In addition, support straps help gently stabilize limbs when performing medical procedures. The unit is positioned within the elephant holding facility at the Kansas City Zoo. The unit was installed in 1994 during renovation of the elephant exhibit, whereupon the elephant management program was changed from free-contact to protected contact. The ERD is utilized for reproductive assessments, semen collection, transabdominal ultrasound, evaluation of integumentary wounds, ophthalmic and aural examination, and administration of injectable medications. However, no elephant had been anesthetized and rotated in the restraint. The affected animal could not be guaranteed to re-enter the ERD once rotated, but would enter and station in the ERD on a daily basis. Because of this, a conspecific was conditioned to allow rotation without the use of sedatives or tranquilizers, to prepare for the actual immobilization. Adjustments in strap placement, cushioning, critical evaluation of mechanical stability, and placement of hydraulic panels allowed staff to prepare for the actual immobilization, minimizing complications. The elephant was conditioned to enter and station in the ERD. After strapping the distal limbs, thorax and caudal abdomen for support, the elephant was immobilized with a combination of 3,000 IU of hyaluronidase (O'Brien Pharmacy, Kansas City, MO USA), 10 mg acepromazine maleate, and 7 mg etorphine hydrochloride (Wildlife Pharmaceuticals Inc., Fort Collins, CO USA) via pole syringe. Close monitoring of induction was performed and when stage III anesthetic plane was achieved, the elephant was rotated into right lateral recumbency, elevating the elephant 6 feet above the floor. No voluntary movement of the animal was noted while the restraint was in motion. Direct arterial blood pressure, indirect oscillometric blood pressure, blood gases, respiratory rate, excursion characteristics, cardiac rate and rhythm, and pulse oximetry was routinely monitored during the procedure. Anesthesia was maintained with intermittent boluses of etorphine hydrochloride. Intravenous physiologic fluids (lactated Ringers solution) were maintained via an i.v. aural catheter, and insufflation with oxygen was provided on a continual basis. Oral examination and palpation demonstrated an incomplete transverse fissure of the left mandibular molar, intact gingival, and proper dental occlusion with the upper arcade.  Digital radiographs of the left mandible were performed based on exposures obtained with a set of skeletonized jaws. Advantages of this diagnostic modality are the immediate imaging results, portability, and digital imaging and storage, and does not require a developer or fixative. Adjustments in radiographic angle and technique were made to obtain the best diagnostic image. Radiographic imaging demonstrated a sequestrum consisting of a fractured enamel plate  ²of the mandibular molar with a fistulous tract that coursed ventrally to communicate through the skin. The elephant was elevated 6 feet above the ground, which presented unique challenges. Because of the relatively small operating space, intubation was not possible, but insufflation was readily achieved and successful based on pulse oximetry trends. A commercial lift was utilized to elevate two large-animal circle anesthetic units to the level of the elephant's head. During immobilization the legs were cushioned and restraint straps removed to lessen the potential for occlusive damage to the tissues. The ERD allows an elephant to be positioned in either right or left lateral recumbency.
Upon completion of diagnostic procedures, the narcotic agent was reversed with 1,400 mg naltrexone hydrochloride (Zoopharm, Laramie, WY USA) administered 25% intravenously and 75% subcutaneously. The elephant awoke within 90 sec and was rotated to a standing position within the restraint. Thereafter, the elephant was confined in the restraint for approximately 45 min, until no untoward effects were likely to occur. The elephant was released from the restraint and resumed normal eating and drinking within 8 hr, and voluntarily entered the restraint within 2 wk following the procedure. The elephant was stable throughout the procedure; however, a predetermined objective for mean arterial blood pressures (<200 MAP) was not achieved. Hyaluronidase was utilized to promote rapid absorption of the narcotic and neuroleptic agents.³ Acetylpromazine was used to maintain peripheral perfusion by reducing the hypertensive effects of etorphine,¹ which has been documented in previous immobilizations of African elephants.^3-5 Etorphine hydrochloride, a powerful narcotic agent, has been successfully used as an immobilizing agent in both wild and captive African elephants.^3-5 Use of an ERD allowed full control of the immobilization, increasing safety for personnel, preventing injury to the elephant, and positioning the left mandible on the dorsal plane. Disadvantages are the elevated height of the elephant, relatively small operating space, and disrupted line of sight communication. A second procedure will be performed in the near future to address the fracture and subsequent sequestrum diagnosed during the first immobilization. The elephant is currently being conditioned to allow restraint in a holding stall that will allow greater access to the oral cavity and surgical manipulation of the affected mandible.
ACKNOWLEDGMENTS
We thank the staff of the Kansas City Zoological Park for their care, concern, and expertise in helping make this procedure a success.
LITERATURE CITED
1 Booth, N.H. Psychotropic agents. In: Booth, N.H., and R.E. McDonald (eds.). Veterinary Pharmacology and Therapeutics.  W.B. Saunders, Co., Philadelphia, PA. P. 329.
2 Fagan, V.D.A., J.E. Oosterhuis, and A. Roocraft. 2001. Captivity disorders in elephants: impacted molars and broken tusks. Der Zoologische Garten 71:281-303.
3 Honeymoon, V.L., G.R. Pettifer, and D.H. Dyson. 1992. Arterial blood pressure and blood gas values in normal standing and laterally recumbent African (Loxodonta africana) and Asian (Elephas maximus)    elephants. J. Zoo Wildl. Med. 23:205-210.
4. Kock, R.A., P. Morkel, and M.D. Kock. 1993. Current immobilization procedures used in elephants. In: Fowler,
M.E. (ed.).  Zoo and Wild Animal Medicine: Current Therapy 3. W.B. Saunders Co., Philadelphia, PA.  Pp. 436-441.
5 Raath, J.P. 1999. Relocation of African elephants. In: Fowler, M.E., and R.E. Miller (eds.). Zoo and Wild Animal Medicine: Current Therapy 4. W.B. Saunders, Co., Philadelphia, PA.  Pp. 525-533.

Waters, W.R., Palmer, M.V., Bannantine, J.P., Greenwald, R., Esfandiari, J., Andersen, P., McNair, J., Pollock, J.M., Lyashchenko, K.P., 2005. Antibody responses in reindeer (Rangifer tarandus) infected with Mycobacterium bovis. Clinical and Diagnostic Laboratory Immunology 12, 727-735.
Abstract: Despite having a very low incidence of disease, reindeer (Rangifer tarandus) are subject to tuberculosis (TB) testing requirements for interstate shipment and herd accreditation in the United States. Improved TB tests are desperately needed, as many reindeer are falsely classified as reactors by current testing procedures. Sera collected sequentially from 11 (experimentally) Mycobacterium bovis-infected reindeer and 4 noninfected reindeer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for antibody specific to M. bovis antigens. Specific antibody was detected as early as 4 weeks after challenge with M. bovis. By MAPIA, sera were tested with 12 native and recombinant antigens, which were used to coat nitrocellulose. All M. bovis-infected reindeer developed responses to MPB83 and a fusion protein, Acr1/MPB83, and 9/11 had responses to MPB70. Other antigens less commonly recognized included MPB59, ESAT-6, and CFP10. Administration of purified protein derivatives for skin testing boosted serum antibody responses, as detected by each of the assays. Of the noninfected reindeer, 2/4 had responses that were detectable immediately following skin testing, which correlated with pathological findings (i.e., presence of granulomatous lesions yet the absence of acid-fast bacteria). The levels of specific antibody produced by infected reindeer appeared to be associated with disease progression but not with cell-mediated immunity. These findings indicate that M. bovis infection of reindeer elicits an antibody response to multiple antigens that can be boosted by skin testing. Serological tests using carefully selected specific antigens have potential for early detection of infections in reindeer.

Wise, D.J., Carter, G.R., Flores, E.F. Laboratory Diagnosis of Viral Infections. Carter, G. R., Wise, D. J., and Flores, E. F. A Concise Review of Veterinary Virology. Ithaca: International Veterinary Information Service (www. ivis. org), 2005; Document No. A3407. 0305.  2005.
Ref Type: Electronic Citation

Wooding, F.B., Stewart, F., Mathias, S., Allen, W.R., 2005. Placentation in the African elephant, Loxodonta africanus: III. Ultrastructural and functional features of the placenta
598. Placenta 26, 449-470.
Abstract: Successful transfer of nutrients to the elephant fetus during pregnancy relies on a variety of placental modifications. Our light and electron microscopical investigations show that the structure is endotheliochorial from implantation to term, with unicellular, never syncytial trophoblast. Light and electron microscope immunocytochemistry shows the restriction of the glucose transporter 1 isoform to the basolateral surfaces of the trophoblast, with the glucose transporter 3 restricted to the apical plasmalemma of the trophoblast. Glucose transport to the fetus therefore requires a sequential use of both isoforms. Light and electron microscope cytochemistry indicate the presence of iron deposits only in the haemophagous zones confirming their iron transport function. No trophoblast areas with high concentrations of Calcium binding protein, specialised for Calcium transport were found. In situ hybridisation demonstrated the presence of IGF-II mRNA in the trophoblast from the earliest stage, with TGFbeta1 and HGF-SF mRNA expressed subsequently but only IGF-II and HGF mRNA present in the second half of pregnancy. The results are briefly discussed in terms of placental growth and function and indicate that the elephant placenta is another example of a unique solution to the variety of problems posed by a resident fetus

Principles of validation of diagnostic assays for infectious diseases.  2004.
Ref Type: Electronic Citation

Brown, J.L., Olson, D., Keele, M., Freeman, E.W., 2004. Survey of the reproductive cyclicity status of Asian and African elephants in North America. Zoo Biology 23, 309-321.
Abstract: The Asian and African elephant populations in North America are not self sustaining, and reproductive rates remain low. One problem identified from routine progestagen analyses is that some elephant females do not exhibit normal ovarian cycles. To better understand the extent of this problem, the Elephant TAG/SSP conducted a survey to determine the reproductive status of the captive population based on hormone and ultrasound evaluations. The survey response rates for facilities with Asian and African elephants were 81% and 71%, respectively, for the studbook populations, and nearly 100% for the SSP facilities. Of the elephants surveyed, 49% of Asian and 62% of African elephant females were being monitored for ovarian cyclicity via serum or urinary progestagen analyses on a weekly basis. Of these, 14% of Asian and 29% of African elephants either were not cycling at all or exhibited irregular cycles. For both species, ovarian inactivity was more prevalent in the older age categories (>30 years); however, acyclicity was found in all age groups of African elephants. Fewer elephant females (B30%) had been examined by transrectal ultrasound to assess reproductive-tract integrity, and corresponding hormonal data were available for about three-quarters of these females. Within this subset, most (B75%) cycling females had normal reproductive-tract morphologies, whereas at least 70% of noncycling females exhibited some type of ovarian or uterine pathology. In summary, the survey results suggest that ovarian inactivity is a significant reproductive problem for elephants held in zoos, especially African elephants. To increase the fecundity of captive elephants, females should be bred at a young age, before reproductive pathologies occur. However, a significant number of older Asian elephants are still not being reproductively monitored. More significantly, many prime reproductive-age (10-30 years) African females are not being monitored. This lack of information makes it difficult to determine what factors affect the reproductive health of elephants, and to develop mitigating treatments to reinitiate reproductive cyclicity.

Brown, J.L., Goritz, F., Pratt-Hawkes, N., Hermes, R., Galloway, M., Graham, L.H., Gray, C., Walker, S.L., Gomez, A., Moreland, R., Murray, S., Schmitt, D.L., Howard, J., Lehnhardt, J., Beck, B., Bellem, A., Montali, R., Hildebrandt, T.B., 2004. Successful artificial insemination of an Asian elephant at the National Zoological Park. Zoo Biology 23, 45-63.
Abstract: For decades, attempts to breed elephants using artificial insemination (AI) have failed despite considerable efforts and the use of various approaches. However, recent advances in equipment technology and endocrine-monitoring techniques have resulted in 12 elephants conceiving by AI within a 4-year period (19982002). The successful AT technique employs a unique endoscope-guided catheter and transrectal ultrasound to deliver semen into the anterior vagina or cervix, and uses the "double LH surge" (i.e., identifying the anovulatory LH (anLH) surge that predictably occurs 3 weeks before the ovulatory LH (ovLH) surge to time insemination. This study describes the 6-year collaboration between the National Zoological Park (NZP) and the Institute for Zoo Biology and Wildlife Research (IZW), Berlin, Germany, that led to the refinement of this AI technique and subsequent production of an Asian elephant calf. The NZP female was the first elephant to be inseminated using the new AI approach, and was the fifth to conceive. A total of six AI trials were conducted beginning in 1995, and conception occurred in 2000. Semen was collected by manual rectal stimulation from several bulls in North America. Sperm quality among the bulls was variable and was thus a limiting factor for AI. For the successful AI, semen quality was good to excellent (75-90% motile sperm), and sperm was deposited into the anterior vagina on the day before and the day of the ovLH surge. Based on transrectal ultrasound, ovulation occurred the day after the ovLH surge. Pregnancy was monitored by serum and urinary progestagen, and serum prolactin analyses in samples collected weekly. Fetal development was assessed at 12, 20, and 28 weeks of gestation using transrectal ultrasound. Elevated testosterone measured in the maternal circulation after 36 weeks of gestation reliably predicted the calf was a male. Parturition was induced by administration of 40 IU oxytocin 3 days after serum progestagens dropped to undetectable baseline levels. We conclude that AI has potential as a supplement to natural breeding, and will be invaluable for improving the genetic management of elephants, provided that problems associated with inadequate numbers of trained personnel and semen donors are resolved.

Dahl, N.J., Olson, D., Schmitt, D., Blasko, D.R., Kristipati, R., Roser, J.F., 2004. Development of an enzyme-linked immunosorbent assay (ELISA) for luteinizing hormone (LH) in the elephant (Loxodonta africana and Elephas maximus). Zoo Biology 23, 65-78.
Abstract: A simple, rapid enzyme-linked immunosorbent assay (ELISA) for the measurement of LH in plasma and serum of elephants (Loxodonta africana and Elephas maximus) has been developed, validated, and used for comparative studies. Purified elephant LH (eleLH) diluted in elephant plasma was used as standards (0.78-50 ng/ml). A monoclonal antibody against the beta-subunit of bovine LH (518B(7)) was used as the capture antibody. The second antibody (a polyclonal rabbit anti-human LH antibody), conjugated to horseradish peroxidase, cleaved a substrate (tetramethyl benzidine), resulting in a color change. The total assay time was approximately 21/2 hr, with incubations at room temperature. Sensitivity was found to be 1.56 ng/ml. Cross-reactivities to elephant FSH and TSH were low: 0.9% and 0.15%, respectively. The accuracy of the assay was demonstrated by comparing the ELISA with a validated eleLH radioimmunoassay (RIA), progesterone data, and  ultrasound observations. Blood samples from 18 Asian and African elephant cows were analyzed with the ELISA and RIA, and an additional 11 cows were used to describe endocrine parameters for LH and progesterone using only RIA. No difference was found in LH peak concentrations between the ELISA and RIA. The time from the progesterone decline to the first LH peak, and the time between the two peaks were similar between species. Asian cows had higher LH peaks than African cows. Ultrasound confirmed the time of ovulation occurring with the second LH peak. Three cows were inseminated and confirmed to be pregnant using this ELISA as a timing device. Instrumentation is not always required, as LH peaks approximating 3 ng/ml can be visually observed. In conclusion, this ELISA can be used as a field test to determine time of ovulation for artificial insemination (AI) or natural breeding of both species of elephants, and thus is an important tool for the preservation of captive populations worldwide.

Hildebrandt, T.B., Hermes, R., Janssen, D.L., Oosterhuis, J.E., Murphy, D., Göritz, F. Reproductive evaluation in wild African elephants prior to translocation. 2004 PROCEEDINGS AAZV, AAWV, WDA JOINT CONFERENCE.  75-76. 2004.
Ref Type: Conference Proceeding
Abstract: Translocations of wild African (Loxodonta africana) elephants have increased significantly since 1993 after Clem Coetzee developed a new method to move adult elephants in Zimbabwe. Since then the technique have been optimized mainly by the staff of the Kruger National Park (KNP) and over 750 elephants in family units and almost 100 mature bulls have been translocated by the KNP capture team.1 The translocations were mainly performed for reducing the number of elephants in KNP and for stocking other reserves. Few elephants were also moved for overseas export to international zoological institutions. However, each elephant translocation is always a logistic challenge and is extremely costly. Therefore, it is very important to select the right elephants or elephant groups for the future translocation. If the main goal of a translocation is the establishment of a new breeding group, it is especially important to select infertile individuals and highly pregnant females which could have a miscarriage due to the transport stress. The IZW team developed a field applicable portable ultrasound technique which allows the reproductive ev ry Killmar (ZSSD), and Randy Rieches (ZSSD).

LITERATURE CITED
1. Hofmeyr, M. 2003. Translocation as a management tool for control of elephant populations. Managing African Elephant Populations: Act or Let Die. Beekbergen, The Netherlands, 6.-7.Nov., Pp. 38-39.
2. Hildebrandt T. B., F. Göritz, N. C. Pratt, D. L. Schmitt, S. Quandt, J. Raath and R. R. Hofmann. 1998. Reproductive assessment of male elephants (Loxodonta africana and Elephas maximus) by ultrasonography. J. Zoo Wildl. Med. 29: 114-128.

Janssen, D.L., Oosterhuis, J.E., Fuller, J., Williams, K. Field technique: A method for obtaining trunk wash mycobacterial cultures in anesthetized free-ranging African elephants (Loxodonta africana). 2004 PROCEEDINGS AAZV, AAWV, WDA JOINT CONFERENCE.  582-583. 2004.
Ref Type: Conference Proceeding
Abstract: The Guidelines for the Control of Tuberculosis in Elephants 2003 (Guidelines) of the National tuberculosis Working Group for Zoo and Wildlife Species were written to protect the health and safety of captive elephants together with their handlers and the viewing public.1 The Guidelines specifically address the display and transport of captive elephants but do not address the unique situation of free-living elephants being imported and subsequently displayed to the public.

Although the Guidelines describe a technique for collecting and handling a trunk wash in a trained, standing, non-anesthetized elephant, it does not describe a similar technique for anesthetized elephants in lateral recumbency. In an attempt to detect active mycobacterial infection in a group of 3 male and 8 female free-ranging African elephants scheduled for import into the United States, a technique was developed for collecting trunk washes in recumbent,  anesthetized elephants for mycobacterial culture.

A South African game-capture crew, experienced in translocating elephants, anesthetized elephants in groups via remote drug delivery and from a helicopter. The ground crew accomplished multiple simultaneous procedures including anesthesia maintenance and monitoring, physical and reproductive examinations, collection of general diagnostic and investigative samples, and trunk washes for mycobacterial cultures. This was accomplished while the capture crew was preparing animals for loading into specially designed trailers for transport to a holding boma. Little time was available for any one of procedure with multiple
animals being attended to at one time.

Once an elephant was stable in lateral recumbency, a 3-m foal stomach tube, prepackaged and sterilized, was inserted into the dependent side of the trunk tip. It was then gently fed up the trunk approximately 2.5 m. A 50-ml sample suction trap was attached to the end of the foal tube.The suction trap was then attached to a battery powered, portable aspirator pump designed for emergency medical care. The aspiration pump was activated to collect secretions from the most proximal portion of the trunk. If little or no secretions were collected by this means, the system was disconnected between the sample trap and the foal tube. Then, 100 ml of sterile saline was placed into raised end of the foal tube allowing it to drain toward the tip through gravity. The suction trap and aspiration pump were reattached to collect a sample in the sample trap. Then, the sample trap was replaced with a new trap, and the foal tube was inserted into the oral pharynx for collection of a separate oropharyngeal sample. This same procedure was repeated
with each elephant.

ACKNOWLEDGMENTS
So African veterinarians, Mike Bester, Larry Killmar, Janet Payeur, ARC/OVI, Thomas Hildebrant, Eric Zeehandelar, Kevin Reily, Denise SoFranko.

LITERATURE CITED
1. National tuberculosis Working Group for Zoo and Wildlife Species. 2003. Guidelines for the Control of Tuberculosis in Elephants 2003. USDA-APHIS: http://www.aphis.usda.gov/ac/TBGuidelines2003.pdf

Kautenburger, R., Wannemacher, J., Muller, P., 2004. Multi element analysis by X-ray fluorescence: A powerful tool of ivory identification from various origins. Journal Of Radioanalytical And Nuclear Chemistry 260, 399-404.
Abstract: The 11th CITES Conference in 2000 decided to maintain a total worldwide ban on trade of elephant ivory. The 12th Conference in 2002 decided to allow a conditional trade in stockpiled ivory in 2004. Reopening of the ivory trade would require methods to identify the origin of the ivory so that legal ivory can be distinguished from the black-market ones. In this study, we describe the X-ray fluorescence multi-element analysis of ivory samples from various regions of Africa and Southeast-Asia to characterize ivory of the various elephant populations.

Kinsella, J.M., Deem, S.L., Blake, S., Freeman, A., 2004. Endoparasites of African forest elephants (Loxodonta africana cyclotis) from the Republic of Congo and Central African Republic. Comparative Parasitology 71 , 104-110.
Abstract: Fecal samples were collected from 6 African forest elephants, Loxodonta africana cyclotis, from the Nouabale-Ndoki National Park, Republic of Congo, and the Dzangha-Sangha National Park, Central African Republic. One of the elephants was found freshly dead from natural causes, and 12 species of intestinal parasites (2 bot fly larvae, 1 trematode, and 9 nematodes) were collected during a complete necropsy. In addition, fecal samples revealed the presence of a schistosome, Bivitellobilharzia sp., a tracheal nematode, Mammomonagamus sp., and a complex of intestinal strongylids and ciliates. The nematode genera Decrusia and Equimurbia are reported for the first time from African elephants, and the ciliate genus Latteuria is reported for the first time from wild elephants. The parasite fauna of the African elephant is discussed in the light of recent genetic evidence that the forest and savannah elephants may be separate species.

Liu, C.H., Chang, C.H., Chin, S.C., Chang, P.H., Zhuo, Y.X., Lee, C.C., 2004. Fibrosarcoma with lung and lymph node metastases in an Asian elephant (Elephas maximus)
672. J. Vet. Diagn. Invest 16,  421-423.
Abstract: A case of fibrosarcoma with lung and lymph node metastases in a 54-year-old female Asian elephant (Elephas maximus) is described. After pododermatitis of 2 years duration in the right forefoot, a mass developed in the lateral toenail. At postmortem, metastasis to the right axillary lymph node and both lungs was noted. Microscopic examination of primary and metastatic sites revealed infiltrating bundles of spindle cells, with fairly distinct cell borders, variable amounts of eosinophilic cytoplasm, and elongate or oval nuclei. Tumor cells were often arranged in interwoven bundles and herringbone patterns. Mitotic figures were numerous and frequently bizarre. The diagnosis of fibrosarcoma with lung and lymph node metastases was made on the basis of histologic features and positive immunohistochemical staining for vimentin

Loomis, M.R., Loomis, J.M. Equipment for use in monitoring anesthetized animals in remote geographic locations. 2004 PROCEEDINGS AAZV, AAWV, WDA JOINT CONFERENCE.  499-501. 2004.
Ref Type: Conference Proceeding
Abstract: Monitoring anesthetized animals in remote geographic locations with no electrical power source can be accomplished with the use of commercially available equipment or with modifications of available equipment. The use of portable solar panels to recharge batteries can supply adequate power to operate most equipment. Equipment for monitoring oxygenation, ventilation, cardiac rhythm and rate, blood pressure and core temperature have been successfully used in areas without an electrical grid or electrical generators.

Criteria for Choice of Equipment for Field Use
Size, weight, power requirements, durability and the ability to operate in harsh environmental conditions should be considered when choosing monitoring equipment for field use. Of concern are the power requirement and the source of the power, particularly in areas where there is no power grid or generator available.

Power Source
There are a number of types of rechargeable batteries on the market.1 Nickel metal halide batteries (NiMH) were chosen for use in monitoring equipment in this study (MAHA Powerx 2100mAh, Thomas Distributing, 128 East Wood, Paris, IL 61944). NiMH batteries have several features that make them attractive for remote use.1 They can be recharged 500 to 1000 times, have no memory, have a fairly steady discharge curve and have the least negative environmental impact when disposed of than other available batteries.1 One disadvantage of NiMH batteries is that they have a self discharge rate of 2-3% per day when not in use. AA NiMH batteries produce 1.2 volts.
Battery energy output is measured in milliamp hours (mAh)2. A battery rated at 1700 mAh will produce 1700 mA for 1 hr. Different manufacturers produce batteries with different power outputs. AA NiMH batteries are rated at up to 2400 mAh. The higher the mAh, the greater the output of the battery.

Batteries are charged using fast, smart chargers attached to portable solar panels (iPowerUS fast smart charger, iPower corporation, CA, USA). A fast charger delivers the amount of current necessary to recharge the battery in 1 hr or less. In general, a slower charge rate will extend the overall life of the battery.3 To overcome the deleterious effects of rapidly charging a battery, a smart charger has a current-limiter built into it that reduce the current as the battery is charged, thereby preventing most of the deterioration.3 The fast smart charger is attached to a portable solar panel (Sun Catcher Expedition solar charger, PowerQwest, Inc.3400 Corporate Way, Suite C Duluth, GA 30096 USA) via a 12 volt "cigarette lighter" type plug. The panel produces 25 watts of power, which is more than enough power to charge 8 AA NiMH batteries at a time. Equipment that uses AA or AAA batteries is preferred so that a large number of different sized rechargeable batteries are not required in the field.

Monitoring Equipment
Oxygenation is measured with a pulse oximeter or by arterial blood gas determination using a portable clinical analyzer. Several brands of pulse oximeters have been successfully used and recharged in the field. An Invacare model 3402NV (Sims BCI, Inc., Waukesha, WI 53186) is relatively small, light weight and operates on 6 AA batteries. This oximeter is durable and operates well on rechargeable AA NiMH batteries.

An I-Stat portable clinical analyzer (Heska Corp. 1613 Prospect Parkway, Fort Collins, CO 80525 USA) has been successfully used in the field using rechargeable 9-volt NiMH batteries. A challenge of using the I-Stat in the field is the analyzer's normal operating temperature of 16-30°C (61-86°F). The I-Stat has been kept in the proper operating temperature range by placing it in a 12-volt thermoelectric cooler (Coleman, Spirit Lake, IA 51360, USA). The thermoelectric cooler runs directly off of the solar panel.

Ventilation is measured using capnography or arterial blood gas determination. The criteria for choice of a capnograph include a waveform display, mainstream and sidestream capabilities and powered by rechargeable AA batteries. The Novametrix Tidal Wave model 615 (Novametrix Medical Systems, INC., Wallingford, and CT USA 06492) meets these criteria. The Tidal Wave comes standard with a rechargeable computer-type battery, but can be ordered with a battery tray, which holds 7 AA batteries. This instrument is durable and operates well on rechargeable NiMH batteries. The sidestream capability allows a large gauge needle to be placed in the lumen of a large endotracheal tube for sampling.

Cardiac rate and rhythm are monitored by use of an electrocardiograph (ECG). A compact ECG unit (Heska Vet/ECG 2000, Heska Corp., 1613 Prospect Parkway, Fort Collins, CO 80525 USA) that operates on 3 AAA rechargeable NiMH batteries is durable and dependable in the field. Blood pressure is measured by a direct arterial line or by indirect methods. Of the indirect methods, automated oscillometry has been successfully used in the field. No automated oscillometric blood pressure machine that runs on replaceable batteries could be found. A compact, durable instrument, Oscillomate 9300 (CAS Medical Systems, Inc., 44 East Industrial Blvd., Branford, CT 06405), was modified for field use. A transformer was manufactured which is inserted between the internal battery of the blood pressure monitor and the solar panel. This allows the internal battery of the blood pressure monitor to be recharged directly from the solar panel.

All monitoring equipment, battery chargers and rechargeable NiMH batteries are transported into the field in a backpack that is designed for photographic equipment (Lowepro Supertrecker AW II, Lowepro USA, P.O. Box 6189, Santa Rosa, CA 95406). All of the above equipment has been dependably used to monitor immobilized elephants in a variety of remote habitats in Cameroon, including dry, hot habitat,2 hot humid habitat.

LITERATURE CITED
1. New technology batteries guide: available battery types. http://www.nlectc.org/txtfiles/batteryguide/batype. htm, March, 2004.
2. New technology batteries guide: performance, economics and tradeoffs.http://www.nlectc.org/txtfiles/batteryguide/ba-type.htm, March, 2004.
3. New technology batteries guide: battery chargers and adapters.http://www.nlectc.org/txtfiles/batteryguide/ba-char.htm, March, 2004.
4. Horne, W.A., M.N. Tchamba, and M.R. Loomis. 2001. A simple method of providing intermittent positivepressureventilation to etorphine-immobilized elephants (Loxodonta africana) in the field. J. Zoo Wildl.Med. 32: 519-522.

Stringfield, C.E., Oh, P., Granich, R., Scott, J., Sun, B., Joseph, M., Flood, J., Sedgwick, C.J. Epidemiologic investigation of a Mycobacterium tuberculosis infection of multiple animal species in a metropolitan zoo. 2004 PROCEEDINGS AAZV, AAWV, WDA JOINT CONFERENCE.  46-48. 2004.
Ref Type: Conference Proceeding
Abstract: From 1997 to 2000, six cases of Mycobacterium tuberculosis (TB) infection were diagnosed in three species of animals at, or recently originating from, the Los Angeles Zoo. Restriction fragment length polymorphism (RFLP) analysis showed that five of six animal isolates shared an identical IS6110 pattern, with the sixth differing only by one additional band. A multiinstitutional epidemiologic investigation was conducted to identify and interrupt possible transmission among the animal cases, and to screen personnel for active TB infection and TB skin-test conversion.
Animal Cases
In April and October of 1994, Asian elephant (Elephas maximus) #1 and Asian elephant #2 arrived at the Los Angeles Zoo from a private elephant facility where they had lived together. They were housed together at the zoo until November of 1996 when elephant #2 was returned to the facility for several months before transfer to another zoo. In the spring of 1997, Elephant #1 (30 yr old) died of salmonellosis, with M. tuberculosis found in granulomatous lymph node lesions from the thoracic and abdominal cavities, and Elephant #2 (30 yr old) was found to have a positive trunk wash culture for M. tuberculosis. In July of 1998, one of a closed herd of three Rocky Mountain goats (Oreamnos americanus) consisting of a sire and two offspring, died of pulmonary M. tuberculosis at 6 yr of age. The goat's asymptomatic herdmates were screened and had negative chest radiographs and tracheal wash cultures, but one of the two goats was positive on tuberculin skin-test. In October of 1998, a clinically normal Black rhinocerus (Diceros bicornis) was diagnosed with Mycobacerium tuberculosis after a positive skin test and nasal wash culture. In the winter of 1998, the two remaining goats were evaluated again with negative chest radiographs and tracheal wash cultures. However, 1 yr later, both were humanely euthanatized at 8 and 12 yr of age due to clinical evidence of tuberculosis on chest radiographs (both animals), and active clinical signs in one (neither were able to be orally treated). In January of 2001, a rhino was humanely euthanatized after a protracted illness that was nonresponsive to aggressive treatment. The rhino was found to have severe multifocal hemosiderosis and atypical mycobacterial infection in her lungs, with no M. tuberculosis  cultured. This animal had been treated with oral Isoniazid and Rifampin for 1 yr, cultured routinely, and was never culture positive again.
 Epidemiologic Investigation
Investigators examined medical and location histories of the affected animals, animal handling practices, health-care procedures, and performed an infection control assessment of the animal compounds and health-care facilities (including measuring air flow in the compounds by smoke testing). We conducted a review of zoo employee medical records for evidence of TB symptoms, tuberculin skin-test results, and chest radiograph information. A list of current and former employees was cross-matched with reported TB cases in the California state registry from 1985 to 2000. As part of the annual occupational health screening in June of 2000, zoo employees underwent questioning regarding TB symptoms, received tuberculin skin tests, and completed a questionnaire on medical history, job type, and history of contact with the infected animals.
Epidemiologic Findings
No common cross-species contact outside the animal compounds and no contact with an infectious human were found. The distance at which the public was kept from the animals and the distance of the compounds from each other (the elephant compound was 27 meters from the rhino compound and the goat compound was 90 m from both) suggests that direct transmission was unlikely. No active TB cases in humans were found, and no matches were found in the database of reporte d cases. The RFLP analysis of this strain of M. tuberculosis matched that of three elephants with which #1 and #2 were housed at a private elephant facility from September of 1993-February of 1994.1 We hypothesize that elephants #1 and #2 were infected at the private facility and were shipped with latent M. tuberculosis infection in 1994, subsequently infecting the black rhino and Mountain goats at the Los Angeles Zoo.
Of interest, animal caretaking and animal contact were not associated with a positive tuberculin skin-test, while groundskeepers were found to have an increased risk of tuberculin skin-test conversion compared with other job categories. Employees attending the elephant necropsy and employees who trained elephants were more likely to have tuberculin skin-test conversion than those who did not.
Conclusion
This is the first documented human and veterinary epidemiologic investigation of Mycobacterium tuberculosis affecting multiple species in a zoo. ² No evidence of transmission from humans to animals or active infections in humans were found. Genotyping evidence strongly suggests transmission from one species to another, although no evidence of transmission was discovered. Human tuberculin skin-test conversions associated with the elephants were most likely due to lack of respiratory protection for these employees when the risk of TB infection was not known. The finding that groundskeepers and not animal handlers were associated with a higher risk of tuberculin skin-test conversion was surprising, and we hypothesized that this may have to do with groundskeepers as a group being more likely to have
been born outside of the United States.
Control measures to eliminate the spread of disease to people and animals were undertaken immediately and throughout this outbreak, and no further cases of M. tuberculosis have been diagnosed at the zoo in the past 3 yr despite ongoing surveillance. Four elephants and three rhinos that had direct contact with the infected animals remain TB negative by trunk and nasal wash culture methods as outlined by the USDA for elephant TB surveillance. Methods of indirect transmission in mammalian zoo species and causes of variability in infection and morbidity within and among species warrant further investigation. Ongoing vigilance, occupational health programs and infection control measures in potentially exposed animals are recommended to prevent ongoing transmission of M. tuberculosis in zoo settings.
Acknowledgments
The authors thank the Animal Care and Animal Health staff of the Los Angeles Zoo who cared so well for these animals, and the veterinarians (including consulting pathologists), technicians, and medical records staff who collected, analyzed, and organized the clinical data. We could not have performed this evaluation without Sue Thisdell, Safety Officer at the Los Angeles Zoo; Jothan Staley and Donna Workman-Malcom of the City of Los Angeles Occupational Health Services Division; Lee Borenstein, Elenor Lehnkering, Patrick Ryan, Jeanne Soukup, and Annette Nita of the Los Angeles County Department of Health Services; and Diana Whipple for her RFLP expertise.
LITERATURE CITED
1. Mikota, S.K., L. Peddie, J. Peddie, R. Isaza, F. Dunker, G. West, W. Lindsay, R.S.Larsen, M. D. Salman, D. Chatterjee, J. Payeur, D. Whipple, C. Thoen, D. Davis, C. Sedgwick, R.J. Montali, M. Ziccardi, J. Maslow. 2001. Epidemiology and diagnosis of Mycobacterium tuberculosis in captive asian elephants (Elephas maximus). J. Zoo Wildl. Med. 32: 1-16.
2. Oh, P., R. Granich, J. Scott, B. Sun, M. Joseph, C. Stringfield, S. Thisdell, J. Staley, D. Workman-Malcolm, L. Borenstein, E. Lehnkering, P. Ryan, J. Soukup, A.Nitta, J. Flood. 2002. Human exposure following Mycobacterium tuberculosis infection of multiple animal species in a metropolitan zoo. Emerging Infectious Diseases. 8 (11): 1290-1293.orte

Chakraborty, A., 2003. Necropsy of elephant. In: Das, D. (Ed.), Healthcare, Breeding and Management of Asian Elephants. Project Elephant. Govt. of India, New Delhi, pp. 145-151.

Cheeran, J.V., Nair, N.D., 2003. Techniques and Procedures for Post-Mortem of Elephants. Project Elephant and Central Zoo Authority, New Delhi India.

Czekala, N.M., MacDonald, E.A., Steinman, K., Walker, S., Garrigues, N.W., Olson, D., 2003. Estrogen and LH dynamics during the follicular phase of the estrous cycle in the Asian elephant. Zoo Biology 22, 443-454.
Abstract: Pituitary and corpus luteum hormone patterns throughout the elephant estrous cycle have been well characterized. By contrast, analysis of follicular maturation by measurement of circulating estrogens has been uninformative. This study tested the ability of a urinary estradiol-3-glucuronide radioimmunoassay to noninvasively assess follicular development during the nonluteal phase of the elephant estrous cycle, and to determine the relationship between estrogen production and the "double LH surge." Daily urine and serum samples were collected throughout seven estrous cycles from three Asian elephants, and urine was collected from an additional three females, for a total of 13 cycles. Serum was analyzed for luteinizing hormone (LH), and urine was analyzed for estrogens and progestins. Elephants exhibited a typical LH pattern, with an anovulatory LH (anLH) surge occurring approximately 21 days before the ovulatory LH (ovLH) surge. The urinary estrogen pattern indicated the presence of two follicular waves during the nonluteal phase. The first wave (anovulatory) began 5 days before the anLH surge and reached a maximum concentration the day before the peak. Thereafter, urinary estrogens declined to baseline for 2 weeks before increasing again to peak concentrations on the day of the ovLH surge. Urinary progestins were baseline throughout most of the follicular phase, increasing 2-3 days before the ovLH surge and continuing into the luteal phase. These results support previous ultrasound observations that two waves of follicular growth occur during the nonluteal phase of the elephant estrous cycle. Each wave is associated with an increase in estrogen production that stimulates an LH surge. Thus, in contrast to serum analyses, urinary estrogen monitoring appears to be a reliable method for characterizing follicular activity in the elephant.

Ganswindt, A., Palme, R., Heistermann, M., Borragan, S., Hodges, J.K., 2003. Non-invasive assessment of adrenocortical function in the male African elephant (Loxodonta africana) and its relation to musth. Gen Comp Endocrinol 134, 156-166.
Abstract: German Primate Centre, Department of Reproductive Biology, Kellnerweg 4, 37077 Gottingen, Germany. ganswindt@www.dpz.gdwg.de

Adult male elephants periodically show the phenomenon of musth, a condition associated with increased aggressiveness, restlessness, significant weight reduction and markedly elevated androgen levels. It has been suggested that musth-related behaviours are costly and that therefore musth may represent a form of physiological stress. In order to provide data on this largely unanswered question, the first aim of this study was to evaluate different assays for non-invasive assessment of adrenocortical function in the male African elephant by (i) characterizing the metabolism and excretion of [3H]cortisol (3H-C) and [14C]testosterone (14C-T) and (ii) using this information to evaluate the specificity of four antibodies for determination of excreted cortisol metabolites, particularly with respect to possible cross-reactions with androgen metabolites, and to assess their biological validity using an ACTH challenge test. Based on the methodology established, the second objective was to provide data on fecal cortisol metabolite concentrations in bulls during the musth and non-musth condition. 3H-C (1 mCi) and 14C-T (100 microCi) were injected simultaneously into a 16 year old male and all urine and feces collected for 30 and 86 h, respectively. The majority (82%) of cortisol metabolites was excreted into the urine, whereas testosterone metabolites were mainly (57%) excreted into the feces. Almost all radioactive metabolites recovered from urine were conjugated (86% 3H-C and 97% 14C-T). In contrast, 86% and >99% of the 3H-C and 14C-T metabolites recovered from feces consisted of unconjugated forms. HPLC separations indicated the presence of various metabolites of cortisol in both urine and feces, with cortisol being abundant in hydrolysed urine, but virtually absent in feces. Although all antibodies measured substantial amounts of immunoreactivity after HPLC separation of peak radioactive samples and detected an increase in glucocorticoid output following the ACTH challenge, only two (in feces against 3alpha,11-oxo-cortisol metabolites, measured by an 11-oxo-etiocholanolone-EIA and in urine against cortisol, measured by a cortisol-EIA) did not show substantial cross-reactivity with excreted 14C-T metabolites and could provide an acceptable degree of specificity for reliable assessment of glucocorticoid output from urine and feces. Based on these findings, concentrations of immunoreactive 3alpha,11-oxo-cortisol metabolites were determined in weekly fecal samples collected from four adult bulls over periods of 11-20 months to examine whether musth is associated with increased adrenal activity. Results showed that in each male levels of these cortisol metabolites were not elevated during periods of musth, suggesting that in the African elephant musth is generally not associated with marked elevations in glucocorticoid output. Given the complex nature of musth and the variety of factors that are likely to influence its manifestation, it is clear, however, that further studies, particularly on free-ranging animals, are needed before a possible relationship between musth and adrenal function can be resolved. This study also clearly illustrates the potential problems associated with cross-reacting metabolites of gonadal steroids in EIAs measuring glucocorticoid metabolites. This has to be taken into account when selecting assays and interpreting results of glucocorticoid metabolite analysis, not only for studies in the elephant but also in other species.

Hunt, K.E., Wasser, S.K., 2003. Effect of long-term preservation methods on fecal glucocorticoid concentrations of grizzly bear and african elephant. Physiol Biochem Zool. 76, 918-928.
Abstract: Center for Conservation Biology, Department of Biology, University of Washington, Seattle, WA, 98195-1800, USA.

Kruzic, J.J., Nalla, R.K., Kinney, J.H., Ritchie, R.O., 2003. Crack blunting, crack bridging and resistance-curve fracture mechanics in dentin: Effect of hydration. Biomaterials 24, 5209-5221.
Abstract: Few studies have focused on a description of the fracture toughness properties of dentin in terms of resistance-curve (R-curve) behavior, i.e., fracture resistance increasing with crack extension, particularly in light of the relevant toughening mechanisms involved. Accordingly, in the present study, fracture mechanics based experiments were conducted on elephant dentin in order to determine such R-curves, to identify the salient toughening mechanisms and to discern how hydration may affect their potency. Crack bridging by uncracked ligaments, observed directly by microscopy and X-ray tomography, was identified as a major toughening mechanism, with further experimental evidence provided by compliance-based experiments. In addition, with hydration, dentin was observed to display significant crack blunting leading to a higher overall fracture resistance than in the dehydrated material. The results of this work are deemed to be of importance from the perspective of modeling the fracture behavior of dentin and in predicting its failure in vivo.

Pothiwong, W., Kamonrat, P., Uthaichotiwan, P., 2003. A morphological study and diagnotic ultrasonography of Asian elephant kidney. Thai Journal of Veterinary Medicine 33, 79-88.

Schmitt, D., 2003. Ultrasonography of elephants. In: Das, D. (Ed.), Healthcare, Breeding and Management of Asian Elephants. Project Elephant. Govt. of India, New Delhi, pp. 32-36.

Schmitt, D.L., 2003. Proboscidea (Elephants). In: Fowler, M.E., Miller, R.E. (Eds.), Zoo and Wild Animal Medicine. Elsevier Science USA, pp. 541-550.

Ziccardi, M., Wong, H.N., Tell, L.A., Fritcher, D., Blanchard, J., Kilbourn, A., Godfrey, H.P. Further optimization and validation of the antigen 85 immunoassay for diagnosing mycobacteriosis in wildlife. Proc Amer Assoc Zoo Vet.  219-220. 2003.
Ref Type: Conference Proceeding
Abstract: Mycobacteriosis caused by Mycobacterium bovis,  M. tuberculosis and M. avium has been a well-documented health problem for zoological collections as long ago as the late 19^th century.  Prevalence estimation in these captive wildlife populations, however, has been hampered by diagnostic test methods that are oftentimes difficult or impossible to conduct and/or interpret (due to the requirement for multiple immobilizations for measurement of response), the occurrence of non-specific results with methods such as the intradermal skin test, and/or the near-total lack of validation, optimization and standardization of any of the available test methods in the species of interest.  Additionally, because intradermal skin testing is the primary screening method for many of these species, the ability to compare exposure in captive wildlife with exposure in free-ranging populations has been limited due to the difficulty with follow-up in free-ranging populations.  Lastly, unlike testing methods that use serological techniques, skin testing precludes retrospective studies of banked samples to determine onset of reactivity.

Recently, human tuberculosis researchers working with tuberculosis in humans have developed an immunoassay that detects a serum protein complex (the antigen 85, or Ag85, complex) produced by mycobacteria in the early stages of mycobacterial infections¹.  Previous work has shown that this method is a promising diagnostic tool in the evaluation of tuberculosis exposure in some primate (including orangutan (Pongo pygmaeus), a species known for non-specific tuberculin responses)²  and captive hoofstock species³.  In order to determine the feasibility and applicability of a widespread use of this method for captive and free-ranging wildlife species, we have undertaken a number of pilot studies on different populations of interest, with the goals of optimizing and validating the immunoassay through analysis of serum from known infected and non-infected individuals and through comparisons with other diagnostic methods.  Thus far, we have begun evaluating the applicability of the antigen 85 immunoassay in various avian, primate, rhinoceros and hoofstock species for detecting tuberculosis and/or paratuberculosis (Johne's disease) infections.  Preliminary results, a summary of which will be presented, indicate that this method may be a valuable adjunct to other testing methods (including gamma interferon and multiple-antigen ELISA) to allow a better evaluation of true mycobacterial status in these species.

LITERATURE CITED

1.Bentley-Hibbert, S. I., X. Quan, T. G. Newman, K. Huygen and H. P. Godfrey. 1999.  Pathophysiology of Antigen 85 in patients with active tuberculosis. Infect Immun. 67(2):581-8.
2.Kilbourn, A. M., H. P. Godfrey, R. A. Cook, P. P. Calle, E. J. Bosi, S. I. Bentley-Hibbert, K. Huygen, M. Andau, M. Ziccardi and W. B. Karesh.  2001.  Serum Antigen 85 levels in adjunct testing for active mycobacterial infections in orangutans.  J. Wildl. Dis. 37(1): 65-71.
3.Mangold, B. J., R. A. Cook, M. R. Cranfield, K. Huygen, and H. P. Godfrey.  1999.  Detection of elevated levels of circulating antigen 85 by dot immunobinding assay in captive wild animals with tuberculosis.  J. Zoo Wildl. Med. 30(4): 477-483.

Zuba, J.R., Stetter, M.D., Dover, S.R., Briggs, M. Development of rigid laparoscopy techniques in elephants and rhinoceros. Proc Amer Assoc Zoo Vet.  223-227. 2003.
Ref Type: Conference Proceeding
Abstract: Diagnostic and surgical laparoscopy has become a routine procedure in human and veterinary medicine and has similar potential uses in zoological medicine. Surgical telescopes and fiberoptic cables allow the veterinarian to look inside body cavities of the patient and specialized instruments provide the ability to perform a wide variety of diagnostic and surgical procedures.  Rigid laparoscopy is considered minimally invasive surgery and is associated with a more rapid post-operative recovery rate and an improved prognosis as compared to conventional surgical techniques.³  Laparoscopic surgery is commonly being utilized in horses and other zoo animals for a variety of abdominal surgical procedures including tubal ligation and ovariectomy.^7,8,12  Recent advances in technology now provide the ability to perform laparoscopy in the largest mammalian species maintained in zoological collections.¹¹

A variety of disease problems and reproductive disorders have been documented in the rhinoceros and elephant.  Due to their size and anatomy, many of the standard diagnostic tests available in human and veterinary medicine are not routine in these animals (radiography, advanced imaging techniques, liver biopsy, abdominal tap, etc.) at this time.  Although conventional abdominal surgery has been performed in rhinoceros and elephants^1,5,10 survival rates have been extremely low.  Laparoscopy can provide a variety of additional diagnostic options, and may also provide an avenue for performing surgical procedures that would otherwise be impossible in these animals.

A multi-institutional collaboration has been organized to address common goals with regard to enhancing diagnostic capabilities and improving surgical techniques in elephants and rhinoceros.  Specifically, we are aiming to: Develop laparoscopic techniques and equipment for use in rhinoceros and elephants, which can be used to significantly expand our diagnostic and treatment capabilities in these large mammals. Improve international conservation efforts in both rhinoceros and elephants by helping improve animal health and welfare of these species in captive and free ranging situations.

This collaborative effort has been organized to critically review current equipment, techniques and uses with the ultimate goal of overcoming some of the inherent difficulties with laparoscopy in these large vertebrates.  This includes further development and modification of equipment, investigation of surgical techniques, and expansion of clinical applications.

Cooperation in the development of innovative surgical equipment for the anatomic variety of our zoological species is necessary for the advancement of zoological medicine.  Karl Storz Veterinary Endoscopy of America (KSVEA, Goleta, California 93117, USA) was instrumental in the initial development of this specialized equipment at the San Diego Wild Animal Park and by generously providing research and development for our continuing investigations.  The availability of appropriately sized equipment has limited the application of laparoscopic techniques to animals of a size consistent with the intended species of its production.  The largest laparoscopy equipment commercially available is marketed for use in equine medicine (57 cm telescope and accessories).  This system has been used with limited success in rhinoceros¹¹ and elephants.  In many cases, to adequately visualize and manipulate visceral organs, a longer telescope and associated instrumentation is required.  We continue to work closely with KSVEA on the production of specialized equipment (Table 1).  Standard light source, fiberoptic light cable and electronic insufflator units manufactured for use in domestic large animals have been used successfully in megavertebrates.  Due to its compact size and versatility, the authors suggest the use of a portable, battery operated laparoscopy kit (Techno Pack, KSVEA) (monitor, light source, camera and digital recording device) especially under field conditions.

To date we have evaluated the utility of specially designed laparoscopic equipment in approximately seven elephants and four rhinoceros.  Experiences from these limited cases have identified several technical and procedural challenges, which need to be overcome if megavertebrate laparoscopy is to be successfully performed.¹¹  Some of these challenges are summarized below:

Technical challenges:
·Equine laparoscopic equipment too fragile and/or short for certain surgical applications in rhinoceros and elephant
·Size and disposition of megavertebrates (> 1000 kg)
·Thick, non-pliable, pachydermatous skin puts unusual pressure (and risk of damage) on equipment
·Insufflation and illumination of large abdominal cavity
·Great depth to visualize and physically reach/manipulate large organs
·Thick, redundant, fibro-elastic peritoneum which is difficult to puncture during surgical attempts to enter the abdominal cavity
·Influence of patient positioning:  unable to use conventional laparoscopic positioning techniques in these species
·Both rhinoceros and elephants are hindgut fermentors and have very large and extensive lower intestinal tracts.  Gas dilation of these bowel loops combined with limited positioning options, can make laparoscopic visualization of certain organs a problem

Procedural challenges:
·Cost of developing new and specialized laparoscopic equipment is high; duplicate equipment is not available at this time
·Charismatic nature of megavertebrates makes it difficult to perform surgery on such important and high exposure species 
·Limited clinical cases for testing equipment due to the relatively low number of megavertebrates in zoological facilities
·Use of laparoscopy in zoo mammals may be out of the comfort zone for many veterinarians and curators due to lack of experience with this instrumentation
·Lack of published surgical procedures in these species
·Inability to perform laparoscopic procedure in "surgical suite" as with most other species
·Risks associated with megavertebrate anesthesia and sedation including difficulty in providing safe anesthetic procedures, proper restraint, and safety of personnel

A multidisciplinary, systematic approach has been initiated to critically review current instrumentation and procedures with the objective of overcoming these technical difficulties. 

The initial phase of our project has been to develop laparoscopic techniques and equipment (Table 1) for use in rhinoceros and elephants by utilizing individuals that may have died of natural causes, or live animals with medical conditions that warrant abdominal surgery.  We have already had the opportunity to perform laparoscopic surgery on both live and deceased white rhinoceros, black rhinoceros, African and Asian elephants.  From our initial studies, it is apparent that laparoscopy in these megavertebrates is possible and may greatly enhance our ability to care for and manage these animals in captive and free ranging situations.  The authors have received some funding and institutional support that allows us to travel to institutions which may benefit from the use of this equipment and/or our experience.  We are also interested in those zoological institutions which may find themselves dealing with a terminal case in an elephant or rhinoceros, to please contact us directly and to consider allowing laparoscopy to be conducted on the animal prior to a post-mortem examination.

Ultimately, we envision the results of our studies on captive animals to be applicable to the management and conservation of elephants and rhinoceros in the wild.  A variety of medical disorders are commonly reported in black and white rhinoceros.  Many of these medical conditions are difficult to diagnose, monitor and treat.  Furthermore, there is a paucity of information on the incidence of these disease conditions in free ranging populations.  The use of minimally invasive laparoscopic techniques will greatly enhance our diagnostic abilities in this species and would be extremely valuable to the understanding of medical conditions of captive and free ranging rhinoceros and to conservation efforts overall.

Throughout many parts of Africa, wildlife professionals are seriously concerned about the negative effects large elephant herds are having on the native flora and fauna within parks and reserves.^2,6,9  The historical rangelands of the elephant have become interrupted by national borders and artificial barriers.  Habitats surrounding wildlife parks are increasingly being converted to agricultural lands. The encroachment of human populations has caused a dramatic increase in the number and severity of human-elephant conflicts.^2,9  These conflicts are commonplace in many parts of East and Southern Africa.  Although there have been a variety of plans to reduce human-elephant and elephant environment impacts, little overall success has been achieved in most countries.⁹

Historically elephant population control has primarily been limited to culling and translocation of small groups.^2,6  Immunocontraception has been attempted with a small population of elephants but is not currently realistic in many situations.^4,6  In wildlife parks where large herds of elephants exist there is currently no effective, humane method of population control.

One of our long-range goals is to develop laparoscopic techniques, such as ovariectomy and tubal ligation, in free ranging African elephants that can be used to sterilize reproductive females.  Once these techniques have been developed, it is our intention to train local wildlife veterinarians and health professionals to perform laparoscopic sterilization of elephants in the field and thus provide local wildlife officials with a tool to help manage elephant populations.  It is our hope this will improve conservation efforts across Africa by reducing human-elephant conflicts and helping to save critical ecosystems.

Acknowledgements
This ongoing project is possible due to the generous contributions of the following individuals:  Hans Lunneman, Christopher Chambliss, Michele McCutcheon, Lynn Richardson, Dean Hendrickson, Robin Radcliffe, Rolf Radcliffe, Laurie Gage, Larry Galuppo, Bill Lindsay, John Olsen, Genny Dumonceaux, and the veterinary staffs at the San Diego Wild Animal Park and Disney's Animal Programs.

LITERATURE CITED
1.Byron, H., J. Olsen, M. Schmidt, J. Copeland, and L. Byron. 1985. Abdominal surgery in three adult male Asian elephants. J Am Vet Med Assoc. 187:1236-1237.
2.Chalfota J. and Owen-Smith N. 1996. Options for the management of elephants in northern Botswana. Pachyderm. 22:67-73.
3.Cook, R.A., and D.R. Stoloff. 1999. The application of minimally invasive surgery for the diagnosis and treatment of captive wildlife. In: Fowler, M., Miller, E. (Eds): Zoo and Wild Animal Medicine: Current Therapy 4. W.B. Saunders, Philadelphia, PA. Pp 30-40.
4.Delsink, A.K., van Altena, J.J., Kirkpatrick, J. Grobler, D., and Fayrer-Hosken, R.A. 2002. Field applications of immunocontraception in African elephants (Loxodonta africana).  J Soc.Reprod. Fert. 60: 117-124.
5.Fowler, M.E., and R. Hart. 1973. Castration of an Asian elephant using etorphine anesthesia. J Am Vet Med Assoc. 163: 539-543.
6.Garai, M.E.  2001.  Managing elephants on private reserves in South Africa.  Proceedings of the International Elephant and Rhino Research Symposium. Vienna, Austria. 259-261.
7.Hendrickson, D.A. 2002. New techniques for performing equine laparoscopic ovariectomy. DVM Best Practices Magazine. Oct. 2002.
8.Hendrickson, D.A., and D.G. Wilson. 1996. Instrumentation and techniques for laparoscopic and thoracoscopic surgery in the horse. Vet. Clin. N.A. Equine Pract.12; 2: 235.
9.Hoare, R., Update on the study and management of human-elephant conflict in Africa. Pachyderm. 33: 91-92.
10.Olsen, J., and H. Byron. 1993. Castration of the elephant. In: Fowler, M. (Ed): Zoo and Wild Animal Medicine: Current Therapy 3, 3^rd ed. W.B. Saunders, Philadelphia, PA. Pp. 441-444.
11.Radcliffe R.M., D.A. Hendrickson, G.L. Richardson., J.R. Zuba, and R.W. Radcliffe. 2000. Standing laparoscopicguided uterine biopsy in a southern white rhinoceros (Ceratotherium simum simum). J. Zoo Wildl. Med. 31:201207.
12.Rogerson, D., M. Brown, B. Watt, C. Keoughan, and M. Hanrath. 2002. Hand-assisted laparoscopic technique for removal of ovarian tumors in standing mares. J. Am. Vet. Med. Assoc. 220(10):1503-1507.

See source for Table 1.  Current laparoscopy equipment specifically manufactured for use in megavertebrates.^a
a All equipment was specially manufactured by Karl Storz Veterinary Endoscopy of America (KSVEA, Goleta, California, USA) and is not commercially available at this time.

Bechert, U.S., Southern, S. Monitoring Environmental Stress in African Elephants (Loxodonta africana) through Molecular Analysis of Stress-Activated Proteins. Baer, C. K. Proceedings American Association of Zoo Veterinarians.  249-253. 2002. Milwaukee, Wisconsin.
Ref Type: Conference Proceeding
Abstract: Many disease outbreaks appear to be facilitated by increased stress due to overcrowding, and changing environmental conditions triggered by climate variability and human activities. Currently, the health of populations is typically assessed with the tools of population dynamics: estimations of trends in abundance, mortality, and reproductive rates. However, for populations that have long generation times, this approach is sometimes too slow to provide an early warning about the impact of environmental stressors such as disease, pollution, and anthropogenic activities. We have developed new techniques for detecting chronic physiologic stress and disease in mammals, based on the molecular analysis of the expression patterns of multiple stress-activated proteins and genes. This approach represents a novel tool for health monitoring, and can provide an early warning of increased environmental stress and compromised health in elephants and other mammals. This paper describes a study in progress, in which the molecular analysis of stress is being used to explore correlations between stress level and information regarding population abundance, distribution, habitat needs, human-elephant interactions, and movements of elephants (Loxodonta africana) in the northern Botswana region. This technique will provide a more objective way to assess carrying capacity for African elephants, thus facilitating development of effective management plans for this species.

Geddes, L.A., 2002. Electrocardiograms from the turtle to the elephant that illustrate interesting physiological phenomena. Pacing Clin Electrophysiol 25, 1762-1770.
Abstract: This article describes a collection of ECGs from many species obtained over the past 50 years. Presented are ECGs of species in which the pacemaker is a separate contractile chamber with its own action and recovery potentials. In such species, pacemaker atrial and AV block can be produced. Shortening of the atrial refractory period and the negative inotropic effect can be produced by vagal stimulation. The cardiac electrogram and stroke volume are recorded from the turtle heart. The ECG and respiration were recorded from the snake. ECG records were obtained from the anesthetized and decapitated housefly. ECG records of the rabbit show slowing when the nose encountered irritating vapors. Records from a dog with atrial fibrillation exhibit rhythmic fibrillation frequency changes correlated with respiration. In addition, in a morphinized dog with atrial fibrillation, impulses crossed the AV node only during inspiration. The ECGs of a cow and camel exhibit long P-R intervals and biphasic P waves. Finally the elephant ECG shows a clear U wave following the T wave.

Kumar, G.A., Ghosh, K.N.A., Sreekumaran, T., Chandrasekharan, K., 2002. Reproduction in elephants. Journal of Indian Veterinary Association Kerala 7, 38-40, 48-48.

Mikota, S.K., Maslow, J. Epidemiology and Treatment of Tuberculosis in Elephants: 2002. Baer, C. K. American Association of Zoo Veterinarians Annual Conference.  384-387. 2002. 2002.
Ref Type: Conference Proceeding

Oh, P., Granich, R., Scott, J., Sun, B., Joseph, M., Stringfield, C., Thisdell, S., Staley, J., Workman-Malcolm, D., Borenstein, L., Lehnkering, E., Ryan, P., Soukup, J., Nitta, A., Flood, J., 2002. Human exposure following Mycobacterium tuberculosis infection of multiple animal species in a Metropolitan Zoo. Emerg Infect Dis 8, 1290-1293.
Abstract: From 1997 to 2000, Mycobacterium tuberculosis was diagnosed in two Asian elephants (Elephas maximus), three Rocky Mountain goats (Oreamnos americanus), and one black rhinoceros (Diceros bicornis) in the Los Angeles Zoo. DNA fingerprint patterns suggested recent transmission. An investigation found no active cases of tuberculosis in humans; however, tuberculin skin-test conversions in humans were associated with training elephants and attending an elephant necropsy.

Payeur, J.B., Jarnagin, J.L., Marquardt, J.G., Whipple, D.L., 2002. Mycobacterial isolations in captive elephants in the United States. Ann N Y Acad Sci 969, 256-258.
Abstract: Interest in tuberculosis in elephants has been increasing over the past several years in the United States. Several techniques have been used to diagnose mammalian tuberculosis. Currently, the test considered most reliable for diagnosis of TB in elephants is based on the culture of respiratory secretions obtained by trunk washes.

Pearson, E.G., 2002. Diseases of the hepatobiliary system. In: Smith, B.P. (Ed.), Large Animal Internal Medicine. Mosby, St.Louis, pp. 790-795.

Reilly, J., 2002. Growth in the Sumatran elephant (Elephas maximus sumatranus) and age estimation based on dung diameter. J. Zool. , Lond 258, 205-213.
Abstract: The aim of this study was to investigate age-related growth in the Sumatran elephant Elephas maximus sumatranus and to use the derived relationship to determine the age structure of the wild elephant population in Way Kambas National Park (WKNP), Sumatra. Shoulder height, forefoot circumference and diameter of dung bolus were found to be related to age of captive Sumatran elephants using the Von Bertalanffy growth function. All length measurements were highly correlated with age in the Sumatran elephant and provide growth models for determining the age structure of wild populations. Female captive elephants reached their growth plateau earlier than male elephants who continued growing throughout the ages observed. There was no clear evidence of a secondary growth spurt in male elephants. The growth model relating dung diameter to age was used to predict the age structure of the wild elephant population in WKNP from dung measured along random line transects. The wild elephant population in WKNP is
young and dominated by sub-adults (between 5 and 15 years of age). There are marked differences between the age structure of the population as revealed in the current survey and that reported from previous studies, suggesting that changes have occurred within the population in the intervening period. The use of dung diameter to predict age offers a robust field technique for use in situations where direct observations are limited, and the use of other age estimation methods is impractical. It is easily coupled with dung counts for estimating the size, age structure and biomass of elephant populations, and has considerable potential for investigating the effects of poaching on age structure and identifying where priority action should be directed in human-elephant conflict situations.

Suedmeyer, W.K. Conditioning programs for transabdominal ultrasound gestational monitoring in an eastern black rhinoceros (Dicerosbicornis michaeli), african elephant, (Loxodonta africana), african lion (Panthera leo), and bornean orangutan (Pongo pygmaeus pygmaeus). Proc Amer Assoc Zoo Vet.  50-52. 2002.
Ref Type: Conference Proceeding

Suedmeyer, W.K. Transabdominal ultrasonic Gestational monitoring in an African elephant (Loxodonta africana). Baer, C. K. American Association of Zoo Veterinarians Annual Conference.  219-220. 2002. 2002.
Ref Type: Conference Proceeding

Tresamol, P.V., 2002. Elephant surra. Journal of Indian Veterinary Association Kerala 7, 60.

Tuntivanich, P., Soontornvipart, K., Tuntivanich, N., Wongaumnuaykul, S., Briksawan, P., 2002. Schirmer tear test in clinically normal Asian elephants. Veterinary Research Communications 26, 297-299.
Abstract: The objective of the study was to evaluate normal tear production in captive Asian elephants (Elephas maximus) in Thailand. 44 elephants (80 eyes) were studied. The mean value for a 1-minute Schirmer tear test (STT), without topical anaesthesia, was 34.3±1.7 mm/min, with a range of 14-70 mm/min. There was no significant difference between males and females. STT values varied with age, being lowest in the 0-20 year age group and highest in the 41-60 year age group.

Vijayan, N., Nair, N.D., 2002. Autopsy in elephants. Journal of Indian Veterinary Association Kerala 7, 53-51.

Wise, D.J., Carter, G.R., 2002. Immunology A Comprehensive Review. Iowa State University Press (Blackwell), Ames IA.

Woodford, M.H., Keet, D.F., Bengis, R.G., 2002. A guide to post-mortem procedure and a review of pathological processes identified in the elephant. Post-mortem Procedures for Wildlife Veterinarians and Field Biologists. IUCN, pp. 36-47.

Ball, R.L. Ultrasound Evaluation of the Pleura Space and Associated Connective Tissue in the Asian Elephant (Elephas maximus). A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  245. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Fritsch, G., Hermes, R., Maltzan, J. New Aspects of Sexual Maturation in Male Elephants. A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  25. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Ganswindt, A., Heistermann, M., Hodges, J.K. Faecal Glucocorticoid and Androgen Metabolite Excretion in Male African Elephants (Loxodonta africana). A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  258. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Ganswindt, A., Heistermann, M., Borragan, S., Hodges, J.K., 2001. Assessment of testicular endocrine function in captive African elephants by measurement of urinary and fecal androgens. Zoo Biology 21,  27-36.

Goeritz, F., Hildebrandt, T.B., Hermes, R., Quandt, S., Jewgenow, K., Hofmann, R.R., Hofer, H., Meyer, H.H.D. Results of Hormonal Contraception in Free-Ranging African Elephants. A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  262. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Graham, L., Schwarzenberger, F., Möstl, E., Galama, W., Savage, A., 2001. A versatile ezyme immunoassay for the determination of progestogens in feces and serum. Zoo Biology 20, 227-236.
Abstract: The ability of zoos to monitor the reproductive status of their animals can vastly improve the effectiveness of husbandry/management practices, and noninvasive methods such as fecal steroid analysis are the easiest to apply in a zoo setting. Furthermore, enzyme immunoassay (EIA) is preferred to radioimmunoassay (RIA) as the method of quantifying hormones because EIAs do not involve the use, storage, and disposal of radioactive materials. However, progesterone is excreted in the feces as predominantly unconjugated metabolites (progestogens) and, until recently, antibodies able to cross-react with a variety of progestogens were used primarily in RIAs. An EIA using a broad-spectrum progestogen antibody is described and applied to serum and/or fecal samples from female African elephants, black rhinoceros, white rhinoceros, okapi, and hippopotami. The clear progestogen profiles generated in these species suggest that the described EIA would be as versatile as the RIA using the same antibody and could be a practical and economical alternative to RIAs for monitoring gonadal function via progestogen analysis in zoo species.

Isaza, R. The elephant trunk wash - An update. ProcElephant Mangers Association Annual Conference.  2001.
Ref Type: Conference Proceeding

Lehnhardt, J., Bolling, J., Pratt, N., Joseph, S., Miller, G., Graham, L., Miller, M., Neiffer, D., Hildebrandt, T., Goeritz, F. Elephant Artificial Insemination (AI) in Protected Contact. A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  70. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Miller, M., Neiffer, D., Weber, M., Fontenot, D., Stetter, M., Bolling, J. Salmonella Culture and PCR Results in a Group of Captive African Elephants (Loxodonta africana). A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  83-86. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Montali, R.J., Richman, L.K., Mikota, S.K., Schmitt, D.L., Larsen, R.S., Hildebrandt, T.B., Isaza, R., Lindsay, W.A. Management Aspects of Herpesvirus Infections and Tuberculosis in Elephants. A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  87-95. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding
Abstract: Elephant endotheliotropic herpesvirus (EEHV) infections and tuberculosis have emerged as causes of illness and mortality in captive elephants. Twenty-six confirmed EEHV cases are documented. Since 1995, 7 have occurred in North America, 10 in Europe and 2 in Asia. A PCR test was used to detect the virus in symptomatic animals; a serological test to identify carrier elephants is under development. The African elephant is a potential source of the EEHV that is lethal for Asian elephants. Fatal infections have also occurred in Asian elephants without African elephant contacts. Three of 6 elephants recovered after treatment with antiviral famciclovir; however, more research is needed to improve the usefulness of this drug. Asian elephants that are less than 10-years old and have been moved to another facility and/or have had contact with African elephants are at increased risk for contracting EEHV. Animals traveling between facilities with a history of EEHV cases may be at greater risk. All young elephants should be monitored daily for anorexia, lethargy, body swellings and blue discoloration (bruising) of the tongue, and be trained for blood sampling and potential oral and rectal treatment with famciclovir.
Since 1996, Mycobacterium tuberculosis has affected about 3% of Asian elephants in North America. Most were from 5 U.S. States with some contacts between private herds. Mandatory annual testing for tuberculosis by trunk wash cultures was established in 1998, and 22 culture-positive M. tuberculosis elephants were identified between 1996-2001. Fifteen were treated with anti-tuberculosis drugs and 7 that died or were euthanized were proven to have tuberculosis at necropsy. Antemortem sera was available from 4/7 4 (75%) were strongly ELISA positive. Tuberculosis is uncommon in African elephants but was recently associated with M. bovis in the U.S. and M. tuberculosis in Germany. Conversely, M. bovis tuberculosis, apparently unrecognized in Asian elephants, recently occurred in Germany. Management issues of elephant tuberculosis will be discussed relative to its complex epidemiology and clinical-pathological correlations.

O'Sullivan, T.J., Junge, E., 2001. The use of sonography in the follow-up care of a foot abscess in a female Asian elephant (Elephas maximus). In: Csuti, B., Sargent, E.L., Bechert, U.S. (Eds.), The Elephant's Foot. Iowa State University Press, Ames, Iowa, USA, pp. 115-116.

Schwammer, H. From Artificial Insemination to Birth. A Case Study on African Elephants (Loxodonta africana). A Research Update on Elephants and Rhinos; Proceedings of the International Elephant and Rhino Research Symposium, Vienna, June 7-11, 2001.  289-292. 2001. Vienna, Austria, Schuling Verlag. 2001.
Ref Type: Conference Proceeding

Wyatt, J. Elephant breeding soundness examination forms and database. Kirk Baer, C. and Wilmette, M. W. Proceedings American Association of Zoo Veterinarians, American Association of Wildlife Veterinarians, Association of Reptilian and Amphibian Veterinarians, and the National Association of Zoo and Wildlife Veterinarians Joint Conference 2001.  396-400. 2001. USA, AAZV. 1.
Ref Type: Conference Proceeding
Abstract: Routine use of standardized soundness examination forms for male and female elephants provides data for an inter-relational database.  Through queries we may answer population-based reproductive questions essential for promoting self-sustaining populations. This poster presentation demonstrates breeding soundness examination forms and accompanying database used in a pilot project to evaluate 25 elephants.

Brown, J.L., 2000. Reproductive endocrine monitoring of elephants: an essential tool for assisting captive management. Zoo Biology 19, 347-367.
Abstract: Considerable information now is available about the basic reproductive biology of elephants, especially females. However, as important as this knowledge is, it no longer is enough to simply compile it into a database. The potential exists for using endocrine monitoring techniques to solve real problems. This review summarizes our current knowledge of elephant endocrinology and offers suggestions on how to use the technology to maximize reproductive potential. The oestrous cycle can be monitored through the analysis of serum progestogens, primarily 5alpha-reduced compounds, and consists of an 8- to 12-week luteal phase and a 4- to 6-week inter-luteal period. Proof of ovarian cyclicity currently is mandatory before Species Survival Plan breeding recommendations are approved. However, because many adult females are not cycling normally, the reproductive monitoring of all cows throughout their life span is now encouraged. Complete endocrine evaluations in conjunction with ultrasound examinations and behavioral assessments are needed to identify causes of reproductive failure and develop mitigating treatments. Progestogen analyses also are effective for monitoring pregnancy, but only if longitudinal samples are collected. Alternatively, pregnancy can be diagnosed in occasional samples using serum prolactin or possibly relaxin measurements after 20 weeks of gestation. Parturition can be predicted on the basis of the rapid decrease in progestogens that occurs about 2-5 days before birth. An updated model of ovarian dynamics during the oestrous cycle suggests that two waves of follicular development occur 3 weeks apart during the non-luteal phase, possibly under the control of follicle-stimulating hormone. Each follicular wave culminates in a luteinizing hormone (LH) surge, with the second surge inducing ovulation and corpus luteum formation. The functional significance of the first, anovulatory LH surge is under investigation, but from a practical perspective it can be used to schedule breeding (by artificial insemination or natural mating) to coincide with the ovulatory LH surge. Less is known about the reproductive biology of bulls, aside from the fact that musth is associated with dramatic changes in androgen secretion. Studies are needed to determine whether poor libido and inadequate semen quality observed in some mature elephants are due to testicular steroidogenic dysfunction. When blood samples cannot be collected for routine hormone analysis, gonadal activity can be monitored non-invasively through the measurement of excreted steroid metabolites (males: androgens; females: estrogens, progestogens) in urine and faeces. Lastly, suggestions for future research priorities are provided.

Doi, O., Komatsumoto, M., Terazono, M., Wada, S., 2000. Exfoliative cytology in vaginal vestibule of female Asian elephants: relation to circulating progesterone concentrations. Zoological Science 17, 1303-1309.
Abstract: The oestrous cycle of the Asian elephant (Elephas maximus) was monitored by analysis of exfoliative cytology in the vaginal vestibule and serum progesterone concentrations. Appearance frequency of each 5 exfoliative cells; parabasal, intermediate, superficial anuclear and nuclear cells and leukocytes; on the smear collected from two elephants was calculated, and serum progesterone concentrations were measured by radioimmunoassay. Serum progesterone concentrations changed regularly with the cycle between 14 and 17 weeks. Using spectrum analysis (Yule-Walker method) to appearance frequency of exfoliative cells, it was found that the time when a superficial cell markedly appeared in vaginal vestibule corresponded to the time when serum progesterone concentration was almost negligible. It is suggested that the time when numbers of two kinds of superficial (anuclear and nuclear) cells and parabasal and intermediate cells increase to the smear of the elephant, means the period from pro-oestrus to oestrus and from metoestrus to dioestrus, respectively.

Enoe, C., Georgiadis, M.P., Johnson, W.O., 2000. Estimation of sensitivity and specificty of diagnostic tests and disease prevalence when the true disease state is unknown. Preventive Veterinary Medicine 45, 61-81.

Fleming, G.J., Isaza, R. Thermography evaluation of trunk paralysis in an Asian elephant (Elephas maximus) using digital thermography. Proc. AAZV and IAAAM Joint Conf.  502-503. 2000.
Ref Type: Conference Proceeding

Freeman, K., 2000. Bone marrow evaluation. In: Feldman, B., Zinkl, J.G., Jain, N.C. (Eds.), Schalm's Veterinary Hematology. Lippinicott, Williams & Wilkins, Baltimore,  Maryland, USA, pp. 29-32.

Greiner, M., Gardner, I., 2000. Epidemiologic issues in the validation of veterinary diagnostic tests. Preventive Veterinary Medicine 45, 3-22.

Hermes, R., Olson, D., Goritz, F., Brown, J.L., Schmitt, D.L., Hagan, D., Peterson, J.S., Fritsch, G., Hildebrandt, T.B., 2000. Ultrasonography of the estrous cycle in female African elephants (Loxodonta africana). Zoo Biology 19, 369-382.
Abstract: The endocrinology of the elephant oestrous cycle has been well characterized, but little emphasis has been placed on evaluating corresponding changes in the reproductive tract. Ultrasound was used to document changes in reproductive tract morphology throughout the oestrous cycle in four cycling female African elephants. During a 7-month period, frequent ultrasound examinations (n=190) during the luteal and non-luteal phase were compared with serum progesterone and luteinizing hormone (LH) concentrations during a 7-month period. Ultrasonographic images documented vaginal and cervical oedema and changes in mucus consistency during the non-luteal phase. The cross-sectional diameter of the endometrium showed a dramatic increase during the non-luteal phase and followed cyclic changes. A different pattern of follicular development on the ovary was associated with the two LH surges. Follicle growth associated with the first, anovulatory LH surge was characterized by the formation of multiple small follicles, in contrast to the maturation of a single large follicle at the second, ovulatory LH (ovLH) surge. Ovulation and the subsequent formation of a corpus luteum (CL) were observed only after the ovLH surge. Ultrasound data in combination with endocrine assessments suggest that the African elephant is non-ovulatory, although multiple non-ovulatory luteal structures developed during the late non-luteal phase of each cycle. Both ovulatory CL and non-ovulatory luteal structures were present only through one cycle and regressed at the end of the luteal phase in conjunction with the drop in serum progesterone. We conclude that periodic reproductive-tract ultrasound assessments in association with continued endocrine monitoring of the oestrous cycle should be incorporated into the routine reproductive health assessment of elephants. This information is necessary for determining reproductive fitness before making breeding recommendations. It also has proven to be an invaluable tool for use with assisted reproductive techniques and has enormous potential for evaluating the efficiency of hormonal therapies used to treat reproductive dysfunction.

Hildebrandt, T.B., Goritz, F., Pratt, N.C., Brown, J.L., Montali, R.J., Schmitt, D.L., Fritsch, G., Hermes, R., 2000. Ultrasonography of the urogenital tract in elephants (Loxodonta africana and Elephas maximus): an important tool for assessing female reproductive function. Zoo Biology 19, 321-332.
Abstract: At present, the worldwide captive elephant population is not self-sustaining.  The major reason for low reproductive rates is the heretofore undiagnosed reproductive disorders of nulliparous females of prime breeding age.  Recent advances in ultrasound technology have facilitated the detection of these disorders in non-sedated animals.  Approximately 2000 ultrasonographic examinations were performed in more than 280 captive and wild African and captive Asian female elephants.  The entire urogenital tract was scanned, measured and documented to provide a reference for ultrasound specialists involved in elephant breeding programs. The primary pathological lesions that influenced reproductive rates in these females were uterine tumors and endometrial cysts, and ovarian cysts that resulted in acyclicity.  The detection of these disorders and their stage of development can be used by elephant managers to make decisions approximately which animals to include in breeding programs.  Ultrasonography can be used as an effective tool for assessing the reproductive fitness of female breeding candidates in both African and Asian elephants.

Hildebrandt, T.B., Hermes, R., Pratt, N.C., Fritsch, G., Blottner, S., Schmitt, D.L., Ratanakorn, P., Brown, J.L., Rietschel, W., Goritz, F., 2000. Ultrasonography of the urogenital tract in elephants (Loxodonta africana and Elephas maximus): an important tool for assessing male reproductive function. Zoo Biology 19, 333-345.
Abstract: The success rate of captive elephant breeding programs worldwide is poor. Along with undiagnosed reproductive disorders in females and fatal diseases such as the newly discovered herpesvirus infection, male infertility now is considered a major contributing factor in the failure to maintain self-sustaining captive populations. To address questions related to male reproductive dysfunction, approximately 309 ultrasonographic assessments combined with semen collection were performed in captive (n=10) and wild (n=4) African (Loxodonta africana) and captive (n=61) Asian (Elephas maximus) elephants. Bulls ranged from 4 to 50 years of age and were examined at 9 institutions in North America, 13 in Europe, 2 in Africa, and 7 in Asia. About half of the reproductive assessments were performed in protected contact situations with elephants handled in a restraint device, and half involved assessments of trained Asian bulls managed in free contact. Four wild African and two Asian elephant bulls were evaluated after receiving general anaesthesia. Transrectal ultrasound was used to characterize the morphology and functionality of the entire urogenital tract, including the testes and accessory sex organs. Bulls were categorized on the basis of breeding status (breeders vs. non-breeders) and social history (i.e., type of interaction with conspecifics and keepers). Most of the bulls were non-breeders (designated Types I-V). Type I (n=3 African, 6 Asian) and Type V (n=1 Asian) were immature and castrate, respectively. On the basis of keeper evaluations, Type II bulls (n=2, 4) were subordinate to older cows and keepers, whereas Type III bulls (n=4, 28) were dominated by other bulls. Type IV (n=1, 8) were older bulls of unknown history that exhibited numerous testicular pathologies resulting in poor semen quality. Non-breeding bulls included those that were exposed to females, but failed to breed, as well as those that had no opportunities to breed. Type VI individuals (n=4, 14) were proven breeders. The percentage of observable reproductive tract pathology in adult males was remarkably low (14%), even in older bulls. However, apparent infertility of non-organic cause (i.e., not due to specific anatomical abnormalities) in these otherwise healthy bulls was high (32%). Semen quality varied markedly in ejaculates collected from the same bull, as well as from different bulls. In conclusion, although many of these bulls could serve as semen donors for natural mating or artificial insemination, the inconsistent production of good-quality ejaculates raises questions as to the reliability of these individuals to participate in breeding programs. The apparent inhibitory effect of suppressive social interactions on reproductive potential also needs to be investigated. Ultrasound examinations combined with semen collection should be conducted periodically to estimate the reproductive value of each bull and determine whether altered management strategies are needed to enhance captive breeding.

Hittmair, K.M., Vielgrader, H.D., 2000. Radiographic diagnosis of lameness in African elephants (Loxodonta Africana). Veterinary Radiology and Ultrasound 41, 511-515.
Abstract: Lameness in captive elephants is most commonly caused by pododermatitis or degenerative joint disease. Hard surfaces such as concrete, which produce a damp and cold environment, wet and muddy conditions, as well as restricted movement are the major causes of these problems. Radiography was performed in two African elephants at the Schoenbrunn Zoo in Vienna to determine the cause and extent of lameness. Various radiographic techniques are described for use in trained elephants. Low time settings were used to avoid loss of detail through movement and to minimize exposure while observing radiation safety. A 37-year-old elephant had front limb lameness due to an interdigital abscess. In radiographs of the foot an inhomogenous soft-tissue swelling without involvement of the phalanges was seen. Ultrasonography was helpful in visualizing the fluid-filled abscess. In additional joint radiographs severe degenerative joint disease was identified. A 13-year-old elephant had lameness of the hind limb. Radiographs of the hind limb from the foot to the stifle were made. Open physes and early signs of degenerative joint disease were identified on the radiographs.

Larsen, R.S., Salman, M.D., Mikota, S.K., Isaza, R., Triantis, J. Validation and use of a multiple-antigen ELISA for detection of tuberculosis infections in elephants. Proc. AAZV and IAAAM Joint Conf.  231-233. 2000.
Ref Type: Conference Proceeding

Lyashchenko, K., Singh, M., Colangeli, R., Gennaro, M.L., 2000. A multi-antigen print immunoassay for the development of serological diagnosis of infectious disease. Journal of Immunological Methods 242, 91-100.

Radcliffe, R.M., Hendrickson, D.A., Richardson, G.L., Zuba, J.R., Radcliffe, R.W. Considerations for laparoscopy in megavertebrates. Proceedings AAZV and IAAAM Joint Conference.  81-82. 2000.
Ref Type: Conference Proceeding

Tveden, H., Weiss, D.J., 2000. Classification and laboratory evaluation of anemia. In: Feldman, B., Zinkl, J.G., Jain, N.C. (Eds.), Schalm's Veterinary Hematology. Lippinicott, Williams & Wilkins, Baltimore,  Maryland, USA, pp. 143-15.

Wasser, S.K., Hunt, K.E., Brown, J.L., Cooper, K., Crockett, C.M., Bechert, U., Millspaugh, J.J., Larson, S., Monfort, S.L., 2000. A generalized fecal glucocorticoid assay for use in a diverse array of nondomestic mammalian and avian species. Gen Comp Endocrinol 120, 260-275.
Abstract: Noninvasive fecal glucocorticoid analysis has tremendous potential as a means of assessing stress associated with environmental disturbance in wildlife. However, interspecific variation in excreted glucocorticoid metabolites requires careful selection of the antibody used in their quantification. We compared four antibodies for detecting the major fecal cortisol metabolites in yellow baboons following (3)H cortisol administration, ACTH challenge, and HPLC separation of fecal glucocorticoid metabolites. The most effective antibody (ICN corticosterone RIA; Cat. No. 07-120102) demonstrated relatively high cross-reactivities to the major cortisol metabolites present in feces during peak excretion, following both radiolabel infusion and ACTH challenge. This same antibody also detected increased fecal glucocorticoid metabolites after ACTH administration in the African elephant, black rhinoceros, Roosevelt elk, gerenuk, scimitar-horned oryx, Alaskan sea otter, Malayan sun bear, cheetah, clouded leopard, longtailed macaque, and northern spotted owl. Results suggest that (1) fecal glucocorticoid assays reliably detect endogenous changes in
adrenal activity of a diverse array of species and (2) where comparisons were made, the ICN corticosterone antibody generally was superior to other antibodies for measuring glucocorticoid metabolites in feces.

Bhat, M.N., Manickam, R., Ramkrishna, J., 1999. Screening of captive wild animals for tuberculosis. Indian Veterinary Journal 76, 959-961.
Abstract: The passive haemagglutination (PHA) test was used to test 109 captive elephants (Elephas maximus), and spotted deer (Cervus axis), blackbuck (Antilope cervicapra) and common langurs (Semnopithecus entellus?) (4 of each) for tuberculosis; 51 of the elephants and the 4 langurs were also assessed by the tuberculin test. PHA titres of 1:16 or 1:32 were found in 4 elephants, 1 deer and 2 langurs, but all were apparently healthy except 1 langur that had clinical signs indicative of tuberculosis. There were 4 positive reactors in the tuberculin tests, all elephants, but these animals did not have significant PHA titres. It is concluded that the procedures and reagents used for the diagnosis of tuberculosis in domestic animals are not reliable for testing wild animals.

Brown, J.L., Hildebrandt, T.B., Theison, W., Neiffer, D.L., 1999. Endocrine and ultrasound evaluation of a non-cycling African elephant: identification of an ovarian follicular cyst. Zoo Biology 18, 223-232.
Abstract: The reproductive rate of captive African elephants is low because of logistical difficulties associated with transporting animals for breeding, the danger of maintaining bulls and medical or physiological problems. There also is growing evidence that a significant number of mature female elephants are not experiencing normal estrous cycles. The case described in this report involves the diagnosis and attempted treatment of an ovarian follicular cyst in an African elephant at the Pittsburgh Zoo. On the basis of serum progesterone analysis, the female exhibited regular ovarian cycles from July 1993 through March 1994, but from November 1994 to the present has not shown any evidence of reproductive cyclicity. In April 1996, a large follicular structure was identified on the right ovary using transrectal ultrasound. In an attempt to luteinize the cyst, 500 ug gonadotropin-releasing hormone (GnRH) was administered intravenously in October 1996, which stimulated a modest increase in serum luteinizing hormone (LH) (approximately twofold over baseline), but no resumption of ovarian activity. The elephant was treated again 5 months later with a higher dose of GnRH (5 mg, i.v.) with the same results. An ultrasound evaluation in July 1997 indicated the structure was still present. In October 1997, the female was given human chorionic gonadotropin (hCG; 10,000 IU, i.m.), which induced estrus and breeding, but no ovulation or luteinizing of the cyst, and she remains acyclic. These results suggest that conventional methods developed in other species for treating ovarian follicular cysts may not necessarily be effective in the elephant. It is also important that reproductive age females be monitored via continuous progesterone analysis and occasional reproductive tract ultrasound evaluations to understand better the etiology of ovarian dysfunction so that effective treatments can be developed to induce consistent ovarian activity.

Fies, M., Heistermann, M., Hodges, J.K., 1999. Patterns of urinary and fecal steroid excretion during the ovarian cycle and pregnancy in the African elephant (Loxodonta africana). Gen Comp Endocrinol 115, 76-89.
Abstract: The aims of the present study were to (I) determine the relative abundance of the 5alpha-reduced progestins 5alpha-pregnane-3-ol-20-one (5alpha-P-3OH) and 5alpha-dihydroprogesterone (5alpha-DHP) and progesterone (P4) in African elephant feces and to establish improved fecal progestin assays for monitoring ovarian function; and (ii) describe longitudinal profiles of urinary and fecal progestin and estrogen metabolites during pregnancy. Matched urine and fecal samples were collected weekly from six adult females throughout 18 nonfertile cycles and two complete pregnancies (89 and 93 weeks duration). Fecal samples were lyophilized and extracted with 80% methanol in water and immunoreactive 5alpha-P-3OH, 5alpha-DHP, and P4 and (for pregnant females only) estrone (E1) and estradiol (E2) determined by enzyme immunoassay. Urine samples were hydrolyzed, ether-extracted, and assayed for 5alpha-P-3OH, E1, and E2. HPLC cochromatography of fecal extracts with various radioactive progestin tracers confirmed the presence of large amounts of both 5-reduced progestins (5alpha-P-3OH > 5alpha-DHP) but not of P4. 5-Reduced progestins (but not P4) were excreted in a cyclic pattern and levels were significantly correlated with urinary 5alpha-P-3OH. Fecal 5alpha-P-3OH showed the more pronounced and consistent luteal-phase elevation and a better correspondence to urine with respect to timing of the luteal-phase rise. Fecal and urinary 5-reduced progestins increased gradually during early pregnancy to maximum values around week 40-45. Levels gradually declined during the second half of pregnancy, reaching baseline values 2 days before parturition. Urinary estrogens did not show any cyclic pattern during the preconception period and levels remained low during the first 30 weeks of gestation. Thereafter, there was a rapid 10- to 20-fold increase to maximum values at mid-pregnancy, followed by a gradual decline to birth. There was no mid-pregnancy elevation in fecal estrogens, but there was a modest increase in E1 during the second half of gestation.

Fowler, M.E., Miller, R.E., 1999. Zoo and Wild Animal Medicine Current Therapy 4. W.B. Saunders, Philadelphia.

Gage, L.J., 1999. Radiographic techniques for the elephant foot and carpus. In: Fowler, M.E., Miller, R.E. (Eds.), Zoo and Wild Animal Medicine: Current Therapy 4. W.B. Saunders, Philadelphia,PA,USA, pp. 517-520.

Green, E.M., 1999. Thermography. In: Colahan, P.T., Merritt, A.M., Moore, J.N., Mayhew, I.G. (Eds.), Equine Medicine and Surgery. Mosby, St. Louis MO USA, pp. 1333-1340.

Gual-Sill, F., Pickard, A.R., Holt, W.V., Green, D. Preliminary Results of Non-Invasive Monitoring of the Estrous Cycle in Female Asian Elephants (Elephas maximus) through Fecal Steroid Analysis. 1999 Proceedings American Association of Zoo Veterinarians.  87-92. 1999. 1999.
Ref Type: Conference Proceeding
Abstract: For a number of years, estrous cycle monitoring and pregnancy detection in the Asian elephant has been performed using urinary steroid hormone metabolite analysis; this technique presents some practical problems. Monitoring the reproductive status through fecal steroid analysis is possible in this and many other species. The steroid metabolite profiles of female Asian elephants were monitored by enzyme-linked immunosorbent assay (ELISA), to provide detailed information about the estrous cycle and pregnancy in this species, and to investigate causes of reproductive failure. Fecal and matched urine samples were non-invasively collected regularly for 6 mo from captive female Asian elephants. (n = 4 cyclic; n = 1 acyclic). The samples were frozen at -20ºC. Gas Chromatography and Mass Spectroscopy (GC-MS) procedures were used to investigate the steroid hormone metabolite profile and to identify the major excretory metabolites; no steroid metabolites were found in the concentrated extracted feces of this species using the currently available methodology. The fecal pregnanetriol profile observed in three of the cyclic females showed a clear relation with their matched urinary pregnanetriol profile and a cyclic pattern was demonstrated. Fecal pregnanetriol values increased from an overall mean of 94.67 ng/g of dry feces (+/- 13.24, range 31.5 - 219.12 ng/g) during the inter-luteal period to a luteal phase mean of 334.61 ng/g dry feces (+/- 43.48, range 34.35 - 1035.1 ng/g). All the data collected from the fecal and urinary analysis of pregnanetriol in all five individuals investigated demonstrated a significant relationship between urinary and fecal pregnanetriol. The acyclic individual showed a mean fecal pregnanetriol concentration of 84.91 ng/g (+/- 13.06) and values ranged from 33.17 ng/g to 211.42 ng/g. Fecal steroid hormone metabolite analysis for monitoring estrous cycles in Asian elephants may be used in the future to monitor free-roaming, wild or semi-wild individuals as well as those in captivity to assist reproductive and conservation programs of this highly endangered species.

Hietala, S.K., Gardner, I.A., 1999. Validity of using diagnostic tests that are approved for use in domestic animals for non-domestic species. In: Fowler, M.E., Miller, R.E. (Eds.), Zoo and Wild Animal Medicine, 4^th ed. W.B. Saunders, Philadelphia, pp. 55-57.

Hildebrandt, T.B., Fritsch, G., Hermes, R., Jewgenow, K., Rudolph, M., Maltzan, J., Wiesner, H., Pratt, N.C., Schmitt, D.L., Goritz, F. Ultrasound Monitoring of the Sexual Maturation in the Male Elephant. 1999 Proceedings of the American Association of Zoo Veterinarians.  203-204. 1999. 1999.
Ref Type: Conference Proceeding
Abstract: In general, the reproductive rate of elephants in captivity is low. This is partly because of logistic difficulties associated with transporting these large animals for breeding purposes and there may be physiologic problems which also contribute to this low reproductive rate. In context with a reproductive assessment of potential breeding bulls it appears that many adult bulls of both species (Loxodonta africana and Elephas maximus) are not producing viable sperms and/or sufficient ejaculate. Our current understanding of incomplete sexual maturation or temporary infertility in male elephants is at best fragmentary. The following study was performed for characterizing the physiologic sexual maturation process in young male elephants. Two adolescent individuals of both species have been examined in order to investigate the time of their sexual maturity. The examination utilized transrectal ultrasonography of the urogenital tract, rectal stimulation for the collection of ejaculates as well as blood samples for plasma testosterone determination. The development of the testes, the accessory glands (especially the ampullae), the concentration of the testosterone, the body-height and the success of ejaculation after manual stimulation was documented and evaluated over a 3-yr period. The results were compared with data from other bull elephants which had ultrasonographic examinations or post mortem investigations. The findings of this study led to important conclusions about the characterization of the reproductive status of male elephants by means of ultrasonographic examinations. We established criteria for reproductive soundness in connection with the recruitment of potential semen donors for future artificial insemination projects. The ultrasonographic examination combined with the semen collection were appropriate methods for characterizing the exact state of sexual maturity or for identifying potential reproductive disorders in male elephants.

Hodges, J.K., Fieb, M., Heistermann, M. Non-Invasive Reproductive Assessment in the African Elephant: Applications to Studies in Captivity and in the Wild. Roth, T. L., Swanson, W. F., and Blattman, L. K. Seventh World Conference on Breeding Endangered Species: Linking Zoo and Field Research to Advance Conservation.  1999. Cincinnati, OH. 1999.
Ref Type: Conference Proceeding

Isaza, R., Ketz, C.J., 1999. A Trunk Wash Technique for the Diagnosis of Tuberculosis in Elephants. Verh. ber. Erkrg. Zootiere 39, 121-124.

Mangold, B.J., Cook, R.A., Cranfield, M.R., Huygen, K., Godfrey, H.P., 1999. Detection of elevated levels of circulating antigen 85 by dot immunobinding assay in captive wild animals with tuberculosis. Journal of Zoo and Wildlife Medicine 30, 477-483.
Abstract: Antemortem diagnosis of tuberculosis in captive wild animals is often difficult. In addition to the variability of host cellular immune response, which does not always indicate current active infection, reactivity to saprophytic or other mycobacteria is common and may interfere with the interpretation of the intradermal tuberculin skin test. Furthermore, the immobilization required for administrating the test and evaluating skin reactions in these animals may result in unacceptable levels of morbidity and mortality, of particular concern in individuals of rare or endangered species. Proteins of the antigen 85 (Ag85) complex are major secretory products of actively metabolizing mycobacteria in vitro. Production of these proteins by mycobacteria during growth in vivo could result in increases in circulating levels of Ag85 in hosts with active tuberculosis. A dot blot immunoassay has been used to detect and quantify circulating Ag85 in captive wild animals with tuberculosis. Elevated levels of Ag85 were observed in animals with active tuberculosis as compared with uninfected animals. Study populations included a herd of nyala (Tragelaphus angasi) (n=9) with no history of exposure to Mycobacterium bovis. Serum Ag85 levels ranged from <5 to 15 uU/ml (median, 5 uU/ml). The other group included 11 animals from a mixed collection with a documented history of an M. bovis outbreak. Animals with pulmonary granulomatous lesions (n=3) had serum Ag85 levels ranging from 320 to 1,280 uU/ml (median, 320 uU/ml). Animals with only chronic mediastinal or mesenteric lymphadenitis (n=4) had serum Ag85 levels ranging from <5 to 80 uU/ml (median, <5 uU/ml). This assay could provide an important adjunct to intradermal skin testing for antemortem diagnosis of tuberculosis in nondomestic species.

Morris, D.D., Barton, M.H., 1999. Techniques for evaluating the hemostatic system. In: Colahan, P.T., Merritt, A.M., Moore, J.N., Mayhew, I.G. (Eds.), Equine Medicine and Surgery. Mosby, St. Louis MO USA, pp. 1986-1987.

Sood, R., 1999. Medical Laboratory Technology: Methods and Interpretation. Jaypee Brothers, New Delhi, India.

Walsh, M.T., Thompson, J. Use of thermography as a diagnostic and prognostic tool in selected cetacean conditions. Proceedings of American Association of Zoo Veterinarians.  358. 1999.
Ref Type: Conference Proceeding
Abstract: The measurement of change in core body temperature, and its relation to infection or inflammation, is one of the oldest and most widely recognized diagnostic tools in medicine. The use of a thermometer is considered a basic part of the initial physical exam in most species and is often followed by other more sophisticated techniques to try to isolate the source of illness. With the development of affordable heat sensitive cameras the clinician can now detect general or specific areas of abnormal tissue temperatures. Detectable changes in temperature may be related to superficial tissue involvement or a reflection of heat production at a deeper level. These manifestations may include isolated or general areas involving such conditions as abscess, trauma, cellulitis, dermatitis, tendonitis, myositis, and pyothorax.
A thermographic camera was used in clinical cases in cetaceans to refine previous findings that indicated it's potential applications in diagnosis and prognosis. Individuals which showed clinical signs compatible with trauma, dental disease, and dermal conditions were examined with an EVS DTIS - 500 camera (Emerge Interactive, 10315 102nd Terrace, Sebastian, Fl 32958 USA) and therapy monitored with periodic thermal scans. Dental disease including trauma to oral tissues, periodontal abscess, and mandibular infections could be readily located, temperature measurements taken, and the size of area of involvement noted. Post therapy follow-up illustrated the ability to gauge the effect of therapy as evidenced by temperature decrease and a decrease in the size of the area involved. The clinician can also better determine the length of drug use based on the response. In one individual case it showed the infection from an abscessed tooth spreading down the lingual side of the mandible.External trauma to the skin can be monitored for extent, complications and speed of resolution. Rake marks received from other dolphins have shown an inflammatory response present much longer than expected. A loss of normal temperature can also be used as a clue to the presence of material that may require debridement. Dermatitis is currently being investigated for possible application of this technology. A Tursiops truncatus female with an extensive visual roughening of the skin showed substantial heat in the affected areas of the skin with thermography but no signs of inflammation on bloodwork. The skin inflammation was readily monitored by thermography until total resolution.

Gunasena, K.T., Lakey, J.R., Villines, P.M., Bush, M., Raath, C., Critser, E.S., McGann, L.E., Critser, J.K., 1998. Antral follicles develop in xenografted cryopreserved African elephant (Loxodonta africana) ovarian tissue. Anim Reprod Sci 53, 265-275.
Abstract: The preservation of germ plasm from endangered species could augment captive breeding programs aimed at maintaining genetic diversity. Mammalian female germ plasm (oocytes) is extremely difficult to collect and cryopreserve; however, a promising alternative is the cryopreservation of ovarian tissue. In the present study, athymic nude (nu/nu) Balb/C mice were used to evaluate in vivo viability of cryopreserved ovarian tissue from Institute of Cancer Research genotype (ICR) mice or elephants. Female mice were ovariectomized prior to transplant of cryopreserved-thawed ovarian tissue from ICR mice (n=4) or elephants (n=6). Control mice were sham operated (n=4) or ovariectomized (n=5). Transplants were in the ovarian bursa, enabling in vivo ovulation and pregnancies from allografts. Vaginal cytology was monitored daily, and the intervals between andduration of epithelial cells present in smears were evaluated. Appearance of epithelial cells in sham-operated and allografted mice were at intervals of 4.3+/-0.6 and 3.3+/-0.5 days, lasting for 1.4+/-0.1 and 1.6+/-0.2 days, respectively. Sporadic incidence of epithelial cells in ovariectomized animals occurred at longer intervals (8.6+/-3.8 days). Females with xenografted elephant ovarian tissue demonstrated epithelial cells in vaginal smears at intervals of 4.5+/-1.0 days, for 2.5+/-0.5 days duration, which was significantly longer than the other groups (P < 0.05). Histological evaluation of tissues at the time of epithelial cells in smears demonstrated well-developed antral follicles, although oocytes were of poor morphological appearance or only cumulus-like complexes were seen. The nude mouse model is effective for assessing cryopreserved ovarian tissue xenograft function which can support the development of antral follicles.

Hildebrandt, T., Goritz, F., Pratt, N.C., Schmitt, D., Quandt, S., Raath, J.P., Hofmann, R.R., 1998. Reproductive assessment of male elephants (Loxodonta africana and Elephas maximus) by ultrasonography. Journal of Zoo and Wildlife Medicine 29, 114-128.
Abstract: Transrectal ultrasonography was performed on five wild and two captive male African elephants (Loxodonta africana) and four captive male Asian elephants (Elephas maximus) to develop standards for assessment of reproductive health and status.  The entire internal urogenital tract was visualized ultrasonographically by using a 3.5 MHz or a 7.5 MHz transducer in combination with a probe extension adapted for elephant anatomy.  The findings were verified by postmortem ex situ ultrasound examinations in several individuals of each species.  Each part of the internal urogenital tract was sonographically detectable except for the bulbourethral glands and the cranial portion of the ureters and ductus deferentes, which were only observed in situ in the neonate.  Each structure visualized was measured and described.  The size and morphology of the urogenital structures, especially the accessory glands, were indicative of breeding status, if known.  There was a notable difference between African and Asian males in the size and morphology of the prostate gland and a slight difference in the shape of the ampullae.  No other structure showed significant species differences.  The detection of the location and description of the testes may provide information for modifying present castration procedures.  Furthermore, ultrasound examination of the male accessory glands may aid in the identification of potential semen donors for assisted reproduction programs in captive elephants.

Jacobson, R.H., 1998. Validation of serological assays for diagnosis of infectious diseases. Rev. sci. tech. Off. int. Epiz. 17, 469-486.

Montali, R.J., Spelman, L.H., Cambre, R.C., Chattergee, D., Mikota, S.K. Factors influencing interpretation of indirect testing methods for tuberculosis in elephants. Proceedings AAZV and AAWV Joint Conference.  109-112. 1998.
Ref Type: Conference Proceeding
Abstract: Serologic and other laboratory tests (such as BTB, ELISA, and gamma interferon) are often used in conjunction with the intradermal tuberculin test to detect tuberculosis (TB) in animals.  The skin test is considered the "gold standard" in domestic cattle and humans, and the BTB test has been highly rated for use in cervid species.  However, these indirect methods for TB diagnosis have not been proven valid in most exotic species susceptible to Mycobacterium tuberculosis complex (which includes M. bovis) infection.  In addition, many of the tuberculin skin testing methods used in exotic species are not uniform in terms of tuberculin type(s) and sites used and interpretation of the end points.

Wright, P.F., 1998. International standrads for test methods and reference sera for diagnostic tests for antibody detection. Rev. sci. tech. Off. int. Epiz. 17, 527-533.

 1997. Laboratory Procedures for Veterinary Technicians. Mosby, St. Louis MO.

Furley, C.W., 1997. Tuberculosis in elephants. Lancet British edition 350, 224.
Abstract: Tests on 171 elephants in zoos and circuses in the USA revealed that 33% were positive to one or more skin tests and 11% were positive by ELISA. As there is no standard procedure for testing elephants caution should be used when interpreting the results.

Gage, L.J., Fowler, M.E., Pascoe, J.R., Blasko, D., 1997. Surgical removal of infected phalanges from an Asian elephant (Elephas maximus). Journal of Zoo and Wildlife Medicine 28, 208-211.
Abstract:  A 40-yr-old female Asian elephant (Elephas maximus) developed cellulitis in her left front leg.  A draining tract behind the lateral nail of her left front foot was discovered.  This lesion was treated by aggressive irrigation using a variety of disinfectant solutions.  Radiographically, there was degeneration and fragmentation of the distal phalanx of the fifth digit and patterns suggestive of osteomyelitis of the second (middle) phalanx.  The fragments of the distal phalanx and the affected portion of the second phalanx were removed surgically.  Six months after surgery the incision had healed but  fistulous tract remained on the palmar surface of the foot.  The tract extended to the second phalanx, and there was radiographic evidence of osteomyelitis in the second phalanx and the distal portion of the proximal phalanx.  The remainder of the second phalanx and the distal potion of the proximal phalanx were surgically removed. Aggressive aftercare allowed complete wound closure by second intention.

Gage, L.J. Techniques for radiographing the elephant foot and carpus using a portable equine radiographic unit. Proc. Amer.Assoc. of Zoo Vet.  190. 1997.
Ref Type: Conference Proceeding

Hildebrandt, T.B., Göritz, F., Pratt, N.C., Schmitt, D.L., Lehnhardt, J., Hermes, R., Quandt, S., Raath, J., West, G., Montali, R.J. Assessment of health and reproductive status in African elephants by transrectal ultrasonography. Proc: Am Assoc Zoo Vet Ann Conf.  207-211. 1997.
Ref Type: Conference Proceeding

Kania, S.A., Richman, L.K., Kennedy, M., Montali, R.J., Potgleter, L.N.D., 1997. The isolation, detection, and cross-reactivity of Asian elephant IgG for the development of serological diagnostic tests. Journal of Veterinary Allergy and Clinical Immunology 5, 125-128.
Abstract: Asian elephant (Elephas maximus) IgG was isolated and purified using a recombinant protein A and proteins G affinity matrix and DEAE cibacron blue chromatography. Rabbits were inoculated with elephant IgG to produce anti-Asian elephant IgG. Using an ELISA, it was determined that the anti-Asian elephant sera has strong reactivity with Asian elephant IgG and African elephant (Loxodonta africana) IgG, moderate reactivity with manatee (Trichechus manatus latirostris) IgG and weaker reactivity with IgG from hyrax (Procavia capensis) and black rhinoceros (Diceros bicornis). Commercially available antisera produced against cattle, deer, and rabbit IgG react weakly with Asian elephant IgG.

Keet, D.F., Grobler, D.G., Raath, J.P., Gouws, J., Carstens, J., Nesbit, J.W., 1997. Ulcerative pododermatitis in free-ranging African elephant (Loxodonta africana) in the Kruger National Park. Onderstepoort Journal of Veterinary Research 64, 25-32.
Abstract: The occurrence of severe lameness in adult African elephant bulls in a shrub Mopane (Colophospermum mopane) ecosystem was investigated. Large ulcers in the soles of at least one front foot were seen in each of the recorded cases. Microscopically, the lesion can be described as a severe, chronic-active, ulcerative, bacterial pododermatitis (complicated by hypersensitivity/septic vasculitis). A variety of bacteria were isolated from these lesions as well as from regional lymph nodes. Streptococcus agalactiae was the most consistent isolate, while Dichelobacter nodosus, the only organism known to be involved with foot disease in domestic ruminants, was isolated from two cases. Contributory factors such as body mass, portal of entry and origin of potential pathogens may have predisposed to the development of the lesions.

Mikota, S.K., Maslow, J. Theoretical and technical aspects of diagnostic techniques for mammalian tuberculosis. Proceedings, American Association Zoo Veterinarians.  162-165. 1997.
Ref Type: Conference Proceeding

Montali, R.J., Hildebrandt, T., Goritz, F., Hermes, R., Ippen, R., Ramsay, E.C., 1997. Ultrasonography and pathology of genital tract leiomyomas in captive Asian elephants: implications for reproductive soundness.  Verh. ber. Erkrg. Zootiere 38, 199-204.

 1996. Principles of validation of diagnostic assays for infectious diseases. Manual of Standards for Diagnostic Tests and Vaccines. Office International des Epizooties (O.I.E.), Paris, pp. 8-15.

Briggs, M., Phillips, L.G., Kimball, V.P., Valli, V.E. Bone marrow collection in the Asian elephant (Elephas maximus). Proceedings American Association of Zoo Veterinarians.  431-433. 1996.
Ref Type: Conference Proceeding
Abstract: The collection of bone marrow is a common practice for the diagnosis of a variety of disease in mammals and birds.  These include disease which can cause any type of blood dyscrasias, anemias, thrombocytopenias, and/or leukopenias.  Examples of a few of these diseases include feline leukemia, feline lymphosarcoma, lymphoma, bovine leukosis, aplastic anemias of many etiologies, equine infectious anemia, and various types of leukemia.  Dependent upon the species involved, marrow is routinely collected from a variety of locations, such as the crest of the tibia in psittacines, or the ribs, iliac crest, or sternum in equids, to the dorsal spinous processes or sternum in the cow. Although in these species there is seldom a  problem with collection, obtaining marrow from some of the megavertebrates such as the elephant or rhinoceros proves to be problematic due to both the restraint of the animals and their size.  This paper describes a simple and effective way to obtain quality bone marrow samples from an Asian elephant (Elephas maximus) and thus likely applicable to other megavertebrate species.

Dalovision, J.R., Montenegro-James, S., Kemmerly, S.A., Genre, C.F., Chambers, R., Pankey, G.A., Failla, D.M., Haydel, K.G., Hutchinson, L., Lindley, M.F., Praba, A., Eisenach, K.D., Cooper, E.S., 1996. Comparison of the amplified Mycobacterium tuberculosis (MTB) direct test, aplicor MTB PCR and IS6, 110-PCR for detection of MTB in respiratory specimens. Clin. Infect. Dis. 23, 1099-1106.

Gardner, I.A., Hietala, S., Boyce, W.M., 1996. Validity of using serological tests for diagnosis of diseases in wild animals. Rev. Sci Tech. Off. Int. Epiz. 15, 323-335.

Hama, N., Murata, K., Yasuda, S., Shimada, A., Sakai, H., Yanai, T., 1996. An autopsy case of an Asian elephant (Elephas maximus) which died without clinical signs. Japanese Journal of Zoo and Wildlife Medicine 1, 49-53.

Hildebrandt, T., Goritz, F., Pratt, N.C., Quandt, S., Lehnhardt, J., Montali, R.J., Pitra, C., 1996. Ultrasonography as a tool to evaluate the reproductive tract in female Asian elephants ultrasound in elephants. J. Ultras. Med 15, 59.

Sandin, R.L., 1996. Polymerase chain reaction and other amplification techniques in mycobacteriology. Clinical Mycobacteriology 16, 617-639.

Sarma, K.K., Kalita, D., Dutta, B., Barua, S.K., 1996. Determination of mean arterial pressure (MAP) in Asian elephant (Elephas maximus). Indian Veterinary Journal 73, 777-778.

Schwarzenberger, F., Mostl, E., Palme, R., Bamburg, E., 1996. Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild, and zoo animals. Animal Reproduction Science 42, 515-526.

Wasser, S.K., Papageorge, S., Foley, C., Brown, J.L., 1996. Excretory fate of estradiol and progesterone in the African elephant (Loxodonta africana) and patterns of fecal steroid concentrations. General and Comparative Endocrinology 102, 255-262.
Abstract: We developed and validated a noninvasive method to quantify fecal estrogens and progestins as a tool for monitoring long-term ovarian activity in free-ranging African elephants. The lag times between iv injection of [(3)H]estradiol and [(14)C]progesterone and peak excretion of radioactivity in urine and feces were approximately 4 hr and 48 hr, respectively. The majority of progesterone metabolites recovered was excreted in feces (55%) versus urine (45%), whereas comparatively little of the recovered estradiol metabolites were excreted in feces (5%) compared to urine (95%). Intrasample variation in fecal hormone concentrations was extremely high but could be substantially reduced by extracting well-mixed fecal powder from freeze-dried samples, taken from the central or premixed portion of the wet sample. This method resulted in a close correspondence between matched serum and fecal progestins (mean correlation =0.81, range 0.61-0.94) collected from five nonpregnant adult females over a 7-month period. Fecal estrogen profiles were more ambiguous, tending to overlap with those of fecal progestins. We conclude that analyses of fecal progestins can provide an effective, noninvasive means of characterizing ovarian activity in free-ranging African elephants.

Brown, J.L., Wemmer, C.M., Lehnhardt, J., 1995. Urinary Cortisol Analysis for Monitoring Adrenal Activity in Elephants. Zoo Biology 14 , 533-542.
Abstract: Cortisol was measured in dichloromethane-extracted elephant urine using an ¹²⁵I solid-phase radioimmunoassay (RIA). The cortisol RIA was validated by demonstrating 1) parallelism between dilutions of pooled urinary extracts and the standard curve, 2) significant recovery of exogenous cortisol added to elephant urine, and 3) a relationship between changes in the peripheral and urinary cortisol after an adrenocorticotropin hormone (ACTH) challenge. One African (Loxodonta africana) and one Asian (Elephas maximus) elephant were given three injections of ACTH (1.25 mg) at 2 h intervals. Serum cortisol increased four- to eightfold within 30 min after the first injection and peaked (nine- to twelvefold increase) after the second injection. Serum concentrations began to decline 2-3 h after the last injection but were still approximately fourfold higher than baseline at the end of the collection period (hour 8). In the urine, cortisol concentrations were increased in the first sample postinjection (1.5 - 4 h) and peaked twenty- to fortyfold by ~6 h. Urinary cortisol remained elevated at 8 h, but returned to baseline by the following morning. Analysis of high performance liquid chromatography fractions of extracted urine revealed that immunoactivity was associated with free cortisol (~90% of total immunoactivity) and a more polar, unidentified metabolite. A method for preserving urine was developed to allow storing unfrozen samples. One pool of urine from each of one African and two Asian elephants was divided into aliquots, placed in tubes containing absolute ethanol (10%), sodium azide (0.1%) or distilled water (control), and frozen after 0, 1 , 2, 3, 4, 6, 8, 10, 12, and 24 weeks of storage at ~25^ºC. In unpreserved samples, cortisol concentrations were reduced 46% by 2 weeks and 95% by 24 weeks. In contrast, ethanol- and sodium azide-preserved samples retained 100 and 95% of cortisol immunoactivity through 8 weeks and 93 and 85% of activity through 12 weeks, respectively. We infer from these data that changes in urinary cortisol excretion in the elephant reflect fluctuations in adrenal activity and may be a useful indicator of stress. Additionally, urine samples can be collected and stored unfrozen for at least 2 months before any appreciable loss in cortisol immunoactivity occurs, a finding potentially useful to field application of this technique.

Hildebrandt, T., Goritz, F., 1995. Sonographischer nachweis von leiomyomen im genitaltrakt weiblicher elefanten. verh. ber Erkrankg. Zootiere 37, 287-294.

Hildebrandt, T.B., Goritz, F. Transrectal ultrasonography for ovary and pregnancy in Indian elephant. Verh ber Erkrg Zootiere.  261-268. 1995.
Ref Type: Conference Proceeding

Papageorge, S., Wasser, S.K., Foley, C., Brown, J. Fecal steroid analysis: validation of extraction and radioimmunoassay for estradiol and progestagens in African elephants (Loxodonta africana) and analysis of fecal samples utilizing a validated method. Joint Conf AAZV/WDA/AAWV.  447. 1995.
Ref Type: Conference Proceeding

Watve, M.G., 1995. Helminth Parasites of Elephants: Ecological Aspects. In: Daniel, J.C. (Ed.), A Week with Elephants; Proceedings of the International Seminar on Asian Elephants. Bombay Natural History Society; Oxford University Press, Bombay, India, pp. 289-295.
Abstract: The helminth parasites of free ranging as well as captive elephants of the Mudumalai Wildlife Sanctuary were studied quantitatively by analysing over 200 dung samples and 7 autopsy examinations. The prevalence and intensities of infection were high in both captive as well as wild elephants. The helminth communities of both were, however, species poor as compared to other mammalian host species. The high prevalence and intensities are thought to be related to the absence of predation and the low species diversity may be a result of absence of other closely related host species. The age and sex of individuals sampled did not affect their parasite loads significantly. The faecal propagule densities were significantly greater during the dry season as compared to the wet season. Stronglid nematodes of the genus Quilonia dominated the helminth communities. Tapeworm infection was significantly greater in captive elephants than the wild ones. The possible reasons for this difference are discussed.

Hildebrand, T., Göritz, F., 1994. Einsatz der transrektalen Sonographie zur Beurteilung des Genitaltraktes weiblicher Elefanten. Imaging 61(suppl 2), 98.

Komoin-Oka, C., Truc, P., Bengaly, Z., Formenty, P., Duvallet, G., Lauginie, F., Raath, J.P., N'-Depo, A.E., Leforban, Y., 1994. A study of the prevalence of trypanosome infections in different species of wild animals in Comoe National Park Cote d'Ivoire: preliminary results of a comparison of three diagnostic methods. Revue d'Elevage et de Medecine Veterinaire des Pays Tropicaux 47, 189-194.
Abstract: Microscopic examination of thin smears, an ELISA, and kit for in vitro isolation of trypanosomes (KIVI) were compared for diagnosis of trypanosomiasis in 3 elephants, 53 African buffaloes, 12 roan antelope (Hippotragus equinus), 9 hartebeest (Alcelaphus buselaphus), 19 waterbuck (Kobus ellipisiprymnus), 61 Kob (Kobus kob), and 6 wart hogs (Phacochoerus aethiopicus). In the 82 animals on which all 3 tests were carried out, 20% were positive by the thin smear method, 50% by the ELISA and 80% by the KIVI test. The trypanosomes were probably T. brucei, T congolense and T. vivax but these were not identified.

Mikota, S.K., Sargent, E.L., Ranglack, G.S., 1994. Medical Management of the Elephant. Indira Publishing House, West Bloomfield MI.

Fowler, M.E., 1993. Zoo and Wild Animal Medicine Current Therapy 3. W.B. Saunders, Philadelphia.

Chakraborty, A., Chaudhury, B., 1992. Pathology of Fasciola jacksoni infestation in elephants. Indian Journal of Veterinary Pathology 16, 98-101.
Abstract: Fasciola jacksoni infection was discovered in 2 out of 3 elephants autopsied at Assam State Zoo, India, during 1985 to 1989. The parasites were attached to biliary epithelium. Microscopy demonstrated that the biliary epithelium was distorted by necrotic tissue which contained erythrocytes and ova of F. jacksoni. The epithelium was analyzed by X-ray microanalysis, which showed that the infected epithelium contained aluminum, silicon, calcium and iron, while non-infected, normal biliary epithelium contained only phosphorus and sulfur. Scanning electron microscopy demonstrated that both the dorsal and ventral surfaces of the parasite possessed spines.

Kharchenko, V.A., Marunchin, A.A., 1992. Helminths of mammals in the Kiev zoological park. Vestnik Zoologii 3, 61-63.
Abstract: Necropsy of 6 animals that died in the Kiev Zoo, Ukraine, revealed the presence of Trichuris trichiura and Subulura distans in Macaca nemestrina, Prosthenorchis elegans in Saimiri sciureus, Murschidia murchida and Hawkesius hawkesi in Elephas maximus and T. globulosa in Giraffa camelopardalis. No helminths were found in Equus hemionus and Felis lynx. The deaths of M. nemestrina and S. sciureus were attributed to the helminth infections. The results of the examination of faeces of other zoo animals for helminth ova are also presented.

Schaedler, J.M., Krook, L., Wootton, J.A., Hover, B., Brodsky, B., Naresh, M.D., Gillette, D.D., Madsen, D.B., Horne, R.H., Minor, R.R., 1992. Studies of collagen in bone and dentin matrix of a Columbian mammoth (late Pleistocene) of central Utah. Matrix 12, 297-307.
Abstract: A Columbian mammoth, Mammuthus columbi, was excavated at an elevation of 9000 feet in Huntington Canyon, Emery County, Utah. Radiocarbon dates on the skeleton indicated death approximately 11,200 years ago. The skeleton was removed from postglacial, Late Quaternary, lake sediments deposited as glacial runoff approximately 9500 years ago. The bones and teeth were especially well preserved in a saturated lake bed. After excavation the bones and teeth were preserved by controlled desiccation, without hardeners, over a period of 9 months. Microradiography, light and electron microscopy, medium and high angle X-ray diffraction, amino acid analysis and cyanogen bromide peptide mapping were undertaken to evaluate the packing, organization, and preservation of collagen in bone and dentin of this mammoth. Microradiography and light microscopy showed that the bone consisted of especially well preserved compact and trabecular bone, and electron microscopy of demineralized bone and tusk showed that the matrix consisted of lamellae of densely packed cylindrical collagen fibrils. Cell remnants with intact nuclei, with or without a nucleolus, as well as variable lengths of plasma membrane were occasionally present on the surface of bony trabecula. Remnants of odontoblast processes were present in some dentin tubules. High and low angle X-ray diffraction demonstrated that the demineralized matrix contained native collagen molecules and amino acid analysis showed that the composition was comparable to that of type I collagen. Cyanogen bromide peptide mapping indicated that the major peptides of type I collagen were present and had the same electrophoretic mobility as that of type I collagen of demineralized Asian elephant bone and rat tail tendon. Abstract truncated at 250 words.

Vijayan, N., Gangadharan, B., Rajan, A., 1992. An autopsy study on certain diseases of captive elephants. Indian Journal of Wildlife Health Management 1, 16-22.

John, M.C., Nedunchelliyan, S., Raghvan, N., 1991. Tuberculin testing in Indian elephants. Indian Journal of Veterinary Medicine 11, 48-49.

Kramer, B., Teixeira, M., Hattingh, J., 1991. The histology of the adrenal gland of the African elephant, Loxodonta africana. South African Journal of Zoology 26, 193-198.
Abstract: The histology, particularly the ultrastructural cytology, of the adrenal gland of the African elephant, Loxodonta africana, is virtually unknown. Tissue from 14 adult male and female elephants was processed for light and transmission electron microscopy. The gland is surrounded by a thick capsule composed of an outer layer of dense connective tissue and an inner layer in which smooth muscle fibres predominate. Below the layer of smooth muscle, a continuous layer of relatively undifferentiated "capsular" cells occur. Where the capsular cells abut on the zona glomerulosa, they appear to be differentiating into glomerulosa cells, as small lipid droplets are present in their cytoplasm. The cortex is divided into three zones as is found in the adrenal glands of other mammals. Large amounts of collagenous and reticular tissue support the secretory cells, which have a marked lipid content. With electron microscopy, the cortical cells show features typical of steroid-producing cells. The medulla is characterized by an outer region of pale-staining chromaffin-positive (adrenaline) cells and an inner region of intensely staining chromaffin-positive (noradrenaline) cells. The latter cells contain granules of different sizes and structure.

Loypetjra, P., 1991. Electrocardiography of the wildlife animals. Thai Journal of Veterinary Medicine 21, 175-186.
Abstract: The electrocardiogram of wildlife animals was recorded using hexaxial lead system.  The animals were seventeen crocodiles, one gibbon, three lorises, three tigers, four elephants and one binturong.  All of them were conscious during the measurement.      The Lead II of electrocardiogram was used in evaluating heart rate, rhythm and measuring the amplitude, time interval and segment.  Standard limb leads were employed to calculate mean electrical axis of the ventricles.      Electrocardiographic features of P, QRS and T waves in all species were normal without slurring or notching.  The values of heart rate per minute of crocodile, gibbon, loris, tiger, elephant, and binturong were 43-65, 166, 125-214, 90-154, 35-49 and 150, respectively.  The duration of P wave in crocodile was between 0.051-0.179 sec, QRS wave was 0.108-0.158 sec, P-R interval was between 0.24-0.42 sec, and Q-T interval was between 0.282-0.454 sec.  P wave duration in gibbon, loris, tiger, elephant and binturong were nearly the same which were 0.04 to 0.08 sec.  The mean electrical axes were between 66 deg-72 deg, 57 deg, 110 deg, 70 deg-85 deg, 40 deg-90 deg and 120 deg in crocodile, gibbon, loris, tiger, elephant and binturong, respectively.      The large variation of ECG values within species was thought to be the unrestrained and exciting effects.  However, this was considered to be normal and could be found in each species.

Sreekumar, K.P., Nayar, K.N.M., Pillai, M.G.R., Sadanandan, K.P., Nayar, S.R., Chandrasekharan, K., Nirmalan, G., 1991. Electrocardiographic studies before, during and after athirathra yajna in animals. Journal of Veterinary and Animal Sciences 22, 112-115.

Wolfe, R., Cathey, M.A., Roberts, F.K., 1991. The latex test revisited. Arthritis and Rheumatism 34, 951-959.
Abstract: Rheumatoid factor (RF) testing by latex fixation in 8,287 outpatients yielded a sensitivity of 81.6% and 78.0% at titers of 1:20 and 1:80, respectively, and a specificity against noninflammatory rheumatic disorders (NIRD) of 96.6%  and 97.7% and against NIRD plus inflammatory disorders of 95.2% and 96.8%, respectively.  The predictive value of a positive test result at the clinic prevalence rate for rheumatoid arthritis (RA) (16.4%) was approximately 80%, and was 70% at 10% prevalence and 10% at 1% prevalence.  No associations of RA with age or sex were found in non-RA patients.  RF titers increased minimally with age in RA patients and were higher in men than in women.  This study suggests that latex testing is far more specific than has been believed and that the titer is not spuriously increased with age.

Haagsma, J., Eger, A. ELISA for diagnosis of tuberculosis and chemotherapy in zoo and wildlife animals. Proceedings of American Association of Zoo Veterinarians , 107-110. 1990.
Ref Type: Conference Proceeding
Abstract: The aim of this study was to improve the diagnosis of bovine tuberculosis in zoo and wildlife animals, in particular by using an Enzyme-Linked Immunosorbent Assay (ELISA). In addition, suspected cases of tuberculosis (TB) with a positive skin test and /or ELISA were treated with antituberculosis drugs. The diagnosis of TB in animals is based primarily on the intradermal tuberculin test, corresponding with cellular immune response. Although this test has practical disadvantages in zoo animals, the application is still of high value. For this purpose tuberculins with a well controlled high potency and specificity should be used. In order to diagnose hypergic or anergic animals it is recommended to use PPD tuberculin with double strength (2 mg tuberculoprotein per ml) or to double the dose (0.2 ml instead of 0.1 ml), so that about 10,000 I.U. are applied. A strict interpretation scheme can increase the efficacy of the test, in particular in the comparative test. In order to improve the diagnosis, we have studied for some years the use of the ELISA which corresponds with humoral immunity.

Harris, C.R. In search of a cervix. Proc.Ann.Elephant Workshop 10.  43-50. 1989.
Ref Type: Conference Proceeding

Hegel, G.V., Hanichen, T., Mahnel, H., Wiesner, H., 1989. Warts (papilloma/sarcoid) in elephant. Erkrankungen der Zootiere 31, 201-205.
Abstract: Warts ( Papilloma, Sarcoid) in  Elephants  ( Hegel,G.)1989; translated from German by Gerda Martin. Papilloma virus - from the group Papova virus - is considered  an etiological agents of wart- like skin changes in cattle, sheep, mountain goat, and rabbit. (ROSENBERGER,1970; ROLLE and MAYR, 1984). Equine sarcoid (PALMER. 1985) found in horses is most likely caused by bovine papilloma virus. The alternate name is based on clinical and morphological differences in the actual papilloma. In the initial stage, the sarcoid is similar to that of the papilloma; however in later stages, tumorous decay on the surface of the epidermis, and proliferation of the mesenchymal part of the tumor in the subcutis dominate (DIET and WIESNER, 1982). Wart- like changes in the skin of elephants as described by PILASKI et al (1987, 1988), proved to be caused by Herpes virus.  Such skin changes in elephants are not rare and require treatment since size and volume of the excrescences  may cause functional disturbances in the patient. Even if the animal's  general   well being  is not impaired, the importance of esthetics and hygiene should not be disregarded in a place where there are spectators and visitors (zoo, circus). The following paper reports findings of wart- like skin changes in elephants. Observations and Therapy In the elephants  kept in the Hellabrunn  Zoo, no case of papilloma or similar skin tumors had occurred since 1972. First case: In 5-28 - 1987, a ca. 18 month old female L.a. named " Sabi" arrived In Hellabrunn. This animal had a wart- like thickening of 1 cm at the dorsal end of the trunk. After 8 weeks, more of those such skin changes appeared on trunk and lower lip without  impairment in general well being. Treatment consisted of  one daily, subcutaneous injection of 1 amp. Chelidonium D7 (DHU Chelidonium majus L.), and application of fresh ??Schoellkraut  juice dabbed onto the warts but was unsuccessful.  After a change of treatment was made: 10 drops of Thuja D4 (DHU Thuja occidentalis L) and 20 drops Acidum nitricum D12 (DHU Acidum nitricum), orally, once a day, at separate times of the day, there remained, after 2 weeks, a wart on the lower lip the size of a cherry pit, and the before mentioned  wart on the dorsal end of  the trunk had now grown to the size of a cherry. Even the strength of Thuja LM 6 (DHU Thuja occidentalis L.)  20 drops, oral, the growth of the wart on the dorsal end of the trunk, now with a diameter of 5 cm, could not be stopped: Exstirpation had to be performed. Frequent  sucking had promoted strong ulceration.  A secondary infection  had set in,  the surface showed granular  tissue exuding blood and pus.

On 10 -  6 -  1987 the growth was exstirpated and tissue was sent for virolog. and histolog. examination. In addition, tissue was removed from a fresh small wart for vaccine. During the operation the animal was immobilized (anesthetic: 0.3 ml Immobilon* (large animal Immobilon Rc* - Vet. Ltd.),  10mg Xylacin, 150 IE Hyaluronidase i. m.). There were no complications during recovery. Two  weeks post op., the first vaccination was given, followed by a second vacc. four weeks there after, of 5.0 ml, subcut.., of an auto vaccine developed by the Institute for Medical Microbiology, Dept. of Infectious and Epidemic Medicine. In February 1988, there occurred another bout with wart- like growth on the ventral part of the trunk, lower jaw, shoulders and feet, some with a diameter of 15 mm.  From the sedated young animal tissue was taken from several newly grown warts for the manufacture of auto vaccine (sedation: "Hellabrunner Mischung" / 150 IE Hyaluronidase). After 10 days, the first vaccination was given, and by the time of the second vacc." Sabi" was free of externally visible skin changes.

On 6. 6. 1988, "Sabi" fell ill again. Over night she was covered with 48 warts,  with  diameters from 2mm - 15 mm on trunk and head, and 10 more on the chest.The attempt to "ice"  the warts with liquid nitrogen was not successful. Instead, coagulation of ca. 20 of the larger warts was used. The monopolar coagulation electrode of the Erbotom F 2 (Erbe Elektromedizin) coagulates reaching deeply  into the healthy zone of the surrounding tissue. As before, tissue for the manufacture of the auto vaccine was taken, as well as  0.5 ml of blood from the ear vein for the manufacture of a "own- blood"  nosode. (Large animal, premedication: 20 mg Xylazin i.m., 20 minutes later : 0.5 ml Immobilon R (large animal Immobilon R c - Vet Ltd.) and 150 IE Hyaluronidase i.m. The following day, "Sabi" was given the "own- blood" nosode at a strength of C5 (20 drops daily).In addition, she was vacc. once again. Since "Sabi" was free of warts at the time of the second vaccination -   given 4 weeks after the first - the "own- blood" treatment was discontinued. Shortly there after, however, several new warts cropped up (diameter ca. 1 cm), so that the "own- blood"  treatments were continued. Since that time "Sabi" has had no recurrences.Second case : The Indian elephant cow (E. maximus) , named "Dirndl" , age ca. 22 years, had been kept in the box next to "Sabi" since "Sabi's" arrival. They kept trunk contact. On 5-2-1988, "Dirndl" showed on the distal trunk a substantially increased raised area ca. 2 x 2 cm oozing blood. It seemed to be an injury from a metal rope used in off limiting. The wound was disinfected and treated twice a day with chloromycetin spray with Gentian violetR (Parke Davis).  After  one week  the growth had increased substantially  and on the surface,  it had a cauliflower-like ulcerated  appearance.Upon light touch or movement of the dorsal  trunk, blood appeard spontaneously.   Four days later, the growth was exstirpated, while the animal was standing. (Sedation: 2.2 ml Hellabrunn mixture / 150 Hyaluronidase i. m.) . The attempt to close the skin of the trunk over the wound failed because the tension in that area was too great.  The surface of the wound was cauterized and treated with ChloromycetinSpray with Gentian violet R (Parke Davis). Tissue for pathological and histological examination was sent out.  One week after the operation,  the area of the wound was  highly swollem and the wound was infected. Treatment: Several times a day, an  ablution with a 0.1 % Rivanol solutionnR (Asid - 2 Aethoxy-6.9-diamin  acridinlactat)  and application of Sulfonamid-Codliver oil salve (WDT = Sulfadimidin- Sodium- cod liver oil).  In addition, analogous to "Sabi" , once daily 20 drops of "own-blood" nosode,  potency C 5  given orally. Three weeks post. op., there could be clearly distinguished a limited relapse, an area of  6 x 9 cm rising  ca. 2 cm  above the healthy skin of the trunk. The surface looked like the first growth.  It was extirpated under general anesthetic  (Premed.:80 mg Xylazin i. m., 20 min. later: 1.8 ml ImmobilionR and 150 IE Hyaluronidase). In addition, the whole wound was coagulated  by monopolar coagulation electrode as above. Daily for 4 weeks, the wound  was brushed with a 1:5 wood tar -alcohol - solution.There were no complications during recovery. After 5 weeks , all that could be seen was a ca. 1.5 cm long small scar on the skin of the trunk.

Histomorphological Findings:  Fixation with formalin, embedding in paraffin; stain: Hemalaun-Eosin, connective tissue stain  in the manner of Masson. The histomorphological  findings based on the tissue samples of "Sabi" and "Dirndl"  are the  same, and agree  with the findings of 3 other skin tumor tissue taken from elephants of different origin (tab. 1). The tumors  consist mainly of fibroplastic cells  with more or less  abundant collagen fibers and blood vessels. The boundaries from the adjacent corium and lower skin is largely indistinct.  In all larger neoplasties , the covering epidermis has been preserved  at margins only due to superficial ulceration.  Here the P. acuta aseptica diffusa borders  are irregular and strongly profiled, the epithelium is acanthoid and hyperkeratotic. The nuclei of tumor cells  are considerably anisomorphic, some have gigantic nuclei. Mitosis is frequent. Due to the ulcerated epidermis , there is deep infiltration with infectious cells. Virological findings: From the extirpated tissue taken from the African Elephant "Sabi" ca. 3 g was homogenated, in addition, the cells were "opened" by defrosting and ultrasound, and the "cleared" tissue suspension was analyzed  for free virus particles after concentration and negative-contrasting with  electron microscopy .  At the same time, small tissue samples of 2 mm  from deeper epidermis layers  were fixed as usual for the ultrahistological exam , embedded in epoxy  resin, and ultra thin slices were scanned by the electron microscope. No papilloma virus was found  in the concentrated, cell free tissue extract or  the ultrathin slices of tissue samples .No virus particle of  any kind was found.

Discussion
To  show papilloma by culturing cannot be done since  no species of this genus can  be propagated  in cell cultures  with the exception of its original host. The failed  attempt to prove their presence with the electronmicroscope does not exclude a papilloma virus etiology in tumors. When virus particles are viewed in higher concentrations, the electron microscopic  proof is successful. Using ultrahistologyical methods the particles in cell nuclei can only be found when the few cells  of specific skin cells  are in the virus propagation stage. In the case of virally induced papilloma however, a true virus propagation is not necessary. In the last few years, it was found that equine sarcoid  can be caused  by bovine papilloma virus. But it was only the genome of the virus which could be isolated by means of gene technology (ALTMANN, 1980; HAUSEN, 1980); the virus itself could not.  The oncogenetic potency of the virus in heterologic hosts , without true virus production, has been established.  A broader spectrum of hosts  for , at least , the papilloma virus in cattle seems to be the case.  And a bovine papilloma induced skin fibromatose in  (a) horse has been reported (LANCASTER, 1979). This virus can also appear in wild 'cud chewers, perhaps even carnivores. It is in part also related to the human papilloma virus. The possibility of transfer to humans (LANCASTER 1982) as well as other mammals such as elephants  has not been proved but is probable. In comparing the histological findings of the 5 skin growths with those of the viral fibropapilloma in cattle and horse (called equine sarcoid here), the relative immaturity of the tumorous tissue is evident. It compares to the so- called sarcoid in horses. The sarcoid-like structure and the indistinct  separation from healthy tissue  speaks for a virus etiology and  morphologically a relapse can be expected. This occurred in both of the clinically described cases.

A differential diagnosis excludes a Herpes virus infection, as described by PILASKI et al. (1987, 1988) in elephants on the basis of different histological findings. Inclusions could not be found in any of the cases. The warts on the elephants were clinically similar to the well known sarcoids in horses (DIETZ and WIESNER, 1982). The two sick animals were in "trunk contact" occupying adjacent boxes. Almost one year after the arrival of  "Sabi" who had warts, "Dirndl" fell sick. That points to the infectious nature of warts. The relapse after the first operation on "Dirndl" suggests that the extirpation of the growths was not complete. This may be related to the fact that the animal was standing  and only sedated. In contrast , the extirpation of the "relapse" was carried out on a fully immobilized animal and with the use of the Erbotom F 2  for coagulation including the adjacent tissue.  We know of various 'wart therapies' in human medicine with differing success. The various treatments employed in the one and one half years of "Sabi's" illness can be labled neither successful, nor unsuccessful. The use of auto vaccine which is analogous  to a "stable specific " vaccine in the treatment of papilloma in cattle, could  perhaps have triggered the recurrence of warts at the  conclusion of the vaccination treatments. That would favor the etiology of a virus 'picture.' The influence of the 'burn' or extirpation of a single or more growths which returned, in the surrounding growths cannot be determined. It remains inconclusive if the use of the "own- blood" nosode C 5 aided the successful therapy , since the necessity  to fight a recurrence had not yet occurred.

Kalemera, M.C., 1989. Observations on feeding preference of elephants in the Acacia tortilis woodland of Lake Manyara National Park, Tanzania. African Journal of Ecology 27, 325-333.
Abstract: The dry season feed preferences of elephants [Loxodonta africana] were recorded in the Acacia tortilis (Forsk) Hyne woodlands of Lake Manyara National Park in northern Tanzania from 1980 to 1981. At least 35 plant species were eaten varying from trees to small herbs. Elephants mainly browsed in the woodland but also grazed at the lake shore. Feeding on woody species appeared to be in proportion to abundance. The relatively higher amount of grazing during the dry season was related negatively to periods of high rainfall. When rainfall was low, lake levels dropped increasing the availability of lake-shore grassland whereas during years of high rainfall the grassland was flooded and elephants resorted to browsing as a major form of foraging.

Wissdorf, H., Poulsen-Naatrup, C., 1989. Presentation of the Vena caudalis centralis, a further important anatomical structure for venipuncture in Asian elephant situated dorsally in the tail. Anatomia Histologia Embryologia. 13, 279-280.

Hromadka, J. Observations made on an Asian elephant (Elephas maximus) during gestation. Proc.Ann.Elephant Workshop 9.  93-105. 1988.
Ref Type: Conference Proceeding

Karesh, W.B., Smith, F., Frazier-Taylor, H., 1987. A remote method for obtaining skin biopsy samples. Conservation Biology 1, 261-262.

Rubel, A. Physiological and pathological conditions associated with reproduction of female Asian elephants at the Zurich zoo. Proc.1st.Intl.Conf.Zool.Avian Med.  379. 1987.
Ref Type: Conference Proceeding

Fowler, M.E., 1986. Zoo and Wild Animal Medicine. W.B. Saunders, Philadelphia.

Jensen, J. Paralumbar kidney biopsy in a juvenile African elephant. Proc.Am.Assoc.Zoo Vet.  17. 1986.
Ref Type: Conference Proceeding
Abstract: Same case as Ref # 305.

Kahl, V.A.L. The study of the reproductive cycle of the African elephant (Loxodonta africana) through correlation of behavior and cytology with serum and urinary hormonal patterns.  1986. Nebraska, Omaha, USA, University of Nebraska.
Ref Type: Thesis/Dissertation

Meijler, F.L., van der Tweel, L.H., 1986. Electrocardigrams of 10 elephants and a killer whale in Harderwijk. Ned Tijdschr Geneeskd 130, 2344-2348.

Allen, J.L., Welsch, B., Jacobson, E.R., Turner, T.A., Tabeling, H., 1984. Medical and surgical management of a fractured tusk in an African elephant. Journal of the American Veterinary Medical Association 185, 1447-1449.

Schmidt, M.J., 1981. Technic and applications of venipuncture in the elephant. In: Mellen, J., Littlewood, A. (Eds.), Recent developments in research and husbandry at the Washington Park Zoo. Washington Park Zoo, Portland, Oregon, pp. 63.

Folk, G.E., Folk, M.A., 1979. Physiology of large mammals by implanted radio capsules. In: Amlaner, Jr.C.J., Macdonald, D.W. (Eds.), A handbook on biotelemetry and radio tracking. Pergamon Press, New York, pp. 33-43.

Hall-Martin, A.J., Ruther, H., 1979. Application of stereo photogrammetric techniques for measuring African elephants. Koedoe 22, 187-198.
Abstract: Measurements of shoulder height and back length of African elephants were obtained by means of stereo photogrammetric techniques.  A pair of Zeiss UMK 10/1318 cameras, mounted on a steel frame on the back of a vehicle, were used to photograph elephants in the Addo Elephant National Park, Republic of South Africa.  Several modifications of nomal photogrammetry procedure applicable to the field situation (eg. control points) and the computation of results (eg. relative orientation) are briefly mentioned.  Six elephants were immobilized after being photographed and the measurements obtained from them agreed within a range of 1 cm-10 cm with the photogrammetric measurements.

Mill, J., Kuntze, A., 1978. ECG studies in healthy elephants and in one diseased elephant (Elephas maximus). Erkrankungen der Zootiere 14, 315-326.

Nevill, G.F., Crompton, W.G., Hennessy, M.A., Watson, P.F., 1976. Instrumentation for artificial insemination in the African elephant  Loxodonta africana. International Zoo Yearbook 16, 166-171.

Jainudeen, M.R., Scheurmann, E., 1975. Diseases of the working elephant (Elephas maximus) in Sri Lanka (Ceylon) with special reference to diagnostic and therapeutic possibilities within the
country. Deutsche Tierarztliche Wochenschrift 82, 355-359.

Watson, P.F., D'Souza, F., 1975. Detection of oestrus in the African elephant (Loxodonta africana). Theriogenology 4, 203-209.
Abstract: Swabs of mucus and cells from the reproductive tract of a 15 year old female African elephant in captivity were examined. Daily samples were obtained over a 1-year period by means of a probe designed to penetrate the urogenital sinus to a depth of 90 cm.  Dried smears of mucous material showed ferning patterns at intervals of approximately 16 days.  Dried spots of supernatant from washing of the swabs also showed intense ferning at 16 day intervals, but with greater regularity. Smears were stained and examined for the presence of squamous cells over a 4-month period.  Results indicate a regular occurrence of certification at approximately 15-day intervals. These observations indicate that the oestrous cycle of this elephant has a duration of approximately 16 days.  This is the first detailed study of the oestrous cycle in the African elephant, knowledge of which is essential for artificial breeding.

Maberry, M.B., 1972. Diagnosis of pregnancy in Asiatic elephant. Journal of Zoo and Wildlife Medicine 3, 31-33.

Jainudeen, M.R., Eisenberg, J.F., Tilakertne, N., 1971. Oestrous cycle of the Asiatic elephant, Elephas maximus, in captivity. Journal of Reproduction and Fertility 27, 321-328.
Abstract: During the course of a programme to breed the Asiatic elephant, Elephas maximus, in captivity, the oestrous cycles of eleven adult females were studied.  Two methods were used for detecting oestrus: (1) daily testing with a male elephant, and (2) urogenital smear cytology.  Overt signs of oestrus were not observed but "standing" oestrus was observed in ten animals. The duration of oestrus ranged from 2 to 8 days with a mode of 4 days.  Oestrous cycles in six animals ranged from 18 to 27 days with a mean of 22 days.  Urogenital smear cytology failed to indicate accurately the onset of behavioural oestrus but increases in the number of cornified cells may occur before, during and slightly after behavioural oestrus.  Considerable mating activity occurred during oestrus.  A description of mating behaviour is presented.  The findings are discussed in relation to breeding elephants in captivity and to the phenomenon of temporal gland activity.

Geddes, L.A., Hoff, H.E., Cohen, B.S., 1967. The electrocardiogram of an elephant. The Southwestern Veterinarian 20, 211-216.

Jayasinghe, J.B., Fernando, S.D.A., Brito-Babapulle, L.A.P., 1964. The electrocardiogram of a baby elephant. American Heart Journal 67 , 388-390.

Jayasinghe, J.B., Fernando, S.D.A., Brito-Babapulle, L.A.P., 1963. The electrocardiographic patterns of Elephas maximus -- the elephant of Ceylon. British Veterinary Journal 119, 559-564.

Jayasinghe, J.B., Brito-Babapulle, L.A.P., 1961. A report on the electrocardiogram of the Ceylon elephant. Ceylon Veterinary Journal 9 , 69-70.

Hashimoto, Y., Yamauchi, S., Yasunobo, E., 1956. Dissection of an elephant. Bulletin University Osaka Prefecture series B 6, 30-52.

Ayer, A.A., Mariappa, D., 1952. A radiographic study of ossification in the Indian elephant fetus. Journal of the Anatomical Society of India 1, 3-10.

Hill, W.C.O., 1938. The external and radiological anatomy of a foetal Asiatic elephant. Ceylon Journal of Science 21, 31-43.

White, P.D., Jenks, J.L., Benedict, F.G., 1938. The electrocardiogram of the elephant. American Heart Journal 16, 744-750.
Abstract:  An analysis has been made of the electrocardiograms of nine circus elephants with heart rates ranging from 24 to 53 per minute (average of 35 to 40).  Relatively low amplitude of the P-, QRS, and T-waves was found in the three classical leads (with greatest excursions in Lead I), despite accurate standardization which was made easy by the remarkably low resistance invariably found (often only 200 to 300 ohms in any given lead).  The various time intervals (P-R of 0.28 to 0.41 sec, QRS of 0.12 to 0.18 sec, and Q to T time -- duration of systole -- of 0.59 to 0.79 sec) were beyond the measurements to be expected at slow heart rates in the case of mammals of average size like man, and may be explained by the immense size of the elephant's heart with its longer paths of impulse conduction and its greater bulk of contracting muscle.

Forbes, A., Cobb, S., Cattell, M., 1921. An electrocardiogram and an electromyogram in an elephant. American Journal of Physiology 55, 385-389.

Bonvicini, A., 1897. Necropscopia di una elefantessa Cisti d'echinococco nel fegato e nei polmoni. Bologna.
Abstract: Note: Citation may be incomplete.  May be a book.

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