immune

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.

Knauf, S., Blad-Stahl, J., Lawrenz, A., Schuerer, U., Wehrend, A., 2009. Plasma preparation and storage for African elephants (Loxodonta africana). Journal of Zoo and Wildlife Medicine 40, 71-75.
Abstract: The use of plasma as a life-saving tool for neonatal African elephants (Loxodonta africana) that failed passive transfer of immunoglobulins is proposed. The methodology of blood sampling, plasma extraction, and plasma storage is described. Values for cellular component sedimentation and biochemical parameters of extracted plasma that was collected from 2 female elephants is presented. The proposal for a central plasma bank for elephants in European zoos is suggested.

Landolfi, J.A., Schultz, S.A., Mikota, S.K., Terio, K.A., 2009. Development and validation of cytokine quantitative, real time RT-PCR assays for characterization of Asian elephant immune responses
71. Vet. Immunol. Immunopathol. 131, 73-78.
Abstract: Infectious disease is an important factor in Asian elephant health and long-term species survival. In studying disease pathogenesis, it is important to consider not only the pathogen, but also the effectiveness of the host immune response. Currently, there is a paucity of information available on elephant immune function. Measurement of cytokine levels within clinical samples can provide valuable information regarding immune function during health and disease that may elucidate disease susceptibility. To develop tools for assessment of elephant immune function, Asian elephant partial mRNA sequences for interleukin (IL)-2, IL-4, IL-10, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and beta-actin were determined. Sequence information was then utilized to design elephant-specific primers and probes for quantitative, real time, RT-PCR assays for the measurement of cytokine mRNA. Greater than 300bps of Asian elephant mRNA sequence were determined for each cytokine of interest. Consistent and reproducible, real time, RT-PCR assays with efficiencies of greater than 93% were also developed. Assay sensitivities ranged from less than 1 to 5000 DNA copies with the exception of IL-12, which had a sensitivity of 42,200 copies. Employment of molecular techniques utilizing mRNA-based detection systems, such as real time, RT-PCR, facilitate sensitive and specific cytokine detection and measurement in samples from species for which commercial reagents are not available. Future studies utilizing these techniques to compare elephant immune function during health and in the face of infection will be useful for characterizing the contribution of the elephant immune system to disease

Looringh van Beeck, F.A., Reinink, P., Hermsen, R., Zajonc, D.M., Laven, M.J., Fun, A., Troskie, M., Schoemaker, N.J., Morar, D., Lenstra, J.A., Vervelde, L., Rutten, V.P., van, E.W., Van, R., I, 2009. Functional CD1d and/or NKT cell invariant chain transcript in horse, pig, African elephant and guinea pig, but not in ruminants
109. Mol. Immunol. 46, 1424-1431.
Abstract: CD1d-restricted invariant natural killer T cells (NKT cells) have been well characterized in humans and mice, but it is unknown whether they are present in other species. Here we describe the invariant TCR alpha chain and the full length CD1d transcript of pig and horse. Molecular modeling predicts that porcine (po) invariant TCR alpha chain/poCD1d/alpha-GalCer and equine (eq) invariant TCR alpha chain/eqCD1d/alpha-GalCer form complexes that are highly homologous to the human complex. Since a prerequisite for the presence of NKT cells is the expression of CD1d protein, we performed searches for CD1D genes and CD1d transcripts in multiple species. Previously, cattle and guinea pig have been suggested to lack CD1D genes. The CD1D genes of European taurine cattle (Bos taurus) are known to be pseudogenes because of disrupting mutations in the start codon and in the donor splice site of the first intron. Here we show that the same mutations are found in six other ruminants: African buffalo, sheep, bushbuck, bongo, N'Dama cattle, and roe deer. In contrast, intact CD1d transcripts were found in guinea pig, African elephant, horse, rabbit, and pig. Despite the discovery of a highly homologous NKT/CD1d system in pig and horse, our data suggest that functional CD1D and CD1d-restricted NKT cells are not universally present in mammals

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

Perelygin, A.A., Zharkikh, A.A., Astakhova, N.M., Lear, T.L., Brinton, M.A., 2008. Concerted evolution of vertebrate CCR2 and CCR5 genes and the origin of a recombinant equine CCR5/2 gene. J. Hered. 99, 500-511.
Abstract: Chemokine receptors (CCRs) play an essential role in the initiation of an innate immune host response. Several of these receptors have been shown to modulate the outcome of viral infections. The recent availability of complete genome sequences from a number of species provides a unique opportunity to analyze the evolution of the CCR genes. A phylogenetic analysis revealed that the CCR2 gene evolved in concert with the paralogous CCR5 gene, but not with another paralogous gene, CCR3, in the opossum, platypus, rabbit, guinea pig, cat, and rodent lineages. In addition, evidence of concerted evolution of the CCR2 and CCR5 genes was observed in chicken and lizard genomes. A unique CCR5/2 gene that originated by unequal crossing over between the CCR2 and CCR5 genes was detected in the domestic horse. The CCR2, CCR5, and CCR5/2 genes were mapped to ECA16q21 using fluorescent in situ hybridization (FISH). Single-nucleotide polymorphisms identified in the equine CCR5 gene and characterized within 5 horse breeds provide haplotype markers for future case/control studies investigating the genetic bases of horse susceptibility to infectious diseases

Lynn, D.J., Bradley, D.G., 2007. Discovery of alpha-defensins in basal mammals. Dev. Comp Immunol. 31, 963-967.
Abstract: Alpha-defensins are essential molecules of the innate immune system that have broad spectrum antimicrobial activity against a range of bacteria and viruses. To date, alpha-defensins have only been identified in the Euarchontoglires branch of the mammals. This has led to speculation that alpha-defensins may be specific to this group, a somewhat surprising finding, given their importance in the immune system. The mammalian genome project provided us with the opportunity to search for alpha-defensins in previously unexamined mammalian superorders. Using hidden Markov model (HMM) profile searching, we report the discovery of alpha-defensins in the African savanna elephant, the lesser hedgehog tenrec, and the nine-banded armadillo genomes representing two of the most basal mammalian superorders, Afrotheria and Xenarthra. Furthermore, we identify an alpha-defensin-like gene in the gray short-tailed opossum, suggesting that alpha-defensins may have evolved much earlier than previously thought, before the divergence of placental mammals and marsupials approximately 130 mya

Cooper, D.W., Larsen, E., 2006. Immunocontraception of mammalian wildlife: Ecological and immunogenetic issues. Reproduction 132, 821-828.
Abstract: Immunocontraception involves stimulating immune responses against gametes or reproductive hormones thus preventing conception. The method is being developed for the humane control of pest and overabundant populations of mammalian wildlife. This paper examines three fundamental issues associated with its use: (1) the difficulties of obtaining responses to self-antigens, (2) the likely evolution of genetically based non-response to immunocontraceptive agents, and (3) the possible changes in the array of pathogens possessed by the target species after generations of immunocontraception. Our review of the literature demonstrates that the barriers to an effective immunocontraceptive are at present very basic. Should they be overcome, the effects of immunocontraception on the immunogenetic constitution of wildlife populations through the selection for nonresponders must be examined. We suggest that the attempt to use the animal's own immune system to modulate reproduction may be incompatible with the basic biological function of protection against infectious disease. Research programs on mammalian immunocontraception should involve measurement of the heritability of non-response and an assessment of the likely change in the response of the contracepted population to possible pathogens.

Dangolla, A., Ekanayake, D.K., Rajapakse, R.P., Dubey, J.P., Silva, I.D., 2006. Seroprevalence of Toxoplasma gondii antibodies in captive elephants (Elephaus maximus maximus) in Sri Lanka
516. Veterinary Parasitology 137, 172-174.
Abstract: Serum samples collected during August 2003-June 2004 from 45 privately owned captive and 8 elephants from the Pinnawala Elephant Orphanage were tested for the presence of antibodies against Toxoplasma gondii using the direct modified agglutination test (MAT). Antibodies were found in sera of 14 of 45 (32%) privately owned elephants with titers of 1:25 in three, 1:50 in three, 1:100 in three, 1:200 in three, and 1:400 in three elephants. The elephants from Pinnawala Elephant Orphanage were seronegative. This is the first report of T. gondii seroprevalence in elephants in Sri Lanka

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.

Isaza, R., Davis, R.D., Moore, S.M., Briggs, D.J., 2006. Results of vaccination of Asian elephants (Elephas maximus) with monovalent inactivated rabies vaccine. American Journal of Veterinary Research 67, 1934-1936.
Abstract: OBJECTIVE: To evaluate the humoral immune response of Asian elephants to a primary IM vaccination with either 1 or 2 doses of a commercially available inactivated rabies virus vaccine and evaluate the anamnestic response to a 1-dose booster vaccination. ANIMALS: 16 captive Asian elephants. PROCEDURES: Elephants with no known prior rabies vaccinations were assigned into 2 treatment groups of 8 elephants; 1 group received 1 dose of vaccine, and the other group received 2 doses of vaccine 9 days apart. All elephants received one or two 4-mL IM injections of a monovalent inactivated rabies virus vaccine. Blood was collected prior to vaccination (day 0) and on days 9, 35, 112, and 344. All elephants received 1 booster dose of vaccine on day 344, and a final blood sample was taken 40 days later (day 384). Serum was tested for rabies virus-neutralizing antibodies by use of the rapid fluorescent focus inhibition test. RESULTS: All elephants were seronegative prior to vaccination. There were significant differences in the rabies geometric mean titers between the 2 elephant groups at days 35, 112, and 202. Both groups had a strong anamnestic response 40 days after the booster given at day 344. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirmed the ability of Asian elephants to develop a humoral immune response after vaccination with a commercially available monovalent inactivated rabies virus vaccine and the feasibility of instituting a rabies virus vaccination program for elephants that are in frequent contact with humans. A 2-dose series of rabies virus vaccine should provide an adequate antibody response in elephants, and annual boosters should maintain the antibody response in this species

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

Oni, O., Wajjwalku, W., Boodde, O., Chumsing, W., 2006. Canine distemper virus antibodies in the Asian elephant (Elaphas maximus)
407. Veterinary Record 159, 420-421.

Perelygin, A.A., Lear, T.L., Zharkikh, A.A., Brinton, M.A., 2006. Comparative analysis of vertebrate EIF2AK2 (PKR) genes and assignment of the equine gene to ECA15q24-q25 and the bovine gene to BTA11q12-q15
416. Genet. Sel Evol. 38, 551-563.
Abstract: The structures of the canine, rabbit, bovine and equine EIF2AK2 genes were determined. Each of these genes has a 5' non-coding exon as well as 15 coding exons. All of the canine, bovine and equine EIF2AK2 introns have consensus donor and acceptor splice sites. In the equine EIF2AK2 gene, a unique single nucleotide polymorphism that encoded a Tyr329Cys substitution was detected. Regulatory elements predicted in the promoter region were conserved in ungulates, primates, rodents, Afrotheria (elephant) and Insectifora (shrew). Western clawed frog and fugu EIF2AK2 gene sequences were detected in the USCS Genome Browser and compared to those of other vertebrate EIF2AK2 genes. A comparison of EIF2AK2 protein domains in vertebrates indicates that the kinase catalytic domains were evolutionarily more conserved than the nucleic acid-binding motifs. Nucleotide substitution rates were uniform among the vertebrate sequences with the exception of the zebrafish and goldfish EIF2AK2 genes, which showed substitution rates about 20% higher than those of other vertebrates. FISH was used to physically assign the horse and cattle genes to chromosome locations, ECA15q24-q25 and BTA11q12-15, respectively. Comparative mapping data confirmed conservation of synteny between ungulates, humans and rodents

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

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.

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

Delves, P.J., Roitt, I.M., 2005. Vaccines for the control of reproduction--status in mammals, and aspects of comparative interest
592. Dev. Biol. (Basel) 121, 265-273.
Abstract: The objective of producing vaccines which target elements of the reproductive system to control fertility has been pursued for many years. Of the many targets for such vaccines, several sperm-associated antigens have been proposed for antibody-mediated intervention before fertilization but the very abundance of antigen to be neutralized has been a barrier. Zona pellucida antigens associated with the surface of the oocyte have also been targeted and used successfully for control of 'wild' elephant populations but worries concerning immunopathologically-mediated tissue damage have been mooted. Vaccines using human chorionic gonadotropin (hCG) which is required for the implantation and maintenance of the fertilized egg, although of interest for the development of fertility control in human populations, has no relevance in the context of the present conference because external fertilization of fish eggs is independent. The pathways by which gonadotropin-releasing hormone (GnRH) secreted by the hypothalamus promote release of luteinizing (LH) and follicle-stimulating hormone (FSH) which govern the physiological maturation and maintenance of the reproductive organs, provide many targets for immunological intervention. Most consistent success has been reported using GnRH-based vaccines which are immunosterilizing in a variety of mammalian species such as pigs, rodents and white-tailed deer. The fact that the structure of the decapeptide, GnRH, has been maintained over so many years of evolution and been conserved across so many animal species, encourages the view that a strategy for control of sexual maturation in fish based upon stimulation of GnRH antibodies may well prove to be a practical proposition, provided the formulation of an appropriate highly immunogenic vaccine can be achieved

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.

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

Natiello, M., Lewis, P., Samuelson, D., 2005. Comparative anatomy of the ciliary body of the West Indian manatee (Trichechus manatus) and selected species. Vet. Ophthalmol. 8, 375-385.
Abstract: OBJECTIVE: To examine the anatomy of the ciliary body in the West Indian manatee (Trichechus manatus), paying close attention to its vascularization and to compare to those of its distant relative, the African elephant (Loxodonta africana), the amphibious hippopotamus (Hippopotamus amphibius) and the aquatic short-finned pilot whale (Globicephala macrorhynchus). PROCEDURE: Specimens from each species were preserved in 10% buffered formalin, and observed stereomicroscopically before being embedded in paraffin, sectioned and stained by Masson trichrome, hematoxylin and eosin, and periodic acid-Schiff for light microscopic evaluation. RESULTS: The network of blood vessels in the ciliary processes of the West Indian manatee appear to have an intricate pattern, especially with regard to venous outflow. Those of the elephant are slightly less complex, while those of the hippopotamus and whale have different vascular patterns within the ciliary body. Musculature within the ciliary body is absent within the manatee and pilot whale. CONCLUSIONS: In general, there appears to be a direct relationship between the increased development of vasculature and the loss of musculature within the ciliary bodies of the aquatic and amphibious mammals presently studied. Specifically, the ciliary body of the West Indian manatee has a comparatively unique construction, especially with regard to its vasculature.

Naz, R.K., Gupta, S.K., Gupta, J.C., Vyas, H.K., Talwar, A.G., 2005. Recent advances in contraceptive vaccine development: a mini-review
577. Hum. Reprod. 20, 3271-3283.
Abstract: Contraceptive vaccines (CV) may provide viable and valuable alternatives to the presently available methods of contraception. The molecules that are being explored for CV development either target gamete production [luteinizing hormone-releasing hormone (LHRH)/GnRH, FSH], gamete function [sperm antigens and oocyte zona pellucida (ZP)], and gamete outcome (HCG). CV targeting gamete production have shown varied degrees of efficacy; however, they either affect sex steroids causing impotency and/or show only a partial rather than a complete effect in inhibiting gametogenesis. However, vaccines based on LHRH/GnRH are being developed by several pharmaceutical companies as substitutes for castration of domestic pets, farm and wild animals, and for therapeutic anticancer purposes such as in prostatic hypertrophy and carcinoma. These vaccines may also find applications in clinical situations that require the inhibition of increased secretions of sex steroids, such as in uterine fibroids, polycystic ovary syndrome, endometriosis and precocious puberty. CV targeting molecules involved in gamete function such as sperm antigens and ZP proteins are exciting choices. Sperm constitute the most promising and exciting target for CV. Several sperm-specific antigens have been delineated in several laboratories and are being actively explored for CV development. Studies are focused on delineating appropriate sperm-specific epitopes, and increasing the immunogenicity (specifically in the local genital tract) and efficacy on the vaccines. Anti-sperm antibody (ASA)-mediated immunoinfertility provides a naturally occurring model to indicate how a vaccine might work in humans. Vaccines based on ZP proteins are quite efficacious in producing contraceptive effects, but may induce oophoritis, affecting sex steroids. They are being successfully tested to control feral populations of dogs, deer, horses and elephants, and populations of several species of zoo animals. The current research for human applicability is focused on delineating infertility-related epitopes (B-cell epitopes) from oophoritis-inducing epitopes (T-cell epitopes). Vaccines targeting gamete outcome primarily focus on the HCG molecule. The HCG vaccine is the first vaccine to undergo Phase I and II clinical trials in humans. Both efficacy and lack of immunopathology have been reasonably well demonstrated for this vaccine. At the present time, studies are focused on increasing the immunogenicity and efficacy of the birth control vaccine, and examining its clinical applications in various HCG-producing cancers. The present article will focus on the current status of the anti-sperm, anti-ZP, anti-LHRH/GnRH and anti-HCG vaccines

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.

Weissengruber, G.E., Egerbacher, M., Forstenpointner, G., 2005. Structure and innervation of the tusk pulp in the African elephant (Loxodonta africana)
617. Journal of Anatomy 206, 387-393.
Abstract: African elephants (Loxodonta africana) use their tusks for digging, carrying and behavioural display. Their healing ability following traumatic injury is enormous. Pain experience caused by dentin or pulp damage of tusks seems to be negligible in elephants. In this study we examined the pulp tissue and the nerve distribution using histology, electron microscopy and immunhistochemistry. The results demonstrate that the pulp comprises two differently structured regions. Randomly orientated collagen fibres characterize a cone-like part lying rostral to the foramen apicis dentis. Numerous nerve fibres and Ruffini endings are found within this cone. Rostral to the cone, delicate collagen fibres and large vessels are orientated longitudinally. The rostral two-thirds of the pulp are highly vascularized, whereas nerve fibres are sparse. Vessel and nerve fibre distribution and the structure of connective tissue possibly play important roles in healing and in the obviously limited pain experience after tusk injuries and pulp alteration. The presence of Ruffini endings is most likely related to the use of tusks as tools

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

Agnew, D.W., Munson, L., Ramsay, E.C., 2004. Cystic endometrial hyperplasia in elephants
741. Vet. Pathol. 41, 179-183.
Abstract: Most captive female elephants are nulliparous and aged and many have endometrial disease, factors that may hinder fertility. This study characterized the pathologic features and demographic distribution of endometrial lesions from 27 captive Asian (Elephas maximus) and 13 African elephants (Loxodonta africanus), 12- to 57-years of age. The principal lesion was marked cystic and polypoid endometrial hyperplasia (CEH), present in 67% of Asian and 15% of African elephants ranging from 26 to 57 years. The lower prevalence in African elephants likely reflects their younger age range in this study. Fourteen of 15 affected elephants with breeding information were nulliparous. These results suggest that CEH and polyps are common in aged nulliparous elephants, and the severity of these lesions may impair fertility. These findings will be useful in the interpretation of ultrasonographic findings during reproductive examinations of potential breeding cows. Also, breeding programs should focus on younger animals

Jones, C.J., Wooding, F.B., Mathias, S.S., Allen, W.R., 2004. Fetomaternal glycosylation of early placentation events in the African elephant Loxodonta africana
740. Placenta 25, 308-320.
Abstract: During implantation in the African elephant (Loxodonta africana), fetal trophoblast displaces the surface uterine epithelium and superficially penetrates the uterine glands. This limited invasion is followed by the upgrowth of blunt fingers of endometrial stroma, covered with trophoblast and containing capillaries that subsequently vascularize the growing placenta. We have used lectin histochemistry to compare the glycosylation of maternal endothelial cells in the endometrium with those growing within the trophoblastic processes of a 2 g embryo (approximately 125 days' gestation), and also examine changes in the endometrial glands associated with trophoblastic invasion. Maternal vessels at the apices of the trophoblast-covered stromal upgrowths showed increased expression of terminal N-acetyl galactosamine, N-acetyl glucosamine oligomers, some sialic acids, and tri/tetra-antennate non-bisected complex N-linked glycan, as indicated by increased lectin staining. The areas of increased staining were also more resistant to neuraminidase digestion. Invaded glands had distended walls composed of flattened epithelial cells, some of which showed heavy lectin staining suggestive of intracellular glycan accumulation. The vascular changes suggest that new maternal capillary growth is accompanied by alterations in surface glycosylation. This may be the result of increased glycosyl transferase activity associated with cell proliferation and may also indicate the expression of significantly increased anti-adhesive molecules preventing blood stasis and egress of maternal immunocompetent cells into the fetal compartment

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

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

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.

Schweitzer, M., Hill, C.L., Asara, J.M., Lane, W.S., Pincus, S.H., 2002. Identification of immunoreactive material in mammoth fossils. J Mol Evol 55, 696-705.
Abstract: Department of Microbiology, Montana State University, Bozeman, MT 59717, USA. schweitzer@montana.edu

The fossil record represents a history of life on this planet. Attempts to obtain molecular information from this record by analysis of nucleic acids found within fossils of extreme age have been unsuccessful or called into question. However, previous studies have demonstrated the long-term persistence of peptides within fossils and have used antibodies to extant proteins to demonstrate antigenic material. In this study we address two questions: Do immunogenic/antigenic materials persist in fossils? and; Can fossil material be used to raise antibodies that will cross-react with extant proteins? We have used material extracted from a well-preserved 100000-300000-year-old mammoth skull to produce antisera. The specificity of the antisera was tested by ELISA, western blotting, and immunohistochemistry. It was demonstrated that antisera reacted specifically with the fossils and not the surrounding sediments. Reactivity of antisera with modern proteins and tissues was also demonstrated, as was the ability to detect evolutionary relationships via antibody-antigen interactions. Mass spectrometry demonstrated the presence of amino acids and specific peptides within the fossil. Peptides were purified by anion-exchange chromatography and sequenced by tandem mass spectrometry. The collagen-derived peptides may have been the source of at least some of the immunologic reactivity, but the antisera identified molecules that were not observed by mass spectrometry, indicating that immunologic methods may have greater sensitivity. Although the presence of peptides and amino acids was demonstrated, the exact nature of the antigenic material was not fully clarified. This report demonstrates that antibodies may be used to obtain information from the fossil record.

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

Barber, M.R., Lee, S.M., Steffens, W.L., Ard, M., Fayrer-Hosken, R.A., 2001. Immunolocalization of zona pellucida antigens in the ovarian follicle of dogs, cats, horses and elephants. Theriogenology 55, 1705-1717.
Abstract: A comparative evaluation of the location of immunoreactive porcine zona pellucida (pZP) glycoproteins was performed with polyclonal rabbit anti-pZP antibodies on ovarian sections of the dog, cat, horse and elephant. For this, formalin (light microscopy) and glutaraldehyde (transmission electron microscopy [TEM]) fixed ovarian sections were incubated with antibodies raised against highly purified pZP. Staining patterns were determined with diaminobenzidine (DAB) at the light level. The dog ZP had a distinct staining distribution that is characterized by intense staining around the periphery of the ZP and the oolemma and less dense staining throughout the width of the ZP. In dog follicles that contained multiple oocytes, there were oocytes of identical and dissimilar stages. Cat ovarian sections showed uniform staining of the ZP. Horse results showed uniform staining of ZP and ooplasm, and granulosa cells (GC). Elephant sections showed staining of the ZP with dense staining at the oolemma, as well as staining of the ooplasm. In all species the staining of the ZP was not evident until GC differentiation. In all cases there was no staining of ovarian tissue with control normal rabbit serum. Specific staining patterns of ZP were evaluated by TEM and immunogold staining. The immunogold-linked anti-pZP antibodies stained the ZP matrix in all species. There was staining of ooplasm organelles suggesting that ZP secretion originates from the oocyte of the dog and cat. In addition, follicular and ZP measurements were taken that allowed accurate characterization of follicle stage. These findings suggest that in all 4 species the ZP is recognized by anti-pZP antibodies and there is also evidence to suggest the possible origins of ZP glycoproteins.

Barber, M.R., Fayrer-Hosken, R.A., 2000. Possible mechanisms of mammalian immunocontraception. J Reprod Immunol 46, 103-124.
Abstract: Ecological and conservation programs in ecosystems around the world have experienced varied success in population management. One of the greatest problems is that human expansion has led to the shrinking of wildlife habitat and, as a result, the overpopulation of many different species has occurred. The pressures exerted by the increased number of animals has caused environmental damage. The humane and practical control of these populations has solicited the scientific community to arrive at a safe, effective, and cost-efficient means of population control. Immunocontraception using zona pellucida antigens, specifically porcine zona pellucida (pZP), has become one of the most promising population control tools in the world today, with notable successes in horses and elephants. A conundrum has risen where pZP, a single vaccine, successfully induces an immunocontraceptive effect in multiple species of mammals. This review describes the most current data pertaining to the mammalian zona pellucida and immunocontraception, and from these studies, we suggest several potential mechanisms of immunocontraception.

Fayrer-Hosken, R.A., Grobler, D., van Altena, J.J., Bertschinger, H., Kirkpatrick, J.F., 2000. Immunocontraception of African elephants. Nature London 407, 6801.
Abstract: Sum: Based on a South African trial of 41 adult females, it is argued that pZP immunocontraception is a humane method to control elephant populations without behavioural side effects.

Lewis, M.H., Gluck, J.P., Petitto, J.M., Hensley, L.L., Ozer, H., 2000. Early social deprivation in nonhuman primates: long-term effects on survival and cell-mediated immunity. Biol Psychiatry 47, 119-126.
Abstract: BACKGROUND: Early differential social experience of non-human primates has resulted in long-term alterations in behavior and neurobiology. Although brief maternal separation has been associated with changes in immune status, the long-term effects on survival and immune function of prolonged early social deprivation are unknown. METHODS: Survival rates were examined in rhesus monkeys, half of which had been socially deprived during their first year of life. Repeated measures of immune status were tested in surviving monkeys (18-24 years old). Peripheral blood T, B, and natural killer lymphocytes, macrophages, and monocytes were measured by flow cytometry. Functional cellular immune activity measures included T-cell proliferative responses to mitogens (concanavalin and phytohemagglutinin), T-cell memory response to tetanus toxoid antigen, T-cell-dependent B-cell proliferative responses to mitogen (PWM) and natural killer cell cytotoxic activity. RESULTS: Despite identical environments following isolation, early social deprivation resulted in a significantly decreased survival rate, males being particularly vulnerable to early death. Early social deprivation was associated with a decrease in the ratio of helper to suppressor T cells, and a significant increase in natural killer cell number and in natural killer cell activity in the surviving monkeys. No differences in T- or B-lymphocyte proliferation following mitogen or tetanus toxoid antigen stimulation were observed. CONCLUSIONS: Prolonged early social deprivation of non-human primates profoundly affected mortality and resulted in lifelong effects on cell-mediated immune status.

Lin, Y.N., Wong, W.K., 2000. Sero-prevalence of Japanese Encephalitis virus in various species of animals in Singapore - a preliminary study. Singapore Journal of Primary Industries 28, 57-61.
Abstract: A study was conducted to determine exposure of local animals to Japanese encephalitis virus (JEV) 9 years after the removal of its main amplifying host animals in Singapore. 295 serum samples from local (40 dogs, 10 cats, 10 wild boars, 40 chickens, 10 dairy cattle, 10 dairy goats, 55 crows, 44 domestic pigs) and imported (10 ducks, 28 chickens, 5 babirusas, 11 pot-bellied pigs, 5 bearded pigs, 1 Ankole cattle, 1 Asian elephant, 2 orangutans, 3 long-tailed macaques, 1 springbok, 2 reticulated pythons, 1 mouflon, 1 red-eared terrapin, 1 Cape hunting dog, 1 siamang, 1 leopard cat, 1 puma and 5 bats) were screened for JEV antibodies. Imported exotic animals like babirusas, pot-bellied pigs, bearded' pigs, Ankole cattle, Asian elephant and orangutans were seropositive for JEV. High titres and exposure rates were observed in 72.7% of pot-bellied pigs and 100% of the babirusas and bearded pigs with titres of 430.5, 256 and 256, respectively. Antibodies were not detected in the other species of wildlife tested. Among local animals, 100% of goats had moderate antibody levels (GMT=34) while 70% of local cattle had low (GMT=9,8) titres. Ten pet dogs were seronegative while military and stray dogs had 20 and 40% exposure rates, respectively. Local ducks and chickens had low exposure rates (10%, GMT=32 and 0%, respectively) while imported ducks and chickens from Malaysia had high rates (80%, GMT_90.5 and 35.7%, GMT=48.5%, respectively). The cats and crows were seronegative for JEV antibodies. 89% of domestic sows (GMT=217) and 20% of gilts (GMT=16) were seropositive while weaners were seronegative. Wild boars from Pulau Tekong also had high exposure rates (100%, GMT=238.9). It is concluded that a low level of JEV is currently maintained in certain animal populations in Singapore.

Fayrer-Hosken, R.A., Bertschinger, H.J., Kirkpatrick, J.F., Grobler, D., Lamberski, N., Honneyman, G., Ulrich, T., 1999. Contraceptive potential of the porcine zona pellucida vaccine in the African elephant (Loxodonta africana). Theriogenology 52, 835-846.
Abstract: Immunocontraception has been successful in controlling free-roaming equids; however, what is the potential for the immunocontraceptive control of the African elephant (Loxodonta africana)? The porcine zona pellucida (pZP) glycoproteins share antigenic domains with the African elephant zona pellucida (elZP) glycoproteins, and anti-zona pellucida serum antibodies have been successfully stimulated. To determine the cross-reactivity of the pZP and elZP, immunocytochemistry was evaluated by light and electron microscopy. Specifically, the binding of polyclonal antibodies against total heat-solubilized-porcine zona pellucida to fixed elephant ovary sections was evaluated. The elZP of primary, secondary and tertiary follicles was recognized by the rabbit-anti-pZP serum, but there was no apparent recognition of the primordial follicles. The ability of anti-pZP antibodies to recognize the elZP demonstrates that there is molecular homology between the pZP and elZP glycoproteins. This homology makes the African elephant a candidate for pZP immunocontraception. Three captive elephants were vaccinated with 400 micrograms pZP with a synthetic trehalose dicorynomycolate (S-TDCM) adjuvant. The elephants received 2 boosters of 600 micrograms pZP at 4 wk and 10 m.o. after the primary vaccination. The vaccinated female elephants developed significant (P < 0.05) titers to pZP over prevaccination levels. These levels persisted for 12 to 14 m.o. after the third vaccination. This preliminary evidence shows that the female elephant can develop significant serum antibody levels to pZP. These levels of antibodies are comparable to those required in horses for successful immunocontraception. Thus, porcine zona pellucida immunocontraception might be used to control elephant populations.

Bhat, M.N., Manickam, R., Nedunchelliyan, S., Jayakumar, V., 1998. Detection of leptospirial antibodies in the sera of elephants. Indian Veterinary Journal 75, 201-203.
Abstract: Leptospirosis is an infectious disease of man and animals, caused by antigenically distinct members of the genus Leptospira. Upadhya et al (1979) detected antibody against Leptospira valbuzzi and L. pyrogenes in the sera of elephants. Arora (1994) detected antibodies against L. pomona in a sambar and black buck. In the present study, a serological survey was undertaken to detect antibodies against seven serovars of Leptospira sp. in elephants (Elephas maximus), spotted deer (Axis axis), and blackbucks (Antelope cervicapra). Serum samples were collected from 109 elephants in 15 elephant camps in Madras, Karnataka, and the Andaman and Nicobar Islands, and from 4 spotted deer and 4 blackbuck in a zoo in Madras. In 23 (21%) of the elephants, antibodies, at titres of 1:100 to 1:200, were found by microscopic agglutination tests, to 6 serovars of Leptospira interrogans: pomona, icterohaemorrhagiae, grippotyphosa, hebdomadis, hardjo, and canicola. No leptospiral antibodies were found in the serum of the other animals. Clinical signs were present only in the elephants with L. grippotyphosa; the other serovars are reported for the first time in Indian elephants.

Kelly, P.J., Carter, S.D., Azwai, S.M., Cadman, H.F., 1998. Isolation and characterization of immunoglobulin g and IgG subclasses of the African elephant (Loxodonta africana). Comparative Immunology, Microbiology and Infectious Diseases 21, 65-73.
Abstract: Immunoglobulins were precipitated from sera pooled from 5 African elephants (killed for population control) with ammonium sulfate and separated by gel filtration and fast protein liquid chromatography (ion exchange). Analysis of the fractions by SDS-PAGE showed IgG of 150 kDa with up to 5 subclasses, each having heavy chains of 57 kDa and light chains of 27 kDa. Three monoclonal antibodies against human IgG and polyclonal antibodies against canine, bovine, cameline, equine, phocine and feline IgG showed strong cross-reactivity with the African elephant IgG subclasses. No serum molecules corresponding to IgM or IgA could be detected, even when ammonium sulfate precipitation was used at 50% saturation.

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.

Barnard, B.J.H., 1997. Antibodies against some viruses of domestic animals in southern African wild animals. Onderstepoort Journal of Veterinary Research 64, 95-110.
Abstract: Twenty-four species of South African wild animals were tested for the presence of antibodies to the viruses of 16 common diseases of domestic animals around 1993-5. Positive results were obtained for African horse sickness, equine encephalomyelitis virus, equine herpesvirus-1, bovine herpesvirus-1, Allerton disease (Herpes mammillitis; bovine herpesvirus 2), lumpy skin disease, parainfluenza, encephalomyocarditis, bluetongue, Wesselsbron disease, bovine ephemeral fever, and Akabane disease complex. No antibodies could be demonstrated against the viruses of equine influenza, equine infectious anaemia, equine viral arteritis or Rift Valley fever. The negative results support observations that the latter diseases, with the exception of equine viral arteritis, are absent in South Africa. The number of animal species found positive for a specific virus, ranged from 0-16. No antibodies were found in crocodiles or warthogs, whereas antibodies against Wesselsbron and bovine herpesvirus-1 were present in 16 species. Antibodies against viruses of horses were found almost exclusively in zebras and, although elephants reacted to African horse sickness, no neutralizing antibodies against it could be demonstrated in their sera. Zebras were also found to be positive for Wesselsbron and Akabane, which are usually regarded as viruses of ruminants. Antibodies against most viruses were encountered in all vegetation zones in South Africa, but most viruses were more prevalent in the high-rainfall zone in KwaZulu-Natal.

Bhat, M.N., Manickam, R., 1997. Detection of rinderpest antibodies in elephants, spotted deer, and blackbucks. International Journal of Animal Sciences 12, 201-203.

Bhat, M.N., Manickam, R., Kumanan, K., 1997. Serological evidence of bovine herpesviruses 1 and 2 in Asian elephants. Journal of Wildlife Diseases 33, 919-920.
Abstract: Antibodies were detected against bovine herpesviruses 1 (BHV 1) and 2 (BHV 2) in Asian elephants (Elephas maximus) using the passive haemagglutination (PHA) test. The study was conducted during May to December 1994 using sera collected from zoos and national parks in India. Four (4%) of 109 elephant sera had PHA titres ranging from 1:8 to 1:32 against BHV 1. 25 (23%) of the 109 elephant sera had PHA titres ranging from 1:8 to 1:64 against BHV 2. It is concluded that Asian elephants appear to be better reservoirs for herpesviruses which are serologically related to BHV 2.

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.

Kania, S., Kennedy, M., Potgieter, L., Richman, L.K., Montali, R.J. Isolation and Detection of Asian elephant IgG. Proceedings American Association of Zoo Veterinarians. 566-571. 1996.
Ref Type: Conference Proceeding
Abstract: Serological tests of elephant serum have been limited due to a lack of suitable detecting reagents. This has restricted the repertoire of assays for diagnosis as well as determination of species relatedness. To address this problem we isolated Asian elephant IgG, prepared antisera reactive with it, and coupled an IgG fraction of anti-elephant IgG to fluorescein-5-isothiocyanate (FITC) and to biotin. The cross-reactivity of anti-Asian elephant IgG with IgG of other species was determined. Conversely, the reactivity of antisera prepared against IgG of other species was examined to determine their usefulness as diagnostic reagents for Asian elephant studies. To purify Asian elephant IgG, serum was applied to immobilized recombinant protein A/G. Bound IgG was eluted with low pH buffer. The IgG was further purified by column chromatography on diethlyaminoethyl and cibacron blue dye equilibrated with 0.02 M Tris-HCl, pH 8.0 containing 0.028 M sodium chloride. The flow-through (containing IgG) was concentrated using an ultrafilter with a 100 kDa cut-off. The purity of the preparation was established by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 7.5% resolving gel. Antisera reactive with Asian elephant IgG was produced in rabbits. Its specificity was confirmed with western blots prepared by SDS-PAGE of Asian elephant serum. The IgG fraction was isolated from rabbit antisera using immobilized recombinant protein A/G. The IgG fraction (anti-eIgG) was labeled with FITC using a Flurotag FITC conjugation kit (Sigma, St. Louis, MO). Anti-eIgG was also coupled to biotin (succinimidyl-6-[biotinamido] hexanoate) in carbonate-bicarbonate buffer, pH8.0. The FITC conjugate has been useful for indirect fluorescence assays (IFA) of elephant IgG bound to antigens on microscope slides. To determine the reactivity of antibody prepared against elephant IgG with IgG of other species we developed a capture enzyme linked immunosorbent assay (ELISA). Plates were coated with anti-eIgG and then received sera or purified IgG from various species. Bound antibody was detected with biotin labeled anti-Asian elephant IgG followed by peroxidase labeled avidin and 2,2'-Azino-bis(3-ethylbenzthiazoline 6-sulfonic acid) diammonium (ABTS) substrate. The reactivity of commercially available conjugated antisera with elephant IgG was determined by coating ELISA plates with purified IgG followed by peroxidase conjugated antisera and substrate. The reactivity of sera diluted 1/320,000 was determined in the capture ELISA with anti-eIgG and biotin anti-eIgG (Fig. 1). Relative to Asian elephant sera the reactivity of African elephant was 28.2%, Sri Lankan elephant 116.3%, and horse 9.2%. In the IgG capture system (Fig. 2), when purified IgG was added to the plates, the reactivity to bovine IgG was 40.7%, feline IgG 5.2%, and canine IgG 18.5%, relative to Asian elephant IgG. When purified IgG was coated on ELISA plates and detected with conjugated anti-IgG prepared against various IgGs (Fig. 3) the reactivity to elephant IgG of anti-bovine IgG was 65.3%, anti-deer IgG 26.9%, and anti-rabbit IgG 17.8% compared to the homologous Asian elephant IgG reaction. The reactivity of anti-bovine IgG with elephant IgG was only about one third as intense, however, as the homologous bovine reaction (anti-bovine IgG with bovine IgG). We conclude that anti-Asian elephant conjugates will be useful for diagnosis in Asian elephant species and subspecies. The apparent weak cross-reactivity between anti-Asian elephant IgG and African IgG in serum is somewhat surprising. The development of African elephant reagents will permit us to more thoroughly examine the cross-reactivity of African and Asian IgGs.

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.

Van-Aswegen, G., Schoeman, J.H., De-Vos, V., Van-Noorden, S., 1994. The oesophagus and stomach of the African elephant: a histological, immunocytochemical and immunofluorescence study. Onderstepoort Journal of Veterinary Research 61, 223-229.
Abstract: Histological, immunocytochemical and immunofluorescence methods were employed to study the oesophagus and stomach of the elephant. The histological findings were similar to those in monogastric species like pigs and humans. In the mucosa of the stomach, endocrine cells were immunoreactive to gastrin, somatostatin, chromogranin A and serotonin. Nerve cells immunoreactive to somatostatin, bombesin, VIP, PHI and CGRP were detected in the submucosal and myenteric plexus of the stomach. In the stomach, the absence of glucagon cells and the presence of endocrine cells immunoreactive to PYY, are in contrast to the situation in other mammals and need further investigation. Small gastric ulcers were observed in some of the specimens.

Bennet, D., 1993. Immune-based erosive inflammatory joint disease of the dog: canine rheumatoid arthritis. 2. Pathological investigations. Journal of Small Animal Practice 28, 909-928.
Abstract: The pathological features of 30 cases of canine rheumatoid arthritis are described. The principle pathologic feature is a chronic symmetrical polysynovitis. The pathological features of the joints varied in severity. The synovial membrane generally showed villous hypertrophy with aggregates of lymphocytes and plasma cells. Destruction of articular cartilage and bone occurred in association with a replacement granulation tissue which often produced a pannus over the articular surface. Immunofluorescence studies demonstrated complexes of IgG or IgM with C3 in synovial lining cells, macrophages, blood vessel walls and free in the extracellular tissues. IgG and IgM producing plasma cells were also common. Fibrinogen deposites were extensive. The immunofluorescence findings were non-specific but support the concept of an immune complex mediated inflammation within the joints. Investigations for bacterial, mycoplasmal and viral infections of the joints were negative.

Ermel, R.W., Kenny, T.P., Chen, P.P., Robbins, D.L., 1993. Molecular analysis of rheumatoid factors derived from rheumatoid synovium suggests an antigen-driven response in inflamed joints. Arthritis and Rheumatism 36, 380-388.
Abstract: Objective. Understanding the molecular genetic basis for rheumatoid factor (RF) production is necessary to a better understanding of the etiology and pathogenesis of rheumatoid arthritis (RA). We sought to define the genetic basis for RF in RA. Methods. The heavy and light chain variable region genes encoding 4 human monoclonal RF were cloned and sequenced using the polymerase chain reaction and the dideoxynucleotide chain-termination method. Results. The heavy and light chains of the C6 RF and the light chain of the G9 RF were encoded by 3 new RF-related Ig V-region genes. The heavy and light chains of D5 and G4 RF's were identical: most of their mutations caused amino acid substitutions. Conclusions. The RF-related Ig V-region gene repertoire is large and still expanding. The data from D5 and G4 strongly suggest that these 2 RF's arise in an antigen-driven response in rheumatoid synovium. The presumed germline V genes for C6 may represent disease-specific RF-related V genes.

Harth, M., 1993. Gold in rheumatoid arthritis: standard, substitute or sham? Journal of Rheumatology 20, 771-773.

Kelly, P.J., Tagwira, M., Matthewman, L., Mason, P.R., Wright, E.P., 1993. Reactions of sera from laboratory, domestic and wild animals in Africa with protein A and A recombinant chimeric protein AG. Comparative Immunology, Microbiology and Infectious Diseases 16, 299-305.
Abstract: An ELISA was developed to determine the reactivity of peroxidase labelled Protein A and a recombinant Protein A + Protein G construct, to sera from a variety of laboratory, domestic and wild animals from Africa. There was variability in the binding capacity of sera from individuals of the same species, but 4 groups could be recognized. Sera from birds and crocodiles were at most weakly reactive with either Protein A or the chimeric construct. Sera from some domestic animals such as horse, goat and cat, and sera from some wild ungulates including buffalo, wildebeest, waterbuck and impala were reactive with Protein A, but reacted to a much greater degree with the chimeric construct. Sera from larger wild animals such as elephant, rhinoceros and giraffe were strongly reactive with the chimeric protein and moderately reactive with Protein A. Sera from primates and dog, pig, guinea pig and rabbit reacted strongly with both proteins. It was concluded that as chimeric proteins that combine the IgG binding capacities of Protein A and Protein G can be used to detect immunoglobulin from a wide variety of African wild animal species, they may be of great value in seroepidemiological investigations of these animal populations.

Rubin, L.A., Hawker, G.A., 1993. Stress and the immune system: preliminary observations in rheumatoid arthritis using an in vivo marker of immune activity. Arthritis and Rheumatism 3, 204-207.

van Schaardenburg, D., Hazes, J.M.W., de Boar, A., Zwinderman, A.H., Meijers, K.A.E., Breedveld, F.C., 1993. Outcome of rheumatoid arthritis in relation to age and rheumatoid factor at diagnosis. Journal of Rheumatology 20, 45-52.
Abstract: Our retrospective followup study reports the outcome of rheumatoid arthritis (RA) in relation to age (under 60 vs 60 years and over) and rheumatoid factor status at diagnosis. A sample of 130 adult patients with RA was assessed at a mean of 5.6 years after diagnosis. At final evaluation disease activity and radiographic damage in seropositive patients were similar in both age groups, but functional capacity was markedly lower in the older onset group, indicating lower functional reserves in this group. In seronegative patients the outcome was favorable in both age groups, especially in the older patients. Seropositive patients in both age groups had more disease activity, a lower functional capacity and more radiographic damage than seronegative patients; these differences were greater in the older onset patients. The mortality in patients with RA compared to the general population (standardized mortality ratio, SMR) was higher in seropositive patients (SMR 2.78, 98% CI 1.70-4.13) but not in seronegative patients (SMR 0.45, 95% CI 0.08-1.13). The relative risk of dying was 6 times higher in seropositive patients than in seronegative patients (95% CI 1.7-20.9).

Vaughan, J.H., 1993. Pathogenetic concepts and origins of rheumatoid factor in rheumatoid arthritis. Arthritis and Rheumatism 36, 1-6.

Binepal, V.S., Wariru, B.N., Davies, F.G., Soi, R., Olubayo, R., 1992. An attempt to define the host range for African horse sickness virus (Orbivirus, Reoviridae) in east Africa, by a serological survey in some Equidae, Camelidae, Loxodontidae and Carnivore. Vet. Microbiol. 31, 19-23.
Abstract: A survey was carried out in horse, zebra, elephant, camel, sheep and goat and wild carnivore sera for virus-serum neutralizing antibody to the nine type strains of African horse sickness virus. Antibody was found amongst the horse, zebra and elephant sera to all nine different strains. No antibody was detected in any sera from camels, sheep and goats. None was found in sera from hyaena and jackals in this series but had been detected earlier.

Cedillo, L., Gil, C., Mayagoitia, G., Giono, S., Cuellar, Y., Yanez, A., 1992. Experimental arthritis induced by Mycoplasma pneumoniae in rabbits. Journal of Rheumatology 19, 344-347.
Abstract: Experimental arthritis in rabbits was induced by M. pneumoniae. We compared it with the arthritis produced by well known animal arthritogenic agents (M. pulmonis and M. arthritidis). Mycoplasmas were detected in the knee joint by different techniques. M. pneumoniae and M. pulmonis produced a chronic arthritis. Live M. pneumoniae and M. pulmonis were recovered from the joint during all experiments. No live M. arthritidis was detected. Live mycoplasmas play an important role in acute arthritis. A similar pattern was shown by M. pneumoniae and M. pulmonis. This animal model could be helpful in the study of arthritis induced by a human pathogen mycoplasma.

Harris, E.D., Jr., 1992. Excitement in synovium: the rapid evolution of understanding of rheumatoid arthritis and expectations for therapy. Journal of Rheumatology 19, 3-5.
Abstract: Multiple events give rise to rheumatoid arthritis (RA) and many different elements create an overall pathological effect. It is highly unlikely that a single cause for RA will be identified. A greater appreciation of the multiple events that lead to the development of RA is opening up a number of promising points of intervention with may serve as potential alternatives to the broader-based and often toxic drugs used in RA therapy today.

Jacoby, F. Contribution to the epidemiology of cowpox virus in the Federal Republic of Germany. Untersuchungen zur Epidemiologie des Kuhpockenvirus in der Bundesrepublik Deutschland. 1-140. 1992. Giessen, Germany, Fachbereich Veterinarmedizin, Justus-Liebig-Universitat.
Ref Type: Thesis/Dissertation
Abstract: The indirect immunofluorescence test for antibodies to cowpox orthopoxvirus was positive in 218 of 303 wild rodents (Microtus agrestis, M. arvalis, Apodemus flavicollis, Clethrionomys glareolus and Rattus norvegicus). Attempts to isolate the virus failed. 202 of 277 cats from 58 of 67 locations in Germany, also 61 of 106 cattle and 13 of 38 zoo or circus elephants were also positive.

Kushner, I., Dawson, N.V., 1992. Changing perspecitves in the treatment of rheumatoid arthritis. Journal of Rheumatology 19, 1831-1833.

Panayi, G.S., Lanchrury, J.S., Kingsley, G.H., 1992. The importance of the T cell in initiating and maintaining the chronic synovitis of rheumatoid arthritis. Arthritis and Rheumatism 35, 729-735.

Pruzanski, W., Vadas, P., 1992. Should tetracyclines be used in arthritis? Journal of Rheumatology 19, 1495-1497.

Ramos-Remus, C., Sibley, J., Russell, A.S., 1992. Steroids in rheumatoid arthritis: the honeymoon revisited. Journal of Rheumatology 19, 667-670.

Wither, J., 1992. Molecular aspects of the rheumatic diseases. Journal of Rheumatology 19, 649-650.

Anderson, S.T., Schiller, C.A., 1991. Rheumatoid-like arthritis in a lion tailed macaque. Journal of Rheumatology 18, 1247-1250.
Abstract: Abstract. Very few satisfactory models of rheumatoid arthritis (RA) exist in nonhuman species. It is particularly striking that nonhuman primates have only rarely been described to have disease processes resembling classic RA seen in humans. We describe the case of a lion tailed macaque (Macaca silenus), housed at the National Zoological Park in Washington DC, that had a polyarticular inflammatory arthropathy resembling RA. Gross and histopathological examination of necropsy tissues and radiographic findings strongly suggest a rheumatoid-like disease never before described in this species.

Cheng, H.C., Yamashiro, D., 1991. Synthesis and receptor binding activity of elephant beta- endorphin, a beta-endorphin homolog with highly potent analgesic activity. International Journal of Peptide and Protein Research 38, 66-69.
Abstract: Elephant beta-endorphin and its analog, elephant beta- endorphin(6-31) were synthesized by standard solid phase method. Receptor binding activity showed that elephant beta-endorphin was five to six times more potent than human beta-endorphin in its ability to bind to opiate receptors on rat brain membrane. In a previous study (Wong, C.-L., Wai, M.-K., Cheng, H.-C., Chung, D. & Yamashiro, D (1990) Clinical and Experimental Pharmacology and Physiology 16, 33-37), tail flick test for intracerebroventricularly administered beta-endorphin showed that the antinociceptive potency of elephant beta-endorphin was seven to eight times higher than that of human beta-endorphin in mice. Results from both studies suggest that elephant beta-endorphin was a much more potent antinociceptive agent than human beta- endorphin in tail flick test and its higher analgesic activity might be due to its higher affinity for opiate receptors in the brain.

Clark, H.W., 1991. The potential role of mycoplasmas as autoantigens and immune complexes in chronic vascular pathogenesis. American Journal of Primatology 24, 235-243.

Healey, L.A., Wilske, K.R., 1991. Evaluating combination drug therapy in rheumatoid arthritis. Journal of Rheumatology 18, 641-642.

Jarjour, W.N., Jeffries, B.D., Davis, J.S., Welch, W.J., Mimura, T., Winfield, J.B., 1991. Autoantibodies to human stress proteins. Arthritis and Rheumatism 34, 1133-1138.
Abstract: Unselected sera from patients with various rheumatic, inflammatory bowel, and autoimmune skin diseases (n=268) were examined against human cell lysate by immunoblotting procedures, to determine the prevalence of autoantibodies to stress proteins (heat-shock proteins) hsp60 (homolog of Escherichia coli groEL and Mycobacterial 65K antigens), hsp73, and hsp90. Using standard, sensitive and specific assay conditions, IgG and IgM autoantibodies to these stress proteins were not demonstrable, or were detected infrequently, in sera from control subjects (n=36) and from patients with rheumatoid arthritis, Sjogren's syndrome, ankylosing spondylitis, Reiter's syndrome, systemic lupis erythematosus, and systemic sclerosis. Autoantibodies to hsp60 were relatively more common (>= 20% of sera) in patients with mixed connective tissue disease, polymyositis/dermatomyositis, psoriatic arthritis, inflammatory bowel disease, epidermolysis bullosa acquista, and bullous pemphigoid. Anti-hsp73 autoantibodies were detected in 20% or more of the sera from patients were Lyme disease and ulcerative colitis. Taken together, these data extend the spectrum of autoimmune and inflammatory diseases in which humoral anti-stress protein reactivity develops. However, the paucity of humoral autoreactivity to stress proteins in patients with systemic lupis erythematosus and rheumatoid arthritis argues against a direct role of anti-stress protein autoantibodies in the pathogenesis of these disorders.

Vulfovich, Yu.V., 1991. Mycoplasm arthritogenicity and human mycoplasma-induced arthritis. Vestnik Adademii Meditsinskikh Nauk SSSR 1991, 6-9.

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.

Sabin, J.E., 1990. Joseph Hersey Pratt's cost-effective class method and its contemporary application. Psychiatry 53, 169-184.

Schwartz, B.D., 1990. Infectious agents, immunity, and rheumatic diseases. Arthritis and Rheumatism 33, 457-465.

Sironi, G., Caniatti, M., Caniatti, M., 1990. Immunohistochemical detection of papillomavirus structural antigens in animal hyperplastic and neoplastic epithelial lesions. Journal of Veterinary Medicine Series A 37 , 760-770.
Abstract: One hundred and seventy two hyperplastic and neoplastic epithelial lesions from 8 different mammalian and 1 avian species were tested with an immunohistochemical technique to detect papillomavirus structural antigens. Selected lesions were diagnosed histologically as papilloma, fibropapilloma, equine sarcoid, squamous cell carcinoma, basalioma, epulis, keratoacanthoma, trichoepithelioma, pilomatrixoma, epidermal inclusion cyst, and hyperkeratotic or acanthotic epidermal lesions. Positive nuclear staining was detected in 14 out of 23 papillomas, 8 out of 32 fibropapillomas and in 1 out of 3 hyperplastic epidermal lesions. Positive samples were found in 5 of 8 mammalian species. Selected samples were also examined by transmission electron microscopy. In 4 samples papillomavirus was seen. In two other samples, negative with immunoperoxidase technique, papovavirus-like particles were observed.

Wong, C.L., Wai, M.-K., Cheng, H.-C., Chung, D., Yamashiro, D., 1990. Preliminary study on the antinociceptive effect of elephant beta-endorphin. Clinical and Experimental Pharmacology and Physiology 17, 33-37.
Abstract: 1. Intraventricular administration of human beta-endorphin and elephant beta-endorphin significantly prolonged the tail flick response tested 30 min later. However, elephant beta-endorphin was about 7-8 times more potent than human beta-endorphin in the tail flick test. 2. beta-Endorphin antagonized the antinociceptive effect of both human beta-endorphin and elephant beta-endorphin by the same extent. Naloxone also antagonized the antinociceptive effects of the beta-endorphins but it was less effective than beta-endorphin. 3. Human beta-endorphin and elephant beta-endorphin were of equal potency in inhibiting the abdominal constriction response induced by intraperitoneal (i.p.) acetic acid. Both beta-endorphin and naloxone antagonized these effects of the beta-endorphins with naloxone being more effective. 4. The present study showed that different opioid receptor subtypes may be involved in the tail flick test and the abdominal constriction test. Furthermore, elephant beta-endorphin was a better antinociceptive agent than human beta-endorphin in the tail flick test.

Pattnaik, B., Venkataramanan, R., 1989. Detection of virus-infection-associated (VIA) antibody in serum of animals susceptible to foot-and-mouth-disease virus. Indian Journal of Animal Sciences 59, 356-357.
Abstract: Foot and mouth disease virus (FMDV) infection in tissue culture and in animals lead to the production of a small heat-labile virus infection associated (VIA) antigen. Sera from two elephants tested, were positive for VIA antibodies in the double-immunodiffusion test. Of 180 cattle serum samples from animals without known vaccination history, 64 were positive for VIA antibody. Of the VIA negative 59 cattle all were negative for FMDV neutralizing antibody. Virus carriers can be detected within regularly vaccinated herds by monitoring the VIA antibody.

Schmidt, M.J. Zinc deficiency, presumptive secondary immune deficiency and hyperkeratosis in an Asian elephant: A case report. Proc.Am.Assoc.Zoo Vet. 23-31. 1989.
Ref Type: Conference Proceeding
Abstract: Zinc deficiency in an Asian elephant caused a secondary immune deficiency, and skin lesions which included superinfected vesiculobullae above the toenails and hyperkeratosis on the extensor surfaces of both elbows and on the tail. The elephant responded to therapy with an immune stimulant drug, but the chronic recurring skin lesions did not heal until after zinc supplementation was added to the diet. Additional excerpt: Dramatic improvement was noted within two weeks after the elephant was started on 2 g zinc carbonate per day. Lesions resolved by eight weeks. Subsequently, the dietary zinc level was adjusted from 21.56 mg/kg of feed to 53.6 mg/kg of feed on a dry matter basis.

Vulfovich, Yu.V., Gorina, L.G., Mitchenko, A.F., Goncharova, S.A., Gamova, N.A., Neustroeva, V.V., 1989. Mycoplasma and rheumatoid arthritis in children. Vestnik Adademii Meditsinskikh Nauk SSSR 1989, 82-84.

Wiegeshaus, E., Balasubramanian, V., Smith, D.W., 1989. Immunity to tuberculosis from the perspective of pathogenesis. Infect Immun 57 , 3671-3676.

Arnett, F.C., Edworthy, S.M., Bloch, D.A., McShane, D.J., Fries, J.F., Cooper, N.S., Healey, L.A., Kaplan, S.R., Liang, M.H., Luthra, H.S., Medsger, T.A., Jr., Mitchell, D.M., Neustadt, D.H., Pinals, R.S., Schalller, J.G., Sharp, J.T., Wilder, R.L., Hunder, G.O., 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis and Rheumatism 31, 315-324.
Abstract: The revised criteria for the classification of rheumatoid arthritis (RA) were formulated from a computerized analysis of 262 contemporary, consecutively studied patients with RA and 262 control subjects with rheumatic diseases other than RA (non-RA). The new criteria are as follows: 1) morning stiffness in and around joints lasting at least 1 hour before maximal improvement; 2) soft tissue swelling (arthritis) of 3 or more joint areas observed by a physician; 3) swelling (arthritis) of the proximal interphalangeal, metacarpophalangeal, or wrist joints; 4) symmetric swelling (arthritis); 5) rheumatoid nodules; 6) the presence of rheumatoid factor; and 7) radiographic erosions and/or periarticular osteopenia in hand and/or wrist joints. Criteria 1 and 4 must have been present for at least 6 weeks. Rheumatoid arthritis is defined by the presence of 4 or more criteria, and no further qualifications (classic, definite or probable) or list of exclusions is required. In addition, a "classification tree" schema is presented which performs equally well as the traditional (4 of 7) format. The new criteria demonstrated 91-94% sensitivity and 89% specificity for RA when compared with non-RA rheumatic disease control subjects.

Clark, H.W., Coker-Vann, M.R., Bailey, J.S., Brown, T.M., 1988. Detection of mycoplasmal antigens in immune complexes from rheumatoid arthritis synovial fluids. Annals of Allergy 60, 394-398.
Abstract: This study was directed towards the detection of suspected antigenic microbial fragments in the immune complex (IC) fraction from chronic inflammatory disorders of the delayed type allergy. Mycoplasmas as the microbial prototype and joint fluid from the rheumatoid host were investigated. Protein-A affinity chromatography was used to isolate the immunoglobulin complex (IgG-IC) in six synovial fluids obtained from rheumatoid arthritis patients. The IgG-IC was digested with pepsin to further purify and obtain F(ab)2 complexes with greater specificity. The F(ab)2 complexes were dissociated and electrophoresed by SDS-PAGE and analyzed by immunoblotting using affinity purified rabbit antisera to six reference strains of human mycoplasmas. The presence of trace amount of mycoplasma antigens in the immune complex fractions was indicated by specific banding with antisera to M. pneumoniae, M. arthritidis, M. hominis, M. fermantans, and M. salivarium in one or more of the six synovial fluid fractions. The ELISA and immunoblot assays of seroconversion in rabbits immunized with the synovial fluid fractions also indicated the presence of mycoplasmal antigens.

Bennet, D., 1987. Immune-based erosive inflammatory joint disease of the dog: canine rheumatoid arthritis. I. Clinical, radiological and laboratory investigations. Journal of Small Animal Practice 28, 779-797.
Abstract: The features of 30 cases of canine rheumatoid arthritis are described. The disease is a chronic symmetrical polyarthritis characterized by erosive, destructive changes within the joint. The latter can be identified on radiographs by loss of mineral, the presence of discrete erosions or an irregular joint margin. Increased periarticular soft tissue is common and periosteal new bone is not unusual. The most obvious clinical feature is generalized stiffness particularly after rest. Joints are often thickened and painful on manipulation. a third of cases present with pyrexia, lethargy and inappetence in addition to lameness. Synovial fluid evaluation shows an increased number of white cells, most of which are polymorphs; the mucin clot is poor. The ESR is generally increased and rheumatoid factor (an antiglobulin auto-antibody) is present in 73 per cent of cases. Treatment is often unrewarding, although many dogs can cope for considerable periods of time.

Gaskin, J.M., Andresen, T.L., Olsen, J.H., Schobert, E.E., Buesse, D., Lynch, J.D., Walsh, M., Citino, S., Murphy, D., 1987. Encephalomyocarditis in zoo animals: Recent experiences with the disease and vaccination. Proceedings of the 1st International Conference on Zoological and Avian Medicine 491.
Abstract: Encephalomyocarditis (EMC), a specific viral infection caused by a group of antigenically related viruses in the family Picornaviridae, a genus of Cardiovirus, continues to be a source of sporadic mortality loss in zoo animals in Florida. Deaths in a young Nyala antelope, 2 chimpanzees, 3 llamas, a two-toed sloth, 3 ringtail lemurs, a ruffed lemur, and an orangutan have recently been confirmed by virus recovery. Experimental vaccine trials were initiated in pygmy goats, Barbados sheep, and white mice using B-propiolactone inactivated virus preparations. Various adjuvants, including aluminum hydroxide, mineral oil, and dimethyl dioctadecyl ammonium bromide (DDAB) were used to enhance the immune responses to inactivated virus. The vaccine preparations produced varying levels of hemagglutinations-inhibition (HI) antibodies in the immunized animals. Experimental challenge of unvaccinated weaned pigs, pygmy goats, and Barbados sheep demonstrated that, although they seroconverted, they did not become ill when exposed to the virulent EMC virus strains used in this study. Laboratory mice, however, proved to be very susceptible when exposed to these same strains, and either died acutely or developed posterior paresis and paralysis subsequent to challenge. All experimental vaccine preparations protected mice against challenge. In vaccinated goats and sheep, the oil-emulsion-adjuvanted and DDAB-adjuvanted vaccines produced the highest and most persistent HI antibody titers. Sera obtained from African elephants were screened for HI antibodies to EMC virus. Ninety-three African elephant sera from the Kruger National Park in the Republic of South Africa had titers of less than 10 hemagglutination-inhibition units (HIU) while 4 of 76 imported juvenile African elephants had titers from 10-40 HIU and the rest had no titer. EMC virus infections are apparently acquired in Florida from reservoir hosts and HI titers of 40 HIU or higher indicate subclinical infection with the virus. Experimental vaccines may help prevent EMC in susceptible species; HI responses to vaccination in various exotic species are being evaluated.

Johnson, B., Burton, M., Qualls, C.W., Jr., 1986. Interstitial pulmonary fibrosis in an African elephant. Journal of the American Veterinary Medical Association 189, 1188-1190.

Phillips, P.E., 1986. Infectious agents in the pathogenesis of rheumatoid arthritis. Seminars in Arthritis and Rheumatism 16, 1-10.

Clark, H.W., Bailey, J.S., Brown, T.M., 1985. Medium-dependent Properties of Mycoplasmas. Diagn Microbiol Infect Dis 3, 283-294.
Abstract: Without a cell wall, the morphology, growth rate, and composition of mycoplasmas are culture media-dependent with variable properties best described as environmentally related. The adaptation of mycoplasmas to either a tissue cell or cell-free culture media, with dependency upon specific animal or plant products for survival, has led to investigations of their human host-related properties. The influence of culture media on the antibiotic sensitivities of mycoplasmas was measured by use of three different broths in two different assay systems. The variable results indicate that the inhibition of mycoplasma protein synthesis or growth may also by host-tissue dependent. The addition of noninhibitory penicillins to different culture media was found to affect the composition and antigenicity of some mycoplasmas. Using the complement fixation test, we found some human sera that were more reactive than rabbit antisera to mycoplasmas cultured in human synovial broth or in myelin-enriched broth. Mycoplasmas cultured in human lung broth and pig lung broth had media-dependent antigenicity. The antigenicity and the growth of mycoplasmas were found to depend on the proteolytic enzymes used to provide the essential peptides in tissue broths. The media-affected mycoplasmas indicate the presence of species-, strain-, and tissue-specific antigen sites that may determine immunopathogenicity in the genetically susceptible host.

Cole, B.C., Washburn, L.R., Taylor-Robinson, D., 1985. Mycoplasma-induced arthritis. In: Razin, S., Barile, M.F. (Eds.), The Mycoplasmas. Volume IV. Mycoplasma pathogenicity. Academic Press, New York, pp. 107-160.

Brown, T.M., Bailey, J.S., Iden, K.I., Clark, H.W., 1982. Antimycoplasma approach to the mechanism and the control of rheumatoid disease. In: Sorenson, J.R.J. (Ed.), Inflammatory diseases and copper. Humana Press, pp. 391-407.

Clark, H.W., Laughlin, D.C., Brown, T.M., 1981. Rheumatoid arthritis in elephants -- a review to date. Proceedings American Association of Zoo Veterinarians 95-99.

Clark, H.W., Laughlin, D.C., Bailey, J.S., Brown, T.M., 1980. Mycoplasma species and arthritis in captive elephants. Journal of Zoo and Wildlife Medicine 11, 3-15.
Abstract: Sixty-seven elephants (62 Elephas maximus and 5 Loxodonta africana) from three circus groups and five zoos were examined serologically and cultured for mycoplasma in a search for arthritogenic agents previously unrecognized in this animal species. In two groups of elephants, 28 of the 35 female genital tracts cultured were found to be colonized by one or more strains of mycoplasma. More than half of the elephants had complement fixing antibodies to one or more of the new mycoplasma isolates. Lameness and other rheumatoid disorders were found associated with rheumatoid factor activity and changes in mycoplasma antibody titers. In view of the arthritogenic activity of mycoplasma in other species, these new findings suggested the clinical significant of mycoplasma in elephants and the need for investigation, especially in relation to the high incidence of rheumatoid-type disorders observed in these captive elephants.

Prager, E.M., Wilson, A.C., Lowenstein, J.M., Sarich, V.M., 1980. Mammoth albumin. Science 209, 287-289.
Abstract: Serum albumin was detected immunologically in muscle from a mammoth that died about 40,000 years ago. Rabbits injected with ground mammoth muscle produced antibodies that react strongly with elephant albumin, weakly with sea cow albumin, and still more weakly or not at all with other mammalian albumins. Since elephant albumin elicited antibodies with the same specificity, some of the surviving mammoth albumin molecules evidently have antigenic sites identical to those on native elephant albumin. Much of the mammoth albumin has, however, undergone postmortem change. The small amount of soluble albumin extractable from mammoth muscle is heterogeneous in size, charge, and antigenic properties.

Cole, B.C., Cassell, G.H., 1979. Mycoplasma infections as models of chronic joint inflammation. Arthritis and Rheumatism 22, 1375-1381.

Kuntze, A., Hunsdorff, P., 1979. Further haematological and biochemical findings (Ca, inorganic P, Mg, Na, K, Fe, glucose, enzymes) with reference to iron deficiency anaemia in young elephants (Elephas maximus)]. Erkrankungen der Zootiere 13, 147-150.
Abstract: Summary: Regular haematological checks were applied to young elephants with anaemia to study the therapeutic effects of oral and parenteral iron medication. Additional serum electrolyte values are reported, with some of them discussed. They are relating to calcium, phosphorus, magnesium, sodium, potassium, glucose, SGOT, SGPT, and alkaline phosphatase

Wilkes, E., Meek, E.S., 1979. Rheumatoid arthritis: Review of searches for an infectious cause. Part II. Infection 7, 192-197.

Wilkes, E., Meek, E.S., 1979. Rheumatoid arthritis: Review of searches for an infectious cause. Part I. Infection 7, 125-128.
Abstract: No distinctive pattern has yet emerged from the acumulated mass of results that would provide a generally acceptable hypothesis of the etiology of rheumatoid arthritis. A number of immunologic aberrations have been described, but there has been no identification of a key immunologic defect that might link together the various components of the immune response into an agreed pattern. The possiblity of a persistent antigenic stimulus arising from an infection cannot be confirmed or refuted. If a virus is involved, it would seem more likely to be a "slow" virus rather than a commonly recognized form, but there is no strong candidate of this type in view. Despite the fact that mycoplasmas are undoubtedly arthritogenic in other species, their role as an atiologic agent in rheumatoid arthritis has not been proven. The idea that bacterial cell wall peptidoglycan may provide a persistent stimulus has much to offer, but it is not possible at this stage to accept peptidoglycan as a recognized etiologic factor. This suggestion will, however, undoubtedly stimulate much further investigation.

Brown, T.M., Clark, H.W., 1978. Rheumatoid inflammation -- Part I. Inflo (Arthritis Institute) 11, 1-2.

Clark, H.W., Bailey, J.S., Laughlin, D.C., Brown, T.M., 1978. Isolation of mycoplasma from the genital tracts of elephants. Zentralblatt fur Bakteriologie,Parasitenkunde,Infektionskrankheiten und Hygiene 1. Abt. Originale 241, 262.

Alexander, J.W., Begg, S., Dueland, R., Schultz, R.D., 1976. Rheumatoid arthritis in the dog: clinical diagnosis and management. Journal of the American Animal Hospital Association 12, 727-734.

Clark, H.W., Bailey, J.S., Brown, T.M. Mycoplasma hypersensitivity reactions. Proceedings of the Society for General Microbiology 111, 171. 1976.
Ref Type: Abstract
Abstract: Many immunological disorders that apparently are cell-mediated have no known aetiologic antigens other than tissue-related autoantibodies. The human host is challenged continually by many microbial antigens including several types of mycoplasmas. The immunologic response to mycoplasma antigens is dependent upon several factors other than colonizability and cytopathogenicity. Mixed microbial infections can have an augmentive or suppressive effect on the human host cell-mediated immunity (CMI). Mycoplasma can stimulate the thymus-derived 'T' cells and the bone marrow 'B' cell systems as indicated by various CMI responses such as the migratory inhibitory factor, delayed-type skin reactions, lymphocyte transformations, and humoral antibody reactions in the human host. Investigations of the mycoplasma hypersensitivity reactions in chronic rheumatoid disorders have included several factors such as long-term monitoring of CMI responses and obscured foci of mycoplasma antigens that would distinguish them from the acute-convalescent responses. In addition to the effects of therapeutic agents (immunosuppressants), physiological changes (hormonal), and environmental factors (trauma) on CMI, the 'T' cell derived anti-IgG rheumatoid factor can neutralize the humoral mycoplasma antibodies. Recent studies indicate that the frequent and variable anergic responses observed in rheumatoid disorders are dependent upon both the test mitogen and the mycoplasma antigen as well as the host lymphocytes. Tissue inflammation resulting from antigen-antibody hypersensitivity reactions, apparently occurs when the CMI responsive host is challenged by mycoplasma reinfection or antigen released from a tissue focus. The incorporation of specific tissue antigens by mycoplasma is another factor influencing their reactions in systemic "autoimmune" disorders and may require the challenging antigenic precursors to be cultured in specific human tissue media. The identification of the sensitizing and challenging antigens also includes the appraisal of mycoplasma exoantigens and exoenzymes, such as DNase, released into the tissues as well as the physiologically optimum fractions.

Newton, C.D., Lipowitz, A.J., Halliwell, R.E., Allen, H.L., Biery, D.N., Schumacher, H.R., 1976. Rheumatoid arthritis in dogs. Journal of the American Veterinary Medical Association 168, 113-121.

Pedersen, N.C., Pool, R.C., Castles, J.J., Weisner, K., 1976. Noninfectious canine arthritis: rheumatoid arthritis. Journal of the American Veterinary Medical Association 169, 295-303.
Abstract: Chronic unremitting, generally symmetric, erosive arthritis was studied in 8 dogs. The disease had clinical, serologic, radiographic, and pathologic changes similar to those of rheumatoid arthritis of man. The condition occurred mainly in smaller breeds of dogs, with time of onset from 8 months to 8 years of age. Characteristic radiographic changes were seen in the first several weeks to several months after the appearance of the initial lameness. Synovial fluid contained an increased number of neutrophils, and synovial fluid and synovial tissues were sterile for anaerobic and aerobic bacteria, mycoplasma, chlamydia, and viruses. Corticosteroids were therapeutically ineffective in all of the cases; however, corticosteroids, cyclophosphamide and azathioprine were effective when used in combination in several dogs.

Newton, C.D., Lipowitz, A.J., 1975. Canine rheumatoid arthritis: A brief review. Journal of the American Animal Hospital Association 11, 595-599.

Brown, T.M., Clark, H.W., Bailey, J.S., 1974. Natural occurance of rheumatoid arthritis in great apes -- a new animal model. Proceedings of the Zoological Society of the Philadelphia Centennial Symposium on Science and Research 43-79.

Sokoloff, J., 1972. The pathology of rhematoid arthritis and allied disoders. In: Hollander, J.L., McCarty, D.J. (Eds.), Arthritis and Allied Conditions. Lea and Febiger, Philadelphia,PA, pp. 309-332.

Vaughan, J.H., 1972. The rheumatoid factors. In: Hollander, J.L., McCarty, D.J. (Eds.), Arthritis and Allied Conditions. Lea and Febiger, Philadelphia,PA, pp. 153-171.

Basson, P.A., McCully, R.M., de Vos, V., Young, E., Kruger, S.P., 1971. Some parasitic and other natural diseases of the African elephant in the Kruger National Park. Onderstepoort Journal of Veterinary Research 38 , 239-254.

Brown, T.M., Clark, H.W., Bailey, J.S., Gray, C.W., 1970. A mechanistic approach to treatment of rheumatoid type arthritis naturally occuring in a gorilla. Trans. Am. Clin. and Climat. Assoc. 82, 227-247.

Ruddy, S., Austen, K.F., 1970. The complement system in rheumatoid synovitis. Arthritis and Rheumatism 13, 713-723.
Abstract: Stoichiometric hemolytic assays were used to measure the activities of the first four reacting components of the complement sequence in synovial fluids from patients with seropositive or seronegative rheumatoid arthritis or degenerative joint disease. The pattern of component reductions in the seropositive rheumatoid arthritis fluids was consistent with activation of the complement system by an intra-articular immunologic process.

Bartfield, H., 1969. Distribution of rheumatoid factor in non-rheumatoid states. Annals of the New York Academy of Science 168, 30-40.

Mongan, E.S., Atwater, E.C., 1968. A comparison of patients with seropositive and seronegative rheumatoid arthritis. Medical Clinics of North American 52, 533-538.

Cave, A.J.E., Aumonier, F.J., 1964. Lymph node structure in an Asiatic elephant. Journal of the Royal Microscopic Society 82, 251-255.

Cave, A.J.E., Aumonier, F.J., 1962. Elephant and rhinoceros lymph node histology. Journal of the Royal Microscopic Society 80, 209-214.
Abstract: The histology is described, for the first time, of certain lymph nodes of the African Elephant (Loxodonta africana Blumenbach), of the Great Indian Rhinoceros (Rhinoceros unicornis Linn.) and of the African Black Rhinoceros (Diceros bicornis Linn.): reference is made to the nodes of the African White Rhinoceros (Ceratotherium simum Gray). All nodes studied prove to be haemolymph organs.

Mellors, R.C., Nowoslowski, A., Korngold, L., Sengson, B.L., 1961. Rheumatoid factor and the pathogenesis of rheumatoid arthritis. Journal of Experimental Medicine 113, 475-483.

Mellors, R.C., Nowoslowski, A., Korngold, L., 1961. Rheumatoid arthritis and the cellular origin of rheumatoid factors. American Journal of Pathology 39, 533-546.

Ropes, M.W., 1959. Diagnostic criteria for rheumatoid arthritis: 1958 revision. Annals of the Rheumatic Diseases 18, 49-53.

Heyman, A., Sheldon, W.H., Evans, L.D., 1953. Pathogenesis of the Jarisch-Herxheimer reaction. British Journal of Venereal Diseases 28 , 50.

Brown, T.M., Wichelhausen, R.H., Robinson, L.B., Merchout, W.R., 1949. The in vivo action of aureomycin on pleuropneumonia-like organisms associated with various rheumatic diseases. Journal of Laboratory and Clinical Medicine 34, 1404-1410.

Ishigami, T., 1918. The influence of psychic acts on the progress of pulmonary tuberculosis. Am. Rev. Tuberc. 2, 470-484.

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