Meeting report: Spontaneous lesions and diseases in wild, captive-bred, and zoo-housed nonhuman primates and in nonhuman primate species used in drug safety studies.

The combination of loss of habitat, human population encroachment, and increased demand of select nonhuman primates for biomedical research has significantly affected populations. There remains a need for knowledge and expertise in understanding background findings as related to the age, source, strain, and disease status of nonhuman primates. In particular, for safety/biomedical studies, a broader understanding and documentation of lesions would help clarify background from drug-related findings. A workshop and a minisymposium on spontaneous lesions and diseases in nonhuman primates were sponsored by the concurrent Annual Meetings of the American College of Veterinary Pathologists and the American Society for Veterinary Clinical Pathology held December 3-4, 2011, in Nashville, Tennessee. The first session had presentations from Drs Lowenstine and Montali, pathologists with extensive experience in wild and zoo populations of nonhuman primates, which was followed by presentations of 20 unique case reports of rare or newly observed spontaneous lesions in nonhuman primates (see online files for access to digital whole-slide images corresponding to each case report at http://www.scanscope.com/ACVP%20Slide%20Seminars/2011/Primate%20Pathology/view.apml). The minisymposium was composed of 5 nonhuman-primate researchers (Drs Bradley, Cline, Sasseville, Miller, Hutto) who concentrated on background and spontaneous lesions in nonhuman primates used in drug safety studies. Cynomolgus and rhesus macaques were emphasized, with some material presented on common marmosets. Congenital, acquired, inflammatory, and neoplastic changes were highlighed with a focus on clinical, macroscopic, and histopathologic findings that could confound the interpretation of drug safety studies.

Chronic polymyositis associated with disseminated Sarcocystosis in a captive-born rhesus macaque.

A 2-year-old, captive-born, clinically healthy male, rhesus macaque, was euthanatized as part of an experimental study. At necropsy, diffuse pale streaking of the trunk, lumbar, and limb muscles were noted macroscopically. On histology, numerous elongated cysts that contained crescent-shaped basophilic spores were found in the fibers of skeletal muscles. Scattered affected myofibers were degenerate and accompanied by eosinophilic-to-granulomatous inflammation. Sarcocysts had prominent villus-like projections with the morphology of a type 11 sarcocyst wall similar to Sarcocystis neurona but possessing many more villus microtubules than is reported for S. neurona. In addition, bradyzoites were very long, up to approximately 12 microm in length. The protozoa were consistent with a Sarcocystis sp., based on histology and ultrastructure, however, a definitive identification of the species was not possible. Nonspecific immunohistochemical crossreaction with Sarcocystis cruzi antisera was observed. The 18S ribosomal deoxyribonucleic acid sequence showed 91% similarity to Sarcocystis hominis, 90% similarity to Sarcocystis buffalonis, and 89% similarity to Sarcocystis hirsuta. Interestingly, the ITS1 sequence showed very little homology to any sequence in GenBank, suggesting that this is possibly a unique Sarcocystis sp. Sarcocystosis is often considered an incidental finding, particularly in wild-caught animals, with little clinical significance. However, as demonstrated in this report and others, disseminated sarcocystosis can occur in captive-born rhesus macaques with or without clinical signs. In some cases interference with research results can occur; including death in fulminant cases.

Chronic eosinophilic dermatitis associated with persistent feline herpes virus infection in cheetahs (Acinonyx jubatus).

A chronic ulcerative and eosinophilic dermatitis occurred in 20 captive cheetahs (Acinonyx jubatus) with persistent feline herpes virus 1 (FHV1) infection. Affected animals had erythematous, ulcerated plaques primarily on the face and forelegs in sites of contact with lachrymal and salivary secretions. The dermatitis was characterized by dense infiltrates of eosinophils and plasma cells and pseudoepitheliomatous hyperplasia. Rare keratinocytes within the lesions had nuclei with marginated chromatin and small eosinophilic inclusions composed of herpes virus nucleocapsids. Virus isolated from lesions was confirmed to be FHV1. Lesions persisted and progressed unless removed by cryoexcision. The occurrence of this unusual reaction to FHV1 in approximately 5% of captive North American cheetahs suggests a species propensity for a Th2-dominant response to herpes virus infection. This atypical immune reaction may indicate a heritable trait or modulation of the immune response by other factors such as chronic stress.

Mycobacterium marinum dermatitis and panniculitis with chronic pleuritis in a captive white whale (Delphinapterus leucas) with aortic rupture.

A 16-year-old female white whale, Delphinapterus leucas, died after nearly 18 months of chronic lymphopenia and pyogranulomatous dermatitis. Necropsy revealed rupture of the aorta with hemorrhage into the cranial mediastinum and between fascial planes of the ventral neck musculature. Multiple foci of ulcerative dermatitis and panniculitis were present across the thorax and abdomen and surrounded the genital folds. In addition, there was a chronic proliferative pleuritis with over 20 liters of histiocytic exudate in the thoracic cavity. Acid-fast bacteria consistent with Mycobacterium sp. were identified in sections of skin lesions and in cytospins of pleural exudate. Cultures of pleura and 1 skin lesion collected at necropsy yielded sparse growth of an acid-fast bacillus with colony characteristics and morphology consistent with Mycobacterium marinum. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis confirmed the presence of M. marinum DNA in samples of skin. This is the first documented occurrence of mycobacteriosis in a white whale and is a unique presentation of mycobacterial dermatitis and panniculitis with chronic pleuritis in a cetacean. The improved PCR-RFLP protocol utilized in this case unifies techniques from several protocols to differentiate between species of Nocardia and rapidly growing mycobacteria clinically relevant to aquatic animals.

Mycobacterium tuberculosis in zoo and wildlife species.

Tuberculosis caused by Mycobacterium tuberculosis and M. tuberculosis-like organisms has been identified in a wide range of species, including non-human primates, elephants and other exotic ungulates, carnivores, marine mammals and psittacine birds. Disease associated with M. tuberculosis has occurred mostly within captive settings and does not appear to occur naturally in free-living mammals. Mycobacterium tuberculosis probably originated as an infection of humans, but from the zoonotic standpoint, non-human primates, Asian elephants and psittacine birds have the potential to transmit this disease to humans. However, the overall prevalence of disease in these susceptible species is low and documented transmissions of M. tuberculosis between animals and humans are uncommon. Mycobacterium tuberculosis causes progressive pulmonary disease in mammals and a muco-cutaneous disease in parrots. In all cases, the disease can disseminate and be shed into the environment. Diagnosis in living animals is based on intradermal tuberculin testing in non-human primates, culturing trunk secretions in elephants, and biopsy and culture of external lesions in parrots. Ancillary testing with deoxyribonucleic acid probes and nucleic acid amplification, and enzyme-linked immunosorbent assays have been adapted to some of these species with promising results. Additionally, new guidelines for controlling tuberculosis in elephants in the United States of America, and programmes for tuberculosis prevention in animal handlers have been established.

Epidemiology and diagnosis of Mycobacterium tuberculosis in captive Asian elephants (Elephas maximus).

The deaths of two Asian elephants (Elephas maximus) in August 1996 led the United States Department of Agriculture to require the testing and treatment of elephants for tuberculosis. From August 1996 to September 1999. Mycobacterium tuberculosis infection was confirmed by culture in 12 of 118 elephants in six herds. Eight diagnoses were made antemortem on the basis of isolation of M. tuberculosis by culture of trunk wash samples; the remainder (including the initial two) were diagnosed postmortem. We present the case histories, epidemiologic characteristics, diagnostic test results, and therapeutic plans from these six herds. The intradermal tuberculin test, enzyme-linked immunosorbent assay serology, the blood tuberculosis test, and nucleic acid amplification and culture are compared as methods to diagnose M. tuberculosis infection in elephants.

Subcutaneous atypical mycobacteriosis in captive tiger quolls (Dasyurus maculatus).

From July 1989 to October 1998, 9/37 (24%) adult captive tiger quolls (Dasyurus maculatus) were diagnosed with atypical mycobacterial infection involving the subcutis and skin. Females were more often affected than males (seven females, two males). Grossly, lesions presented as focal thickenings, plaques, and abscesses within the subcutis, often with fistulous tracts. The subcutis and skin overlying cervical and thoracic regions were the primary sites of infection. Cytology of subcutaneous impression smears from all nine affected tiger quolls revealed pyogranulomatous inflammation admixed with several acid-fast bacilli. Histologically, all tiger quolls had nodular to diffuse pyogranulomatous panniculitis and cellulitis. Small numbers of acid-fast bacilli were noted histologically in 7/9 (78%) animals. Skin cultures from seven tiger quolls were positive for one or more different Runyon group IV mycobacteria. The disease described in these tiger quolls is similar to subcutaneous atypical mycobacteriosis of humans and domestic animals.

Clinical and pathological findings of a newly recognized disease of elephants caused by endotheliotropic herpesviruses.

The unique clinical and pathological findings in nine Asian (Elephas maximus) and two African (Loxodonta africana) elephants from North American Zoos with a highly fatal disease caused by novel endotheliotropic herpesviruses are described. Identification of the viruses by molecular techniques and some epidemiological aspects of the disease were previously reported. Consensus primer polymerase chain reaction (PCR) combined with sequencing yielded molecular evidence that confirmed the presence of two novel but related herpesviruses associated with the disease, one in Asian elephants and the second in African elephants. Disease onset was acute, with lethargy, edema of the head and thoracic limbs, oral ulceration and cyanosis of the tongue followed by death of most animals in 1 to 7 days. Pertinent laboratory findings in two of three clinically evaluated animals included lymphocytopenia and thrombocytopenia. Two affected young Asian elephants recovered after a 3 to 4 wk course of therapy with the anti-herpesvirus drug famciclovir. Necropsy findings in the fatal cases included pericardial effusion and extensive petechial hemorrhages in the heart and throughout the peritoneal cavity, hepatomegaly, cyanosis of the tongue, intestinal hemorrhage, and ulceration. Histologically, there were extensive microhemorrhages and edema throughout the myocardium and mild, subacute myocarditis. Similar hemorrhagic lesions with inflammation were evident in the tongue, liver, and large intestine. Lesions in these target organs were accompanied by amphophilic to basophilic intranuclear viral inclusion bodies in capillary endothelial cells. Transmission electron microscopy of the endothelial inclusion bodies revealed 80 to 92 nm diameter viral capsids consistent with herpesvirus morphology. The short course of the herpesvirus infections, with sudden deaths in all but the two surviving elephants, was ascribed to acute cardiac failure attributed to herpesvirus-induced capillary injury with extensive myocardial hemorrhage and edema.

Idiopathic hemorrhagic vasculopathy syndrome in seven black rhinoceros.

Idiopathic hemorrhagic vasculopathy syndrome (IHVS) was diagnosed in 7 black rhinoceros; this newly described syndrome is characterized by severe body swelling in conjunction with a rapid and profound decrease in Hct. The disorder may be acute or chronic, may recur, and is potentially fatal. Five of the rhinoceros survived an initial episode of IHVS, and 2 of these 5 survived a recurrent episode of IHVS. Two rhinoceros died during treatment of IHVS. Treatment protocols varied, but all 7 rhinoceros received broad-spectrum antibiotics, because an infectious cause was suspected. All rhinoceros also received nonsteroidal antiinflammatory drugs and supportive care. Idiopathic hemorrhagic vasculopathy syndrome has many similarities to other vasculopathies of domestic animals, such as equine purpura hemorrhagica, but it also appears to have unique identifying features. It has been hypothesized that IHVS may be an immune response to an as yet unidentified infectious agent. Thorough and extensive testing has not identified the potential causative agent, nor the factors that predispose some black rhinoceros to developing IHVS. Further research into the rhinoceros immune system is ongoing and should help elucidate the mechanisms through which IHVS develops.

Evaluation of a multiple-antigen enzyme-linked immunosorbent assay for detection of Mycobacterium tuberculosis infection in captive elephants.

Mycobacterium tuberculosis has become an important agent of disease in the captive elephant population of the United States, although current detection methods appear to be inadequate for effective disease management. This investigation sought to validate a multiple-antigen enzyme-linked immunosorbent assay (ELISA) for screening of M. tuberculosis infection in captive elephants and to document the elephant's serologic response over time using a cross-sectional observational study design. Serum samples were collected from 51 Asian elephants (Elephas maximus) and 26 African elephants (Loxodonta africana) from 16 zoos and circuses throughout the United States. Infection status of each animal was determined by mycobacterial culture of trunk washes. Reactivity of each serum sample against six antigens was determined, and the linear combination of antigens that accurately predicted the infection status of the greatest number of animals was determined by discriminant analysis. The resulting classification functions were used to calculate the percentage of animals that were correctly classified (i.e., specificity and sensitivity). Of the 77 elephants sampled, 47 fit the criteria for inclusion in discriminant analysis. Of these, seven Asian elephants were considered infected; 25 Asian elephants and 15 African elephants were considered noninfected. The remaining elephants had been exposed to one or more infected animals. The specificity and sensitivity of the multiple-antigen ELISA were both 100% (91.9-100% and 54.4-100%, respectively) with 95% confidence intervals. Mycobacterium bovis culture filtrate showed the highest individual antigen specificity (95%; 83.0-100%) and sensitivity (100%; 54.4-100%). Serum samples from 34 elephants were analyzed over time by the response to the culture filtrate antigen; four of these elephants were culture positive and had been used to calculate the discriminant function. Limitations such as sample size, compromised ability to ascertain each animal's true infection status, and absence of known-infected African elephants suggest that much additional research needs to be conducted regarding the use of this ELISA. However, the results indicate that this multiple-antigen ELISA would be a valuable screening test for detecting M. tuberculosis infection in elephant herds.

Canine distemper in terrestrial carnivores: a review.

Canine distemper virus is a member of the genus Morbillivirus in the family Paramyxoviridae. Canine distemper has been recorded in domestic dogs for centuries. It is now recognized as a worldwide problem of carnivores and has the second highest fatality rate of any infectious disease, after rabies, in domestic dogs. The importance of this disease in nondomestic animals has become evident with vaccine-induced infections in a variety of species and large-scale epidemics in captive and free-ranging felids. To date, canine distemper has been reported in all families of terrestrial carnivores: Canidae, Felidae, Hyaenidae, Mustelidae, Procyonidae, Ursidae, and Viverridae. Veterinarians, including those working with nondomestic carnivores, should be familiar with the clinical signs, diagnosis, and clinical management of this disease.

Use of famciclovir for the treatment of endotheliotrophic herpesvirus infections in Asian elephants (Elephas maximus).

Two juvenile Asian elephants (Elephas maximus) presented with an acute onset of facial edema and lethargy. Examination of the oral cavity of each animal revealed cyanosis of the tip and distal margins of the tongue suggestive of endothelial inclusion body disease (EIBD) of elephants. Whole-blood samples were obtained, and polymerase chain reaction tests confirmed the presence of elephant herpesvirus. The animals were administered famciclovir (Famvir, SmithKline Beecham Pharmaceuticals, Philadelphia, Pennsylvania 19101, USA), a potent human anti-herpesvirus drug, in the course of their disease, and recovery followed a treatment regime of 3-4 wk. These are the first known cases of elephants surviving EIBD.

Common cuttlefish (Sepia officinalis) mortality at the National Zoological Park: implications for clinical management.

Six out of seven cuttlefish acquired by the Smithsonian National Zoological Park in July 1998 died before 1 November 1998. Postmortem examinations showed mantle ulcers, secondary bacterial infections, inanition, and cuttlebone fractures. The surviving cuttlefish developed a progressive focal mantle ulcer, was treated with oral chloramphenicol intermittently for 9 wk, and maintained a normal appetite and growth rate until death at 7 mo of age. The National Zoological Park pathology database showed signalments, histories, and causes of mortality of 186 common cuttlefish, each 1-14 mo old, that received gross and histologic examinations; for example, the largest group of cuttlefish of known sex, age, and body weight at postmortem were 7-9 mo old and weighed an average of 376.2 g (males, n = 18) and 299.0 g (females, n = 15). Many cuttlefish had multiple pathologic diagnoses. Significant diseases included inflammation and secondary bacterial infections, especially gastrointestinal, cardiovascular, respiratory, reproductive, and ophthalmic, and septicemia due to Vibrio spp. or other gram-negative bacteria. Mantle lesions, including ulceration/dermatitis, abscess/granuloma, necrosis/fibrosis/cellulitis, and laceration/abrasion/erosion, were also identified, along with inanition, cuttlebone lesions, and trauma. Mantle lesions were associated with secondary bacterial infections and death. On the basis of this information, if captive cuttlefish behavior creates risk for development of mantle lesions, administration of antibiotics effective against gram-negative bacteria may delay or halt disease progression. Cuttlefish exhibits require proper design, husbandry, economic resources, and staffing to minimize disease syndromes and mortality.

Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus.

Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentis belonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known as Cryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed.

Novel endotheliotropic herpesviruses fatal for Asian and African elephants.

A highly fatal hemorrhagic disease has been identified in 10 young Asian and African elephants at North American zoos. In the affected animals there was ultrastructural evidence for herpesvirus-like particles in endothelial cells of the heart, liver, and tongue. Consensus primer polymerase chain reaction combined with sequencing yielded molecular evidence that confirmed the presence of two novel but related herpesviruses associated with the disease, one in Asian elephants and another in African elephants. Otherwise healthy African elephants with external herpetic lesions yielded herpesvirus sequences identical to that found in Asian elephants with endothelial disease. This finding suggests that the Asian elephant deaths were caused by cross-species infection with a herpesvirus that is naturally latent in, but normally not lethal to, African elephants. A reciprocal relationship may exist for the African elephant disease.

Abdominal lipomatosis attributed to tall fescue toxicosis in deer.

Five adult female Eld's deer died acutely or were euthanatized because of clinical signs including anorexia, signs of depression, and uremia. On necropsy, these deer had large masses of necrotic abdominal fat constricting the ureters, causing hydroureter and hydronephrosis. The herd from which these deer originated was maintained on pastures consisting primarily of tall fescue, samples from which were subsequently confirmed to be infected with an endophytic fungus that is known to cause similar lesions in cattle. A retrospective study of deaths in this herd revealed a sharp increase in incidence of abdominal lipomatosis since 1994. Physical examinations on the herd revealed > 90% of females to be affected. Endophyte-infected tall fescue forage was concluded to be a major factor in the development of lipomatosis in these deer. Other contributing factors were considered. Lesions caused by endophyte-infected fescue can be severe, and this disease can develop in nondomestic species.

Primary Mycobacterium avium complex infections correlate with lowered cellular immune reactivity in Matschie's tree kangaroos (Dendrolagus matschiei).

The National Zoological Park has maintained a breeding colony of Matschie's tree kangaroos (Dendrolagus matschiei) since 1975 with a documented history and continued prevalence of Mycobacterium avium complex (MAC) infections. No evidence of immunosuppressive retrovirus infections or loss of heterozygosity that may have led to an immune dysfunction in these animals was found. Isolates of MAC organisms from affected tree kangaroos and from their environment had no common restriction fragment DNA types. Cellular immune reactivity in apparently healthy tree kangaroos was 3- to 6-fold lower than in humans and other marsupial and eutherian mammals, as determined by lymphocyte proliferative assays. Thus, while MAC infections are typically opportunistic in humans and other mammals, tree kangaroos commonly develop primary progressive disease with MAC from random sources. Comparative information derived from this study should benefit both the endangered tree kangaroo and humans with immunosuppressive disorders that lead to mycobacterial infections.

Follicle-oocyte atresia and temporal taphonomy in cold-stored domestic cat ovaries.

In vitro oocyte maturation followed by in vitro fertilization (IVM/IVF) success in the domestic cat remains inferior to commonly studied livestock or laboratory species. The objectives here were (1) to histologically assess atresia status of freshly excised follicle/oocyte complexes, and (2) to evaluate taphonomic change (deterioration after excision) of these complexes after ovarian cold storage for up to 48 h. After excision of 50 ovarian pairs, one ovary was preserved immediately and the other stored in phosphate buffered saline (4 degrees C) for 4, 8, 12, 24, or 48 h before fixation and examination. Ovaries were classified as luteal if prominent corpora lutea (CL) were present or as follicular if antral follicles and no CL were present. Two classes of follicle-oocyte complexes (preantral and antral) were microscopically evaluated. Of the 2,280 complexes examined, 64.3% demonstrated clear evidence of slight to severe degeneration, with various stages being described and photographed for the first time. There was no histological evidence indicating distinctive morphological differences between oocytes recovered from follicular versus luteal donors. Storage of whole ovaries in cold saline inhibited taphonomic changes for 48 h after excision. In summary, there is marked variability in the number and quality of follicle populations in cat ovaries. A high percentage of full-sized follicular oocytes are undergoing atresia at any given time. However, additional gross degeneration as a result of cold-storage appears modest for up to 48 h. Nonetheless, this high level of natural atresia in the cat likely contributes to comparatively lower IVM/IVF success than in other species.

Endocrine neoplasia in New World primates.

Of 1,106 New World primates necropsied from the National Zoological Park (Washington, D.C.) and the Department of Comparative Pathology, Johns Hopkins University School of Medicine (Baltimore, Maryland) 22 (1.9%) animals were identified with 27 neoplasms. Of this group, nine animals (two females, seven males) had a total of 13 endocrine neoplasms. All animals were adults, with an age range of 2.7-25 years (average, 12.1 years). Seven were Callitrichidae and two were Cebidae. The adrenal gland was the most affected organ, with seven (53.8%) neoplasms, followed by the pituitary and thyroid gland with two (15.4%) cases each, and the pancreas and parathyroid gland with one tumor (7.7%) each. All neoplastic disorders were benign. Immunocytochemistry assays for growth hormone, adrenocorticotropic hormone, prolactin, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone, and chromogranin A were performed on two pituitary neoplasms. Pheochromocytoma was the most frequent neoplasm, representing 5 (38.4%) of the 13 neoplasms. The remaining were thyroid cystadenoma (two, 15.4%), corticotrophic cell pituitary adenoma (two, 15.4%), adrenal ganglioneuroma (one, 7.7%), adrenal cortical adenoma (one, 7.7%), parathyroid chief-cell adenoma (one, 7.7%), and pancreatic islet-cell adenoma (one, 7.7%).