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.

Retrospective study of orthovoltage radiation therapy for nasal tumors in 42 dogs.

Megavoltage radiation therapy currently is the standard of care for dogs with nasal tumors. Some studies report that surgery and adjunctive orthovoltage radiation therapy result in longer control of these tumors than does megavoltage radiation therapy alone. This study reports less effective control of nasal tumors in dogs treated with surgery and orthovoltage radiation than previously observed, supporting the superiority of megavoltage radiation therapy for these tumors. In addition, this study suggests 2 new prognostic indicators for dogs with nasal tumors and describes toxicity associated with surgery and orthovoltage therapy. Forty-two dogs with nasal tumors were treated with surgical cytoreduction and 48 Gy orthovoltage radiation therapy administered in twelve 4-Gy fractions. Median survival was 7.4 months. One- and 2-year survival rates were 37% and 17%, respectively. Dogs with facial deformity had shorter survival than those without deformity (P = .005). Dogs with resolution of clinical signs after treatment had longer survival than those with chronic nasal signs (P = .0001). Acute radiation toxicity was moderate to severe for skin and eye and negligible for oral mucosa. Toxicity healed within 1 month after radiation therapy. Late toxicity was mild, but 70% of evaluable dogs experienced persistent ocular signs. Only 39% of dogs achieved a disease-free period.

Cloning of a beta-hemolysin gene of Brachyspira (Serpulina) hyodysenteriae and its expression in Escherichia coli.

Brachyspira (Serpulina) hyodysenteriae induces a mucohemorrhagic diarrheal disease in pigs. The production of a beta-hemolysin has been considered a major virulence attribute of this organism. Previous reports have failed to correlate a specific cloned gene sequence with a purified beta-hemolytic protein sequence. Thus, questions still remain concerning the structural gene sequence of the hemolysin. To answer this question unequivocally, the beta-hemolytic toxin was purified from extracts of log-phase spirochetes, and the N-terminal amino acid sequence was determined (K-D-V-V-A-N-Q-L-N-I-S-D-K) and compared with the translated sequences of previously cloned genes, tlyA to tlyC. The lack of homology between tlyA to tlyC translated sequences and the purified beta-hemolytic toxin sequence resulted in the study that is reported here. A degenerate probe was designed based on the N-terminal amino acid sequence of the purified beta-hemolysin and used to screen a B. hyodysenteriae genomic library. Three overlapping clones were identified, and one was sequenced to reveal an open reading frame coding for a putative 8.93-kDa polypeptide containing the N-terminal sequence of the purified beta-hemolysin. To distinguish this gene from the tlyA to tlyC genes, it has been designated hlyA. A hemolysis-negative Escherichia coli strains containing hlyA was beta-hemolytic on blood agar media. Also, the hemolytic activity of the recombinant protein had identical protease and lipase sensitivities and electrophoretic mobility to those of native B. hyodysenteriae beta-hemolysin. Based on sequence analysis, the translated protein had a pI of 4.3, an alpha-helical structure, and a phosphopantetheine binding motif. Hybridization analysis of genomic DNA indicated that the hlyA gene was present in B. hyodysenteriae and B. intermedia but was not detected in B. innocens, B. pilosicoli, or B. murdochii under high-stringency conditions. The location of hlyA on the chromosomal map was distinct from the locations of tlyA, tlyB, and tlyC.

Reduction in inflammation following blockade of CD18 or CD29 adhesive pathways during the acute phase of a spirochetal-induced colitis in mice.

Colitis develops in mice infected with Brachyspira (Serpulina) hyodysenteriae. Numerous granulocytes (PMNs) are evident in cecal tissue sections 24-48 h post-infection. The role of PMNs was assessed by utilizing monoclonal antibodies specific for CD18 or CD29 to block PMN recruitment. Macroscopic lesions were less severe in mice treated with either monoclonal antibody compared to lesions observed in isotype control-treated mice. While these monoclonal antibodies may inhibit extravasation of other leukocytes, the central role of PMNs was further demonstrated in that colitis was reduced following neutrophil depletion. There was less edema and epithelial erosions in ceca of mice receiving anti-Ly6G, -CD18 or -CD29 monoclonal antibody compared to mice receiving the control. Moreover, there was a significant reduction in PMN infiltration in tissues of mice treated with anti-CD18. The reduction in infiltrating PMNs did not result from downregulation of neutrophil chemoattractant MIP-2 expression in anti-CD18-treated mice. In contrast, PMN recruitment into the cecum was apparently CD29-independent. It is noteworthy that the number of PMNs observed in anti-CD18-treated mice was significantly higher than observed in non-infected mice. The data provide evidence for a threshold number of PMNs necessary for lesion development and indicate that CD18, but not CD29, adhesive pathways are crucial for PMN recruitment in bacterial colitis.

Pathogenicity of West Nile virus in chickens.

In the fall of 1999, West Nile virus (WNV) was isolated for the first time in the Western Hemisphere during an outbreak of neurologic disease in humans, horses, and wild and zoo birds in the northeastern United States. Chickens are a potential reservoir for WNV, and little is known about the pathogenicity of WNV in domestic chickens. Seven-week-old chickens derived from a specific-pathogen-free flock were inoculated subcutaneously with 1.8 x 10(3) 50% tissue culture infectious dose of a crow isolate of WNV in order to observe clinical signs and evaluate the viremic phase, gross and microscopic lesions, contact transmission, and immunologic response. There were no observable clinical signs in the WNV-inoculated chickens during the 21-day observation period. However, histopathologic examination of tissues revealed myocardial necrosis, nephritis, and pneumonitis at 5 and 10 days postinoculation (DPI); moderate to severe nonsuppurative encephalitis also was observed in brain tissue from one of four inoculated birds examined at 21 DPI. WNV was recovered from blood plasma for up to 8 DPI. Virus titers as high as 10(5)/ml in plasma were observed at 4 DPI. Fecal shedding of virus was detected in cloacal swabs on 4 and 5 DPI only. The WNV also was isolated from myocardium, spleen, kidney, lung, and intestine collected from chickens euthanatized at 3, 5, and 10 DPI. No virus was isolated from inoculated chickens after 10 DPI. Antibodies specific to WNV were detected in inoculated chickens as early as 5 DPI by the plaque reduction neutralization test and 7 DPI by the indirect fluorescent antibody test. Chickens placed in contact with inoculated chickens at 1 DPI lacked WNV-specific antibodies, and no WNV was isolated from their blood plasma or cloacal swabs throughout the 21 days of the experiment.

Clinical, bacteriologic, serologic, and pathologic features of infections with atypical Taylorella equigenitalis in mares.

OBJECTIVE: To characterize clinical, serologic, bacteriologic, cytologic, and pathologic endometrial responses of mares to 2 donkey-origin atypical bacterial isolates resembling Taylorella equigenitalis. DESIGN: Prospective in vivo study. ANIMALS: 10 healthy mares. PROCEDURE: Mares in estrus (2/group) were inoculated by intrauterine infusion with 2 isolates of classic T equigenitalis or 2 isolates of atypical Taylorella sp or were sham-inoculated. Bacteriologic, serologic, clinical, uterine, cytologic, and pathologic endometrial responses were assessed 4, 11, 21, 35, and 63 days after inoculation and on day 111 in mares with positive culture results on day 63. RESULTS: One atypical isolate failed to cause infection. The second atypical isolate and both classic T equigenitalis isolates induced similar transient metritis and cervicitis. Both classic isolates and 1 atypical isolate induced anti-T equigenitalis complement-fixing antibodies detectable at day 11. Classic isolates and an atypical isolate provoked intense neutrophilic endometritis followed by a resolving, subacute, neutrophilic-mononuclear endometrial response. The atypical isolate and classic isolates were recovered from the uterus, clitoral fossa, or clitoral sinus of one or both exposed mares for as long as 111 days. CONCLUSIONS AND CLINICAL RELEVANCE: Atypical Taylorella sp infections should be considered as a differential diagnosis of equine infertility in US-origin mares, even those not exposed to stallions from countries where contagious equine metritis occurs. The origins and prevalence of atypical Taylorella sp infection in US horses and donkeys are undetermined.

A comparison of the morphologic effects of Serpulina hyodysenteriae or its beta-hemolysin on the murine cecal mucosa.

Studies were carried out to compare the early morphologic changes in the cecal mucosa of mice either infected with Serpulina hyodysenteriae or exposed to the beta-hemolysin of S. hyodysenteriae. Sixty-five 12-24-week-old C3H/HeOuJ mice were infected with S. hyodysenteriae by gastric intubation. Two mice were necropsied every hour for 30 hours following infection. S. hyodysenteriae was isolated from the cecal contents of each mouse at all time points. Macroscopic lesions were first apparent at 14 hours postinfection (PI), and light microscopic lesions were first apparent at 10 hours PI, earlier than has been previously reported. Ultrastructural changes, first evident at 6 hours PI, included disarray and loss of microvilli and terminal web, with dilatation of intercellular spaces. Luminal bacteria were translocated through epithelial cells to the lamina propria, where capillaries exhibited changes indicative of increased permeability. In another experiment, solutions containing between 2,500 and 25,000 hemolytic units of purified S. hyodysenteriae hemolysin were placed within the lumen of surgically closed murine ceca (n = 10); ceca were collected for examination 3 hours following treatment. Ultrastructural changes consisted of loss of microvilli and terminal web and marked vacuolation and exfoliation of epithelial cells. Significant numbers of necrotic and apoptotic epithelial cells were present, and epithelial cells internalized moderate numbers of bacteria. The hemolysin of S. hyodysenteriae induces some of the same early ultrastructural changes in the cecal epithelium of mice as occur following infection with S. hyodysenteriae. Based on the observed bacterial translocation, luminal bacteria also appear to play a unique role in lesion development in this model.

Morphologic and temporal characterisation of lesions in an enhanced murine model of Serpulina hyodysenteriae infection.

This laboratory has previously reported a murine model of Serpulina hyodysenteriae infection in which mice fed a defined diet, Teklad 85420 (TD), developed caecal lesions more consistently than mice fed a conventional rodent chow (CRC). The objectives of the current studies were to characterise and compare the time of onset of lesions, the morphological nature and severity of lesions and the extent of colonisation by S. hyodysenteriae in mice fed the two diets. In the first of two experiments, 50 C3H/HeJ and 50 C3H/HeOuJ mice were fed either TD or CRC and then half of each group was infected with S. hyodysenteriae. Mice (n = 5) from each group were killed and examined on days, 1, 2, 4, 9 or 17 after infection. Each mouse was examined grossly and microscopically and assigned lesion scores based on lesion severity. The second experiment was designed in an identical way to the first, but had slightly smaller group sizes (n=20). Mice (n=4) were killed for necropsy at the same five time points after infection and their caeca were homogenised and examined by quantitative bacteriology with media selective for S. hyodysenteriae. There were no differences in any finding due to mouse strain. Group lesion scores over the entire experimental period were significantly higher in mice fed TD (mean total lesion index = 13) than in mice fed CRC (mean total lesion index = 8.8). Lesions were also temporally distributed in a significantly different manner in that they appeared earlier (day 1) and persisted longer in the TD-fed mice in comparison to CRC-fed mice. Furthermore, lesions of equivalent severity from each treatment group presented identical microscopic features. Finally, quantitative bacteriological results indicated that there was no significant difference in the number of cfu of S. hyodysenteriae isolated from mice fed TD and those fed CRC. These results demonstrate that the characteristic severe lesions associated with S. hyodysenteriae infection in mice can occur 1 day after oral challenge in mice fed Teklad diet 85420. Bacteriological results further indicate that the enhancement of lesion formation in this model is not due to any significant effect of the diet on numbers of spirochaetes in the caeca of infected mice.