Journal of Veterinary Diagnostic Investigation

The likelihood that a study will yield statistically significant results depends on the chosen sample size. Surveillance and diagnostic situations that require sample size calculations include certification of disease freedom, estimation of diagnostic accuracy, comparison of diagnostic accuracy, and determining equivalency of test accuracy. Reasons for inadequately sized studies that do not achieve statistical significance include failure to perform sample size calculations, selecting sample size based on convenience, insufficient funding for the study, and inefficient utilization of available funding. Sample sizes are directly dependent on the assumptions used for their calculation. Investigators must first specify the likely values of the parameters that they wish to estimate as their best guess prior to study initiation. They further need to define the desired precision of the estimate and allowable error levels. Type I (alpha) and type II (beta) errors are the errors associated with rejection of the null hypothesis when it is true and the nonrejection of the null hypothesis when it is false (a specific alternative hypothesis is true), respectively. Calculated sample sizes should be increased by the number of animals that are expected to be lost over the course of the study. Free software routines are available to calculate the necessary sample sizes for many surveillance and diagnostic situations. The objectives of the present article are to briefly discuss the statistical theory behind sample size calculations and provide practical tools and instruction for their calculation.

Antemortem biopsy of the rectal mucosa was evaluated as a method for the preclinical diagnosis of chronic wasting disease (CWD) in a herd of ranch-raised Rocky Mountain elk (Cervus elaphus nelsoni) quarantined because of exposure to CWD. Biopsy samples were obtained from 41 elk during the winter of 2005–2006 and from 26 elk from that herd still alive and available for testing during the winter of 2006–2007. Samples were examined for PrP^CWD, the protein marker for CWD infection, by immunohistochemistry. PrP^CWD was detected in follicles of the rectoanal mucosa–associated lymphoid tissue in biopsy samples from 1 elk with clinical signs of chronic wasting disease and 5 clinically normal elk. The diagnosis was confirmed in all 6 animals by postmortem analysis of brain and peripheral lymph nodes. PrP^CWD was also observed in the submucosal plexus and myenteric plexus of the enteric nervous system, and in close association with nonmyelinated mucosal and submucosal nerve fibers. In antemortem rectal biopsy samples from positive animals, immunostaining was consistently observed in approximately 60% of the mucosa-associated lymphoid tissue follicles if 10 or more total follicles per biopsy were present for evaluation. Most antemortem biopsy samples obtained from elk younger than 6.5 years contained at least 10 follicles per rectal mucosal biopsy. These findings support the analysis of antemortem biopsy of the rectal mucosa samples as part of an integrated strategy to manage chronic wasting disease in Rocky Mountain elk.

A multiplex polymerase chain reaction (mPCR) assay was developed for detection and characterization of pathogenic Escherichia coli that cause diarrhea and edema disease in swine. The mPCR assay was designed as a single reaction for detecting 5 different adhesins (K88, K99, 987P, F41, and F18), 3 enterotoxins (LT, STaP, and STb), and the Shiga toxin (Stx2e) associated with porcine pathogenic E. coli. The specificity of the mPCR assay was evaluated by comparison with results from previous analysis of 100 porcine isolates characterized by colony blot hybridization with DNA probes for the 5 adhesins and 4 toxin genes. There was complete agreement between the 2 methods. The mPCR assay for E. coli pathogens isolated from swine was further evaluated by examination of strains containing virulence factors that are known to have different antigenic subtypes or DNA sequence variations. It was found that the mPCR assays targeting genes encoding for K88 and F18 amplified products with the appropriate sizes from strains containing genes for different K88 and F18 antigenic subtypes; mPCR assays targeting the gene encoding for STaP amplified product from only STaP-positive but not STaH-positive isolates; and mPCR assays targeting the gene encoding for the Stx2 amplified products from only Stx2-positive and not Stx1-positive isolates. Similarly, mPCR assays targeting the gene encoding for LTI did not produce the appropriate product from strains containing genes for LTII. The mPCR assays are simple to perform, and they should be useful for diagnosis of porcine colibacillosis, including the genotypic characterization of E. coli isolates from pigs with diarrhea or edema disease.

Accurate diagnosis of Mycobacterium bovis infection (bovine tuberculosis [bTB]) in live animals is notoriously problematic. The aim of the present study was to evaluate the diagnostic potential of 2 new serologic tests (multiantigen print immunoassay [MAPIA] and lateral flow immunoassay rapid test [RT]) in comparison with mycobacterial culture of tracheal washes for determining M. bovis infection status in a free-ranging population of wild meerkats (Suricata suricatta). During a longitudinal study lasting 2.5 years, 240 individually identifiable meerkats were each sampled up to 8 times under anesthesia every 3 months. Diagnostic accuracy was determined through Bayesian and maximum likelihood estimations of sensitivity, specificity, and likelihood ratios for each diagnostic test when used independently and in parallel to classify the disease status of individual meerkats in the absence of a gold standard. Culture of tracheal washes was highly specific (0.99; 95% confidence interval [CI] = 0.77, 1.00) but of low sensitivity (0.36; 95% CI = 0.24, 0.50) for diagnosing M. bovis-infected individuals. The longitudinal nature of the study with repeated sampling of the same individual animals served simultaneously to improve chances of detecting infection and increase confidence in a negative result in individual animals repeatedly testing negative. Although MAPIA and RT were individually of limited diagnostic use, interpreting the results of these 2 tests in parallel produced estimates of sensitivity (0.83; 95% CI = 0.67, 0.93) and specificity (0.73; 95% CI = 0.62, 0.82) high enough to usefully inform decision making when determining exposure to bTB in wild meerkats and potentially other species in which bTB poses a diagnostic challenge.

The objective of the present study was to determine whether offspring of dams infected with Mycobacterium avium subsp. paratuberculosis (Map) have an increased risk for Map infection. Antemortem and postmortem disease surveillance data were used to identify positive and test-negative ruminants born at the Zoological Society of San Diego (ZSSD) between 1991 and 2007 and to estimate cumulative lifetime incidence. A matched case-control study, nested within the population, was conducted and conditional logistic regression analyses were used to quantify the association between infection status of offspring and their dams. Cases (infected ruminants, n = 47) were matched to controls (test-negative ruminants, n = 152) by species, birth date, birth enclosure, and follow-up time to control for confounding factors. The overall cumulative lifetime incidence of infection was estimated at 2.2%, but it decreased over time and varied by species. There was a significant association between infection status of offspring and their dams (odds ratio [OR] = 6.8, P < 0.01), which is consistent with studies in domestic livestock species. The association was stronger for animals whose dam was diagnosed within 2 years of their birth (OR = 9.0, P < 0.01) than for animals whose dam was diagnosed more than 2 years after their birth (OR = 6.0, P < 0.01) compared to animals with test-negative dams. For positive animals born to a positive dam, 85.3% of the Map infections were attributable to having a positive dam. For the entire population of ZSSD ruminants, 36.8% of the cases were attributable to having a positive dam. Findings will help guide future management of Map infection in zoo ruminant populations.

A total of 745 slaughtered pigs were examined during routine meat inspection for suspected tuberculous lesions. Specimens from suspected lesions were collected for conventional mycobacteriologic examinations. Suspected mycobacterial colonies were subjected to molecular typing based on the Mycobacterium species-specific intergenic spacer (IGS) target. The study resulted in detection of suspected lesions in 110 (14.8%) carcasses, from which only 67 specimens produced suspected mycobacterial colonies. Conventional examination was only able to identify 56 isolates as Mycobacterium species, which was confirmed by polymerase chain reaction amplification of the IGS target. Interestingly, out of these, 18 and 12 isolates were Mycobacterium tuberculosis and Mycobacterium bovis, respectively. Sequence analysis of IGS resolved the identities of 10 of the 11 conventionally unidentified isolates as being 4 different nontuberculous Mycobacterium species. The last isolate was proposed as a non-Mycobacterium species and was confirmed by its identification as Rhodococcus equi based on the 16S ribosomal DNA sequence analysis. The study described the isolation of Mycobacterium tuberculosis from pigs and revealed high burden of infection with both tuberculous and nontuberculous mycobacterial species among pigs in Egypt. In addition, the study showed the usefulness of IGS sequence analysis as a reliable molecular tool that would be useful for further epidemiologic and public health studies.

Clinical and Laboratory Standards Institute interpretive breakpoints for in vitro susceptibility tests that predict mecA-mediated oxacillin resistance in Staphylococcus pseudintermedius isolates from animals have been changed twice in the past decade. Moreover, there are no counterpart recommendations for human isolates of S. pseudintermedius. Individual medical and veterinary laboratories variably use interpretive breakpoints identical to those recommended for use with Staphylococcus aureus or identical to those recommended for use with coagulase-negative staphylococci. The purpose of the current study was to examine correlations between oxacillin disk diffusion, oxacillin gradient diffusion, oxacillin microbroth dilution, and cefoxitin disk diffusion tests used to predict mecA-mediated resistance in S. pseudintermedius and to retrospectively estimate, from disk diffusion zone diameter measurements, the prevalence and rate of increase of oxacillin resistance among canine S. pseudintermedius isolates submitted to a veterinary teaching hospital laboratory. Oxacillin disk diffusion zone diameters of ≤17 mm and oxacillin minimum inhibitory concentrations of ≥0.5 μg/ml were highly correlated with detection of mecA in canine S. pseudintermedius isolates by polymerase chain reaction. MecA-mediated resistance among S. pseudintermedius isolates from dogs increased from less than 5% in 2001 to near 30% in 2007. More than 90% of the methicillin-resistant S. pseudintermedius isolates in 2006 and 2007 were also resistant to representatives of ≥4 additional antimicrobial drug classes. Cefoxitin disk diffusion with the resistance breakpoint set at ≤24 mm significantly underestimated the presence of mecA in S. pseudintermedius.

Although cytauxzoonosis has historically been nearly 100% fatal in domestic cats, increasing number of reports of infected cats that demonstrate less-severe disease suggest the existence of different strains of Cytauxzoon felis. To test this hypothesis, the genetic variability of C. felis was examined in blood samples from naturally infected domestic cats from Arkansas and Georgia by using the first and second ribosomal internal transcribed spacer regions (ITS1, ITS2) as markers to assess genotypic variability. In addition, the clinical outcome of infection (survival vs. fatal disease) was analyzed. Within the C. felis ITS1 region, there were a total of 8 single nucleotide polymorphisms (SNP) and a single nucleotide insertion. Within the ITS2 region, there were a total of 4 SNPs and a single 40 base pair insertion. When taken together, the ITS1 and ITS2 sequence data defined a total of 11 different sequences and 3 unique genotypes. One unique ITS1-ITS2 genotype was detected in samples submitted exclusively from Arkansas, and a second unique genotype was submitted exclusively from Georgia. There was a significant association between infection with C. felis that contained particular ITS genotypes and survival of the infected domestic cat. The identification of unique C. felis genotypes obtained from different geographic areas and the association of particular ITS genotypes with the outcome of infection suggest the existence of parasite strains that may vary in pathogenicity to the domestic cat and offer an explanation for the survival of some infected cats in more recent case studies.

Scrapie, a transmissible spongiform encephalopathy (TSE), is a naturally occurring fatal neurodegenerative disease of sheep and goats. The current study documents incubation periods, pathologic findings, and distribution of abnormal prion proteins (PrP^Sc) by immunohistochemistry and Western blot in tissues of genetically susceptible and resistant neonatal lambs inoculated with pooled brain homogenates from 13 U.S. origin scrapie-affected ewes. Nine Suffolk lambs with genotypes AA/RR/QQ (n = 5) and AA/RR/QR (n = 4) at codons 136, 154, and 171, respectively) were orally inoculated, within 12 hr of birth, with 1 ml of a 10% (w/v) brain homogenate prepared from scrapie-affected sheep brains. Inoculated animals were euthanized when advanced clinical signs of scrapie were observed. All QQ sheep developed clinical signs of scrapie, with a mean survival time of 24 months. Spongiform lesions in the brains and PrP^Sc deposits in the central nervous system and lymphoid tissues were present in these sheep. None of the QR sheep succumbed to the disease. A previous study that used a larger volume (30 ml of 10% brain suspension) of the same inoculum in 4-month-old Suffolk lambs of susceptible genotype documented longer survival periods (average 32 months), and only 5 of 9 inoculated sheep developed scrapie. Findings of this study suggest that orally exposed neonatal lambs of a susceptible (QQ) genotype exhibit a higher attack rate and shorter incubation period than older (4-month-old) lambs exposed to a larger dose (30×) of the same inoculum.

During the period from January 2002 to March 2007, infections by melanized fungi were identified with greater frequency in aquarium-maintained leafy seadragons (Phycodurus eques)and weedy seadragons (Phyllopteryx taeniolatus), pivotal species to the educational and environmental concerns of the aquarium industry and conservation groups. The objective of this study was to characterize the pathology and identify fungi associated with phaeohyphomycotic lesions in these species. Samples from 14 weedy and 6 leafy seadragons were received from 2 institutions and included fresh, frozen, and formalin-fixed tissues from necropsy and biopsy specimens. Fresh and frozen tissues were cultured for fungi on Sabouraud dextrose agar only or both Sabouraud dextrose agar and inhibitory mold agar with gentamicin and chloramphenicol at 30°C. Isolates were processed for morphologic identification and molecular sequence analysis of the internal transcribed spacer region and D1/D2 domains of the large subunit ribosomal RNA gene. Lesions were extensive and consisted of parenchymal and vascular necrosis with fungal invasion of gill (11/20), kidney (14/20), and other coelomic viscera with or without cutaneous ulceration (13/20). Exophiala sp. isolates were obtained from 4 weedy and 3 leafy seadragons and were identified to species level in 6 of 7 instances, namely Exophiala angulospora (1) and a novel species of Exophiala (5), based on nucleotide sequence comparisons and phylogenetic analyses. Disseminated phaeohyphomycosis represents an important pathologic condition of both weedy and leafy seadragons for which 2 species of Exophiala,1a novel species, have been isolated.

The objective of the current study was to evaluate whether blood plasma progesterone (P₄) measurements with a time-resolved fluorescent antibody test (TR-FAT) kit designed for humans was applicable for goats. The first experiment was designed to verify whether the concentrations of P₄ measured by TR-FAT can be used to monitor the estrous and ovarian activity in goats (n = 14). Blood samples (322) were collected, and the ovaries were scanned using ultrasonography. The second experiment was carried out on 4 goats (60 samples) and designed to compare the TR-FAT with radioimmunoassay (RIA). The time interval between the lowest concentrations of P₄ assayed by TR-FAT was 21 ± 0.3 days and did not differ significantly from the length of the interestrous interval. The highest concentrations of P₄ were confirmed by detection of corpus luteum. During estrus, the mean concentration did not differ significantly between both methods. Significant differences were present during the luteal phases; however, the profiles of P₄ assayed by both methods followed a similar pattern. Regression analysis showed a correlation between the 2 methods (r = 0.98; r 2 = 0.96; P < 0.0001). The Bland-Altman plot showed that all averages were within the 95% limits of agreement; however, the differences between both methods tend to be greater as the average increases. The results demonstrated that the TR-FAT method can be applied to monitor estrous cycles in goats through measurements of plasma P₄ concentrations. Moreover, not only does the TR-FAT meet the requirements for safety, but it is also a method of high throughput, rapidity, and simplicity.

The IDEXX Swine influenza virus H1N1 and H3N2 enzyme-linked immunosorbent assays (ELISAs) are used worldwide, but their capacity to detect antibodies to European Swine influenza viruses (SIVs) has not been documented. A total of 313 well-defined sera from SIV seronegative pigs and pigs experimentally infected with European SIVs were used to compare the performance of both ELISAs and the hemagglutination inhibition (HI) test. The ELISAs largely failed to detect pigs that had been infected with H1N1 (0/42 positive in H1N1 ELISA) or H3N2 only (9/18 positive in H3N2 ELISA; group 1). Higher ELISA detection rates were found after consecutive infection of pigs with either H1N1 or H3N2 and 1 other subtype (7/40 and 11/22 positive in H1N1 and H3N2 ELISA, respectively; group 2). Of 39 pigs that had been vaccinated twice with 1 of 4 commercial SIV vaccines (group 3), 25 tested positive in the H1N1 and 4 in the H3N2 ELISA. Pigs that had received a single vaccination after a prior infection with H1N1 and/or H3N2 (group 4) were more frequently positive than group 1 or 3 pigs (23/24 and 15/24 positive in H1N1 and H3N2 ELISA, respectively). Both the H1N1 and H3N2 ELISA showed a low sensitivity (39% and 35%, respectively) relative to the HI test. Because pigs in the field are frequently infected and/or vaccinated with multiple SIV subtypes and variants, they are more likely to test positive in the ELISAs. However, the interpretation of ELISA results will be difficult, and HI remains the method of choice.

Recently, screening tests for monitoring the prevalence of transmissible spongiform encephalopathies specifically in sheep and goats became available. Although most countries require comprehensive test validation prior to approval, little is known about their performance under normal operating conditions. Switzerland was one of the first countries to implement 2 of these tests, an enzymelinked immunosorbent assay (ELISA) and a Western blot, in a 1-year active surveillance program. Slaughtered animals (n = 32,777) were analyzed in either of the 2 tests with immunohistochemistry for confirmation of initial reactive results, and fallen stock samples (n = 3,193) were subjected to both screening tests and immunohistochemistry in parallel. Initial reactive and false-positive rates were recorded over time. Both tests revealed an excellent diagnostic specificity (> 99.5%). However, initial reactive rates were elevated at the beginning of the program but dropped to levels below 1% with routine and enhanced staff training. Only those in the ELISA increased again in the second half of the program and correlated with the degree of tissue autolysis in the fallen stock samples. It is noteworthy that the Western blot missed 1 of the 3 atypical scrapie cases in the fallen stock, indicating potential differences in the diagnostic sensitivities between the 2 screening tests. However, an estimation of the diagnostic sensitivity for both tests on field samples remained difficult due to the low disease prevalence. Taken together, these results highlight the importance of staff training, sample quality, and interlaboratory comparison trials when such screening tests are implemented in the field.

Cases of tuberculosis due to Mycobacterium avium subsp. avium in 52 adult red deer (Cervus elaphus) from a farm were studied using different diagnostic techniques. Immunological probes consisted of the comparative cervical tuberculin (CCT) skin test, the interferon-γ (IFN-γ) assay, and 2 enzyme-linked immunosorbent assays (ELISAs) employing either avian purified protein derivatives or protoplasmatic antigen (PPA-3) as antigens. Three of the animals were euthanized due to severe weakness, loss of weight, and emaciation. Macroscopically, the 3 animals showed tuberculous lesions located mainly in lymph nodes of the digestive system and small intestine but also in other organs and lymph nodes. Polymerase chain reaction was carried out on samples from the 3 deer using primers to detect IS901, IS900, and IS6110, specific for Mycobacterium avium subsp. avium, Mycobacterium avium subsp. paratuberculosis, and Mycobacterium tuberculosis complex, including Mycobacterium bovis, respectively. The last 2 agents cause pathologies very similar to avian tuberculosis in deer. The 3 deer were strongly positive by both ELISAs, slightly positive by the IFN-γ test, and 1 of 2 was positive by the CCT test. As with domestic ruminants, ELISA could detect deer in an advanced stage of infection, with large numbers of mycobacteria.

Neospora caninum is a parasite responsible for abortion in cows and neuromuscular disease in dogs. Serology is the most widely used technique to evaluate the prevalenceof N. caninum in different host populations. A sandwich enzyme-linked immunosorbent assay (ELISA) based on the use of an anti-SRS2 monoclonal antibody was evaluated against the indirect fluorescent antibody test for 100 canine sera and against a well-characterized ELISA for 102 bovine sera. In cattle sera, the relative sensitivity and relative specificity were 100%. In dog sera, the relative specificity and relative sensitivity were 94% and 86%, respectively. The kappa value was 1 for bovine sera and 0.77 for canine sera. The seroprevalence was 3.9% in bovine sera and 21–23% in canine sera. The SRS2 sandwich ELISA was considered a valuable tool in both species.

Measurements of glycated proteins such as serum fructosamine, glycated hemoglobin, and glycated albumin (GA) are increasingly used to complement serum glucose concentration for better management of diabetes mellitus. For example, the degree of glycemic control in diabetic cats can be determined by evaluating fructosamine concentration. Unfortunately, fructosamine tests are currently not performed in Japan, and as such, the measurement of GA may serve as a replacement test. The objectives of the current study were 2-fold. First, serum GA and fructosamine level were evaluated for positive correlation in cats as a preliminary gauge on whether serum GA use is applicable. Second, a GA percentage reference range was determined from healthy control cats for possible future diagnostic use. A positive correlation was determined for fructosamine and GA in both normal and diabetic cats. Moreover, the serum GA percentage reference interval based on control cats was determined to be 7.5–13.9% (95% nonparametric interfractile interval). Interestingly, no significant difference in serum GA percentages was observed between samples from diabetic cats with excellent glycemic control and control cats. However, good, fair, and poor glycemic control diabetic cats resulted in a significant increase in serum GA percentages in comparison to control cats. Therefore, these results indicate that serum GA may be a useful glycemic control indicator that could substitute for fructosamine to monitor glycemic control in diabetic cats.

Concurrent tracheal hypoplasia and discrete subaortic stenosis are described in a 12-week-old Rottweiler puppy that presumably died of pulmonary edema. A brief literature review and comparison to previously published cases of tracheal hypoplasia in other breeds is presented along with a description of a subaortic septal ridge and comparison to the analogous condition in humans.

A 6-year-old, 30-kg, female German Shepherd Dog, living in a leishmaniasis enzootic area, was presented with a severe rear limb motor disorder and a medical history of acute onset of fever. Routine hematology indicated neutrophilia. Spinal survey radiographs were consistent with osteomyelitis and discospondylitis. Because of the poor clinical prognosis and the painful nature of the lesions, the dog was euthanized at the owners' request. At necropsy, T13-L1 vertebrae had large areas of necrosis within the vertebral bodies. Histopathological findings were consistent with chronic, severe, fungal osteomyelitis and discospondylitis. Polymerase chain reaction identified Scedosporium apiospermum, a eutrophic filamentous fungus now recognized as an emerging agent of severe infections in immunosuppressed human patients.

A case of atypical mucosal xanthomas in a 14-year-old Shi-Tzu dog is reported. Grossly, 1–6-mm granular plaques or slightly elevated papillary nodules were found in the oral cavity (dorsal and ventral sides of the tongue, inner upper lip) and in the upper digestive tract (esophagus, stomach). Microscopically, subepithelial aggregates of large foamy cells were found in strong association with papillary epithelial hyperplasia. By immunohistochemistry, the majority of these cells tested positive for lysozyme and ACM1. The cells had cytoplasmic lipid content that stained positively with oil red O. These findings confirmed a monocyte/macrophage lineage. On the basis of macroscopic observations, microscopic changes, and the absence of a clinical metabolic defect, the condition in this dog appears similar to that of humans with oral verruciform xanthoma. The pathogenesis of these xanthomas in the dog remains obscure, although this condition appears to affect people and dogs with an Asian origin.

A malignant schwannoma of the right acoustic nerve of a dog is described. The neoplasm was found during necropsy of a 12-year-old, spayed, female Labrador Retriever that experienced a sudden onset of severe pain in the neck and upon opening the mouth. Concurrent mild hind limb ataxia also was present. The tumor had infiltrative growth, and the spindle-shaped neoplastic cells were arranged in sheets and concentric whorls. Immunohistochemical staining of the neoplastic spindle cells was positive for S-100 antigen, neuron-specific enolase, and glial fibrillary acidic protein.

Cryptococcus gattii was isolated from a 1.5-year-old dog with systemic cryptococcosis in Oregon. The dog had no link to Vancouver Island or British Columbia, Canada. Samples from a nasal swab and from a granulomatous mass within the cranial cavity were pooled for culture. Colonies on Sabouraud dextrose agar were mucoid and exhibited bimorphic morphology, melanin-pigmented and unpigmented. Pigmented colonies were encapsulated budding spherical yeast, whereas unpigmented colonies were of unencapsulated ovoid budding yeast. In addition to defective melanin production, the unpigmented colony type exhibited defective mating. Genetic analysis by high-resolution multilocus sequence typing revealed that the 2 isolates are genetically identical at 8 unlinked loci tested and that the 2 isolates are both the VGIIa Vancouver Island major genotype. Findings are consistent with expansion of the Vancouver Island outbreak onto the mainland Pacific Northwest region of the United States.

Bovine viral diarrhea virus (BVDV) is an important pathogen that primarily infects ruminants, leading to several clinical problems including abortion. BVDV-specific antibodies were reported in a wide range of hosts within domestic and wildlife animal populations, and serological studies also indicated BVDV infection in buffaloes. The purpose of this study was to analyze the presence of BVDV in 2 water buffalo (Bubalus bubalis) herds with a history of abortion. Virus isolation from aborted fetuses and from maternal buffy coat and the molecular characterization of the isolates confirmed the presence of BVDV in these animals. The sequence analysis based on the 5′ UTR and N^pro coding regions of the Pestivirus genome revealed that the isolates belong to subgenotype 1b of BVDV. The findings of this study also suggest a possible role of BVDV in causing congenital infection in water buffalo. Its presence in fetal tissues as well as in maternal blood raises questions about the possible development of clinical disease or its influence in abortions in water buffalo.

A case of malignant melanoma in a 7-year-old, intact, black, male Huacaya alpaca with a history of a chronic, nonhealing wound involving the left external nostril, weight loss, and inappetence is described. Malignant melanoma was diagnosed by histology of punch biopsy specimens from a mass on the maxilla associated with the nonhealing wound and from a mass in the submandibular region. The alpaca was humanely euthanized 10 days after the diagnosis on the basis of the poor prognosis and rapid clinical deterioration. At postmortem examination, the alpaca had an ulcerated, multilobulated, black pigmented mass (8.0 cm × 6.0 cm × 4.0 cm) that infiltrated the left rostral maxilla extending into the marrow space and into the left nasal cavity. Numerous, discrete, coalescing masses were present in the subcutaneous tissue of the submandibular area, peritracheal connective tissue, pericardium, and diaphragmatic parietal pleura and were disseminated throughout the pulmonary parenchyma. The masses were diffusely black on cut surface and exuded black pigment. Histologically, all masses were composed of spindloid to polygonal cells with indistinct cell borders and moderate amounts of cytoplasm that contained abundant fine, black granules (melanin), confirming metastasis of a primary mucocutaneous melanoma. To the authors' knowledge, this is the first report of a malignant melanoma involving bone in a New World camelid.

Bovine viral diarrhea virus (BVDV) is an emerging infectious pathogen of concern to the alpaca industry. A 4-month-old, intact, male alpaca cria was diagnosed as persistently infected with BVDV on the basis of repeated positive antemortem polymerase chain reaction (PCR) and virus isolation (VI) assays and negative serologic titers to BVDV. Immunohistochemistry, real-time reverse transcription PCR, and VI performed on tissues collected at necropsy demonstrated disseminated BVDV-1b infection. Virus was detected in multiple tissues, including parotid salivary gland, testes, prostate, kidneys, skin, and gastrointestinal tract. Demonstration of BVDV in previously unreported tissues suggests additional potential routes of BVDV transmission in alpacas.

A mixture of ketamine, xylazine, and butorphanol was inadvertently injected into the right carotid artery of a 1-year-old alpaca. Injection was followed by a brief period of recumbency and seizure activity. The alpaca recovered, but was euthanatized 72 hr later because of development of progressive neurologic deficits. Pathologic findings were confined to the right cerebrum, meninges, thalamus, and hippocampus. Cerebrocortical edema with astrocytic reaction, perivascular hemorrhage and neutrophilic infiltration, and fibrinoid necrosis of vasculature within the meninges and thalamus were the most prominent lesions. Neuronal necrosis was mild. Astrocytic reaction within the right cerebral cortex was confirmed with immunohistochemistry for glial fibrillary acidic protein.

A 1-cm-diameter nodule was identified in the left inguinal mammary gland of a 9-year-old male maned wolf (Chrysocyon brachyurus). The mass was surgically excised and examined histologically. Microscopically, the neoplasm consisted of papillary proliferations of epithelial cells on well-defined fibrovascular stalks. A myoepithelial layer was located between the single layer of epithelial cells and the fibrovascular stalk. This histologic appearance was compatible with a diagnosis of simple ductal mammary papilloma. Immunohistochemical staining was positive for p63, cytokeratins AE1/AE3, and estrogen receptors. The clinical and histologic observations in the present case indicate that male maned wolves may develop mammary tumors that are similar to those observed in domestic dogs and humans.

A 4-year-old female Richardson's ground squirrel (Spermophilus richardsonii) presented with multicentric nodules arising from the skin of the middle of the tail and lumbosacral regions. Histologically, the nodules were composed of a proliferation of spindloid to pleomorphic cells that sometimes formed sheets and fascicular to storiform patterns. Diffuse infiltration of eosinophils was also noted. The results of immunohistochemistry indicated positive labeling for vimentin, mast cell tryptase, c-kit, and Ki-67. Toluidine blue stain revealed fine, metachromatic, cytoplasmic granules. The histologic diagnosis was mast cell tumor. The neoplasm recurred and metastasized to the right lumbar lymph node 1 month later.

A sarcomatoid carcinoma was diagnosed in the lung of a 10-year-old captive Egyptian fruit bat (Rousettus aegyptiacus). Both carcinomatous and sarcomatous cytologic phenotypes were identified histologically. Cells of both types stained positive for pancytokeratin and S-100. Stromal cells stained positively for muscle actin. No staining for vimentin was noted in either neoplastic or normal internal control tissues. To the authors' knowledge, this is the first report of a pulmonary sarcomatoid carcinoma in a bat, and only the third report of sarcomatoid carcinoma outside the human literature.

Helcococcus ovis is a newly established species in the genus Helcococcus. The clinical significance of this organism in sheep has not been reported. In the current report, isolation of H. ovis from a 6-month-old mixed-breed ewe lamb that died of respiratory disease is described. Pathologic examination revealed severe, focally extensive, chronic necrotizing pleuritis with intralesional coccobacilli and mild, multifocal, subacute mucopurulent bronchopneumonia, indicating a bacterial etiology. A Gram-positive bacterium was isolated in heavy growth from the lung tissue. DNA sequence analysis on the 16S rDNA gene demonstrated that the isolate was H. ovis. To the authors' knowledge, this is the first report of isolation of H. ovis associated with infection in sheep with pleuritis and bronchopneumonia.

Postmortem examination of a free-range white-tailed deer (Odocoileus virginianus) revealed severe emaciation, bilateral firm proliferation of the metatarsal diaphyses, and a large intrathoracic mass associated with the accessory lung lobe. Smaller masses were evident in the abomasum, duodenum, omentum, and the capsular surface of the liver. Microscopically, the masses were similar and were diagnosed as eosinophilic granulomas with intralesional fungal hyphae characteristic of Zygomycetes spp. Fungal hyphae were identified as Conidiobolus incongruus by 18S ribosomal RNA sequencing on fresh lung tissue. Furthermore, the proliferative lesions of the metatarsal bones along with the intrathoracic mass were compatible with hypertrophic osteopathy.

A 10-year-old, female, pregnant alpaca (Vicugna pacos) presented with a discolored, swollen right eye. The eye did not respond to treatment and was enucleated. Sectioning of the fixed eye revealed an ocular mass replacing the aqueous and vitreous humors, with multiple regions of bone formation. Histopathological, immunohistochemical, and transmission electron microscopy confirmed the tumor was an intraocular melanoma with bone formation. Osteogenic melanoma is a rare variant of melanoma not reported in the eye before.