Veterinary Pathology

Genotype-dependent differences in Marek's disease (MD) susceptibility were identified using 14-day-old line N and 6₁ (resistant) and 15I and 7₂ (susceptible) inbred chickens infected with HPRS-16 MD virus (MDV). All line 7₂ chickens developed progressive MD. Line 15I had fluctuating MD-specific clinical signs and individuals recovered. A novel histologic scoring system enabled indices to be calculated for lymphocyte infiltration into nonlymphoid organs. All genotypes had increased mean lesion scores (MLSs) and mean total lesion scores after MDV infection. These differed quantitatively and qualitatively between the genotypes. Lines 6₁ and 7₂ had a similar MLS distribution in the cytolytic phase, although scores were greater in line 7₂. At the time lymphomas were visible in line 7₂, histologic lesions in line 6₁ were regressing. AV37⁺ cells were present in similar numbers in all genotypes in the cytolytic phase, suggesting that neoplastically transformed cells were present in all genotypes regardless of MD susceptibility. After the cytolytic phase, AV37⁺ cell numbers increased in lines 7₂ and 15I but decreased in lines 6₁ and N. In the cytolytic and latent phases, in all genotypes, most infiltrating cells were CD4⁺. After this time, line 7₂ and 15I lesions increased in size and most cells were CD4⁺; line 6₁ and N lesions decreased in size and most cells were CD8⁺. In all genotypes, AV37 immunostaining was weak in lesions with many CD8⁺ cells, suggesting that AV37 antigen expression or AV37⁺ cells were controlled by CD8⁺ cells. The rank order, determined by clinical signs and pathology, for MD susceptibility (highest to lowest) was 7₂ > 15I > 6₁ > N.

Quinolones and magnesium deficiency cause similar lesions in joint cartilage of young animals. Chondrocytes cultivated in the presence of quinolones and in Mg-free medium show severe alterations in cytoskeleton and decreased ability to adhere to the culture dish. We investigated whether Mg²⁺ supplementation can prevent quinolone-mediated effects on chondrocytes in vitro. Chondrocytes cultivated in Dulbecco's modified Eagle's medium/HAM's F-12 medium were treated with ciprofloxacin (80 and 160 μg/ml) and enrofloxacin (100 and 150 μg/ml). Mg²⁺ was added at a concentration of 0.0612 mg/ml (MgCl) and 0.0488 mg/ml (MgSO₄) or a triple dose. In addition, cells were cultivated in Mg-free medium and accordingly treated with Mg²⁺ supplementation. After 5 days in culture, the number of adherent cells per milliliter was determined. The number of chondrocytes in quinolone-treated groups decreased to 12-36% that of the control group within the culture period. With Mg²⁺ supplementation, the number of attached cells increased to 40-70% that of control cells. The threefold dose of Mg²⁺ led to better results than did the single dose. Cell proliferation tested by immunohistochemical staining with Ki67 (clone MIB5) decreased from 70% in control groups to 55%, 48%, and 30% in enrofloxacintreated groups in a concentration dependent manner (50, 100, and 150 μg/ml). Addition of Mg²⁺ did not increase the rate of cell proliferation. These results suggest that a great part of quinolone-induced damage is due to magnesium complex formation, as Mg²⁺ supplementation is able to reduce the effects in vitro. However, quinolone effects on cell proliferation seem to be an independent process that is not influenced by magnesium supplementation.

Direct bird-to-human transmission, with the production of severe respiratory disease and human mortality, is unique to the Hong Kong-origin H5N1 highly pathogenic avian influenza (HPAI) virus, which was originally isolated from a disease outbreak in chickens. The pathobiology of the A/chicken/Hong Kong/ 220/97 (H5N1) (HK/220) HPAI virus was investigated in chickens, turkeys, Japanese and Bobwhite quail, guinea fowl, pheasants, and partridges, where it produced 75-100% mortality within 10 days. Depression, mucoid diarrhea, and neurologic dysfunction were common clinical manifestations of disease. Grossly, the most severe and consistent lesions included splenomegaly, pulmonary edema and congestion, and hemorrhages in enteric lymphoid areas, on serosal surfaces, and in skeletal muscle. Histologic lesions were observed in multiple organs and were characterized by exudation, hemorrhage, necrosis, inflammation, or a combination of these features. The lung, heart, brain, spleen, and adrenal glands were the most consistently affected, and viral antigen was most often detected by immunohistochemistry in the parenchyma of these organs. The pathogenesis of infection with the HK/220 HPAI virus in these species was twofold. Early mortality occurring at 1-2 days postinoculation (DPI) corresponded to severe pulmonary edema and congestion and virus localization within the vascular endothelium. Mortality occurring after 2 DPI was related to systemic biochemical imbalance, multiorgan failure, or a combination of these factors. The pathobiologic features were analogous to those experimentally induced with other HPAI viruses in domestic poultry.

Formalin-fixed paraffin-embedded archival tissues of seven adult cats of both sexes that died after being experimentally infected with Yersinia pestis were examined light microscopically to characterize the lesions. The cats were exposed in two groups using two routes of infection: ingestion of Y. pestis-infected rodent or a subcutaneous injection of Y. pestis to simulate a flea bite. Immunohistochemistry was performed on tissues from all organ systems from a representative cat from each group to determine the distribution of Y. pestis bacilli during infection. In all seven cats, bubonic plague lesions were seen. The lesions of pneumonic plague were present in two cats. Septicemic plague was confirmed in all seven cats by bacteriologic culture. Aggregations of bacteria were seen in lymphoid tissue in all cats and in lung tissues from the two cats with pneumonic plague. The most consistent histologic finding was necrosuppurative inflammation in the lymph nodes. Invariably, Y. pestis bacteria were present in large numbers at affected sites. Orally infected cats had more numerous lesions in the lymph nodes of the head and neck regions. These experimentally induced cases of feline plague document that cats are unique among carnivores in exhibiting bubonic, pneumonic, and septicemic plague following exposure to Y. pestis. The lesions of the orally infected cats were consistent with those previously described for naturally occurring Y. pestis infections in cats and corroborate the contention that cats most commonly contract plague by eating Y. pestis-infected rodents and not via flea bite. The histopathology of Y. pestis disease in these cats is comparable to that described for human plague.

Gastric mucosal hypertrophy/nodular hyperplasia occurs as a consequence of Helicobacter infection in mice and humans. The pathogenesis of this hyperplastic response is not understood. To determine the role of host cellular immunity in gastric mucosal hypertrophy/hyperplasia, 6-8-week-old female euthymic BALB/c (n = 30) or NIH athymic nude (n = 40) mice were inoculated with H. heilmannii. Equal numbers of uninoculated mice of each strain served as controls. Mice from each group were euthanatized at 0.5, 6, 12, and 18 months postinoculation (PI). Lymphoplasmacytic gastritis and lymphoid follicle development were less severe in nude mice than in euthymic mice at < 6 months PI. The prevalence of gastritis at 0.5, 6, 12, and 18 months PI was 0%, 17%, 67%, and 88%, respectively, in infected nude mice and 33%, 83%, 71%, and 100%, respectively, in infected BALB/c mice. CD4⁺ T cells in infected nude mice were evident at ≥ 6 months PI but were less numerous than in infected BALB/c mice at comparable time intervals. However, numbers of CD4⁺ T cells increased substantially throughout the experiment in infected BALB/c mice. The prevalence of nodular mucosal hyperplasia at 0.5, 6, 12, and 18 months PI was 0%, 0%, 33%, and 20%, respectively, in infected nude mice and 0%, 33%, 80%, and 80%, respectively, in infected BALB/c mice. Nodular hyperplasia occurred in association with the appearance of chronic lymphoplasmacytic inflammation and CD4⁺ T cells at 12 and 18 months PI in nude mice. H. heilmannii-associated gastritis and mucosal remodeling is reduced in immunodeficient mouse strains lacking normal CD4⁺ T cell numbers as compared with the response in immunocompetent mice. Additionally, CD4 immunocompetence is an integral aspect of the mucosal hypertrophy/nodular hyperplasia in experimental H. heilmannii-associated disease of mice.

Various pathogens gain access to the intestinal wall via specialized cells, the M cells, found among the follicle-associated epithelial cells overlying the domes of the Peyer's patches. The present study was undertaken to examine the uptake of live Mycobacterium avium subsp. paratuberculosis in the distal small intestine of goat kids. Following laparotomy, distal small intestinal segments of five goats were ligated and injected with bacterial suspension. After 1 hour, the intestinal segments were excised and fixed for light and electron microscopic studies. M. a. paratuberculosis organisms were observed by transmission electron microscopy at locations in the intestinal wall, suggesting transcellular transportation through the M cells. The organisms were present both in the cytoplasm of the M cells and in the cytoplasm of intraepithelial leukocytes found in M-cell pockets. Intercellular bacteria between M cells were occasionally seen. Bacteria were not observed in association with the absorptive epithelium. This study indicates that in goat kids, M. a. paratuberculosis enters the intestinal wall primarily through the M cells in the follicle-associated epithelium of the Peyer's patches.

Twenty-eight histologically confirmed cases of porcine leptomeningitis were examined retrospectively, with focus on the pathology of the inner and middle ear, brain, and vestibulocochlear nerve. Tissues were evaluated by histology and immunohistochemistry for Streptococcus suis serotype 2 antigen, and the bacteriologic results were recorded. Exudative otitis interna was diagnosed in 20/28 pigs (71%). The lesions primarily affected the perilymphatic ducts, with consistent involvement of the scala tympani. Perineuritis of the vestibulocochlear nerve was seen in all but four of the ears affected with otitis interna. Immunohistochemically, S. suis serotype 2 antigen was demonstrated in the leptomeningeal, perineural, and labyrinthine exudates in 11 cases. Otitis media was diagnosed in 10/28 pigs (34%), but evidence of extension to the inner ear was not observed. The findings were highly similar to descriptions of meningogenic labyrinthitis in humans and in laboratory animal models. Otitis interna in pigs can also develop via the meningogenic route and is not always, as generally stated, tympanogenic.

Twenty feline vaccine-associated sarcomas were examined by transmission electron microscopy. Tumors contained pleomorphic spindle cells, histiocytoid cells, and giant cells. Most tumors contained myofibroblasts, which had morphologic features similar to those of fibroblasts. These cells were further distinguished by subplasmalemmal dense plaques and thin cytoplasmic actin myofilaments organized as elongated bundles concentrated at irregular intervals forming characteristic dense bodies. Intracellular crystalline particulate material was found in 5 of the 20 tumors. Energy dispersive X-ray spectroscopy was used to identify the crystalline material within one tumor as aluminum-based. One tumor from a feline leukemia virus-infected cat contained budding and immature retroviral particles.

We analyzed the localization of gold particles, expression of immunogenic protein, and histopathologic changes after vaccinating guinea pigs and mice with a DNA vaccine to the Ebola virus glycoprotein administered by cutaneous particle bombardment. Gold particles were deposited in all layers of the epidermis and in the dermis. Those in the epidermis were lost as the damaged layers sloughed, while those in the dermis were phagocytized by macrophages. Glycoprotein was demonstrated by immunohistochemistry primarily in keratinocytes in the epidermis and hair follicle epithelium and less frequently in dermal macrophages, fibroblasts, sebocytes, and cells that appeared to be Langerhans cells. The number of cells that expressed glycoprotein increased between 4 and 8 hours postvaccination, then decreased to near zero by 48 hours. The vaccine sites were histologically divisible into three zones. The central portion, zone 1, contained the most gold particles in the dermis and epidermis and had extensive tissue damage, including full-thickness epidermal necrosis. Zone 2 contained fewer gold particles in the epidermis and dermis and had less extensive necrosis. The majority of cells in which glycoprotein was expressed were in zone 2. Zone 3 contained gold particles only in the epidermis and had necrosis of only a few scattered cells. Regeneration of the epidermis in damaged areas was evident at 24 hours postvaccination and was essentially complete by day 5 in the mice and day 10 in the guinea pigs. Inflammatory changes were characterized by hemorrhage, edema, and infiltrates of neutrophils initially and by infiltrates of lymphocytes and macrophages at later times. In zone 1, inflammation affected both the epidermis and dermis. Peripherally, inflammation was relatively limited to the epidermis. CD3-positive dendritic epidermal cells were demonstrated in the epidermis and superficial hair follicles of unvaccinated immunocompetent mice and beige mice but not of SCID mice. These cells disappeared from all but the most peripheral portions of the vaccine sites of vaccinated mice within 24 hours. They reappeared slowly, failing to reach numbers comparable with unvaccinated mice by 35 days postvaccination. The epidermis of control guinea pigs also had CD3-positive cells, but they did not have dendrites. These findings should contribute to a better understanding of the mechanisms operating in response to DNA vaccination by particle bombardment.

Eight newborn calves showing ataxia were necropsied and examined histologically. Six of seven cerebrospinal fluid samples collected from these animals had neutralizing antibody for Akabane virus (AKV). All examined calves had nonsuppurative encephalomyelitis, localized mainly in the midbrain and spinal cord. Corresponding to the encephalitic lesion, AKV antigen was demonstrated in neuroglial cells in the brain stem and neuronal cells in the ventral horn of the spinal cord. This is the first study to demonstrate AKV antigen by immunohistochemistry in naturally infected newborn calves.

An 8-year-old male Tibetan Terrier showed prolonged astasia, complete paralysis, ticlike signs, and seizure and died 2 months after the onset of symptoms. Histopathologically, there was moderate to severe infiltration of pleomorphic histiocytic mononuclear cells bilaterally in the basiarachnoidal and ventricular areas of the brain. The spinal dura mater, arachnoidal space, and leptomeninges were also affected by infiltrative proliferation of these mononuclear cells. The infiltrating cells had the morphologic characteristics of histiocytes but exhibited moderate pleomorphism and atypia, with abundant mitotic figures. With immunohistochemistry and lectin histochemistry, most of the infiltrating cells were positive for lysozyme and lectin RCA-1 and negative for glial fibrillary acid protein, suggesting that they were of monocytic/histiocytic-origin. Positive proliferating cell nuclear antigen immunostaining demonstrated that most nuclei of the histiocytic cells were in the S phase of the cell cycle, consistent with a proliferating population of cells. Based on these findings, the case was diagnosed as diffuse leptomeningeal malignant histiocytosis.

A 12-year-old Appaloosa gelding was referred to the Texas Veterinary Medical Center with a history of chronic diarrhea and weight loss. At necropsy, numerous oval, craterlike ulcers were observed throughout the small intestine. Histologically, these lesions were composed of a neoplastic proliferation of round cells with intracytoplasmic phosphotungstic acid-hematoxylin-positive granules. The tumor cells stained positively for the CD3 antigen and negatively for a B-cell marker. A diagnosis of large granular lymphoma was based on the morphologic and immunohistochemical characteristics of the neoplasm. The postmortem presentation of this case depicted unusual multifocal, ulcerative lymphomatous lesions throughout the small intestine without involvement of the regional lymph nodes. The histologic and ultrastructural morphology of the neoplastic lymphocytes was similar to that in previously reported cases of abdominal equine large granular lymphomas, but in this case the neoplasm was restricted to the small intestine.

Epulides account for 0-7.8% of tumors in surveys of feline oral neoplasms. A review of the literature revealed no reports of multiple epulides in cats. Multiple, concurrent epulides were diagnosed microscopically in 13 cats. Fibromatous and ossifying epulides were diagnosed in 11 of 13 cats and fibromatous epulides were diagnosed in 2 of 13 cats. Microscopically, these epulides were nonencapsulated, well-vascularized, infiltrative, highly cellular neoplasms that expanded the gingiva and were composed of haphazardly arranged, spindle-shaped to stellate cells amid a dense, collagenous stroma. Osseous foci were a feature in the fibromatous and ossifying epulides. The mitotic rate was low and there was marked hyperplasia of the overlying gingiva with a prominent downgrowth of epithelial cords. These tumors recurred in 8 of 13 cats following surgical excision. While uncommon, multiple epulides in cats have a high incidence of recurrence but do not appear to have metastatic potential.

Actinobacillus equuli is a rare cause of peritonitis in adult horses. Septicemia and peritonitis due to A. equuli were diagnosed at necropsy in an 8-year-old Saddlebred mare. The origin of the infection was not known; however, small necrotic colonic mucosal lesions presumed to have been caused by phenylbutazone treatment may have allowed bacterial invasion. A good response to antimicrobial treatment has been documented in the small numbers of previously reported acute cases of peritonitis. Because it is potentially treatable, it is important for pathologists and clinicians to identify horses with A. equuli peritonitis.

The large intestines of 21 raccoons (Procyon lotor; 11 wild caught, 10 laboratory confined) were examined for the presence of intestinal spirochetes. Light microscopy of sections stained with hematoxylin and eosin and Warthin-Starry stain showed the presence of spiral shaped organisms deep within the lumina of intestinal glands at the ileocolonic junction of 16 raccoons (76% prevalence). All laboratory-confined, grouphoused raccoons harbored the organisms, but only 6/11 (55% prevalence) live-trapped raccoons were positive for these spirochetes. The organisms were free in the glandular lumina, and there were no microscopic lesions. Two types of spirochetes were identified in the colonic glands: a slender spirochete 10-13 μm in length, 0.3 mm in diameter, and possessing long, thin tapered ends and a larger, regularly waved spiral organism (0.5 μm in diameter). The slender spirochete did not resemble any of the known spirochete genera and failed to grow on medium used to propagate oral treponemes and members of the genus Brachyspira.

Sarcomas associated with injection sites are a rare but important problem in cats. Immunohistochemical detection of p53 protein may correlate to mutation of the p53 tumor suppressor gene, a gene known to be important in oncogenesis. The expression of nuclear p53 protein in 40 feline injection site-assocated sarcomas was examined by immunohistochemical staining. In 42.5% (17/40), tumor cell nuclei were stained darkly; in 20% (8/40), tumor cell nuclei were stained palely; and in 37.5% (15/40), tumor cell nuclei were unstained. Immunohistochemical detection of p53 protein in a proportion of injection site-associated sarcomas suggests that mutation of the p53 gene may play a role in the pathogenesis of these tumors.

Cryptosporidium spp. infection was associated with aural-pharyngeal polyps in three iguanas (Iguana iguana). All iguanas were presented for masses protruding from the ear canal, and the disease was characterized by a chronic clinical course. The masses consisted of nests of cystic glands surrounded by abundant fibrous connective tissue and lined by hyperplastic cuboidal to pseudostratified columnar epithelium that was moderately to heavily colonized by cryptosporidial organisms. Electron microscopy revealed that the majority of organisms were trophozoites.

Ten cecal tumors were identified during the postmortem examination of seven horse carcasses at slaughter (one horse had three tumors). The multinodular and hemorrhagic tumors ranged from 1 to 10 cm in diameter and consisted of spindle cells arranged in thin, interconnected trabeculae that were often separated by sinuses filled with mucinous fluid, erythrocytes, and siderophages. Spindle cells of all tumors were immuno-positive for vimentin, neuron-specific enolase, and c-kit protein but lacked reactivity with antibodies to glial fibrillary acidic protein, S100 protein, and desmin. In one tumor, spindle cells diffusely bound antibodies to synaptophysin. Most tumors contained focal reactivity to smooth muscle actin antibodies; one tumor reacted diffusely. Ultrastructurally, tumor cells were connected by desmosome-like structures and exhibited extended cell processes; some contained dense core neurosecretory granules. These equine stromal tumors appeared to share some characteristics with human gastrointestinal stromal tumors.