Veterinary Pathology

The common marmoset (Callithrix jacchus) is a small New World primate native to Brazil that has been used extensively in biomedical research. A retrospective analysis of archived hematoxylin and eosinstained tissue sections and clinical records was conducted at the New England Primate Research Center on 86 marmosets more than 1 year of age that were euthanized during the past decade because of morbidity and failure to thrive. Approximately 17% (15 of 86) were found to have amyloid deposits in one or more organs, including the liver, adrenal glands, kidneys, and intestine. This material was shown by amino acid sequence analysis to be composed of serum amyloid A (SAA)-related protein. This type of amyloidosis, designated AA or “secondary,” is associated typically with an inflammatory process that induces elevated levels of the SAA amyloidogenic precursor molecule. Notably, there were no significant pathologic differences or other distinguishing features in animals with amyloid versus those without; furthermore, on the basis of the limited number of serum specimens available for analysis, the SAA concentrations in the two groups were comparable, thus suggesting the possible inheritable nature of the disorder. In this respect, the common marmoset provides a unique experimental model for study of the pathogenesis and treatment of AA and other forms of systemic amyloidosis.

Posterior paresis/paralysis in farmed mink is responsible for significant morbidity and mortality, with individual farms reporting the loss of as many as 700 animals each year. Although this disease has been recognized by North American mink farmers for approximately 40 years, there are few published reports focusing on this entity. The objective of this study was to investigate the etiology and pathogenesis of the disease. Complete necropsy examinations were done on 40 clinically affected mink, ranging from 7 to 10 weeks of age, and on three normal animals in the same age range from two mink farms. Thirty-two of the 40 clinically affected animals had an isolated vertebral lesion characterized by bone lysis and proliferation that usually was centered on an intervertebral disk space in the midthoracic area. An inflammatory reaction, composed primarily of neutrophils, was present within the vertebral sections in 25 of the 40 affected animals (62.5%), and the presence of gram-positive cocci was confirmed in 8 of 10 animals (80%) in which bacterial organisms were observed histologically. Bacterial cultures from 15 affected animals yielded Streptococcus sp. from the intervertebral disk space in 13 of 15 (86.7%) animals and from heart blood in 6 of 8 (75%). A farm visit revealed no history or evidence of traumatic wounds as a source of infection in these animals, and the diet appeared to be adequate for skeletal development. We conclude that posterior paresis/paralysis in farmed mink is associated with bacterial diskospondylitis, likely occurring secondary to bacteremia/septicemia.

Immunohistochemistry, the standard method for diagnosing amyloid A (AA) amyloidosis, is limited in animals because it requires a large array of animal-specific anti-AA antibodies, not commercially available. The Shtrasburg method (SH method) is a highly specific and sensitive technique, helping in the diagnosis and determination of AA amyloidosis in humans. The aim of this study is to determine whether the SH method is applicable in the diagnosis of AA amyloidosis in a variety of animals. Tissue samples were obtained from animals suffering from spontaneous or experimentally induced AA amyloidosis (mice, hamsters, guinea pigs, cheetahs, cats, cows, ducks, a dog, a goose, a chicken, and a turaco). Detection of the amyloid and quantitative evaluation were performed using Congo red staining, and specific AA typing was performed by the potassium permanganate technique. The studied tissues were subjected to the SH method, which confirmed the AA nature of the amyloid deposit, by displaying in polyacrylamide gel electrophoresis protein bands consistent with the molecular weight of the species-specific AA, in all the animals examined, except mice, hamsters, and guinea pigs. N-terminal analysis of these bands corroborated their AA origin. We conclude that the SH method may be used as an ancillary simple tool for the diagnosis of AA amyloidosis in a large number of domestic and wild animals. Moreover, our findings further increase the feasibility of applying this method in humans.

Ten dogs with neuroendocrine carcinoma of the liver were selected for inclusion in the study. Clinical signs were anorexia (7), vomiting (5), polydipsia/polyuria (3), icterus (2), lethargy (2), weight loss (2), paresis (1), ataxia (1), weakness (1), collapse (1), and urinary tract infection (1). Hematologic and biochemical abnormalities included anemia (2/8), leukocytosis (4/8), high liver enzyme activity (serum alkaline phosphatase, 7/9; alanine transaminase, 7/9; aspartate transaminase, 8/9), and high total bilirubin (6/9). Grossly, the tumors were diffuse, involving all liver lobes in six dogs, and two dogs had various-sized nodules in addition to diffuse involvement. Histologically, there were eight tumors with solid or trabecular pattern (group A), one tumor with cords or rows of neoplastic cells (group B), and one tumor with multiple rosette-like structures (group C). immunohistochemical studies revealed that all 10 neoplasms were positive for at least one of the endocrine markers used: neuron-specific enolase (NSE; 8/10), synaptophysin (5/10), and chromogranin-A (3/10). A panel of NSE, chromagranin-A, and synaptophysin detected 100% of the tumors in our series. Electron microscopy confirmed the diagnosis by the presence of intracytoplasmic neurosecretory granules in the two examined cases. our results show that neuroendocrine markers commonly used in humans can be used for the diagnosis of hepatic neuroendocrine carcinoma in dogs, preferably a panel of synaptophysin, chromagranin-A, and NSE because chromogranin-A alone is not as useful in dogs as in humans.

Phenobarbital (PB) therapy is frequently associated with elevated serum alanine aminotransferase (ALT) and alkaline phosphatase (AP) activities in dogs without clinical signs of liver disease. The goal of this study was to determine if increased serum ALT and AP activities in clinically healthy PB-treated epileptic dogs are due to hepatic enzyme induction or to subclinical liver injury. Liver biopsies were obtained from 12 PB-treated dogs without clinical signs of liver disease but with elevated serum ALT and/or AP activities or both. Liver biopsies were obtained from eight healthy control dogs not receiving PB. Biopsies were evaluated histopathologically (all dogs) and liver homogenates were assayed for ALT (all dogs) and AP (six treated dogs, all controls) activities. As a positive control, liver cytochrome P4502B, an enzyme known to be induced by PB, was measured by benzyloxyresorufin-O-dealkylase activity and immunoblotting (five treated dogs, all controls). Serum AP isoenzyme analyses were performed. Results showed that ALT and AP activities in liver homogenates were not increased in treated dogs compared with controls, whereas the positive control for induction, CYP2B, was dramatically increased in treated dogs. Histopathological examination of liver biopsies revealed more severe and frequent abnormalities in treated dogs compared to controls, but similar types of abnormalities were found in both groups. Serum AP isoenzyme analyses in treated dogs demonstrated increased corticosteroid-induced and liver isoenzyme activities compared to controls. Results do not support induction of ALT or AP in the liver as the cause of elevated serum activities of these enzymes due to PB.

Aspiration of lytic bone lesions is an excellent diagnostic test in the initial evaluation of primary bone neoplasia. However, cytologically, it can be difficult to differentiate osteosarcoma (OSA) from other bone neoplasms, including fibrosarcoma, chondrosarcoma, synovial cell sarcoma, and plasma cell myeloma. The purpose of this study is to determine the sensitivity and specificity of alkaline phosphatase (ALP) staining to differentiate OSA from other tumors that express vimentin by immunocytochemistry or immunohistochemistry. ALP is a hydrolytic enzyme present in multiple tissues including liver, kidney, intestine, placenta, and bone. Hypothetically, neoplasms actively producing bone should be specifically positive for ALP staining. Unstained, cytologic specimens were incubated for 8-10 minutes with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt-phosphatase substrate. A positive reaction stains the membrane of the cells gray to black. Samples were counterstained with a Romanowsky's stain to determine whether the sample was of representative cellularity. A total of 61 vimentin-positive neoplasms have been evaluated and confirmed histopathologically. Tumors that expressed vimentin and were positive for ALP included 33 OSAs, one multilobular tumor of bone, one amelanotic melanoma, and one chondrosarcoma. Tumors that expressed vimentin and were negative for ALP included chondrosarcomas (three of four), multiple fibrosarcomas, and multiple synovial cell sarcomas. The sensitivity is 100%, and the specificity is 89%. In conclusion, ALP appears to be a highly sensitive and fairly specific marker in the diagnosis of OSA.

Skin lesions are a frequent manifestation of Leishmania infantum infections in Mediterranean countries. This study demonstrates by real-time reverse transcriptase-polymerase chain reaction the local cytokine response in skin biopsies from Leishmania-infected dogs (n = 10). As controls, we investigated skin biopsies from healthy (n = 10) and fleabite hypersensitive dogs (n = 10). We established a quantitative PCR to determine the parasite burden in biopsies. The objective was to elucidate whether a correlation exists between parasite number, histologic response, and T helper-1 (TH1)/T helper-2 (TH2) cytokine expression in lesional skin of naturally infected dogs. In Leishmania-infected dogs, interleukin-4 (IL-4), tumor necrosis factor α (TNF-α) and interferon-γ (IFN-γ) messenger RNA production was significantly higher than controls. Furthermore, dogs with a high Leishmania burden had a significantly higher IL-4 expression, whereas no difference was noted with regard to expression of other cytokines. By comparing the pattern of inflammation and cytokine expression, a clear trend became evident in that levels of IL-4, TNF-α, and IFN-γ were elevated in biopsies with a periadnexal nodular pattern and in biopsies where the severity of the periadnexal infiltrate was equal to the perivascular to interstitial infiltrate. Expression of IL-4, IL-13, and TNF-α was slightly increased in biopsies where plasma cells prevailed on lymphocytes, whereas expression of IFN-γ was moderately higher when lymphocytes were predominating. In summary, the present study demonstrates that the local immune response in naturally occurring leishmaniasis includes TH1 as well as TH2 cytokine subsets. Furthermore, respective data suggest that increased expression of the TH2-type cytokine IL-4 is associated with both severe clinical signs and a high parasite burden in the skin lesions.

Intraneural perineurioma is an extremely rare condition characterized by perineurial cell proliferation within peripheral nerve (PN) sheaths. In the veterinary field, this entity has been reported only in a dog. We examined multiple enlargements of PNs in 11 chickens (Gallus gallus domesticus) (9 Japanese bantams and 2 specific pathogen-free White Leghorn), which were inoculated with an avian leukosis virus (ALV) causing so-called fowl glioma. All chickens clinically exhibited progressive leg paralysis. Lumbosacral plexus, brachial plexus, and/or spinal ganglion were commonly affected, and these nerves contained a diffuse proliferation of spindle cells arranged concentrically in characteristic onion bulb-like structures surrounded by residual axons and myelin sheaths. The spindle cells were immunohistochemically negative for S-100α/β protein. Electron microscopy revealed that these cells were characterized by short bipolar cytoplasmic processes, occasional cytoplasmic pinocytotic vesicles, and discontinuous basal laminae. These features are consistent with those of intraneural perineurioma. Furthermore, the specific sequence of the ALV was detected in the PN lesions of 8/11 (73%) birds by polymerase chain reaction. These results indicate that the multiple intraneural perineuriomas of chicken may be associated with the ALV-A causing fowl glioma.

Over 100 free-ranging adult California sea lions (Zalophus californianus) and one Northern fur seal (Callorhinus ursinus), predominantly adult females, were intoxicated by domoic acid (DA) during three harmful algal blooms between 1998 and 2000 in central and northern California coastal waters. The vector prey item was Northern anchovy (Engraulis mordax) and the primary DA-producing algal diatom was Psuedonitzschia australis. Postmortem examination revealed gross and histologic findings that were distinctive and aided in diagnosis. A total of 109 sea lions were examined, dying between 1 day and 10 months after admission to a marine mammal rehabilitation center. Persistent seizures with obtundation were the main clinical findings. Frequent gross findings in animals dying acutely consisted of piriform lobe malacia, myocardial pallor, bronchopneumonia, and complications related to pregnancy. Gross findings in animals dying months after intoxication included bilateral hippocampal atrophy. Histologic observations implicated limbic system seizure injury consistent with excitotoxin exposure. Peracutely, there was microvesicular hydropic degeneration within the neuropil of the hippocampus, amygdala, pyriform lobe, and other limbic structures. Acutely, there was ischemic neuronal necrosis, particularly apparent in the granular cells of the dentate gyrus and the pyramidal cells within the hippocampus cornu ammonis (CA) sectors CA4, CA3, and CA1. Dentate granular cell necrosis has not been reported in human or experimental animal DA toxicity and may be unique to sea lions. Chronically, there was gliosis, mild nonsuppurative inflammation, and loss of laminar organization in affected areas.

Tissues were obtained at necropsy from the nasal vestibule, turbinates, nasopharynx, trachea, tracheobronchial bifurcation, and lung from each of 10 clinically healthy calves persistently infected (PI) with bovine viral diarrhea virus (BVDV) serotype 2a. Tissues from the nasal vestibule were obtained by biopsy from five additional PI calves. Formalin-fixed tissues were processed for immunohistochemistry to localize the distribution of BVDV throughout the respiratory tract. Antigen distribution and intensity were subjectively evaluated. Throughout the respiratory tract, mononuclear leukocytes, vascular smooth muscle, and endoneural and perineural cells had BVDV immunoreactivity (BVDV-IR). Multifocally, squamous and ciliated columnar epithelium throughout the respiratory tract contained weak to moderate BVDV antigen. Viral antigen was not seen in goblet cells. BVDV-IR in mixed tubuloalveolar glands of the nasal cavity was weak to strong in serous secretory cells and ductular epithelium. Chondrocytes of the concha often contained BVDV antigen diffusely. Nasal mucus-secreting and tracheobronchial glands multifocally contained weak viral signal. In all cases, alveolar macrophages had moderate to strong BVDV-IR, whereas BVDV-IR in alveolar epithelial cells was weak to moderate. BVDV was present in interalveolar leukocytes and mesenchymal cells. Results indicate that serous secretions of the nasal cavity, productive viral infection of epithelium, and infected leukocytes in respiratory secretions are likely major sources of infectious BVDV from PI calves. The presence of BVDV antigen in respiratory epithelium is, at least, indirect support for the notion that this virus predisposes PI cattle to secondary microbial infections.

The immunohistochemically determined estrogen receptor (ER) α (ERα) and progesterone receptor (PR) status, as well as recognized, well-accepted prognostic indicators and host factors were prospectively analyzed in 84 cases of primary canine mammary carcinoma for their effect on disease-free period (recurrence free, metastasis free, or combined) (DFP) after an observation period of 18 months. The presence of one or both receptors, as well as tumor size, lymph node status, histologic grading, intravascular growth, and necrosis, were of prognostic value for DFP. In multivariate analysis, only tumor size and histologic grading proved to be independent prognosticators. None of the host factors analyzed were of prognostic value for DFP. ERα, PR, or both were detected in 173 out of 228 tumors: 70 ERα and PR; 5 ERα only; 98 PR only. Statistically significant differences regarding the presence of one or both receptors were observed between benign and malignant tumors and between complex, mixed, and simple histologic subtypes of benign and malignant tumors. In the group of malignant tumors (n = 155), the presence of one or both receptors was more frequent in tumors smaller than 3 cm, without lymph node metastasis, with tubulopapillary rather than solid patterns of growth among simple carcinomas, of histologic grades I and II, without both intravascular growth and necrosis, and with lymphocyte cell infiltrates. The most frequent groups of hormone receptors-positive tumors were the ERα-positive and PR-positive group among benign and the ERα-negative and PR-positive group among malignant tumors.

A spontaneous case of renal heterotopia involving the lung parenchyma of a free-living, adult, female common dolphin (Delphinus delphis), which was found stranded alive on the North Adriatic Sea coast of Italy, is reported in this study. The lesion, slightly visible from the macroscopic point of view, had the histologic appearance of a ''foreign tissue island,'' which was poorly demarcated from the surrounding pulmonary tissue. Within such an island, several regularly shaped and apparently mature kidney glomeruli and tubules could be observed, with no evidence of secondary tissue reaction. To the best of our knowledge, this should be the first description of heterotopic kidney tissue occurrence in the lung of any domestic or wild animal species.

A cutaneous mass at the base of the retroauricular region of a 4-year-old, female Golden Retriever was examined pathologically. Histologically, the mass formed multiple nodules consisting of a proliferation of large clear cells with abundant cytoplasm. Mitotic figures among the neoplastic cells were very sparse. The large clear cells were intensely positive for cytokeratins (AE1/AE4, cytokeratin 8 and 18) and moderately positive for lysozyme and contained periodic acid-Schiff-positive granules in the cytoplasm. In addition, small flat cells lined the islands of neoplastic large clear cells, and these were strongly positive for alpha-smooth muscle actin and vimentin, and some were positive for cytokeratin (AE1/AE4), suggesting they were myoepithelial cells. No local recurrence or metastasis has been recognized during the 18 months since surgical excision. On the basis of these findings, the present tumor was diagnosed as apocrine sweat gland adenoma, clear cell variant. There have been few previous reports of canine apocrine adenomas showing a clear cell morphology.

In November 2002, a 2-year-old, spayed Maltese Terrier in central Mississippi was presented for an acute illness characterized by uncontrolled hyperactivity that rapidly progressed to generalized tremors, ataxia, and intermittent hyperthermia. Postmortem examination after a 2-week course revealed mild, multifocal, nonsuppurative meningo encephalitis, with focal necrosis in the medulla. Reverse transcriptase-nested-polymerase chain reaction for West Nile virus (WNV) was positive on brain and negative on other tissues. Immunohistochemistry was negative on all tissues. The clinical, postmortem, and laboratory findings are consistent with acute encephalitis due to WNV infection. WNV infection should be considered in dogs showing signs of encephalitis when and where WNV and mosquito vectors occur.

Epithelioid trophoblastic tumor (ETT) is an unusual type of trophoblastic tumor, with features resembling carcinoma. In this study, we describe a 4-year-old cynomolgus monkey (Macaca fascicularis) showing, at necropsy, a lobulated mass replacing the left ovary and several nodular lesions within the lungs. Histologically, the mass in the ovary and lung metastases were characterized by nests of epithelioid cells, with intermingled, occasional, multinucleate tumor cells consistent with syncytiotrophoblasts and moderate amount of eosinophilic, hyaline-like material. Immunohistochemically, the tumor cells were diffusely positive for cytokeratins (AE1/AE3) and inhibin-α, but only focal immunoreactivity was observed for human chorionic gonadotropin, whereas placental alkaline phosphatase was always negative. On the basis of morphology and immunohistochemical reactivity, tumor cells were identified as intermediate trophoblast.

A clinically normal, 3-year-old female rhesus monkey (Macaca mulatta), which was part of a routine toxicology study, had a mass in the right ovary with metastases to the adjacent mesentery and lungs. The histologic features and immunohistochemistry results were consistent with the diagnosis of choriocarcinoma. Neoplastic cell types included cytotrophoblast (positive for cytokeratin), syncytiotrophoblast (positive for human chorionic gonadotropin), and extravillous trophoblast (positive for human placental lactogen). Because the neoplasm was present in the ovary, the uterus was normal, and the animal was not currently pregnant, this was considered a primary ovarian neoplasm of germ cell origin. The monkey had elevated serum levels of chorionic gonadotropin at the beginning of the study, indicating that, as in women, choriocarcinomas in monkeys can be associated with increased gonadotropin levels and that the tumor was preexisting at the start of the toxicology study.

Hepatocellular carcinoma (HCC) with metastasis to the spleen in a Holstein cow was studied by histopathologic and immunohistochemical methods. The tumor was characterized by a pseudoglandular (acinar) pattern with an associated fibrous stroma. Individual cells often had a “hepatoid” appearance but were interspersed with scattered cells exhibiting a clear, periodic acid-Schiff (PAS)-positive cytoplasm and small eccentric nuclei. This pattern was present in nodules found in both liver and spleen. Moreover, hepatoid tumor cells were positive for alpha-fetoprotein. Immunohistochemical studies suggest that myofibroblasts were responsible for the production of fibrous septa surrounding the pseudoglandular structures of bovine HCC. In summary, our histologic and immunohistochemical findings support a diagnosis of primary HCC with splenic metastasis. Furthermore, the associated stromal response appears to be of a myofibroblast origin. The primary etiology of bovine HCC and the significance of the intralesional, PAS-positive clear cells remain undetermined.

Abdominal ultrasound examination in an 11-year-old, intact, female Labrador dog with hepatic disease revealed a nodular swelling of the left adrenal gland. Hyperadrenocorticism was suspected, but endocrine tests were negative. At the owner's request, an adrenalectomy was performed. Grossly, a nodular mass protruded from the external surface of the left adrenal gland and in cut section was hemorrhagic and effaced the cortical and medullary regions. Histologic examination revealed a cortical neoplasm with medullary involvement. The mass was composed of well-differentiated adipose cells, megakaryocytes, hematopoietic cells, and macrophages containing hemosiderin deposits. A diagnosis of cortical adrenal myelolipoma was made.