Veterinary Pathology

Observational studies are the basis for much of our knowledge of veterinary pathology and are highly relevant to the daily practice of pathology. However, recommendations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offer advice on planning and conducting an observational study with examples from the veterinary pathology literature. Investigators should recognize the importance of creativity, insight, and innovation in devising studies that solve problems and fill important gaps in knowledge. Studies should focus on specific and testable hypotheses, questions, or objectives. The methodology is developed to support these goals. We consider the merits and limitations of different types of analytic and descriptive studies, as well as of prospective vs retrospective enrollment. Investigators should define clear inclusion and exclusion criteria and select adequate numbers of study subjects, including careful selection of the most appropriate controls. Studies of causality must consider the temporal relationships between variables and the advantages of measuring incident cases rather than prevalent cases. Investigators must consider unique aspects of studies based on archived laboratory case material and take particular care to consider and mitigate the potential for selection bias and information bias. We close by discussing approaches to adding value and impact to observational studies. Part 2 of the series focuses on methodology and validation of methods.

The epithelial-mesenchymal transition (EMT) is a dynamic process linked to metastasis in many tumor types, including mammary tumors. In this study, we evaluated E-cadherin and vimentin immunolocalization in primary canine mammary carcinomas (20 cases) and their respective metastases, as well as their relationship with the core regulators SNAIL/SLUG. To assess the number of cells undergoing the process of EMT, we quantitated double-positive (E-cadherin⁺/vimentin⁺) cells using immunofluorescence, via cell counting and image analysis. In addition, SNAIL/SLUG expression was evaluated by established immunohistochemical methods. Primary tumors had significantly more E-cadherin⁺/vimentin⁺ co-expression than their paired respective lymph node or distant metastasis, respectively. Furthermore, the percentage of E-cadherin⁺/vimentin⁺ cells in grade II and III carcinomas was significantly higher than in grade I tumors. Primary tumors had significantly higher SNAIL/SLUG expression when analyzed based on the percentage of positive cells compared with their respective distant metastases in pairwise comparisons. An inverse correlation was noted between SNAIL/SLUG immunoreactivity and percentage of E-cadherin⁺/vimentin⁺ immunopositive cells in primary tumor samples when SNAIL/SLUG immunoreactivity was grouped into 2 categories (high versus low) based on percentage-positive staining. These results show a positive correlation between E-cadherin⁺/vimentin⁺ cells and higher tumor grade, establish differences between primary tumor and their respective metastases, and provide further support that EMT plays a critical role in the metastasis of canine mammary carcinoma. Furthermore, these data suggest that modulation of this process could provide greater therapeutic control and provide support for further research to determine if E-cadherin⁺/vimentin⁺ co-immunoreactivity imparts predictive value in the clinical outcome of patients with canine mammary carcinomas.

Oligodendroglioma is a common brain tumor in dogs, particularly brachycephalic breeds. Oligodendrocyte precursor cells (OPCs) are suspected to be a possible origin of oligodendroglioma, although it has not been well elucidated. In the present study, 27 cases of canine brain oligodendrogliomas were histologically and immunohistochemically examined. The most commonly affected breed was the French Bulldog (n = 19 of 27, 70%). Seizure was the most predominant clinical sign (n = 17 of 25, 68%). The tumors were located mainly in the cerebrum, particularly in the frontal lobe (n = 10 of 27, 37%). All cases were diagnosed as anaplastic oligodendroglioma (AO) and had common histologic features characterized by the proliferation of round to polygonal cells with pronounced atypia and conspicuous mitotic activity (average, 10.7 mitoses per 10 high-power fields). Honeycomb pattern (n = 5 of 27, 19%), myxoid matrix (n = 10, 37%), cyst formation (n = 6, 22%), necrosis (n = 19, 70%), pseudopalisading (n = 5, 18.5%), glomeruloid vessels (n = 16, 59%), and microcalcification (n = 5, 19%) were other histopathologic features of the present tumors. Immunohistochemically, the tumor cells were positive for Olig2 in all cases and for other markers of OPCs in most cases, including SOX10 (n = 24 of 27, 89%), platelet-derived growth factor receptor α (n = 24, 89%), and NG2 (n = 23, 85%). The present AO also consisted of heterogeneous cell populations that were positive for nestin (n = 13 of 27, 48%), glial fibrillary acidic protein (n = 5, 19%), doublecortin (n = 22, 82%), and βIII-tubulin (n = 15, 56%). Moreover, cultured AO cells obtained from 1 case retained expression of OPC markers and exhibited multipotent characteristics in a serum culture condition. Overall, the findings suggest that transformed multipotent OPCs may be a potential origin of canine AO.

Canine splenic lymphoid nodules are currently classified as indolent lymphomas (marginal zone lymphoma [MZL], mantle cell lymphoma [MCL]) or nodular hyperplasia (lymphoid [LNH] or complex [CNH] type). Their differentiation can be difficult on morphology, because of similar histologic appearance and poorly defined diagnostic criteria. Thirty-five surgical samples of splenic lymphoid nodules were reviewed in order to assess the diagnostic contribution of immunophenotyping, proliferative activity and clonality (PARR) in differentiating between hyperplastic and neoplastic lesions. Proliferative activity was evaluated by double immunolabeling for Ki-67 and CD79a, in order to separately assess the proliferative activity of B cells and non-B cells. Definitive diagnoses were MZL (n = 11), MCL (n = 4), LNH (n = 10), and CNH (n = 10). The overall concordance between histology and PARR was above 90%. Lymphomas had a significantly higher percentage of CD79a-positive areas (mean, 36.30%; P = .0004) and a higher B-cell proliferative activity (median Ki-67 index, 5.49%; P = .0012). The threshold value most accurately predicting a diagnosis of lymphoma was ≥28% of B-cell areas, with a Ki-67 index above 3%. Dogs were monitored for a median follow-up time of 870 days (IQR, 569-1225), and no relapses were documented. Overall median survival time was 1282 days. The combination of histology, immunohistochemistry and PARR can improve the diagnostic accuracy for canine splenic lymphoid nodules, although the long-term behavior of these lesions appears similar.

Biopsy samples of colorectal polyps were collected and examined from 67 Miniature Dachshund dogs (including 35 cases with an additional biopsy). Histopathologic diagnoses of the initial biopsy samples were “inflammatory polyp” in 52 cases (78%), “adenoma” in 10 cases (15%), and “adenocarcinoma” in 5 cases (8%). Eight of 10 cases (80%) diagnosed as adenoma also had inflammatory polyp lesions in the same specimen. A second biopsy was performed in 25 cases (48%) initially diagnosed with inflammatory polyp. Pathologic diagnoses for the second biopsy were inflammatory polyp in 11 cases (44%), adenoma in 9 cases (36%), and adenocarcinoma in 5 cases (20%). The number of beta-catenin-positive nuclei in epithelial cells was significantly higher in adenoma (46%) and adenocarcinoma (75%) as compared with inflammatory polyp (6%). Normal epithelial cells and hyperplastic goblet cells in inflammatory polyps showed homogeneous positive cytoplasmic immunoreactivity for adenomatous polyposis coli (APC) antigen. However, APC expression was decreased in areas of intense nuclear beta-catenin expression in adenoma and adenocarcinoma lesions. Foci of cytokeratin 5/6–positive squamous cell-like neoplastic cells showed intense beta-catenin nuclear expression that was similar to squamous morules described in human colorectal tumors. The results of the present study suggest that the inflammatory polyp in Miniature Dachshunds is a progressive disease that may develop into adenoma and/or adenocarcinoma. In addition, immunohistochemical findings suggest that aberrations of APC and beta-catenin expression may be involved in tumor development within the inflammatory polyp lesions.

The biological behavior and immunohistochemical features of feline renal cell carcinoma (RCC) have not been well characterized. In the present study, immunohistochemical examinations were performed in 12 feline cases of RCC. The RCC consisted of solid (n = 2), solid-tubular (n = 2), tubular (n = 3), papillary (n = 2), tubulopapillary (n = 2), and sarcomatoid (n = 1) type lesions. Of the cases with RCC, 1 developed metastatic disease and 6 cases had no evidence of recurrence at 80 to 2292 days after surgery. One papillary-type tumor had cuboidal cells with scant cytoplasm and monomorphic nuclei, and the other had pseudostratified columnar cells with abundant cytoplasm. Immunohistochemistry revealed that the tumor cells in most cases were positive for cytokeratin (CK)7, CK20, KIT, and CD10, with the exception of cases of the solid type with clear cytoplasm (solid anaplastic), papillary type with columnar cells, and sarcomatoid types. A small number of tumor cells in the solid anaplastic and in the sarcomatoid types were positive for aquaporin-1. Increased expression of N-cadherin and Twist along with nuclear accumulation of β-catenin were observed in the sarcomatoid type. These results indicated that CK, KIT, and CD10 are relatively strongly expressed in most feline RCC. The solid anaplastic RCC exhibited CD10 expression with the absence of distal tubule marker expression. Although immunohistochemistry profiles were relatively consistent with those described in human RCC, the histopathologic features were different from those seen in humans. Epithelial-mesenchymal transition (EMT) marker expression in the current cases may suggest the involvement of an EMT-like mechanism in the development of sarcomatoid RCC in cats.

Plasmacytoid and rhabdoid variants of urothelial carcinomas (UCs) of the urinary bladder have been described in humans with plasma cell–like or rhabdoid cellular appearance and aggressive clinical outcome. Canine UC of the bladder is generally classified as papillary/nonpapillary and infiltrating/noninfiltrating with limited information regarding other histological patterns. We report 3 cases of UC of the urinary bladder showing a unique discohesive cellular morphology with malignant behavior resembling the human plasmacytoid and rhabdoid variants of UC, which may raise some difficulties in diagnosis. Epithelial-mesenchymal transition and reduced E-cadherin expression were revealed by immunohistochemistry in 2 cases, possibly explaining the discohesive and invasive behavior of the tumor cells. The findings broaden the morphological spectrum as well as the distinct clinical features of canine UC of the urinary bladder.

Tissue microarray (TMA) is a time- and cost-saving technique allowing the simultaneous immunohistochemical evaluation of multiple tissue samples. The aim of this study was to assess the efficacy of TMA at classifying canine gastrointestinal spindle cell tumors as gastrointestinal stromal tumor (GIST), smooth muscle tumor (SMT), and non-GIST/non-SMT based on the expression of α-smooth muscle actin (α-SMA), desmin, and CD117. Thirty-four cases were investigated on TMAs, sampling 2 cores each. Immunohistochemistry was performed on TMAs and full sections, and the results were compared. Comparing full sections, TMA specificity and sensitivity were 100% and 93.8%, respectively, for α-SMA; 100% and 80.8% for desmin; and 100% and 100% for CD117. TMA allowed the identification of 6 of 6 GISTs, 25 of 26 SMTs, and 2 of 2 non-GIST/non-SMTs. One SMT was misdiagnosed as non-GIST/non-SMT. Based on these results, TMA-based immunohistochemistry is efficient at diagnosing canine gastrointestinal spindle cell tumors and might be applied on large caseloads in a research setting.

Newcastle disease (ND), caused by virulent strains of Newcastle disease virus (NDV), is a devastating disease of poultry worldwide. The pathogenesis of ND in quail is poorly documented. To characterize the ability of virulent NDV strains to replicate and cause disease in quail, groups of 14 two-week-old Japanese quail (Coturnix japonica) were experimentally inoculated with 10⁸ EID₅₀ (embryo infectious dose 50%) units of 1 of 4 virulent NDV strains: 2 isolated from quail (N2, N23) and 2 from chickens (Israel, Pakistan). At day 2 postinfection, noninfected quail (contact group) were added to each infection group to assess the efficacy of virus transmission. Tested NDV strains showed moderate pathogenicity, with highest mortality being 28% for the N2 strain and below 10% for the others. Two N2-inoculated birds showed neurological signs, such as head tremor and ataxia. Microscopic lesions were present in N2-, Israel-, and Pakistan-inoculated birds and consisted of nonsuppurative encephalitis. Contact birds showed no clinical signs or lesions. In both inoculated and contact birds, virus replication was moderate to minimal, respectively, as observed by immunohistochemistry in tissues and virus isolation from oropharyngeal and cloacal swabs. Strains originally isolated from quail resulted in higher numbers of birds shedding in the inoculation group; however, transmission appeared slightly more efficient with chicken-derived isolates. This study shows that virulent NDV strains have limited replicative potential and mild to moderate disease-inducing ability in Japanese quail.

Failure of the cartilage canal blood supply leads to ischemic chondronecrosis which causes osteochondrosis, and osteochondral lesions. Osteochondrosis is a disease with a heritable component and usually occurs under aseptic conditions. Because bacteria can bind to growth cartilage and disrupt the blood supply in pigs and chickens, we considered whether this might play a role in development of equine osteochondrosis. The aim of this study was to examine whether bacteria are present in canals in the growth cartilage of foals with septic arthritis/osteomyelitis, and whether this is associated with osteochondrosis. The material consisted of 7 foals aged 9-117 days euthanized because of septic arthritis/osteomyelitis. The 7 cases had 16 lesions in growth cartilage that were evaluated histologically. Bacteria were present in cartilage canals in foals with septic arthritis/osteomyelitis. Portions of necrotic canals adjacent to bacteria frequently contained neutrophils, termed acute septic canals; or granulation tissue with neutrophils, termed chronic septic canals. Acute and chronic septic canals were associated with ischemic chondronecrosis in the articular-epiphyseal cartilage complex (AECC) of 5 cases and in the physis of 2 cases, and ossification was focally delayed in 5 of those 7 cases. Lesions occurred with and without adjacent osteomyelitis. Bacteria were present in cartilage canals and were associated with focal chondronecrosis in both the AECC and the physis. This establishes sepsis as a plausible cause of some osteochondral lesions in horses. It is recommended that horses with sepsis-related osteochondral lesions may be used for breeding without increasing the prevalence of OCD-predisposing genes in the population.

Chronic liver disease is an important cause of illness in horses, and treatment is mainly supportive. Research into new treatment modalities for humans has shown promising data regarding metallothionein (MT), which has been shown to possess regenerative, antifibrotic, and anti-inflammatory properties. This study aimed to examine the relationship between hepatic MT expression and the histopathologic markers of hepatic inflammation, fibrosis and bile duct proliferation, as well as cellular regeneration in 77 selected cases of chronic liver disease in horses. We hypothesized that higher MT expression would be associated with increased heptocellular proliferation and decreased fibrosis, inflammation, and bile duct proliferation. Hepatocellular MT expression was evaluated with immunohistochemistry. Additionally, cellular regeneration was evaluated with immunohistochemistry for Ki-67, a protein expressed during all active stages of the cell cycle. The severity of inflammation and fibrosis was scored, and bile duct proliferation was assessed by counting bile duct profiles. MT expression was observed in 73 of 77 (94.8%) cases of chronically diseased livers. Ki-67 expression was seen in resident Kupffer cells (n = 42, 54.6%), lymphocytes (n = 39, 50.7%), bile duct epithelium (n = 10, 13.0%), and hepatocytes (n = 8, 10.4%). MT expression was significantly associated with Ki-67 staining in bile duct epithelium and Kupffer cells. Additionally, median MT expression was higher in cases containing lymphocytic infiltrates as compared with cases with no lymphocytic infiltrate (P < .05). These findings are the first known report of MT expression within chronic equine hepatic disease.

Wobbly hedgehog syndrome (WHS) is a leading cause of neurologic disease in African pygmy hedgehogs (APHs; Atelerix albiventris). This study describes the signalment, clinical signs, gross, microscopic, and ultrastructural lesions of WHS in a cohort of 12 pet APHs. Microscopically, lesions consisted of status spongiosus of the white matter, typically bilateral and symmetrical, with myelin degeneration and loss that was accompanied by neuronal/axonal degeneration plus reactive microgliosis and mild, focal astrocytosis and astrogliosis. Lesions were most severe in the cerebellum and medulla oblongata, as well as cervical and thoracic spinal cord. Less affected areas were the corona radiata, corpus callosum, corpus striatum, internal capsule, and the mesencephalon. Ultrastructurally, the lesions consisted of splitting of the myelin sheath at the intraperiod line with subsequent focal expansion, resulting in status spongiosus, disruption, dilatation, rhexis, and phagocytosis. Based on these results, WHS is best described as a “spongy myelinopathy” with widespread central nervous system involvement.

The authors describe pathological and microbiological features of mortalities in a captive breeding colony of Lord Howe Island stick insects (Dryococelus australis) over a period of 18 months. There were 2 peaks of mortality in this period. In the first, insects presented dead with minimal premonitory signs of illness. In the second, affected insects were ataxic with contracted limbs and inability to climb or right themselves. Gross lesions were uncommon but included pigmented plaques on the gut and cloacal prolapse. Histological lesions in both outbreaks indicated a cellular innate immune response including nodulation characterized by Gram-negative bacterial bacilli entrapped within nodules of pigmented hemocytes, and melanization characterized by melanin within hemocyte nodules and around bacteria. Hemolymph culture findings varied and often yielded a mixed growth. Pure growth of Serratia marcescens was cultured in 44% of animals in Outbreak 1, while pure growth of Pseudomonas aeruginosa was cultured in 30% of animals in Outbreak 2. Cases with S. marcescens-positive culture often showed inflammation at the foregut-midgut junction. The frequency of mixed bacterial culture results did not allow firm conclusions about causality to be made, and may indicate primary bacterial infection or increased susceptibility to hemolymph colonization with an opportunistic pathogen. These findings highlight the utility of histopathology combined with ancillary testing when investigating mortality in captive insect colonies.

A striking form of lymphocytic mural folliculitis is described in 6 tigers (Panthera tigris). Clinically, all tigers exhibited regionally extensive chronic, variably waxing and waning alopecia with minimal scaling and crusting most pronounced over the head, neck, and shoulders. More severely affected tigers exhibited marked hyperpigmentation and lichenification. Pruritus was not a feature. Tigers generally lacked signs of systemic illness and clinical pathology findings were unremarkable. Histologic examination of skin biopsies revealed infiltrative lymphocytic mural folliculitis extending the length of the hair follicle. Mild epidermal lymphocytic infiltrates were frequent. The surrounding dermis was histologically unremarkable in 4 of 6 tigers or associated with mild perifollicular and periadnexal mixed inflammation in 2 of 6 tigers. The cause of the mural folliculitis was not identified, and tigers responded poorly to immunomodulatory therapy. Lymphocytic mural folliculitis might be a nonspecific hypersensitivity reaction pattern in tigers, and care should be taken to differentiate this reaction pattern from epitheliotropic T-cell lymphoma.

Chlamydial infections in crocodiles have been described in several countries and in several different species. These are typically associated with severe pharyngitis and conjunctivitis, with death occurring secondary to compromise of the upper respiratory tract due to obstruction of the trachea. A population of ranched Siamese crocodiles in central Thailand experienced an epizootic of sudden death in juvenile animals. The affected animals had fulminant systemic disease primarily involving the liver and spleen but also affecting the kidneys, heart, and the whole of the respiratory tract. Chlamydia sp. were noted in liver and spleen during histopathological examination and confirmed with transmission electron microscopy and polymerase chain reaction (PCR). The sequence of the PCR product suggested a novel Chlamydia sp. of Siamese crocodiles. Crocodile farming represents an important economy in several parts of the world. Epizootics, such as the one described in this manuscript in association with Chlamydia sp., can have devastating impact on the industry and represent a potential zoonosis of significant public health concern. This is the first report of Chlamydia sp. and Aeromonas sobria causing systemic disease in crocodiles as well as the first histopathological and ultrastructural description of Chlamydia infection in Siamese crocodiles.

Idiopathic chronic diarrhea (ICD) is a common ailment affecting captive rhesus macaques (Macaca mulatta). ICD cases are characterized by diarrhea in the absence of commonly identified diarrheal pathogens and multiple recurrences even after supportive therapy. Histologically, the disease is characterized by lymphoplasmacytic colitis. We identified 35 rhesus macaques euthanized for ICD during a 7-month period and described demographic, clinical, histologic, and immunologic commonalities. We found a trend of historic Campylobacter spp. and trichomonad infections. Furthermore, rhesus macaques with ICD demonstrated loss of normal colonic adherent bacterium, identified in this study as Helicobacter macacae; increased abundance of Pentatrichomonas hominis; and increased frequency of colonic serotonin-positive enterochromaffin cells. Interestingly, colonic and ileal T-helper cells of animals with ICD manifested decreased capacity for expression of certain cytokines, in particular interleukin (IL)–4 and IL-13. These data further describe a common ailment and suggest new avenues to identify complex interactions involved in the etiology of recurring diarrhea in young rhesus macaques.

Uterine tumors resembling ovarian sex cord tumors (UTROSCTs) are rare uterine neoplasms that exhibit prominent sex cord-like differentiation. The authors describe 4 cases of UTROSCTs that were identified as incidental lesions in female baboons. All baboons were in good body condition. One animal had a 2-mm-diameter yellow-tan mass in the uterine body along the attachment of the left broad ligament; the other 3 did not have any gross lesions in the uterus. Histologically, the myometrium contained multifocal well-demarcated neoplasms composed of cuboidal to columnar cells arranged in variable arrangements of sheets, nests, cords, trabecular, and retiform patterns that occasionally formed Call-Exner-like bodies. In all cases, the neoplastic cells were diffusely positive for WT-1 and negative for calretinin, CD99, and desmin. One case was positive for inhibin and CD10. To the best of the authors’ knowledge, this is the first report of UTROSCTs in nonhuman primates and in the veterinary literature.