Veterinary Pathology

The accurate distinction of reactive and neoplastic lymphoid proliferations can present challenges. Given the different prognoses and treatment strategies, a correct diagnosis is crucial. Molecular clonality assays assess rearranged lymphocyte antigen receptor gene diversity and can help differentiate reactive from neoplastic lymphoid proliferations. Molecular clonality assays are commonly used to assess atypical, mixed, or mature lymphoid proliferations; small tissue fragments that lack architecture; and fluid samples. In addition, clonality testing can be utilized to track neoplastic clones over time or across anatomic sites. Molecular clonality assays are not stand-alone tests but useful adjuncts that follow clinical, morphologic, and immunophenotypic assessment. Even though clonality testing provides valuable information in a variety of situations, the complexities and pitfalls of this method, as well as its dependency on the experience of the interpreter, are often understated. In addition, a lack of standardized terminology, laboratory practices, and interpretational guidelines hinders the reproducibility of clonality testing across laboratories in veterinary medicine. The objectives of this review are twofold. First, the review is intended to familiarize the diagnostic pathologist or interested clinician with the concepts, potential pitfalls, and limitations of clonality testing. Second, the review strives to provide a basis for future harmonization of clonality testing in veterinary medicine by providing diagnostic guidelines.

Devil facial tumor disease (DFTD) is an emergent transmissible cancer exclusive to Tasmanian devils (Sarcophilus harrisii) and threatening the species with extinction in the wild. Research on DFTD began 10 years ago, when nothing was known about the tumor and little about the devils. The depth of knowledge gained since then is impressive, with research having addressed significant aspects of the disease and the devils’ responses to it. These include the cause and pathogenesis of DFTD, the immune response of the devils and the immune evasion mechanisms of the tumor, the transmission patterns of DFTD, and the impacts of DFTD on the ecosystem. This review aims to collate this information and put it into the context of conservation strategies designed to mitigate the impacts of DFTD on the devil and the Tasmanian ecosystem.

In humans and mouse models, Foxp3⁺ regulatory T cells are known to control all aspects of immune responses. However, only limited information exists on these cells’ role in diseases of other animals. In this review, we cover the most important features and different types of regulatory T cells, which include those that are thymus-derived and peripherally induced, the mechanisms by which they control immune responses by targeting effector T cells and antigen-presenting cells, and most important, their role in animal health and diseases including cancer, infections, and other conditions such as hypersensitivities and autoimmunity. Although the literature regarding regulatory T cells in domestic animal species is still limited, multiple articles have recently emerged and are discussed. Moreover, we also discuss the evidence suggesting that regulatory T cells might limit the magnitude of effector responses, which can have either a positive or negative result, depending on the context of animal and human disease. In addition, the issue of plasticity is discussed because plasticity in regulatory T cells can result in the loss of their protective function in some microenvironments during disease. Lastly, the manipulation of regulatory T cells is discussed in assessing the possibility of their use as a treatment in the future.

Ependymal cells are epithelial support cells that line the central canal and ventricular cavities of the central nervous system, providing the interface between the cerebrospinal fluid and the parenchyma of the brain and spinal cord. The spinal ependymal layer (SEL) is composed of 3 main cell types: tanycytes, ependymocytes, and cerebrospinal fluid–contacting neurons. A fourth cell type, termed the supraependymal cell, is also occasionally described. Cells of the SEL show restricted proliferative capacity in health but display neural stem cell properties both in vitro and in vivo in various disease states. A growing body of literature is devoted to the regenerative roles of the SEL, particularly in the context of spinal cord injury, where mechanical damage to the spinal cord leads to a significant increase in SEL proliferation. SEL-derived cell progeny migrate to sites of injury within the injured spinal cord parenchyma and contribute primarily to glial scar formation. In additional to their role as endogenous neural stem cells, cells of the SEL may be an important source of cytokines and other cell signaling molecules, such as tumor necrosis factor, heat shock proteins, and various growth factors. The SEL has become of recent interest to neuroscience researchers because of its potential to participate in and respond to diseases affecting the spinal cord (eg, traumatic spinal cord injury) and neurodegenerative disease. The intimate association of the SEL with the cerebrospinal fluid makes intrathecal therapies a viable option, and recent studies highlight the potential promise of treatments that augment SEL responses to disease.

Murine noroviruses (MNVs) are highly prevalent in laboratory mice, can cause persistent infections, and have been shown to infect macrophages, dendritic cells, and B cells. To address the potential impact of MNV infection on research outcomes, numerous studies have been conducted with various mouse models of human disease and have generated mixed results, ranging from no impact to significant disease. Many of these studies included histologic evaluations after MNV infection, and these results have similarly been variable in terms of whether MNV induces lesions, despite the fact that localization of MNV by viral culture and molecular techniques have demonstrated systemic distribution regardless of mouse immune status. The aim of this review is to summarize the histologic findings that have been reported with MNV infection in several mouse models. The studies demonstrate that experimental infection of MNV in wild-type mice results in minimal to no histologic changes. In contrast, immunodeficient mice consistently have detectable MNV-induced lesions that are typically inflammatory and, in the most severe cases, accompanied by necrosis. In these, the liver is commonly affected, with more variable lesions reported in the lung, gastrointestinal tract, mesenteric lymph nodes, brain, and spleen. In specific disease models including atherosclerosis, MNV infection had a variable impact that was dependent on the mouse model, viral strain, timing of infection, or other experimental variables. It is important to recognize the reported MNV lesions to help discern the possible influence of MNV infection on data generated in mouse models.

Urothelial carcinoma (UC) is the most common neoplasm of the canine urinary tract. Clinical presentation of UC is shared with several other, more common urinary tract disorders, and this often delays diagnosis of the UC. Definitive diagnosis of UC requires histopathologic examination of a biopsy specimen, but the cost and invasiveness for these diagnostic tests often result in most diagnoses being made on the basis of clinical findings, diagnostic imaging, and cytologic examination of urine sediment. Regardless of the diagnostic process used, most UCs currently are not diagnosed until they are at an advanced clinical stage and so are associated with poor prognosis. Improved methods for earlier and less invasive detection are needed. In a previous study, the authors demonstrated the presence of highly recurrent DNA copy number aberrations (CNAs) in canine UC and hypothesized that detection of these CNAs in tumor cells can be used as a molecular diagnostic for UC. In this study, a multiplexed droplet digital polymerase chain reaction (ddPCR) assay was detected to detect and quantify CNAs of specific regions of canine chromosomes 8, 13, 19, and 36. The assay was effective at differentiating 31 neoplastic and 25 nonneoplastic bladder tissues based on copy number, with 100% sensitivity and specificity in tissue samples. CNAs were also detected by ddPCR in 67% (12 of 18) of urine DNA specimens derived from UC patients. The findings show that ddPCR is a useful molecular technique to detect CNAs and may be used as a noninvasive molecular diagnostic test for canine UC.

Canine liposarcoma is an uncommon soft tissue sarcoma usually arising in the subcutis. While liposarcoma classification in dogs is based solely on histology, in humans it depends on the detection of genetic abnormalities that can lead to specific protein overexpression. This study is an immunohistochemical evaluation of MDM2 and CDK4 expression in canine liposarcoma designed to assess the correlation of these proteins with histologic type, grade, mitotic index and Ki67 labeling index and evaluate their utility in improving tumor classification. Fifty-three liposarcomas were retrospectively collected: 24 were well differentiated liposarcomas (WDL), 16 of which expressed MDM2 and 21 CDK4; 7 were myxoid liposarcomas (ML), 1 of which expressed MDM2 and 5 expressed CDK4; 18 were pleomorphic liposarcomas (PL), all were MDM2 negative and 12 expressed CDK4. Four tumors were morphologically consistent with dedifferentiated liposarcoma (DDL) a subtype described only in humans: 3 expressed MDM2 and 4 expressed CDK4. MDM2 expression correlated with histotype (highly expressed in WDL and DDL) and grade (highly expressed in grade 1 tumors). Histotype correlated with the Ki67 labeling index (lowest in WDL and highest in DDL). A revised classification, considering MDM2 expression, allowed 8 WDL to be reclassified as PL and correlated significantly with mitotic and Ki67 labeling index (both significantly lower in WDL and progressively higher in ML and DDL). These results partially parallel data reported for human liposarcomas, suggesting that WDL and DDL are distinct neoplastic entities characterized by MDM2 expression, which may represent a useful diagnostic and potentially prognostic marker for canine liposarcoma.

Osteosarcoma (OSA) is a malignant heterogeneous primary bone tumor responsible for up to 90% of all primary bone tumors in dogs. In this study, osteocalcin (OC) and osteonectin (ON) immunoreactivity was evaluated in 23 canine OSAs, 4 chondrosarcomas, 4 fibrosarcomas, 2 hemangiosarcomas, and 4 histiocytic sarcomas. The effects of three different decalcification agents (ethylenediaminetetraetic acid [EDTA], formic acid and hydrochloric acid [HCl]) on the immunoreactivity for OC and ON was also assessed. Immunoreactivity to OC was present in 19/23 (83%) cases of OSA and all cases of chondrosarcoma. In three OSAs the extracellular matrix showed immunoreactivity to OC. None of the fibrosarcomas, histiocytic sarcomas or hemangiosarcomas showed immunoreactivity to OC. The sensitivity and specificity for OC in canine OSA in this study was 83% and 71% respectively. For ON, 100% of both OSAs (23/23) and non-OSAs (14/14) showed cytoplasmic immunoreactivity to this antibody, giving a sensitivity of 100% but a complete lack of specificity. There were no significant differences in immunoreactivity for OC and ON between the different decalcification agents used. In conclusion, OC showed high sensitivity for identifying OSA but it failed to distinguish between OSA and chondrosarcoma, and the osteoid produced by neoplastic cells in most cases did not show immunoreactivity to OC. These factors may limit the practical utility of OC in the diagnosis of OSA in dogs when chondrosarcoma is a differential diagnosis. ON showed no specificity in detecting OSA and has little practical application for the diagnosis of OSA in dogs.

Choroid plexus tumors (CPTs) are reported with an increasing incidence in dogs, and they call for a reexamination of histologic features and criteria of classification corresponding to their biological behavior. In this study, the human World Health Organization classification was applied to 16 canine CPTs, and the expression of molecules involved in neoplastic cell adhesion (E-cadherin, N-cadherin), invasion (doublecortin), and proliferation (Ki-67) was investigated. Mitotic index was found to be the main criterion for grading CPTs. Cell density and multilayering of papillae were also statistically associated with histologic grade. Intraventricular spread and parenchymal invasion was observed for tumors showing histologic benign features. E-cadherin was expressed in all CPT grades, independent of tumor invasion. N-cadherin immunolabeling was more expressed in grade I than high-grade CPTs, whereas doublecortin expression was not detected in CPTs. An increasing proliferative activity was observed in relation with histologic grade.

In the past few years, Newcastle disease virus (NDV) strains with epizootic characteristics belonging to subgenotypes VIIi and XIIIb emerged in the Middle East and Asia. In this study, 2 NDV strains—1 representative of subgenotype VIIi isolated in Israel (Kvuzat/13) and 1 representative of subgenotype XIIIb isolated in Pakistan (Karachi/07)—were characterized by intracerebral pathogenicity index and detailed clinicopathologic assessment. The intracerebral pathogenicity index values for Kvuzat/13 and Karachi/07 were 1.89 and 1.85, respectively, classifying these strains as virulent by international standards. In 4-week-old White Leghorn chickens, both strains caused 100% mortality within 4 (Kvuzat/13) and 5 (Karachi/07) days postinfection. Histopathology and immunohistochemistry for NDV nucleoprotein showed that both strains had wide systemic distribution, especially targeting lymphoid organs and mucosa-associated lymphoid tissues in the respiratory and intestinal tracts. Results of the animal experiment confirm that both Kvuzat/13 and Karachi/07 are highly virulent and behaved as velogenic viscerotropic NDV strains.

Thrombomodulin (TM) is a membrane glycoprotein expressed on endothelial cells, which plays a major role in the protein C anticoagulation pathway. In people with inflammation, TM expression can be down-regulated on endothelial cells and a soluble form released into circulation, resulting in increased risk of thrombosis and disseminated intravascular coagulation. TM is present in dogs; however, there has been minimal investigation of its expression in canine tissues, and the effects of inflammation on TM expression in canine tissues have not been investigated. The objective of this study was to evaluate endothelial TM expression in tissues from dogs with systemic inflammatory diseases. A retrospective evaluation of tissue samples of lung, spleen, and liver from dogs with and without systemic inflammatory diseases was performed using immunohistochemistry (IHC) and a modified manual IHC scoring system. TM expression was significantly reduced in all examined tissues in dogs diagnosed with septic peritonitis or acute pancreatitis.

X-linked hereditary nephropathy (XLHN) in Navasota dogs is a spontaneously occurring disease caused by a mutation resulting in defective production of type IV collagen and juvenile-onset renal failure. The study was aimed at examining the evolution of renal damage and the expression of selected molecules potentially involved in the pathogenesis of XLHN. Clinical data and renal samples were obtained in 10 XLHN male dogs and 5 controls at 4 (T0), 6 (T1), and 9 (T2) months of age. Glomerular and tubulointerstitial lesions were scored by light microscopy, and the expression of 21 molecules was investigated by quantitative real-time polymerase chain reaction with selected proteins evaluated by immunohistochemistry. No significant histologic lesions or clinicopathologic abnormalities were identified in controls at any time-point. XLHN dogs had progressive proteinuria starting at T0. At T1, XLHN dogs had a mesangioproliferative glomerulopathy with glomerular loss, tubular necrosis, and interstitial fibrosis. At T2, glomerular and tubulointerstitial lesions were more severe, particularly glomerular loss, interstitial fibrosis, and inflammation. At T0, transforming growth factor β, connective tissue growth factor, and platelet-derived growth factor α mRNA were overexpressed in XLHN dogs compared with controls. Clusterin and TIMP1 transcripts were upregulated in later stages of the disease. Transforming growth factor β, connective tissue growth factor, and platelet-derived growth factor α should be considered as key players in the initial events of XHLN. Clusterin and TIMP1 appear to be more associated with the progression rather than initiation of tubulointerstitial damage in chronic renal disease.

Pulmonary hypertension is a well-known though poorly characterized disease in veterinary medicine. In humans, pulmonary veno-occlusive disease (PVOD) is a rare cause of severe pulmonary hypertension with a mean survival time of 2 years without lung transplantation. Eleven adult dogs (5 males, 6 females; median age 10.5 years, representing various breeds) were examined following the development of severe respiratory signs. Lungs of affected animals were evaluated morphologically and with immunohistochemistry for alpha smooth muscle actin, desmin, CD31, CD3, CD20, and CD204. All dogs had pulmonary lesions consistent with PVOD, consisting of occlusive remodeling of small- to medium-sized pulmonary veins, foci of pulmonary capillary hemangiomatosis (PCH), and accumulation of hemosiderophages; 6 of 11 dogs had substantial pulmonary arterial medial and intimal thickening. Ultrastructural examination and immunohistochemistry showed that smooth muscle cells contributed to the venous occlusion. Increased expression of CD31 was evident in regions of PCH indicating increased numbers of endothelial cells in these foci. Spindle cells strongly expressing alpha smooth muscle actin and desmin co-localized with foci of PCH; similar cells were present but less intensely labeled elsewhere in non-PCH alveoli. B cells and macrophages, detected by immunohistochemistry, were not co-localized with the venous lesions of canine PVOD; small numbers of CD3-positive T cells were occasionally in and around the wall of remodeled veins. These findings indicate a condition in dogs with clinically severe respiratory disease and pathologic features resembling human PVOD, including foci of pulmonary venous remodeling and PCH.

Feline primary cutaneous lymphomas (FPCLs) account for 0.2% to 3% of all lymphomas in cats and are more frequently dermal nonepitheliotropic small T-cell tumors. Emergence of FPCL seems unrelated to feline leukemia virus (FeLV) serological positivity or to skin inflammation. A total of 17 cutaneous lymphomas with a history of vaccine injection at the site of tumor development were selected from 47 FPCLs. Clinical presentation, histology, immunophenotype, FeLV p27 and gp70 expression, and clonality were assessed. A majority of male (12/17), domestic short-haired (13/17) cats with a mean age of 11.3 years was reported. Postinjection time of development ranged from 15 days to approximately 9 years in 5 cats. At diagnosis, 11 of 17 cats had no evidence of internal disease. Lymphomas developed in interscapular (8/17), thoracic (8/17), and flank (1/17) cutaneous regions; lacked epitheliotropism; and were characterized by necrosis (16/17), angiocentricity (13/17), angioinvasion (9/17), angiodestruction (8/17), and peripheral inflammation composed of lymphoid aggregates (14/17). FeLV gp70 and/or p27 proteins were expressed in 10 of 17 tumors. By means of World Health Organization classification, immunophenotype, and clonality, the lesions were categorized as large B-cell lymphoma (11/17), anaplastic large T-cell lymphoma (3/17), natural killer cell–like (1/17) lymphoma, or peripheral T-cell lymphoma (1/17). Lineage remained uncertain in 1 case. Cutaneous lymphomas at injection sites (CLIS) shared some clinical and pathological features with feline injection site sarcomas and with lymphomas developing in the setting of subacute to chronic inflammation reported in human beings. Persistent inflammation induced by the injection and by reactivation of FeLV expression may have contributed to emergence of CLIS.

The histopathologic characteristics of colorectal inflammatory polyps that formed in Miniature Dachshunds were compared with those of other colorectal proliferative lesions, including adenomas and adenocarcinomas. Fifty-three colorectal polypoid lesions were histopathologically classified into inflammatory polyps (26 cases), adenoma (18 cases), and adenocarcinoma (9 cases). All 26 dogs that were diagnosed with inflammatory polyps were Miniature Dachshunds, indicating that colorectal inflammatory polyps exhibit a marked predilection for this breed. The inflammatory polyps had complex histopathologic features and were classified into 3 stages based on their epithelial composition. In early stage (stage 1), the polyps tended to exhibit a thickened mucosa containing hyperplastic goblet cells, dilated crypts filled with a large amount of mucus, and mild lymphocyte and macrophage infiltration. In later stages (stages 2 and 3), more severe neutrophil infiltration, interstitial mucus accumulation, granulation tissue, and occasional osteoid tissue were seen. Also, a few small foci of dysplastic epithelial cells were detected. The hyperplastic goblet cells, which were a major component of the epithelium of the inflammatory polyps, were positive for cytokeratin 20 (CK20), while the dysplastic epithelial cells found in inflammatory polyps (stage 3) and the tumor cells of the adenomas and adenocarcinomas were negative for CK20. These CK20-negative epithelial cells exhibited cytoplasmic and nuclear immunoreactivity for beta-catenin. In addition, the epithelial cells in the inflammatory polyps demonstrated significantly higher cyclooxygenase 2 and fibroblast growth factor 2 expression than did those of the adenomas and adenocarcinomas, suggesting that the arachidonate cascade is involved in the development of colorectal inflammatory polyps in miniature dachshunds.

Two 4-year-old spayed female Siamese cats were seized by the British Columbia Society for the Prevention of Cruelty to Animals after confinement to an abandoned housing unit without food for 9 weeks. One cat was found dead, and the second was euthanized within 24 hours due to neurologic deterioration despite therapy. Polioencephalomalacia of the caudal colliculus, hepatic lipidosis, cachexia, and congestive heart failure with cardiomyocyte atrophy were identified in both cats through postmortem examination and attributed to a prolonged period of starvation. Brain lesions were likely the result of thiamine deficiency (Chastek paralysis), which can be associated with both malnutrition and liver disease. This case highlights the importance of thiamine supplementation during realimentation of cats with hepatic lipidosis. Heart failure resulting from cachexia may have contributed to the death of the first cat and the morbidity of the second cat.

Two horses euthanized for neurologic deficits were diagnosed with hamartomatous myelodysplasia of the spinal cord. One was a 5-week-old Holsteiner colt exhibiting spasms of muscle rigidity in the extensor muscles of the limbs and epaxial muscles, and the other was a 3-year-old Thoroughbred colt exhibiting progressive ataxia and hypermetria in the pelvic limbs. Each had focal disorganization of the white and gray matter of the spinal cord forming a mass interspersed with neurons, glial cells, and disoriented axon bundles. In the Holsteiner colt, the mass was at the level of C₅ and included islands of meningeal tissue contiguous with the leptomeninges. The mass occluded the central canal forming hydromyelia cranial to the occlusion. In the Thoroughbred colt, the mass was at the level of L₁ on the dorsal periphery of the spinal cord and did not involve the central canal.

Cyprinid herpesvirus 2 is a pathogen of goldfish, inducing a disease referred to as herpesviral hematopoietic necrosis. The disease is described so far in Japan, North America, Taiwan, Australia, the United Kingdom, and recently also Italy. Here the authors describe histologic lesions in clinically affected fish in comparison with clinically normal but virus DNA–positive goldfish in Switzerland. While necrosis or enhanced single-cell necrosis in the hematopoietic tissue in the pronephros or mesonephros was evident in dead and sick animals, in clinically normal goldfish, only single-cell necrosis was observed. Virus DNA was demonstrated in dead as well as clinically affected and subclinically infected goldfish by polymerase chain reaction and in situ hybridization. This study identifies the presence of goldfish herpesvirus in Switzerland and highlights the fact that the virus might be more widespread than assumed, as clinically normal goldfish can also carry cyprinid herpesvirus 2, showing histologically similar lesions but of lesser extent and severity.

Secreted WNT proteins control cell differentiation and proliferation in many tissues, and NOTUM is a secreted enzyme that modulates WNT morphogens by removing a palmitoleoylate moiety that is essential for their activity. To better understand the role this enzyme in development, the authors produced NOTUM-deficient mice by targeted insertional disruption of the Notum gene. The authors discovered a critical role for NOTUM in dentin morphogenesis suggesting that increased WNT activity can disrupt odontoblast differentiation and orientation in both incisor and molar teeth. Although molars in Notum^-/- mice had normal-shaped crowns and normal mantle dentin, the defective crown dentin resulted in enamel prone to fracture during mastication and made teeth more susceptible to endodontal inflammation and necrosis. The dentin dysplasia and short roots contributed to tooth hypermobility and to the spread of periodontal inflammation, which often progressed to periapical abscess formation. The additional incidental finding of renal agenesis in some Notum ^-/- mice indicated that NOTUM also has a role in kidney development, with undiagnosed bilateral renal agenesis most likely responsible for the observed decreased perinatal viability of Notum^-/- mice. The findings support a significant role for NOTUM in modulating WNT signaling pathways that have pleiotropic effects on tooth and kidney development.