Veterinary Pathology

Feline mammary carcinomas are highly malignant tumors usually associated with poor outcome. Nevertheless, survival times can differ significantly according to various prognostic factors. The Elston and Ellis (EE) histologic grading system, originally developed for human breast cancer, is commonly used to grade feline mammary carcinomas, although it is not really adapted for this species, hence the need of a more relevant grading system. Although few veterinary studies attempted to validate previously published results in an independent cohort, the aim of our study was to evaluate the prognostic value of different histologic grading systems in feline invasive mammary carcinomas, including the EE grading system applicable to human breast cancers and the modified and newly designed histologic grading systems recently proposed by Mills et al. Survey data and histologic features of 342 feline invasive mammary carcinomas were analyzed with respect to overall and cancer-specific survival. The histological grading system with best prognostic value was the mitotic-modified Elston and Ellis (MMEE) grading system: grade III carcinomas (P = .04, hazard ratio [HR] = 1.46, 95% CI, 1.01–2.11), grade II (P = .03, HR = 1.39, 95% CI, 1.03–1.88), and grade I carcinomas (HR = 1.00, reference), with decreasing hazard ratios significantly were associated with a worse overall survival, independently from the pathologic tumor size (pT ≥ 20 mm: P = .002, HR = 1.45, 95% CI, 1.15–1.83) and positive nodal stage (P = .001, HR = 1.51, 95% CI, 1.18–1.94). This retrospective study validates Mills et al’s proposal to adapt the thresholds for mitotic counts to better assess the histological grade of the highly proliferative mammary carcinomas encountered in the cat.

Johne’s disease is an enteric disease caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Upon ingestion of MAP, it is translocated across the intestinal epithelium and may be killed by intestinal macrophages, or depending on the bacterial burden and immunological status of the animal, MAP may thwart innate defense mechanisms and persist within the macrophage. This study aimed to determine the numbers of macrophages and MAP present in bovine midileal tissue during different stages of infection. Immunofluorescent (IF) labeling was performed on frozen bovine midileal intestinal tissue collected from 28 Holstein dairy cows. The number of macrophages in midileal tissue sections was higher for clinically affected cows, followed by subclinically affected cows and then uninfected control cows. Macrophages were present throughout the tissue sections in clinical cows, including the tunica muscularis, submucosa, and the lamina propria around the crypts and in the villous tips, with progressively fewer macrophages in subclinically affected and control cows. Clinically affected cows also demonstrated significantly higher numbers of MAP and higher numbers of macrophages with intracellular MAP compared to subclinically affected cows. MAP IF labeling was present within the submucosa and lamina propria around the crypts, progressing into the villous tips in some clinically affected cows. Our findings indicate that number of macrophages increases with progression of infection, but a significant number of the macrophages present in the midileum are not associated with MAP.

Salmonella is a major foodborne pathogen and pork is one of the main sources of human salmonellosis. Understanding the pathogenesis and progression of the infection within the host is of interest to establish potential approaches to control the disease in pigs. The present study evaluates factors such as intestinal colonization, fecal shedding, and pathogen persistence by 2 studies using experimental challenge with Salmonella Typhimurium in weaned pigs and euthanasia at different time points (1, 2, and 6 and 2, 14, and 30 days postinfection [dpi], respectively). Histopathology of intestine at early time points (1 dpi and 2 dpi) showed severe damage to the epithelium together with an increase in polymorphonuclear cells and macrophages (P < .001), particularly in jejunum and ileum. Large quantities of Salmonella were detected within the contents of the ileum, cecum, and colon in early infection. Salmonella could also be observed in the medulla of tonsils and mesenteric lymph nodes. From 6 dpi onward, signs of recovery were observed, with progressive restoration of the epithelium, reduction of the inflammatory infiltrate, and elimination of Salmonella from the mucosa. Concentration of Salmonella in feces and ileum content decreased, but shedding did not cease even at 4 weeks after infection. Persistence of the bacteria in mesenteric lymph nodes was identified within the connective tissue at 14 and 30 dpi. Our results demonstrate a recovery of the disease after an initial acute phase but also show persistence within the lumen and surrounding lymphoid tissue. These findings are relevant to developing effective control strategies.

Encephalitis in hamsters, which was induced by equine herpesvirus (EHV)–9, EHV-1 strain Ab4p, and zebra-borne EHV-1, was investigated and compared to assess viral kinetics and identify the progression and severity of neuropathological findings. Hamsters were inoculated with EHV-9, EHV-1 strain Ab4p, and zebra-borne EHV-1 via the nasal route and euthanized at 24, 48, 72, 96, 120, 144, and 168 hours postinoculation (HPI). The inoculated hamsters had mild to severe neurological signs at 60 to 72, 96, and 120 HPI, and the mortality rate was 75%, 0%, and 0% for animals inoculated with EHV-9, EHV-1 strain Ab4p, and zebra-borne EHV-1 viruses, respectively. Inoculated hamsters had varying degrees of rhinitis and lymphoplasmacytic meningoencephalitis, as well as differences in the severity and distribution of cerebral lesions. Furthermore, the cellular distribution of viral antigen depended on the inoculated virus. Neuronal necrosis was widely detected in animals inoculated with EHV-9, while marked perivascular cuffs of infiltrating inflammatory cells and gliosis were detected in animals inoculated with EHV-1 strain Ab4p and zebra-borne EHV-1. In the present study, 3 viruses belonging to the herpesvirus family induced encephalitis after initial propagation in the nasal cavity. These viruses might travel to the brain via the olfactory pathway and/or trigeminal nerve, showing different distributions and severities of neuropathological changes.

Equine herpesvirus 1 (EHV-1) uses equine major histocompatibility complex class I (MHC class I) as an entry receptor. Exogenous expression of equine MHC class I genes in murine cell lines confers susceptibility to EHV-1 infection. To examine the in vivo role of equine MHC class I as an entry receptor for EHV-1, we generated transgenic (Tg) mice expressing equine MHC class I under the control of the CAG promoter. Equine MHC class I protein was expressed in the liver, spleen, lung, and brain of Tg mice, which was confirmed by Western blot. However, equine MHC class I antigen was only detected in bronchiolar epithelium and not in other tissues, using the immunofluorescence method employed in this study. Both Tg and wild-type (WT) mice developed pneumonia 3 days after intranasal infection with EHV-1. The bronchiolar epithelial cells of Tg mice showed more severe necrosis, compared with those in WT mice. In addition, the number of virus antigen-positive cells in the lungs was higher in Tg mice than in WT mice. These results suggest that exogenous expression of equine MHC class I renders mice more susceptible to EHV-1 infection.

Porcine epidemic diarrhea virus (PEDV) induces an often fatal gastrointestinal disease in piglets. In this study, we performed a PEDV infection experiment with the Microminipig, the smallest of experimental minipigs, as a novel small animal model. We orally inoculated a neonatal Microminipig with an intestinal homogenate of a PEDV-infected pig and housed it in a small cage originally designed for rats in an animal biosafety level 2 facility. The infected Microminipig showed the typical signs of porcine epidemic diarrhea (PED), such as watery diarrhea, loss of appetite and weight loss. We also recognized a high amount of excreted PEDV in its rectal swabs and villus atrophy of the small intestine. These results suggest that the Microminipig is a good small animal model for PED, which may contribute to a better understanding of the pathogenesis of PEDV.

Senescent cells accumulate with age but tissue-based studies of senescent cells are limited to selected organs from humans, mice, and primates. Cell culture and xenograft studies have indicated that senescent cells in the microenvironment may play a role in tumor proliferation via paracrine activities. Dogs develop age-related conditions, including in the testis, but cellular senescence has not been confirmed. We hypothesized that senescent cells accumulate with age in canine testes and in the microenvironment of testicular tumors. We tested the expression of the established senescence markers γH2AX and p21 on normal formalin-fixed, paraffin-embedded testes from 15 young dogs (<18 months of age) and 15 old dogs (7–15 years of age) and correlated the findings with age-dependent morphological changes. A statistically significant age-dependent increase in the percentage of p21-expressing cells was observed for testicular fibroblasts (4-fold) and Leydig cells (8-fold). However, p21-expressing cells were still a rare event. In contrast, the percentage of γH2AX-positive cells did not increase with age. P21- and γH2AX-expressing cells were rare in the microenvironments of tumors. Age-dependent morphological changes included an increased mean number of Leydig cells per intertubular triangle (2.95-fold) and a decreased spermatogenesis score. To our surprise, no age-related changes were recorded for interstitial collagen content, mean tubular diameter, and epithelial area. Opposed to our expectations based on previous in vitro data, we did not identify evidence of a correlation between age-associated accumulation of senescent cells and testicular tumor development. Understanding the role of the microenvironment in senescence obviously remains a challenging task.

Digital microscopy (DM) has been employed for primary diagnosis in human medicine and for research and teaching applications in veterinary medicine, but there are few veterinary DM validation studies. Region of interest (ROI) digital cytology is a subset of DM that uses image-stitching software to create a low-magnification image of a slide, then selected ROI at higher magnification, and stitches the images into a relatively small file of the embedded magnifications. This study evaluated the concordance of ROI-DM compared to traditional light microscopy (LM) between 2 blinded clinical pathologists. Sixty canine and feline cytology samples from a variety of anatomic sites, including 31 cases of malignant neoplasia, 15 cases of hyperplastic or benign neoplastic lesions, and 14 infectious/inflammatory lesions, were evaluated. Two separate nonblinded adjudicating clinical pathologists evaluated the reports and diagnoses and scored each paired case as fully concordant, partially concordant, or discordant. The average overall concordance (full and partial concordance) for both pathologists was 92%. Full concordance was significantly higher for malignant lesions than benign. For the 40 neoplastic lesions, ROI-DM and LM agreed on general category of tumor type in 78 of 80 cases (98%). ROI-DM cytology showed robust concordance with the current gold standard of LM cytology and is potentially a viable alternative to current LM cytology techniques.

Articular osteochondrosis (OC) arises due to vascular failure and ischemic chondronecrosis. The aim of the study was to describe the histological and computed tomographic (CT) characteristics of changes in the distal femoral physis of pigs, to determine if they represented OC lesions and if the pathogenesis was the same as for articular OC. The material included 19 male Landrace pigs bred for predisposition to OC. One or 2 pigs were euthanized and CT-scanned at 2-week intervals from 82 to 180 days of age. Material from 10 pigs was available for histological validation. The CT scans revealed 31 lesions confirmed in 3 planes and 1 additional macroscopically visible lesion confirmed in 2 CT planes. Twelve of the lesions were histologically validated. All lesions were compatible with OC. Cartilage canal and eosinophilic streak morphological changes corresponded to failure of end arteries coursing from the epiphysis, toward the metaphysis. The location of lesions was compatible with failure at the point of vessel incorporation into bone. Vascular failure was associated with retention of viable hypertrophic chondrocytes and delayed ossification but not cartilage necrosis. Lesion width ranged from 1.1% to 45.6% of the physis. Several lesions were expected to resolve due to small size and evidence of CT-identifiable, reparative ossification. Angular limb deformity was not detected in any pig. The pathogenesis of physeal OC started with vascular failure that was morphologically identical to articular OC. The heritable predisposition may therefore be the same. The association between lesions and limb deformity should be studied further in older pigs in future.

A neurological disease was investigated in 3 German Shepherd pups from the same litter that failed to grow normally, appeared stiff, were reluctant to move, and were deaf. They developed intermittent seizures and ataxia and had proprioceptive defects. Histopathology showed severe vacuolation of neurons, astrocytes in nervous tissue, renal tubular epithelial cells, and macrophages in nervous tissue, spleen, and liver. Vacuoles appeared empty with no storage material stained by periodic acid–Schiff (PAS) or Sudan black stains, leading to a diagnosis of a lysosomal storage disease and in particular an oligosaccharidosis. Biochemical and genomic studies showed that this was β-mannosidosis, not previously diagnosed in dogs. A c.560T>A transition in exon 4 of the MANBA gene was found, which segregated in these and other family members in a manner consistent with it being the causative mutation of an autosomal recessive disease. This mutation led to substitution of isoleucine to asparagine at position 187 of the 885 amino acid enzyme, a change expected to have functional significance.

This study describes clinical and histopathological features, treatment, and outcome of cats diagnosed with ocular mycobacteriosis. Cases diagnosed from 2012 to 2017 were reviewed for (a) histopathological evidence of ocular (pyo)granulomatous inflammation containing acid-fast bacilli with mycobacterial morphology, (b) positive mycobacterial culture and/or mycobacterial DNA identified by polymerase chain reaction of ocular tissue, or (c) presumed mycobacteriosis based on ophthalmic examination and positive interferon-gamma release assay. Twenty-five cats (31 eyes) were included; 14 cats (17/31 eyes, 55%) were blind at presentation (unilateral: n = 12 cats; bilateral: n = 2 cats); one unilaterally affected cat later became bilaterally blind. Another 5 cats (7/31 eyes, 23%) became blind after initially being bilaterally visual (unilateral: n = 3 cats; bilateral: n = 2 cats). The commonest ocular finding was uveitis (87%). The main histopathological features were granulomatous to pyogranulomatous chorioretinitis with retinal detachment, anterior uveitis, optic neuritis, episcleritis, scleritis, and/or retrobulbar cellulitis. Nineteen cats (76%) had systemic signs, with disseminated disease being diagnosed in 9, defined by interstitial pulmonary disease, generalized lymphadenopathy, and/or nonocular infection. Nine cats were diagnosed with Mycobacterium bovis, 2 with Mycobacterium microti, 1 with Mycobacterium tuberculosis complex, and 1 with Mycobacterium avium-intracellulare complex. The infecting species was unknown in the remaining cats. Combined surgery (enucleation: n = 5 cats; biopsy: n = 3 cats) and systemic treatment with 2 or 3 appropriate antibiotics for 2 to 7 months resulted in remission in 8 of the 10 cats treated; however, the cat treated with dual therapy relapsed after 8 months. A total of 16 cats (64%) were euthanized; 2 were lost to follow-up.

Myocarditis can cause death or permanent heart damage. As epidemiologic and etiopathologic data for canine myocarditis are lacking, we performed a retrospective study using nucleic acid extracted from archived (2007 to 2015) tissues from myocarditis cases and control dogs without myocardial lesions. Heart tissue from pediatric/juvenile and adult dogs was tested with a comprehensive panel of conventional and real-time polymerase chain reaction (PCR) assays targeting recognized agents of canine myocarditis based on a literature review and informed by the comparative epidemiology of human myocarditis. The PCR screen, which included canine parvovirus 2 (CPV-2), canine distemper virus, canine herpesvirus, Borrelia spp, West Nile virus, adenovirus, parainfluenza virus, pneumovirus, respiratory coronavirus, influenza virus, Bartonella spp, Rickettsia spp, Mycoplasma spp, and Neospora caninum, did not detect agents in 35 of 66 cases (53%; 95% confidence interval [CI], 41%–65%) and was frequently negative in adults (21/26); by comparison, agents were not detected in 27 of 57 controls (47%; 95% CI, 35%–60%). Canine distemper virus, herpesvirus, adenovirus, coronavirus, parainfluenza virus, Mycoplasma haemocanis, and N. caninum were occasionally detected in both cases and controls; thus, PCR detection was not considered to indicate causation. We previously reported that CPV-2 continues to be associated with myocarditis in young dogs despite widespread vaccination; in adults, CPV-2 was detected in 2 of 26 cases and 4 of 22 controls. As several agents were similarly detected in cases and controls, it is unclear if these are cardiopathogenic, incidental, or latent. West Nile virus was detected at the analytic limit in 1 adult case. We did not detect Borrelia spp, Bartonella spp, Rickettsia spp, or influenza A virus in the myocarditis cases. These data demonstrate the limitations of current targeted diagnostic tests and the need for additional research to identify unknown agents and develop testing strategies for canine myocarditis.

Pathologic features of 12 cats with naturally acquired systemic hypertension and concomitant hypertensive encephalopathy were analyzed. All cats demonstrated acute onset of signs localized to the forebrain and/or brainstem, including stupor, coma, and seizures. All cats had systemic hypertension, ranging from 160 to 300 mm Hg. Gross lesions were identified in 4 of 12 cases, including caudal herniation of the cerebrum and cerebellum, sometimes with compression of the rostral colliculus and medulla. Histologically, all cases featured bilaterally symmetrical edema of the cerebral white matter. Associated vascular lesions, especially arteriolar hyalinosis, were also observed. Concurrent lesions were chronic tubulointerstitial nephritis (11/12 cases), adenomatous hyperplasia of the thyroid gland (4 cases), hypertensive choroidal arteriopathy (6 cases), and left ventricular hypertrophy (5 cases). This study demonstrates that the typical histologic manifestation of spontaneous hypertensive encephalopathy in cats is bilaterally symmetrical edema of the subcortical cerebral white matter.

Proliferation of ectopic Schwann cells within the central nervous system (CNS) parenchyma (schwannosis) in early life is most commonly associated with human neurofibromatosis type-2 and has been unrecognized in domestic animals. Three foals and a calf, 5 to 11 weeks old, with progressive neurological signs from birth were studied. Histologically, at multiple levels of the spinal cord, all animals had bilateral plaques of proliferative spindle cells, predominantly affecting the white matter adjacent to dorsal and ventral nerve roots and variably extending into the gray matter. Proliferating cells had strong intracytoplasmic immunoreactivity for the Schwann cell markers myelin protein zero and periaxin, highlighting the formation of peripheral nervous system (PNS) myelin within the spinal cord. In all cases, foci of disorganized neural tissue (glioneuronal hamartomas) were present, which in 2 cases formed a mass effect that resulted in syringohydromyelia. Neonatal presentation suggests a congenital maldevelopment of the nervous system, with spontaneous invasion of PNS-derived Schwann cells into the CNS.

Chlamydia pneumoniae is a ubiquitous pathogen causing disease in humans, mammals, birds, reptiles, and amphibians. Since 2012, C. pneumoniae infection has caused neurologic disease and mortality in a breeding colony of endangered Houston toads (Anaxyrus houstonensis) at the Houston Zoo. The purpose of this report is to present the histopathologic and ultrastructural characteristics of C. pneumoniae infection in Houston toads. Fourteen cases were evaluated by histopathology and 1 case was evaluated by electron microscopy. The major histopathologic finding was necrotizing and histiocytic polioencephalomyelitis and ganglionitis. Bacteria formed intracytoplasmic inclusions within neurons but frequently extended into the surrounding tissue from necrotic cells. Ultrastructural evaluation showed the bacteria formed reticulate and elementary bodies characteristic of Chlamydia spp.

We identified multiple extraintestinal cystacanths during routine postmortem examination of 3 small Indian mongooses and 2 African green monkeys from the Caribbean island of St. Kitts. In mongooses, cystacanths were encysted or free in the subcutaneous tissue, skeletal muscle, or peritoneal or pericardial cavities, whereas in the monkeys, they were in the cavity and parietal layer of the, tunica vaginalis, skeletal muscle, and peritoneal cavity. Morphological, histological, and molecular characterization identified these cystacanths as Oncicola venezuelensis (Acanthocephala: Oligacanthorhynchidae). There was minimal to mild lymphoplasmacytic inflammation associated with the parasite in the mongooses and moderate inflammation, mineralization, hemorrhage, and fibrosis in the connective tissue between the testis and epididymis in 1 monkey. We identified a mature male O. venezuelensis attached in the aboral jejunum of a feral cat, confirming it as the definitive host. Termites serve as intermediate hosts and lizards as paratenic hosts. This report emphasizes the role of the small Indian mongoose and African green monkey as paratenic hosts for O. venezuelensis.

During a screen for vascular phenotypes in aged laboratory mice, a unique discrete phenotype of hyaline arteriolosclerosis of the intertubular arteries and arterioles of the testes was identified in several inbred strains. Lesions were limited to the testes and did not occur as part of any renal, systemic, or pulmonary arteriopathy or vasculitis phenotype. There was no evidence of systemic or pulmonary hypertension, and lesions did not occur in ovaries of females. Frequency was highest in males of the SM/J (27/30, 90%) and WSB/EiJ (19/26, 73%) strains, aged 383 to 847 days. Lesions were sporadically present in males from several other inbred strains at a much lower (<20%) frequency. The risk of testicular hyaline arteriolosclerosis is at least partially underpinned by a genetic predisposition that is not associated with other vascular lesions (including vasculitis), separating out the etiology of this form and site of arteriolosclerosis from other related conditions that often co-occur in other strains of mice and in humans. Because of their genetic uniformity and controlled dietary and environmental conditions, mice are an excellent model to dissect the pathogenesis of human disease conditions. In this study, a discrete genetically driven phenotype of testicular hyaline arteriolosclerosis in aging mice was identified. These observations open the possibility of identifying the underlying genetic variant(s) associated with the predisposition and therefore allowing future interrogation of the pathogenesis of this condition.