Organisation of the Atlantic salmon (Salmo salar) thymus and its content of Ig-expressing cells.

The thymus of fishes is located as a dual organ in a rostrodorsal projection within the gill chamber and is covered by the operculum. The histological organization of the teleost fish thymus displays considerable diversity, particularly in salmonids where a clear distinction between the thymus cortex and medulla is yet to be defined. Recent interest has focused on the role of B cells in thymic function, but the presence of these cells within the salmon thymus remains poorly understood. In this morphological study, we applied in situ hybridization to investigate developing Atlantic salmon thymi for the expression of recombination activating (Rag) genes 1 and 2. We identified the location of the cortex, aligning with the previously described inner zone. Expression of IgM and IgD transcripts was predominantly observed in cells within the outer and subcapsular zones, with lesser expression in the cortex and inner zone. IgT expression was confined to a limited number of cells in the inner zone and capsule. The location of the thymus medulla could not be established. Our results are discussed in the context of the recently identified lymphoid organs, namely the intrabranchial lymphoid tissue (ILT) and the salmon bursa.

Eptesipox virus-associated lesions in naturally infected big brown bats.

Bats have many unique qualities amongst mammals; one of particular importance is their reported tolerance to viruses without developing disease. Here, the authors present evidence to the contrary by describing and demonstrating viral nucleic acids within lesions from eptesipox virus (EfPV) infection in big brown bats. One hundred and thirty bats submitted for necropsy from Saskatchewan, Canada, between 2017 and 2021 were screened for EfPV by polymerase chain reaction (PCR); 2 had amplifiable poxvirus DNA. The lesions associated with infection were oral and pharyngeal ulcerations and joint swelling in 2/2 and 1/2 cases, respectively. These changes were nonspecific for poxvirus infection, although intracytoplasmic viral inclusion bodies within the epithelium, as observed in 2/2 bats, are diagnostic when present. Viral nucleic acids, detected by in situ hybridization (ISH), were observed in the epithelium adjacent to ulcerative lesions from both cases and within the joint proliferation of 1 case. A new isolate of EfPV was obtained from 1 case and its identity was confirmed with electron microscopy and whole genome sequencing. Juxtanuclear replication factories were observed in most cells; however, rare intranuclear virus particles were also observed. The significance of the presence of virus particles within the nucleus is uncertain. Whole genome assembly indicated that the nucleotide sequence of the genome of this EfPV isolate was 99.7% identical to a previous isolate from big brown bats in Washington, USA between 2009 and 2011. This work demonstrates that bats are not resistant to the development of disease with viral infections and raises questions about the dogma of poxvirus intracytoplasmic replication.

Claw bed inverted squamous papilloma associated with canine papillomavirus type 2 in a dog.

A claw bed inverted squamous papilloma (ISP) presented clinically as a swollen digit in a dog. Canine papillomavirus (CPV) type 2 was amplified by PCR and localised to the papilloma's epidermis using in situ hybridisation. This is the first report demonstrating a claw bed ISP caused by CPV.

Un papillome squameux inversé de la matrice unguéale est décrit cliniquement comme un gonflement du doigt chez un chien. Le papillomavirus canin (CPV) de type 2 a été amplifié par PCR et localisé dans l'épiderme du papillome par hybridation in situ. Il s'agit du premier rapport faisant état d'un papillome squameux inversé de la matrice unguéale par le CPV.

Um caso de papiloma escamoso invertido no leito ungueal em um cão apresentando aumento de volume em um dígito. O vírus do papiloma canino (CVP) Tipo 2 foi amplificado por PCR e localizado na epiderme do papiloma utilizando hibridização in situ. Este foi o primeiro relato demonstrando um papiloma escamoso invertido causado por CPV.

Un papiloma escamoso invertido del lecho ungueal se presentó clínicamente como un dedo hinchado en un perro. Se amplificó mediante PCR genoma del virus papiloma canino tipo 2 (CPV) y se localizó en la epidermis el papiloma mediante hibridación in situ. Este es el primer reporte de caso que demuestra la existencia de un papiloma escamoso invertido del lecho ungueal causado por CPV.

Granulomatous mural folliculitis in 16 domestic goats: Infection with malignant catarrhal fever viruses and colocalization with ovine herpesvirus-2 using in situ hybridization.

Granulomatous mural folliculitis (GMF) is an uncommon reaction pattern occasionally observed in nonadapted ruminant hosts infected with malignant catarrhal fever viruses. This report characterizes GMF and concurrent cutaneous lesions in 16 goats with crusting dermatitis using histochemistry including hematoxylin and eosin, periodic acid-Schiff, and Grocott's methenamine silver, and immunohistochemistry for CD3, CD20, ionized calcium binding adaptor molecule 1, and cytokeratin AE1/3. Infiltrates in all 16 GMF cases consisted of macrophages and fewer T lymphocytes, and variably included eosinophils, multinucleated histiocytic giant cells, and/or neutrophils. Formalin-fixed paraffin-embedded skin and fresh skin samples from caprine GMF cases were tested using pan-herpesvirus nested conventional polymerase chain reaction (PCR) and partial sequencing, ovine herpesvirus-2 (OvHV-2) real-time PCR, and OvHV-2 colorimetric in situ hybridization (ISH). Five of 16 goats with GMF (31%) were PCR positive for malignant catarrhal fever viruses, including caprine herpesvirus 3 in 1 goat and OvHV-2 in 4 goats. Three goats also had positive intranuclear OvHV-2 hybridization signal in follicular keratinocytes, among other cell types, localized to areas of GMF. Herpesviruses were not detected in the formalin-fixed paraffin-embedded skin of 9 goats without GMF. This case series describes relatively frequent detections of malignant catarrhal fever viruses in the skin of goats with GMF, including the first report of caprine herpesvirus 3, and localizes OvHV-2 infected follicular keratinocytes within areas of GMF.

DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 1A IN ARCHIVAL TISSUE USING RNASCOPE® IN SITU HYBRIDIZATION.

Hemorrhagic disease due to elephant endotheliotropic herpesvirus infection (EEHV-HD) is an important cause of calf mortality in managed and free-ranging Asian (Elephas maximus) and African elephant (Loxodonta spp.) populations. Consequently, infection has profound implications for elephant population growth and sustainability. The mechanisms of disease caused by EEHV (i.e., infection, dissemination, shedding, latency) are relatively undefined, in part because of a lack of robust validated assays for detecting viral gene products in relevant samples. To address this issue, we used RNAscope® in situ hybridization (ISH) based on EEHV1A DNA polymerase and terminase genes to detect EEHV1A RNA in archival formalin-fixed, paraffin-embedded Asian elephant heart and tongue from PCR-confirmed cases (n = 4) of EEHV-HD and Asian elephants (n = 2) that died from other causes. EEHV1A-positive cases had positive hybridization signal in endothelial cell nuclei of both tissues for both DNA polymerase and terminase. EEHV-negative cases lacked signal. In positive cases, the number of positive nuclei was manually assessed to provide an estimate of the viral load and compare sensitivity of the two probes. In all cases, heart had greater signal than tongue for both probes (Wilcoxon rank test; P ≤ 0.01). Overall, terminase hybridization signal was greater than DNA polymerase signal (Wilcoxon rank test; P ≤ 0.01). Results indicate RNAscope ISH is a valuable tool for detection of EEHV in archival samples and for confirming infection. Additionally, the terminase gene is the optimal target and heart is preferable to tongue for detection in cases of EEHV-HD. Results will inform future investigations of viral tropism in EEHV-HD cases due to EEHV1A.

Localization of goose haemorrhagic polyomavirus in naturally infected geese using in situ hybridization.

Goose haemorrhagic polyomavirus (GHPV) is the aetiological agent of haemorrhagic nephritis enteritis of geese (HNEG), a fatal disease that impacts geese and has been recorded only in Europe. The present study describes the first clinical cases of HNEG in Taiwan and the phylogenesis of Taiwanese GHPV, and it elucidates the pathogenesis of GHPV infection using in situ hybridization (ISH). The genomes of Taiwanese GHPV were highly similar to the previously reported strains. The diseased geese showed various degrees of vascular damage, especially in the digestive tract. The affected geese in the early stage showed transmural haemorrhagic enteritis in the intestine. In the middle to late stages, the most obvious lesion was hypoxic necrosis of renal tubules around intralobular central veins. Mineralization deposited in the kidney and systemic gout were also found. ISH revealed GHPV DNA in the vascular endothelial cells throughout the body, but not in the parenchymal cells of organs. Accordingly, the pathogenesis of GHPV infection was consistent with viral tropism in the endothelial cells. Specific attack of vascular endothelium by GHPV resulted in endothelial cell necrosis and subsequent increases of blood vessel permeability, as well as secondary circulation disorders, such as oedema, haemorrhage, and ischaemic necrosis in the adjacent parenchyma. RESEARCH HIGHLIGHTS Cell tropism of GHPV is determined by in situ hybridization. The tropism results in vascular dysfunction and subsequent pathobiology. Haemorrhagic nephritis and enteritis of geese described outside Europe for the first time.

Lethal infection caused by Tetratrichomonas gallinarum in black swans (Cygnus atratus).

BACKGROUND: Tetratrichomonas gallinarum is parasitic protozoa with a wide host range. However, its lethal infection is rare reported. CASE PRESENTATION: Here, we described the first lethal cases of T. gallinarum infection in black swans in China. Five black swans died within a week in succession without obvious symptoms except mild diarrhea. At necropsy, severe lesions were observed in caeca with thickened caecal walls and hemorrhages in the mucosa. A large number of moving trophozoites were found in the contents of the cecum by microscopic examination. The livers were enlarged with multiple bleeding spots on the surface. Histopathology of the livers showed mononuclear cell infiltration and moderate hyperplasia of fibrous tissue. The histopathology of the cecum showed that the villi of the cecum were edematous. Finally, the presence of T. gallinarum was determined by specific PCR andin-situ hybridization assay. Additionally, common pathogens that can cause similar symptoms were excluded. CONCLUSIONS: The death of the black swan was caused by T. gallinarum, suggesting that the parasite might be a new threat to the Cygnus birds.

Experimental porcine astrovirus type 3-associated polioencephalomyelitis in swine.

Porcine astrovirus type 3 (PoAstV3) is an emerging virus in the family Astroviridae that has been recently associated with polioencephalomyelitis/encephalitis. Herein, we describe the experimental oral and intravenous inoculation of an infectious central nervous system (CNS) tissue homogenate containing PoAstV3 to cesarean-derived, colostrum-deprived pigs, and the subsequent development of clinical signs, histologic lesions, specific humoral immune response, and detection of viral particles by electron microscopy (EM) and viral RNA by RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) and in situ hybridization (ISH). IgG against a portion of the PoAstV3 ORF2 capsid was first detected at 7 days post-inoculation (DPI) in 2 of 4 inoculated animals and in all inoculated animals by 14 DPI. At 21 and 28 DPI, 2 of 4 inoculated animals developed ataxia, tetraparesis, and/or lateral recumbency. All inoculated animals had histologic lesions in the CNS including perivascular lymphoplasmacytic cuffs, multifocal areas of gliosis with neuronal necrosis, satellitosis, and radiculoneuritis, and PoAstV3 RNA as detected by RT-qPCR within multiple anatomic regions of the CNS. Consistent viral structures were within the soma of a spinal cord neuron in the single pig examined by EM. Of note, PoAstV3 was not only detected by ISH in neurons of the cerebrum and spinal cord but also neurons of the dorsal root ganglion and nerve roots consistent with viral dissemination via axonal transport. This is the first study reproducing CNS disease with a porcine astrovirus strain consistent with natural infection, suggesting that pigs may serve as an animal model to study the pathogenesis of neurotropic astroviruses.

Dissecting the localization of Tilapia tilapinevirus in the brain of the experimentally infected Nile tilapia, Oreochromis niloticus (L.).

Tilapia tilapinevirus or tilapia lake virus (TiLV) is an emerging virus that inflicts significant mortality on farmed tilapia globally. Previous studies reported detection of the virus in multiple organs of the infected fish; however, little is known about the in-depth localization of the virus in the central nervous system. Herein, we determined the distribution of TiLV in the entire brain of experimentally infected Nile tilapia. In situ hybridization (ISH) using TiLV-specific probes revealed that the virus was broadly distributed throughout the brain. The strongest positive signals were dominantly detected in the forebrain (responsible for learning, appetitive behaviour and attention) and the hindbrain (involved in controlling locomotion and basal physiology). The permissive cell zones for viral infection were observed mostly to be along the blood vessels and the ventricles. This indicates that the virus may productively enter into the brain through the circulatory system and widen broad regions, possibly through the cerebrospinal fluid along the ventricles, and subsequently induce the brain dysfunction. Understanding the pattern of viral localization in the brain may help elucidate the neurological disorders of the diseased fish. This study revealed the distribution of TiLV in the whole infected brain, providing new insights into fish-virus interactions and neuropathogenesis.

Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden.

BACKGROUND: Passerine birds are frequently infected with diverse haemosporidian parasites. While infections are traditionally considered benign in wild birds, recent studies demonstrated mortalities of passerine species due to exo-erythrocytic development of the parasites, which can damage organs in affected hosts. However, exo-erythrocytic development remains insufficiently investigated for most haemosporidian species and thus little is known about the virulence of tissue stages in wild passerine birds. The aim of the present study was to investigate natural haemosporidian infections in deceased Eurasian blackbirds (Turdus merula) and song thrushes (Turdus philomelos) and to determine parasite burden and associated histological effects. METHODS: For molecular analysis, blood and tissue samples from 306 thrushes were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR. For the detection of parasite stages in organ samples, tissue sections were subjected to chromogenic in situ hybridization (CISH) using genus- and species-specific probes targeting the rRNAs of parasites. Exo-erythrocytic parasite burden was semi-quantitatively assessed and histological lesions were evaluated in haematoxylin-eosin-stained sections. RESULTS: By PCR, 179 of 277 Eurasian blackbirds and 15 of 29 song thrushes were positive for haemosporidians. Parasites of all three genera were detected, with Plasmodium matutinum LINN1 and Plasmodium vaughani SYAT05 showing the highest prevalence. CISH revealed significant differences in exo-erythrocytic parasite burden between lineages in Eurasian blackbirds, with P. matutinum LINN1 frequently causing high exo-erythrocytic parasite burdens in various organs that were associated with histological alterations. Song thrushes infected with P. matutinum LINN1 and birds infected with other haemosporidian lineages showed mostly low exo-erythrocytic parasite burdens. Two Eurasian blackbirds infected with Leucocytozoon sp. TUMER01 showed megalomeronts in various organs that were associated with inflammatory reactions and necroses. CONCLUSION: This study suggests that P. matutinum LINN1, a common lineage among native thrushes, regularly causes high exo-erythrocytic parasite burdens in Eurasian blackbirds, which may result in disease and mortalities, indicating its high pathogenic potential. The findings further illustrate that the same parasite lineage may show different levels of virulence in related bird species which should be considered when assessing the pathogenicity of haemosporidian parasite species. Finally, the study provides evidence of virulent Leucocytozoon sp. TUMER01 infections in two Eurasian blackbirds caused by megalomeront formation.

Gene expression of prostaglandin EP4 receptor in three canine carcinomas.

BACKGROUND: Chronic inflammation mediated by the cyclooxygenase enzymes, specifically their product prostaglandin E2 (PGE2), can result in the development of cancer. PGE2 promotes cell proliferation, apoptosis, and angiogenesis through interaction with its specific receptors (EP1 receptor - EP4 receptor [EP1R-EP4R]). In multiple human cancers, the expression of EP4R is associated with the development of malignancy and a poor prognosis. The expression of EP4R has not yet been evaluated in canine tumors. The aim of this study was to characterize the mRNA gene expression of EP4R (ptger4) in canine squamous cell carcinoma (SCC), apocrine gland anal sac adenocarcinoma (AGASACA), and transitional cell carcinoma (TCC). Archived tumor samples of canine cutaneous SCC (n = 9), AGASACA (n = 9), and TCC (n = 9), and matched archived normal tissue controls were evaluated for mRNA expression of canine EP4R using RNA in situ hybridization (RNAscope®). Quantification of RNAscope® signals in tissue sections was completed with an advanced digital pathology image analysis system (HALO). Data was expressed as copy number, H-index, and percent tumor cell expression of EP4R. RESULTS: In all canine SCC, AGASACA, and TCC samples evaluated, strong universal positive expression of EP4R was identified. For SCC and AGASACA, mRNA EP4R expression was statistically higher than that of their respective normal tissues. The TCC tissues displayed significantly less mRNA EP4R expression when compared to normal bladder mucosa. CONCLUSIONS: These results confirm the mRNA expression of canine EP4R in all tumor types evaluated, with SCC and AGASACA displaying the highest expression, and TCC displaying the lowest expression. This study also represents the first reported veterinary evaluation of EP4R expression using the novel in situ hybridization technique, RNAscope®.

Bovine Babesiosis Diagnosed in Formalin-Fixed, Paraffin-Embedded Tissues by Using In Situ Hybridization.

Bovine babesiosis, caused by Babesia divergens, is in general a rare disease in Europe. Nonetheless, local outbreaks can cause severe economic damage, and postmortem identification represents a diagnostic challenge. During a recent outbreak in May 2018 in northern Germany, 21 animals of a herd of 150 cattle died within 40 days having had clinical signs of fever and hemoglobinuria. Gross examination of 4 of the 21 deceased animals revealed a tick infestation, jaundice, and dark brown staining of urine and kidneys. Histologically, there were iron-positive deposits, hyperplasia of the red pulp of the spleen, and centrilobular necrosis of hepatocytes. In several locations, small basophilic granules suggestive of intraerythrocytic parasites were visible in hematoxylin-eosin- and Giemsa-stained sections. Peripheral blood smears from a living cow from the herd and polymerase chain reaction (PCR) of feeding ticks revealed B. divergens infection. In situ hybridization (ISH) was applied on formalin-fixed, paraffin-embedded (FFPE) tissue of the necropsied cattle to confirm babesiosis in these animals postmortem. Digoxigenin-labeled DNA probes were generated based on a specific nucleotide sequence for B. divergens, obtained by PCR and sequencing of DNA isolates from infected Ixodes ricinus ticks from deceased cattle. ISH using these probes allowed postmortem diagnosis of B. divergens infection in routinely fixed FFPE tissues.

Polioencephalomyelitis in Domestic Swine Associated With Porcine Astrovirus Type 3.

In the past decade, different members of the genus Mamastrovirus have been associated with outbreaks of neurologic disease in humans, cattle, sheep, mink, and, most recently, porcine astrovirus 3 (PoAstV3) in swine. We performed a retrospective analysis of 50 cases of porcine neurologic disease of undetermined cause but with microscopic lesions compatible with a viral encephalomyelitis to better understand the role and pathogenesis of PoAstV3 infection. Nucleic acid was extracted from formalin-fixed paraffin-embedded (FFPE) tissue for reverse transcription quantitative polymerase chain reaction (RT-qPCR) testing for PoAstV3. In addition, 3 cases with confirmed PoAstV3-associated disease were assayed by RT-qPCR to investigate PoAstV3 tissue distribution. PoAstV3 was detected in central nervous system (CNS) tissue via RT-qPCR and in situ hybridization in 13 of 50 (26%) FFPE cases assayed. PoAstV3 was rarely detected in any tissues outside the CNS. Positive cases from the retrospective study included pigs in various production categories beginning in 2010, the earliest year samples were available. Based on these results, PoAstV3 appears to be a recurring putative cause of viral encephalomyelitis in swine that is rarely detected outside of the CNS at the time of clinical neurologic disease, unlike other common viral causes of neurologic disease in swine.

Herpesvirus Encephalitis in a Little Blue Penguin (Eudyptula minor).

An 11-day-old little blue penguin (Eudyptula minor) died unexpectedly. Prior to hatching, the egg experienced trauma and resultant defects were repaired. The chick hatched without complication and was clinically normal prior to death. Necropsy revealed congested lungs. Histologic examination showed moderate nonsuppurative encephalitis with focally extensive neuronal necrosis and intranuclear inclusions in neurons within necrotic foci. Herpesvirus DNA was detected in brain tissue with a generic herpesvirus polymerase chain reaction. Sanger sequencing demonstrated 100% and 98% sequence homology to sphenicid alphaherpesvirus 1 and penguin herpesvirus 2, respectively. In situ hybridization demonstrated large amounts of herpesvirus nucleic acid in intranuclear inclusions and neuronal nuclei. Combined histology, polymerase chain reaction, Sanger sequencing, and in situ hybridization results were most consistent with herpesviral encephalitis, most likely caused by sphenicid alphaherpesvirus 1. To our knowledge, this is the first report of a herpesvirus infection causing encephalitis in a penguin and the first report of herpesvirus in this species.

A Subset of Equine Gastric Squamous Cell Carcinomas Is Associated With Equus Caballus Papillomavirus-2 Infection.

Squamous cell carcinoma (SCC) is the most common neoplasm of the equine stomach. However, the mechanisms underlying malignant transformation are unknown. As Equus caballus papillomavirus-2 (EcPV-2) is a likely cause of some genital SCCs, we hypothesized that EcPV-2 is associated with a subset of equine gastric SCCs. To this aim, we performed polymerase chain reaction (PCR) and in situ hybridization (ISH) for EcPV-2 E6/ E7 oncogenes on 11 gastric SCCs and on gastric samples from 15 control horses with no SCC. PCR for EcPV-2 was positive in 7/11 (64%) gastric SCCs; non-SCC gastric samples were all negative. Intense hybridization signals for EcPV-2 E6/E7 nucleic acid were detected by ISH within tumor cells in 5/11 (45%) gastric SCCs, including distant metastases. No hybridization signals were detected within any of the non-SCC gastric cases. This study provides support for a potential association between EcPV-2 infection and a subset of equine gastric SCC.

Prevalence of Equus caballus Papillomavirus Type-2 Infection and Seropositivity in Asymptomatic Western Canadian Horses.

Equus caballus papillomavirus type 2 (EcPV-2) has been recognized as a potential cause of a subset of genital squamous cell carcinomas (SCCs) in horses. In the current study, we measured EcPV-2 seropositivity in 50 healthy horses from Western Canada, and these were compared to a herd of horses with known EcPV-2 exposure. Second, the presence of EcPV-2 DNA was measured using EcPV-2-specific PCR (polymerase chain reaction), performed on a variety of tissues collected at necropsy from 70 horses that lacked any history, gross, or histologic evidence of neoplasia or papillomavirus-associated disease. EcPV-2-specific RNA in situ hybridization (R-ISH) was performed on PCR-positive samples to identify the specific tissues infected. The prevalence of asymptomatic infection with EcPV-2 in Western Canadian horses was 20/70 (29%). Exposure to EcPV-2 as measured by seropositivity was 18/50 (36%). EcPV-2 positivity by anatomic location, as measured by R-ISH, was as follows: penis 10/29 (35%), vulva 5/34 (15%), eyelid 8/68 (12%), oral mucosa 7/65 (11%), skin from muzzle 7/68 (10%), and retropharyngeal lymph node 2/64 (3%). The youngest horses with EcPV-2 infection, based on PCR, were fetuses, suggesting for the first time that vertical transmission of EcPV-2 occurs in horses. The current study observed an increased prevalence of EcPV-2 as compared to previous studies. We suggest that this difference is due to our use of biopsies in place of superficial swabs. We propose that EcPV-2 infection in asymptomatic horses is more common than previously reported and that the virus' role in equine genital SCCs may be more complex than originally thought.

Prevalence and Prognostic Impact of Equus caballus Papillomavirus Type 2 Infection in Equine Squamous Cell Carcinomas in Western Canadian Horses.

Equus caballus papillomavirus type-2 (EcPV-2) has been proposed as a causal factor in equine genital squamous cell carcinoma (SCC). This study had 2 objectives: first, calculate the frequency of papillomavirus (PV) and EcPV-2 infection in papillomas, carcinomas in situ (CIS), and SCCs in Western Canadian horses; and second, determine if EcPV-2 status of equine SCCs is associated with overall survival (OS). EcPV-2 status of 115 archived tissue samples, spanning 6 years, was determined using broad spectrum (MY09/11) and EcPV-2-specific polymerase chain reaction (PCR) assays, EcPV-2-E6/E7 chromogenic RNA in situ hybridization (R-ISH), and amplicon sequencing. A retrospective survey gathered data on history, outcome, breeding, treatment, and rationales of referring veterinarians when managing PV-associated diseases. Histologic grade and completeness of surgical margins of SCCs were also considered. EcPV-2 DNA was identified in 10/58 (17%) SCC, 8/27 (30%) papillomas, 0/5 CIS, and 0/11 lesions identified as "other." Overall, 18/101 (18%) of these lesions were positive for EcPV-2. EcPV-2 was identified in 10/35 (29%) SCCs arising from genital tissues but in 0/22 SCCs from other locations. There was no association between breeding history and EcPV-2 status of genital SCCs. EcPV-2 status of genital SCCs was not associated with OS (P = .76). The strongest negative predictors of OS were a lack of treatment (P < .01) and recurrence post-treatment (P < .01). Weaker predictors of OS included older age at time of diagnosis (P = .02). Completeness of margins at surgical excision, concurrent disease, treatment type, anatomic location of the SCC (anogenital vs other), and histologic grade of the SCC did not influence OS (P > .1).

Ibex-Associated Malignant Catarrhal Fever in Duikers (Cephalophus Spp).

Eight duikers, representing 3 different species cohoused in a single zoological collection, died in a 10-month period. Black, red-flanked, and yellow-backed duikers were affected, appearing clinically with a combination of anorexia, diarrhea, ataxia, tremors, and/or stupor, followed by death within 72 hours of onset of clinical signs. Consistent gross findings were pulmonary ecchymoses (8/8), generalized lymphadenomegaly (6/8), ascites (5/8), and pleural effusion (4/8). Dense lymphocyte infiltrates and arteritis affected numerous tissues in most animals. Ibex-associated malignant catarrhal fever (MCF) viral DNA was detected in all cases by polymerase chain reaction and in situ hybridization. Identical ibex-MCF virus sequence was detected in spleen of a clinically healthy ibex (Capra ibex) housed in a separate enclosure 35 meters away from the duikers.

Enterospora nucleophila (Microsporidia) in Gilthead Sea Bream (Sparus aurata): Pathological Effects and Cellular Immune Response in Natural Infections.

Enterospora nucleophila is a microsporidian responsible for an emaciative disease in gilthead sea bream (Sparus aurata). Its intranuclear development and the lack of in vitro and in vivo models hinder its research. This study investigated the associated lesions, its detection by quantitative polymerase chain reaction, and the cellular immune response of naturally infected fish. The intensity of infection in the intestine was correlated with stunted growth and reduced body condition. At the beginning of the outbreaks, infection prevalence was highest in intestine and stomach, and in subsequent months, the prevalence decreased in the intestine and increased in hematopoietic organs and stomach. In heavy infections, the intestine had histologic lesions of enterocyte hypercellularity and proliferation of rodlet cells. Infected enterocytes had E. nucleophila spores in the cytoplasm, and a pyknotic nucleus, karyorhexis or karyolysis. Lymphocytes were present at the base of the mucosa, and eosinophilic granule cells were located between the enterocytes. In intestinal submucosa, macrophage aggregates containing spores were surrounded by lymphocytes and granulocytes, with submucosal infiltration of granulocytes. Macrophage aggregates appeared to develop into granulomata with necrotic areas containing parasite remnants. Immunohistochemistry revealed mast cells as the main type of granulocyte involved. Abundant IgM+ and IgT+ cells were identified by in situ hybridization in the submucosa when intracytoplasmic stages were present. This study describes the lesions of E. nucleophila in gilthead sea bream, an important aquaculture species.

The first reptilian circovirus identified infects gut and liver tissues of black-headed pythons.

Viral metagenomic analysis of the liver of a black headed python (Aspidites melanocephalus) euthanized for a proliferative spinal lesion of unknown etiology yielded the first characterized genome of a reptile-infecting circovirus (black-headed python circovirus or BhPyCV). BhPyCV-specific in situ hybridization (ISH) showed that viral nucleic acids were strongly expressed in the intestinal lining and mucosa and multifocally in the liver. To investigate the presence of this virus in other snakes and its possible pathogenicity, 17 snakes in the python family with spinal disease were screened with ISH yielding a second BhP positive in intestinal tissue, and a Boelen's python (Morelia boeleni) positive in the liver. BhPyCV specific PCR was used to screen available frozen tissues from 13 of these pythons, four additional deceased pythons with and without spinal disease, and fecal samples from 37 live snakes of multiple species with unknown disease status. PCR detected multiple positive tissues in both of the ISH positive BhP and in the feces of another two live BhP and two live annulated tree boas (Corallus annulatus). Preliminary analysis indicates this circovirus can infect BhPs where it was found in 4/5 BhPs tested (2/2 with spinal disease, 2/3 live with unknown status), Boelen's python (1/2 with spinal disease), and annulated tree boa (2/6 live with unknown status) but was not detected in other python species with the same spinal lesions. This circovirus' causal or contributory role in spinal disease remains speculative and not well supported by these initial data.