A putatively novel papillomavirus associated with cutaneous plaques and squamous cell carcinoma in captive North American snow leopards (Panthera uncia).

Cutaneous plaques and squamous cell carcinoma (SCC) are common in captive North American snow leopards (SLs) (Panthera uncia). Our objective was to determine whether these lesions are potentially associated with papillomavirus(es). Polymerase chain reaction (PCR) was performed on 3 cutaneous plaques using degenerate primers for papillomaviruses. A putatively novel papillomavirus was identified that shared 76% sequence identity to Felis catus papillomavirus 2. Specific PCR for this virus was performed on 5 cutaneous SCC samples and 7 normal skin samples, which were all positive. In situ hybridization for this putatively novel virus was performed, which revealed strong hybridization signals within hyperplastic cells in cutaneous plaques (n = 3) and within neoplastic cells in cutaneous SCC samples (n = 5). No hybridization signals were identified within normal skin. Ultimately, identification of a causal viral agent in the development of plaques and SCC in SLs will help guide therapeutic intervention and lay the foundation for development of prophylactic vaccines.

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.

HOPX+ injury-resistant intestinal stem cells drive epithelial recovery after severe intestinal ischemia.

Intestinal ischemia is a life-threatening emergency with mortality rates of 50%-80% due to epithelial cell death and resultant barrier loss. Loss of the epithelial barrier occurs in conditions including intestinal volvulus and neonatal necrotizing enterocolitis. Survival depends on effective epithelial repair; crypt-based intestinal epithelial stem cells (ISCs) are the source of epithelial renewal in homeostasis and after injury. Two ISC populations have been described: 1) active ISC [aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 (LGR5+)-positive or sex-determining region Y-box 9 -antigen Ki67-positive (SOX9+Ki67+)] and 2) reserve ISC [rISC; less proliferative; homeodomain-only protein X positive (HOPX+)]. The contributions of these ISCs have been evaluated both in vivo and in vitro using a porcine model of mesenteric vascular occlusion to understand mechanisms that modulate ISC recovery responses following ischemic injury. In our previously published work, we observed that rISC conversion to an activated state was associated with decreased HOPX expression during in vitro recovery. In the present study, we wanted to evaluate the direct role of HOPX on cellular proliferation during recovery after injury. Our data demonstrated that during early in vivo recovery, injury-resistant HOPX+ cells maintain quiescence. Subsequent early regeneration within the intestinal crypt occurs around 2 days after injury, a period in which HOPX expression decreased. When HOPX was silenced in vitro, cellular proliferation of injured cells was promoted during recovery. This suggests that HOPX may serve a functional role in ISC-mediated regeneration after injury and could be a target to control ISC proliferation.NEW & NOTEWORTHY This paper supports that rISCs are resistant to ischemic injury and likely an important source of cellular renewal following near-complete epithelial loss. Furthermore, we have evidence that HOPX controls ISC activity state and may be a critical signaling pathway during ISC-mediated repair. Finally, we use multiple novel methods to evaluate ISCs in a translationally relevant large animal model of severe intestinal injury and provide evidence for the potential role of rISCs as therapeutic targets.

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.

Use of a bipolar sealing device to seal partial cystectomy with and without augmentation with a single-layer simple continuous suture pattern in an ex vivo canine model.

OBJECTIVE: To evaluate the ability of a bipolar sealing device (BSD) to seal canine bladder tissue and to determine the influence of suture augmentation on resistance to leakage of sealed partial cystectomies. STUDY DESIGN: Ex vivo, simple randomized study. SAMPLE POPULATION: Urinary bladders harvested from canine cadavers (n = 23). METHODS: Partial cystectomy of the cranial third of each bladder was performed with a BSD. This seal was augmented with a simple continuous pattern of 4-0 polydioxanone in half of the specimens. A pressure transducer inserted through the ureter measured intraluminal pressure at initial leakage and catastrophic failure as dyed saline was infused via a catheter inserted through the urethra. Initial leakage pressure and pressure at catastrophic failure were compared between sutured and nonsutured sealed partial cystectomies. RESULTS: Sutured sealed cystectomies showed initial leakage at lower pressures compared to non-sutured cystectomies (8.6 vs. 17.7 mm Hg; P = .0365) but were able to sustain greater pressures at catastrophic failure (34.3 vs. 21.8 mm Hg; P = .007). Catastrophic failure occurred along the seam of all nonsutured sealed cystectomies and at the suture holes in 10 of the 12 sutured bladders. CONCLUSION: Partial cystectomies were effectively sealed with a BSD in this canine cadaveric bladder model. Augmentation with a simple continuous suture pattern increased the pressure at which catastrophic leakage occurred but lowered initial leak pressure. CLINICAL SIGNIFICANCE: This study provides evidence supporting the evaluation of BSD use for partial cystectomy in live animals.

Localization of Felis catus Papillomavirus Type 2 E6 and E7 RNA in Feline Cutaneous Squamous Cell Carcinoma.

Findings from polymerase chain reaction-based methods have suggested a role of Felis catus papillomavirus 2 (FcaPV-2) in the development of feline cutaneous squamous cell carcinoma (SCC). However, because polymerase chain reaction cannot localize deoxyribonucleic acid or ribonucleic acid within the lesion, it is difficult to differentiate a coincidental FcaPV-2 infection and a causative association. Given that a key event in the pathogenesis of human papillomavirus-induced cancer is the expression of viral E6 and E7 oncogenes, localization of FcaPV-2 E6 and E7 transcription within neoplastic cells in feline SCCs would support a causative role for this papillomavirus. Therefore, RNAscope in situ hybridization was used to localize FcaPV-2 E6 and E7 transcripts in 18 formalin-fixed paraffin-embedded samples of cutaneous SCC. Positive signals were present within 5 of 9 samples (56%) from ultraviolet-protected sites and 0 of 9 samples from ultraviolet-exposed sites. In the 4 in situ hybridization-positive samples that contained adjacent hyperplastic skin, hybridization patterns in these regions were characterized by intense nuclear signals within the superficial epidermis and punctate signals within the basal epithelial layers. However, within the 5 SCCs, punctate signals were present within all layers of the epidermis, with progressive loss of intense nuclear signals within the superficial epidermis. This hybridization pattern is consistent with unregulated E6 and E7 transcription and decreased viral replication and is similar to the pattern observed in human papillomavirus-induced cancers as they progress from hyperplastic lesions containing productive infections to nonproductive neoplasms. These findings support a causative role for FcaPV-2 in the pathogenesis of feline SCC.

Generalized papillomatosis in three horses associated with a novel equine papillomavirus (EcPV8).

BACKGROUND: Currently, seven equine papillomaviruses (PV) are known and are associated with one of three different and distinct clinical presentations. HYPOTHESIS/OBJECTIVES: To report the clinical, histopathological and immunohistochemical findings in horses with generalized papillomatosis associated with a novel equine PV, Equus caballus papillomavirus 8 (EcPV8). ANIMALS: Three client-owned quarter horses. METHODS: Case report, retrospective. RESULTS: Dozens to thousands of papillomas involved the axilla, inguinal area and proximal limbs as well as the ventral and lateral neck, thorax and abdomen. Lesions were sometimes confluent in ventral areas. Fewer lesions were on the face, ears, distal limbs and genitalia. Plaque-type papillomas were small, 0.5 to 1.5 cm in diameter and hyperkeratotic. Histologically, plaque-type papillomas prominently involved follicular infundibula. Immunohistochemical findings demonstrated PV antigen in superficial keratinocyte nuclei. PCR using degenerate primers for the PV L1 gene and sequencing of amplicons revealed PV DNA sequences that were 98% identical for all three cases, but shared <70% identity to other PVs. Horses were otherwise healthy; serum immunoglobulin levels and peripheral blood lymphocyte phenotyping did not identify a known immunodeficiency syndrome. Lesions nearly completely resolved after 1.5 years in one horse and persisted for two years in another, despite intralesional human IFN-alpha treatment. The oldest horse was lost to follow-up. CONCLUSION AND CLINICAL IMPORTANCE: A novel equine papillomavirus (EcPV8) is associated with a distinct, plaque-type, generalized papillomatosis. Papillomas persisted for months to years, with or without treatment.

Does hepatic steatosis have an impact on the short term hepatic response after complete attenuation of congenital extrahepatic portosystemic shunts? A prospective study of 20 dogs.

OBJECTIVE: To evaluate the relationship between hepatic steatosis and increase in liver size and resolution of shunting after surgical attenuation of congenital extrahepatic portosystemic shunts in dogs. STUDY DESIGN: Prospective study. ANIMALS: Dogs (n = 20) with congenital extrahepatic portosystemic shunts. METHODS: Shunts were attenuated using ameroid ring constrictors. Portal blood flow and liver volume were evaluated using computed tomography before and ≥8 weeks after surgery. Hepatic steatosis was quantified by stereological point counting of lipid droplets and lipogranulomas (LG) in liver biopsies stained with Oil-red-O. Associations between steatosis and preoperative liver volume, liver growth after surgery, and development of acquired shunts were evaluated. RESULTS: Acquired shunts developed in 2 dogs (10%). Dogs with larger preoperative liver volumes relative to bodyweight had fewer lipid droplets per tissue point (P = .019). LG per tissue point were significantly associated with age: 0.019 ± 0.06 for dogs <12 months versus 0.25 ± 0.49 for dogs >12 months (P = .007). There was a significant positive association between liver growth after surgery and the number of LG/month of age in dogs >12 months (P = .003). There was no association between steatosis, presence of macrosteatosis, the number of LG or development of acquired shunts. CONCLUSIONS: This preliminary study suggests that the presence of hepatic lipidosis and LG has no demonstrable effect on development of acquired shunts or the magnitude of increase in liver volume after attenuation of congenital extrahepatic portosystemic shunts in dogs.

Complete genome sequence of canine papillomavirus type 9.

Papillomaviruses are nonenveloped, double-stranded DNA viruses that are associated with both benign and malignant tumors in animals and humans. We report the complete genome sequence of canine papillomavirus type 9 isolated from a solitary pigmented plaque on a mixed-breed bloodhound.

Complete genome sequence of canine papillomavirus type 10.

Papillomaviruses are epitheliotropic, nonenveloped, circular, double-stranded DNA viruses within the family Papillomaviridae that are associated with benign and malignant tumors in humans and animals. We report the complete genome sequence of canine papillomavirus type 10 identified from a pigmented plaque located on the head of a mixed-breed bloodhound.

Clinicopathological findings of canine seborrhoeic keratosis with comparison to pigmented viral plaques.

BACKGROUND: Seborrhoeic keratoses (SKs) are common benign epidermal neoplasms in humans and are rarely diagnosed in the dog. These circumscribed, raised, variably pigmented plaques arise in middle aged to older humans, with a focal or multicentric distribution; although common, the underlying cause of these lesions is not known. Although less common in the dog, the lesions are similar and have features that overlap with papillomavirus-associated pigmented viral plaques. HYPOTHESIS/OBJECTIVES: Seborrhoeic keratoses in the dog are negative for canine papillomavirus. ANIMALS: Eleven cases of SK from a 12 year period were reviewed. METHODS: This was a retrospective study of the histopathological findings and case histories. Complete clinical records following collection of the skin biopsy were available in five of 11 cases. Immunohistochemistry was performed for all cases; PCR analysis was carried out for papillomavirus in six cases. RESULTS: Histologically, SKs had an exophytic to mildly endophytic epidermal proliferation, creating a papillomatous to acanthotic, hyperkeratotic, frequently pigmented plaque. There was an absence of hypergranulosis or viral cytopathic effect; PCRs for canine papillomavirus within the formalin-fixed paraffin-embedded skin biopsies were negative. No breed, sex or site predilection was recognized. The mean age at biopsy of the lesions was 8.8 years (range 5-14 years). CONCLUSIONS AND CLINICAL IMPORTANCE: Histopathological features and negative papillomavirus status distinguish SK as an important differential diagnosis for pigmented viral plaques in dogs.

A subset of equine oral squamous cell carcinomas is associated with Equus caballus papillomavirus 2 infection.

The aetiology of oral squamous cell carcinoma (SCC) in horses is unknown, but papillomavirus infection as well as chronic periodontal disease are suspected to play a pathogenic role. In humans, some oropharyngeal cancers develop in association with human papillomaviruses. Equus caballus papillomavirus 2 (EcPV2) is suspected to play a causal role in the development of equine genital SCC. Given that association, we hypothesized that EcPV2 is associated with the development of oral SCC in horses. We performed standard polymerase chain reaction (PCR) and in-situ hybridization (ISH) for EcPV2 on 31 formalin-fixed paraffin-embedded equine oral SCCs (lingual, gingival, palate) and 10 equine non-SCC oral samples. PCR for EcPV2 was positive in 10/31 (32%) oral SCCs while all non-SCC oral samples were negative. Intense hybridization signals for EcPV2 nucleic acid were detected by ISH within neoplastic epithelial cells in 8/31 (26%) oral SCCs but not in the adjacent normal oral mucosa. No hybridization signals were detected within control samples. This study provides additional support for a pathogenic association of EcPV2 in oral SCC in horses.

Papillomaviral skin diseases of humans, dogs, cats and horses: A comparative review. Part 2: Pre-neoplastic and neoplastic diseases.

Papillomaviruses (PVs) are well recognized to cause pre-neoplastic and neoplastic diseases in humans. Similarly, there is increasing evidence that PVs play a significant role in the development of pre-neoplastic and neoplastic diseases of the haired skin of dogs and cats, and the mucosa of horses. As the mechanisms by which PVs cause neoplasia are well studied in humans, it is valuable to compare the PV-induced neoplasms of humans with similar PV-associated neoplasms in the companion animal species. In the second part of this comparative review, the pre-neoplastic and neoplastic diseases thought to be caused by PVs in humans, dogs, cats, and horses are described. This includes PV-induced cutaneous plaques, cutaneous squamous cell carcinomas (SCCs) and mucosal SCCs within the four species. The review concludes with a discussion about the potential use of vaccines to prevent PV-induced diseases of dogs, cats, and horses.

Papillomaviral skin diseases of humans, dogs, cats and horses: A comparative review. Part 1: Papillomavirus biology and hyperplastic lesions.

Papillomaviruses (PVs) cause disease in humans, dogs, cats, and horses. While there are some differences, many aspects of the pathogenesis, presentation, and treatment of these diseases are similar between the four species. In this review, the PV-induced diseases of humans are compared to the similar diseases that develop in the companion animal species. By comparing with the human diseases, it is possible to make assumptions about some of the less common and less well-studied diseases in the veterinary species. In the first part of this review, the PV lifecycle is discussed along with the classification of PVs and the immune response to PV infection. The hyperplastic diseases caused by PVs are then discussed; including PV-induced cutaneous, anogenital, and oral warts within the four species.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Novel amdovirus in gray foxes.

We used viral metagenomics to identify a novel parvovirus in tissues of a gray fox (Urocyon cinereoargenteus). Nearly full genome characterization and phylogenetic analyses showed this parvovirus (provisionally named gray fox amdovirus) to be distantly related to Aleutian mink disease virus, representing the second viral species in the Amdovirus genus.

Canine Papillomavirus 2 E6 Does Not Interfere With UVB-Induced Upregulation of p53 and p53-Regulated Genes.

Cutaneous papillomaviruses are oncogenic viruses that cause severe, persistent infections that can develop into skin cancers within ultraviolet (UV)-exposed skin of immunodeficient individuals, such as those with X-linked severe combined immunodeficiency (XSCID). A canine research model of XSCID exhibits a similar phenotype; these dogs develop severe canine papillomavirus 2 (CPV2) infections that often progress to cancer. Thus, the dog is a natural, spontaneous model to investigate cutaneous papillomavirus infections in immunodeficient patients. The human papillomavirus oncogene E6 contributes to cancer development, in part, by initiating degradation of the tumor suppressor protein p53, or by inhibiting upregulation of p53-dependent genes required within the cell growth arrest and apoptotic pathways, thereby leading to an accumulation of DNA damage required for oncogenesis. Currently, little is known about CPV2, and how it promotes cancer development. The aim of this study was to determine if CPV2 oncogene E6 similarly affects p53 upon activation by UV radiation, as well as the downstream p53-regulated genes necessary to control growth arrest and apoptosis. We determined that cutaneous CPV2 E6 does not degrade p53, or interfere with the upregulation of p53-regulated genes p21, Bax, Bak, or lncRNA-p21, suggesting that CPV2 may use a p53-independent mechanism to contribute to oncogenesis.

Unlocking the Role of a Genital Herpesvirus, Otarine Herpesvirus 1, in California Sea Lion Cervical Cancer.

Urogenital carcinoma in California sea lions (Zalophus californianus) is the most common cancer of marine mammals. Primary tumors occur in the cervix, vagina, penis, or prepuce and aggressively metastasize resulting in death. This cancer has been strongly associated with a sexually transmitted herpesvirus, otarine herpesvirus 1 (OtHV1), but the virus has been detected in genital tracts of sea lions without cancer and a causative link has not been established. To determine if OtHV1 has a role in causing urogenital carcinoma we sequenced the viral genome, quantified viral load from cervical tissue from sea lions with (n = 95) and without (n = 163) urogenital carcinoma, and measured viral mRNA expression using in situ mRNA hybridization (Basescope®) to quantify and identify the location of OtHV1 mRNA expression. Of the 95 sea lions diagnosed with urogenital carcinoma, 100% were qPCR positive for OtHV1, and 36% of the sea lions with a normal cervix were positive for the virus. The non-cancer OtHV1 positive cases had significantly lower viral loads in their cervix compared to the cervices from sea lions with urogenital carcinoma. The OtHV1 genome had several genes similar to the known oncogenes, and RNA in situ hybridization demonstrated high OtHV1 mRNA expression within the carcinoma lesions but not in normal cervical epithelium. The high viral loads, high mRNA expression of OtHV1 in the cervical tumors, and the presence of suspected OtHV1 oncogenes support the hypothesis that OtHV1 plays a significant role in the development of sea lion urogenital carcinoma.

Unexpected but transient tumour enlargement preceded complete regression and long-term control after irradiation of squamous cell carcinoma in a red-eared slider (Trachemys scripta elegans).

A red-eared slider with a chronic non-healing ulcerative shell lesion was diagnosed with cutaneous squamous cell carcinoma (SCC). The animal underwent surgical debulking, and adjuvant hypofractionated radiation therapy. The lesion initially responded, with near complete tumour regression, but then began growing again just a few months after finishing radiotherapy. Then, after several months with no additional tumour-directed therapy, the lesion again regressed. Five years post-irradiation and with no further treatment, the turtle now remains tumour-free. This unusual pattern of disease regression followed by transient growth and then long-term local tumour control suggests either spontaneous remission, or a pseudoprogression-like phenomenon. Careful clinical follow-up and reporting of future cases will aid in determining whether this pseudoprogression-like event was random, versus being a common component of the chelonian response to irradiation of cutaneous SCC.

Abrogation of Constitutive and Induced Type I and Type III Interferons and Interferon-Stimulated Genes in Keratinocytes by Canine Papillomavirus 2 E6 and E7.

Cutaneous papillomaviruses can cause severe, persistent infections and skin cancer in immunodeficient patients, including people with X-linked severe combined immunodeficiency (XSCID). A similar phenotype is observed in a canine model of XSCID; these dogs acquire severe cutaneous papillomavirus infections that can progress to cancer in association with canine papillomavirus type 2 (CPV2). This canine model system provides a natural spontaneous animal model for investigation of papillomavirus infections in immunodeficient patients. Currently, it is unknown if CPV2 can subvert the innate immune system and interfere with its ability to express antiviral cytokines, which are critical in the host defense against viral pathogens. The aim of the current study was to determine if the oncogenes E6 and E7 from CPV2 interfere with expression of antiviral cytokines in keratinocytes, the target cells of papillomavirus infections. We determined that E6 but not E7 interferes with the constitutive expression of some antiviral cytokines, including interferon (IFN)-ß and the IFN-stimulated gene IFIT1. Both E6 and E7 interfere with the transcriptional upregulation of the antiviral cytokines in response to stimulation with the dsDNA Poly(dA:dT). In contrast, while E6 also interferes with the transcriptional upregulation of antiviral cytokines in response to stimulation with the dsRNA Poly(I:C), E7 interferes with only a subset of these antiviral cytokines. Finally, we demonstrated that E7 but not E6 abrogates signaling through the type I IFN receptor. Taken together, CPV2 E6 and E7 both impact expression of antiviral cytokines in canine keratinocytes, albeit likely through different mechanisms.