Case report: Infratentorial Embryonal Tumor with Abundant Neuropil and True Rosettes (ETANTR) in an 8-month-old Maine Coon.

An 8-month-old female Main Coon with a history of recurrent behavioral changes and anorexia was presented with sternal recumbency and depression. Within 5 days, the cat progressively worsened with symptoms of stupor and coma and was euthanized. At post-mortem examination, a solid, grayish infratentorial mass located in the midline rostrally to the cerebellum, was observed. Histologically, highly cellular clusters of small-to-medium undifferentiated cells were intermingled with paucicellular areas with fibrillary eosinophilic (neuropil-like) appearance. Numerous multilayered (ependymoblastic) true rosettes were present. The mitotic activity was frequent (up to 15 mitoses/HPF), involving both undifferentiated cells and rosettes. By immunohistochemistry (IHC), tumor cells were diffusely positive for vimentin, variably for synaptophysin, S-100, and NSE, and focally for NeuN; they were negative for GFAP and CK AE1/AE3. The histological and IHC aspects were consistent with an Embryonal Tumor with Abundant Neuropil and True Rosettes (ETANTR). Embryonal neoplasms of the central nervous system (CNS) are characterized by primitive undifferentiated cells, able to develop toward neuronal, glial, ependymal, and mesenchymal lines. Although extremely rare, juvenile embryonal tumors should be considered in the differentials of CNS disorders in young cats.

Clinicopathologic features and biologic behavior of canine splenic nodules with stromal, histiocytic and lymphoid components.

The term fibrohistiocytic nodule has been discouraged in favor of specific pathologic entities, including complex nodular hyperplasia, splenic stromal sarcoma and histiocytic sarcoma. Nevertheless, the diagnosis of splenic lesions with mixed stromal, histiocytic and lymphoid components still remains a challenge due to lack of straightforward histologic criteria. Misestimation of the biologic behavior of these lesions may lead to detrimental consequences on the clinical management of patients. In this study, we retrospectively evaluated the clinicopathologic features and outcome of canine splenic nodular lesions with mixed components, to identify prognostic factors and histologic criteria of malignancy. Thirty-seven cases were included. Immunohistochemistry did not allow for further subclassification. Nine (24.3%) dogs died from disease-related causes after a median of 234 days (range, 48-1,247). One-, 2- and 3-year disease-specific survival rates were 80, 60, and 43%, respectively. When considering nodules with stromal cell atypia and at least one of mitotic count ≥9, presence of karyomegaly/multinucleated cells and lymphoid component <40%, half of these dogs died of disease-related causes with a median disease-specific survival time of 548 days (95% CI, 0-1216). In the remaining dogs, no disease-related death was reported (P < 0.001). Canine splenic nodular lesions with mixed stromal, histiocytic and lymphoid components and histologic criteria of malignancy may behave aggressively, leading to distant metastasis and death. In the absence of further criteria aiding their classification, and to better characterize their biologic behavior, we encourage the distinction of these complex splenic tumors from conventional sarcomas and histiocytic sarcomas.

Role of Rotifers in Betanodavirus Transmission to European Sea Bass Larvae.

Marine invertebrates such as rotifers or Artemia, frequently used for fish larvae feeding, can be a potential source of pathogens. It has been demonstrated that Artemia can act as a nervous necrosis virus (NNV)-vector to Senegalese sole larvae. Therefore, in this study, we aimed to clarify the role of rotifers in NNV transmission to sea bass larvae following an oral challenge. Our results showed that sea bass larvae fed on a single dose of rotifers retaining NNV displayed clinical signs, mortality, and viral replication similar to the immersion challenge, although the course of the infection was slightly different between the two infection routes. Furthermore, we also demonstrated that rotifers can internalize NNV particles due to their filtering nature and maintain virus viability since viral particles were detected by immunohistochemistry, immunofluorescence, and cell culture within the rotifer body. However, viral quantification data suggested that rotifers are not permissive to NNV replication. In conclusion, this research demonstrated NNV horizontal transmission through rotifers to sea bass larvae, highlighting the importance of establishing strict routine controls on live food to prevent the introduction of potential pathogens to hatcheries.

Localization and genotyping of canine papillomavirus in canine inverted papillomas.

Numerous canine papillomaviruses (CPVs) have been identified (CPV1-23). CPV1, 2, and 6 have been associated with inverted papillomas (IPs). We retrieved 19 IPs from 3 histopathology archives, and evaluated and scored koilocytes, inclusion bodies, giant keratohyalin granules, cytoplasmic pallor, ballooning degeneration, and parakeratosis. IHC targeting major capsid proteins of PV was performed, and CPV genotyping was achieved by PCR testing. Tissue localization of CPV DNA and RNA was studied by chromogenic and RNAscope in situ hybridization (DNA-CISH, RNA-ISH, respectively). IPs were localized to the limbs (50%), trunk (30%), and head (20%), mainly as single nodules (16 of 19). In 15 of 19 cases, immunopositivity was detected within the nuclei in corneal and subcorneal epidermal layers. PCR revealed CPV1 in 11 IPs and CPV2 DNA in 3 IPs. Overall, 14 of 17 cases were positive by both DNA-CISH and RNA-ISH, in accord with PCR results. A histologic score >5 was always obtained in cases in which the viral etiology was demonstrated by IHC, DNA-CISH, and RNA-ISH. IHC and molecular approaches were useful to ascertain the viral etiology of IPs. Although IHC is the first choice for diagnostic purposes, ISH testing allows identification of PV type and the infection phase. RNA-ISH seems a promising tool to deepen our understanding of the pathogenesis of different PV types in animal species.

Prognostic Value of PD-L1, PD-1 and CD8A in Canine Diffuse Large B-Cell Lymphoma Detected by RNAscope.

Immune checkpoints are a set of molecules dysregulated in several human and canine cancers and aberrations of the PD-1/PD-L1 axis are often correlated with a worse prognosis. To gain an insight into the role of immune checkpoints in canine diffuse large B-cell lymphoma (cDLBCL), we investigated PD-L1, PD-1 and CD8A expression by RNAscope. Results were correlated with several clinico-pathological features, including treatment, Ki67 index and outcome. A total of 33 dogs treated with chemotherapy (n = 12) or chemoimmunotherapy with APAVAC (n = 21) were included. PD-L1 signal was diffusely distributed among neoplastic cells, whereas PD-1 and CD8A were localized in tumor infiltrating lymphocytes. However, PD-1 mRNA was also retrieved in tumor cells. An association between PD-L1 and PD-1 scores was identified and a higher risk of relapse and lymphoma-related death was found in dogs treated with chemotherapy alone and dogs with higher PD-L1 and PD-1 scores. The correlation between PD-L1 and PD-1 is in line with the mechanism of immune checkpoints in cancers, where neoplastic cells overexpress PD-L1 that, in turn, binds PD-1 receptors in activated TIL. We also found that Ki67 index was significantly increased in dogs with the highest PD-L1 and PD-1 scores, indirectly suggesting a role in promoting tumor proliferation. Finally, even if the biological consequence of PD-1+ tumor cells is unknown, our findings suggest that PD-1 intrinsic expression in cDLBCL might contribute to tumor growth escaping adaptive immunity.

Chromogenic in situ hybridization for the detection of lambda and kappa immunoglobulin light chains as a potential auxiliary diagnostic technique in canine plasmacytomas.

The heterogeneous morphologic features of canine plasmacytomas (PCTs) can make their differentiation from other round cell tumors challenging. Immunohistochemistry (IHC) for lambda (λ) and kappa (к) immunoglobulin (Ig) light chains is often equivocal because of high background staining. The chromogenic in situ hybridization (CISH) technique for light chains has shown higher sensitivity compared to IHC in human plasma cell tumors. Therefore, we aimed to validate automated CISH for light chains in canine tissues and to evaluate its diagnostic potential in canine PCTs, in conjunction with routinely used IHC markers. CISH for light chains demonstrated a clear signal in plasma cell populations of canine control tissues (lymph nodes, lymphoplasmacytic inflammation) showing a polyclonal pattern with a prevalence of λ-producing cells. CISH detected monotypic light chain expression in 33 of 53 (62%) PCTs, 31 expressing λ and 2 expressing к. CISH was more sensitive than IHC for λ light chain (58% vs. 47%, respectively) and more easily interpretable given the absence of confounding background staining. The absence of CISH staining for both λ and к in a considerable subset of tumors may be the result of lower light chain production by neoplastic cells. Multiple myeloma oncogene 1 (MUM1) was expressed by all but 2 PCTs (96%), which showed λ expression by CISH and IHC. The identification of poorly differentiated canine PCTs requires the assessment of a panel of IHC markers, with the potential support of CISH for Ig light chains.

CD30 Cross-Reactivity and Expression in Feline Normal Tissues and Lymphomas.

CD30 is a transmembrane glycoprotein of the tumor necrosis factor receptor superfamily included in the diagnostic algorithm of human cutaneous, anaplastic large cell and Hodgkin lymphomas and represents an optimal therapeutic target for CD30+ tumors. Similar diagnostic and therapeutic approaches are largely missing for feline lymphomas. Cross-reactivity of the antihuman CD30 receptor clone Ber-H2 was investigated in feline lymphomas. Comparative analysis of feline and human CD30 identified 61% identity of the amino acid sequence, with 100% identity of the main sequence of the epitope targeted by the antibody (RKQCEPDYYL). CD30 expression in normal feline tissues was restricted to rare lymphoid cells in perifollicular and interfollicular lymph node areas and in the thymic medulla. In feline lymphoma, CD30 was expressed in 4 of 33 (13%) T-cell lymphomas, 3 of 22 (14%) B-cell lymphomas, and 5 of 7 (71%) mixed-cell lymphomas, showing diffuse (1/5) or multifocal (4/5) positivity restricted to neoplastic multinucleated lymphoid cells and binucleated cells consistent with Reed-Sternberg-like cells. Based on the human classification system, cell morphology, expression of multiple markers (mixed cell components), and CD30 positivity, these cases were considered most consistent with classical Hodgkin-like lymphoma (HLL). The other 2 mixed-cell lymphomas were CD30 negative and thus most consistent with either T-cell-rich large B-cell lymphoma (TCRLBCL) or nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). These findings provide multiple data supporting the cross-reactivity of the Ber-H2 anti-CD30 clone in feline tissues and give evidence of the usefulness of CD30 in the diagnostic evaluation of feline lymphoma.

Quantitative real time polymerase chain reaction (qRT-PCR) and RNAscope in situ hybridization (RNA-ISH) as effective tools to diagnose feline herpesvirus-1-associated dermatitis.

BACKGROUND: Felid herpesvirus type 1 (FHV-1)-associated dermatitis is characterized by facial and nasal involvement; clinical and histopathological manifestations may overlap with other dermatitides. OBJECTIVE: To evaluate the realibility of qRT-PCR-2- ΔΔC q and RNAscope in situ hybridization (RNA-ISH) methods to diagnose FHV-1-associated dermatitis, in formalin-fixed paraffin-embedded (FFPE) tissues. ANIMALS: Sixteen FFPE samples from cats with facial dermatitis and four controls were studied. METHODS AND MATERIALS: Based on histopathological features, cases were separated into: Group 1, samples with herpetic dermatitis (four); Group 2, samples with nonherpetic facial dermatitis (six); Group 3, samples with facial dermatitis of ambiguous nature (allergic or viral) (six); and Group 4, samples from healthy cats (four). A relative quantification using the 2- ΔΔC q method was used to estimate the "upregulation" of each FHV-1 target viral gene copies (glycoprotein-B and thymidine-kinase) relative to reference gene. Detection of FHV-1 mRNA was performed using the RNAscope 2.5 detection kit. RESULTS: By 2- ΔΔC q analysis, upregulation of both FHV-1 genes was observed in all samples from Group 1 and two of six from Group 3. No upregulation was identified in samples from groups 2 and 4. Positive mRNA hybridization signal was observed in all cases from Group 1 and two cases of Group 3. No positivity was observed in samples from groups 2 and 4. CONCLUSIONS AND CLINICAL IMPORTANCE: QRT-PCR 2-ΔΔCq analysis and RNA-ISH can identify the FHV-1 genome as causative agent of the associated dermatitis, even where inclusion bodies are not detectable. Both techniques are functional in retrospective studies, have greater specificity than conventional PCR, and may be proposed for research and diagnostic purposes.

Felis catus Papillomavirus Types 1, 2, 3, 4, and 5 in Feline Bowenoid in Situ Carcinoma: An In Situ Hybridization Study.

Several studies based on histopathology or molecular investigations suggest a causal relation between Felis catus papillomavirus (FcaPV-2) infection and bowenoid in situ carcinoma (BISC) in cats. Nevertheless, data on distribution of viral DNA for different F. catus papillomavirus types (FcaPV-1, 2, 3, 4, 5) in precancerous skin lesions are lacking. In this study, incisional and excisional skin biopsies from 18 cats with BISC were investigated for the presence of FcaPV DNA by quantitative polymerase chain reaction (qPCR) and chromogenic in situ hybridization (CISH) using specific probes to detect each of the FcaPVs that have been identified so far. By qPCR analysis, 15 of 18 samples were positive for FcaPV-2, 2 were positive for FcaPV-4, and 1 sample was negative for all FcaPVs studied. Two cases were positive for FcaPV-5 by qPCR only. FcaPV-1 and FcaPV-3 were not detected by either method. CISH positivity for FcaPV-2 and FcaPV-4 was 100% concordant with qPCR. FcaPV-2 CISH signal was observed as nuclear dots within grouped neoplastic keratinocytes in 12 BISCs and in the perilesional skin of 9 biopsies. In 3 of these 9 cases, the signal was not observed within the BISC. FcaPV-4 CISH positivity was detected only within BISCs in 2 cases. The overall rate of concordance for FcaPV detection between PCR and CISH was 97.8%. This study suggests that CISH is a reliable method to detect FcaPV-2 and FcaPV-4 infection in cats and provides useful information on the type, rate, and localization of infected cells.

Detection and Prognostic Relevance of Circulating and Disseminated Tumour Cell in Dogs with Metastatic Mammary Carcinoma: A Pilot Study.

In human breast cancer, both circulating tumour cells (CTCs) in peripheral blood and disseminated tumour cells (DTCs) in the bone marrow are predictive of short survival and may be used as liquid biopsy to guide therapy. Herein we investigate, for the first time, the feasibility to quantify CTCs and DTCs in canine metastatic mammary carcinoma (MMC) with the automated CellSearch platform, which identifies tumour cells by immune-magnetic enrichment and fluorescent labelling. Using this approach before start of treatment, we could detect at least 1 CTC per 7.5 mL of peripheral blood in 12 out of 27 evaluable samples (44.4%) and at least 1 DTC per 1 mL of bone marrow in 11 out of 14 evaluable samples (78.6%). Conversely, we did not find any CTCs in the healthy, negative control dogs (n = 5) that we analysed in parallel. Interestingly, the levels of CTCs/DTCs and the prevalence of positive dogs closely resemble results obtained by CellSearch assay in metastatic breast cancer patients at diagnosis. Moreover, in the canine cohort, the presence of CTCs was significantly associated with poor outcome. These observations identify the first actionable marker in veterinarian oncology to guide treatment of canine MMC. Furthermore, our findings have important implications for human research, since it reinforce the value of canine MMC as model useful to speed up pharmacological studies with primary endpoint of overall survival, given the reduced life-span of the canine species.

Bilateral microphthalmia and aphakia associated with multiple eye abnormalities in a free-living European red deer calf (Cervus elaphus).

A free-living European red deer calf (Cervus elaphus) was euthanized due to bilateral microphthalmia. Lens was missing, replaced by proliferating squamous epithelial cells; hyperplastic squamous cells, sebaceous and mucinous glands were observed within the cornea with the characteristics of inclusion cyst. Findings were consistent with congenital microphthalmia/aphakia, with multiple eye abnormalities.

Microchip-associated fibrosarcoma in a cat.

A 9-year-old, neutered male cat was presented for a subcutaneous mass on the neck. After surgical removal of the mass, a pet identification microchip was found within the tumour. Histological examination of the mass revealed typical features of the feline postinjection sarcoma. The cat had never received injections at the tumour site; all routine vaccinations were administered in the hindlimbs. Few cases of sarcomas developing at the site of microchip application have been reported in animals, although the contributory role of vaccine administrations has not been ruled out. This is the first report of a microchip-associated fibrosarcoma in a cat. Adherence to American Association of Feline Practitioners vaccination guidelines, avoiding the interscapular area, enabled confirmation of the definitive aetiology of the neoplasia.

Malignant mesothelioma of the tunica vaginalis testis in a dog: histological and immunohistochemical characterization.

Histologic and immunohistochemical features of a malignant mesothelioma of the testicular tunica vaginalis in a 9-year-old male mixed-breed dog are reported. The dog had a large scrotal swelling determined by ultrasonography to be a heterogeneous irregular mass characterized by a mixed echogenic pattern and located in the left vaginal canal of the testis. Histologically, the neoplasm was nodular, was poorly demarcated, and consisted of pleomorphic cells that were associated with a significant desmoplastic reaction. By immunohistochemistry, neoplastic cells stained positively for cytokeratin and vimentin and were immunoreactive with an antimesothelial cell monoclonal antibody. Malignant mesotheliomas of tunica vaginalis testis are rare in mammalian species, including dogs.

A case of coccygeal chondroid chordoma in a cat: morphological and immunohistochemical features.

In August 2007, a 4-year-old, intact, female Domestic Shorthair cat was examined for a mass on the tip of the tail. Histological examination performed after apical caudectomy revealed a neoplasm affecting the distal part of the last coccygeal vertebra. The neoplasm consisted of lobules of physaliferous cells surrounding cartilaginous tissue and a central core of trabecular bone. A diagnosis of chondroid chordoma was made based on histomorphological features and immunohistochemical results. Chondroid chordoma has been previously reported in humans, rats, ferrets, and mink. To the authors' knowledge, chondroid chordoma has not been reported in cats. Neither recurrence nor metastasis was reported 7 months after surgery.

Multiple feather follicle cysts in a Moroseta hen (Gallus gallus).

An 8-month-old white feathered, black skinned Moroseta hen was presented for examination because of numerous 2 mm- to 30 mm-diameter irregularly shaped, hard nodules in the skin of the head, wings, back, and abdomen. The nodules were confined to the skin and did not involve subcutaneous tissues. Nodules consisted of dilated feather follicles packed with a caseous tan-to-pale-yellow material admixed with feather remnants. Histologically, affected feather follicles were markedly dilated and filled with laminated keratin debris. Necrosis of the epidermis and perifollicular lymphocyte infiltration was also present. Bacteriologic investigation of internal organs was negative, while secondary bacteria, Proteus spp. and Bacillus spp., were isolated from skin nodules. A concomitant lice infestation of Menopon spp., as well as leg mange caused by Cnemidocoptes spp., were also present. These bacterial isolates and parasites were not related to the disease condition. The condition observed was differentiated from benign feather follicle tumors, and a diagnosis of multiple feather follicle cysts was made. In addition, a breed predisposition was hypothesized.

PrPSc in salivary glands of scrapie-affected sheep.

The salivary glands of scrapie-affected sheep and healthy controls were investigated for the presence of the pathological prion protein (PrP(Sc)). PrP(Sc) was detected in major (parotid and mandibular) and minor (buccal, labial, and palatine) salivary glands of naturally and experimentally infected sheep. Using Western blotting, the PrP(Sc) concentration in glands was estimated to be 0.02 to 0.005% of that in brain. Immunohistochemistry revealed intracellular depositions of PrP(Sc) in ductal and acinar epithelia and occasional labeling in the lumina of salivary ducts. The presence of PrP(Sc) in salivary glands highlights the possible role of saliva in the horizontal transmission of scrapie.

Mediastinal lymphoma in a badger (Meles meles).

A case of mediastinal lymphoma in a free-living, adult male European badger (Meles meles), from Trento Province (northern Italy) is described. A yellow-red mass delimited by a thick fibrous capsule completely occupied the anterior mediastinum. Based on the histologic and immunohistochemical findings, a diagnosis of mediastinal lymphocytic lymphoma was made. Documentation of neoplasms in free-living species is important for comparison with domestic animals and humans and may give insight into epidemiology of these diseases. To the authors' knowledge, this is the first report of a mediastinal lymphoma in a badger.