A Combined Approach for Detection of Ovine Small Ruminant Retrovirus Co-Infections

Jaagsiekte retrovirus (JSRV)-induced ovine pulmonary adenocarcinoma (OPA) is an important ovine respiratory disease in Switzerland. Furthermore, ovine lungs with OPA frequently exhibited lesions suggestive of maedi-visna virus (MVV) or caprine arthritis encephalitis virus (CAEV) infection, indicating that co-morbidities might occur. Lungs and pulmonary lymph nodes were sampled from suspected OPA cases, inflammatory lung lesions and control lungs (total of 110 cases). Tissues were (a) processed for histology and immunohistochemistry (IHC), and (b) underwent DNA extraction and real-time PCR for JSRV, MVV and CAEV. Peptide sequences were used to generate virus-specific customized polyclonal antibodies. PCR-positive OPA cases and formalin-fixed and paraffin-embedded MVV- and CAEV-infected synovial cell pellets served as positive controls. Fifty-two lungs were histologically diagnosed with OPA. Histological evidence of MVV/CAEV infection was detected in 25 lungs. JSRV was detected by PCR in 84% of the suspected OPA cases; six were co-infected with MVV and one with CAEV. MVV was detected by PCR in 14 cases, and four lungs were positive for CAEV. Three lungs had MVV/CAEV co-infection. In IHC, JSRV was detected in 91% of the PCR-positive cases, whereas MVV and CAEV immunoreactivity was seen in all PCR-positive lungs. Although PCR showed a higher sensitivity compared to IHC, the combined approach allows for investigations on viral cell tropism and pathogenic processes in co-morbidities, including their potential interdependency. Furthermore, an immunohistochemical tool for specific differentiation of MVV and/or CAEV infection was implemented

‘Left, right, left’ – a practical guide for forensic veterinary pathologists on identification of cattle and pig limbextremities

The identification of left and right fore or hind limbs can be very challenging in porcine or bovine cases sent from slaughterhouses for forensic surveys, especially when dissected below the carpal or tarsal joints. This short practical guide should be considered as an aid in the process of documentation and investigation of forensic farm animal cases

Indocyanine-based near-infrared lymphography for real-time detection of lymphatics in a cat with multiple mast cell tumours

Case summary An 11-year-old female domestic shorthair cat was presented with cutaneous mast cell tumours (MCTs) localised at the right temporal region, the left buccal region and on the third digit of the right thoracic limb. Staging was negative and locoregional lymph nodes appeared normal, based on clinical findings. During surgery, real-time indocyanine green (ICG)-based lymphography was performed to detect the cutaneous draining pattern of all the primary MCTs. ICG was injected intracutaneously in four quadrants around each tumour, and a clear lymphogram was visible shortly after injection. Using near-infrared lymphography (NIR-L) for guidance, all lymphadenectomies were performed in 12 mins or less, with a maximal incision length of 3.5 cm. The smallest resected node was 0.9 cm in diameter. All MCTs were classified as low-grade cutaneous MCT. All four ICG-positive lymph nodes were considered premetastatic or metastatic. The only ICG-negative resected node was also negative for tumour cells. No complications related to NIR-L were recorded. Relevance and novel information This is the first description of NIR-L in a cat with MCT. Application was straightforward and ICG enrichment only occurred in the metastatic nodes, suggesting correct identification of lymphatic draining patterns. Of note, as previously described in dogs, we did detect nodal metastasis, despite low-grade primary tumours. The clinical relevance should be evaluated in future studies

Strain diversity in Mycobacterium avium subsp. paratuberculosis-positive bovine fecal samples collected in Switzerland

Paratuberculosis or Johne’s disease is a chronic intestinal disease in domestic and wild ruminants. It affects global dairy economy and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The objective of this study was to analyze strain diversity in MAP-positive fecal samples by using a particular single nucleotide polymorphism (SNP) distinguishing between cattle (C-) and sheep (S-) type MAP and analysis of SNPs within gyrA and gyrB genes differentiating between Types I, II, and III. Moreover, mycobacterial interspersed repetitive unit and variable-number tandem repeat (MIRU-VNTR) analysis using eight established loci was performed. A total of 90 fecal samples from diseased animals presenting diarrhea and/or weight loss, originating from 59 bovine herds across 16 cantons of Switzerland were screened by PCR for the MAP-specific F57 and IS900 genes and were further subtyped. 96.7% and 3.3% of the samples contained C- and S-type MAP, respectively. Ten INRA Nouzilly MIRU-VNTR (INMV) profiles, with a discriminatory index of 0.802, calculated based on 65 epidemiological independent genotypes, were detected: INMV 1 (33.8%), INMV 2 (23.1%), INMV 6 (16.9%), INMV 9 (9.2%), INMV 116 (4.6%), INMV 3 (3.1%), INMV 5 (3.1%) and INMV 72 (1.5%), including two novel INMV profiles, namely INMV 253 (3.1%; S-type III) and INMV 252 (1.5%; C-type). INMV 1, INMV 2, and INMV 6 comprised almost 75% of the F57- and IS900-positive samples. Typing data from 11 herds suggest that there are some herds with intra-herd diversity of genotypes. The results of this study indicate a heterogeneity of MAP in Switzerland

Morphological and molecular characterization of a new mycobacterium avium Subsp. paratuberculosis S-type strain Genotype in goats

Paratuberculosis is a chronic bacterial disease of global importance mainly in domestic and wild ruminants, caused by Mycobacterium avium subsp. paratuberculosis (MAP). In goats, paratuberculosis is mostly caused by the "C-type" (cattle) and in a few cases by the "S-type" (sheep) strain of MAP. In 2017, a caprine S-type III isolate with a new VNTR profile was identified in a Swiss alpine region. In 2018, new caprine isolates with the same novel VNTR profile originating from a farm of a close by neighboring valley were analyzed. Here we report on this MAP S-type III outbreak in a Swiss dairy goat farm in which we investigated the pathological changes, distribution and genotype of MAP tissue homogenates. Full necropsy and histological examination were undertaken on two female adult goats with a history of weight loss and intermitting diarrhea. Routine and special stains were applied to characterize the morphological changes. DNA was extracted from 33 different tissue samples and tested for MAP by qPCR targeting IS900 and F57. Subtyping was performed, using the variable number tandem repeats (VNTR) and mycobacterial interspersed repetitive units (MIRU) approach. The goats showed moderate to marked emaciation and displayed typical clinical features of paratuberculosis. A moderate granulomatous enteritis and regional lymphadenitis with a small to moderate number of acid-fast bacteria within macrophages was detected. MAP detection was mainly restricted to the gastrointestinal tract, mesenteric and hepatic lymph nodes. Subtyping the S-type isolates using a panel of eight established MIRU-VNTR loci identified a new genotype, INMV 218

NSs protein of Schmallenberg virus counteracts the antiviral response of the cell by inhibiting its transcriptional machinery

Bunyaviruses have evolved a variety of strategies to counteract the antiviral defence systems of mammalian cells. Here we show that the NSs protein of Schmallenberg virus (SBV) induces the degradation of the RPB1 subunit of RNA polymerase II and consequently inhibits global cellular protein synthesis and the antiviral response. In addition, we show that the SBV NSs protein enhances apoptosis in vitro and possibly in vivo, suggesting that this protein could be involved in SBV pathogenesis in different ways

Standardisation of canine meningioma grading: Inter‐observer agreement and recommendations for reproducible histopathologic criteria

The human grading system is currently applied to canine meningioma, although it has not been validated in dogs. The present study focused on standardising the human grading system applied to canine meningioma. Four veterinary neuropathologists graded 186 canine meningiomas as follows: Grade I tumour, with 50% of the tumour; (6) Report necrosis, small cells, hypercellularity and macronucleoli, even when focal; (7) Report anaplasia if multifocal

Mutations in the Schmallenberg virus Gc glycoprotein facilitate cellular protein synthesis shutoff and restore pathogenicity of NSs deletion mutants in mice

Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach higher titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro. Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild type SBV as has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an IFN antagonist. Therefore SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by the infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins while the ability to control the production of IFN maps to the NSs protein. Importance The identification of viral determinants of pathogenesis is key to the development of prophylactic and interventions measures. In this study we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc glycoprotein can restore pathogenicity of attenuated mutants resulting from deletions or mutations in the non-structural protein NSs. Our findings highlight the fact that careful consideration should be taken when designing live attenuated vaccines based on deletions of non-structural proteins since single mutations in the viral glycoproteins appear to revert attenuated mutants to virulent phenotypes

An Engineered IFNγ-Antibody Fusion Protein with Improved Tumor-Homing Properties

Interferon-gamma (IFNγ) is one of the central cytokines produced by the innate and adaptive immune systems. IFNγ directly favors tumor growth control by enhancing the immunogenicity of tumor cells, induces IP-10 secretion facilitating (CXCR3+) immune cell infiltration, and can prime macrophages to an M1-like phenotype inducing proinflammatory cytokine release. We had previously reported that the targeted delivery of IFNγ to neoplastic lesions may be limited by the trapping of IFNγ-based products by cognate receptors found in different organs. Here we describe a novel fusion protein consisting of the L19 antibody, specific to the alternatively spliced extra-domain B of fibronectin (EDB), fused to a variant of IFNγ with reduced affinity to its cognate receptor. The product (named L19-IFNγ KRG) selectively localized to tumors in mice, showed favorable pharmacokinetic profiles in monkeys and regained biological activity upon antigen binding. The fusion protein was investigated in two murine models of cancer, both as monotherapy and in combination with therapeutic modalities which are frequently used for cancer therapy. L19-IFNγ KRG induced tumor growth retardation and increased the intratumoral concentration of T cells and NK cells in combination with anti-PD-1