[Diagnostic value of the BRAF variant V595E in urine samples, smears and biopsies from canine transitional cell carcinoma]. / Diagnostische Aussagekraft der BRAF-Mutation V595E in Urinproben, Ausstrichen und Bioptaten beim kaninen Übergangszellkarzinom.

OBJECTIVE: Transitional cell carcinoma (TCC) is the most common malignant tumour of the canine urinary tract. Previously, the mutation of the BRAF gene V595E was identified in approximately 85 % of canine TCC cases by DNA sequencing of TCC tumour cells, both in frozen and paraffin-embedded tissue sections, as well as in urine. The objective of this study was to establish these methods in cytological smears and to investigate the prevalence of BRAF mutation V595E in canine TCC in our cohort of patients. MATERIAL AND METHODS: Biopsy samples (n = 43), urine (n = 48) and/or cytological smears (n = 31) from 66 dogs with TCC (n = 33), urinary bladder polyps (n = 7), cystitis (n = 23) or without bladder diseases (n = 3), submitted for routine diagnostics, were selected. DNA isolation from paraffin material, urine and cytological smears was performed using commercially available kits. Exon 15 was examined for the presence of the BRAF mutation c.1784T>A by Sanger sequencing. RESULTS: In 39/43 paraffin-embedded biopsies and 38/48 urine samples, a sufficient amount of good quality DNA was isolated. DNA isolation and sequencing were successful in 16/18 smears with a high cell count, but not in the 10/13 smears with low cellularity. In all cases from which different sample materials were available, the results of BRAF analysis were identical in paraffin-embedded tissue, cytological smears and/or urine. In 22/31 dogs (70.9 %) with TCC, the presence of the BRAF mutation was confirmed, whereas it could not be detected in animals without pathological findings or with cystitis or with a polyp. CONCLUSION AND CLINICAL RELEVANCE: BRAF mutation analysis is a new and good method to be able to mostly confirm a diagnosis of TCC in uncertain cases. Non-invasive diagnostic samples, including urine and urine sediment containing sufficient numbers of relevant cells as well as cytology aspirates and formalin-fixed biopsies can be used for analysis. However, it is important to note that only a positive identification of the mutation is diagnostic. Further research is necessary to investigate prognostic and therapeutic relevance of the variant and how this genetic analysis can be used as an early detection method for TCC.

Detection of porcine circovirus type 2 and its association with PMWS in wild boars and domestic pigs in Germany: a histopathological, immunohistochemical and molecular biological study.

Beside domestic pigs wild boars can also be affected by postweaning multisystemic wasting syndrome (PMWS). For the first time a nationwide survey of wild boars (n = 356) and domestic pigs (n = 340) was carried out in Germany by histopathology, immunohistochemistry (IHC) and quantitative PCR (qPCR). Whereas 102/340 domestic pigs were immunoreactive for PCV2 antigen in at least one examined tissue, only 8/356 wild boars reacted positively. Similar findings could be found in qPCR: all domestic pigs showed viral DNA in at least one tissue, while in the examined tissues of 170 wild boars PCV2-DNA was not detectable. The specimens were examined histologically for histiocytosis and depletion of lymphocytes, both typical for PMWS. Based on these findings, six wild boars and 69 domestic pigs were assumed to be affected by PMWS.

Distribution of ORF2 and ORF3 genotypes of porcine circovirus type 2 (PCV-2) in wild boars and domestic pigs in Germany.

Porcine circovirus 2 (PCV-2), the essential infectious agent in PCVD (porcine circovirus diseases) circulates at high rates among domestic pig and wild boar populations. Wild boars may be viremic and shed the virus with excretions and secretions, and thus serve as a reservoir for domestic pig PCV-2 infection. We hypothesize that PCV-2 strains circulating in wild boars and in domestic pigs are significantly different and thus, partially independent. To prove this hypothesis, the present study investigated by sequence analysis the distribution of ORF2 and ORF3 genotypes of the PCV-2 genome within wild boars (n=40) and domestic pigs (n=60) from overlapping greater areas of Germany. The genotypes were compared with PCV-2 sequences from the Genbank database. The dominating genotype in domestic pigs was PCV-2b (98.4% of infected pigs), while only 4.8% of them were infected with PCV-2a. The corresponding prevalences of PCV-2a and -2b genotypes in wild boars were 58% and 70%, respectively. When also ORF3 genotypes were taken into account, more than 50% of wild boar PCV-2 genotypes were rare among German and European domestic pigs. In conclusion, these data provide evidence for a certain independence of PCV-2 infections in both species and a low chance for domestic pigs to be infected with PCV-2 of wild boar origin. On the other hand, PCV-2 genotypes specific for domestic pigs are also common in wild boars, although at lower frequencies, suggesting the spread of domestic pig PCV-2 to the wild boar population.

Qualitative and quantitative distribution of PCV2 in wild boars and domestic pigs in Germany.

Porcine circovirus 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), has been detected in North American and European wild boars at prevalences arguing for high circulation rates among populations. Systematic data on the qualitative distribution of PCV2 infections and on PCVD (PCV2 diseases) in wild boars are rare, however, and quantitative data about viral loads are missing. To be able to judge the PCV2/PCVD situation in wild boars, evaluation of the nationwide qualitative and quantitative distribution of PCV2 and PCVD in Germany was the objective of the present study. Wild boar samples were compared with domestic pig samples of the same greater areas, including tonsils, lungs, spleen, Lnn. bronchiales and Lnn. mesenterici of 349 wild boars and 348 domestic pigs. All of the wild boars and 308 of the domestic pigs have been apparently free of PCVD, 40 of the domestic pigs had been rejected from slaughter due to health problems (i.e. wasting). Tissues were examined by pathohistology, immunohistology (IHC), nested PCR (nPCR and quantitative PCR (qPCR). One wild boar (0.3%) and 8.7% of the domestic pigs were classified as PCVD-affected, based on pathohistology and IHC. PCV2 DNA was detected in 63.1% and 45.4% of the wild boars by nPCR and qPCR, respectively, and in 100% and 98.8% of the domestic pigs. PCV2 loads differed significantly between wild boars (average: 10(2.8) PCV2 genomes/microg extracted sample DNA) and domestic pigs (average: 10(4.2) PCV2 genomes/microg of sample DNA). The qualitative detection of PCV2 DNA in tissues of wild boars and domestic pigs was abundant and not of any pathological relevance. The overall load of PCV2 in domestic pigs was relatively high and borderline with respect to PCVD, and there was no difference between apparently healthy pigs and pigs rejected from slaughter in this respect. Most of the wild boars were infected with PCV2 at loads less relevant for PCVD.