Papillomaviruses in ruminants: An update.

Papillomaviruses (PVs) are complex viruses which infect the skin or mucosae of a broad range of amniotes worldwide. They cause benign or malignant lesions depending on environmental factors, virus oncogenicity and the location of infection. Bovine papillomaviruses (BPVs) are the second most studied PVs beyond human PVs. In the past few years, genetic characterization of animal PVs has increased due to the availability of new techniques, which simplified the sequencing of entire genomes. Therefore, this review aims to provide an update of the current epidemiology, classification and genome features of ruminant PVs (mainly BPVs) affecting animals worldwide. The review also aimed to clarify the key differences between the high-risk Delta papillomaviruses and the seemingly low-risk Xi, Epsilon, Dyoxi and Dyokappapillomavirus as well as the recently described PVs BPV18, 19, 21 and PpuPV1 that belongs to an unclassified genus.

Viruses associated with congenital tremor and high lethality in piglets.

The recently described atypical porcine pestivirus (APPV) has been associated with congenital tremor (CT) type A-II in piglets in different countries. Another important neurological pathogen of pigs is porcine teschovirus (PTV), which has been associated with non-suppurative encephalomyelitis in pigs with severe or mild neurological disorders. There have been no reports of APPV and/or PTV coinfection associated with CT or encephalomyelitis in Brazilian pig herds. The aim of this study was to describe the pathological and molecular findings associated with simultaneous infection of APPV and PTV in piglets with clinical manifestations of CT that were derived from a herd with high rates of CT-associated lethality. In 2017, three piglets from the same litter with CT died spontaneously. The principal pathological alterations in all piglets were secondary demyelination and hypomyelination at the cerebellum, brainstem and spinal cord confirmed by histopathology and luxol fast blue-cresyl violet stain. Additional significant pathological findings included multifocal neuronal necrosis, neuronophagia and gliosis found in the cerebral cortex and spinal cord of all piglets, while atrophic enteritis and mesocolonic oedema were observed in some of them. APPV and PTV RNA were detected in the central nervous system of affected piglets, and PTV was also detected in the intestine and faeces. The pathological alterations and molecular findings together suggest a dual infection due to APPV and PTV at this farm. Moreover, the combined effects of these pathogens can be attributed to the elevated piglet mortality, as coinfections involving PTV have a synergistic effect on the affected animals.

Pneumonia due to Talaromyces marneffei in a Dog from Southern Brazil with Concomitant Canine Distemper Virus Infection.

The pathological and molecular findings associated with Talaromyces marneffei-induced pneumonia with concomitant infection by canine distemper virus (CDV) are described in a dog. The principal pathological alteration occurred in the lungs. Histopathology confirmed multifocal granulomatous pneumonia associated with numerous intralesional and intracellular septate fission cells consistent with T. marneffei. A molecular assay designed to amplify a partial fragment of the 18S rRNA gene of T. marneffei provided positive results from two fungal cultures derived from the lung. Sequencing and phylogenetic analyses confirmed the results of polymerase chain reaction (PCR). Furthermore, antigens of the CDV N protein were identified within the bronchial epithelium by immunohistochemistry and a PCR assay amplified the CDV N gene from hepatic and pulmonary fragments. Collectively, the pathological and molecular techniques confirmed a diagnosis of T. marneffei-induced pneumonia with concomitant infection by CDV. These findings represent the first description of pulmonary penicilliosis in the dog and extend the geographical niche of this emerging infectious pathogen. In this case, infection by CDV may have induced immunosuppression, which facilitated the development of pulmonary penicilliosis.

Brazilian strain of bovine respiratory coronavirus is derived from dual enteric and respiratory tropism.

Bovine coronavirus (BCoV) is a pathogen related to enteric and respiratory diseases in cattle worldwide. Enteric (BECoV) strains of BCoV are predominant in South America, and genetic investigations have been conducted to identify its relationship with isolates of respiratory origin (BRCoV). In this study, we used a BRCoV strain (BR-UEL11) derived from an outbreak of respiratory disease in feedlot cattle in southern Brazil, and compared the partial sequence of the polymorphic region of Spike (which was detected and sequenced by two distinct reverse transcription-polymerase chain reactions) with those of other BCoV strains. The phylogenetic relationship of BR-UEL11 with Brazilian BCoV, which is associated with calf diarrhea and winter dysentery (enteric, BECoV; respiratory, BRCoV), and classical reference prototypes was analyzed. The analysis showed that the BRCoV strains from Brazil clustered with a clade that was distinct from most isolates associated with calf diarrhea (BECoV) and ancestral prototype strains such as Mebus, Nebraska, and LYVB. Furthermore, the BRCoV strains from Brazil clustered with a clade that contained recent strains associated with winter dysentery, showing 98-99% nucleotide identity with those strains. These results suggested that the Brazilian BCoV evolved from being solely enteric to a dual enteric and respiratory tropic virus.

Pathological, Immunohistochemical and Molecular Findings Associated with Senecavirus A-Induced Lesions in Neonatal Piglets.

This study investigated the cause of the mortality of piglets with cutaneous, enteric and neurological disorders from seven pig farms located in different geographical regions of Brazil. Twelve 1- to 5-day-old piglets were submitted for pathological evaluation. The principal gross findings included faint rib impressions on the pleural surface of the lungs (n = 9), diphtheritic glossitis (n = 6) and ulcerative lesions at the coronary band (n = 5). Histopathology revealed interstitial pneumonia (n = 12), myocarditis (n = 6), diphtheritic glossitis (n = 3), encephalitis (n = 3) and atrophy of intestinal villi with vacuolation of the superficial epithelial cells (n = 6). Immunohistochemistry with monoclonal antibodies specific for Senecavirus A (SenV-A) demonstrated immunoreactivity of the choroid plexus of the cerebrum, degenerate epithelium of ulcerative lesions of the tongue, the urothelium of the kidney and urinary bladder, and the superficial cells of the intestine. Reverse transcriptase polymerase chain reaction (PCR), PCR and/or quantitative PCR assays were used to investigate viral agents associated with vesicular and/or enteric diseases. Antigens and RNA of SenV-A were identified in multiple tissues of all piglets; molecular assays for all other viruses evaluated yielded negative results. These findings confirm the participation of SenV-A in the multiple lesions observed in these piglets. Several theories are proposed: SenV-A may be eliminated via the urinary system, neurological disease may occur due to initial invasion of choroid plexus, enteric disease may be related to atrophy and fusion of villi of the small intestine, and vertical transmission could be a form of dissemination.

Isolation and molecular characterization of Leptospira borgpetersenii serovar Hardjo strain Hardjobovis in the urine of naturally infected cattle in Brazil.

Most epidemiologic studies on bovine leptospirosis are based on serological tests that use antibodies against several serotypes, including the serovar Hardjo, which is widespread and considered to be the most adapted to bovine hosts. However, using only serological studies is not sufficient to identify and distinguish species of leptospires. The aim of this study was report the first isolation in Brazil of two strains serovar Hardjo obtained in urine samples from naturally infected cows in a small Brazilian dairy herd and find the genetic species and consequently the type strain Hardjobovis by molecular characterization. Fifteen dairy cows with a history of reproductive failure, such as abortion and infertility, were selected. Urine samples obtained from each animal were immediately seeded in tubes containing Ellinghausen-McCullough-Johnson-Harris culture medium. The identification of the isolates was performed by Multilocus variable-number tandem-repeat analysis (MLVA) technique and phylogenetic analysis of partial sequence of gene sec Y. From the 15 urine samples evaluated, two Leptospira were found and identified as the Londrina 49 and Londrina 54 strains. The MLVA profiles and sequencing of gene sec Y characterized the isolates as L. borgpetersenii serovar Hardjo strain Hadjobovis because it has different genetic pattern of Leptospira interrogans serovar Hardjo strain Hardjoprajitno. Therefore, more studies are needed including isolation and molecular characterization from regional strains to obtain a better knowledge about epidemiology of serovar Hardjo in bovine which may assist in future strategies of prevention and control of bovine leptospirosis.

Molecular detection and phylogenetic relationship of wild-type strains of canine distemper virus in symptomatic dogs from Uberlândia, Minas Gerais / Detecção molecular e relação filogenética de cepas selvagens do vírus da cinomose canina em cães sintomáticos oriundos de Uberlândia, Minas Gerais

This study investigated the occurrence of canine distemper virus (CDV) by evaluating the presence of viral RNA within urine samples of dogs from Uberlândia, MG, with clinical manifestations suggestive of infection by CDV by targeting the CDV N gene. Of the clinical samples collected ( n =33), CDV viruria was detected in 45.5%. Five dogs died spontaneously; all had characteristic CDV-associated histopathological alterations and demonstrated CDV viruria. Statistical analyses revealed that the age, gender, breed, or the organ system of the dog affected had no influence on the occurrence of canine distemper. Myoclonus and motor incoordination were the most significant neurological manifestations observed. A direct association was observed between keratoconjunctivitis and dogs with CDV viruria. These findings suggest that CDV viruria in symptomatic dogs might not be age related, and that symptomatic dogs can demonstrate clinical manifestations attributed to CDV without viruria identified by RT-PCR. Additionally, the results of the sequence identities analysed have suggested that all Brazilian wild-type strains of CDV currently identified are closely related and probably originated from the same lineage of CDV. Nevertheless, phylogenetic analyses suggest that there are different clusters of wild-type strains of CDV circulating within urban canine populations in Brazil.

A presença do ácido nucleico (RNA) do vírus da cinomose canina (CDV) foi avaliada por meio da amplificação parcial do gene N pela técnica RT-PCR realizada em urina de cães provenientes de Uberlândia, Minas Gerais, que apresentavam sinais clínicos sugestivos de cinomose. Das 33 amostras de urina avaliadas, o CDV foi identificado em 45,5%. Em cinco cães que morreram espontaneamente, além da excreção do CDV na urina, foram observadas alterações histopatológicas associadas à infecção por esse vírus. Análises estatísticas demonstraram que a idade, gênero, raça e o sistema orgânico comprometido dos cães avaliados não exerceram influência no diagnóstico da cinomose canina. Mioclonia e incoordenação motora foram as manifestações neurológicas que apresentaram frequência de ocorrência significativa (P<0,05). Uma associação direta foi observada entre a presença de ceratoconjuntivite e a identificação de virúria pelo CDV. Esses achados sugerem que a excreção do CDV pela urina em cães com sinais clínicos compatíveis com cinomose pode não ser relacionada com a idade do animal, e que animais sintomáticos podem apresentar manifestações clínicas atribuídas ao CDV, porém sem a caracterização de virúria por RT-PCR. Adicionalmente, análises filogenéticas sugerem que várias cepas de CDV podem estar circulando em populações caninas de áreas urbanas no Brasil.

Transplacental Transmission of Ovine Herpesvirus 2 in Cattle with Sheep-associated Malignant Catarrhal Fever.

Sheep-associated malignant catarrhal fever (SA-MCF) is an important infectious disease of ruminants worldwide that is caused by ovine herpesvirus 2 (OvHV-2). OvHV-2 is transmitted predominantly by contact between infected and susceptible hosts, while the documentation of vertical transmission is rare. This report presents the pathological and molecular findings associated with transplacental transmission of OvHV-2 in cattle. Two Girolanda cows with corneal oedema, lethargy, mucopurulent nasal discharge and ulcerative stomatitis died spontaneously; one of these was pregnant with a 4-month-old fetus. Significant pathological findings included widespread lymphoplasmacytic necrotizing vasculitis and lymphoplasmacytic accumulations in several organs of both cows and the fetus. A polymerase chain reaction that targeted the tegument protein gene of OvHV-2 amplified viral DNA from the brain of the pregnant cow and her fetus, as well as from the kidney of the pregnant cow. The pathological findings observed in the cow and her fetus, together with the presence of OvHV-2 DNA in tissues of these animals, are suggestive of transplacental transmission of OvHV-2 in SA-MCF in cattle.

Senecavirus A: An Emerging Vesicular Infection in Brazilian Pig Herds.

Vesicular diseases are clinically and economically important infections that affect farm animals. North American studies have suggested that Senecavirus A infection might be associated with a vesicular disease in pigs known as porcine idiopathic vesicular disease (PIVD). In the beginning of 2015, outbreaks of porcine vesicular disease have occurred in six Brazilian states from three geographical regions. Official diagnostic tests were performed with negative results for classical vesicular diseases of compulsory reporting. This study investigated Senecavirus A infection in PIVD outbreaks in which other aetiological agents were ruled out. A primer set was designed to amplify a 542-bp product size of VP3/VP1 region of Senecavirus A genome in RT-PCR assay. Primer specificity was analysed in silico and in porcine biological specimens. For this, clinical specimens were collected from eight pig herds affected with PIVD, including vesicular fluid (n = 4) and swabs (n = 7) and scrapings of ruptured vesicles and ulcerative lesions (n = 5) from weaned and adult pigs. Clinically healthy animals (n = 52) of PIVD-affected and non-affected pig herds also were evaluated for Senecavirus A infection. The 16 samples from PIVD-affected animals were positive for Senecavirus A in the RT-PCR assay, while none of the clinically healthy pigs were detected with the virus. Sequencing analysis revealed high nucleotide (87.6-98.5%) and amino acid (95-99.4%) similarities to SVV-01 prototype and other Senecavirus A strains from North American pigs. Primer set presented herein was suitable for molecular characterization of Senecavirus A. The results suggest that Senecavirus A was the aetiological agent of the vesicular disease outbreaks in the evaluated pig herds. This is the first study to report the Senecavirus A infection in clinically affected pigs outside of North America. Senecavirus A was considered a novel emerging pathogen associated with an important vesicular disease in Brazil.

Phylogenetic analyses of the hemagglutinin gene of wild-type strains of canine distemper virus in southern Brazil.

This study examined the phylogenetic relationship of strains of canine distemper virus (CDV) collected from Paraná State, Brazil, based on the hemagglutinin gene. Urine samples were collected from 4 dogs from northern Paraná State that demonstrated clinical manifestations of canine distemper. The participation of CDV was initially confirmed by RT-PCR targeting the nucleocapsid protein, after which the complete hemagglutinin gene was sequenced from each sample. Sequences were deposited in and compared with those already in GenBank. Phylogenetic analyses, using amino acid and nucleotide sequences based on the hemagglutinin gene, demonstrated that these strains of CDV are closely related to those from the Europe 1 lineage of CDV, with marked differences from other recognized geographical clusters of CDV isolates and from the vaccine strains. The strains of CDV from this region of southern Brazil appear to be related to those from Europe 1.

Molecular detection and characterization of hepatitis E virus in naturally infected pigs from Brazilian herds.

The aim of this study was to investigate the presence of swine hepatitis E virus (HEV) from pigs of different production categories and from different pig farms in South Brazil. A total of 170 porcine faecal samples from breeder sows, boars, suckling piglets, weaned and growing pigs were collected from 14 pig farms. The faecal samples were screened by nested RT-PCR using primers targeting the ORF2 region of HEV genome. The samples that were positive from this screening were used in a nested RT-PCR targeting the ORF1 region. The screening detected HEV RNA in 62.5% of the pig farms and in 15.3% of the faecal samples. In 15 faecal samples, it was possible to amplify the HEV RNA with both the ORF1 and ORF2 regions. The phylogenetic analyses obtained for both ORFs confirmed that all of the Brazilian swine HEV isolates clustered with genotype 3b, the same genotype described previously in humans in Brazil.

Canine distemper virus and Toxoplasma gondii co-infection in dogs with neurological signs / Co-infecção pelo vírus da cinomose canina e Toxoplasma gondii em cães com sinais neurológicos

O presente estudo relata a ocorrência de co-infecção entre o vírus da cinomose canina (CDV) e Toxoplama gondii em cães com sinais neurológicos. Amostras de soro e tecido nervoso (pos-mortem) de 21 cães, suspeitos de cinomose canina foram analisadas pela Reação de Imunofluorecência indireta (RIFI) para pesquisa de anticorpos contra T. gondii e N. caninum e por RT-PCR para CDV. Dezessete (80,9 por cento) cães foram positivos para o CDV pela RT-PCR e 8 (38,1 por cento) foram positivos para anticorpos contra T. gondii. Sete cães (41,1 por cento) apresentaram-se positivos para ambos agentes, caracterizando processo de co-infecção. Somente 1 (4,7 por cento) cão foi soropositivo para N. caninum (RIFI=100), entretanto este mesmo animal foi positivo para T. gondii (RIFI=4096) e para CDV (RT-PCR).

VP6 gene diversity in Brazilian strains of porcine group C rotavirus.

Group C rotavirus (RV-C) has been found in Brazilian pig herds; however, wild-type strains have not yet been characterized. We made a molecular analysis of a region of gene 5 in Brazilian RV-C strains. Stool samples from 11 piglets (diarrheic and with normal consistency) positive for the RV-C VP6 gene in an RT-PCR assay were sequenced. A 270-bp amplicon of nine sequences was analyzed. All sequences showed high identity to the Cowden strain of the porcine RV-C prototype and 81.3 to 94.3% to each other (230 nucleotide fragment). Three Brazilian strains were classified in the Cowden group, while the other six showed higher heterogeneity (84.3 to 87.3%) with the prototype strain. Four clusters were formed in the dendrogram, including one human, one bovine, and two porcine clusters; one of these was formed by the six Brazilian strains described in this study. The Brazilian RV-C strains described here did not show any association with the year of collection, the presence of diarrhea, the age of the pig, or the geographical region of herd origin. This strongly suggests that these heterogeneous strains are widely spread in Brazilian pig herds. We conclude that there is genetic polymorphism in the VP6 gene of porcine RV-C strains in Brazil.

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains.

Group B rotaviruses (RV-B) were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8) were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

Group B rotaviruses (RV-B) were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8) were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7 percent nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8 percent identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8 percent. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

Molecular analysis of the bovine coronavirus S1 gene by direct sequencing of diarrheic fecal specimens.

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. The BCoV S protein plays a fundamental role in viral attachment and entry into the host cell, and is cleaved into two subunits termed S1 (amino terminal) and S2 (carboxy terminal). The present study describes a strategy for the sequencing of the BCoV S1 gene directly from fecal diarrheic specimens that were previously identified as BCoV positive by RT-PCR assay for N gene detection. A consensus sequence of 2681 nucleotides was obtained through direct sequencing of seven overlapping PCR fragments of the S gene. The samples did not undergo cell culture passage prior to PCR amplification and sequencing. The structural analysis was based on the genomic differences between Brazilian strains and other known BCoV from different geographical regions. The phylogenetic analysis of the entire S1 gene showed that the BCoV Brazilian strains were more distant from the Mebus strain (97.8% identity for nucleotides and 96.8% identity for amino acids) and more similar to the BCoV-ENT strain (98.7% for nucleotides and 98.7% for amino acids). Based on the phylogenetic analysis of the hypervariable region of the S1 subunit, these strains clustered with the American (BCoV-ENT, 182NS) and Canadian (BCQ20, BCQ2070, BCQ9, BCQ571, BCQ1523) calf diarrhea and the Canadian winter dysentery (BCQ7373, BCQ2590) strains, but clustered on a separate branch of the Korean and respiratory BCoV strains. The BCoV strains of the present study were not clustered in the same branch of previously published Brazilian strains (AY606193, AY606194). These data agree with the genealogical construction and suggest that at least two different BCoV strains are circulating in Brazil.

Molecular analysis of the bovine coronavirus S1 gene by direct sequencing of diarrheic fecal specimens

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. The BCoV S protein plays a fundamental role in viral attachment and entry into the host cell, and is cleaved into two subunits termed S1 (amino terminal) and S2 (carboxy terminal). The present study describes a strategy for the sequencing of the BCoV S1 gene directly from fecal diarrheic specimens that were previously identified as BCoV positive by RT-PCR assay for N gene detection. A consensus sequence of 2681 nucleotides was obtained through direct sequencing of seven overlapping PCR fragments of the S gene. The samples did not undergo cell culture passage prior to PCR amplification and sequencing. The structural analysis was based on the genomic differences between Brazilian strains and other known BCoV from different geographical regions. The phylogenetic analysis of the entire S1 gene showed that the BCoV Brazilian strains were more distant from the Mebus strain (97.8 percent identity for nucleotides and 96.8 percent identity for amino acids) and more similar to the BCoV-ENT strain (98.7 percent for nucleotides and 98.7 percent for amino acids). Based on the phylogenetic analysis of the hypervariable region of the S1 subunit, these strains clustered with the American (BCoV-ENT, 182NS) and Canadian (BCQ20, BCQ2070, BCQ9, BCQ571, BCQ1523) calf diarrhea and the Canadian winter dysentery (BCQ7373, BCQ2590) strains, but clustered on a separate branch of the Korean and respiratory BCoV strains. The BCoV strains of the present study were not clustered in the same branch of previously published Brazilian strains (AY606193, AY606194). These data agree with the genealogical construction and suggest that at least two different BCoV strains are circulating in Brazil.

RT-PCR em pools de soros sangüíneos para o diagnóstico da infecção aguda e de animais persistentemente infectados pelo vírus da diarréia viral bovina / RT-PCR in pools of bovine blood serum to detect acute infection and persistently infected animals with bovine viral diarrhea virus

Utilizou-se a técnica da RT-PCR para a detecção da região 5' UTR do genoma do vírus da diarréia viral bovina (BVDV) em pools de soros sangüíneos provenientes de um rebanho, constituído por 226 animais, que apresentava distúrbios da reprodução. A partir das amostras individuais de soro e de acordo com a categoria dos animais e o número de animais por categoria foram formados 10 pools (A a J) de soros. A primeira avaliação revelou a amplificação de um produto com 290pb nas reações referentes aos grupos D (35 vacas) e H (25 bezerros lactentes) que, após o desmembramento em amostras individuais, resultou na identificação de 11 vacas lactantes e 12 bezerros em amamentação positivos. Para a identificação de animais persistentemente infectados (PI) entre os 23 positivos na primeira avaliação, realizou-se a segunda colheita de soros sangüíneos, três meses após. A RT-PCR das amostras individuais de soro revelou resultado positivo em cinco bezerros. Em dois, foi possível isolar o BVDV em cultivo de células MDBK. A especificidade das reações da RT-PCR foi confirmada pelo seqüenciamento dos produtos amplificados a partir do soro de uma vaca com infecção aguda, de um bezerro PI e das duas amostras do BVDV isoladas em cultivo celular. A utilização da RT-PCR em pools de soros sangüíneos demonstrou ser uma estratégia rápida de diagnóstico etiológico e de baixo custo tanto para a detecção de infecção aguda quanto de animais PI.

The 5' untranslated region of the bovine viral diarrhea virus (BVDV) genome was detected by RT-PCR assay in pools of blood sera samples collected from a cattle herd (n=226 animals) with reproductive failures. Based on the classes of animal and the number of animals per class, the individual blood serum samples were distributed in 10 sera pools (A to J). During the first evaluation a 290bp amplicon was amplified in reactions from groups D (35 cows) and H (25 sucking calves). The individual analysis of serum from groups D and H resulted in positive reactions in serum samples from 11 cows and 12 calves. For the identification of persistently infected (PI) animals, three months after the first examination, blood serum samples from 23 positive animals were reevaluated by RT-PCR, resulting in five positive calves. In two of these calves the BVDV was isolated in MDBK cell culture. The specificity of RT-PCR amplicons from one cow with acute infection, one PI calf, and two wild type BVDV strains isolated in cell culture were confirmed by nucleotide sequencing. The use of RT-PCR in pools of blood sera proved to be a quick and low cost strategy for the etiological diagnosis of the acute infection as well as to detect PI animals thereby favoring the implementation of control and prophylaxis measures.

Avaliação da urina e de leucócitos como amostras biológicas para a detecção ante mortem do vírus da cinomose canina por RT-PCR em cães naturalmente infectados / Evaluation of the urine and leucocytes as biological samples for ante mortem detection of canine distemper virus by RT-PCR assay in naturally infected dogs

Urine and leucocytes were comparatively evaluated as clinical samples for ante mortem detection of the canine distemper virus (CDV) by a reverse transcription-polymerase chain reaction (RT-PCR) assay. One hundred and eighty eight dogs with clinical symptoms of distemper, were distributed in three groups. The group A was constituted of 93 dogs with systemic signs of distemper; the group B by 11 dogs with neurological signs, and the group C by 84 dogs that presented simultaneously systemic and neurological signs. In 66.5 percent (125/188) of the dogs was amplified an amplicon with 287 base pair of the CDV nucleoprotein gene. In 60.8 percent (76/125) of the animals the CDV was detected simultaneously in the urine and leucocytes, and in 39.2 percent (49/125) of the dogs just a type of clinical sample (urine: n=37; leucocytes: n=12) was positive. These results demonstrate that the different forms of clinical distemper disease can hinder the choice of only one type of clinical sample to carry out the ante mortem etiological diagnosis of CDV infection, and false-negative results can be generated.