RESUMO 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.

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.

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%) cães foram positivos para o CDV pela RT-PCR e 8 (38,1%) foram positivos para anticorpos contra T. gondii. Sete cães (41,1%) apresentaram-se positivos para ambos agentes, caracterizando processo de co-infecção. Somente 1 (4,7%) cão foi soropositivo para N. caninum (RIFI=100), entretanto este mesmo animal foi positivo para T. gondii (RIFI=4096) e para CDV (RT-PCR).

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.

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.

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.

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% (125/188) of the dogs was amplified an amplicon with 287 base pair of the CDV nucleoprotein gene. In 60.8% (76/125) of the animals the CDV was detected simultaneously in the urine and leucocytes, and in 39.2% (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.

Fragmentos com 721 pares de bases do gene da hemaglutinina (H) do vírus da cinomose canina (CDV), amplificados pela RT-PCR a partir de três estirpes vacinais (Snyder Hill, Onderstepoort e Rockborn) e de 27 amostras de campo, provenientes de cães com cinomose, foram clivados com as endonucleases Hinf I and Rsa I. A seleção das enzimas foi realizada por meio de análises in silico de seqüências do CDV depositadas em bases públicas de dados. Tanto as estirpes vacinais quanto as amostras selvagens do CDV apresentaram com a enzima Hinf I o mesmo perfil de restrição, confirmando a identidade do fragmento amplificado pela RT-PCR, uma vez que todas as estirpes com seqüências disponíveis (GenBank) têm sítios de restrição para essa enzima nas mesmas posições. O perfil de restrição das estirpes vacinais Snyder Hill e Onderstepoort, que diferem entre si, foi confirmado com a enzima Rsa I que também clivou a estirpe Rockborn nas mesmas posições que a estirpe Snyder Hill. Todas as 27 amostras de campo do CDV apresentaram com a enzima Rsa I o mesmo perfil de restrição, indicando conservar os mesmos sítios de restrição para essa enzima. O perfil das amostras de campo foi diferente daquele obtido nas três estirpes vacinais. Os perfis de restrição do gene que codifica a hemaglutinina do CDV, gerados pela enzima Rsa I, sugerem diferenças moleculares entre as estirpes vacinais e as selvagens circulantes na região norte do estado do Paraná e abrem a perspectiva da elaboração de análises moleculares comparativas mais complexas, como o seqüenciamento de todo o gene H, de estirpes do CDV identificadas em diferentes regiões brasileiras.

The restriction fragment length polymorphism (RFLP) assay of a 721 bp fragment from hemagglutinin (H) gene of canine distemper virus (CDV) amplified by RT-PCR was analyzed with Hinf I and Rsa I enzymes. Clinical samples from 27 dogs with natural canine distemper infection, and three vaccine (Snyder Hill, Onderstepoort and Rockborn) CDV strains were analyzed. All RT-PCR amplified product from CDV wild-type and vaccine-strains had the same RFLP pattern with Hinf I enzyme showing the amplicon specificity. The RFLP pattern for CDV vaccine-strains generated with Rsa I enzyme was the expected by in silico analysis. All 27 wild-type CDV strains present the same Rsa I enzyme RFLP pattern that was different from vaccine-strains pattern, suggesting molecular differences between vaccine-strains and wild-type of CDV from dogs population in North region of Paraná State/Brazil. These results open the perspectives of the accomplishment of comparative molecular analysis, such as sequencing of the whole gene H, of CDV wild-type strains identified in different Brazilian regions.

A presença do vírus da cinomose canina (CDV) foi avaliada pela reação em cadeia da polimerase, precedida de transcrição reversa (RT-PCR), em 87 amostras de urina de cães que apresentavam sinais clínicos sugestivos de cinomose. Os animais foram distribuídos em três grupos. No grupo A foram incluídos 41 cães com alterações sistêmicas; no grupo B, 37 cães com alterações neurológicas; e no grupo C, nove cães com alterações sistêmicas e neurológicas simultâneas. O grupo D (controle) foi composto por 20 cães assintomáticos. Os resultados da RT-PCR foram correlacionados com a forma clínica da infecção e com as alterações hematológicas encontradas. Foi possível a amplificação parcial do gene da nucleoproteína do CDV em 41 (47,1%) das 87 amostras de urina provenientes de cães com sinais clínicos sugestivos de cinomose. Todas as amostras obtidas de animais assintomáticos foram negativas na RT-PCR. Amostras positivas foram encontradas nos três grupos de animais com sinais clínicos na proporção de 51,2% (24/41), 29% (11/37) e 100% (9/9) para os grupos A, B e C, respectivamente. A leucocitose foi a alteração hematológica mais freqüente nos três grupos de cães com sinais clínicos porém, não foi possível estabelecer correlação entre o resultado da RT-PCR e as alterações hematológicas. Os resultados demonstraram que, independente da forma de apresentação clínica, a técnica da RT-PCR realizada em urina pode ser utilizada no diagnóstico ante mortem da infecção pelo CDV.

The urine of 87 dogs with clinical signs suggestive of canine distemper was analyzed by RT-PCR for detection of canine distemper virus (CDV) nucleoprotein gene. The samples were allotted to the following groups: group A- with 41 dogs with systemic symptoms, group B- with 37 dogs with neurological signs, and group C- with 9 dogs with simultaneous systemic and neurological clinical signs. Group D (control) included 20 assymptomatic dogs. A chi2 was used to test RT-PCR results according to clinical form and hematological characteristics. The RT-PCR was positive for CDV in 47% (41/87) of the urine samples from dogs with clinical signs. All samples from assymptomatic dogs were RT-PCR negatives. Positive samples were found in all groups of dogs with distemper symptoms according to the following propositions: 51.2% (21/41), 29% (11/37) and 100% (9/9) for groups A, B and C, respectively. In all clinical forms (groups A, B and C) leucocytosis was the most frequent observed hematological alteration. No relationship between RT-PCR results and hematological changes was observed. The results showed that independently of the clinical stage of the illness the RT-PCR based on urine sample can be applied for ante mortem diagnosis of CDV.

Avaliou-se o potencial adjuvante do cloreto de dimetildioctadecilamônio (DDA cloreto) em estimular as respostas imune humoral e celular, do tipo hipersensibilidade cutânea tardia (DTH), em cobaios que receberam preparações de antígeno contendo o herpesvírus bovino tipo 1 (BHV-1) inativado. Os animais foram vacinados com o BHV-1 em cinco diferentes formulações, representadas pelos grupos: A- adjuvante completo / incompleto de Freund, B- hidróxido de alumínio (Al(OH)3), C- DDA cloreto, D- associação Al(OH)3 / DDA cloreto e E- BHV-1 em solução aquosa sem adjuvante. Os animais do grupo F, grupo-controle negativo, foram inoculados apenas com meio de cultivo celular. Os maiores títulos de anticorpos neutralizantes do BHV-1, expressos em log2, foram obtidos nos grupos D, A e C, com títulos de 6,25, 6,0 e 5,25, respectivamente. Na avaliação da DTH, os maiores aumentos na espessura da dobra da pele foram observados nos grupos A (2,4mm), C (1,8mm) e D (1,1mm). O DDA cloreto, utilizado tanto de forma isolada quanto em associação, determinou a potencialização das respostas imunológicas humoral e celular de cobaios imunizados com o BHV-1 inativado.

The adjuvanticity of dimethyl dioctadecyl ammonium chloride (DDA chloride) to induce humoral and cell mediated (delayed type hypersensitivity - DTH -) immune responses was assessed in guinea pigs that received antigen preparations containing inactivated bovine herpesvirus type 1 (BHV-1). The animals were vaccinated with five different formulations, containing BHV-1 represented by groups: A- complete/incomplete Freund adjuvant, B- aluminum hydroxide (Al(OH)3), C- DDA chloride, D- association Al(OH)3/DDA and E- BHV-1 in aqueous solution without adjuvant. Group F was the negative control group and the animals received only cell culture medium. The higher titers of BHV-1 neutralizing antibodies, expressed in log2, were obtained in groups D, A and C with values of 6.25, 6.0 and 5.25, respectively. In the DTH assessment, the higher increase in skin thickness was observed in group A (2.4mm), C (1.8mm) and D (1.1mm). These results showed that DDA chloride used alone or in association to other substance boosted the humoral and cell mediated immune responses in guinea pigs immunized with inactivated BHV-1.

Dez amostras de urina de cães que apresentavam sinais clínicos sistêmicos e neurológicos indicativos da infecção pelo vírus da cinomose canina (CDV) foram analisadas pela técnica da reação em cadeia pela polimerase precedida de transcrição reversa (RT-PCR) para a detecção do RNA do CDV. O exame histopatológico foi realizado em fragmentos de cérebro, cerebelo e bexiga. Como controle negativo foram colhidos fragmentos de órgãos e urina de quatro cães sem sinais clínicos de doença infecciosa e que morreram por outras causas. Todos os cães (9/10) positivos em RT-PCR apresentaram alterações histológicas no cérebro e cerebelo, características de encefalite aguda (5/9) ou crônica (4/9) compatíveis com as causadas pelo CDV. Um dos cães com alterações clínicas neurológicas semelhantes às observadas em cinomose foi negativo na RT-PCR e apresentou alterações histopatológicas inespecíficas. Nas amostras de bexiga não foram observadas lesões histológicas. Todas as amostras biológicas provenientes dos cães-controle foram negativas na RT-PCR (urina) e não apresentaram alterações histológicas (fragmentos de órgãos). O trabalho evidenciou a especificidade da RT-PCR no diagnóstico precoce e ante mortem na infecção pelo CDV.

The reverse transcription-polymerase chain reaction (RT-PCR) was used to detect canine distemper virus (CDV) RNA in 10 urine samples from dogs that had died with clinical systemic and neurological signs indicative of CDV infection. Brain, cerebellum and urinary bladder fragments were collected for histopathological examination. For the negative control, urine and organ fragments were collected at necropsy from four dogs without clinical symptoms of infectious diseases that had died from other causes. The dogs positives in RT-PCR (9/10) presented histological lesions in the brain and cerebellum characteristic of acute (5/9) or chronic (4/9) encephalitis compatible with those caused by CDV. One of the dogs with neurological signs similar to those observed in canine distemper was negative in RT-PCR and presented non-specific histopathological alterations that could be associated to others nervous system diseases. Although the RT-PCR detected the CDV in urine of nine dogs with clinical signs of distemper, no histopathological alterations were observed in any of the urinary bladder samples. All the biological samples from the control dogs were negative in RT-PCR and did not present any histopathological alterations in organ. Besides describing the most characteristic histopathological lesion found in the central nervous system of dogs with acute and chronic encephalitis due to canine distemper, this study also showed the specificity of the RT-PCR technique in early and ante mortem diagnosis of CDV infections.

Cytotoxin production was studied in 60 Serratia marcescens strains isolated from hospitalized patients. Association of cytotoxic activity with serotype, source of isolation and presence of plasmids was also evaluated. Thirteen of the 60 S. marcescens strains produced a cytotoxic effect on Vero cells. These strains were isolated from distinct clinical sources and classified into seven different serotypes (O1:H7; O4:NM; O10:NT; O19:NM; O6,14:H4; O6,14:NM and O6,14:H1). No relationship was observed between cytotoxic activity and clinical source or serotypes of the strains. Plasmids from five cytotoxin-producing S. marcescens strains were transferred to E. coli K12/711. The transconjugants did not exhibit cytotoxicity, indicating that the cytotoxic effect is not plasmid-mediated among these strains. Although a cytotoxic activity was demonstrated in filtrates of some S. marcescens strains, further studies should be performed to assess the role of this toxin in pathogenesis