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(AU)

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(AU)

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).(AU)

Effect of indole acetic acid on the in vitro activation and survival of bovine (Bos indicus) preantral follicles

The objective of this study was to evaluate the development of preantral follicles from the Nelore breed of Bos indicus after in vitro culture of ovarian cortices in different concentrations of indole acetic acid (IAA). In addition, this study investigated the possible association of mammalian genes with IAA activity in bovine preantral follicles using an in silico approach. Ovaries (N = 8) from Nelore heifers were collected, and the ovarian cortex was divided into 14 fragments were cultured individuallu for two or siz days in Minimum Essential Medium (MEM) with 0, 10, 40, 100, 500 or 1000 ng/ml of IAA. Follicles were classified as primordial or as developing (primary and secondary) follicles. The in silico approach to search for auxinresponsive candidate genes was performed with bioinformaties tools, such as GenBank and SWISS-PROT database, Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Clustal W program. Compared to the control culture, the percentage of primordial follicles was reduced (P < 0,05) and the percentage of developing follicles was increased ( P < 0,05) after 2 or 6 days of culture in all media tested. Furtherimore, culture of the ovarian cortex for 6 days reduced the percentage of healthy, morphologically normal follicles when compared with the control (P < 0,05). In contrast, cultures supplemented with 10 ng/ml of IAA were the only samples that had similar (P < 0,05) percentages relative to the control group. Finally, we found a mammalian gene that was homologous to the plant gene, ROOTY, which may be involved in the oocyte response to IAA. We conclude that the 10 ng/ml concentration of IAA promoted follicular survival and activation of nelore bovine preantral follicles after 6 days of culture in vitro.(AU)

Effect of indole acetic acid on the in vitro activation and survival of bovine (Bos indicus) preantral follicles

The objective of this study was to evaluate the development of preantral follicles from the Nelore breed of Bos indicus after in vitro culture of ovarian cortices in different concentrations of indole acetic acid (IAA). In addition, this study investigated the possible association of mammalian genes with IAA activity in bovine preantral follicles using an in silico approach. Ovaries (N = 8) from Nelore heifers were collected, and the ovarian cortex was divided into 14 fragments were cultured individuallu for two or siz days in Minimum Essential Medium (MEM) with 0, 10, 40, 100, 500 or 1000 ng/ml of IAA. Follicles were classified as primordial or as developing (primary and secondary) follicles. The in silico approach to search for auxinresponsive candidate genes was performed with bioinformaties tools, such as GenBank and SWISS-PROT database, Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Clustal W program. Compared to the control culture, the percentage of primordial follicles was reduced (P < 0,05) and the percentage of developing follicles was increased ( P < 0,05) after 2 or 6 days of culture in all media tested. Furtherimore, culture of the ovarian cortex for 6 days reduced the percentage of healthy, morphologically normal follicles when compared with the control (P < 0,05). In contrast, cultures supplemented with 10 ng/ml of IAA were the only samples that had similar (P < 0,05) percentages relative to the control group. Finally, we found a mammalian gene that was homologous to the plant gene, ROOTY, which may be involved in the oocyte response to IAA. We conclude that the 10 ng/ml concentration of IAA promoted follicular survival and activation of nelore bovine preantral follicles after 6 days of culture in vitro.

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.(AU)

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.(AU)

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% (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.(AU)

Perfil de restrição de um fragmento do gene da hemaglutinina amplificado pela RT-PCR a partir de estirpes vacinais e selvagens do vírus da cinomose canina / Restriction pattern of a hemagglutinin gene amplified by RT-PCR from vaccine strains and wild-type canine distemper virus

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.(AU)

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.(AU)

Efeito do dimetildioctadecilamônio na resposta imune humoral e celular de cobaios inoculados com o herpesvírus bovino-1 inativados / Effect of dimethyl dioctadecyl ammonium chloride on the humoral and cellular response to inactivated bovine herpesvirus-1 in guinea pigs

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.(AU)

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.(AU)

Lesões histológicas no sistema nervoso central de cães com encefalite e diagnóstico molecular da infecção pelo vírus da cinomose canina / Histopathological lesions in the central nervous system of dogs with encephalitis and molecular diagnosis of canine distemper virus infection

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. (AU)

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.(AU)

Detecção do gene da nucleoproteína do vírus da cinomose canina por RT-PCR em urina de cães com sinais clínicos de cinomose / Detection of canine distemper virus nucleoprotein gene by RT-PCR in urine of dogs with distemper clinical signs

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.(AU)

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 c2 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.(AU)