Meningoencefalite em bovinos causada por herpesvírus bovino-5 no Mato Grosso do Sul e São Paulo

Quinze focos de meningoencefalite por herpesvírus bovino-5 (BHV-5) foram diagnosticados entre agosto de 1993 e dezembro de 1996, sendo 14 provenientes do estado do Mato Grosso do Sul e um do estado de São Paulo. A doença ocorreu em diversos municípios e em diferentes épocas do ano. Foram afetados bovinos de 6 a 60 meses de idade, com uma morbidade de 0,05% a 5% e letalidade próxima a 100%. Os sinais clínicos foram exclusivamente nervosos e o curso da enfermidade variou de 1 a 15 dias. As principais lesões histológicas detectadas foram meningite e encefalite difusa com malacia do córtex cerebral e presença de corpúsculos de inclusão intranucleares em astrócitos e neurônios. O vírus foi isolado do cérebro de 11 de um total de 12 animais, e sua identidade confirmada por imunoperoxidase, utilizando-se anticorpos monoclonais específicos. Os surtos de encefalite por BHV-5 representam 5% dos diagnósticos realizados em bovinos pelo Hospital Veterinário da Universidade Federal do Mato Grosso do Sul. Os resultados deste trabalho evidenciam a importância da doença no Mato Grosso do Sul e indicam a necessidade de incluir a encefalite por BHV-5 no diagnóstico diferencial de outras doenças do sistema nervoso de bovinos frequentes no Estado

Patterns and Processes of Mycobacterium bovis Evolution Revealed by Phylogenomic Analyses

Mycobacterium bovis is an important animal pathogen worldwide, parasitizing wild and domesticated vertebrate live stocks as well as humans. We investigated evolutionary patterns and processes in 38 sequenced genomes of M. bovis from the Americas, Europe, Asia, and Africa. We found that US strains are evolving distinctly (though with low magnitude) from most South American lineages regarding core-coding size, GC-content, and codon usage bias, and we further employ agreement to this pattern as proxy for phylogenetic reliability. Three data types were used for tree inference: 1. Single copy core-coding genes (core-coding); 2. Indel blocks coded as numeric characters (indels); 3. Gene presence/absence similarly coded (pangenome). Core-coding´s performance was below expected regarded we used 2,000+ genes, but indels and pangenome individually estimated reasonable trees, with HGT rates as high as 200x the DNA substitution rate. In addressing why core-coding performed poorly, we excluded phylogenetic method bias, homologous recombination, positive, and purifying selection as possible causes, whilst transient retention of random mildly deleterious mutations due to recent diversification, and also ongoing pseudogeneization, may be plausible. Although total evidence´s retention index was as good as presence/absence data individually in identifying US samples, it additionaly found a South American clade with more lineages excluding only two Brazilian samples. We further inferred divergence times using indels + pangenome due to lack of informativeness of core-coding, recovering relatively similar date estimates to previous studies on the origin of M. bovis (< 10,000 y) regardless of the dating prior (strict vs. uncorrelated clock, uniform vs. exponential clock, different age/rate standard deviation priors), evincing the suitability and unsuspected robustness of using strictly genomic-wide presence/absence data to dating, while also decisively rejecting a strict clock for our dataset. All trees supported a relatively basal position of African strains, which were isolated from Homo sapiens, suggesting it was an important region for early diversification and confirming we were one of the earliest hosts of the species. Evolution of US strains was affected by lateral acquisition of 57 exclusive genes from, including genes important for virulence. A clade of three US localities, distant from one another, is under even faster evolutionary rates supported by core-coding, indels, and pangenome.Fil: Patané, José S.L.. Universidade de Sao Paulo; BrasilFil: Martins, Joaquim. Universidade de Sao Paulo; BrasilFil: Castelão, Ana Beatriz. Universidade Federal do Mato Grosso do Sul; BrasilFil: Nishibe, Christiane. Universidade Federal do Mato Grosso do Sul; BrasilFil: Montera, Luciana. Universidade Federal do Mato Grosso do Sul; BrasilFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zumárraga, Martín José. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cataldi, Ángel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias; ArgentinaFil: Junior, Antônio Fonseca. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Roxo, Eliana. Instituto Biológico de São Pablo; BrasilFil: Osório, Ana Luiza A. R.. Universidade Federal do Mato Grosso do Sul; BrasilFil: Jorge, Klaudia S.. Universidade Federal do Mato Grosso do Sul; BrasilFil: Thacker, Tyler C.. United States Department of Agriculture; Estados UnidosFil: Almeida, Nalvo F.. Universidade Federal do Mato Grosso do Sul; BrasilFil: Araújo, Flabio R.. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Setubal, João C.. Universidade de Sao Paulo; Brasil. Biocomplexity Institute of Virginia Tech; Estados Unido

Patterns and Processes of Mycobacterium bovis Evolution Revealed by Phylogenomic Analyses

Mycobacterium bovis is an important animal pathogen worldwide, parasitizing wild and domesticated vertebrate live stocks as well as humans. We investigated evolutionary patterns and processes in 38 sequenced genomes of M. bovis from the Americas, Europe, Asia, and Africa. We found that US strains are evolving distinctly (though with low magnitude) from most South American lineages regarding core-coding size, GC-content, and codon usage bias, and we further employ agreement to this pattern as proxy for phylogenetic reliability. Three data types were used for tree inference: 1. Single copy core-coding genes (core-coding); 2. Indel blocks coded as numeric characters (indels); 3. Gene presence/absence similarly coded (pangenome). Core-coding´s performance was below expected regarded we used 2,000+ genes, but indels and pangenome individually estimated reasonable trees, with HGT rates as high as 200x the DNA substitution rate. In addressing why core-coding performed poorly, we excluded phylogenetic method bias, homologous recombination, positive, and purifying selection as possible causes, whilst transient retention of random mildly deleterious mutations due to recent diversification, and also ongoing pseudogeneization, may be plausible. Although total evidence´s retention index was as good as presence/absence data individually in identifying US samples, it additionaly found a South American clade with more lineages excluding only two Brazilian samples. We further inferred divergence times using indels + pangenome due to lack of informativeness of core-coding, recovering relatively similar date estimates to previous studies on the origin of M. bovis (< 10,000 y) regardless of the dating prior (strict vs. uncorrelated clock, uniform vs. exponential clock, different age/rate standard deviation priors), evincing the suitability and unsuspected robustness of using strictly genomic-wide presence/absence data to dating, while also decisively rejecting a strict clock for our dataset. All trees supported a relatively basal position of African strains, which were isolated from Homo sapiens, suggesting it was an important region for early diversification and confirming we were one of the earliest hosts of the species. Evolution of US strains was affected by lateral acquisition of 57 exclusive genes from, including genes important for virulence. A clade of three US localities, distant from one another, is under even faster evolutionary rates supported by core-coding, indels, and pangenome.Fil: Patané, José S.L.. Universidade de Sao Paulo; BrasilFil: Martins, Joaquim. Universidade de Sao Paulo; BrasilFil: Castelão, Ana Beatriz. Universidade Federal do Mato Grosso do Sul; BrasilFil: Nishibe, Christiane. Universidade Federal do Mato Grosso do Sul; BrasilFil: Montera, Luciana. Universidade Federal do Mato Grosso do Sul; BrasilFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zumárraga, Martín José. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cataldi, Ángel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias; ArgentinaFil: Junior, Antônio Fonseca. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Roxo, Eliana. Instituto Biológico de São Pablo; BrasilFil: Osório, Ana Luiza A. R.. Universidade Federal do Mato Grosso do Sul; BrasilFil: Jorge, Klaudia S.. Universidade Federal do Mato Grosso do Sul; BrasilFil: Thacker, Tyler C.. United States Department of Agriculture; Estados UnidosFil: Almeida, Nalvo F.. Universidade Federal do Mato Grosso do Sul; BrasilFil: Araújo, Flabio R.. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Setubal, João C.. Universidade de Sao Paulo; Brasil. Biocomplexity Institute of Virginia Tech; Estados Unido

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis

Nested-PCR for <i>Mycobacterium bovis</i> TbD1 and culture results of 229 bovine and bubaline tissue homogenates.

<p>CITT = Comparative intradermal tuberculin test.</p><p>LCT = Lesions compatible with tuberculosis.</p><p>NVL = No visible lesions.</p><p>*Confirmed by PCR with primers JB21 and JB22 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091023#pone.0091023-Rodriguez1" target="_blank">[15]</a>.</p><p>**p-value for chi-square of linear trend <0.001.</p><p>Different lowercase letters in rows indicate significant differences (p<0.05) within the paired comparisons between culture and nested-PCR.</p><p>Different capital letters in the columns indicate proportions significantly different (p<0.05).</p

Bacterial standard strains used for evaluation of analytical specificity or analytical sensitivity of the nested-PCR.

<p>*Fundação Oswaldo Cruz, Instituto Nacional de Controle de Qualidade em Saúde.</p