Polymorphisms in the Prion Protein Gene of cattle breeds from Brazil / Polimorfismos no gene da proteína priônica em bovinos no Brasil

One of the alterations that occur in the PRNP gene in bovines is the insertion/deletion (indel) of base sequences in specific regions, such as indels of 12-base pairs (bp) in intron 1 and of 23- bp in the promoter region. The deletion allele of 23 bp is associated with susceptibility to bovine spongiform encephalopathy (BSE) as well as the presence of the deletion allele of 12 bp. In the present study, the variability of nucleotides in the promoter region and intron 1 of the PRNP gene was genotyped for the Angus, Canchim, Nellore and Simmental bovine breeds to identify the genotype profiles of resistance and/or susceptibility to BSE in each animal. Genomic DNA was extracted for amplification of the target regions of the PRNP gene using polymerase chain reaction (PCR) and specific primers. The PCR products were submitted to electrophoresis in agarose gel 3% and sequencing for genotyping. With the exception of the Angus breed, most breeds exhibited a higher frequency of deletion alleles for 12 bp and 23 bp in comparison to their respective insertion alleles for both regions. These results represent an important contribution to understanding the formation process of the Brazilian herd in relation to bovine PRNP gene polymorphisms.(AU)

Uma das mudanças que ocorrem no gene PRNP em bovinos é a inserção e/ou deleção (indels) de sequências de bases, em determinadas regiões como, por exemplo, as indels de 12 pares de bases (pb) no íntron 1 e 23pb na região promotora. O alelo de deleção de 23pb está relacionado com a suscetibilidade à Encefalopatia Espongiforme Bovina (EEB), assim como a presença do alelo de deleção de 12pb. Neste estudo foi genotipada a variabilidade de nucleotídeos da região promotora e íntron 1 do gene PRNP em bovinos das raças Angus, Canchim, Nelore e Simental, para identificar os perfis genotípicos de resistência e/ou suscetibilidade à EEB de cada animal. Foi realizada a extração de DNA genômico para amplificação das regiões alvo do gene PRNP, por meio da reação em cadeia de polimerase (PCR) utilizando-se primers específicos. Os produtos da PCR foram submetidos à eletroforese em gel de agarose a 3%, e sequenciamento para a realização da genotipagem. Com exceção da raça Angus, a maioria das raças apresentaram maiores frequências do alelo de deleção tanto para 12pb como 23pb, em comparação com seus respectivos alelos de inserção, para as duas regiões. Esses resultados abrem caminhos para o conhecimento de como o rebanho brasileiro está sendo formado com relação aos polimorfismos do gene PRNP bovino.(AU)

Detecção de Brucella abortus em tecidos bovinos utilizando ensaios de PCR e qPCR / Detection of Brucella abortus in bovine tissue using PCR and qPCR

Objetivou-se no presente estudo avaliar as técnicas reação em cadeia da polimerase (PCR) e PCR em Tempo Real (qPCR) para detectar Brucella abortus, a partir de tecidos bovinos com lesões sugestivas de brucelose. Para isto, 21 fragmentos de tecidos bovinos coletados em abatedouros de Mato Grosso do Sul foram processados e submetidos ao cultivo microbiológico e extração do DNA genômico para realização das reações de PCR e qPCR. No cultivo microbiológico, oito amostras apresentaram crescimento bacteriano e cinco foram confirmadas como B. abortus por PCR. Diretamente das amostras de tecido, DNA do gênero Brucella (oligonucleotídeos IS711) foi detectado em 13 (61,9 por cento) amostras de tecido e 17 (81 por cento) amostras de homogeneizado. Já com os oligonucleotídeos espécie-específicos BruAb2_0168F e BruAb2_0168R, 14 (66 por cento) amostras de tecido e 18 (85,7 por cento) amostras de homogeneizado foram amplificadas. Seis amostras positivas na PCR espécie-específica foram sequenciadas e o best hit na análise BLASTn foi B. abortus. Na qPCR, 21 (100 por cento) amostras de tecidos e 19 (90,5 por cento) amostras de homogeneizado foram positivas para B. abortus. Dez amostras de DNA de sangue bovino de rebanho certificado livre foram utilizadas como controle negativo nas análises de PCR e qPCR utilizando-se os oligonucleotídeos BruAb2_0168F e BruAb2_0168R. Na PCR nenhuma amostra amplificou, enquanto que na qPCR 2 (20 por cento) amplificaram. Conclui-se que as duas técnicas detectam a presença de B. abortus diretamente de tecidos e homogeneizados, porém a qPCR apresentou maior sensibilidade. Os resultados obtidos indicam que a qPCR pode representar uma alternativa rápida e precisa para a detecção de B. abortus diretamente de tecidos, e ser utilizada em programas de vigilância sanitária, por apresentar sensibilidade e especificidade satisfatórias.

The aim of the study was to evaluate the technical polymerase chain reaction (PCR) and Real-Time PCR (qPCR) to detect Brucella abortus from bovine tissues with suggestive lesions of brucellosis. For this, 21 fragments of bovine tissues collected at abattoirs of Mato Grosso do Sul were processed and subjected to microbiological culture and extraction of genomic DNA to perform the PCR reactions and qPCR. Eight samples of microbiological culture showed bacterial growth and five samples were confirmed as B. abortus by PCR. DNA of Brucella (IS711 primers) was detected in 13 (61.9 percent) directly from tissue samples and 17 (81 percent) from tissue homogenate samples. With the species-specific set of primers BruAb2_0168F and BruAb2_0168R, 14 (66 percent) tissue samples and 18 (85.7 percent) tissue homogenate samples were positive. Six positive samples in the species-specific PCR were sequenced and the best hit in the BLASTn analysis was B. abortus. By qPCR, 21 (100 percent) tissue samples and 19 (90.5 percent) tissue homogenate samples were positive for B. abortus. Ten samples of DNA from bovine blood from an accredited-free herd were used as negative control in PCR and qPCR analysis using the primers BruAb2_0168F and BruAb2_0168R, and no one amplified by PCR, whereas two samples were amplified by qPCR (20 percent). In conclusion, both techniques detect the presence of B. abortus directly from tissues and homogenized, but the qPCR showed high sensitivity. The results indicate that qPCR can represent an alternative tool for faster and more accurate detection of B. abortus directly from tissues, and use in health surveillance programs by presenting satisfactory sensitivity and specificity.

Avaliação genética das vacinas contra a brucelose bovina comercializadas no Brasil / Genetic evaluation of vaccine against bovine brucellosis marketed in Brazil

A prevenção contra infecções causadas por Brucella abortus em bovinos é realizada por meio da administração das amostras vacinais B19 e RB51. Existem relatos de que estas vacinas podem causar aborto em fêmeas vacinadas. Portanto, toda a ocorrência de aborto em animais vacinados merece um estudo aprofundado sobre a causa. No Brasil, não há registro sobre a origem das amostras B19 e RB51 utilizadas na produção das vacinas comerciais. Assim, um estudo para verificar possíveis mutações em relação às amostras referência USDA B19 e USDA RB51 de B. abortus se faz necessário, devido às amostras vacinais poderem reverter a sua virulência. Objetivou-se com este estudo caracterizar genotipicamente as amostras vacinais B19 e RB51 comercializadas no Brasil. A metodologia utilizada foi a genotipagem de genes marcadores destas amostras vacinais, por meio da amplificação pela reação em cadeia da polimerase. Os resultados obtidos permitiram a identificação do genótipo das vacinas comerciais B19 e RB51 disponíveis e utilizadas em bovinos no Brasil. A ausência de mutações nas vacinas testadas corrobora com a qualidade genética das mesmas, quanto à estabilidade dos genes analisados.

Vaccine strains B19 and RB51 are administered to cattle for prevention against infection by Brucella abortus. However, there are reports that these vaccines can cause miscarriages. Thus, every miscarriage among vaccinated animals should be thoroughly studied to determine the cause. In Brazil, there are no records on the origin of B19 and RB51 samples used in the preparation of commercial vaccines. Therefore, a study is needed to determine possible mutations in relation to the USDA reference samples of B. abortus due to the fact that vaccine samples could revert to the virulence of the disease. The aim of the present study was to perform a genotype analysis of vaccine strains B19 and RB51 used in Brazil. The methodology was based on the genotyping of marker genes of these vaccine strains by amplification using polymerase chain reaction. The results allowed the identification of the genotype of the B19 and RB51 commercial vaccine available for use on cattle in Brazil. The absence of mutations in the samples tested confirmed the genetic quality of the vaccines and stability of genes analyzed.

Real-time polymerase chain reaction based on msa2c gene for detection of Babesia bovis.

This paper reports a quantitative real-time polymerase chain reaction (q-PCR) based on the msa2c gene and standardized with Platinum SYBR Green/ROX for the detection of Babesia bovis in cattle. The msa2c q-PCR amplified a DNA fragment with average dissociation temperature of 77.41°C (± 0.25°C). No amplification was detected when DNA from B. bigemina, A. marginale or Bos taurus was used as the template. The detection limit of the msa2c q-PCR was 1000 copies per ml of blood sample, with a linear correlation between the number of msa2c copies and threshold cycle. The comparison between msa2c q-PCR and conventional PCR for cytochrome b revealed 88.8% agreement, with a Kappa index of 0.75. In the comparison between msa2c q-PCR and an enzyme-linked immunosorbent assay (ELISA) with semi-purified B. bovis antigen, agreement was 96.3% and the Kappa index was 0.91. The agreement between three tests was 85.8%. The msa2c q-PCR detected a higher number of positive cattle than conventional PCR in an enzootically stable area, but did not differ significantly from ELISA. No significant differences were detected between the three diagnostic tests with cattle from an enzootically unstable area. All animals raised on a tick-free facility were negative for B. bovis in the three tests. These results suggest that msa2c q-PCR is a useful test for the detection of B. bovis infection.

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale.

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.

Evidência molecular de brucella sp. em ozotoceros bezoarticus (veado campeiro) do Pantanal sul-mato-grossense / Molecular evidence of brucella sp. in deer (ozotoceros bezoarticus) of the southern Pantanal

A presença de Brucella spp. entre animais silvestres pode influenciar a taxa de reprodução destes hospedeiros, além de atuarem como fonte de infecção natural para os animais domésticos e humanos. O objetivo deste estudo foi identificar a presença de Brucella spp. em 44 amostras de sangue de veado campeiro (Ozotoceros bezoarticus) do Pantanal do Sul-Mato-Grossense, utilizando a técnica de PCR. Observou-se que 20,4 por cento (9/44) das amostras foram positivas. A sequência consenso de nucleotídeo obtida no sequenciamento do isolado de veado campeiro apresentou 514 pb e 95 por cento de identidade com virB5 de B. abortus (best hits acesso nr AF226278, e-value 0.0), já na análise filogenética a amostra de Brucella isolada de veado campeiro apresentou-se muito próximo de B. suis. A alta porcentagem de amostras positivas sugere que a brucelose pode ser um problema entre os veados campeiros na área estudada e que estes animais podem representar riscos para outros animais domésticos e silvestres.

The presence of Brucella spp. in wild animals can influence their reproduction rate and may be a source of infection for domestic animals and humans. The objective of this study was to identify the presence of Brucella spp. in 44 blood samples from the deer Ozotoceros bezoarticus in the southern Pantanal of Sul-Mato-Grossense, using the PCR technique. It was seen that 20.4 percent (9/44) of the samples were positive. The consensus sequence was obtained by sequencing these samples, which then showed 514 pb and 95 percent of identity with gene virB5 of B. abortus (best hits accession nr AF226278, e-value 0.0). The phylogenetic analysis of the sample isolated from deer revealed the Brucella to be very close to B. suis. The high percentage of positive samples suggests that brucellosis may be a concern in deer within the studied area, and that these animals may poses a risk for other domestic and wild ones.

The Brucella abortus phosphoglycerate kinase mutant is highly attenuated and induces protection superior to that of vaccine strain 19 in immunocompromised and immunocompetent mice.

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that can function as virulence factors to better understand the host-pathogen interplay. Herein, we identified the gene encoding the phosphoglycerate kinase (PGK) of B. abortus strain 2308. To test the role of PGK in Brucella pathogenesis, a pgk deletion mutant was constructed. Replacement of the wild-type pgk by recombination was demonstrated by Southern and Western blot analyses. The B. abortus Delta pgk mutant strain exhibited extreme attenuation in bone marrow-derived macrophages and in vivo in BALB/c, C57BL/6, 129/Sv, and interferon regulatory factor-1 knockout (IRF-1 KO) mice. Additionally, at 24 h postinfection the Delta pgk mutant was not found within the same endoplasmic reticulum-derived compartment as the wild-type bacteria, but, instead, over 60% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP1. Furthermore, the B. abortus Delta pgk deletion mutant was used as a live vaccine. Challenge experiments revealed that the Delta pgk mutant strain induced protective immunity in 129/Sv or IRF-1 KO mice that was superior to the protection conferred by commercial strain 19 or RB51. Finally, the results shown here demonstrated that Brucella PGK is critical for full bacterial virulence and that a Delta pgk mutant may serve as a potential vaccine candidate in future studies.

Molecular and antigenic characterisation of ribosomal phosphoprotein P0 from Babesia bovis.

Babesia bovis is a tick-borne pathogen that remains an important constraint for the development of cattle industries in tropical and subtropical regions of the world. Effective control can be achieved by vaccination with live attenuated phenotypes of the parasite. However, these phenotypes have a number of drawbacks, which justifies the search for new, more efficient immunogens based mainly on recombinant protein technology. In the present paper, ribosomal phosphoprotein P0 from a Brazilian isolate of B. bovis was produced and evaluated with regard to conservation and antigenicity. The protein sequence displayed high conservation between different Brazilian isolates of B. bovis and several Apicomplexa parasites such as Theileria, Neospora and Toxoplasma. IgG from cattle experimentally and naturally infected with B. bovisas well as IgG1 and IgG2 from naturally infected cattle reacted with the recombinant protein. IgG from cattle experimentally infected with Babesia bigemina cross-reacted with B. bovis recombinant P0. These characteristics suggest that P0 is a potential antigen for recombinant vaccine preparations against bovine babesiosis.

Desenvolvimento e avaliação de uma cepa knockout de Brucella abortus obtida pela deleção do gene virB10 / Development and evaluation of a strain of Brucella abortus gotten by the knockout of the virB10 gene

Brucella spp. são bactérias gram-negativas, intracelulares facultativas que são patogênicas para muitas espécies de mamíferos causando a brucelose, uma zoonose difundida mundialmente. Por isso a busca de alternativas de controle mais eficientes se faz necessário como o desenvolvimento de novas cepas que possam ser testadas como potenciais imunógenos. Neste estudo realizou-se a deleção do gene virB10 da cepa S2308 de Brucella abortus gerando uma cepa knockout provavelmente incapaz de produzir a proteína nativa correspondente. O gene virB10 faz parte de um operon que codifica para um sistema de secreção do tipo IV, essencial para a sobrevivência intracelular e multiplicação da bactéria em células hospedeiras. A deleção foi realizada pela construção do plasmídeo suicida pBlue:virB10:kan e eletroporação deste em células eletrocompetentes de B. abortus S2308, ocorrendo a troca do gene selvagem pelo gene interrompido, com o gene de resistência a canamicina, por recombinação homóloga dupla. Camundongos BALB/c foram inoculados com as cepas S19, RB-51, ΔvirB10 de B. abortus e B. abortus S2308 selvagem; os resultados demonstraram que camundongos BALB/c inoculados com S19 e camundongos BALB/c inoculados com S2308 apresentaram queda mais rápida de linha de tendência, quando comparadas aos demais grupos, para recuperação bacteriana (RB) e peso esplênico (PE) respectivamente. Os grupos que receberam ΔvirB10 S2308 de B. abortus e RB-51 demonstraram comportamento semelhante para ambas as características. Na sexta semana após a inoculação, os resultados para RB (log de UFC ± desvio padrão) e PE (peso esplênico ± desvio padrão), respectivamente, mostraram: grupos inoculados com as cepas S2308 (4,44±1,97 e 0,44±0,11), S19 (1,83±2,54 e 0,31±0,04), RB-51 (0,00±0,00 e 0,20±0,01) e ΔvirB10 S2308 (1,43±1,25 e 0,19±0,03). Considerado o clearance bacteriano, todos os grupos diferiram...

Brucella spp. are intracellular facultative gram-negative bacteria which are pathogenic for many species of mammals, causing brucellosis, a worldwide spread zoonosis. Therefore the search for more efficient alternatives of control, as the development of new potential immunogens is necessary. In this study, we knockouted virB10 from Brucella abortus S2308 strain, generating a mutant strain probably incapable to produce the corresponding native protein. The gene virB10 is part of an operon that codifies for type IV secretion system, which is essential for the intracellular survival and multiplication of the bacteria in host cells. The knockout was carried through by the construction of the suicidal plasmid pBlue: virB10: kan and eletroporation in eletrocompetent cells of B. abortus S2308, leading to the exchange of the wild gene for the interrupted gene, containing the gene of resistance to kanamycin, for double homologous recombination. BALB/c mice were inoculated with S19, RB-51, ΔvirB10 strains of B. abortus and S2308 wild strain; the results demonstrated that the BALB/c mice inoculated with S19 and BALB/c mice inoculated with S2308 presented faster fall of trend line, when compared with the too much groups, for bacterial recovery (BR) and esplenic weight (EW) respectively. The groups that received ΔvirB10 S2308 B. abortus and RB-51 demonstrated similar behavior for both the characteristics. In the sixth week postinoculation, the results for BR (log UFC ± standart deviations) and EW (esplenic weight ± standart deviations), respectively, showed: groups inoculated with strains S2308 (4,44±1,97 and 0,44±0,11), S19 (1,83±2,54 and 0,31±0,04), RB-51 (0,00±0,00 and 0,20±0,01) and ΔvirB10 S2308 (1,43±1,25 and 0,19±0,03). Considered the bacterial clearance, all the groups differed statistical from the group that received S2308 (p<0,0001), the group inoculated...

[Detection of antibodies against Anaplasma sp. in small ruminants from the semi-arid region against Pernambuco State, Brazil]. / Detecção de anticorpos para Anaplasma sp. em pequenos ruminantes no semi-árido do Estado de Pernambuco, Brasil.

This paper reports the detection of antibodies against Anaplasma sp. in goats and sheep from the semi-arid region from Pernambuco State, Brazil, using an enzyme-linked immunosorbent assay with recombinant MSP5 of Anaplasma marginale. Sera from 243 goats and 68 sheep from Ibimirim municipality were analyzed and frequencies of antibodies of 11.93% (29/243) and 16.17% (11/68) were found for goats and sheep, respectively. The epidemiological relevance of the findings was discussed.

Detecção de anticorpos para Anaplasma sp. em pequenos ruminantes no semi-árido do estado de Pernambuco, Brasil / Detection of antibodies against Anaplasma sp. in small ruminants from the semi-arid region against Pernambuco State, Brazil

Neste trabalho é descrita a detecção de anticorpos para Anaplasma sp. em caprinos e ovinos da região do semi-árido do Estado de Pernambuco, Brasil, utilizando-se um ensaio de imunoadsorção enzimática baseado em MSP5 recombinante de Anaplasma marginale. Foram analisados soros de 243 caprinos e 68 ovinos provenientes do município de Ibimirim, e observadas freqüências de anticorpos de 11,93 por cento (29/243) e 16,17 por cento (11/68) para caprinos e ovinos, respectivamente. A importância epidemiológica dos achados foi discutida.

This paper reports the detection of antibodies against Anaplasma sp. in goats and sheep from the semi-arid region from Pernambuco State, Brazil, using an enzyme-linked immunosorbent assay with recombinant MSP5 of Anaplasma marginale. Sera from 243 goats and 68 sheep from Ibimirim municipality were analyzed and frequencies of antibodies of 11.93 percent (29/243) and 16.17 percent (11/68) were found for goats and sheep, respectively. The epidemiological relevance of the findings was discussed.

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu.

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.

[Transcription of genes of membrane proteins of Brazilians isolates of Anaplasma marginale]. / Transcrição de genes de proteínas de membrana de isolados brasileiros de Anaplasma marginale.

This work shows the transcription profile of membrane protein genes in three Brazilian isolates of Anaplasma marginale (Rio Grande do Norte, Pernambuco-Zona da Mata, and Pernambuco-Sertão). RNA was purified from cattle blood experimentally-infected with the three isolates of A. marginale. After reverse transcription, genes omp1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, and 14; opag1-3; virB3, 9, and 10; am097, 197, 254, 854, and 956 were amplified by PCR, with specific primers. Transcripts were detected for all genes, except omp2, 3 e opag3 in all isolates and for omp7 in one out of the three isolates analyzed. Absence of transcription for opag3 and omp7 diverge from the North American isolates of A. marginale. Reasons for such differences were discussed.

Transcrição de genes de proteínas de membrana de isolados brasileiros de Anaplasma marginale / Transcription of genes of membrane proteins of Brazilians isolates of Anaplasma marginale

Este trabalho demonstra o padrão de transcrição de genes de proteínas de membrana em três isolados brasileiros de A. marginale (Rio Grande do Norte, Pernambuco-Zona da Mata e Pernambuco-Sertão). O RNA foi purificado a partir de sangue de bovinos infectados experimentalmente com os três isolados de A. marginale. Após transcrição reversa, os genes omp1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13 e 14; opag1-3; virB3, 9, 10; am097, 197, 254, 854 e 956 foram amplificados por PCR, com oligonucleotídeos iniciadores específicos. Detectaram-se transcritos para todos os genes analisados, exceto omp2, 3 e opag3 em todos os isolados e do gene omp7 em um dos isolados estudados. A ausência de transcrito para os genes opag3 e omp7 diverge do observado em isolados americanos da riquétsia. Possíveis razões para essas diferenças são discutidas.

This work shows the transcription profile of membrane protein genes in three Brazilian isolates of Anaplasma marginale (Rio Grande do Norte, Pernambuco-Zona da Mata, and Pernambuco-Sertão). RNA was purified from cattle blood experimentally-infected with the three isolates of A. marginale. After reverse transcription, genes omp1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, and 14; opag1-3; virB3, 9, and 10; am097, 197, 254, 854, and 956 were amplified by PCR, with specific primers. Transcripts were detected for all genes, except omp2, 3 e opag3 in all isolates and for omp7 in one out of the three isolates analyzed. Absence of transcription for opag3 and omp7 diverge from the North American isolates of A. marginale. Reasons for such differences were discussed.

ELISA com MSP5 recombinante truncada para detecção de anticorpos contra Anaplasma marginale em bovinos / ELISA based on recombinant truncated MSP5 for detection of antibodies against Anaplasma marginale in cattle

Os objetivos deste estudo foram produzir e solubilizar a proteína MSP5 recombinante truncada de Anaplasma marginale, e avaliar seu desempenho em um ensaio de imunoadsorção enzimática indireto (ELISA) para detecção de anticorpos contra a riquétsia. O gene msp5, exceto a região N-terminal hidrofóbica, foi amplificado por PCR, clonado em plasmídeo pTrcHis-TOPO e expresso em Escherichia coli. A solubilização da proteína recombinante foi avaliada em diferentes pHs e concentrações de uréia. A sensibilidade e a especificidade do ensaio foram avaliados testando-se 66 soros de animais infectados experimentalmente com A. marginale e 96 soros negativos, com o estado de infecção destes animais confirmado por PCR. Um total de 1.666 amostras de soros bovino, provenientes do Brasil - Rio Grande do Sul (73), Mato Grosso do Sul (91), Pernambuco (86), Bahia (314) e Minas Gerais (267)-, Uruguai (32) e Costa Rica (803) foram testadas nos ELISAs com MSP5 truncada e com MSP1a recombinantes e a concordância entre os dois testes foi avaliada. O ELISA indireto com MSP5 truncada foi capaz de detectar animais infectados com 96,97 por cento de sensibilidade e 100 por cento de especificidade. Nos animais infectados experimentalmente, o ELISA detectou anticorpos do 12° até o último dia de observação (37° dia). Os ELISAs para MSP5 e MSP1a apresentaram concordância de 95,67 por cento, com índice kappa de 0,81. Os resultados discordantes apresentaram uma diferença significativa (p <0,001). Anticorpos contra A. marginale foram detectados em animais de todas as regiões estudadas. O ELISA com MSP5 recombinante truncada apresentou bom desempenho na detecção de anticorpos contra A. marginale, com alta sensibilidade e especificidade, representando uma importante ferramenta para o diagnóstico da anaplasmose bovina em estudos epidemiológicos.

The objective of this study was the production and solubilization of recombinant truncated MSP5 of Anaplasma marginale and the evaluation of its performance in an enzyme-linked immunosorbent assay (ELISA), to detect antibodies against the rickettsia in cattle. The fragment of msp5 gene, except the hydrophobic N-terminal region, was amplified by PCR, cloned in pTrcHis-TOPO plasmid and expressed in Escherichia coli. Solubilization of the recombinant protein was evaluated in different pHs and concentrations of urea. The sensibility and specificity of the assay were evaluated with 66 sera from cattle experimentally-infected and 96 sera from cattle free of A. marginale defined by polymerase chain reaction for msp5 gene. Serum samples from 1,666 cattle from Brazil - states of Rio Grande do Sul (73), Mato Grosso do Sul (91), Pernambuco (86), Bahia (314) and Minas Gerais (267), Uruguay (32) and Costa Rica (803), were tested by ELISAs with recombinant truncated MSP5 and with recombinant MSP1a, and the agreement between both ELISAs was calculated. ELISA with recombinant truncated MSP5 protein detected infected animals with sensibility of 96.97 percent and specificity of 100 percent. In cattle experimentally-infected, the ELISA detected antibodies from the 12th day post-infection (DPI) to the end of the experiment, at the 37th DPI. The agreement between the ELISAs with truncated MSP5 and MSP1a antigens was 95.67 percent, with a kappa index of 0.81. Disagreement results showed significative difference (p <0.001). Antibodies for A. marginale were detected in animals of the all the region analyzed. The ELISA with recombinant truncated MSP5 showed a good performance in ELISA for detention of antibodies against A. marginale, with high sensitivity and specificity, representing an important tool for the diagnosis of anaplasmose bovine in epidemiological studies.

The role of the vacB gene in the pathogenesis of Brucella abortus.

Brucella species are important zoonotic pathogens affecting a wide variety of mammals. Therefore, the identification of new Brucella virulence factors is of great interest in understanding bacterial pathogenesis and immune evasion. In this study, we have identified Brucella abortus vacB gene that presents 2343 nucleotides and 781 amino acids and it shows 39% identity with Shigella flexneri vacB gene that encodes an exoribonuclease RNase R involved in bacterial virulence. Further, we have inactivated Brucella vacB by gene replacement strategy generating a deletion mutant strain. In order to test the role of Brucella vacB in pathogenesis, BALB/c and interferon regulatory factor-1 (IRF-1) knockout (KO) mice received Brucella vacB mutant, the virulent parental strain 2308 or the vaccine strain RB51 and the bacterial CFU numbers in spleens and mous survival were monitored. Our results demonstrated that the B. abortus DeltavacB mutant and the wild type strain 2308 showed similar CFU numbers in BALB/c mice. Additionally, IRF-1 KO mice that received either the vacB mutant or S2308 strain died in 12-14 days postinfection; in contrast, all animals that received the RB51 vaccine strain survived for 30 days postinoculation. In summary, this study reports that the vacB gene in B. abortus has no impact on bacterial pathogenesis.

Role of Toll-like receptor 4 in induction of cell-mediated immunity and resistance to Brucella abortus infection in mice.

Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) during Brucella abortus infection. CHO reporter cell lines transfected with CD14 and TLRs showed that B. abortus triggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived from Brucella rough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A from Rhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers of Brucella were monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks following B. abortus infection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance to B. abortus infection and that S lipid A has potent adjuvant activity.

Molecular and immunological characterisation of recombinant Brucella abortus glyceraldehyde-3-phosphate-dehydrogenase, a T- and B-cell reactive protein that induces partial protection when co-administered with an interleukin-12-expressing plasmid in a DNA vaccine formulation.

To identify antigens of Brucella spp. that are potentially involved in stimulating a protective T-cell-mediated immune response, previous studies identified 10 clones from a Brucella abortus 2308 genomic library with primed lymphocytes as probes. One selected positive clone (182) contained an insert of 1.2 kb which was identified, sequenced and characterised. The deduced amino acid sequence of the open reading frame (ORF) revealed 82% and 81% identity to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes from Agrobacterium tumefaciens and Xanthobacter flavus, respectively. Southern blot analysis demonstrated that the gap gene is present in only one copy in the Brucella genome. B. abortus GAPDH was then expressed in Escherichia coli as a fusion protein with the maltose-binding protein (MBP). To demonstrate the functional activity of Brucella GAPDH, E. coli gap mutants were transformed with a Brucella pMAL-gap construct. Genetic complementation was achieved and as a result E. coli mutants were able to grow on glucose or other carbon source medium. The humoral and cellular immune responses to the recombinant (r) GAPDH were characterised. In Western blots, sera from naturally infected cattle and sheep showed antibody reactivity against rGAPDH. In response to in-vitro stimulation by rGAPDH, splenocytes from mice vaccinated with rGAPDH or B. abortus S19 were able to produce gamma-interferon and tumour necrosis factor-a but not interleukin (IL)-4. Furthermore, gap associated with murine IL-12 gene in a DNA vaccine formulation partially protected mice against experimental infection.

Induction of a th1-type of immune response but not protective immunity by intramuscular DNA immunisation with Brucella abortus GroEL heat-shock gene.

The immunogenicity and protective efficacy of a DNA vaccine encoding the GroEL heat-shock gene from Brucella abortus was tested in BALB/c mice immunised by intramuscular (i.m.) needle injection or epidermally by gene gun. The Brucella GroEL gene was amplified by PCR and cloned into two different mammalian expression vectors pCMV-link and pCMV-tPA. The D17 cell line was transfected with both constructs and GroEL transcripts were detected by Northern blot. To determine the level of protein synthesised, transfected cell lysates were then submitted to Western blot. The non-secreted form of the recombinant GroEL produced by the pCMV-link construct was detected in much greater amount than the secreted form of the protein produced by the pCMV-tPA construct. After immunisation, a strong anti-GroEL IgG response was detected in mice vaccinated by i.m. injection or gene gun only when the pCMV-link/ GroEL plasmid was used. Regarding the pattern of immune response induced, i.m. needle injection raised a predominantly Th1 response with mostly IgG2a-specific anti-GroEL and high levels of IFN-gamma produced by splenic T cells. Gene gun immunisation induced a ThO type of immune response in mice characterised by a high IgG1/IgG2a ratio, and IL-4 and interferon (IFN)-gamma production. Even though a distinct pattern of immune response was generated depending upon the immunisation route used, neither method engendered a significant level of protection with the GroEL DNA vaccine.