First report and whole-genome sequencing of Pseudochrobactrum saccharolyticum in Latin America.

The Brucellaceae family comprises microorganisms similar both phenotypically and genotypically, making it difficult to identify the etiological agent of these infections. This study reports the first isolation, identification, and characterization of Pseudochrobactrum saccharolyticum (strain 115) from Latin America. Strain 115 was isolated in 2007 from a bovine in Brazil and was initially classified as Brucella spp. by classical microbiological tests and bcsp31 PCR. The antimicrobial susceptibility of strain 115 was tested against drugs used to treat human brucellosis by minimal inhibitory concentration test. Subsequently, the whole genome of the strain was sequenced, assembled, and characterized. Phylogenetic trees built from 16S rRNA and recA gene sequences enabled the classification of strain 115 as Pseudochrobactrum spp. Phylogenomic analysis using Single Nucleotide Polymorphisms and Average Nucleotide Identity allowed the classification of the strain as P. saccharolyticum. Additionally, a Tetra Correlation Search identified one related genome from the same species, which was compared with strain 115 by analyzing genomic islands. This is the first identification and whole-genome sequence of P. saccharolyticum in Latin America and highlights a challenge in the diagnosis of bovine brucellosis, which could be solved by including the sequencing of 16S rRNA and recA genes in routine diagnostics.

Allele and genotype frequency for milk beta-casein in dairy cattle in the northern region of Tocantins State, Brazil / Frequências alélicas e genotípicas para beta-caseína do leite em bovinos leiteiros da microrregião de Araguaína, Tocantins

At present, there is a concern about the quality of milk and diseases related to its consumption, as it can generate discomfort and allergic reactions in some individuals due to its protein components. Thus, the present study was developed to identify the allele and genotype frequencies of genes for ß-casein, A1 and A2, in dairy herds in the region of Araguaína-TO, Brazil. Genetic material from 421 animals (crossbred dairy cattle in lactation) was used. All animals were numbered for identification, and DNA samples were extracted from hair bulbs. Samples for two markers from the polymorphic regions were characterized and confirmed by real-time PCR using the ABI Prism® 7500 Sequence Detection System (Applied Biosystems). Allele and genotype frequencies were determined using the TaqMan™ detection system, where the primer and probe release different fluorescence signals for each allele of the polymorphism. The sampled herd showed frequencies of 28.27% for the A1 allele and 71.73% for the A2 allele. Genotype frequencies were 52.96% (223/421) for A2A2; 37.53% (158/421) for the A1A2 genotype; and 9.50% (40/421) for the A1A1 genotype. The frequency of the A1 allele for ß-casein in dairy herds from the northern region of Tocantins was low and is per the results of previous studies. Although the A2A2 genotype of ß-casein had a high relative frequency, the A1A2 genotype is still rather frequent, warranting greater selection pressure.(AU)

Atualmente existe uma preocupação em relação à qualidade e doenças que estão relacionadas ao consumo de leite, pois o mesmo pode gerar desconfortos e reações alergicas em alguns indivíduos devido aos seus constituintes protéicos. Assim, o presente estudo teve como objetivo identificar a frequência alélica e genotípica de genes para beta caseína, A1 e A2, em rebanhos leiteiros da região de Araguaína-TO. Foram utilizados material genético de 421 animais (bovinos leiteiros mestiços em lactação), e todos os animais foram numerados para identificação e amostras de DNA foram extraídas de bulbo de folículos pilosos. As amostras para dois marcadores das regiões polimórficas foram caracterizadas e confirmadas por PCR em tempo real, usando um sistema de detecção de sequências ABI Prism® 7500 (Applied Biosystems). As frequências alélicas e genotípicas foram determinadas utilizando o sistema de detecção TaqMan ™, no qual o primer e a sonda emitem diferentes sinais de fluorescência para cada alelo do polimorfismo. Observou-se frequência do alelo A1 de 28,27%, e do alelo A2 de 71,73% no rebanho amostral. A frequência genotípica de A2A2 foi de 52,96% (223/421), com genótipo A1A2 de 37,53% (158/421), e de 9,50% (40/421) animais com genótipo A1A1. A frequência do alelo A1 para beta-caseína em rebanhos leiteiros da região norte do Tocantins foi baixa e seguiu a mesma tendência já observada em estudos anteriores. Os genótipos A2A2 da beta-caseína apresentaram frequência relativa alta, entretanto o genótipo A1A2 ainda é bastante frequente, necessitando de maior pressão de seleção.(AU)

Phylogenetic Group of Escherichia coli Isolates from Broilers in Brazilian Poultry Slaughterhouse.

The aim of the study was to determine the phylogenetic groups of E. coli strains isolated from seemingly healthy broiler and broiler condemned suspected of colibacillosis in a Brazilian slaughterhouse. Samples from respiratory tract and edible giblets (liver and heart) of broilers with and without macroscopic lesions of colibacillosis were collected at slaughter. There were 84 strains isolated from broilers condemned of which 11 were obtained from swabs of the heart, 7 from the liver, and 66 from the respiratory tract. Of the 53 E. coli strains isolated from broilers not condemned, 5 were isolated from the heart, 4 from the liver, and 44 from the respiratory tract. E coli strains were tested via PCR for phylogenetic groups A, B1, B2, C, D, E, and F. Phylogroups A and B1 were the most common phylogroups of E. coli obtained from healthy and sick-appearing broiler carcasses. The results of the study showed that phylogroups B2 and E were associated with the heart samples and phylogroup A was associated with respiratory tract samples, phylogroup B1 with not condemned carcass, and phylogroup D with liver samples.

Reduced Susceptibility to Rifampicin and Resistance to Multiple Antimicrobial Agents among Brucella abortus Isolates from Cattle in Brazil.

This study aimed to determine the susceptibility profile of Brazilian Brucella abortus isolates from cattle to eight antimicrobial agents that are recommended for the treatment of human brucellosis and to correlate the susceptibility patterns with origin, biotype and MLVA16-genotype of the strains. Screening of 147 B. abortus strains showed 100% sensitivity to doxycycline and ofloxacin, one (0.68%) strain resistant to ciprofloxacin, two strains (1.36%) resistant to streptomycin, two strains (1.36%) resistant to trimethoprim-sulfamethoxazole and five strains (3.40%) resistant to gentamicin. For rifampicin, three strains (2.04%) were resistant and 54 strains (36.73%) showed reduced sensitivity. Two strains were considered multidrug resistant. In conclusion, the majority of B. abortus strains isolated from cattle in Brazil were sensitive to the antimicrobials commonly used for the treatment of human brucellosis; however, a considerable proportion of strains showed reduced susceptibility to rifampicin and two strains were considered multidrug resistant. Moreover, there was no correlation among the drug susceptibility pattern, origin, biotype and MLVA16-genotypes of these strains.

Biotyping and genotyping (MLVA16) of Brucella abortus isolated from cattle in Brazil, 1977 to 2008.

Brucellosis is a worldwide distributed zoonosis that causes important economic losses to animal production. In Brazil, information on the distribution of biovars and genotypes of Brucella spp. is scarce or unavailable. This study aimed (i) to biotype and genotype 137 Brazilian cattle isolates (from 1977 to 2008) of B. abortus and (ii) to analyze their distribution. B. abortus biovars 1, 2 and 3 (subgroup 3b) were confirmed and biovars 4 and 6 were first described in Brazil. Genotyping by the panel 1 revealed two groups, one clustering around genotype 40 and another around genotype 28. Panels 2A and 2B disclosed a high diversity among Brazilian B. abortus strains. Eighty-nine genotypes were found by MLVA16. MLVA16 panel 1 and 2 showed geographic clustering of some genotypes. Biotyping and MLVA16 genotyping of Brazilian B. abortus isolates were useful to better understand the epidemiology of bovine brucellosis in the region.

Evaluation of Brucella abortus S19 vaccines commercialized in Brazil: immunogenicity, residual virulence and MLVA15 genotyping.

Live attenuated Brucella abortus S19 is the most effective vaccine against brucellosis in cattle. The assessment of the immunological parameters is essential to guarantee the biological quality of live anti-bacteria vaccines. The evaluation of genetic stability of live bacterial vaccines is also important in quality control. The aims of the present study were to compare (i) the immunogenicity and residual virulence, and (ii) the genotypic profile (MLVA15) of the eight S19 vaccines commercialized in Brazil to the USDA S19 reference strain. Two batches of each of the eight S19 commercial vaccines used in Brazil (A-H) were tested. They were submitted to the potency and residual virulence in vivo tests recommended by OIE and typed by the multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) described for Brucella spp. Our results demonstrated that all S19 vaccines commercialized in Brazil would be approved by Brazilian and OIE recommendations for potency and residual virulence. Furthermore, the S19 vaccine is genetically very homogeneous, as all but two batches (from the same manufacturer) tested showed identical MLVA15 profile. The two batches with different profiles presented six repeat units in locus Bruce07, instead of the five found in all other strains, including the USDA S19 reference strain. Although presenting a slightly different profile, this vaccine was also protective, as demonstrated by the immunogenicity and residual virulence assays performed. Therefore, the commercial Brazilian S19 vaccines were in accordance to Brazilian and international standards for immunogenicity and residual virulence tests. Moreover, our results also show that MLVA could be a useful inclusion to the list of in vitro tests required by the official control authorities to be applied to the commercial S19 vaccines, as an efficient assay to guarantee the quality and stability of the vaccine strains.

PCR Multiplex fluorescente para detecção de bactérias em sêmen bovino / Fluorescent Multiplex PCR for detection of bacteria in semen bovine

Este estudo teve como objetivo avaliar o limiar de detecção da técnica de PCR multiplex fluorescente aliada a eletroforese capilar na detecção de agentes infecciosos em amostras de sêmen experimentalmente contaminadas com concentrações decrescentes das bactérias Brucella abortus, Leptospira interrogans sorovar pomona, Campylobacter fetus e Haemophilus somnus. Amostras de sêmen bovino foram experimentalmente contaminadas com concentrações decrescentes de bactérias obtidas através de diluições seriadas na base 10 de modo a obter-se amostras contendo desde 1 vez até 10-7 bactérias/mL a partir da concentração inicial de Leptospira pomona, Brucella abortus, Campylobacter fetus e Haemophilus somnus. As diluições foram efetuadas individualmente para cada bactéria, bem como nas diferentes concentrações necessárias para a padronização do teste de multiplex PCR. As extrações de DNA de todas as soluções contendo espermatozóides e bactérias analisadas no presente estudo foram realizadas segundo protocolo descrito por Heinemann et al. (2000). Os produtos de PCR multiplex foram avaliados por eletroforese em gel de poliacrilamida 8% e separação eletroforética por sistema capilar em equipamento automático de análise de fragmentos de DNA MegaBace. Observou-se a amplificação de fragmentos de 193pb, 330pb, 400pb e 415pb a partir do DNA de B. abortus, L. pomona, H. somnus, C. fetus, respectivamente. Na análise por eletroforese capilar de produtos da PCR multiplex do DNA para detecção simultânea dos quatro patógenos observou-se a sinal de positividade até a diluição de 10-3 bactérias/mL vezes da concentração inicial da solução estoque de cada bactéria. A técnica de PCR multiplex aliada à eletroforese capilar foi usada pela primeira vez para o diagnóstico direto de quatro bactérias patogênicas no sêmen, demonstrando ser um método rápido na detecção de bactérias causadoras de doenças reprodutivas.

This study aimed to evaluate the threshold of detection of fluorescent multiplex PCR coupled with capillary electrophoresis for detection of infectious agents in semen samples from experimentally infected with decreasing concentrations of the bacteria Brucella abortus, Leptospira interrogans serovar pomona, Campylobacter fetus and Haemophilus somnus. Samples of bovine semen were experimentally infected with decreasing concentrations of bacteria obtained from serial dilutions in the base 10 so as to obtain samples containing a long time until 10-7 bacteria/mL from the initial concentration of Lepstospira pomona, Brucella abortus, Haemophilus somnus and Campylobacter fetus. The dilutions were made individually for each bacterium, as well as in different concentrations needed to standardize the multiplex PCR test. DNA extractions of all solutions containing sperm and bacteria analyzed in this study were performed according to protocol described by Heinemann et al. (2000). The multiplex PCR products were analyzed by electrophoresis on 8% polyacrylamide gel and capillary electrophoretic separation system for automated equipment in the analysis of DNA fragments MegaBACE. We observed amplification of fragments of 193pb, 330pb, 415pb and 400bp from the DNA of B. abortus, L. pomona, H. somnus, C. fetus respectively. The analysis by capillary electrophoresis of multiplex PCR products of DNA for simultaneous detection of the four pathogens was observed by detecting the dilution of 10-3 bacteria / mL times the initial concentration of the stock solution of each bacterium. The multiplex PCR coupled with capillary electrophoresis was first used for the direct diagnosis of four pathogenic bacteria in semen, proving to be a rapid method to detect bacteria that cause reproductive disorders.

Caracterização das proteínas de superfície de membrana externa da sorovariedade Hardjo isolada de bovinos em Minas Gerais / Characterization of outer membrane proteins of serovar Hardjo isolated from cattle in Minas Gerais, Brazil

Foram identificadas diferenças no perfil protéico das amostras de Sponselee, Norma e Hardjoprajitno, com bandas protéicas entre 175, 47 kDA e 12,10 kDa. A amostra Sponselee foi a que apresentou maior número de bandas (12), seguida da Norma que apresentou 11 bandas e de Hardjoprajitno que apresentou 9 bandas. Todas as bandas observadas na amostra Sponselee possuíam correspondentes nas outras duas amostras. A amostra Norma não apresentou uma banda em torno de 35,77 kDa e a Hardjoprajitno não apresentou bandas em torno de 89,59 kDa, 35,77 kDa e 12,10 kDa. O reconhecimento dessas proteínas por soros hiperimunes contra cada uma das amostras também mostrou diferenças, sendo que o maior número de proteínas reconhecido em todas as amostras por todos os soros se encontrou entre 35,83 kDa e 29,19 kDa. Os soros contra bovino na amostra Norma só reconheceu proteínas de baixa massa molecular nas amostras Norma (6,80 kDa) e Hardjoprajitno (6,80 kDa e 5,30 kDa). Soro bovino contra a amostra Hardjoprajitno reconheceu uma proteína de 44,33 kDa todas às amostras e proteínas de 4,22 kDa nas amostras Sponselee e Norma e de 10,49 kDa e 6,16 kDa na Hardjoprajitno. As diferentes proteínas identificadas poderiam se constituir em alvos específicos para o desenvolvimento de testes diagnósticos e vacinas contra leptospirose bovina.

Differences in the protein profile of Leptospira sp. strains Sponselee, Norma and Hardjoprajitno were observed, with bands ranging from 175.47 kDa to 12.10 kDa. Strain Sponselee presented a 12-band profile, while strain Norma showed 11 and strain Hardjoprajitno showed 9 bands in the profile. All bands observed in Sponselee strain profile could match bands in the other two strains. Strain Norma lacks a band at 35.77 kDa and strain Hardjoprajitno lacks the bands at 89.59 kDa, 35.77 kDa and 12.10 kDa. The recognition profile from hyperimmune sera was also different for the studied serovar Hadjo strains. The majority of recognized proteins was in the range of 35.83 kDa to 29.19 kDa. Cattle sera against strain Norma only recognized low molecular mass proteins in strains Norma (6.80 kDa) and Hardjoprajitno (6.80 kDa and 5.30 kDa). Bovine sera against strain Hardjoprajitno recognized a 44.33 kDa protein in all studied strains and proteins of 4.22 kDa in strains Sponselee and Norma and of 10.49 kDa and 6.16 kDa in strain Hadjoprajitno. The different identified proteins could become specific targets to the development of diagnostic tests and vaccines against bovine leptospirosis.

Anticorpos anti-Brucella canis e anti-Brucella abortus emcães de Araguaína, Tocantins / Antibodies anti-Brucella canis and anti-Brucella abortus in dogs from Araguaína, Tocantins,Brazil

Os objetivos deste estudo foram determinar a soroprevalência da infecção por Brucella canis e Brucella abortus e avaliaros possíveis fatores de risco associados à infecção em cães no município de Araguaína, Tocantins. Soros de 374 cães,pertencentes à zona urbana do município de Araguaína-Tocantins, foram analisados pelas técnicas de imunodifusãoem ágar gel (IDGA), para pesquisa de anticorpos contra Brucella canis, e antígeno acidificado tamponado (AAT)e polarização fluorescente (FPA) para detecção de anticorpos contra Brucella abortus. Dos 374 soros testados parapresença de anticorpos contra B. abortus, 21 foram reagentes no AAT, entretanto todos foram negativos pela FPA. Àprova do IDGA 167 animais foram reagentes resultando em uma prevalência para B. canis de 44,53% (IC 95%; 39,43 a49,72). A avaliação de possíveis fatores de risco associados à soropositividade para B. canis não revelou a existência derelação entre a infecção e as variáveis individuais estudadas. Assim, o presente estudo permite concluir que não houveanimais infectados por B. abortus e que a infecção por B. canis está disseminada nos cães do município de Araguaína,Tocantins.

The aims of the present study were to determine the seroprevalence of infection by Brucella canis and Brucella abortusand to evaluate possible risk factors for infection in dogs from Araguaína, Tocantins, Brazil. Sera from 374 dogs, of theurban zones of the municipality, from both sexes, were submitted to the agar-gel immunodiffusion for Brucella canisantibodiesand to rose Bengal test (AAT) and fluorescence polarization assay (FPA) for Brucella abortus-antibodies.From the 374 tested dogs, 21 reacted in the AAT, but no one was positive in the FPA. The seroprevalence of B. canisinfection found in Araguaína, Tocantins, Brazil, was 44.53% (95% IC; 39.43 to 49.72). No association was found amongseropositivity for B. canis and the risk factors studied. Thus, data from the present study showed that there was noinfection by B. abortus among dogs in the sample and that infection by B. canis is widespread and at high prevalence inAraguaína, Tocantins, Brazil.