Molecular detection of Burkholderia mallei in different geographic regions of Brazil.

Glanders is a contagious disease of equids caused by the Gram-negative bacterium Burkholderia mallei. In Brazil, the disease is considered to be reemerging and has been expanding, with records of equids with positive serology in most of the federative units. However, there are few reports describing the genotypic detection of the agent. This study demonstrated the detection of B. mallei by species-specific PCR directly from tissues or from bacterial cultures, followed by amplicon sequencing in equids (equines, mules, and asinines) with positive serology for glanders in all five geographic regions of Brazil. The molecular evidence of B. mallei infection in serologically positive equids in this study expands the possibility of strain isolation and the conduction of epidemiological characterizations based on molecular information. The microbiological detection of B. mallei in cultures from nasal and palate swabs, even in equids without clinical manifestations, raises the possibility of environmental elimination of the agent.

Unraveling the Complexity of the Rhomboid Serine Protease 4 Family of Babesia bovis Using Bioinformatics and Experimental Studies.

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host−pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.

Molecular characterization of Burkholderia mallei strains isolated from horses in Brazil (2014-2017).

Glanders is an infectious zoonosis caused by Burkholderia (B.) mallei that mainly affects equids. The objective of this work was to provide additional knowledge on the diversity of the strains circulating in Brazil. Six Burkholderia mallei isolates obtained during necropsies of glanderous horses between 2014 and 2017 in two different states (Pernambuco and Alagoas) were analyzed by polymerase chain reaction-high-resolution melting (PCR-HRM). While four strains (9902 RSC, BM_campo 1, BM_campo 3 and UFAL2) clustered in the L3B2 branch, which already includes the Brazilian 16-2438_BM#8 strain, two strains (BM_campo 2.1 and BM_campo 2.2) clustered within the L3B3sB3 branch, which mostly includes older isolates, from Europe and the Middle East. Whole genome sequencing of two of these strains (UFAL2 and BM_campo 2.1), belonging to different branches, confirmed the HRM typing results and refined the links between the strains, including the description of the L3B3Sb3Gp1SbGp1 genotype, never reported so far for contemporary strains. These results suggest different glanders introduction events in Brazil, including a potential link with strains of European origin, related to colonization or trade.

Genomic analysis of an outbreak of bovine tuberculosis in a man-made multi-host species system: A call for action on wildlife in Brazil.

We report on a 15-year-long outbreak of bovine tuberculosis (bTB) in wildlife from a Brazilian safari park. A timeline of diagnostic events and whole-genome sequencing (WGS) of 21 Mycobacterium bovis isolates from deer and llamas were analyzed. Accordingly, from 2003 to 2018, at least 16 animals, from eight species, died due to TB, which is likely an underestimated number. In three occasions since 2013, the deer presented positive tuberculin tests, leading to the park closure and culling of all deer. WGS indicated that multiple M. bovis strains were circulating, with at least three founding introductions since the park inauguration in 1977. Using a previously sequenced dataset of 71 M. bovis genomes from cattle, we found no recent transmission events between nearby farms and the park based on WGS. Lastly, by discussing socio-economic and environmental factors escaping current regulatory gaps that were determinant of this outbreak, we pledge for the development of a plan to report and control bTB in wildlife in Brazil.

Genetic Diversity and Potential Paths of Transmission of Mycobacterium bovis in the Amazon: The Discovery of M. bovis Lineage Lb1 Circulating in South America.

Bovine tuberculosis (bTB) has yet to be eradicated in Brazil. Herds of cattle and buffalo are important sources of revenue to people living in the banks of the Amazon River basin. A better understanding of Mycobacterium bovis (M. bovis) populational structure and transmission dynamics affecting these animals can significantly contribute in efforts to improve their sanitary status. Herein, we sequenced the whole genome of 22 M. bovis isolates (15 from buffalo and 7 from cattle) from 10 municipalities in the region of the Lower Amazon River Basin in Brazil and performed phylogenomic analysis and Single Nucleotide Polymorphism (SNP)-based transmission inference to evaluate population structure and transmission networks. Additionally, we compared these genomes to others obtained in unrelated studies in the Marajó Island (n = 15) and worldwide (n = 128) to understand strain diversity in the Amazon and to infer M. bovis lineages. Our results show a higher genomic diversity of M. bovis genomes obtained in the Lower Amazon River region when compared to the Marajó Island, while no significant difference was observed between M. bovis genomes obtained from cattle and buffalo (p ≥ 0.05). This high genetic diversity is reflected by the weak phylogenetic clustering of M. bovis from the Lower Amazon River region based on geographic proximity and in the detection of only two putative transmission clusters in the region. One of these clusters is the first description of inter-species transmission between cattle and buffalo in the Amazon, bringing implications to the bTB control program. Surprisingly, two M. bovis lineages were detected in our dataset, namely Lb1 and Lb3, constituting the first description of Lb1 in South America. Most of the strains of this study (13/22) and all 15 strains of the Marajó Island carried no clonal complex marker, suggesting that the recent lineage classification better describe the diversity of M. bovis in the Amazon.

Bovine tuberculosis in safari park in Brazil

ABSTRACT: Bovine tuberculosis (bTB) is an infectious disease caused by Mycobacterium bovis, affecting domestic animals, wild animals and humans. In captivity, for wild animals, bTB represents a risk to animal keepers and zoo visitors, in addition to the possibility of spreading the infection to domestic animals or through the trade of infected wild animals. Sambar (Cervus unicolor), red deer (Cervus elaphus) and fallow deer (Dama dama) from a safari park in the State of Rio Grande do Sul, Brazil, showed a clinical condition of dyspnea and weight loss. Some animals died and showed lesions suggestive of tuberculosis (LST), which were confirmed by histopathology. After the interdiction of the safari park by the state veterinary authorities, 281 deer were euthanized with the authorization of the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA). Retropharyngeal and submandibular lymph nodes and viscera were collected from 21 animals, which were grown in Stonebrink medium for up to 90 days. After DNA extraction from the bacterial colonies, PCR was performed for targets flanking the region of differentiation 4 (RD4). Of the 21 samples, 14 (66.7%) presented LST with a granulomatous appearance, a whitish coloration, and caseous or calcified consistency, and seven samples (33.3%), showed no lesions. In the culture of 14 samples with LST, 13 (92.8%) presented bacterial growth compatible with M. bovis. In the cultivation of the seven samples without LST, four (57.1%) presented colonies compatible with M. bovis. PCR and DNA sequencing of the PCR amplicons detected as positive all the 17 (100%) bacteriological cultures suggestive of M. bovis, thus confirming the outbreak of bTB in deer. Decisions about positive tested and suspicious animals should be taken based on the evaluation of the risk of transmission to the rest of the zoological animals, animal welfare, conservation considerations and, the zoonotic potential of this pathogen.

RESUMO: A tuberculose bovina (bTB) é uma doença infecciosa causada por Mycobacterium bovis, afetando animais domésticos, animais selvagens e humanos. Para animais selvagens em cativeiro, a bTB representa um risco para os tratadores de animais e visitantes do zoológico, além da possibilidade de espalhar a infecção para animais domésticos ou por meio do comércio de animais silvestres infectados. Cervídeos sambar (Cervus unicolor), veado-vermelho (Cervus elaphus) e gamo (Dama dama) de um parque safári no Estado do Rio Grande do Sul, Brasil, mostraram uma condição clínica de dispneia e perda de peso. Alguns animais morreram e apresentaram lesões sugestivas de tuberculose (LST), as quais foram confirmadas por histopatologia. Após a interdição do parque safári pelas autoridades veterinárias estaduais, 281 veados sofreram eutanásia com a autorização do Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA). Os linfonodos retrofaríngeos e submandibulares e vísceras foram coletados de 21 animais, que foram cultivados em meio Stonebrink por até 90 dias. Após extração de DNA das colônias bacterianas, foi realizada PCR para alvos que flanqueavam a região de diferenciação 4 (RD4). Das 21 amostras, 14 (66,7%) apresentaram LST com aspecto granulomatoso, coloração esbranquiçada e consistência caseosa ou calcificada, e sete amostras (33,3%) não apresentaram lesões. Na cultura de 14 amostras com LST, 13 (92,8%) apresentaram crescimento bacteriano compatível com M. bovis. No cultivo das sete amostras sem LST, quatro (57,1%) apresentaram colônias compatíveis com M. bovis. A PCR e o sequenciamento de DNA dos fragmentos de PCR detectaram como positivo todas as 17 (100%) culturas bacteriológicas sugestivas de M. bovis, confirmando assim o surto de bTB em cervídeos. As decisões sobre animais positivos testados e suspeitos devem ser tomadas com base na avaliação do risco de transmissão para o restante dos animais zoológicos, bem-estar animal, considerações de conservação e no potencial zoonótico desse patógeno.

Genomic analysis of an outbreak of bovine tuberculosis in a man-made multi-host species system: a call for action on wildlife in Brazil

We report on a 15-year-long outbreak of bovine tuberculosis (bTB) in wildlife from a Brazilian safari park. A timeline of diagnostic events and whole-genome sequencing (WGS) of 21 Mycobacterium bovis isolates from deer and llamas were analyzed. Accordingly, from 2003 to 2018, at least 16 animals, from eight species, died due to TB, which is likely an underestimated number. In three occasions since 2013, the deer presented positive tuberculin tests, leading to the park closure and culling of all deer. WGS indicated that multiple M. bovis strains were circulating, with at least three founding introductions since the park inauguration in 1977. Using a previously sequenced dataset of 71 M. bovis genomes from cattle, we found no recent transmission events between nearby farms and the park based on WGS. Lastly, by discussing socio-economic and environmental factors escaping current regulatory gaps that were determinant of this outbreak, we pledge for the development of a plan to report and control bTB in wildlife in Brazil.

Bovine tuberculosis in safari park in Brazil / Tuberculose bovina em parque safári no Brasil

Bovine tuberculosis (bTB) is an infectious disease caused by Mycobacterium bovis, affecting domestic animals, wild animals and humans. In captivity, for wild animals, bTB represents a risk to animal keepers and zoo visitors, in addition to the possibility of spreading the infection to domestic animals or through the trade of infected wild animals. Sambar (Cervus unicolor), red deer (Cervus elaphus) and fallow deer (Dama dama) from a safari park in the State of Rio Grande do Sul, Brazil, showed a clinical condition of dyspnea and weight loss. Some animals died and showed lesions suggestive of tuberculosis (LST), which were confirmed by histopathology. After the interdiction of the safari park by the state veterinary authorities, 281 deer were euthanized with the authorization of the "Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis" (IBAMA). Retropharyngeal and submandibular lymph nodes and viscera were collected from 21 animals, which were grown in Stonebrink medium for up to 90 days. After DNA extraction from the bacterial colonies, PCR was performed for targets flanking the region of differentiation 4 (RD4). Of the 21 samples, 14 (66.7%) presented LST with a granulomatous appearance, a whitish coloration, and caseous or calcified consistency, and seven samples (33.3%), showed no lesions. In the culture of 14 samples with LST, 13 (92.8%) presented bacterial growth compatible with M. bovis. In the cultivation of the seven samples without LST, four (57.1%) presented colonies compatible with M. bovis. PCR and DNA sequencing of the PCR amplicons detected as positive all the 17 (100%) bacteriological cultures suggestive of M. bovis, thus confirming the outbreak of bTB in deer. Decisions about positive tested and suspicious animals should be taken based on the evaluation of the risk of transmission to the rest of the zoological animals, animal welfare, conservation considerations and, the zoonotic potential of this pathogen.(AU)

A tuberculose bovina (bTB) é uma doença infecciosa causada por Mycobacterium bovis, afetando animais domésticos, animais selvagens e humanos. Para animais selvagens em cativeiro, a bTB representa um risco para os tratadores de animais e visitantes do zoológico, além da possibilidade de espalhar a infecção para animais domésticos ou por meio do comércio de animais silvestres infectados. Cervídeos sambar (Cervus unicolor), veado-vermelho (Cervus elaphus) e gamo (Dama dama) de um parque safári no Estado do Rio Grande do Sul, Brasil, mostraram uma condição clínica de dispneia e perda de peso. Alguns animais morreram e apresentaram lesões sugestivas de tuberculose (LST), as quais foram confirmadas por histopatologia. Após a interdição do parque safári pelas autoridades veterinárias estaduais, 281 veados sofreram eutanásia com a autorização do Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA). Os linfonodos retrofaríngeos e submandibulares e vísceras foram coletados de 21 animais, que foram cultivados em meio Stonebrink por até 90 dias. Após extração de DNA das colônias bacterianas, foi realizada PCR para alvos que flanqueavam a região de diferenciação 4 (RD4). Das 21 amostras, 14 (66,7%) apresentaram LST com aspecto granulomatoso, coloração esbranquiçada e consistência caseosa ou calcificada, e sete amostras (33,3%) não apresentaram lesões. Na cultura de 14 amostras com LST, 13 (92,8%) apresentaram crescimento bacteriano compatível com M. bovis. No cultivo das sete amostras sem LST, quatro (57,1%) apresentaram colônias compatíveis com M. bovis. A PCR e o sequenciamento de DNA dos fragmentos de PCR detectaram como positivo todas as 17 (100%) culturas bacteriológicas sugestivas de M. bovis, confirmando assim o surto de bTB em cervídeos. As decisões sobre animais positivos testados e suspeitos devem ser tomadas com base na avaliação do risco de transmissão para o restante dos animais zoológicos, bem-estar animal, considerações de conservação e no potencial zoonótico desse patógeno.(AU)

Characterization of GASA-1, a new vaccine candidate antigen of Babesia bovis.

Surface proteins bound to the cell membrane by glycosylphosphatidylinositol (GPI) anchors are considered essential for the survival of pathogenic protozoans. In the case of the tick-transmitted hemoparasite Babesia bovis, the most virulent causative agent of bovine babesiosis, the GPI-anchored proteome was recently unraveled by an in silico approach. In this work, one of the identified proteins, GASA-1 (GPI-Anchored Surface Antigen-1), was thoroughly characterized. GASA-1 is 179 aa long and has the characteristic features of a GPI-anchored protein, including a signal peptide, a hydrophilic core and a hydrophobic tail that harbors a GPI anchor signal. Transcriptomic analysis shows that it is expressed in pathogenic and attenuated B. bovis strains. Notably, the gasa-1 gene has syntenic counterparts in B. bigemina and B. ovata, which also encode GPI-anchored proteins. This is highly unusual since all piroplasmid GPI-anchored proteins described so far have been found to be species-specific. Sequencing of gasa-1 alleles from B. bovis geographical isolates originating from Argentina, USA, Brazil, Mexico and Australia showed over 98 % identity in both nucleotide and amino acid sequences. A recombinant form of GASA-1 (rGASA-1) was generated in E. coli and anti-rGASA-1 antibodies were raised in mice. Fixed and live immunofluorescence assays showed that GASA-1 is expressed in in vitro cultured B. bovis merozoites and surface-exposed. Moreover, incubation of B. bovis in vitro cultures with anti-GASA-1 antibodies partially, but significantly, reduced erythrocyte invasion, indicating that this protein bears neutralization-sensitive antibody epitopes. Splenocytes of rGASA-1-inoculated mice showed a specific proliferative response when exposed to the recombinant protein, indicating that GASA-1 bears T-cell epitopes. Finally, sera from a group of B. bovis-infected cattle reacted with the recombinant protein, demonstrating that GASA-1 is expressed during natural infection of bovines with B. bovis, and suggesting that it is immunodominant. The high degree of conservation among B. bovis isolates and the presence of syntenic genes in other Babesia species suggest a relevant role of GASA-1 and GASA-1-like proteins for parasite survival, especially considering that, due to their surface location, they are exposed to the selection pressure of the host immune system. The highlighted features of GASA-1 make it an interesting candidate for the development of vaccines against bovine babesiosis.

Haemonchus sp. in beef cattle in Brazil: species composition and frequency of benzimidazole resistance alleles.

The aim of the present study was to investigate the occurrence of Haemonchus contortus and Haemonchus placei in beef cattle and the frequency of single nucleotide polymorphisms associated with benzimidazole (BZ)-resistance in Haemonchus spp. in Brazil. For such, fecal samples were collected from 61 beef cattle ranches in 11 Brazilian states. Third-stage larvae (L3) were produced for morphological species identification and DNA extraction. PCR was performed for the analysis of the isotype 1 ß-tubulin gene and the products were sequenced to confirm the presence of H. placei and H. contortus. For each field population, pyrosequencing assays were performed to quantify the frequency of the F167Y, E198A and F200Y polymorphisms in the isotype-1 ß-tubulin gene. The results of the morphometric analysis of 2345 larvae showed that H. placei was present on all ranches. The analysis of the isotype 1 ß-tubulin gene confirmed 100% prevalence for H. placei and 23.7% for H. contortus. Pyrosequencing assays demonstrated single-nucleotide polymorphisms (SNPs) associated with BZ-resistance in all three codons (F167Y, E198A and F200Y) of the isotype 1 ß-tubulin gene in H. placei field populations. Frequencies of resistance-associated alleles above background (≥ 15%) were found for at least one codon in 11.4% of the field isolates and maximum frequencies of 30, 21 and 29% were found for codons 167, 198 and 200, respectively, on individual ranches. This study confirms the presence of H. contortus in beef cattle in the major livestock farming states in Brazil and demonstrates that genotypes associated with BZ resistance are present in field populations of Haemonchus spp..

Molecular characterization of Mycobacterium bovis infection in cattle and buffalo in Amazon Region, Brazil.

The aim of this study was to characterize Mycobacterium bovis from cattle and buffalo tissue samples, from two Brazilian states, and to analyse their genetic diversity by spoligotyping. Tissue samples from tuberculosis suspect animals, 57 in Amazonas State (12 cattle and 45 buffaloes) and six from Pará State (5 cattle and one buffalo) from slaughterhouses under State Veterinary Inspection, were isolated in culture medium Stonebrink. The positive cultures were confirmed by PCR and analysed by the spoligotyping technique and the patterns (spoligotypes) were identified and compared at the Mycobacterium bovis Spoligotype Database (http://www.mbovis.org/). There was bacterial growth in 44 (69.8%) of the tissues of the 63 animals, of which PCR for region of differentiation 4 identified 35/44 (79.5%) as Mycobacterium bovis. Six different spoligotypes were identified among the 35 Mycobacterium bovis isolates, of which SB0295, SB1869, SB0121 and SB1800 had already been described in Brazil, and SB0822 and SB1608 had not been described. The most frequent spoligotype in this study (SB0822) had already been described in buffaloes in Colombia, a neighbouring country of Amazonas state. The other identified spoligotypes were also described in other South American countries, such as Argentina and Venezuela, and described in the Brazilian states of Rio Grande do Sul, Santa Catarina, São Paulo, Minas Gerais, Mato Grosso do Sul, Mato Grosso and Goiás, indicating an active movement of Mycobacterium bovis strains within Brazil.

Matrix Assisted Laser Desorption Ionization-Time-of-Flight mass spectrometry identification of Mycobacterium bovis in Bovinae.

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.

Epidemiological Study of Mycobacterium bovis Infection in Buffalo and Cattle in Amazonas, Brazil.

Bovine Tuberculosis (BTB) is an endemic disease in about one hundred countries, affecting the economy causing a decrease in productivity, condemnation of meat, and damaging the credibility on international trade. Additionally, Mycobacterium bovis the major causative agent for BTB can also infect humans causing a variety of clinical presentations. The aim of this study was to determine BTB prevalence and the main risk factors for the Mycobacterium bovis prevalence in cattle and buffalos in Amazonas State, Brazil. Tissue samples from 151 animals (45 buffalo and 106 cattle from five herds with buffalo only, 22 herds with cattle only, and 12 herds with buffalo and cattle) were obtained from slaughterhouses under State Veterinary Inspection. M. bovis were isolated on Stonebrink medium. The positive cultures were confirmed by polymerase chain reaction (PCR) testing. The apparent herd and animal prevalence rates were 56.4 and 5.40%, respectively. Regarding animal species, the apparent prevalence rates were 3% in cattle and 11.8% in buffalo. Generalized Linear Mixed Models (GLMM) with random effect were used to assess the association with risk factors on the prevalence. Species (buffalo), herds size (>100 animals) and the presence of both species (buffalo and cattle) in the herd were the major risk factors for the infection by Mycobacterium bovis in the region. The findings reveal an urgent need for evidence-based effective intervention to reduce BTB prevalence in cattle and buffalo and prevent its spread to the human population. Studies are needed to understand why buffalo are more likely to be infected by M. bovis than cattle in Amazon. Recommendations for zoning, use of data from the inspection services to generate information regarding BTB focus, adoption of epidemiological tools, and discouragement of practices that promote the mixing of cattle and buffalo, were made.

Survey of Salmonella spp. in beef meat for export at slaughterhouses in Brazil / Pesquisa de Salmonella spp. em carcaças bovinas durante o processamento em abatedouros-frigoríficos exportadores

The aim of the present study was to investigate the presence of Salmonella spp. in samples collected from beef meat at three points of the slaughter line (after skinning, washing and cooling) at three slaughterhouses in Brazil that export meat. Detection was based on ISO 6579:2002 and confirmed by PCR and qPCR. The isolates were typified using slide agglutination tests and PFGE. The antibiotic sensitivity profile was determined using the disk diffusion method. Contamination was detected in only one slaughterhouse. The overall frequency of contamination by Salmonella spp. was 6.7% of carcasses (6/90) and 2.6% of carcass surface samples (7/270). All isolates were confirmed by PCR and qPCR. The serological analysis and the PFGE showed a single profile: Typhimurium. The strains demonstrated 100% susceptibility to ampicillin, cefotaxime, ciprofloxacin, chloramphenicol, gentamicin and tetracycline. Positive carcasses after cooling pose a direct risk to consumers, since the meat is considered ready to be marketed after this process.(AU)

O objetivo deste trabalho foi investigar a presença de Salmonella spp. em amostras coletadas de carcaças de bovinos, em três pontos da linha de abate (após a esfola, lavagem e refrigeração) de três frigoríficos exportadores no Brasil. A detecção foi realizada pela ISO 6579:2002, e confirmada por PCR e qPCR. Os isolados foram tipificados por testes de soroaglutinação e PFGE e avaliado o perfil de sensibilidade aos antibióticos pelo método de difusão em disco. A contaminação foi detectada em apenas um abatedouro-frigorífico. As contaminações das carcaças (n=90) e amostras de carne (n=270) por Salmonella spp. foram 6 (6,7%) e 7 (2,6%), respectivamente. Todos os isolados foram confirmados por PCR e qPCR. A análise sorológica e o PFGE mostraram um único perfil: Typhimurium. As cepas apresentaram 100% de suscetibilidade à ampicilina, cefotaxima, ciprofloxacina, cloranfenicol, gentamicina e tetraciclina. As carcaças positivas após a refrigeração apresentam um risco direto para o consumidor, uma vez que, após este processo, a carne está pronta para ser comercializada.(AU)

False-negative reactions to the comparative intradermal tuberculin test for bovine tuberculosis / Reações falso-negativas ao teste cervical comparativo para tuberculose bovina

According to the Brazilian National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), the routine tests for the diagnosis of bovine tuberculosis in the country are the simple intradermal tuberculin test (SITT) of the Ministry of Agriculture, Livestock and Food Supply (MAPA), the caudal fold test and the comparative intradermal tuberculin test (CITT). The latter is also used as a confirmatory test. A group of 53 animals from three dairy herds in a focal area for bovine tuberculosis, that were submitted to depopulation in the state of Rio Grande do Sul, were submitted to the CITT. Tissues were cultured and the resulting colonies were confirmed by PCR and DNA sequencing. Among the 53 animals analyzed using the CITT, 32 (60.4%) were negative, 14 (26.4%) were positive and seven (13.2%) results were inconclusive. The CITT detected 11 of the 39 animals with culture-confirmed M. bovis infection as positive. Among the total of 14 uninfected animals based on cultures, the CBT detected eight as negative. Thus, the CITT demonstrated sensitivity of 28.2% and specificity of 57.1% for the population sampled. A total of 24/32 (75.0%) of the animals with negative CITT results were culture positive (confirmed by PCR) and were considered false negatives based on the CITT. The maintenance of these false-negative animals in herds has serious implications for the control of the disease, since they can be a source of infection. The addition of complementary tests could help identify such animals and increase the odds of diagnostic success.(AU)

No Brasil, segundo o Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Animal (PNCEBT), do Ministério da Agricultura, Pecuária e Abastecimento (MAPA), os testes de rotina para o diagnóstico de tuberculose bovina são o teste cervical simples (TCC), o teste da prega caudal (TPC) e o teste cervical comparativo (TCC), sendo que o último também é utilizado como teste confirmatório. Um grupo de 53 animais oriundos de três rebanhos leiteiros de área de foco para tuberculose bovina que foram submetidos a vazio sanitário no Rio Grande do Sul foi submetido ao TCC. Os tecidos destes animais foram cultivados e as colônias resultantes confirmadas por PCR e sequenciamento de DNA. Dos 53 animais analisados no TCC, 32 (60,4%) foram negativos, 14 (26,4%) positivos e sete (13,2%) inconclusivos, com base no PNCEBT. O TCC detectou como positivos 11 dos 39 animais com infecção por M. bovis confirmada por cultivo. Do total de 14 animais não infectados, baseado na cultura, o TCC detectou oito como negativos. Assim, o TCC apresentou, para a população amostrada, sensibilidade de 28,2% e especificidade de 57,1%. Um total de 24/32 (75,0%) dos animais negativos ao TCC foi positivo no cultivo (confirmado por PCR), sendo considerados falso-negativos ao TCC. A manutenção destes animais falso-negativos nos rebanhos tem sérias implicações para o controle da enfermidade, já que os mesmos podem ser fonte de infecção. A adição de testes complementares poderia auxiliar na identificação destes animais, aumentando a cobertura diagnóstica.(AU)

Identificação por espectrometria de massa MALDI-TOF de Salmonella spp. e Escherichia coli isolados de carcaças bovinas / MALDI-TOF mass spectrometry identification of Salmonella spp. and Escherichia coli isolated from bovine carcasses

O objetivo deste trabalho foi introduzir a técnica de espectrometria de massa com fonte de ionização e dessorção a laser assistida por matriz e analisador de tempo-de-voo (MALDI-TOF) para incrementar o método tradicional microbiológico na detecção de Salmonella spp. e Escherichia coli em carcaças bovinas. Foram avaliadas 270 amostras de 90 carcaças de bovinos. Para isolamento de Salmonella spp. e E. coli, foram utilizadas, respectivamente, as metodologias descritas na ISO 6579:2002 e no Compendium of Methods for the Microbiological Examination of Foods. As análises por MALDI-TOF foram realizadas a partir de isolados cultivados em ágar nutriente ou em caldo triptona de soja, provenientes das amostras com características bioquímicas positivas (n=7), inconclusivas (n=4) e negativas (n=85) para Salmonella spp. e bioquímicas positivas (n=37) e negativas (n=85) para E. coli. Os perfis de massas foram adquiridos com o espectrômetro de massas MALDI-TOF Autoflex III SmartBeam e os espectros brutos foram processados usando o programa MALDI Biotyper (Bruker Daltonics). De acordo com a identificação preliminar, com base na morfologia das colônias e nas reações bioquímicas, sete isolados foram considerados positivos para Salmonella spp. Através do MALDI Biotyper, esses sete isolados foram classificados como pertencentes ao gênero Salmonella e, além disso, identificados como S. enterica. Quatro isolados que apresentaram características fenotípicas não usuais e resultados inconclusivos nos testes bioquímicos para Salmonella foram identificados como pertencentes aos gêneros Citrobacter e Proteus após análise por MALDI. Para E. coli, 37 amostras foram positivas pelos testes bioquímicos da espécie, o que foi confirmado por MALDI Biotyper. A metodologia MALDI-TOF permitiu a rápida confirmação da identidade de Salmonella spp. e E. coli, podendo ser utilizada para detecção desses microrganismos em isolados bacterianos de carcaças bovinas.(AU)

The aim of this study was to introduce matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry to improve the traditional microbiological method for the detection of Salmonella spp. and Escherichia coli in beef carcasses. Two hundred seventy samples from 90 beef carcasses were evaluated. The methodologies described in ISO 6579:2002 and in the Compendium of Methods for the Microbiological Examination of Foods were used for Salmonella spp. and E. coli isolation, respectively. MALDI-TOF analysis were performed on tryptone soya broth suspension isolates or directly from nutrient agar colonies, from the positive, inconclusive or negative biochemically tested samples for Salmonella and E. coli. Mass profiles were acquired on an Autoflex III SmartBeam MALDI-TOF mass spectrometer and the raw spectra were processed using the MALDI Biotyper software (Bruker Daltonics). According to the preliminary identification based on colony morphology and the biochemical reactions, seven isolates were positive for Salmonella spp. Through MALDI Biotyper these seven isolates were also classified as belonging to the genus Salmonella and further identified as S. enterica. Four isolates showing unusual phenotypic characteristics and inconclusive results in biochemical tests for Salmonella were identified as belonging to Citrobacter and Proteus genera after MALDI analysis. Regarding Escherichia coli, 37 were positive for species biochemical testing which MALDI Biotyper confirmed. MALDI-TOF methodology allowed rapid Salmonella spp. and E. coli identity confirmation and may be used to detect these microrganisms within bacterial isolates from beef carcasses.(AU)

Diagnóstico histopatológico e molecular de lesões sugestivas de tuberculose em búfalos abatidos nos municípios de Macapá e Santana, estado do Amapá / Histopathological and molecular diagnosis of lesions suggestive of tuberculosis in buffaloes slaughtered in the municipalities of Macapá and Santana, Amapá state, Brazil

Este estudo teve como objetivo avaliar lesões sugestivas de tuberculose em búfalos abatidos em matadouros oficiais no Estado do Amapá, Brasil, a fim de confirmar o diagnóstico de tuberculose por avaliação histopatológica e molecular. As amostras de tecido de 20 búfalos que apresentavam lesões sugestivas de tuberculose, dos municípios de Macapá e Santana, foram coletadas. As amostras foram divididas em duas partes: uma delas foi fixada em formalina a 10% tamponada e rotineiramente processadas para avaliação histopatológica, coradas pela hematoxilina-eosina e Ziehl-Neelsen; e o outra parte foi usado para Nested-PCR para o complexo de Mycobacterium tuberculosis (CMT) e para Mycobacterium bovis. As lesões macroscópicas sugestivas de tuberculose foram observadas nos pulmões, linfonodos brônquicos, mediastínicos, retrofaríngeos e submandibulares, fígado e pleura. Histopatologicamente, todas as amostras apresentaram lesões sugestivas de tuberculose, caracterizadas por granulomas compostos por grande quantidade de infiltração de células epitelióides, células de Langerhans e linfócitos, margeando um centro necrótico, calcificado ou não, rodeado por cápsula de tecido conjuntivo fibroso. Bacilos álcool-ácido resistentes foram observados nos tecidos de 3/20 (15%) búfalos. Com relação à detecção molecular, 13/20 (65%) bubalinos apresentaram amostras de tecidos positivos: 6 foram positivos nas Nested-PCRs para CMT e M. bovis, um foi positivo apenas na Nested-PCR para CMT, e 6 foram positivos apenas na Nested-PCR para M. bovis. Os resultados deste estudo demonstram a importância de diagnosticar a tuberculose em búfalos na região e apontam para a necessidade de implementar medidas eficazes para controlar e erradicar a enfermidade.(AU)

This study aimed to evaluate suggestive lesions of tuberculosis in buffaloes slaughtered in official slaughterhouses in the State of Amapá, Brazil, in order to confirm the diagnosis of tuberculosis by histopathological and molecular evaluation. Tissue samples of 20 buffaloes showing lesions suggestive of tuberculosis, from the municipalities of Macapá and Santana, were collected. The samples were divided into two parts: one was fixed in 10% buffered formalin and routinely processed for histopathological evaluation, stained by hematoxylin-eosin and Ziehl-Neelsen; and the other was used for Nested-PCRs for Mycobacterium tuberculosis complex (MTC) and for Mycobacterium bovis. Gross lesions suggestive of tuberculosis were observed in the lungs, bronchial, mediastinic, retropharyngeal and submandibular lymph nodes, liver and pleura. Histopathologically, all samples showed lesions suggestive of tuberculosis, characterized by granulomas composed of large amount of infiltration of epithelioid cells, Langhans cells and lymphocytes, bordering a necrotic core, calcified or not, surrounded by a fibrous connective tissue capsule. Acid-fast bacilli were observed in the tissues of 3/20 (15%) buffaloes. With regards to the molecular detection, 13/20 (65%) buffaloes showed positive tissue samples: 6 were positive both in the MTC and M. bovis Nested-PCRs, one was positive only in the MTC Nested-PCR, and 6 were positive only in the M. bovis Nested-PCR. The results of this study demonstrate the importance of diagnosing TB in buffaloes in the region and point to the requirement to implement effective measures to control and eradicate the disease.(AU)

Multilaboratory Evaluation of a Novel Lateral Flow Immunochromatographic Assay for Confirming Isolation of Mycobacterium bovis from Veterinary Diagnostic Specimens.

A novel lateral flow immunochromatographic device (LFD) was evaluated in several veterinary diagnostic laboratories. It was confirmed to be specific for Mycobacterium bovis and M.caprae cells. The performance of the novel LFD was assessed relative to the confirmatory tests routinely applied after culture (spoligotyping or quantitative PCR [qPCR]) in each laboratory; liquid (MGIT or BacT/Alert) and/or solid (Stonebrink, Coletsos, or Lowenstein-Jensen) cultures were tested. In comparison to spoligotyping of acid-fast-positive MGIT cultures, percent agreement between positive LFD and spoligotyping results was excellent in two United Kingdom laboratories (97.7 to 100%) but lower in the Spanish context (76%), where spoligotyping was applied to MGIT cultures previously confirmed to be positive for M. tuberculosis complex (MTBC) by qPCR. Certain spoligotypes of M. bovis and M. caprae were not detected by the LFD in Spanish MGIT cultures. Compared to qPCR confirmation, the agreement between positive LFD and qPCR results was 42.3% and 50% for BacT/Alert and MGIT liquid cultures, respectively, and for solid cultures, it ranged from 11.1 to 89.2%, depending on the solid medium employed (Coletsos, 11.1%; Lowenstein-Jensen, 55.6%; Stonebrinks, 89.2%). Correlation between the novel LFD and BD MGIT TBc Identification test results was excellent when 190 MGIT cultures were tested (r = 0.9791; P < 0.0001), with the added benefit that M. bovis was differentiated from another MTBC species in one MGIT culture by the novel LFD. This multilaboratory evaluation demonstrated the novel LFD's potential utility as a rapid test to confirm isolation of M. bovis and M. caprae from veterinary specimens following culture.

Draft Genome Sequences of Two Mycobacterium bovis Strains Isolated from Beef Cattle in Paraguay.

This work reports the draft genome sequences of the Mycobacterium bovis strains M1009 and M1010, isolated from the lymph nodes of two infected cows on a beef farm in Paraguay. Comparative genomics between these strains and other regional strains may provide more insights regarding M. bovis epidemiology in South America.