Glanders and brucellosis in equids from the Amazon region, Brazil.

Glanders and brucellosis are zoonotic infectious diseases that affect equids in several countries worldwide. On Marajó Island (Amazon region of Brazil), Marajoara and Puruca horses, which are well adapted to the climatic and territorial adversities of the region, play a fundamental role in the local economy and in the sociocultural lives of the population. However, these animals have undergone a drastic reduction in number, markedly due to precarious veterinary care, unknown causes of morbidity and mortality, and disordered crossing with other breeds introduced to the island. Thus, this study aimed to investigate the occurrence of glanders and brucellosis in equids on a property located in the municipality of Soure, Marajó Island (Brazil). Serum samples were collected from 388 animals (357 horses and 31 mules), maintained in an extensive breeding system, in a property that was also extensively breeding buffaloes, goats, and sheep, with contact among species. The sera were tested for glanders using an indirect ELISA (ELISAi), and the results were confirmed by immunoblotting. The diagnosis of brucellosis was made using the Rose Bengal test (RBT) and confirmed through the Serum Agglutination test (SAT) and 2-mercaptoethanol test. In the case of glanders, 2.31% (9/388) of animals were positive in ELISAi test, of which eight had results confirmed by immunoblotting, representing 2.06% seropositivity in the entire herd. For brucellosis, serum samples from 6.7% (26/388) horses were reactive in the RBT, of which 4.12% (18/388) had a titer ≥50 and 2.06% (8/388) had a titer ≥100 in the SAT. This is the first study to report the occurrence of glanders and equine brucellosis in the municipality of Soure/Marajó Island. Monitoring the occurrence of such diseases is extremely important since they affect the herds economically and zootechnically, in addition to their high zoonotic potential. The number of animals sampled in this study, as well as the way they are raised and managed, is representative of the total equid population of the island. These results, combined with previous studies on buffaloes, indicate that these diseases are endemic in the Marajo Island.

Comparative study of Mycobacterium bovis primary isolation methods

Abstract For the definitive diagnosis of bovine tuberculosis, isolation of the etiologic agent is required. However, there is no consensus on the best methodology for isolation of Mycobacterium bovis in Brazil. This study evaluated the most used decontaminants and culture media in the country, in order to identify the best combination for the Brazilian samples. Three decontaminants - 2% sodium hydroxide (w/v), 0.75% hexadecylpiridinium chloride (w/v) and 5% sulphuric acid (v/v) and four culture media - 7H11 Middlebrook with additives and OADC supplement “A” (7H11 A), the same media with another supplement trademark (7H11 B), tuberculosis blood agar (B83) and Stonebrink's medium were compared. Regarding the isolation, there were no significant differences between the decontaminants and media combinations, except 7H11A combined to any decontaminant. However, the mean colonies score was significantly greater when the samples were decontaminated with 5% sulphuric acid and inoculated in 7H11 B or SB, without significant difference between them, although colonies appeared earlier on 7H11B than on SB. The trademark of OADC supplement influenced the isolation rate and the number of isolated colonies in Middlebrook 7H11. An incubation time of four weeks was required to detect all positive samples in 7H11 B after decontamination with 5% sulphuric acid but there was an increase in the number of colonies until the sixth week of incubation. Overall, the best strategy for the primary isolation of M. bovis from Brazilian samples was the decontamination with 5% sulphuric acid (final concentration) and inoculation in Middlebrook 7H11 medium formulated with OADC supplement “B”.

Molecular Typing of Mycobacterium bovis from Cattle Reared in Midwest Brazil.

Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain data on isolated M. bovis strains and assist in the control and eradication program for BTB, a cross sectional descriptive molecular epidemiology study in the Brazilian Midwest was conducted. Through spoligotyping and 24-loci MIRU-VNTR methods, 37 clinical isolates of M. bovis circulating in the region were analyzed, 10 isolated from the state of Mato Grosso, 12 from the state of Mato Grosso do Sul and 15 from the state of Goiás. The spoligotyping analysis identified 10 distinct M. bovis profiles (SB0121 n = 14, SB0295 n = 6, SB0140 n = 6, SB0881 n = 3, SB1144 n = 2, SB1145 n = 2, SB0134 n = 1, SB1050 n = 1, SB1055 n = 1, SB1136 n = 1) grouped in six clusters and four orphan patterns. The MIRU-VNTR 24-loci grouped the same isolates in six clusters and 22 unique orphan patterns, showing higher discriminatory power than spoligotyping. When associating the results of both techniques, the isolates were grouped in five clusters and 24 unique M. bovis profiles. Among the 24-loci MIRU-VNTR evaluated, two, ETR-A and QUB 11b loci, showed high discriminatory ability (h = ≥ 0.50), while MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21 and QUB 26 loci showed moderate ability (h = 0.33 or h = 0.49) and were the most effective in evaluating the genotypic similarities among the clinical M. bovis isolate samples. Herein, the 29 patterns found amongst the 37 isolates of M. bovis circulating in the Brazilian Midwest can be due to the animal movement between regions, municipalities and farms, thus causing the spread of various M. bovis strains in herds from Midwest Brazil.

Comparison of nine DNA extraction methods for the diagnosis of bovine tuberculosis by real time PCR / Comparação de nove métodos de extração de DNA para diagnóstico de tuberculose bovina por PCR em tempo real

ABSTRACT: Bovine tuberculosis is an infectious disease with a high impact on the cattle industry, particularly in developing countries. PCR is a very sensitive method for detection of infectious agents, but the sensitivity of molecular diagnosis is largely dependent on the efficiency of the DNA extraction methods. The objective of this study was to evaluate DNA extraction methods for direct detection of Mycobacterium bovis in bovine tissue. Nine commercial kits for DNA extraction were evaluated when combined with two real time PCRs. The DNeasy Blood & Tissue Kit from QIAGEN showed better performance and sensitivity followed by the DNA Mini Kit RBC and FTA Elute Micro Card. Results suggested that, even when the analytical sensitivity of the qPCR is very high, the extraction method can influence the diagnostic sensitivity.

RESUMO: A tuberculose bovina é uma doença infecciosa com um alto impacto na pecuária, particularmente em países em desenvolvimento. A PCR é um método muito sensível para a detecção de agentes infecciosos, mas a sensibilidade do diagnóstico molecular é em grande parte dependente da eficiência dos métodos de extração de DNA. O objetivo deste estudo foi avaliar métodos de extração de DNA para detecção direta de Mycobacterium bovisem tecido bovino. Nove kits comerciais para extração de DNA foram avaliados, quando combinados com duas PCRs em tempo real. O Kit Dneasy Blood & Tissue da Qiagen apresentou melhor desempenho e sensibilidade, seguido dos kits DNA Mini RBC e FTA Elute Micro Card (protocolo modificado com digestão enzimática prévia). Os resultados sugerem que, mesmo quando a sensibilidade analítica do qPCR é muito elevada, o método de extração pode influenciar na sensibilidade de diagnóstico.

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil.

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.