Comparison of three decontamination methods for Mycobacterium bovis isolation.

SIXTY SAMPLES OF TISSUE FRAGMENTS WITH LESIONS SUGGESTIVE OF TUBERCULOSIS FROM BOVINE ABATTOIRS, KEPT IN SATURATED SOLUTION OF SODIUM BORATE, WERE SUBJECTED TO FOUR TREATMENTS: 4% NaOH (Petroff Method), 12 % H2SO4 and 1.5% HPC (1-Hexadecylpyridinium Chloride) decontamination, and physiological saline solution (control). The HPC method showed the lowest contamination rate (3%) when compared to control (88%, p<0.001), NaOH (33%, p<0.001) and H2SO4 (21.7%, p<0.002). Regarding the isolation success, the HPC method was better (40%) than the control (3%, p<0.001), NaOH (13%, p=0.001) and H2SO4 (1.7%, p<0.001) methods. These results indicate that HPC is an alternative to the Petroff method.

Identification of an IS6110 insertion site in plcD, the unique phospholipase C gene of Mycobacterium bovis.

The IS6110 repetitive element is present in multiple copies in most Mycobacterium tuberculosis complex bacteria, except for Mycobacterium bovis strains, which usually contain a single copy of IS6110 located on a 1.9 kb PvuII fragment of the direct repeat region. IS6110 transposition can disrupt coding regions and is a major force of genomic variation. In a previous work it was demonstrated that phospholipase C genes are preferential loci for IS6110 transposition in M. tuberculosis clinical strains. Bacterial phospholipase C enzymes participate in pathogenic mechanisms used by different organisms, and have been implicated in intracellular survival, cytolysis and cell-to-cell spread. Four phospholipase C genes (plcA, plcB, plcC and plcD) were detected in the genomes of M. tuberculosis, Mycobacterium africanum, Mycobacterium microti and 'Mycobacterium canettii'. M. bovis and the vaccine strain M. bovis Bacillus Calmette-Guérin contain only the plcD gene. In the present work, the existence of IS6110 insertions within plcD, the unique phospholipase C gene of M. bovis, has been investigated by PCR, Southern blot hybridization and sequencing analysis. In 18 (7.3 %) of 245 isolates analysed, the plcD gene was interrupted by the insertion of one copy of IS6110, which in all cases was transposed in the same orientation and at the same position, 1 972 894, relative to the genome of M. bovis AF2122/97. These 18 isolates were distributed in 6 different spoligotype patterns and contained 4 to 8 IS6110 copies. In contrast, strains showing an intact plcD gene contained one (87 %), two (9.4 %) or three (2.4 %) IS6110 copies, and only a single isolate (1.2 %) had four IS6110 copies. The implications of plcD gene disruption in M. bovis have not been fully investigated, but no differences in the organ distribution of the disease were detected when animals infected with strains from the same spoligotype patterns bearing plcD : : IS6110 and intact plcD were compared.

European 1: a globally important clonal complex of Mycobacterium bovis.

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle.

Comparison of three decontamination methods for Mycobacterium bovis isolation / Comparação de três métodos de descontaminação para isolamento de Mycobacterium bovis

Sixty samples of tissue fragments with lesions suggestive of tuberculosis from bovine abattoirs, kept in saturated solution of sodium borate, were subjected to four treatments: 4 percent NaOH (Petroff Method), 12 percent H2SO4 and 1.5 percent HPC (1-Hexadecylpyridinium Chloride) decontamination, and physiological saline solution (control). The HPC method showed the lowest contamination rate (3 percent) when compared to control (88 percent, p<0.001), NaOH (33 percent, p<0.001) and H2SO4 (21.7 percent, p<0.002). Regarding the isolation success, the HPC method was better (40 percent) than the control (3 percent, p<0.001), NaOH (13 percent, p=0.001) and H2SO4 (1.7 percent, p<0.001) methods. These results indicate that HPC is an alternative to the Petroff method.

Sessenta amostras de fragmentos de tecidos com lesões sugestivas de tuberculose provenientes de abatedouros bovinos, conservadas em solução saturada de borato de sódio, foram submetidas a quatro tratamentos: descontaminação através dos métodos NaOH 4 por cento (Método Petroff), H2SO4 12 por cento e HPC (Cloreto de hexadecilpiridínio) 1,5 por cento, e solução salina (controle). O método HPC apresentou a menor proporção de contaminação (3 por cento), em relação ao controle (88 por cento, p<0,001), NaOH (33 por cento, p<0,001) e H2SO4 (21,7 por cento, p=0,002). Em relação ao sucesso no isolamento, o método HPC apresentou o melhor resultado (40 por cento), em relação ao controle (3 por cento, p<0,001), NaOH (13 por cento, p=0,001) e H2SO4 (1,7 por cento, p<0,001). Os resultados indicam que o HPC é uma alternativa à utilização do método Petroff.

Comparison of methods for mycobacteria isolation from swine feces

Swine mycobacteriosis is an important cause of carcass condemnation at abattoirs. One of the best ways to recognize the etiologic agent involved, in live animals, is the fecal isolation, as 94 percent of the lesions are located in the digestive tract. Therefore, the goal of the present study was to compare the performance of four decontamination methods followed by inoculation in three different culture media, totalizing twelve procedures of mycobacteria search from swine fecal samples experimentally contaminated. The swine feces were artificially contaminated with 0.02 g of Mycobacterium avium, PIG-B strain, and subjected to mycobacteria isolation trial. The protocols used were: 1) modified Petroff or basic method; 2) modified Lowenstein-Jensen or acidic method; 3) modified Petroff or basic method with re-suspension in Amphotericin B; 4) modified Lowenstein-Jensen or acid method with re-suspension in Amphotericin B, followed by inoculation in Petragnani, Lowenstein-Jensen and Lowenstein-Jensen medium with antibiotics (Penicillin G and Nalidixic acid). There was a difference (p<0.05) between the mycobacterial recovery percentages from swine feces. The acid method with re-suspension in Amphotericin B solution and inoculation in Lowenstein-Jensen medium with antibiotics showed the best results (87 percent of mycobacteria recovery).

As micobacterioses suínas são responsáveis por condenações de carcaças em abatedouro e uma das melhores formas de se conhecer os agentes envolvidos nos animais vivos é o isolamento a partir das fezes, pois em 94 por cento das vezes, as lesões localizam-se no trato digestivo. Assim sendo, o presente estudo teve por objetivo comparar o desempenho de quatro métodos de descontaminação com semeadura em três diferentes meios de cultura, totalizando doze procedimentos na pesquisa de micobactérias a partir de amostras de fezes de suínos contaminadas experimentalmente. Amostras de fezes de suínos foram contaminadas artificialmente com 0,02g de Mycobacterium avium, estirpe de PIG-B, e submetidas à tentativa de isolamento de micobactérias, utilizando-se os seguintes protocolos de descontaminação: 1) Petroff modificado ou método básico; 2) Lowenstein-Jensen modificado ou método ácido; 3) Petroff modificado ou método básico e ressuspensão com anfotericina B; 4) Lowenstein-Jensen modificado ou método ácido e ressuspensão com anfotericina B; com subseqüente semeadura em meios de Petragnani, Lowenstein-Jensen e Lowenstein-Jensen com antibióticos (Penicilina G e Ácido nalidíxico). Houve diferença entre os percentuais de recuperação de micobactérias a partir das fezes de suínos (p<0,05) e o método ácido com ressuspensão em solução de anfotericina B e semeadura em meio de Lowenstein-Jensen com antibióticos apresentou os melhores resultados (87 por cento de recuperação de micobactérias).

Isolation of bovine coronavirus (BCoV) in monolayers of HmLu-1 cells

A partir de 20 amostras fecais de bezerros, com quadro clínico de diarréia, positivas para BCoV por hemaglutinacão/inibicão da hemaglutinacão (HA/HI), procedeu-se o isolamento viral em monocamadas de células da linhagem HmLu-1. Até a quinta passagem seriada, 13 apresentaram efeito citopático do tipo sincicial, semelhante à amostra padrão Kakegawa de BCoV. Ao serem submetidas a uma reacão de soroneutralizacão com gamaglobulina anti-coronavírus bovino, 8 delas foram consideradas positivas, uma vez que o efeito citopático foi neutralizado. Ao serem tituladas e submetidas à reacão de soroneutralizacão em microplacas, apenas 3 delas puderam ser confirmadas como positivas. As células da linhagem HmLu-1 mostraram-se permissivas para o isolamento de BCoV, todavia a baixa intensidade na replicacão viral demonstrou ser necessário o desenvolvimento de novas metodologias para se poder alcancar esse intuito e a confirmacão do isolamento por soroneutralizacão.

Seroprevalence of antibodies against group A rotavirus in cattle from a pioneer frontier in Brazilian Amazon

Rotavirus is a worldwide etiologic agent of diarrhea, responsible for large economic losses. We studied the seroprevalence of antibodies to group A rotavirus in cattle in 67 smallholder farms from Uruará municipality, using counterimmunoelectroosmophoresis with the NCDV strain as a standard antigen. Prevalence of positive smallholder farms was 95.6-100 percent. Significant differences were seen between age groups when the seropositivity rose from the youngest to the oldest groups and between females and males older than 1 year, when the seropositivity was higher in the first group