Pathogenicity and molecular characteristics of infectious bronchitis virus (IBV) strains isolated from broilers showing diarrhoea and respiratory disease.

Abstract 1. The possibility that infectious bronchitis virus (IBV) variants isolated from broilers with enteric and respiratory problems have a different tropism and pathological outcome from those IBV strains causing classical respiratory disease was investigated. 2. IBV variants were isolated from broiler flocks with enteric and respiratory problems in two regions of Brazil. The USP-10 isolate, of enteric origin, was inoculated via the oral oroculonasal routes into IBV-antibody-free broilers and specific pathogen-free (SPF) chickens to determine tissue tropism and pathogenicity and compared with an IBV variant (USP-50) isolated from chickens showing signs of respiratory disease only. 3. Both USP-10 and USP-50 strains caused similar pathological patterns by either route of inoculation. Both variants were detected in respiratory and non-respiratory tissues, including the kidney, intestine and testis. 4. Broilers were more susceptible to infection than SPF chickens, and seroconversion was detected in all of the chicks.

Evaluation of four DNA extraction protocols for Brucella abortus detection by PCR in tissues from experimentally infected cows with the 2308 strain.

This study compared 4 protocols for DNA extraction from homogenates of 6 different organs of cows infected with the Brucella abortus 2308 strain. The extraction protocols compared were as follows: GT (guanidine isothiocyanate lysis), Boom (GT lysis with the carrying suspension diatomaceous earth), PK (proteinase K lysis), and Santos (lysis by boiling and freezing with liquid nitrogen). Positive and negative gold standard reference groups were generated by classical bacteriological methods. All samples were processed with the 4 DNA extraction protocols and amplified with the B4 and B5 primers. The number of positive samples in the placental cotyledons was higher than that in the other organs. The cumulated results showed that the Santos protocol was more sensitive than the Boom (p=0.003) and GT (p=0.0506) methods and was similar to the PK method (p=0.2969). All of the DNA extraction protocols resulted in false-negative results for PCR. In conclusion, despite the disadvantages of classical bacteriological methods, the best approach for direct diagnosis of B. abortus in organs of infected cows includes the isolation associated with PCR of DNA extracted from the cotyledon by the Santos or PK methods.

Tuberculosis determined by Mycobacterium bovis in captive waterbucks (Kobus ellipsiprymnus) in São Paulo, Brazil

Two waterbucks from São Paulo Zoo Foundation exhibited respiratory symptoms in July 2004. After euthanasia, granulommas in lungs and mediastinic lymph nodes were observed. Acid-fast bacilli isolated were identified as Mycobacterium bovis spoligotype SB0121 by PRA and spoligotyping. They were born and kept in the same enclosure with the same group, without any contact to other species housed in the zoo. This is the first detailed description of M. bovis infection in Kobus ellipsiprymnus.

Tuberculosis determined by Mycobacterium bovis in captive waterbucks (Kobus ellipsiprymnus) in SÃo paulo, Brazil.

Two waterbucks from São Paulo Zoo Foundation exhibited respiratory symptoms in July 2004. After euthanasia, granulommas in lungs and mediastinic lymph nodes were observed. Acid-fast bacilli isolated were identified as Mycobacterium bovis spoligotype SB0121 by PRA and spoligotyping. They were born and kept in the same enclosure with the same group, without any contact to other species housed in the zoo. This is the first detailed description of M. bovis infection in Kobus ellipsiprymnus.

Evaluation of DNA extraction protocols for Brucella abortus pcr detection in aborted fetuses or calves born from cows experimentally infected with strain 2308 / Avaliação de diferentes protocolos de extração de DNA para detecção de Brucella abortus a partir de abortos ou de bezerros nascidos de vacas experimentalmente infectadas com estirpe 2308

The objective of the present study was to improve the detection of B. abortus by PCR in organs of aborted fetuses from infected cows, an important mechanism to find infected herds on the eradication phase of the program. So, different DNA extraction protocols were compared, focusing the PCR detection of B. abortus in clinical samples collected from aborted fetuses or calves born from cows challenged with the 2308 B. abortus strain. Therefore, two gold standard groups were built based on classical bacteriology, formed from: 32 lungs (17 positives), 26 spleens (11 positives), 23 livers (8 positives) and 22 bronchial lymph nodes (7 positives). All samples were submitted to three DNA extraction protocols, followed by the same amplification process with the primers B4 and B5. From the accumulated results for organ, the proportion of positives for the lungs was higher than the livers (p=0.04) or bronchial lymph nodes (p=0.004) and equal to the spleens (p=0.18). From the accumulated results for DNA extraction protocol, the proportion of positives for the Boom protocol was bigger than the PK (p<0.0001) and GT (p=0.0004). There was no difference between the PK and GT protocols (p=0.5). Some positive samples from the classical bacteriology were negative to the PCR and viceversa. Therefore, the best strategy for B. abortus detection in the organs of aborted fetuses or calves born from infected cows is the use, in parallel, of isolation by classical bacteriology and the PCR, with the DNA extraction performed by the Boom protocol.

O objetivo do presente estudo foi aperfeiçoar a detecção de Brucella abortus pela PCR em homogeneizados de órgãos de fetos abortados por vacas infectadas, importante mecanismo para descobrir focos da doença na fase de erradicação. Assim, foram comparados diferentes protocolos de extração de DNA, visando à detecção de B. abortus pela PCR em amostras clínicas colhidas de abortos ou de bezerros oriundos de vacas desafiadas com a estirpe 2308 de B. abortus. Para tanto, foram construídos dois grupos padrão ouro com base na bacteriologia clássica, constituídos por: 32 pulmões (17 positivos), 26 baços (11 positivos), 23 fígados (8 positivos) e 22 linfonodos bronquiais (7 positivos). Todas essas amostras foram submetidas a três protocolos de extração de DNA, seguidos do mesmo processo de amplificação com os primers B4 e B5. Nos resultados acumulados por órgão, a proporção de positivos nos pulmões foi maior que a encontrada nos fígados (p=0,04) e nos linfonodos bronquiais (p=0,004), e igual a verificada nos baços (p=0,18). Nos resultados acumulados por método de extração de DNA, a proporção de positivos para o protocolo de Boom foi maior que a verificada para o PK (p<0,0001) e GT (p=0,0004). Não houve diferença entre os protocolos PK e GT (p=0,5). Algumas amostras positivas ao isolamento foram negativas à PCR e vice-versa. Assim, a melhor estratégia para se pesquisar B. abortus em tecidos de fetos abortados ou de bezerros nascidos de vacas infectadas é a utilização, em paralelo, do isolamento e da PCR, com extração do DNA pelo método do Boom.

Evaluation of DNA extraction protocols for Brucella abortus pcr detection in aborted fetuses or calves born from cows experimentally infected with strain 2308.

The objective of the present study was to improve the detection of B. abortus by PCR in organs of aborted fetuses from infected cows, an important mechanism to find infected herds on the eradication phase of the program. So, different DNA extraction protocols were compared, focusing the PCR detection of B. abortus in clinical samples collected from aborted fetuses or calves born from cows challenged with the 2308 B. abortus strain. Therefore, two gold standard groups were built based on classical bacteriology, formed from: 32 lungs (17 positives), 26 spleens (11 positives), 23 livers (8 positives) and 22 bronchial lymph nodes (7 positives). All samples were submitted to three DNA extraction protocols, followed by the same amplification process with the primers B4 and B5. From the accumulated results for organ, the proportion of positives for the lungs was higher than the livers (p=0.04) or bronchial lymph nodes (p=0.004) and equal to the spleens (p=0.18). From the accumulated results for DNA extraction protocol, the proportion of positives for the Boom protocol was bigger than the PK (p< 0.0001) and GT (p=0.0004). There was no difference between the PK and GT protocols (p=0.5). Some positive samples from the classical bacteriology were negative to the PCR and vice-versa. Therefore, the best strategy for B. abortus detection in the organs of aborted fetuses or calves born from infected cows is the use, in parallel, of isolation by classical bacteriology and the PCR, with the DNA extraction performed by the Boom protocol.