Assessment of immunity against avian colibacillosis induced by an aroA mutant containing increased serum survival gene in broilers

Colibacillosis is an important disease in the poultry industry which causes serious economic damages. As it is suggested that vaccination is one of the means to control colibacillosis, we tried to investigate the vaccine potential of a aroA derivative of an O78:K80 avian pathogenic Escherichia coli containing increased serum survival gene. 490 chicks were selected as follows: For assessment of virulence of aroA mutant, 30 chicks were divided into three groups and injected with 0.5ml of PBS or bacterial suspension containing either10(7)colony forming units (CFU) of mutant or parent strains via subcutaneous route. Macroscopic lesions and mortality rate were recorded in different groups during the week after challenge. For assessment of safety and immunogenicity of the aroA mutant, three groups of 20 chicks were vaccinated by aerosol administration of 250 ml of suspension containing 10(8) CFU of mutant strain at days 1 and 14, while the two other groups received PBS or wild type strain. Macroscopic lesions and mortality rate were recorded in different groups until day 21. To determine whether the vaccination is protective against challenges or not, the chickens were vaccinated at days 1 and 14 and challenged intramuscularly with either a homologous or heterologous strains at day 21. Macroscopic lesions and mortality rate were recorded in different groups during the week after challenge. The results revealed that the aroA mutant was slightly virulent, however it was safe and did not cause mortality, lesions or weight loss after vaccination. Antibody responses were similar in the control and mutant groups and vaccination did not induce a significant humoral immunity. The mutant could not protect chickens against both homologous and heterologous challenges. This could be due to several factors such as the high amount of maternal antibodies in the first two weeks of life, and the vaccination procedure.

Molecular genetic differentiation of avian Escherichia coli by RAPD-PCR

Escherichia coli is one of the most important bacterial avian pathogens and a common inhabitant of the gastrointestinal tract of animals. Most pathogenic E. coli can not be differentiated biochemically or by classic microbiologic methods. Molecular typing methods, particularly PCR, facilitated epidemiological and ecological studies of bacteria. Here we describe the application of a random amplified polymorphic DNA- polymerase chain reaction (RAPD-PCR) for molecular genetic differentiation of E. coli isolates in Iran. In this study 58 E. coli isolates including 4 standard strains, 3 food originated isolates, 33 avian isolates, 8 isolates form diarrheic calves and 10 isolates from unweaned diarrheic lambs were analyzed by RAPD-PCR using primer 1247(5/-AAG AGC CCG T-3/). The RAPD analysis showed that these isolates could be grouped into 33 RAPD types and avian isolates were discriminated into 29 genotypes. It was shown that the primer could not differentiate E. coli isolated from lambs. Discriminatory index for entire isolates was 0.912 and for avian isolates was 0.990. We concluded that RAPD-PCR can be used as a method for molecular differentiation of E. coli isolates.

Escherichia coli é um dos patógenos aviários mais importantes e um habitante comum do trato gastrointestinal de animais. A maioria das cepas patogênicas não pode ser diferenciada por métodos bioquímicos ou outros métodos microbiológicos clássicos. Métodos de tipagem molecular, particularmente PCR, têm facilitado os estudos epidemiológicos e ecológicos a respeito desse microrganismo. Nesse estudo, descrevemos a aplicação do RAPD-PCR para a diferenciação molecular de isolados de E.coli do Irã. No estudo, 58 isolados, incluindo 4 isolados padrão, 3 isolados de alimentos, 33 isolados de aves, 8 isolados de bezerros diarréicos e 10 isolados de carneiros diarréicos foram analisados por RAPD-PCR com o primer 1247 (5'-AAG AGC CCG T-3'). A análise mostrou que esses isolados podiam ser agrupados em 33 tipos RAPD, sendo os isolados de aves agrupados em 29 genótipos diferentes. Verificou-se que o primer utilizado não diferenciou os isolados de carneiros. O índice discriminatório para todos os isolados foi 0,912 e para os isolados de aves foi 0,990. Concluiu-se que o RAPD-PCR pode ser usado como método para diferenciação molecular de isolados de E. coli.