Development And Applification Of PCR Assay For Detection Of Bordetella Bronchiseptica In Rabbits

Rabbit bordetella bronchiseptica is caused by bordetella bronchiseptica(Bb), which also calls rabbit bronchus septicemic bacillus or Byrd, it is a kind of gram-negative aerobes and parasitic on respiratory ciliated epithelial cell in livestock, wild animals, and experimental animals, can cause respiratory disease in rabbits, pigs, horses, dogs, rats and other mammals, infected animals mainly characterized by rhinitis and bronchopneumonia. In recent years, People were infected with bordetella bronchiseptica, especially in young children and old people. In our country, the bacterium infection rate is higher in the group of rabbit, whether young immature rabbits, rabbits or breeding rabbits, which led to huge economic loss in rabbit industry.To develop a simple, rapid, sensitive and specific PCR detective method for bordetella bronchiseptica. A pair of specific primers was designed to amplify FHA gene of bordetella bronchiseptica in rabbits. The results are:1. Isolation and identification of Bordetella bronchiseptica from Rabbit:Bacteria were isolated and identified from nasal cavities, lungs, and livers of rabbits in a rabbit farm around Chuzhou in Anhui province. Through morphological, Microscopic examination of stained preparation, cultural, reactional traits, biochemical characteristics, and 16SrRNA PCR, these bacteria were identified as Rabbit bordetella Bronchiseptica. Medicinal sensitivity tests of bacteria showed these bacteria had highly susceptible to Doxycycline, Florfenicol, Gentamicin, and Amikacin enzyme, and medium sensitivity to Kanamycin, Streptomycin, Midecamycin, but it was resistant to Ampicillin, Furaxone, Lincmycin and Nitrofurantoin. The results of artificial infection experiment showed that Laboratory Rabbits infected the bacteria.2. Development of PCR Assay for Detection of Bordetella Bronchiseptica in Rabbits:DNA was extracted from the bordetella bronchiseptica in rabbits. With a pair of primers designed to amplify flagellin gene of bordetella bronchiseptica according to the sequence(AF111796) of filamentous hemagglutinin(FHA),a. fragment of 734bp in length was obtained based on optimum system.Amplified fragment was designated as FHA-734,comparing with NC002929, NC018828and NC019382,the nucleotide homology was 92.9%～96.6%. The optimal PCR system was:MgCl2:1.0μl, temperature:58℃, cycle number:30.3. Applification of PCR Assay:92 samples of nasal mucus from rabbits from Anhui and Jiangsu province were examined by the developed PCR assay, the positive rate was 63.1%, but the rate of agglutinating test was 33.7%. Rabbits were tested by PCR assay, the positive rates were 90% in young immature rabbits,85% in rabbits and 80% in breeding rabbits.