| Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiological agent of porcine contagious pleuropneumoniae, which is characterized by sudden death or growth retardation during the peracute, acute and chronical course of disease. Pigs survived infection may serve as source for nonimmune pig herd. To date, 15 serovars of bivar I and biovar II were documented and stronger virulence was observed in biovar I . The difference of virulence among the isolates contributes to the different severity of the disease. To control and prevent the disease, it will be helpful to know the disease origin and dynamics, and production and application of serotype specific vaccine, which is based on serological and etiological investigations which provide information about predominant serovars. Unfortunately, the commercial availability of serotyping sera and antigens, the fundamental materials for A.pleuropneumoniae research, is very limited currently in China. Thus, the preparation of the above reagents and development of typing system are very important in disease control and prevention.In this report, to prepare the sera for serotyping, 13 serotypes of A. pleuropneumoniae reference strains were cultured, harvested and inactivated prior to being emulsed with adjuvants for raising antibodies in rabbits. The specificity, titer and shelf life of the 13 sera were measured by slide agglutination test, agar diffusion test and counter immunodiffusion assay. The results indicated that the sera possessed good specificity, and can be stored at 4℃ for 1 month, at -20℃ and -80℃ for one year without significant variation in quality. The materials and method were applied to verify five PCR positive field strains and were shown to be serotype 2, 3 and 8. These results revealed that the prepared serum and relevant assay could be used for serotyping field isolates.For the purpose of serologically investigating and typing the possible prevalent A.pleuropneumoniae strains via serum detection, the serotype specific antigens of 14 serotype of A. pleuropneumoniae reference strains were produced from antigen containing supernatant after washing and centrifugtion, sonication or phenol extraction. The titration and specificity of antigens were assessed by slide agglutination test, agar diffusion test and counter immunodiffusion assay. 155 serum samples collected from Hubei, Hunan, Jiangxi, Henan and Anhui provinces were assayed with newly prepared antigens. The results revealed the antigens can be used to differentiate serotypes of A. pleuropneumoniae.Genotyping is a newly developed technique for bacterial typing and could be used totype bacteria, in particular those untypable strains in conventional serological typing formats. In this test, a PCR system was developed on basis of genes encoding the three RTX (apx), outer membrane proteins (omlA), and transferring binding protein (tbpB) of A. pleuropneumoniae. We found that the resultant patterns could distinguish the reference strains for serovars 1, 2, 3, 4, 5, 6, 10, 12 and 13 of biovar I of A. pleuropneumoniae. However, neither the reference strains for serovars 7? 8 and 15 nor the reference strains for serovars 9 and 11 could be distinguished. No amplified products were found when a field isolate of each of the following were examined - Escherichia coli, Bordetella bronchiseptica, Salmonella spp, Pasteurella spp and Streptococcus suis. Complete agreement was obtained between serotyping and genotyping when 12 field isolates of serovars 2, 3 and 10 were examined. The method can also be used to directly detect A. pleuropneumoniae infection in lung and tonsil tissue. The detection results of total 42 tonsils and 126 lungs showed the potential clinical application of the genotyping system because it could not only detect infection but also verify the serotype of A. pleuropneumoniae in tissues. Besides, the ability to sort the untypable strains may be complementary to the serotyping system. Inaddition, the detection is performed in shorter time than serological test.In conclusion, the serotyping sera... |
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