Molecular Epidemiological Study On Actinobacillus Pleuropneumoniae

The ApxIVA gene , a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. A pair primer were designed to specifically amplified a 650 bp fragment.The PCR result of specificity assay showed that the reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae were positive, No positive results appeared in other bacterial species closely related to A. pleuropneumoniae.The sensitivity result showed that as little as 500 of Actinobacillus pleuropneumoniae could be detected by PCR.The detection results for clinical samples tissue showed that Actinobacillus pleuropneumoniae DNA could be found in trachea, nasal cavitiesor and tonsils.The high diagnostic sensitivity and specificity of PCR will make it useful in field diagnostic work and epidemic investigation. The ApxIV gene , a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. A pair of primers were designed for amplifying this 650bp fragment in PCR experiments.The PCR product was labeled with digoxigenin as DNA probe for detection of Actinobacillus pleuropneumoniae.The hybridization assay result showed that the reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae were positive, No cross-hybridization was detected with DNAs from other bacterial species closely related to A. pleuropneumoniae.The sensitivity result showed that as few as 10×102 of Actinobacillus pleuropneumoniae could be detected by DIG-labeled probe. The detection results for clinical samples tissue showed that Actinobacillus pleuropneumoniae could be found in tonsils ,trachea and lung.The high sensitivity and specificity of the DIG-labeled probe will make it useful in field diagnostic work and epidemic investigation. 20 strains of Actinobacillus pleuropneumoniae were isolated from different districts of Shandong province. Medicine sensitivity results showed that most of the isolated strains were sensitive to cephalothin , amikacinum,gentamicin ,and kanamycini. resistance to penicillin , nitrofurantoinum and vancomycin. This study provide theoretical foundation for prevention and treatment for Porcine pleuropneumonia. 20 field strains of Actinobacillus pleuropneumoniae were isolated in lung, tonsils and trachea from 85 pigs with severe pleuropneumonia in different districts of Shandong province. Slide agglutination test and PCR was used to determine their serotype and Apx toxins type respectively. The results of slide agglutination test were indicated that there were 5 serotypes among the 20 field strains, serotype 7(6/20) and serotype 5 (5/20) were the dominant , the other serotype were serotype 3(4/20),serotype 4(3/20) and serotype 8(2/20).The PCR results indicated that there were four kinds of Apx toxins in the 20 field strains , among them, serotype 3,4,8 contain ApxⅡ,ApxⅢand ApxⅣ, serotype 5 contain ApxⅠ, ApxⅡand ApxⅣ, serotype 7 contain ApxⅡand ApxⅣ, ApxⅡand ApxⅣexisted in all the 20 field strains .The results of Apx sequence analysis indicated that the homogeneity of Apx gene were higher than 99% in different serotype APP if they had same type of Apx. The pathogenicity was different when mice inoculated with APP which contain different Apx , serotype 5 APP was very virulent ,for it contain ApxI and ApxⅡ.This study establish the foundation for control Porcine infectious pleuropneumonia in immune prevention , subunit vaccine and PCR diagnosis based on ApxⅣgene. PCV-2 (Porcine circovirus type-2, PCV-2) and PRRSV (Porcine reproductive and respiratory syndrome virus , PRRSV) were detected by PCR from 253 porcine pleuropneumonia samples and 125 clinically healthy lung samples were collected from different districts of Shandong province .The results showed that 171 samples for PCV-2 and 101 samples for PRRSV were positive. The positive ratio were 67.5% and 40% , respectively. the co-infection number of PCV-2 and PRRSV was 68 in 253 samples, the positive ratio was 26.8%. While in the clinically healthy samples, only 21 positive samples for PCV-2 and 12 samples for PRRSV were detected, the positive ratio were 16.8% and 9.6% , respectively , no co-infection samples were found. Statistical result showed significant difference between positive ratio for PCV-2 and PRRSV in Porcine pleuropneumonia samples and in clinically healthy samples. The above results demonstrated that there maybe some relationship between the infection of porcine pleuropneumonia and PCV-2 and PRRSV in pigs.