Serotype Identification Of Haemophilus Parasitae Isolated From Hebei Province And Establishment And Application Of Indirect ELISA

Haemophilus parasuis(HPS)is a common bacterium in the upper respiratory tract of pigs.When the body’s immunity decreases,it may invade the body and lead to systemic diseases characterized by cellulosic serositis,pericarditis,arthritis,and meningitis.And It is also known as Gl?sser’s disease.It affects pigs for 4-7 weeks old weaner and will cause serious losses to the pig husbandry.In this study,a total of 107 sick pigs and suspected H.parasuis infections in Hebei Province were collected from September 2017 to July 2018.The bacteria were isolated,gram staining and satellite growth test were conducted to preliminarily determine whether the isolates were suspected of HPS.The PCR(polymerase chain reaction)test is performed on the bacteria suspected of HPS,and the PCR product is recovered and sequenced,and the isolated bacteria are determined to be HPS and the serotypes at the molecular level.A total of 23 strains of HPS were isolated,including 3 strains of serovar 1,5 strains of serovar 4,4 strains of serovar 5,3 strains of serovar 7 and 4 strains of serovar 14 with 4 strains of untyped,and the isolation rate was 21.49%.Comparing the storage time of HPS under different conditions,the results showed that the colonies could be stored in the refrigerator at 4 °C for 8-10 days directly after TSA scribing.Using the protective agent for the TSB bacteria solution was treated with 30% glycerol,HPS can be stored at-20 °C for 45-60 days and at-80 °C for more than 90 days.In order to facilitate the serological investigation of the prevalence of HPS in Hebei,this study established an indirect ELISA serological diagnosis method for HPS.Although HPS has multiple serotypes,all serotypes are capable of synthesizing Cytolethal distending toxin(CDT),which includes three subunits CdtA,CdtB and CdtC.Compared through DNAstar software,CdtC has the best immunogenicity.In this study,the Cytolethal distending toxin C subunit CdtC protein of Haemophilus parasuis was expressed by E.coli.The protein was purified by a special kit.The purified protein was denaturated and identified as having good antigenicity by Western blot.Using the purified protein as a coating antigen,an ELISA method was established and optimal reaction conditions were optimized.The optimal working conditions were as follows: recombinant CdtC protein was coated at a concentration of 1 μg/mL,100 μL per well,and incubated at 37 ° C for 2 hours;5% skim milk powder was used as a blocking solution,200 μL per well,and blocked at 37 ° C for 1 hour;The serum dilution ratio was 1:200,and incubation was carried out for 1 hour at 37 ° C;the enzyme-labeled secondary antibody was diluted 1:10000,and incubated at 37 ° C for 1 hour;the color development time was 10 minutes.Thirty samples of negative serum were detected by the established method,and the critical value of positive was calculated to be 0.420,which was positive when the value was higher than that.The critical value of negative is 0.341,less than that which is negative.Samples between the two values should be retested.The specificity of the established method was tested using positive serum of common pig disease,and the results showed that the established method has better specificity.The method established in the batch and batch repetitive test identified the method with good repeatability,and the intra and inter-assay coefficient of variation was within 10%.Using this method,561 serum samples collected from September 2017 to July 2018 were tested,with a positive rate of 39.9%.In this study,the seroserovar identification of HPS isolated from Hebei Province was carried out,and an indirect ELISA method for antibody detection of HPS was established.This lays the foundation for the investigation and prevention of HPS in Hebei Province.