| Swine bacterial diseases are caused by multiple bacteria that have a variety of pathogens,very strong transmissibility,different kinds of serotypes,complicated virulence factors,highly antibiotic resistance rates,and coinfection,which lead to a great threat to the Chinese pig industry.Thus,the control of bacterial diseases is becoming more and more difficult due to the high antibiotic resistance in bacteria,which also has become a key factor that limits the development of the Chinese pig industry.Haemophilus parasuis,as a kind important bacterial pathogen,also severely affects the health of pigs,which can lead to polyserositis,meningitis,bronchopneumonia,and arthritis.Meanwhile,Haemophilus parasuis has complicated virulence factors and a very high isolation rate among the pigs in China.However,the pathogenic mechanism is still not very clear.So,it is very urgent to investigate the pathogenesis mechanism of Haemophilus parasuis.Quorum sensing system is a very important system that can regulate not only the bacterial function,but also the communication among bacteria,which also affects the biological characteristics,biofilm formation,antibiotic resistance,and virulence of bacteria.So,it is important to illuminate the pathogenesis mechanism through the study on quorum sensing system,providing the new strategy to control the disease of Haemophilus parasuis.At first,we analyzed the epidemiological features and antibiotic resistance of Haemophilus parasuis isoalates based on the bacteria isolated from Chinese pig farms.Then,we illustrated the function of quorum sensing system on biological characteristics,biofilm formation,and virulence of Haemophilus parasuis.At last,RNA-Seq was used to verify the change caused by quorum sensing system in Haemophilus parasuis.Meanwhile,we also revealed the heat resistant mechanism of Haemophilus parasuis and the receptor of quorum sensing system in Haemophilus parasuis from the results of RNA-Seq.The main contents were as follows: 1.The epidemiological features and antibiotic resistance of pathogenic bacteria in Chinese pig farms.To supply clinical guidelines for the control of swine bacterial diseases,we investigated and analyzed the epidemiological features and antibiotic resistance of the isolated bacteria.In total,44,175 samples from 16 provinces of China had been collected from 2013 to 2017.The results showed that the main bacteria in Chinese pig farms were Streptococcus suis(38.0%),Haemophilus parasuis(21.9%),Pasteurella multocida(7.7%),Escherichia coli(14.4%),Actinobacillus pleuropneumoniae(0.7%),Bordetella bronchiseptica(3.4%),Salmonella(5.1%),and E.rhusiopathiae(2.0%).The isolation rates of different strains and serotypes of Streptococcus suis and Haemophilus parasuis have obviously changed from 2013 to 2017.For example,Streptococcus suis(15.3%-18.6%),Escherichia coli(4.3%-9.9%),Actinobacillus pleuropneumoniae(0.1%-0.5%),and Salmonella(1.6%-3.7%)displayed increasing isolation rates,while those of Haemophilus parasuis(11.6%-7.3%)and E.rhusiopathiae(1.6%-0.6%)were the decreasing trends.But,the Streptococcus suis and Haemophilus parasuis were still the top two bacteria among all isolated bacteria.The prevalent serotypes of Haemophilus parasuis had changed from serotype 4 to serotype 5,and the proportion of Streptococcus suis serotype 2 also decreased 20% from 52.4% in 2013 to 33.6% in 2017.Meanwhile,the seasonal prevalence characteristics of Streptococcus suis,Haemophilus parasuis,and Pasteurella multocida showed that Streptococcus suis and Haemophilus parasuis had a higher isolation rate in the hot season.But,February,March,April,and October were the main seasons for the isolation of Pasteurella multocida.In addition,antimicrobial susceptibility tests indicated that Streptococcus suis,Haemophilus parasuis,and Pasteurella multocida presented very highly and increasing resistance rates to eight kinds of common antibiotics.2.The function of Lux S/AI-2 quorum sensing system in Haemophilus parasuis.Using Haemophilus parasuis serotype 2 wild type strain as parental strain,the lux S deletion mutant(?lux S)and its complemented strain(C-lux S)were constructed to verify the biological changes that might be related with quorum sensing system in Haemophilus parasuis.Then,the effect of quorum sensing system on biological characteristics,biofilm formation,and virulence of Haemophilus parasuis were evaluated based on comparative study of ?lux S and C-lux S strains.Compared with wild type HPS2 and C-lux S strains,?lux S strain showed lower responses to heat resistance(declined 23.0%)and oxidative stress(declined 25.7%),lower ability to autoagglutination,hemagglutination and tissue burdens of bacteria,and the adherent ability to PK-15 cells and the virulence to mice declined about 117 and 10 times,respectively.Whereas,the stronger ability to osmotic stress(increased 23.7%)and biofilm formation was observed in ?lux S strain.On the basis of the above data,the involvement of quorum sensing system in the regulation of physiological function in Haemophilus parasuis was identified.3.The transcriptome of quorum sensing system deficient Haemophilus parasuis.The supernatant of HPS2,?lux S,and C-lux S strains were collected at the end of the logarithmic phase followed by RNA extraction and removal of host DNA.The sequencing results of HPS2,?lux S,and C-lux S strains were obtained via RNA sequencing.At last,data filtering,multiple sequence alignment analysis,and enrichment analysis of differentially expressed genes were done.The results showed that 232 genes were up-regulated and 212 genes were down-regulated in ?lux S strain,compared with that of HPS2 strain.The biological process of these differentially expressed genes often focused on carbon metabolism,RNA degradation,and citrate cycle(TCA cycle).The biological functions of these differentially expressed genes often focused on transport,protease hydrolysis,transmembrane transport,transmembrane signal reception,and signal receptor activity.Meanwhile,the potential heat resistant mechanism of Haemophilus parasuis and receptor protein “Rbs B1” of quorum sensing system were identified through transcriptomics analysis of HPS2,?lux S,and C-lux S strains.4.Quorum sensing system involved the heat resistant mechanism of Haemophilus parasuis.The transcriptional results of HPS2,?lux S,and C-lux S strains showed that many differentially expressed genes might be related with the heat resistant mechanism of Haemophilus parasuis.To verify these results,RT-q PCR was used to detect the expression level of these differentially expressed genes.The results showed that the expression level of rse A gene was up-regulated and rpo E,rse B,deg S,clp P,and htr A genes were down-regulated in ?lux S strain,compared with HPS2 strain.The heat resistant mechanism that Haemophilus parasuis was associated with quorum sensing system which regulated the expression level of rse A,rpo E,rse B,deg S,clp P,and htr A genes and htr A gene was the target gene that play a key role on the heat resistant mechanism of Haemophilus parasuis.Both ?rse A and ?rpo E strains were constructed to verify the function of rse A and rpo E genes in the heat resistant mechanism.The results showed that rse A gene could negatively regulate the heat resistant ability of Haemophilus parasuis and rpo E gene had a reverse result.5.The function of Rbs B1 receptor protein in quorum sensing system of Haemophilus parasuis.The transcriptional analysis results of HPS2,?lux S,and C-lux S strains showed that the rbs B1 gene was down-regulated in ?lux S strain.The rbs B1 gene in Haemophilus parasuis had very high homology of 72.4% and 73.3% with rbs B1 genes in A.actinomycetemcomitans(IDH781)and H.influenzae(86-028NP),respectively,indicating of having similar function with rbs B1 gene in A.actinomycetemcomitans and H.influenzae.To verify the hypothesis,HPS2 rbs B1 mutant strain was constructed and Rbs B1 protein was expressed.These results showed that the degradation speed of AI-2 molecule in the supernatant of ?rbs B1 strain is significantly slower than that of in HPS2 strain,and the ability that Rbs B1 protein absorbed and transported AI-2 molecule was in a dose-dependence manner,which indicating that Rbs B1 protein was the receptor of quorum sensing system of Haemophilus parasuis.Therefore,these findings provided more information about Rbs B1 protein based on the regulatory pathway of quorum sensing in Haemophilus parasuis.In conclusion,the research provided us detail epidemiological features and antibiotic resistance of pathogenic bacteria in Chinese pig farms,illustrated the regulation effect of quorum sensing system in Haemophilus parasuis,explored the heat resistant mechanism of Haemophilus parasuis and receptor protein of quorum sensing system,which contribute to the disease control of Haemophilus parasuis and the further research of quorum sensing system. |
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