| Pasteurella multocida usually colonizes the upper respiratory tracts of a wide specterum of domestic and wild animals,including poultries and other wild birds,pigs,cattle and baffoles,rabbits,cats(domestic house cats as well as large wild cats),dogs,goats,chimpanees,marine mammals,etc.,and generally leads to chronic and acute infections with significant morbidity and mortality.This important Gram-negative bacterium possesses a number of virulence factors contributing to its pathogenesis.Currently known virulencefactors cover capsule,lipopolysaccharides(LPS),fimbriae,adhesion proteins,toxins,iron regulation and iron acquisition proteins,sialic acid metabolism,hyaluronidases,and outer membrane proteins(OMPs).Among them,the capsule and LPS form the basis for differentiation of different P.multocida strains,which are serologically classified into five serogroups(A,B,D,E,F)and 16 serotypes(1-16),and/or genotypically,five capsular genotypes(A,B,D,E,F)and eight LPS genotypes(L1-L8),respectively.It has been widely documented that the prevalence of P.multocida displays a certain level of host predilection,but the molecular basis for this host predilection is completely unknown.In addition,the molecular determinants for P.multocida phylogeny remains unclear.In pigs,P.multocida commonly causes clinical symptoms such as labored breath,atrophic rhinitis,pneumonia,septicemia,and even death.Infections from P.multocida are still significant threats to pig industry of China.However,this pathogenic bacterium does not receive as much attention as the other high-impact pathogenic microorganisms such as porcine reproductive and respiratory syndrome virus,pseudorabies virus,and Streptococcus suis.While a couple of previous studies investigated porcine P.multocida from clinic during the past decades,some data are too old to represent the profile of P.multocida circulating in China in recent years.Most genetic characteristics of P.multocida strains prevalent in Chinese pigs such as the epidemic LPS genotypes,MLST genotypes as well as the epidemic virulence factor-associated genes remins unclear.In this study,we performed a molecular detection on P.multocida isolates from pigs suffered and/or died from respiratory disease in China between December 1,2013 and December 31,2017,using capsular genotyping,LPS genotyping,MLST genotyping and virulence genotyping based on the detection of different virulence gene profiles.After that,we selected several P.multocida strains isolated herein for whole genome sequencing.Together with those publicaly available genome sequences of P.multocida strains originated from different hosts,we performed comparative analyses to screen genes associated with host predilection.Our aim is to understand the genetic characteristics of P.multocida isolates from different hosts and provide insights into molecular basis of the bacterial host predilection and phylogeny.Main findings of this study are listed as follows: 1.Capsular genotypes,LPS genotypes as well as MLST genotypes of P.multocida strains prevalent in pigs in China between December 1,2013 and December 31,2017A total of 262 strains of P.multocida were recovered from tissue samples of pigs suffered and/or died from respiratory disease in China between December 1,2013 and December 31,2017.Capsular typing using multiplex PCR assays determined 128 isolates producing type A capsule,112 isolates producing type D capsule,7 isolates producing type F capsule,and 15 isolates having the nontypable capsule.Percentage of each types of capsule was 48.85%,42.75%,2.67%,5.73%,respectively.To understand the LPS genotypes of these epidemic porcine P.multocida isolates,a total of 136 of the 262 isolates were selected randomly for LPS genotyping using multiplex PCR methods.The assays only identified two types of LPS genotypes,L3 and L6.There were 34 isolates producing LPS genotype L3 while the rest 102 producing LPS genotype L6,with a corresponding percentage of 25.00% and 75.00%,respectively.When combining the capsular genotype with the LPS genotype,four types of capsule: LPS genotype groups(A: L3,A: L6,D: L6,F: L3)were found within the 136 porcine P.multocida isolates.The number of isolates within each of the capsule: LPS genotype groups was 29,37,63,2,with corresponding percentages of 21.32%,27.21%,46.32%,1.47%,respectively.To understand the MLST genotypes of the porcine P.multocida currently circulationg,a total of 80 isolates were futher selected from the 136 porcine strains for multilocus sequence tying.A total of seven MLST genotypes were identified: ST3,17 strains(21.25%);ST10,22 strains(27.50%);ST11,34 strains(42.50%);ST12,2 strains(2.50%);ST16,2 strains(2.50%);ST74,1 strain(1.25%);ST75,2 strains(2.50%).When combining the capsular genotype,LPS genotype with the MLST genotype,there were 8 types of capsule: LPS: MLST genotypes among the 80 porcine P.multocida isolates: A: L3: ST3,16 strains(20.00%);A: L3: ST74,1 strain(1.25%);A: L6: ST10,21 strains(26.25%);D: L6: ST10,1 strain(1.25%);D: L6: ST11,34 strains(42.50%);D: L6: ST16,2 strains(2.50%);D: L6: ST75,2 strains(2.50%);F: L3: ST122,2 strains(2.50%).2.The distribution of main virulence factors-associated genes among porcine P.multocida circulating in ChinaA total of 23 kinds of virulence factors-associated genes(VFGs)including ptf A,fim A,hsf-1,hsf-2,pfh A,tad D,tox A,exb B,exb D,ton B,hgb A,hgb B,Fur,tbp A,nan B,nan H,pm HAS,omp A,omp H,oma87,plp B,sod A,and sod C were detected among the 262 porcine P.multocida isolated herein,using PCR assays.The results showed that several VFGs such as ptf A,fim A,exb B,ton B,Fur,omp A,omp H,oma87,sod A,sod C were broadly characteristic of the porcine P.multocida isolates,as their detection rates were higher than 90.00%.Conversely,the detection rates of tox A and tbp A were lower than 10%.Other VFGs including hsf-1,pfh A,tad D,hgb A,hgb B,nan B,nan H,pm HAS were moderately detected and their detection rates were located between 30.00%~80.00%.In addition,during virulence genotyping,the distribution of some VFGs displayed a certain level of “genotypepreference”.Among those VFGs,tad D and pm HAS were more associated with capsular genotypes than LPS genotypes;while pfh A,hgb A and hgb B were more associated with LPS genotypes than capsular genotypes.However,some VFGs including tox A,hsf-1 and nan B were associated with specific “capsule: LPS gentopes”.The tox A gene was only found in D: L6 strains,while the detection rate of hsf-1 among D: L6 strains was significantly higher than its detection among strains of other capsule: LPS gentopes.The detection of nan B was significantly associated with capsule: LPS gentope A: L6.3.Experimental pathogenicity and complete genome characterization of a pig origin P.multocida serogroup F isolate HN07To invertigate the pathogenicity of pig origin P.multocida serogroup F isolate,a representive strain HN07 was used to challenge mice,chickens,rabbits and pigs,respectively.Our results showed that the isolate was more highly pathogenic to mice than a toxigenic pig origin P.multocida serogroup D strain HN06.However,strain HN07 did not induce severe clinical signs in chickens,even at an infective dose of ~109 colony-forming units(CFU),suggesting that this isolate had low pathogenicity in chickens.HN07 caused severe clinical signs that ultimately led to death in the rabbits challenged with ~105 CFU;the main pathological changes noted were characteristic of fibrinopurulent pneumonia and hemorrhagic pneumonia,thus demonstrating that the porcine isolate is virulent to rabbits.In the pig experiments,the animals challenged intratracheally with HN07 became pyrexial with labored breathing at 12 hours post-infection.Most pigs died from respiratory failure and the main pathological findings included evidence of exudative bronchopneumonia,interstitial pneumonia and hemorrhagic pneumonia.Whole genome resequencing and comparative genomic analysis identified a novel integrative conjugative element ICEpmcn07 which carries an intact type IV secretion system(T4SS)in P.multocida HN07 genome.To our knowledge,this is the T4 SS found in P.multocida for the first time,and the presence of ICEpmcn07 in HN07 genome might affect the virulence of HN07 to pigs.In addition,six VFGs were found to be present in all of the three chicken-virulent P.multocida strains P1059,X73 and GX-Pm but absent in the two chicken-avirulent strains HN07 and Pm70.The missing of these genes might address the question why HN07 is avirulent to chicken.4.Whole genome resequencing of P.multocida strains originated from pigsA total of 45 porcine P.multocida strains isolated herein were randomly selected for whole genome resequencing on a Illumina Hiseq PE150 platform.Sequences assemble via SPAdes generated approximately 18~66 contigs,with average sizes between 35.0 bp and 130.4 bp,and N50 sizes between 160.99 bp and 631.49 bp for the sequenced P.multocida strains.Predicted sizes of the draft genomes composed of those contigs ranged from 2.22 Mbp to 2.49 Mbp,with average G+C contents ranging from 40.04% to 40.67%.The number of open redsing frames(ORFs)was between 2087 and 2391,which include 2027~2330 coding DNA sequences(CDSs),46~53 t RNA genes,and 7~10 r RNAs.5.Capsular genotypes,LPS genotypes,and MLST genotypes of P.multocida from different hostsTo understand the genetic characteristics of P.multocida strains from different species,s total of 109 P.multocida strains including 16 isolates from avian species,20 isolates from bovine species,56 isolates from porcine species,and 17 isolates from rabbit species were analyzed in this study.Capsular typing and LPS genotyping showed that type A was the mostly dominate capsular genotype among the 16 P.multocida strains from avian species(93.75%),and the common LPS genotypes were L1(56.25%)and L3(25.00%);the most commonly identified capsule: LPS genotype among the 16 avian isolates was A: L1(56.25%),followed by A: L3(18.75%).Among the 20 P.multocida strains from bovine species,the common capsular genotypes were A(40.00%)and B(60.00%);the mostly commonly LPS genotypes was L2(60.00%),followed by L3(35.00%);the most commonly identified capsule: LPS genotype was B: L2(60.00%),followed by A: L3(35.00%).The common capsular genotypes among the 56 P.multocida strains from porcine species were A(48.21%)and D(42.86%),the common LPS genotypes were L6(66.07%)and L3(32.14%),while the common capsule: LPS genotypes were D: L6(42.86%),A: L3(25.00%),and A: L6(23.21%).For the 17 P.multocida strains from rabbit species,type A(94.12%)was the dominate capsular genotype,type L3(76.47%)was the mostly common LPS genotype,and A: L3(76.47%)was the mostly dominant capsule: LPS genotypes.RIRDC MLST assigned 8 kinds of MLST genotypes(ST8,ST9,ST27,ST53,ST60,ST129,ST156,ST159)among the 16 avian P.multocida,and ST129(43.75%)was the predominate MLST genotype.The 20 bovine P.multocida were assigned into 4 kinds of MLST genotypes(ST65,ST79,ST80,ST122).For these genotypes,ST122(60.00%)was the most dominant.For the 56 porcine P.multocida,a total of 8 kinds of MLST genotypes(ST7,ST9,ST13,ST27,ST50,ST74,ST122,ST287)were figured out,and ST50(37.50%),ST13(23.21%)and ST74(21.43%)were the common MLST genotypes.The 17 rabbit P.multocida were assigned into 5 kinds of MLST genotypes(ST9,ST27,ST50,ST204,and ST298),and ST9(52.94%)was the most prevalent sequence type.When combining MLST genotypes with capsular types and LPS genotypes,the results showed that a capsular: LPS: MLST genotype A: L1: ST129(43.75%)was predominant among the 16 avian isolates;and B: L2: ST122(60.00%)as well as A: L3: ST79(30.00%)were predominate among the 20 bovine isolates.For the 56 porcine isolates,D: L6: ST50(37.50%)was the predominate genotype;while for the 17 rabbit isolates,A: L3: ST9(52.94%)was the predominate.6.Comparative genomic analyses of P.multocida from different hostsPan-genomic analysis identified 4256 gene clusters among the 109 P.multocida strains from different host species.Among these gene clusters,approximately 1806 genes were predicted as core genes(genes present in 99%-100% of the strains),1841 genes were identified as dispensable gene clusters(genes present in and more than 2 strains but less than 99% of the strains),and 609 genes were identified as strain-specific genes.To figure out the so-called “host predilection” related genes,we further classified the dispensable genes according the following criterions.Initially,we defined a “host predilection related gene” as one present among the strains from one host but absent in strains from all the other hosts.Based on this criterion,there was no host predilection related gene screened.Therefore,we defined a “host predilection related gene” as one gene present among 90% of the strains from one host but absent in 90% of the strains from all the other hosts.However,there was still no genes obtained from the strategy.Finally,we defined a “host predilection related gene” as one gene present among 80% of the strains from one host but absent in 80% of the strains from all the other hosts.Using this criterion,five genes were isolated from the porcine P.multocida strains and one gene was identified from the avian,bovine,and/or rabbit P.multocida strains,respectively.To understand the distribution of the virulence factor-associated genes(VFGs)across the genomes of P.multocida strains originated from different hosts,the total 4256 gene clusters encoded within the 109 strains were annotated using the bacterial Virulence Factor Database(VFDB).Using this strategy,a total of 432 putative VFGs were identified,and they were assigned into 366 VFG categories.The presence of a VFG in a genome was determined based upon the BLAST score ratio(BSR)analysis with a normalized ratio ? 0.8.Following this criterion,it is interesting to find that there is no significant difference on the distribution of VRFs among P.multocida strains from different hosts.The distribution difference of VFGs between different P.multocida strains was mainly reflected on those responsible for the synthesis of capsule,LPS and fimbrial low-molecular-weight proteins;however,they also did not show significant difference on distribution among P.multocida isolates from different hosts.Instead,our data demonstrated that the distribution of some VFGs displayed a certain level of “LPS: MLST genotype-preference”(Figure 7).For example,the hyaluronidase encoding gene pm HAS was absent in the genotypes L6: ST50,L6: ST287,and L2: ST122 strains;the surface fibrils encoded by the gene hsf-2 was missing in genotypes L6: ST50 and L3: ST13 strains.7.Phylogeny of P.multocidaThe phylogenetic trees were generated based on the single nucleotide polymorphisms(SNPs)across the whole genome sequences of the 109 P.multocida from different hosts comparing against the reference P.multocida ATCC 43137 genome,and on the SNPs within the single copy core genes among the comparison genomes,respectively.Using both strategies,it is clearly showed that it is clearly showed that P.multocida strains with the same LPS plus MLST genotypes were phylogenetically clustered together,which suggest that the LPS genotype plus the MLST genotype were the phylogenetical determinants of P.multocida. |
PDF Full Text Request