| Porcine cytomegalovirus(PCMV) can lead to the disease,which mainly causes pneumonia and rhinitis of piglets.PCMV is a kind of conditional pathogenic microorganism,which is easy to be misdiagnosed and mistreated.With the development of the pig industry,the disease has a trend of spread.It is great practical significance to investigate the molecular epidemiology of the disease and to understand the infection rate and epidemic law.Pasteurella multocida(Pm) is the pathogen of Fowl Cholera(FC)and one of the main pathogens causing economic losses in poultry industry.At present,most of the researches on Pm are focused on isolation,identification and antimicrobial susceptibility test,There are fewer studies on the host preference of Pm,the encoded antigens and the unstable effect of clinical antibiotics from the perspective of whole genome.1?Molecular pidemiological investigation of PCMV According to the sequence of PCMV glycoprotein B(g B)gene registered in Gen Bank,two pairs of specific primers were designed and PCR detection method was established.And we investigated the molecular epidemiology of PCMV in Jiangxi Province,and analyzed the sequence of g B bioinformatics.A total of 169 samples of suckling piglets and nursery pigs were tested,12 samples were positive,the positive rate is 7.1%,this shows that the infection rate of PCMV is lower in the dead pigs,and it is more concentrated in the dead pigs with obvious respiratory symptoms.2?Analysis of g B sequence Two full-length pigs g B gene sequences were cloned,sequencing results showed that the sequence length was 2,580 bp,more than 99% similar to the PCMV g B sequence logged in Gen Bank.Bioinformatics analysis shows that PCMV can be divided into two evolutionary branches,JX-1 is located in the same evolutionary branch with most of the domestic isolates,JX-2 is in the same evolutionary branch as the foreign isolates.The nucleotide and amino acid sequences of these two g B sequences have multiple site variations,Three nucleotides were missing from the nucleotide sequence of JX-1 strain at 1130-1133,There are 8 nucleotide variations and 5 amino acid variations,Evolutionary analysis shows that PCMV and human herpesvirus are in the same evolutionary branch,and there may be cross reaction between them.3?Isolation and identification of Pm form poultry We carried out the isolation,identification and drug sensitivity test of the pathogenic bacteria of the dead ducks and chickens suspected of avian cholera,and select sensitive drugs for clinical treatment according to the results.Two strains of bacteria isolated from dead duck and chicken liver are Pm,named the isolated bacteria as: Pm-jx-01(duck)and Pm-jx-02(chicken).Pmjx-01 is highly sensitive to spectinomycin,florfenicol and ampicillin,resistance to metronidazole,ceftriaxone,cefotaxime,amoxicillin,compound minocycline and streptomycin.Pm-jx-02 is highly sensitive to enrofloxacin,penicillin,cefotaxime,amoxicillin,ofloxacin and ceftriaxone,Resistance to ampicillin,compound Xinnuoming,florfenicol,oxytetracycline and amikacin.4?Sequencing and analysis of the whole genome of Pm from poultry Using the third generation DNA sequencing technology,the whole genome of Pm-jx-01 and Pm-jx-02 was sequenced,and two circular chromosome sequences were obtained by splicing.The total length of Pm-jx-01 gene is 2,302,632 bp,GC content is 40.35%,and there are 2,117 coding genes.The total length of Pm-jx-02 gene is 2,321,968 bp,GC content is 40.42%,and there are 2,099 coding genes,only Pm-jx-01 strain has the protein gene encoding the participation in the muticellular organismal process,according to nucleotide sequence comparison and TGPYF analysis,both strains are Pm.There are 279-280 virulence factors in these two isolates.There are 6 classes and 10 resistance genes on the chromosome of Pm-jx-01 strain,as Aminoglycosides(ant3ia?aph33ib?aph3ia?aph6id),?-lactamase(bl2d?oxa1),Chloramphenicols(cata11),sulfonamides(sul2,sul3),tetracyclines(tetb)and macrocyclic lipids(mef B),ICE-1 in chromosome contains 5 drug resistance genes.There are only two classes and two resistance genes in Pm-jx-02 strain,as Chloramphenicols(cata11)and tetracyclines(tetb).According to the results of drug sensitivity test,the main reason for the drug resistance of Pm-jx-01 is the presence of the encoded drug resistance gene in its genome,the resistance gene of Pm-jx-02 is not the main resistant mechanism.Comparative genomics showed that Pm-jx-02 had 600 gene clusters,1,257 single copy genes,113 unique genes.The results of horizontal transfer element analysis showed that there are one ICE-1and four is elements in Pm-jx-01 strain,and there are two secondary metabolites of bacteriocin(bacteriocin)and adhesion protein(LAP).The insertion integration sequence is concentrated between 1350k-1400 k in the genome.Pm-jx-02 has only one is element,one specific gene and a secondary metabolite with unknown function.The insertion integration sequence is distributed throughout the genome.Pan-genome analysis of 12 Pm strains,it was found that with the increase of the number of sequenced genomes,the pan-genome size also increased rapidly,but the core genome decreased.This suggests that the genome of Pm should be an open pan-genome,which has a strong ability to obtain foreign genes.The number of pan-genome and core genome of Pm-jx-01 is more than that of Pm-jx-02.It can be inferred that Pm-jx-01 is easier to mutate than Pm-jx-02.In order to provide data support for the understanding of PCMV infection and prevention and control measures,and to provide further research ideas for the study of drug resistance,pathogenicity and variation mechanism of Pm,we have carried out preliminary molecular epidemiological investigation on PCMV in pig farms in Jiangxi Province,and carried out drug sensitivity test,genome-wide sequencing,analysis and annotation on two clinical isolates of avian Pm. |
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