Study On Three Etiologic Characteristic Of Infected Goats And Analysis Of PCR-RFLP

Contagious caprine pleuropneumonia (CCPP), a severe, acute, and highly contagious respiratory disease, affects all ages and is characterized by high fever, coughing, high morbidity and mortality. Respiration is accelerated and painful. The gross lesions of the disease are typically limited to the thoracic cavity and characterized by fibrinous pleuropneumonia, lung hepatization, and accumulation of pleural fluid. In this study, samples were taken from infected goats in the area of Yubei and Beibei in Chongqing and Shannan in Tibet. The lung, nasal cavity and pleural fluid were the main collected position, followed by heart-blood, liver, spleen, kidney and lymph nodes. Some pathogens were isolated using modified Hartleys medium, ordinary culture medium, culture medium containing cony blood and LB medium. Fourteen strains of pathogens were isolated from 68 goats died from CCPP, of which there were four strains of mycoplasma, five strains of P. mirabilis and five strains of E.coli. The pathogens of CCPP and secondary infection were isolated and identified from the infected goats; cloning and sequencing, animal test, antibiotic susceptibility test and biological characteristics of pathogens were also carried out, which provide theoretical basis for the prevention and control of CCPP.1 Isolation and identification of mycoplasmaTo study the etiologic agent of CCPP, the pathogens had been isolated from the lungs of the infected goats using modified Hartleys medium. The biochemical tests including Carbohydrate termentation, Arginine hydrolysis, Tetrazolium, Film and spot, Requirement of sterin, animal test and antibiotic susceptibility test were carried out followed by the PCR amplification and analysis of a specific fragment of 16S rDNA referring to the members of the M. mycoides cluster. Four field strains, named BB3, BB7,BB42 and YB2 respectively, were isolated from the infected goats. The results showed that the strains BB7 and BB42 shared the same biochemical properties with type strain PG3, whereas BB3 and YB2 were distinct from PG3 in biochemical characteristics; The field strains BB7 and YB2 had certain pathogenicity to mice whereas the strains BB3 and BB42 showed no pathogenicity; The field strains were most sensitive to ceftiofur sodium, more sensitive to tylosin, erythromycin and florfenicol, but least sensitive to lincomycin; PCR products with 548 bp in length were acquired from both field strains and the type strain, indicating the field strains are members of the M. mycoides cluster.2 Isolation, identification, cloning and sequencing of P.mirabilis from goatTo isolate and identify pathogens, which were isolated from the infected goats using ordinary culture medium, culture medium containing cony blood and LB medium. Swarming behavior assay, biochemical test, animal test and antibiotic susceptibility test were carried out.16S rDNA genes of field strains were amplified by PCR, cloned and sequenced. There were five strains isolated from the infected goats. The results showed that the swarming migration of field strains began after being cultured 2.5 h-3.5 h, the swarming migration distance mainly increaseed with the incubation time, the swarming migration velocity increased between 2.5 h and 4.5 h, whereas decreased between 5 h and 5.5 h; With the same incubation time, the swarming migration distance was farthest on 0.5% LB medium, but the distance gradually shortened on 1.0%,1.5% and 2.0% LB medium respectively, indicating that swarming migration distance was negatively correlated with agar concentration; The field strains shared similar biochemical characteristics with P.mirabilis and had strong pathogenicity to mice, the LD50 in mice was between 2.5000×107 cfu·mL-1 and 4.4453×107 cfu·mL-1; The field strains were sensitive to amikacin and ceftazidime, whereas showed no sensitivity to ciprofloxacin, enrofloxacin, norfloxacin, kanamycin, gentamicin and florfenicol; The fragment of 1 535 bp in size were applified. The DNA sequencing showed 99.5%-99.8% similarity with P.mirabilis for 16S rDNA, which indicate that the field strains are P.mirabilis.3 Cloning and prediction of protein structure of virulence factors zapA gene of P.mirabilis from goatTo clone zapA gene, which is an important virulence factor of P.mirabilis, and predict its deduced protein. In this study, zapA gene was subjected to PCR amplification, molecular cloning and DNA sequencing; the gene sequence was then applied to bioinformatics program to predict the signal peptide, transmembrane domain, secondary structure and B cell epitopes of its deducted protein. DNA sequencing showed that the zapA gene was 1476 bp in size, encoding 491 amino acid residues. The gene shared 99.59% homology with reference strain in both nucleotide and its encoded amino acid sequence. The bioinformatic analysis supported the existence of signal peptide, denied the presence of transmembrane domain and predicted the B cell epitopes of ZapA locating in or near amino acids 69-71,78-84,136-139,197-199,353-356,371-373 and 472-474. These results provide the theoretical basis for the expression of ZapA and development of genetic engineering vaccine.4 Analysis of P.mirabilis by PCR-RFLPTo study the polymorphism of P.mirabilis, both 16S rDNA and zapA gene were analyzed with PCR-PFLP after digestion by specific restriction enzymes.16S rDNA and zapA gene of the field strains were amplified by PCR respectively, the PCR products were purified with gel extraction kit.16S rDNA gene of the strains were digested with restriction enzymes EcoRⅠand HindⅢ, and zapA gene were digested with restriction enzymes Ball, DraⅠand VspⅠ; Restriction Map showed that there were 868 bp,667 bp fragments with restriction enzyme EcoRⅠ; 1462 bp,73 bp fragments with HindⅢ; 633 bp,474 bp,369 bp fragments with BalⅠ; 796 bp,627 bp,49 bp,4 bp fragments with DraⅠ, and 864 bp,411 bp,115 bp,86 bp fragments with VspⅠ, which shared the same restriction enzyme fragments with standard strain. The segments from PCR-RFLP analysis displayed no disparity with reference strain both in number and size. All the results indicate that the field strains are members of P.mirabilis with no polymorphism.5 Isolation, identification, cloning and sequencing of E.coli from goatTo isolate and identify pathogens, which were isolated from the infected goats using ordinary culture medium and culture medium containing cony blood. Biochemical test, animal test and antibiotic susceptibility test were also carried out.16S rDNA genes of field strains were amplified by PCR, cloned and sequenced. Five strains of pathogens, which had similar biochemical characteristics with E.coli, were isolated; All field strains had pathogenicity to mice, the LD50 in mice was between 4.8395×10' cfu·mL-1 and 1.0375×108 cfu·mL-1; The field strains were sensitive to Trobicin and furazolidone, whereas had no sensitivity to ceftriaxone sodium, cefoperazone, cephazolin, ampicillin, carbenicillin, doxycycline and florfenicol; The DNA sequencing showed 99.5%-99.8% similarity with E.coli for 16S rDNA, which indicate that the field strains are E.coli.