Isoiation And Identification Of Arcanoba Cterium Pyogenes And The Development Of The Pcr Based Detective Method

Arcanobacterium pyogenes, a Gram-positive, nonmotile, rod-shaped bacterium, often cause purulent infection opportunistically, especially under the condition of the low immunity and commonly found abcess-caused in the mucous membranes of variety of animals. Then, this bacteria can secondary provoke several diseases, such as dairy mastitis, bovine metritis, renal abscess, liver abscess, turkey osteomyelitis, sow endometritis, piglet arthritis and swine pneumonia and acute respiratory infections. The production performance degradation and the bodies’ weakened resistance caused by the abcessed disease of Arcanobacterium pyogenes are harmful to the livestock industry. For the study of the etiology of the calf disease with the main symptoms of severe infection and mortality in large-scale dairy farm, an autopsy to the diseased calf was done.Two bacterial isolated from the calves lung tissues and the isolated bacteria was Arcanobacterium pyogenes based on the traditional bacteriological identification (morphological characteristics, staining characteristics, cultural characteristics and biochemical characteristics, etc.) and Biolog automated rapid microbial identification system identification and16SrRNA analysis. The results of pathogenicity test and antimicrobial sensitivity test showed that the isolated strains had a strong pathogenicity to mice and only a few types of antibiotics such as cefazolin, ofloxacin were sensitive. The antimicrobial sensitivity test provides a reference for clinical selection of the antibiotics.To rapid diagnosis of the Arcanobacterium pyogenes infection, a novel PCR method was developed for detection of the PLO gene. The sensitivity and specificity was high with a minimum detectable bacterium9.2×103CFU/mL and without cross-reactivity to other bacteria, such as Escherichia coli, Brucella. So there is a potential use in the clinical of this novel PCR method. Testing on clinical samples showed that the PCR assay had the same detection rate as the traditional bacteriological identification.To study the pathogenicity of the isolated bacteria, three virulence factors of pyolysin (PLO), collagen binding protein (cbpA) and Neuraminidases (nanH) gene were cloned based on the reference sequences in the GeneBank, and among them, seven primes were synthesized and used for PCR amplification. With the plo, cbpA and nanH gene sequencing, the homology was analyzed with reference strains. The results showed that the homology was more than98%based on the plo gene and85%based on the cbpA the nanH gene sequences. The phylogenetic tree analysis further identified the isolated bacteria was Arcanobacterium pyogenes. Therefore, the gain of the three virulence factors was helpful for research of the characteristics of pathogenicity of the bacteria.In summary, two strains of Arcanobacterium pyogenes was isolated and identified, and the antimicrobial sensitivity test provides a reference for clinical choice of the antibiotics. The clone and sequences analysis of three main virulence factors have provided the materials for study the pathogenicity of the bacteria. At last a novel and rapid PCR method was developed for detection of the Arcanobacterium pyogene infection.