Isolation, Identification and Antibiotics Resistance of Corynebacterium pseudotuberculosis from Goats

  Objective  To identify the pathogen that caused the death of goats in a farm in Fujian for prevention and treatment of Pseudotuberculosis.

  Method   Aseptically collected specimens of lung tissues from the dead goats were used to isolate the microbes for morphological observation and strain identification. DNA of the isolates were extracted, and the specific and 16S rRNA universal primers selected for PCR. The 16S rRNA amplified fragment was cloned, sequenced and analyzed. Biochemical, drug sensitivity, and animal challenge tests were performed on the isolate for taxonomic identification.

  Results  The isolated strain was cultured on an agar medium containing 10% defibrated sheep blood to grow into a colony with a diameter of about 1 mm, a central bulge, a milky white color, an irregular edge, and a beta hemolysis ring. It did not grow well on a nutrient agar nor a MacConkey agar medium. The 16S rRNA sequencing showed an up to 100% homology with Corynebacterium pseudotuberculosis on GenBank by the NCBI online software Blast. Like the specific PCR of C.pseudotuberculosis, that of the isolate also had the same amplified targeted bands. In addition, the biochemical test on the isolate duplicated the characteristics of C. pseudotuberculosis shown in the Berger's Bacterial Identification Manual. Hence, the isolated pathogen named as FJ-PN was verified to be C. pseudotuberculosis. A challenge test on mice with an intraperitoneal or subcutaneous injection of 0.3 mL of 4×10⁵ CFU·mL^-1 of the isolate died within 40 h. Furthermore, two goats injected subcutaneously with 2 mL of 4×10⁵ CFU·mL^-1 and intranasal inoculated simultaneously with 1 mL of 4×10⁵ CFU·mL^-1 of the isolate died within 72 h. The drug sensitivity test showed that the strain was highly sensitive to clarithromycin, ciprofloxacin, penicillin, tetracycline, levofloxacin, ofloxacin, and gentamicin, slightly sensitive to erythromycin, chloramphenicol, ampicillin, cefotaxime, cefoxitin, and rifampicin, and resistant to streptomycin or trimethoprim.

  Conclusion  The pathogenic bacteria isolated from the diseased goats was identified as C. pseudotuberculosis. It had a strong pathogenicity with varying degrees of drug resistance to antibiotics.