Pathogen Analysis Of Dairy Cattle Abortion In Some Areas Of Hebei Province And The Establishment Of PCR Applying To Dignosing Bovine Neosporosis

A lot of infectious diseases, which caused dairy cattle abortion, result in serous lose both in reproduction and milk produce, and have become the most important factor that hinder the development of the cattle industry. There are mainly Brucellosis, Bovine viral diarrhea-Mucosal disease, Toxoplasmosis, and Neosporosis in these infectious diseases. In this study, the specimens were firstly collected from the bovine's blood, clinical specimens and abortus in eight large Hebei cattle fields during March 2004 to February in 2006. These specimens were detected for Brucella - Bovine viral diarrhea virus, Toxoplasma gondii, and Neospora caninum, respectively. Using the combining method of serologic, bacterial and clinic diagnosis, the positive rate of Brucellosis is 5.2%. By IHA test, the positive rate of Toxoplasmosis is 1.7%. By employing the sandwich enzyme-linked immunosorbent assay (sandwich ELISA) method to detec BVDV antigen, which was established in our laboratory, the positive result is 31.3%. Using ELLISA method to detect the N.caninum antibody in cattle serum, positive rate is 19.8%. However, no N.caninum antibody was detected from all of Toxoplasma gondii antibody positive serum;Toxoplasma gondii antibody also was not detected from all of N.caninum antibody positive serum. On the base of these, the PCR testing technique was established to diagnose N.caninum. Based on the sequence alignment of the Nc-5 gene of N.caninum in GenBank, a pair of primers were designed and synthesized. The expected amplified fragment of 350bp was abtained from DNA in blood sample with positity of N.caninum antibody using proteinase K, salting-out and phenylic acid/Trichloromethane by PCR. The amplified product was examined by 2% agar-gel electrophoresis, and was purified by using agarose gel DNA purificatin kit. The result showed that the genes of Nc-5 amplified was about 350bp. The purified pruduct was digested with the corresponding restriction enzymes Msp I, and was detected by electrophoresis. The result showed that the fragment of 350bp was cut into 218bp and 132bp, as expected one. The purified product was cloned to pMD18-T vector. The cloned plasmids were transformed into JM109. The colonies were selected on ampicillin-containing agar plates. Then the plasmids were isolated from several ndependent clones. The specific recombinant plasmid was identified with a pair of primers by PCR.The positive recombinant plasmid can be cloned sequenced by Sanger' s dideoxy sequencing method. The specificity of PCR experiment comparing the positive serum of Brucella, Bovine viral diarrhea virus, Toxoplasma gondii to the positive serum of Neospora caninum,the results were negative. The PCR sensitivity experiments to prove that the highest dilution can amount to 1:40, namely, the template DNA content amounts to the 2.5 mgs/ L, explaining that the PCR diagnosis technique has the higher sensitivity.It is proved that the detected method can diagnose quickly and accurately N. caninum in cattle.