Molecular diagnosis of neosporosis using milk

• Main Authors: Ming-Yi Chien, 簡銘毅
• Other Authors: 莊士德
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/05059531193645299448

碩士 === 國立中興大學 === 獸醫學系暨研究所 === 101 === Neosporosis is an infectious disease caused by Neospora caninum (N. caninum) which is a new species of protozoan parasite recognized in 1988 and has been considered as a major pathogen that causes cattle abortion and serious economic impact in dairy industry in many countries. Cattle infected with neosporsis usually show no obvious clinical signs but result in high percentage of vertical transmission. Without an efficient drug or vaccine against neosporosis, testing and culling are major methods to lower the economic impact. Milk has been shown thought to be similar to blood sample in detection of antibody. However, serological examination is not as convincing as molecular examination. The purpose for this research is to examine the N. caninum DNA in cattle and goat milk, especially colostrums, and also to develop Loop-mediated isothermal amplification PCR (LAMP) for the rapid and simple diagnosis of neosporosis using the milk samples. A total of 582 goat milk samples, obtained from 560 goats in 5 farms, and 55 bovine milk samples, obtained from 54 cows in 6 dairy farms, were examined by conventional PCR and nested PCR for the presence of N. caninum DNA. Among the 582 goat milk, 3 were positive by the conventional PCR method and 130 positive by the nested PCR, respectively. Of 130 positive milk samples, 122 were colostrums, which is about 93%. Among the 55 bovine milk, none was positive by the conventional PCR method but 10 were positive by nested PCR. Of 10 bovine positive milk samples, 9 were colostrums, that is 90%. All products amplified by conventional PCR and nested PCR were sequenced to confirm that the amplicons were that of the DNA of N. caninum. This is the first finding of N. caninum DNA in goat milk. In addition, the observation of a high percentage of colostrum samples positive for N. caninum DNA in not only caprine milk but also in bovine milk indicates that colostrum can be a major and good sample source for diagnosis of N. caninum. A set of primers designated NCHU-5 was constructed for detecting N. caninum by LAMP method. The NCHU-5 primers showed good result for detecting N. caninum DNA in milk samples. The NCHU-5 primers did not cross-react with Toxoplasma gondii, Cryptosporidium parvum and Giardia lamblia. It was demonstrated that NCHU-5 primers has good specificity for N. caninum DNA. By adding SYBR green I to the LAMP products, the resulting color reaction can be determined visually and thus give a rapid and simple diagnosis for the test sample. Thus, NCHU-5 could be used for rapid diagnosis of N. caninum in either milk or aborted fetus in the field.