Detection of Mycobacterium avium subsp. paratuberculosis from cattle and buffaloes in Egypt using traditional culture, serological and molecular based methods

Background: Johne's disease (JD) caused by Mycobacterium avilan subsp. paratuberculosis (MAP) represents a real threat to the agriculture and dairy food industries and believed to be a potential public health problem. Signs of infection in ruminant include weight loss, diarrhea, decreased milk production, and eventually death. The definition of an infected animal based either on the presence of anti-MAP antibodies, or positive bacterial culture. No treatment for the disease exists and controlling the disease is difficult due to its long latent period. JD is a worldwide problem and multiple studies in many countries have been carried out to determine the prevalence of MAP infections. Although some primary non intensive studies confirm presence of JD in Egypt, the disease is currently neglected by the official Egyptian veterinary agencies. There is no official data, no national control program, and no used vaccine. Aim: This study aimed to evaluate three conventional diagnostic methods for MAP under the Egyptian circumstances with a general aim to determine the appropriate strategy to develop a JD control program. These methods were pooled fecal culture, humoral response and insertion sequence 1S900 targets polymerase chain reaction (1S900 PCR). Materials and Methods: Fecal and serum samples (500 each) were collected from Holstein-Friesian cattle and buffaloes housed in five Egyptian governorates. Fecal samples were examined for MAP on the basis of a strategic pooling procedure and performed on Herrold's Egg Yolk Agar Medium (HEYM). Smears were prepared from developed colonies and stained using a Ziehl-Neelsen (ZN) technique. The identity of developed colonies was further confirmed by PCR analysis of1S900 sequence. Sera from both culture-positive and culture-negative animals were evaluated individually for humoral response. Results: Out of 50 pooled specimens, 34 (68%) fecal cultures were positive for MAP. Scrum positive samples of culture positive animals were 63/340 (18.5%). No serum positive were detected in the samples from culture-negative animals. DNA extractions from colonies of 34 culture-positive pooled samples were tested with the PCR targeted to IS900 and revealed 18 isolates (52.94%) were positive. Conclusion: The present study has succeeded in isolating and identifying of MAP from Egyptian cattle and buffaloes in five governorates. The results confirm the epizoology of JD in Egypt and encourage the decision-makers to start creating a program to control it. Pooling procedure for MAP culture is an efficient and reliable method. However, the findings suggest the importance ()fusing both ELISA and fecal culture in MAP diagnosis and control.