Evaluation the virulence of Mycobacterium bovis isolated from milk samples through histopathological study in laboratory animals

Introduction: Mycobacterium bovis has a broad host range, and it is the principal agent responsible for tuberculosis (TB) in bovine, domestic and wild mammals. M. bovis also infects human, causing zoonotic TB through ingestion, inhalation and, less frequently by contact with mucous membranes and broken skin. Zoonotic TB was formerly an endemic disease, usually transmitted to man by consumption of raw cow's milk. It is indistinguishable clinically or pathologically from TB caused by M. tuberculosis . Objective: The aims of this study were, to isolate and identified M. bovis from raw milk samples by different methods, and evaluate the virulence of M. bovis in laboratory animals (Rabbit). Materials and methods: To conduct the study, ninety three cow's milk samples were collected from farms around Baghdad governorate. The decontamination of milk samples was firstly carried out, then samples were subjected to routine tests which include, direct smear for Ziehl Neelsen acid fast stain, culture, each sample was cultured on Lowenstein Jensen media with Sodium pyruvite (All cultures incubated on 37°C for 4–10 weeks with continuous observation), and biochemical testes as Nitrate reduction test, Niacin paper strip test and pyrazinamidase test, were employed to diagnose and identified the bacteria. Beside molecular assay was used to confirm the identification of the isolates by Polymerase Chain Reaction (PCR) using specific primers for M. bovis . The virulence of these isolates were investigated through inoculate it in group of laboratory animals consist of 8 rabbit in addition to other group of 4 animals as control (inoculate with Phosphate Buffer Saline). The animals were scarified after 6 weeks of inoculation, post- mortem examination was carried out, smears were taken from lesions, and tissue samples were collected from lymph nodes and different organs. Results: The results revealed five isolates of M. bovis in direct smear by acid fast Ziehl-Neelsen stain, while eight isolates observed by culture, the colonies appeared with characteristic feature of cream color, rough, and with irregular edge. The molecular assay using PCR technique confirmed the diagnosis of eight positive isolates in smears and culture. The virulence of these isolates were investigated through the pathological effects appeared in inoculated rabbit which showed lesions scattered mainly in lymph nodes and different organs as lung, liver, spleen and kidney when compared with control group which were naive. Beside the infiltration of mononuclear cells in the internal organs particularly in the lungs. The result of histopathological examination clarified the virulence of M. bovis isolates, and its impact on tissue and organs of the rabbit. Conclusion: Our study conclude the presence of M. bovis isolates in milk in high percentage pause important source of tuberculosis infection for human being.