Alifonova K. Beetles-pests of grain stocks: a reservoir and a factor of transmission of mycobacteria tuberculosis

The scientific work is devoted to the establishment of the epizootological role of rice weevil (Sitophilus oryzae (Linnaeus, 1763), Coleoptera, Curculionidae) as a potential reservoir of M. bovis in the process of emergence and spread of tuberculosis. The duration of accumulation inside the insect body and the release of dissociative and pathogenic forms of mycobacteria tuberculosis into the environment by the weevil was studied. The effect of passage through the organism of the weevil on the morphology and biological properties (culture, tinctorial, biochemical, virulence) of the pathogen was determined. In the present study, the research was conducted using dissociative (117 and 118 passages) and virulent (passage №100) strains of M. bovis stored in the museum of the Department of Infectious Diseases of the State Academy of Environmental Health and the subcultures obtained from them. The optimal temperature of cultivation of the pathogen was determined upon obtaining the subculture. It was found that the studied strains have stable properties that are preserved when inoculated into fresh culture medium. The acceleration of the primary growth of subcultures of the 100th passage by 2.1 times, 117th passage by 1.1-1.3 times, and a decrease in the growth of subculture 118th passage by 2.0-2.3 times compared to the original cultures were noted. The viability of mycobacteria was determined in the obtained subcultures by counting colony-forming units and found that they were suitable for the next stages of the experiment. The ability of the rice weevil to mechanically transfer viable mycobacteria on the body surface was determined. By studying the period of carriage of the tuberculosis pathogen by the beetles, we proved the ability to store mycobacteria in the insect's body for a long time. In particular, in the first series of the experiment, when working with a dissociative strain for 50 days, the pathogen was detected in the homogenized beetle fluid and in the flushes from the surface of the weevil's body with a gradual decrease in the number of microbial cells in the microscope field of view. When sowing the homogenized homogenate of infected beetles on the nutrient medium, the growth of the experimental mycobacteria was observed only by day 30 after the start of the experiment, indicating a loss of viability of mycobacteria after a longer stay in the beetle's body. In the second series of the experiment, when rice weevil was infected with a virulent strain of mycobacteria, the pathogen was detected in smears and homogenized beetle fluid up to day 30 inclusive, and during microscopy of body surface washings - up to day 20. Similarly to the first series of the experiment, when the homogenized material was inoculated on the culture medium, growth was observed only in the tubes inoculated on the 30th day of the experiment. After studying the duration of mycobacterial isolation and contamination of environmental objects by rice weevils, it was found that during infection with dissociative forms of M. bovis, during microscopic examination, grain suspensions were detected by day 12 of the experiment (up to the third transplantation of beetles), and growth on the nutrient medium was recorded within 30 days after the start of the experiment (i.e., up to the fifth transplantation of beetles). In the experiment with the pathogenic strain, mycobacteria were detected in the homogenized beetle homogenate similarly up to day 12, and growth on the medium was detected up to day 20 after infection. In terms of morphology and culture properties, the pathogen isolated during the two series of the experiment was identical to the original culture. At each stage of the experiment, the isolated cultures were subjected to molecular genetic identification with confirmation of species affiliation. Genotyping of the cultures was performed using the VNTR method. It was proved that the length of amplicon fragments obtained as a result of PCR with primers targeting the VNTR locus was identical to the M. bovis strains in all the experimental samples. The passage of the pathogen through the rice weevil organism did not cause genome changes that can be identified by VNTR genotyping, i.e., the number of tandem repeats in the variable loci remained unchanged. Investigating the biochemical properties of the experimental cultures, it was found that the passage of mycobacteria through the rice weevil increases enzymatic activity. The results of the dissertation can be used in the research work of the departments and in the educational process for the training of specialists in the specialty 211 "Veterinary Medicine", in the isolation of mycobacteria of tuberculosis .