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Tuberculosis Pathogen of Human and Animals

#3219


The Molecular-Genetic Polymorphism of Tuberculosis Pathogen of Human, Cattle, Pigs and Poultry and Studying of Channels of Infection in Live Farming

Tech Area / Field

  • AGR-DIS/Disease Surveillance/Agriculture
  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • BIO-MIB/Microbiology/Biotechnology

Status
8 Project completed

Registration date
21.03.2005

Completion date
19.01.2007

Senior Project Manager
Weaver L M

Leading Institute
All-Russian Research Veterinarian Institute, Russia, Tatarstan, Kazan

Supporting institutes

  • Central Tuberculosis Research Institute, Russia, Moscow

Collaborators

  • Canadian Food Inspection Agency / Mycrobacterial Diseases Centre of Expertise, Canada, ON, Ottawa

Project summary

Unfavorable epidemic situation for TB has been reported worldwide. This is true for both developed and developing countries. High growth rates of TB incidence have been reported in many countries. Similar TB situation is observed in cattle-breeding, which causes significant economical damage and dramatically threatens human health. Mycobacterium tuberculosis can persist as well in the human as in the animal’s organism. Disease is transmitted in two directions – from humans to animals and back.

Like in others countries, we use epizootologic and clinical diagnostic methods, but the main life-time diagnosis of TB in cattle is done by tuberculin skin test. Its specificity is still questionable due to para-allergic reactions conditioned by animals sensitizing by atypical mycobacteria.

So, to exclude or confirm the diagnosis, cattle with positive reaction to tuberculin are slaughtered in order to perform postmortem examination and laboratory studies of biological materials from the animals.

However, initial changes at TB have no expressed specific morphology. Usually initial tuberculosis changes are confirmed by laboratory methods – microscopy and culture. We also use biological tests – reproduction of TB in guinea-pigs. Each of these methods has significant disadvantages – either low sensitivity or long duration.

Molecular-genetic methods are more specific and sensitive. They allow not only quickly (within 1-2 days) detect M. tuberculosis, but also identify its strain by genetic markers. Since TB of cattle, and mycobacteriosis of pigs and poultry are considered socially dangerous, we propose to investigate animals and humans infection with M. tuberculosis, M. bovis, and M. avium (M. avium are isolated from HIV- infected patients, domestic pigs and poultry). Currently specific genome sequences of M. tuberculosis, M. bovis and M. avium have been detected. Based on this, PCR test-systems can be developed. So, it is urgent to develop and implement new molecular-genetic methods to detect and identify mycobacteria. This could significantly reduce time period to obtain post slaughter diagnosis results and exclude mistakes in diagnosing TB by tuberculin skin tests.

Earlier mycobacteriologists demonstrated that М. tuberculosis, М. bovis and M. avium could be isolated from both humans and animals. However, nobody studied the infection transmission chain or showed whether humans got TB from cattle or animals got mycobacteria from humans. The same uncertainty remains with epidemiologic studies of infections caused by M. avium in humans, pigs and poultry.

To trace channels of mycobacterial infection we should perform molecular-genetic typing of mycobacterial strains. We know from the publications (including our works) that polymorphism exists by several generic markers (IS6110, spacer sequences of DR-region – spoligotyping, VNTR, MIRU, etc.) of tuberculosis complex mycobacteria strains. Our (CTRI of RAMS, Moscow) investigations showed that M.tuberculosis strains (revealed from patients from different regions of Russia) with the same genetic characteristic prevalented (belonged to Beijing spoligotype, W-family and has 42435 VNTR type).

Investigation of polymorphism of M. avium strains has been started. However, similar researches into genotyping of M. bovis and M. avium strains isolated from cattle, pigs and birds were never performed in Russia.

So, the proposed research into genotyping of M. tuberculosis, M. bovis and M. avium isolated from maintenance staff, animals and poultry of the same large cattle farms in the Russian regions with adverse TB situation would allow to determine genetic polymorphism of mycobacteria strains and trace transmission ways.

The goal of the investigation: to establish a concept of development of epidemics and epizootics of bovine, human and bird’s TB based on molecular and epidemiologic studies in the cattle farms with adverse TB situation in Republic of Tatarstan.

To achieve the goal we plan to use the following methods:

We shall isolate mycobacteria from animals with positive tuberculin test and TB patients from adverse TB situation farms specialized in pigs, poultry and cattle breeding. The isolates will be identified by traditional methods. Drug susceptibility testing of M. tuberculosis, M. bovis and M. avium will be done.

Then we shall develop and test PCR test-systems with specific oligonucleotide probes, labeled with fluorescent markers for non-phoresis detection of PCR results. For this we shall use M. tuberculosis, M. bovis and M. avium strains isolated from the animals and patients and previously identified by traditional bacteriology methods.

The PCR test-system efficiency will be estimated after post slaughter diagnosis of pathological materials from cattle, pigs and poultry.

The mycobacterial strains will be genotyped by restriction fragment length polymorphism (RFLP) of insertion sequences (IS) typical for tuberculosis complex and avium-complex mycobacteria. In case of polymorphism restricted by IS-elements we shall use other methods of genetic typing based on PCR.

We shall analyze degree of genetic relationship of M. tuberculosis, M. bovis and M. avium strains isolated from patients and animals of the same farms.

Based on comparative results of epizootologic and epidemiologic studies and genotyping of mycobacteria within the same farms we shall conclude on TB infection transmission ways between humans and animals.


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