The thesis is devoted to chlamydia of farm animals and fish, in the thesis presents the results of the development of diagnostic tools for the identification and species differentiation of pathogens of animal Chlamydia, which caused by Chlamydia-like bacteria, using the PCR method based on 16S rRNA gene.
At the initial stage, the most significant species of bacteria of the order Chlamydiales, isolated from mammals and fish and not included in the family Chlamydiaceae, were identified: Parachlamydia acanthamoebae, Waddlia chondrophila, Candidatus Clavochlamydia salmonicola, Candidatus Piscichlamydia salmonis. The next stage was bioinformation studies. Of the 119,875 primary sequences belonging to 296 strains of 45 bacterial species of the Chlamydiales order presented in international databases, 2,227 nucleotide sequences of the 16S rRNA, 16S–23S rRNA, RNase P, MOMP genes were subjected to detailed analysis. As a result of gene analysis, the 16S rRNA gene was selected to search for hypervariable regions. The intraspecific alignment of the 16S rRNA gene showed single nucleotide substitutions, however, the level of intraspecific homology was 99%. By interspecific comparison of the 16S rRNA gene of phylogenetically close species of bacteria of the order of Chlamydiales, variable regions were found, on the basis of which the design of species-specific oligonucleotide primers was developed for each species (Waddlia chondrophila, Parachlamydia acanthamoebae, Candidatus Clavochlamydia salmonicola, Candidatus Piscichlamydia salmonis). In the process of the primer selection, we used the computer program MEGA 7 and the online service “primer-BLAST”. The primers were designed in such a way that they were able to adapt for real-time PCR. Checking for possible hybridization of the developed primers with the genomes of other organisms, both of conditionally pathogenic organism and pathogens of other diseases, was carried out using the BLAST online service.
The third stage was verification for analytical specificity that performed by amplification of the control DNA samples of nine species included in the Chlamydiales order and it showed the absence of PCR products except products of the expected size and only in case of presence of DNA of the corresponding species. As a result of restriction analysis of PCR products using AluI, TasI and Hin1II endonucleases (Thermo Fisher Scientific, USA), fragments of the expected length were formed, which confirmed the identity of the PCR products.
The optimal annealing temperature during amplification using reagents Thermo Fisher Scientific (USA) is a temperature of 60°C. DNA was isolated from clinical material using the “DNA express” reagent, LITECH. The control samples for checking and testing the developed oligonucleotide primers for the indication and species differentiation of Chlamydia-like bacteria were samples of the control DNA of Parachlamydia acanthamoebae, Waddlia chondrophila, Chlamydia avium, Chlamydia pecorum, Chlamydia abortus, Chlamydia psittaci, Chlamydia suis, Chlamydia caviae, Candidatus Clavochlamydia salmonicola and Candidatus Piscichlamydia salmonis obtained from European reference laboratories. The analytical sensitivity of the developed PCR ranged from 98.95–99.95%, the accuracy and specificity were close to 100%, the diagnostic efficiency was 99.3–99.8%, and the diagnostic value was 83.00–96.43%, due to optimal amplification conditions and the ratio of the reaction mixture in a certain interval of the method, which was ≥ 0.20–0.02 ng/μl. Epizootological screening, in the framework of clinical trials, was carried out on biological material taken from cattle and pigs in farms of the Poltava region and brown trout (Salmo trutta) samples taken from fisheries in the Lviv region. In total, about 300 samples from cattle, 28 samples from pigs, 80 samples of trout were studied. DNA of Chlamydia-like bacteria Waddlia chondrophila (44%) and Parachlamydia acanthamoebae (14.3%) were found in clinical samples of cattle. DNA of Chlamydia-like bacteria Candidatus Piscichlamydia salmonis (13.8%) and Candidatus Clavochlamydia salmonicola (2.5%) were found in samples taken from trout. DNA of Chlamydia-like bacterium Waddlia chondrophila (28.6%) was first detected in samples taken from pigs. In the studied material, the prevalence of Chlamydia-related organisms was higher than representatives of Chlamydia genus.
The data of experimental and clinical trials indicate the appropriateness of using the developed diagnostic methods for epizootological studies and species differentiation of Chlamydia pathogens.
