PCR-test for identification and species differentiation of Waddlia chondrophila

In recent times, the number of species of chlamydia associated with a number of inflammatory diseases in animals has expanded considerably. Moreover, in addition to chlamydia, attention was attracted by Сhlamydia-like organisms, which are not only dangerous for animals, but also carry a zoonotic threat. Currently, the most studied Chlamydia-like organisms are Waddlia chondrophila and Parachlamydia acanthamoebae. They are associated with diseases of the reproductive and respiratory systems in cattle and humans. Considering the danger to animals and the zoonotic threat of chlamydia-like organisms along with the absence of tools to discover them in Ukraine, the aim of our work was to develop a PCR test for the identification and species differentiation of Waddlia chondrophila. Conservative 16S rRNA genes were chosen as target genes for developing a PCR test system for identifying Waddlia chondrophila. Primers were selected specifically to be able to create multiplex combinations with the previously developed PCR test for Parachlamydia acanthamoebae. Both primers were designed with the same physical characteristics to provide simultaneous amplification under the same conditions in single or multiplex PCR. For the specificity evaluation of the primers, a panel of following DNA samples was used: Waddlia chondrophila, Parachlamydia acanthamoebae, Chlamydia avium, Chlamydia pecorum, Chlamydia abortus, Chlamydia psittaci, Chlamydia suis, Chlamydia caviae, Clavochlamydia salmonicola, Piscichlamydia salmonis. PCR product of 88 base pairs (b.p.) was formed during amplification only when the Waddlia chondrophila control DNA was present in the sample, as was expected. The small size of the PCR product theoretically allows the use of this pair of oligonucleotide primers for real-time PCR tests. After testing on clinical samples, developed PCR test system for identifying and species differentiation of Waddlia chondrophila can be used by scientists for extensive monitoring, by veterinary medicine doctors to clarify the diagnosis, and might be introduced into the practice of laboratories of veterinary and humane medicine.