PCR-test for identification and species differentiation of Waddlia chondrophila


Keywords: Waddlia chondrophila, primers, chlamydia, zoonoses, PCR, Chlamydia-like organisms, Chlamydia-related bacteria

Abstract

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.

Downloads

Download data is not yet available.

References

Barkallah, M., Gharbi, Y., Hassena, A.B., Slima, A.B., Mallek, Z., Gautier, M., Greub, G., Gdoura, R., & Fen-dri, I. (2014). Survey of infectious etiologies of bovine abortion during mid- to late gestation in dairy herds. PloS One, 9, e91549. doi: 10.1371/journal.pone.0091549.

Baud, D., Goy, G., Osterheld, M.-C., Borel, N., Vial, Y., Pospischil, A., & Greub, G. (2011). Waddlia chon-drophila: from bovine abortion to human miscarriage. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am, 52(12), 1469–1471. doi: 10.1093/cid/cir205.

Blumer, S., Greub, G., Waldvogel, A., Hässig, M., Thoma, R., Tschuor, A., Pospischil, A., & Borel, N. (2011). Waddlia, Parachlamydia and Chlamydiaceae in bovine abortion. Vet. Microbiol, 152, 385–393. doi: 10.1016/j.vetmic.2011.05.024.

Borel, N., Ruhl, S., Casson, N., Kaiser, C., Pospischil, A., & Greub, G. (2007). Parachlamydia spp. and Related Chlamydia-like Organisms and Bovine Abortion. Emerg. Infect. Dis., 13(12), 1904–1907. doi: 10.3201/eid1312.070655.

Burnard, D., & Polkinghorne, A. (2016). Chlamydial infections in wildlife–conservation threats and/or res-ervoirs of ‘spill-over’ infections? Vet. Microbiol., 196, 78–84. doi: 10.1016/j.vetmic.2016.10.018.

Corsaro, D., Venditti, D., & Valassina, M. (2002). New parachlamydial 16S rDNA phylotypes detected in hu-man clinical samples. Res. Microbiol., 153(9), 563–567. https://www.ncbi.nlm.nih.gov/pubmed/12455703.

Dilbeck, P.M., Evermann, J.F., Crawford, T.B., Ward, A.C., Leathers, C.W., Holland, C.J., Mebus, C.A., Lo-gan, L.L., Rurangirwa, F.R., & McGuire, T.C. (1990). Isolation of a previously undescribed rickettsia from an aborted bovine fetus. J. Clin. Microbiol., 28(4), 814–816. https://www.ncbi.nlm.nih.gov/pubmed/2185269.

Dilbeck-Robertson, P., McAllister, M.M., Bradway, D., & Evermann, J.F. (2003). Results of a new serologic test suggest an association of Waddlia chondrophila with bovine abortion. J. Vet. Diagn. Investig. Off. Publ. Am. Assoc. Vet. Lab. Diagn. Inc, 15(6), 568–569. doi: 10.1177/104063870301500609.

Goy, G., Croxatto, A., Posfay-Barbe, K.M., Gervaix, A., & Greub, G. (2009). Development of a real-time PCR for the specific detection of Waddlia chondrophila in clinical samples. Eur. J. Clin. Microbiol. Infect. Dis. Off. Publ. Eur. Soc. Clin. Microbiol., 28(12), 1483–1486. doi: 10.1007/s10096-009-0804-7.

Greub, G., Boyadjiev, I., La Scola, B., Raoult, D., & Martin, C. (2003). Serological hint suggesting that Parachlamydiaceae are agents of pneumonia in polytraumatized intensive care patients. Ann. N. Y. Acad. Sci., 990, 311–319. https://www.ncbi.nlm.nih.gov/pubmed/12860644.

Haider, S., Collingro, A., Walochnik, J., Wagner, M., & Horn, M. (2008). Chlamydia-like bacteria in respirato-ry samples of community-acquired pneumonia pa-tients. FEMS Microbiol. Lett. 281, 198–202. doi: 10.1111/j.1574-6968.2008.01099.x.

Ks'onz, I.M. (2012). Hlamidiozy tvaryn: monografija, 1st ed. Orijana, Poltava (in Ukrainian).

Taylor-Brown, A., Vaughan, L., Greub, G., Timms, P., & Polkinghorne, A. (2015). Twenty years of research in-to Chlamydia-like organisms: a revolution in our un-derstanding of the biology and pathogenicity of mem-bers of the phylum Chlamydiae. Pathog. Dis., 73, 1–15. doi: 10.1093/femspd/ftu009.

Zezekalo, V.K., Peredera, S.B., Buslik, T.V., & Pochernyaev, K.F. (2018). PCR-test system specific identification Parachlamydia acanthamoebae. Scientific Messenger of Lviv National University of Veterinary Medicine and Biotechnologies, 20(92), 101–104. doi: 10.32718/nvlvet9220 (in Ukrainian).

Zezekalo, V.K., Peredera, S.B., & Shherbakova, N.S. (2019). Onovlennja taksonomichnoi' klasyfikacii' mikroorganizmiv porjadku Chlamydiales. Visnyk Pol-tavs'koi' derzhavnoi' agrarnoi' akademii', 1(24), 207–216 (in Ukrainian).

Abstract views: 0
PDF Downloads: 0
Published
2019-04-02
How to Cite
Zezekalo, V., Pedera, S., Buslik, T., & Pochernyaev, K. (2019). PCR-test for identification and species differentiation of Waddlia chondrophila. Scientific Messenger of LNU of Veterinary Medicine and Biotechnologies. Series: Veterinary Sciences, 21(93), 143-148. https://doi.org/10.32718/nvlvet9325