Molecular diagnosis of bovine genital campylobacterosis: comparative study of three PCR techniques
Degree Final Project defended by Natalia Lopez García
September 15th, 2021
Campylobacter fetus is the causative agent of bovine genital campylobacteriosis (BGC), a venereal disease that causes infertility in cattle. It is also a zoonotic pathogen. Traditionally, culture and identification through biochemical tests are considered the “gold standard” technique to identify C. fetus. The main drawback of this methodology is its low sensitivity since C. fetus has very demanding growth conditions. In addition, biochemical tests for identifying both species and subspecies are scarce and frequently throw ambiguous results.
There are different molecular techniques described in the scientific literature, such as the polymerase chain reaction (PCR), which allows the identification of C. fetus. However, currently, there is no consensus to determine which is the most appropriate for diagnosing bovine genital campylobacteriosis. The primary aim of this work is to evaluate and compare three of these PCRs, whose genetic targets are the gyrB (Persson et al., 2012), cpn60 (Chaban et al., 2009), and cstA (Hum et al., 1997) genes, to establish which of them is the best diagnostic tool for the detection of CGB. An initial set-up of the PCR techniques was carried out, then analytical sensitivity and specificity were assessed, and finally, analyses of 100 previously selected samples from preputial bull washes were carried out to evaluate and compare their diagnostic efficiency. The PCR based on the gyrB gene (Persson et al., 2012) performed best, reaching a detection limit for C. fetus of 10 fg (equivalent to five copies of the genome), and it was also the technique that detected a higher number of positive samples in the analysis of preputial bull washes (8/100). Both the PCR described by Chaban et al. (2009) and Hum et al. (1997) obtained a DNA detection limit of 100 fg (equivalent to fifty copies of the genome) and detected the same positive samples from preputial washes (3/100).
The technique described by Persson et al. (2012) is the one that has presented the best results in this study. Considering the advantages that PCR can have over culture, it is necessary to carry out more studies to validate a molecular technique that represents a valid alternative and to be able to perform comparative studies based on a standard methodology.
