Bovine campylobacteriosis in bulls: insights in the conventional and molecular diagnosis.

Campylobacter fetus is a gram-negative motile bacterium, with two subspecies relevant to cattle health: C. fetus subsp. venerealis (Cfv) and C. fetus subsp. fetus (Cff). Both subspecies are associated with reproductive losses in cattle. In this study, we evaluated the identification of C. fetus for the diagnosis of bovine campylobacteriosis through bacteriological culture, direct immunofluorescence (DIF) and molecular tests in preputial smegma (PS) samples of three Angus bulls challenged with Cfv, Cfv biovar intermedius (Cfvi) or Cff, respectively, in an experiment imitating the natural infection. Two DNA extraction protocols were tested (in-house thermal extraction and commercial kit). Aspiration and scraping collection for PS were compared by conventional tests. Additionally, bacteremia was also evaluated in blood samples. Bulls were challenged by natural mating with heifers that had been experimentally infected with C. fetus subspecies; which led to infection. The Cfv- and Cfvi-bulls were positive for at least 9 months. Although Cff is not considered a venereal strain, in this study it was transmissible to bull from heifers experimentally infected, as evidenced by its colonization and persistence in the preputial cavity for 5 to 6 months. This finding suggests a potential risk of dissemination within herds. The results obtained by bacteriological culture or direct immunofluorescence (DIF) showed no significant differences, regardless the sampling device used (aspiration with Cassou pipette, metal and plastic scraper). C. fetus qPCR, on the other hand, yielded better results with an in-house DNA extraction method than with a commercial kit (75% vs 66.6%). Furthermore, qPCR diagnosis was more efficient than culture (66.6%) or DIF (56%). Bacteremia in whole blood samples was negative by qPCR and bacteriological culture in all samples. Altogether, this study demonstrated the transmission of Cff from heifers to bull and also showed that PCR-based methods are promising for the diagnosis of Bovine Genital Campylobacteriosis from clinical samples of PS.

Isolation of Campylobacter fetus subsp. venerealis from seminal vesicle of a naturally challenged bull.

Campylobacter fetus is a well-recognized pathogen that affects reproductive rate in cattle. In the present study, two Angus bulls were kept (39 days) separately with a group of heifers experimentally infected with Campylobacter fetus subsp. venerealis (Cfv) and Campylobacter fetus subsp. venerealis biovar intermedius (Cfvi), respectively. Each bull resulted infected post-mating by its respective strain (Cfv and Cfvi). Semen samples collected from each bull at days 39, 82, 132 and 269 resulted positive for C. fetus by bacteriological culture and/or direct immunofluorescence (DIF) test, and confirmed by polymerase chain reaction (PCR) from colonies isolated. Diagnosis resulted better with bacteriological culture (100%) compared to DIF (37,5%). Campylobacter fetus was isolated from seminal vesicle and preputial mucosa by bacteriological culture and confirmed by PCR and DIF test from colonies previously isolated from these tissues (day 276). Microscopic lesions detected in both bulls showed moderate diffuse subepithelial lymphoplasmacytic postitis. None of the seminal vesicle presented relevant microscopic lesions. To our knowledge this is the first report of isolation of C. fetus from seminal vesicles in a bull. The experimental model herein described, mimicks the natural infection and constitutes a promising alternative for future studies of campylobacteriosis in cattle.