Bovine genital campylobacteriosis: isolation, identification and virulence profiling of Campylobacter fetus subsp. venerealis in a small animal model

Abstract

Bovine Genital Campylobacteriosis is a significant disease in Australian beef and dairy herds. The causative organism, Campylobacter fetus subsp. venerealis is a microaerophilic bacterium that often resides asymptomatically in bull penile sheaths and is passed on to females during mating. Virulent strains of the bacterium can cause subfertility and late term abortions in infected females. It has the potential to cause large economic losses and is often difficult to diagnose by culture due to the slow-growing, fastidious bacterium becoming overgrown by contaminants. The gold standard for detection is culture and biochemical testing for identification.Isolation and identification methodologies for C. fetus subsp. venerealis have not been reviewed for many years partly due to the lack of an Australian culture collection. Determining the variation in transport media viability, culture phenotyping, addition of newer phenotypic tests or the evaluation of strain virulence relies on the availability of cultures for analysis. Therefore, the objective of this study was to use a shared culture collection to identify best practice for transport media and isolation methodologies, define the phenotypic profile through refined, standardised methods and develop a pregnant guinea pig model to facilitate future studies of the pathogenicity, abortion mechanism and vaccine efficacy of C. fetus subsp. venerealis.This study biochemically characterised 152 Campylobacter-like isolates to determine the breadth of variability, and to test innovative diagnostic tools for phenotypic characterisations. Strains of C. fetus subsp. venerealis were used to determine viability in the commonly recommended Lander's transport and enrichment medium (TEM) as well as the recently described Thomann transport and enrichment (TTE) medium in vitro and in vivo. The in vitro experiment also aimed to determine the efficacy of the two transport media with subsequent sub-culture onto: sheep blood agar (SBA) with and without filtration (0.45 µm) and Campylobacter selective agar. Incubation temperature effect was also studied, by comparing TEM enrichment at 25°C and 37°C. The in vivo cattle field trial found no significant difference in the isolation of C. fetus subsp. venerealis between Lander's and TTE (p=0.1037), while a higher number of thermophilic Campylobacter cultures were isolated from TTE media. In vitro, both TTE and Lander's media were able to sustain C. fetus subsp. venerealis growth for up to 7 days at either 25°C or 37°C, and allow enrichment in spiked mixed cultures with either P. vulgaris or C. jejuni. Campylobacter selective agar appeared to produce the most consistent positive results from spiked mixed cultures, while SBA was overgrown in most instances and 0.45 µm filtration produced inconsistent results and required a high starting concentration (10 5 CFU/ml). Therefore, diagnostically the use of either TTE or Lander's TEM for transport of samples iii followed by plating onto Campylobacter selective agar was optimal for isolation of ...

Time, temperature and media: the three keys to improve the recovery of Campylobacter fetus subsp. venerealis from preputial bull samples.

“…Cfv is a fastidious bacteria (WHOA, 2021); for this reason, we limited the use of Digralsky loop to 5 (Hedderich et al, 2011). At this point, it is important to indicate that the bacterial count was relatively variable between duplicates as previously reported (Koya, 2016).…”

“…From sampling to laboratory (experiment 1): evaluation of transport media, temperature and time of conservation for C. fetus A total of four transport media were compared (Table 1): i) Weybridge (De Lisled, 1982), where the 5uorouracil and cycloheximide were replaced by amphotericin B as previously suggested (Koya, 2016;Martin et al, 2002;Murinda et al, 2006), ii) Lander (Lander, 1990b), where the 5-uoruracil was replaced by amphotericin B as previously suggested (Koya, 2016;Martin et al, 2002;Murinda et al, 2006), iii) Thomann (Harwood et al, 2009), and iv) commercial Stuart transport medium modi ed (Thermo Fisher Scienti c, UK). Phosphate buffered saline (PBS) was used as a control media to assess the viability of Cfv without other compounds as antibiotics and nutrients.…”

“…limited(Chaban et al, 2013;Koya, 2016;Monke et al, 2002), what can impact the probability of isolation of the pathogen from clinical samples decreasing the diagnosis e ciency of BGC through culture.Different strategies have been developed to overcome these drawbacks(Chaban et al, 2013;Hum et al, 1994; Lander, 1990a, b;Monke et al, 2002). However, to our knowledge, a standardized protocol to maximize the recovery of Cfv from preputial bull samples has not been established, thus limiting the reliability of BCG culture-based diagnosis.…”

Foodborne Campylobacter species: Taxonomy, Isolation, Virulence Attributes and Antimicrobial Resistance

“…Diarrhea lasting 5-15 days is the most common clinical sign in dogs <6 months old. Occasionally, the diarrhea becomes chronic and may be accompanied by fever and increased the white blood cells` counts [68].…”

Bovine Campylobacteriosis and its Economic Importance: A Review

“…Bovine genital Campylobacteriosis are often difficult to diagnose by culture due to the slow-growing, fastidious bacterium becoming overgrown by contaminants. The gold standard for detection is culture and biochemical testing for identification [6]. The most common diagnostic methods are culture, direct fluorescent antibody test (DFA T), ELISA, PCR.…”