The bull is the main target for diagnosis and management of BGC. However, there are still difficulties to establish effective preventive and control strategies (Silveira et al. 2018).
As previously reported (Garcia et al 2021a), an experimental model mimicking natural infection can be exploited to study different diagnostic approaches.
The bulls infected with the venereal subspecies (Cfv y Cfvi) were positive, according to the diagnostic tests used, for at least 9 months. This result reinforces its known carrier status between breeding seasons (Campero et al. 2017; Silveira et al. 2018). Regarding the bull exposed to Cff contact, the infection was persistent for at least 5 months and allowed passive or active mechanical transmission by traditional (3 months) or continuous breeding systems. Importantly, this is also a risk for cattle reproductive health.
Although researchers have previously isolated Cff from preputial samples of experimentally and naturally infected bulls (Botelho et al. 2014; Marcellino et al. 2015), it is still unclear if this bacterium is a venereal pathogen in cattle. However, Cff is associated with infertility and induces abortion (Mshelia et al. 2010; Balzan et al. 2020; García et al. 2021b). These pieces of evidence suggest that bulls could have a significant role in the dissemination and maintenance of Cff within herds.
The results from our study demonstrated the transmission from heifers to bulls and the bacterial infection was characterized by colonization and persistence in the preputial cavity. Although this study includes the assessment of only one strain of Cff, our findings show that this subspecies should be considered in BGC diagnosis of bulls because of the potential risk in disseminating the bacteria among heifers or cows. In this line, differential diagnosis could not be mandatory.
The lower persistence of Cff could be due to its low adaptation in the prepuce, probably because of the lack of surface proteins, genomic islands and mobile elements that predominate in Cfv and enhance long term survival in the urogenital tract (Gorkiewicz et al. 2010; Sprenger et al. 2017; Nadin-Davis et al. 2021). Particularly, the variant Cfvi had a higher rate of recuperation from PS than others variants. This could be associated to different genetic profiles regarding genes affecting colonization and attachment capacity (Nadin-Davis et al. 2021).
Bacteriological culture showed higher efficiency detecting the venereal strains (Cfv and Cfvi), with higher bacteriological recuperation of Cfvi. The detection through culture was more efficient in our study in relation to other reports (63–67% vs. 33–38%) (McMillen et al. 2006; Guerra et al. 2014).
Regarding the DIF test, there were significant differences between Cfvi in relation to Cff and Cfv. This may be explained by a higher concentration of the bacterium in the prepuce and may be associated with a more virulent strain. In effect, the higher detection rates of Cfvi through all the diagnostic tests reinforce the idea of a higher bacterium concentration.
Previous studies have reported detection variations, from 69.4–92.6%, with DIF test for Cfv (Figueiredo et al. 2002; Campero et al. 2017) and these percentage values are coincident with that of the present research (72%). In our study, the DIF detection rate decreased when Cff was included in the analysis (63%). The detection of this subspecies has not been previously assessed through this technique and thus we cannot make any comparison. Even though the detection rate of DIF is moderate (56–67%), this technique is still widely used because of its low cost, simplicity and fast processing (Campero et al. 2017; Silveira et al. 2018; Balzan et al. 2020).
No significant differences were detected between culture and DIF. This is in contrast with studies that showed significantly better results by bacteriological culture, which were associated with a low bacterial concentration or inadequate sampling (Marcellino et al. 2015). The moderate concordance between bacteriological culture and DIF opens the debate around the use of both tests in herds with endemic BGC, in order to reduce false negative bulls.
In accordance with this study, some researchers have suggested that the detection efficiency of PS collection by DIF or bacteriological culture seems to be independent of the devices used (Soto and di Rocco 1982; Ramos et al. 1986), while others have described better results with scraping devices (Tedesco et al. 1977; McMillen et al. 2006). According to McMillen et al. (2006), the scraping method would increase the possibility of isolating C. fetus at low concentrations within the preputial crypts, since friction against the mucosa generates detachment of epithelial cells along with the bacteria. As mentioned before, in the present study the results were independent of the device used. Thus, according to our results and those of some other researchers, the use of the device should be based on which is most practical for the veterinarian depending on the length of the preputial cavity. More studies should be made to resolve these discrepancies and found the most efficient device for this purpose.
Our study showed better results for the detection of C. fetus by qPCR (66.6%-75%) than through conventional diagnostic tests (culture and DIF; 66.6% and 56%, respectively). This is in accordance with previous studies (McMillen et al. 2006; Guerra et al. 2014; Del Piazzo et al. 2021; Polo et al. 2021; Mederos et al. 2022). Variations in the molecular technique associated with genotyping failures, which may be due to different local strain variants, cross-reactions, and contaminants inhibiting the reaction (such as blood, urine, or pus) may limit the effectiveness of PCR, mainly in clinical samples (Willoughby et al. 2005; McMillen et al. 2006; Schulze et al. 2006; Spence et al. 2011; Polo et al. 2021). The inclusion of a TaqMan probe-based qPCR in the study was due to its high sensitivity (Iraola et al. 2016). Culture fastidious growth and lower capacity to process large volumes of samples, as well as the lower sensitivity of DIF, makes qPCR an adequate and promising diagnostic tool for C. fetus detection. The In-house DNA extraction method resulted in higher C. fetus detection. Indeed, its lower cost and fast processing makes this method a practical option.
The detection of subspecies of these bacteria continues to cause problems. This is due to the use of sequences or genomic island present both in Cff and Cfv, or even in other species, for PCR assays designed to discriminate between C. fetus subspecies. The use of these common sequences, needless to say, leads to false positives (Moolhuijzen et al. 2009; Abril et al. 2010; Spence et al. 2011; Silva et al. 2020; Polo et al. 2021).
More studies are needed to know the implication of Cff in the reproductive performance in cattle. However, given the difficulties in subspeciation and its association with abortion, we suggest that all three subspecies should be diagnosed as etiological agents of BGC.
The differences on detection between samplings for either diagnostic test could be associated with variations of local immunity and bacterial concentration in the bulls, as well as strain differences (virulence factors) (Van Bergen et al. 2005; Mshelia et al. 2007). Because of these dissimilar factors and other variations, such as sampling and diagnostic test effectiveness, two or more scrapings are needed (Guerra et al. 2014; Del Piazzo et al. 2021; Mederos et al. 2022). Similarly, in our study concordance between tests varied from weak to strong, leading to false negative bulls.
Campylobacter spp., mainly Cff, is associated with bacteremia and extraintestinal infections in humans (Monno et al. 2004) and ruminants (Sahin et al. 2017). Here we were unable to detect any C. fetus in blood. This suggests different tropism associated with dissimilar C. fetus pathogenesis in bulls and humans, as suggested for Cff, which causes abortion in cows, ewes, alpacas, among others (Fenwick et al. 2000; Bidewell et al. 2016; Campero et al. 2017; Sahin et al. 2017).