INTRODUCTION

Bovine genital campylobacteriosis (BGC) is an infectious disease affecting cattle. The Campylobacter genus is responsible for this disease. They are Gram-negative, spiral- shaped, motile bacteria. Currently, 32 Campylobacter species are recognized (^{Chukwu et al., 2019}) which Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv) affect the reproductive system of cattle (^{Chiapparrone et al., 2014}). The disease is considered a mandatory reportable disease, included in List B of terrestrial animal diseases according to the World Organization for Animal Health (WOAH), formerly OIE (^{Tshipamba et al., 2020}). In ruminants Cff has been shown that the pathogen affects the enteric system, especially the intestine (^{Li et al., 2022}), on the other hand, bulls with Cfv are reservoir because the bacteria live and adapt in preputial cysts, while in cows colonizations are in vagina, cervix and uterus ((^{Cagnoli et al., 2020} and ^{Lúcio et al., 2019}) inducing proinflammatory processes (^{Campos-Múzquez et al., 2021}) due to internalization in cells of the endometrial epithelium (^{Campos-Múzquez et al., 2019}) causing abortions (^{Clune et al., 2022}); C. fetus can also affect humans and has been reported to induce systemic infections (^{Adhikari et al., 2022}) endocarditis (^{Lynch et al., 2022}) and meningitis (^{Fernández et al., 2022}). Bacteriological isolation and the use of biochemical tests play an important role for the identification of C. fetus and differentiation between Cff and Cfv, as they are considered the gold standard, however, the implementation of these culture techniques is difficult, as Campylobacter requires more demanding in vitro culture conditions than other bacterial genera, in addition, the culture is poor in reliability and reproducibility (^{Iraola et al., 2016}). Different methods can be used for Cfv identification, such as antigen-antibody reaction as Enzyme-Linked Immunoassays (ELISA), Direct Immunofluorescence (DI) (^{Dorsch et al., 2022}) and molecular methods as Pulsed Field Gel Electrophoresis (PFGE), Amplified Fragment Length Polymorphism (AFLP), multilocus sequence typing and Polymerase Chain Reaction (PCR). Currently PCR is the test with the most promising alternative to efficiently detect Cfv from field samples (^{Chaban et al., 2012}). The objective of this study was to detect the presence of C. fetus by PCR in smegma from bulls used as breeders in the central zone of Tamaulipas state.

MATERIAL AND METHODS

RESULTS

Thirty-eight bulls were sampled; one sample was obtained from each; 31 from preputial lavage and 7 from semen. 18.4% were positive samples (7/38) (Figure 1), of these, two were from semen and five from preputial lavage. Of the 7 positive samples, 5 PCR products were subjected to sequencing. All sequences were compared considering the bacterium with accession number JF901335.1 in Genbank.

Figure 1 2.5% agarose gel with the PCR products. Lane M: 100 mpb (BIOLINE, USA); lanes 1-9: Cfv positive; lane 8: negative control; lane 9: positive control Cff 764 bp and Cfv 233 bp 

The nucleotide sequences of the amplicons obtained from field samples by PCR were identical to those found in the GenBank database. The analysis showed between 99.17 and 97.87% identity with other Cfv (Fig. 2). These results demonstrated that Cfv is present in herds in central Tamaulipas, and that Cfv plays an important role in the reproductive health of cattle.

Figure 2 Alignment of Cfv sequences in bull samples from central Tamaulipas. EMBL-EBI Clustal Omega software was used 

DISCUSSION

It is documented that BGC affects cattle herds; pathology caused by Campylobacter fetus (C. fetus) subspecies venerealis (Cfv). BGC is one of the main causes of infertility and abortion in cattle, sheep and goats; and its presence has been confirmed in several countries of the American continent such as Argentina, Brazil, Costa Rica, Jamaica and Mexico. However, in Mexico, this pathogen has been little studied in livestock areas. The detection of this pathogenic subspecies in bulls used as sires is of utmost importance for the control of this disease in herds, since they are asymptomatic carriers; and, in addition to being used for direct mating, semen is frequently used for artificial insemination, both fresh and in straws preserved in liquid nitrogen. In addition to infertility problems, Cff is considered the main cause of sperm alterations due to the ability of these bacteria to adhere to the spermatozoa causing damage to the acrosomal membrane as well as to the sperm chromatin, which decreases semen quality and consequently the pregnancy percentage, besides influencing infertility in females due to the transmission of bacteria to the genital tract during natural mating (^{Cagnoli et al., 2020}).

The diagnosis of this disease can be performed by various methods such as bacteriological isolation, serological methods such as ELISA and DI, and molecular assays such as PCR (^{Mshelia et al., 2010}; ^{Clune et al., 2022}). Bacteriological isolation is the least sensitive method, as demonstrated by ^{Groff et al., en 2010}, where they compared this method with PCR, resulting in this molecular method being 8.5 times more sensitive than traditional bacteriological isolation. In this study, PCR was used following the recommendations of other researchers; for the identification of Cfv with the cstA gene and for Cff with the virB11 gene (^{Hum et al., 1997}; ^{Iraola et al., 2012}), given that the primers generated for the identification of these genes can detect specific DNA sequences of Cfv and Cff; with the PCR developed allowed positive cases of CGB to be detected in the study area. Another method that has been used is DI; however, ^{Ferreira en 2002} shows that the technique does not distinguish C. fetus subspecies.

In Mexico, beef cattle have been exported to the United States of America since 1994, and the state of Tamaulipas has been characterized for having one of the largest inventories in the country. In the 2020-2021 cycle, the export of live cattle was 30,896 head to the United States according to data from the Secretariat of Agriculture and Rural Development (SADER). Considering the need to increase the number of animals to supply the demand, it is necessary to have an animal health diagnosis of diseases that affect reproductive parameters. The success of the diagnosis depends largely on sampling; based on previous reports reported by ^{Silveira et al., 2018} and ^{Delpiazzo et al., 2021}.

The prevalence of BGC is variable in different areas of the world, and there are several factors that favor its transmission (^{Hoque et al., 2021}). In Mexico, little has been reported on the presence of Campylobacter in cattle; the only study found in our country was conducted by ^{Barajas en 2013} in tropical areas of Mexico; by means of ELISA test, it revealed a seroprevalence of 21.3% in cattle. The detection method was different and the sampling was lower; the frequency detected in this study was 18.4%, indicating that BGC is present in Tamaulipas cattle herds. It is worth mentioning that Barajas' work was carried out with cattle from the humid tropics of the extension program for cattle ranchers in northern Veracruz, a state adjacent to Tamaulipas and with climatic conditions similar to the study area.

The objective of this study was to detect the presence of C. fetus in bulls from the central zone of Tamaulipas state. From the 7 amplifications suggestive of Cfv by PCR, 5 were evaluated by sequencing. The results obtained in the bioinformatic analysis confirm that the amplification corresponds to a region of the virB11 gene which is a sequence of pathogenicity islands present only in Cfv with 233 bp and absent in Cff, which allows differentiating these two subspecies by molecular method. The results obtained in the present work are consistent with what was previously published by ^{Iraola et al., 2016}, so the epidemiological situation could be at risk due to the spread of this pathogen by the use of carrier bulls for reproductive purposes.

CONCLUSION

In this study, the presence of Campylobacter fetus subsp. venerealis was detected by PCR and corroborated by sequencing with a frequency of 18.4% of the bulls analyzed from the central zone of Tamaulipas state, which represents the first report in Mexico of this bacterium by molecular methods.

ACKNOWLEDGMENTS

To ZVD. Francisco Trejo Meza from The Tamaulipas Regional Livestock Association's Genetic Improvement Center for allowing the sampling of milking bulls.