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

The isolation of Campylobacter fetus subsp. venerealis (Cfv) from clinical samples is the gold standard for confirming cases of bovine genital campylobacteriosis, an important cause of infertility in cattle and a potential public health concern. Furthermore, isolation is also necessary for the development of autologous vaccines, characterization of strains for antimicrobial susceptibility patterns, etc. Nevertheless, the sensitivity of culture methods is usually low, and there is no standardized protocol to maximize the recovery of Cfv from clinical samples. The aim of the current study is to design a protocol for the culture of Cfv from preputial samples by evaluating the combination of different transport, enrichment and culture media considering the impact of certain factors (time between collection and enrichment, temperature, and use of filters). The use of modified Lander's transport medium and storing the sample for 24 h at 21 ± 2 °C led to the highest recovery of Cfv CFUs. In contrast, the storage of the samples during 24-48 h in PBS and Thomann rarely allowed the recovery of Cfv regardless of the temperature. The enrichment medium yielding the best results was Preston (significantly higher recovery than Brucella medium), while Cfv could not be isolated with Bolton. Regarding our diagnostic assay (using Lander as transport medium and Preston as enrichment medium), the best protocol in terms of maximizing Cfv recovery as well as limiting contaminations is to culture the samples in i) solid media Preston or Skirrow, and ii) using 0.65 µm filters and incubating plates at 37 °C in microaerophilic conditions.

What about the bull? A systematic review about the role of males in bovine infectious infertility within cattle herds.

Numerous pathogens affect cow fertility. Nevertheless, little information has been published about microorganisms associated with cattle infertility focusing on bulls. The present review offers a current analysis and highlights potential key aspects on the relevance of bulls in the emergence of infertility problems of infectious origin within herds that are still not completely determined. The present systematic review was conducted using the PubMed, Web of Science, and Scopus databases on December 9, 2022. In total, 2,224 bibliographic records were reviewed and, according to strict inclusion criteria, 38 articles were selected from 1966 to 2022, from which we ranked more than 27 different microorganisms (fungi were not identified). The most cited pathogens were BoHV (described by 26.3% of the papers), Campylobacter fetus (23.7%), Tritrichomonas foetus (18.4%), and BVDV, Ureaplasma spp., and Mycoplasma spp. (10.5% each). Despite the general trend towards an increasing number of publications about bull-infertility problems, a number of pathogens potentially transmitted through both natural breeding and seminal doses given to females and associated with infertility within herds were not ranked in the study (e.g., Chlamydia spp.). This work highlights i) the need to clearly establish the role of certain microorganisms not traditionally associated with reproductive problems in bull infertility (e.g., Staphylococcus spp. or BoHV-4) and ii) the need to perform additional studies on breeding bulls to clarify their role in infertility problems within herds. This would allow monitoring for pathogens that have gone unnoticed and those that are fastidious to diagnose and/or potentially transmitted to females.

Exploiting 16S rRNA-based metagenomics to reveal neglected microorganisms associated with infertility in breeding bulls in Spanish extensive herds.

Bovine infectious infertility represents a problem due to the high impact on animal production and, in many cases, in public health. A lack of information on the characteristics of the bacterial population of the bovine reproductive system can hamper a comprehensive understanding of reproductive pathologies and the role that the microbiome could play. A metagenomic study based on the V3-V4 hypervariable region of the bacterial 16S rRNA gene was performed in 1029 preputial samples from bulls raised in an extensive regimen in Spain (944 from herds with low fertility rates -case group-, and 85 samples from reproductively healthy herds -control group-). The most representative phyla as well as the most 10 abundant bacterial families and their abundance did not show significant differences in both case and control groups. Similarly, the (alpha and beta) diversity of the bacterial populations was similar in both type of herds: the Shannon and Simpson indices show a high diversity of species, while the Bray-Curtis dissimilarity index did not show relevant differences in the bacterial communities. A deeper analysis of the operational taxonomic units showed the presence of one genera, Mycoplasma spp. significantly associated with fertility problems. Our study highlights the promising potential that the application of sequencing techniques (e.g. 16S rRNA-based metagenomics) possesses in examining bovine infertility, as they are able to reveal different pathogens that could go unnoticed using diagnostic approaches for only the main known pathogens.

Molecular detection of Tritrichomonas foetus in bovine samples: a novel real-time polymerase chain reaction (PCR) assay targeting EF1-alpha-Tf1 and a comparative study of published PCR techniques.

The parasite T. foetus causes trichomonosis in cattle but is generally asymptomatic in males. Thus, many bulls carrying the disease go unnoticed, making the detection of T. foetus in bulls an important aspect for its control. Due to drawbacks posed by its cultivation, PCR is a preferred option for diagnostic laboratories. Most published PCR protocols target the genomic region compring the 18S, 5.8S, and 28S rRNA genes and internal transcribed spacers 1 and 2 (rRNA-ITS region), homologous to that of other Tritrichomonas species. There is minimal information on alternative genetic targets and no comparative studies have been published. We compared a protocol based on the microsatellite TfRE (called H94) and five protocols based on the rRNA-ITS region (called M06, M15, G02, G05, and N02). We also designed and evaluated a novel PCR-based assay on the EF1-alpha-Tf1 gene (called V21). The analytical sensitivity and specificity assays for the PCR protocols were performed according to the World Organisation for Animal Health (OIE) directives and the comparative study was performed with a widely used PCR (M06) on clinical samples from 466 breeding bulls. V21 showed a high degree of agreement with our reference M06 (kappa = 0.967), as well as M15 (kappa = 0.958), G05 (kappa = 0.948), and H94 (kappa = 0.986). Protocols H94 and V21 appear to be good approaches for confirming clinical cases in preputial bull samples when genomic regions alternative to rRNA-ITS are required. By contrast, N02 gave false negatives and G02 false positives.

Evaluation of PCR assays for Campylobacter fetus detection and discrimination between C. fetus subspecies in bovine preputial wash samples.

Campylobacter fetus is a zoonotic pathogen found in cattle, in which it is one of the main causes of infectious infertility. Most diagnostic laboratories use PCR as quick easy tool for C. fetus identification. However, there is no standardized PCR assay for C. fetus detection and subspecies differentiation, hindering the comparison of results. In this study, we evaluated selected PCR assays targeting the 16S rRNA, gyrB, cpn60, cstA, cdtB and nahE genes for C. fetus identification and ISCfe1, sapB2, parA and virB11 for subspecies differentiation. Analytical sensitivity and specificity were assessed for each PCR assay, and the assays were then tested on 289 bull preputial samples that had also been analysed by 16S rRNA barcode metagenomics. In total, 41 C. fetus-positive samples were included. The P12 PCR assay targeting the gyrB gene performed best, detecting the pathogen in 95.1% of positive samples. For the discrimination of C. fetus subspecies, we were able to identify a proportion (85.4%) of the C. fetus-positive samples correctly as C. fetus venerealis with at least one subspecies-specific PCR, but C. fetus fetus was not detected in any of the samples tested. Remarkably, C. fetus subspecies amplification was observed following PCR on some samples (33.1%) considered C. fetus-negative, highlighting the need for rigorous criteria for discriminating between C. fetus subspecies, to improve understanding of the role of the two C. fetus subspecies in the epidemiology and pathogenesis of bovine infectious infertility.

Liver Transudate, a Potential Alternative to Detect Anti-Hepatitis E Virus Antibodies in Pigs and Wild Boars (Sus scrofa).

In recent years, cases of hepatitis E virus (HEV) infection have increased in Europe in association with the consumption of contaminated food, mainly from pork products but also from wild boars. The animal's serum is usually tested for the presence of anti-HEV antibodies and viral RNA but, in many cases such as during hunting, an adequate serum sample cannot be obtained. In the present study, liver transudate was evaluated as an alternative matrix to serum for HEV detection. A total of 125 sera and liver transudates were tested by enzyme-linked immunosorbent assay at different dilutions (1:2, 1:10, 1:20), while 58 samples of serum and liver transudate were checked for the presence of HEV RNA by RT-qPCR. Anti- HEV antibodies were detected by ELISA in 68.0% of the serum samples, and in 61.6% of the undiluted transudate, and in 70.4%, 56.8%, and 44.8% of 1:2, 1:10, or 1:20 diluted transudate, respectively. The best results were obtained for the liver transudate at 1:10 dilution, based on the Kappa statistic (0.630) and intraclass correlation coefficient (0.841). HEV RNA was detected by RT-qPCR in 22.4% of the serum samples and 6.9% of the transudate samples, all samples used for RT-qPCR were positive by ELISA. Our results indicate that liver transudate may be an alternative matrix to serum for the detection of anti-HEV antibodies.