PhD Thesis defense by Coral Polo Vaquero at the VISAVET Centre of the Complutense University of Madrid

September 6^th, 2023

In Spain, extensive cattle farming is a sector characterized by its priority orientation towards beef production and whose main objective from a productive point of view is to maximize the number of calves per cow and year. Extensive exploitation systems may be able to use the natural resources of the environment, contributing to the maintenance of resilient ecosystems as long as they are properly managed. In 2020, Spain generated the 24 % of beef within the European Union (EU), being the third main beef producing country. However, the average annual fertility rates of this sector in Spain is lower compared to the rest of the EU (69% vs. 80%, respectively, in 2020), which has a negative impact on farm efficiency. Infectious infertility is especially relevant in extensive cattle, where the male plays an important role since a single male (or a very small group) can be used to breed several females.

The presence of pathogens that cause infertility in males and the venereal transmission of pathogens associated with infertility by males are two essential elements to understand the role of the bull in infectious infertility within the herd. The objective of this thesis is to improve knowledge about the role of the bull as a key element in infertility problems of infectious origin in extensive cattle herds, through the study of pathogens associated with infertility in the male, as well as the evaluation and/or development of new and traditional diagnostic tools, with special attention to those most commonly used, such as culture and PCR (polymerase chain reaction) technique, for the detection of Campylobacter fetus and Tritrichomonas foetus. These are two pathogens of recognized importance associated with bovine infertility, both in Spain and worldwide, for which there are no effective treatments and usually cause asymptomatic infections in bulls that can act as carriers, easily going unnoticed hindering their control.

In this context, a systematic review of the scientific literature was carried out in this Thesis through search strings in PubMed, Scopus, and Web of Science. 38 articles (out of a total of 2,224) were selected from 1966 to 2022, in which the presence of 27 different pathogens was related to reproductive problems in male and male-female herds. The most frequently detected pathogens were: bovine herpesvirus ( BoHV ) (identified by 26.3% of all selected works), C. fetus (23.7%), T. fetus (18.4%), and the virus bovine viral diarrhea (BVDV), Ureaplasma spp ., and Mycoplasma spp . (10.5% each), highlighting the absence of some pathogens with a recognized capacity to cause reproductive problems in cattle, such as Histophilus somni , Aspergillus spp . or Candida spp.

To complete the knowledge about microorganisms related to bull’s infertility, a metagenomic study was carried out on 1029 samples of foreskin washes from males within extensive farms in Spain (one sample per bull, where 944 came from animals from herds with low fertility rates and 85 from animals from reproductively healthy herds). The study was carried out by analyzing the hypervariable region V3-V4 of the gene that encodes the 16S of bacterial rRNA. The absence of bacterial populations potentially associated with infertility was revealed: i) alpha diversity of the Shannon (p-value = 0.745) and Simpson (p-value = 0.403) indices, and ii) beta diversity using the Bray-Curtis index which did not show group differentiation based on reproductive success. However, the individual analysis of the operational taxonomic units (Operational taxonomy Unit , OTU) showed the presence of one genus, Mycoplasma spp. significantly associated with infertility (p-value < 0.001). Likewise, C. fetus was identified in the 2.75 % of the samples, all of them from herds with low fertility. On the contrary, other microorganisms, such as Ureaplasma diversum, were detected in a high percentage of animals from both sample groups, making evident the need to complete the knowledge about the true role of opportunistic pathogens, as well as factors that trigger their potential pathogenic capacity. Lastly, this study highlighted the potential of new available methodological approaches, such as metagenomics, in the study of bovine infectious infertility for detection of microorganisms that may be going unnoticed in routine controls or whose role in infertility is unknown.

Regard to the improvement of diagnostic techniques for detection of microorganisms with a known effect on bovine fertility, this thesis focused on two pathogens, C. fetus and T. foetus. Regarding the diagnosis of C. fetus by PCR, a comparative study of protocols was carried out on ten molecular targets: i) the 16S rRNA genes, gyrB, cpn60, cstA, cdtB and nahE for the identification of the C. fetus species, and ii) the genes ISCfe1, sapB2, parA and virB11 for the differentiation of C. fetus subsp. venerealis (Cfv) and C. fetus subsp. fetus (Cff). A new real-time PCR technique based on a pyrosequencing protocol on the gyrB gene was proposed. This protocol improved the detection rates of C. fetus in preputial samples from bulls (being able to detect 95.1% of positive samples) compared to other previously published PCR protocols. The differentiation of C. fetus subspecies is important for the diagnosis of bovine genital campylobacteriosis (BGC), a notifiable disease caused by Cfv, however, our results demonstrated the difficulty for subspecies discrimination using the PCR techniques evaluated.

Regarding T. foetus (causing trichomonosis, a notifiable disease in Spain), the low number of genetic targets available for its detection by PCR was evidenced, where practically all of these protocols are based on the region that includes the 18S gene, 5.8S, and 28S RNAr and the internal transcription spacers 1 and 2 (rRNA-ITS). This region is highly homologous in the genus Tritrichomonas spp., which could lead cross-reactions with other species. Thus, five published protocols based on this target were selected to evaluate their diagnostic performance. So, the availability of alternative targets for T. foetus confirmation cases on clinical samples may be of great interest. However, only the following have been described: i) the TfRE microsatellite for which a single conventional PCR has been published (also evaluated in this comparative study) and ii) the gene that encodes beta-tubulin 1 that corresponds to a commercial PCR kit without published information. In this context, a new PCR technique based on the EF1-alpha-Tf1 gene was designed and evaluated in this Thesis. This protocol showed high concordance (Cohen`s kappa coefficient = 0.967) with the reference PCR [McMillen and Lew, 2016 (indicated by the WOAH as a valid option for the detection of T. foetus). Likewise, this new technique was able to detect 5.75 copies of the T. foetus genome without non-specific amplifications.

Finally, a culture protocol (reference technique for the diagnosis of BGC) for Cfv was established from clinical bulls samples through a comparative study of five transport media (Lander, Stuart, Weibridge, Thoman and PBS), three enrichment media (Preston, Brucella and Bolton) and three agar media (Blood agar, Preston and Skirrow) under different combinations of temperature (21±2 ºC and 4 ºC) and time (24 h and 48 h) for sample transport, and different plate culture strategies [filters use (0.45 µm and 0.65 µm pore diameter) and culture temperatures (37 ºC and 42 ºC)] with the aim of maximize Cfv recovery limiting the presence of other fast growing microorganisms that could compete with Cfv. The best results were obtained with Lander’s transport medium (where 5-fluorouracil was replaced by amphotericin B) stored up to 24 h at constant temperature of 21±2 ºC, in combination with Preston’s enrichment medium (cultured at 37 ºC for 48 h), and subsequently cultured in Preston or Skirrow agar media incubated at 37 ºC during 4 days in microaerophile atmosphere.

In conclusion, the results of this Thesis demonstrate the need for more studies about the bovine infectious infertility causes focused on the true role of the male, as well as the usefulness of new molecular techniques such as metagenomic analysis for the detection, study, and ultimately, control of microorganisms that may be going unnoticed through diagnostic tools. Similarly, in reference to the diagnostic techniques most commonly used for C. fetus and T. foetus detection (culture and PCR), the limitations of the current tools and the susceptibility of improving it in terms of diagnostic performance have been showed, proposing new protocols for this goal.