Bovine Reproductive Tract Research Articles

Bovine reproductive tract and microbiome dynamics: current knowledge, challenges, and its potential to enhance fertility in dairy cows

The cattle production system focuses on maintaining an animal-based food supply with a lower number of cattle. However, the fecundity of dairy cows has declined worldwide. The reproductive tract microbiome is one of the important factors which can influence bovine fecundity. Therefore, reproductive tract microbiomes have been explored during the estrus cycle, artificial insemination, gestation, and postpartum to establish a link between the micro-communities and reproductive performance. These investigations suggested that microbial dysbiosis in the reproductive tract may be associated with declined fertility. However, there is a scarcity of comprehensive investigations to understand microbial diversity, abundance, shift, and host-microbiome interplay for bovine infertility cases such as repeat breeding syndrome (RBS). This review summarizes the occurrence and persistence of microbial taxa to gain a better understanding of reproductive performance and its implications. Further, we also discuss the possibilities of microbiome manipulation strategies to enhance bovine fecundity.

Structural and network analysis of ADAM9 and ADAM10 and its transcript expression in the male reproductive tract of Bos Taurus

A disintegrin and metalloproteinases (ADAMs) are a family of proteins expressed as transmembrane and secreted proteins in tissues. This study investigated the expression of glycoproteins and transcripts of ADAM9 and ADAM10 in different regions of the bovine male reproductive tract (MRT). Fresh bovine samples were collected from the testis, epididymis, and vas deferens of MRT for this study. Special stains were utilized for identifying glycoproteins depending on the type of mucopolysaccharides in the seminiferous tubules of the testis and epithelial cells of the epididymis and vas deferens. Quantitative real-time PCR (qPCR) was used to assess the mRNA concentrations of ADAMs9 and 10. Additionally, structural and network analysis was performed to investigate the similarity between human and bovine ADAM9 and ADAM10. Variable glycoprotein (acidic and neutral mucopolysaccharides) expression was observed in the seminiferous tubules of the testis and epithelial cells of the epididymis and vas deferens. ADAM9 and 10 mRNA were detected in the testis epididymis and vas deferens, with ADAM9 expression being significantly higher than ADAM10 mRNA expression in the testis and tail of epididymis. Significant similarity between the protein sequence of human and bovine ADAM9 (85.75%) and ADAM10 (96.66%) was observed, while the similarity between protein sequence of ADAM 9 and ADAM10 in humans and bovine was <26%. Bovine ADAM9 showed 4 binding sites, while bovine ADAM10, human ADAM9, and human ADAM10 showed 7 binding pockets, each with considerable variations in the affinity of specific antibody sequences to these binding pockets. Network analysis confirmed that ADAM9 and ADAM10 have critical roles in physiological processes involving cell migration, protease activity, and secretase-related functions. In addition, they have large numbers of hydrogen bonds to most other members of the ADAM family. Our study reports similarities in the expression of glycoproteins and ADAM9 and ADAM10 mRNA in the MRT of Bos Taurus. Structural and network analysis of ADAM9 and ADAM10 suggested functional similarities in these proteins, which are likely to be in facilitating spermatogenesis, transcervical sperm migration, egg-sperm fusion, and fertilization. Further, the similarity between human and bovine ADAM9 and ADAM10 proteins suggests that bovine sperms can be considered as a model system for comparative medicine studies.

Describing the Reproductive Microbiome of Tritrichomonas foetus Chronically Infected Bulls and Diagnostic Collection Device Performance.

Tritrichomonas foetus (T. foetus), the causative agent of bovine trichomoniasis, is an obligate protozoan parasite of the bovine reproductive tract and can be found on the penis, prepuce, and distal urethra of the bull and from the cranial vagina to the oviduct in the infected cow. To date, the microbiome of bulls infected with T. foetus has not been described. The objectives of this study were to (1) describe the preputial and penile microbiome of bulls chronically infected by T. foetus, (2) describe the seminal microbiome of T. foetus-infected bulls, and (3) evaluate different collection devices that could be used for sampling. Eleven bulls naturally infected with T. foetus were utilized for the collection of samples. Samples were obtained during the process of a routine breeding soundness exam utilizing either a dacron swab, pizzle stick, double-guarded swab, or semen collection. The preputial and seminal microbiome of T. foetus-infected bulls was dominated by bacterial members of the phyla Fusobacteriota, Firmicutes, Bacteroidota, Actinobacteria, and Campylobacterota. Semen collection yielded the most microbial diversity; however, there was no significant difference between the four methods (p ≥ 0.05). This study characterizes both the preputial and seminal microbial communities of bulls chronically infected by T. foetus.

Comparison of microbiota structure in reproductive tract of Yanbian cattle and Yanhuang cattle.

Microbiota in the reproductive tract of cattle play a vital role in maintaining normal reproduction. However, the information on microbiota in different parts of reproductive tracts with different genetic background is few. The aim of the present study was to describe and compare the microbiota in vagina, cervix and uterus of Yanbian cattle and Yanhuang cattle. The results showed that microbial diversity increases from the vagina to the uterus. The top three bacterial phyla in bovine reproductive tract were Proteobacteria, Firmicutes and Bacteroidetes, accounting for more than 85%. From the vagina to the uterus, the relative abundance of Proteobacteria gradually decreased, while that of Firmicutes gradually increased. Phylum-level Firmicutes and genus-level UCG_010 were significantly enriched in the uterus of Yanbian cattle and Yanhuang cattle. Comparing the same parts of the two breeds, it was found that there was no significant difference in alpha diversity, but significant differences in beta diversity. In addition, microbiota with significant differences in the relative abundance of the reproductive tract were found. These findings lay a foundation for a comprehensive understanding of the structure of the genital tract microbiota of cows and its regulatory mechanisms.

Modulation of Apoptosis by Bovine Gammaherpesvirus 4 Infection in Bovine Endometrial Cells and the Possible Role of LPS in This Process.

The prevalent pathogens associated with bovine uterine infections are bacteria that appear to increase the host's susceptibility to secondary infections with other bacteria or viruses, among which BoGHV4 is the most frequently found. In this work, the study of the pathways of apoptosis induction was carried out on an experimental model of primary culture of endometrial cells, in order to know the implication of BoGHV4 and the presence of bacterial LPS in the pathogenesis of the bovine reproductive tract. For this, different staining techniques and molecular analysis by RT-PCR were used. The results obtained allowed us to conclude that the level of cell death observed in the proposed primary culture is directly related to the time of viral infection and the presence of LPS in BoGHV4 infection. The apoptosis indices in cells infected with BoGHV4 and BoGHV4 + LPS revealed a maximum that correlated with the appearance of cytopathic effects and the maximum viral titers in the model studied. However, morphological, biochemical, and molecular changes were evident during both early and late stages of apoptosis. These findings provide information on the factors that may influence the pathogenesis of BoGHV4 and help to better understand the mechanisms involved in virus infection.

Repurposing of non-steroidal anti-inflammatory drugs for combination therapies to combat multidrug-resistant S. aureus of bovine reproductive tract origin.

Multidrug-resistant bacteria have become the predominant etiology in bovine female reproductive tract infections and thus require effective treatment approaches. The main goal of this study was the molecular detection of mecA, blaZ, tetK, and aacA-aphD genes in Staphylococcus aureus (S. aureus) responsible for methicillin, beta-lactam, tetracycline, and aminoglycoside resistance respectively. Phylogenetic analysis was conducted to check the homology of staphylococcal genes with NCBI sequences. The in-vitro efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) in combination therapies against MDR S. aureus was evaluated using well diffusion assay and checkerboard method. Vaginal swab samples (n = 384) collected from bovines suffering from endometritis, pyometra, and retained placenta were tested for S. aureus. Results showed a 17.96% overall prevalence. Both phenotypic and genotypic resistance was observed among S. aureus isolates with 50.72% and 37.68% isolates being confirmed as methicillin-resistant (MRSA), 36.23% and 18.84% isolates exhibiting beta-lactam, 40.58%, and 27.54% isolates showing tetracycline, and 33.33% and 36.23% isolates showing aminoglycosides resistance based on disc diffusion and gene confirmation, respectively. Phylogenetic analysis indicated homology with previously reported Pakistani isolates suggesting the possibility of MDR S. aureus transmission within and between animals. Synergy testing indicated that combinations of ceftriaxone-ketoprofen (153.77%), ceftriaxone-meloxicam (149.55%), amoxiclav-flunixin meglumine (106.06%), and oxytetracycline-flunixin meglumine (104.47%) showed synergy on well diffusion assay. Based on the fractional inhibitory concentration index by checkerboard method, oxytetracycline-meloxicam and gentamicin-ketoprofen combinations exhibited synergistic interaction. In conclusion, MDR S. aureus resistance was mitigated in-vitro through the combination of antibiotics (oxytetracycline, gentamicin) with NSAIDs (meloxicam, ketoprofen) that could be used to create therapeutic strategies for bovine reproductive issues.

Effect of different sperm populations on neutrophils extracellular traps (NETs) formation in cattle

In cattle, clinical and subclinical inflammation in the bovine female reproductive tract (FRT) significantly reduces fertility. PMN participate in this FRT-associated inflammation by eliminating pathogens by eliciting various defense mechanisms, with the release of neutrophil extracellular traps NETs) being the latest process discovered. Consistently, human-, bovine- and porcine-derived spermatozoa induce release of NETs in exposed PMN of the same species origin, and thereby decreasing sperm motility through NETs-mediated entrapment. The release of NETs in the presence of different sperm sub-populations is evaluated in this work. Cryopreserved bovine sperm were selected and different sperm populations were used: viable sperm, sperm with oxidative stress, capacitated sperm, and sperm with loss of viability. Isolated PMN of dairy cows were co-incubated with these sperm populations for 4 h. Neutrophil elastase (NE) and DNA were detected by fluorescence microscopy analysis. It was noted that exposed bovine PMN released NETs in the presence of sperm. Moreover, sperm-triggered NETosis resulted different phenotypes of NETs, i. e. spread NETs (sprNETs), diffused NETs (diffNETs) and aggregated NETs (aggNETs). Viable/motile spermatozoa induced a higher proportion of NETotic cells at 15, 60 and 120 min in comparison to controls. In conclusion, all bovine sperm populations in co-culture with PMN generated NETs extrusion while viable sperm activated NETotic cells to a greater extent. With this being an early event in the activation of bovine PMN.

Erratum: The in vitro effects of n-3 fatty acids on immune response regulation of bovine ex vivo endometrial explants

Fatty acids are considered metabolic intermediaries, although new facts indicate they also work as signaling molecules with different roles in the immune response. Based on that, in this study, we investigated the anti-inflammatory effects of n-3 polyunsaturated fatty acids (PUFAs) as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and α-linolenic acid (LNA) in ex vivo bovine endometrial explants. For this, two groups were formed: (1) LPS-challenged and (2) control, both to evaluate the accumulation of proinflammatory cytokines as interleukin 1β (IL1B) and interleukin 6 (IL6). To develop the study, bovine female reproductive tracts from non-pregnant Angus heifers without evidence of reproductive diseases were selected. Endometrial explants were processed and treated for 24 h with EPA, DHA, and LNA in five different concentrations (0μM, 50μM, 100 μM, 200μM and 400 μM) and then, challenged with LPS for 24 h. Supernatants were collected to evaluate the concentration of IL1B and IL6 by ELISA. Explants treated with EPA from control groups reduced the concentrations of ILB (200μM) and IL6 (400 μM), and IL6 (50 μM; 100 μM) from the LPS-challenged group. DHA decreased the accumulation of IL1B and IL6 at 200 μM on explants from the LPS-challenged group, and 200 μM reduced IL6 from the control group. In contrast, explants treated with LNA only reduced the accumulation of IL1B to 400μM (from both groups). In conclusion, the EPA acid is the best anti-inflammatory option to decrease the concentration of both pro-inflammatory cytokines (IL1B and IL6) from LPS-challenged and control groups in bovine endometrial explants; while LNA evidence to be the last option to promote an anti-inflammatory response.

The in vitro effects of n-3 fatty acids on immune response regulation of bovine ex vivo endometrial explants

Fatty acids are considered metabolic intermediaries, although new facts indicate they also work as signaling molecules with different roles in the immune response. Based on that, in this study, we investigated the anti-inflammatory effects of n-3 polyunsaturated fatty acids (PUFAs) as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and α-linolenic acid (LNA) in ex vivo bovine endometrial explants. For this, two groups were formed: (1) LPS-challenged and (2) control, both to evaluate the accumulation of proinflammatory cytokines as interleukin 1β (IL1B) and interleukin 6 (IL6). To develop the study, bovine female reproductive tracts from non-pregnant Angus heifers without evidence of reproductive diseases were selected. Endometrial explants were processed and treated for 24 h with EPA, DHA, and LNA in five different concentrations (0μM, 50μM, 100 μM, 200μM and 400 μM) and then, challenged with LPS for 24 h. Supernatants were collected to evaluate the concentration of IL1B and IL6 by ELISA. Explants treated with EPA from control groups reduced the concentrations of ILB (200µM) and IL6 (400 µM), and IL6 (50 µM; 100 µM) from the LPS-challenged group. DHA decreased the accumulation of IL1B and IL6 at 200 µM on explants from the LPS-challenged group, and 200 µM reduced IL6 from the control group. In contrast, explants treated with LNA only reduced the accumulation of IL1B to 400μM (from both groups). In conclusion, the EPA acid is the best anti inflammatory option to decrease the concentration of both pro-inflammatory cytokines (IL1B and IL6) from LPS-challenged and control groups in bovine endometrial explants; while LNA evidence to be the last option to promote an anti-inflammatory response.

Perspective: Can early embryonic losses be reduced in lactating dairy cows?

Perspective: Can early embryonic losses be reduced in lactating dairy cows?

Bovine neonatal microbiome origins: a review of proposed microbial community presence from conception to colostrum.

In recent years, there has been an influx of research evaluating the roles of the reproductive tract microbiota in modulating reproductive performance. These efforts have resulted in a breadth of research exploring the bovine reproductive tract microbiota. The female reproductive tract microbiota has been characterized during the estrus cycle, at timed artificial insemination, during gestation, and postpartum. Additionally, there are recently published studies investigating in-utero inoculation of the bovine fetus. However, critical review of the literature to understand how the microbial shifts during a dam's lifecycle could impact neonatal outcomes is limited. This review demonstrates a consistency at the phyla level throughout both the maternal, paternal, and neonatal microbiomes. Moreover, this review challenges the current gestational inoculation hypothesis and suggests instead a maturation of the resident uterine microbiota throughout gestation to parturition. Recent literature is indicative of microbial composition influencing metabolomic parameters that have developmental programming effects in feed utilization and metabolic performance later in life. Thus, this review enumerates the potential origins of neonatal microbial inoculation from conception, through gestation, parturition, and colostrum consumption while introducing clear paucities where future research is needed to better understand the ramifications of the reproductive microbiome on neonates.

Effects of plant extracts on the growth of beneficial indigenous lactic acid bacteria (BLAB) for their potential use in preventing bovine reproductive tract infections

Effects of plant extracts on the growth of beneficial indigenous lactic acid bacteria (BLAB) for their potential use in preventing bovine reproductive tract infections

An Update on Novel Taxa and Revised Taxonomic Status of Bacteria Isolated from Domestic Animals Described in 2018 to 2021.

Novel bacterial taxonomy and nomenclature revisions can have significant impacts on clinical practice, disease epidemiology, and veterinary microbiology laboratory operations. Expansion of research on the microbiota of humans, animals, and insects has significant potential impacts on the taxonomy of organisms of clinical interest. Implications of taxonomic changes may be especially important when considering zoonotic diseases. Here, we address novel taxonomy and nomenclature revisions of veterinary significance. Noteworthy discussion centers around descriptions of novel mastitis pathogens in Streptococcaceae, Staphylococcaceae, and Actinomycetaceae; bovine reproductive tract pathogens in Corynebacteriaceae; novel members of Mannheimia spp., Leptospira spp., and Mycobacterium spp.; the transfer of Ochrobactrum spp. to Brucella spp.; and revisions to the genus Mycoplasma.

NR3C1 and glucocorticoid-regulatory genes mRNA and protein expression in the endometrium and ampulla during the bovine estrous cycle

The bovine reproductive tract exhibits changes during the estrous cycle modulated by the interplay of steroid hormones. Glucocorticoids can be detrimental when stress-induced but are relevant at baseline levels for appropriate reproductive function. Here, an analysis of quantitative real-time PCR was performed to study the bovine glucocorticoid-related baseline gene transcription in endometrial and ampullar tissue samples derived from three time points of the estrous cycle, stage I (Days 1–4), stage III (Days 11–17) and stage IV (Days 18–20). Our results revealed expression differences during stages, as expression observed in the ampulla was higher during the post-ovulatory phase (stage I), including the glucocorticoid receptor NR3C1, and some of its regulators, involved in glucocorticoid availability (HSD11B1 and HSD11B2) and transcriptional actions (FKBP4 and FKBP5). In contrast, in the endometrium, higher expression of the steroid receptors was observed during the late luteal phase (stage III), including ESR1, ESR2, PGRMC1 and PGRMC2, and HSD11B1 expression decreased, while HSD11B2 increased. Moreover, at protein level, FKBP4 was higher expressed during the late luteal phase, and NR3C1 during the pre-ovulatory phase (stage IV). These results suggest that tight regulation of the glucocorticoid activity is promoted in the ampulla, when reproductive events are taking place, including oocyte maturation. Moreover, most expression changes in the endometrium were observed during the late luteal phase, and may be related to the embryonic maternal recognition. In conclusion, the glucocorticoid regulation changes across the estrous cycle and may be playing a role on the reproductive events occurring in the bovine ampulla and endometrium.

Adaptive sampling during sequencing reveals the origins of the bovine reproductive tract microbiome across reproductive stages and sexes

Cattle enterprises are one of the major livestock production systems globally and are forecasted to have stable growth in the next decade. To facilitate sustainable live weight production, optimal reproductive performance is essential. Microbial colonisation in the reproductive tract has been demonstrated as one of the factors contributing to bovine reproductive performance. Studies also implied that reproductive metagenomes are different at each stage of the estrous cycle. This study applied Oxford Nanopore Technologies’ adaptive long-read sequencing to profile the bovine reproductive microbiome collected from tropical cattle in northern Queensland, Australia. The microbiome samples were collected from cattle of different sexes, reproductive status and locations to provide a comprehensive view of the bovine reproductive microbiome in northern Australian cattle. Ascomycota, Firmicutes and Proteobacteria were abundant phyla identified in the bovine reproductive metagenomes of Australian cattle regardless of sexes, reproductive status and location. The species level taxonomical investigation suggested that gastrointestinal metagenome and the surrounding environment were potentially the origins of the bovine reproductive metagenome. Functional profiles further affirmed this implication, revealing that the reproductive metagenomes of the prepubertal and postpartum animals were dominated by microorganisms that catabolise dietary polysaccharides as an energy substrate while that of the pregnant animals had the function of harvesting energy from aromatic compounds. Bovine reproductive metagenome investigations can be employed to trace the origins of abnormal metagenomes, which is beneficial for disease prevention and control. Additionally, our results demonstrated different reproductive metagenome diversities between cattle from two different locations. The variation in diversity within one location can serve as the indicator of abnormal reproductive metagenome, but between locations inferences cannot be made. We suggest establishing localised metagenomic indices that can be used to infer abnormal reproductive metagenomes which contribute to abortion or sub-fertility.

The mRNA expression of the three major described cold-inducible proteins, including CIRBP, differs in the bovine endometrium and ampulla during the estrous cycle

The cold-inducible proteins (CIPs) are essential for post-transcriptional gene regulation playing diverse tissue-specific roles in maintaining normal cellular function and morphogenesis. The potential implications of CIPs in reproductive events raise questions about their role in the physiology of the bovine reproductive tract. However, the expression changes of CIPs during the bovine estrous cycle have not been studied so far. Here, we hypothesized that the bovine estrous cycle could affect the mRNA expression of the CIPs and other candidate transcripts in the reproductive tract. This study aimed to examine estrous cycle-dependent mRNA expression patterns in the bovine endometrium and ampulla of three of the major described CIPs (CIRBP, RBM3, SRSF5), a set of inflammatory cytokines (IL-10, IL-18, IL-1β), and other candidate genes (IL-10RA, IL-10RB, BCL2, NLRP3, STAT1, STAT3, STAT5A, STAT6). Endometrial and ampullar tissues were assessed by RT-qPCR. Additionally, the mRNA expression levels were correlated among them and with follicular progesterone and estradiol concentrations. The transcript levels of CIPs increased in the endometrium during stage III (Days 11–17) compared to stage I (Days 1–4) and IV (Days 18–20). In the ampulla, the mRNA expression of CIRBP increased during the late luteal phase (stage III), but no differences in the expression of other CIPs were observed. This study expands the current knowledge regarding mRNA expression in the endometrium and oviductal ampulla of cycling heifers, focusing mainly on the CIPs. A better understanding of the mechanisms within the uterus and oviduct during the estrous cycle is crucial to improving the fertility rate.

81 Reproductive Dysfunction in Cattle Following Vaccination Not a Result of Viral Infection of the Reproductive Tract

Abstract Impairment of bovine reproductive function and decreased pregnancy rates have been reported in modified-live vaccinated females. The objective of this study was to determine if virus could be found in various areas of the bovine reproductive tract following vaccination. Previously vaccinated beef females (n = 50) were administered a single dose of one of three treatments on d0 of the estrous cycle; BoviShield (n = 20), Titanium 5 (n = 20), or ViraShield (n = 10) based on estrus expression. Blood samples were collected on d0, 15, and 30 to evaluate Infectious Bovine-Rhinotracheitis (IBR) and Bovine Viral Diarrhea Virus (BVDV) titers and progesterone concentrations. On d15/16, half the animals from each treatment were non-surgically flushed with sterile saline (Uterine Fluid), and uterine biopsies (Uterine Tissue) were collected. On d17, follicular fluid and granulosa cells were collected by follicle aspirations. Remaining animals had uteri flushed and uterine tissue collected on d29, and follicles aspirated on d31. All samples were evaluated for presence of virus. Data were analyzed using the MIXED procedure of SAS. Sub-luteal progesterone concentrations were observed in 15% of the BoviShield and Titanium treatments on d15 and d30, versus 0% in the ViraShield treatment. Virus isolation and RT-PCR results were negative in all samples for all animals. Titers for BVDV-1 did not differ between treatments (P = 0.99) or treatment by time (P = 0.48). Titers for IBR tended to differ by treatment (P = 0.09) and treatment by time (P = 0.06), while BVDV-2 titers were affected by treatment (P < 0.01) and treatment x time (P < 0.01). Sub-luteal progesterone animals had a rapid rise in IBR and BVDV-2 titers from d0 to d15 and then tended (P < 0.07) to decrease to d30, compared to animals that had normal cycles where titers increased from d0 to d15 and remained elevated on d30. Collectively, these results indicate an alternative mechanism of impacting reproductive efficiency, possibly through an immune response.

Characterization and profiling analysis of bovine oviduct and uterine extracellular vesicles and their miRNA cargo through the estrous cycle.

Extracellular vesicles (EVs) found in various biological fluids and particularly in reproductive fluids, have gained considerable attention for their possible role in cell- to- cell communication. Among, the different bioactive molecules cargos of EVs, MicroRNAs (miRNAs) are emerging as promising diagnostic biomarkers with high clinical potential. Aiming to understand the roles of EVs in bovine reproductive tract, we intended to characterize and profile the EVs of oviduct and uterine fluids (OF-EVs, UF-EVs) and their miRNA across the estrous cycle. Nanoparticle tracking analysis and transmission electron microscopy confirmed the existence of small EV population in OF and UF at all stages, (size between 30 and 200nm; concentration: 3.4×1010 EVs/ml and 6.0×1010 EVs/ml for OF and UF, respectively, regardless of stage). The identification of EV markers (CD9, HSP70, and ALIX proteins) was confirmed by western blot. The miRNA analysis revealed the abundance of 310 and 351 miRNAs in OF-EVs and UF-EVs, respectively. Nine miRNAs were differentially abundant in OF-EVs between stages of the cycle, eight of them displayed a progressive increase from S1 to S4 (p<.05). In UF-EVs, a total of 14 miRNAs were differentially abundant between stages. Greater differences were observed between stage 1 (S1) and stage 3 (S3), with 11 miRNAs enriched in S3 compared to S1. Functional enrichment analysis revealed the involvement of these miRNAs in relevant pathways such as cell signaling, intercellular junctions, and reproductive functions that may be implicated in oviduct and uterus modulation across the cycle, but also in their preparation for embryo/conceptus presence and development.

Evaluation of in vitro Activation of Bovine Endometrial and Vaginal Epithelial and Blood Mononuclear Cells to Produce Nitric Oxide in Response to Mycoplasma bovis, Mycoplasma bovigenitalium and Ureaplasma diversum

Abstract Genital mycoplasmosis is a condition present in bovine production systems, and the most important agents involved are Mycoplasma bovis, Mycoplasma bovigenitalium and Ureaplasma diversum. Some aspects of their pathogenesis remain unclear. This study was designed in order to evaluate their ability to stimulate mononuclear cells from the endometrium, vagina and peripheral blood of cycling and healthy cows to produce nitric oxide (NO). Cellular cultures of endometrial, vaginal and peripheral blood cells from 33 healthy cows were cultivated with Mycoplasma bovis, Mycoplasma bovigenitalium and Ureaplasma diversum originated from the 4th passage in culture broth and the NO production was measured by the Greiss reaction. Confirmation of the presence of mononuclear cells and of the agents during and after the NO assay was done by Giemsa stained smears and further cultivation and detection by PCR reaction. Mononuclear cells from all samples produced NO. Mycoplasma bovigenitalium stimulated higher NO production than the others (p&lt;0.05). Endometrial cells produced less NO than vaginal or blood cultured cells. In conclusion, it seems that Mycoplasma bovis, Mycoplasma bovigenitalium, and Ureaplasma diversum are able to activate mononuclear cells and induce the production of NO, thus suggesting that this pathway is elicited in response to the primary infection by these agents. More studies are necessary to verify why these agents remain in the bovine reproductive tract for long periods and how they reassume deleterious effects.

A comparison of fourteen fully characterized mammalian-associated Campylobacter fetus isolates suggests that loss of defense mechanisms contribute to high genomic plasticity and subspecies evolution.

Campylobacter fetus is currently classified into three main subspecies, but only two of these, C. fetus subspecies fetus and C. fetus subsp. venerealis originate principally from ruminants where they inhabit different niches and cause distinct pathogenicity. Their importance as pathogens in international trade and reporting is also different yet the criteria defining these properties have never been fully substantiated nor understood. The situation is further compromised because the ability to differentiate between these two closely related C. fetus subspecies has traditionally been performed by phenotypic characterisation of isolates, methods which are limited in scope, time-consuming, tedious, and often yield inconsistent results, thereby leading to isolate misidentification. The development of robust genetic markers that could enable rapid discrimination between C. fetus subsp. fetus and subsp. venerealis has also been challenging due to limited differences in the gene complement of their genomes, high levels of sequence repetition, the small number of closed genome sequences available and the lack of standardisation of the discriminatory biochemical tests employed for comparative purposes. To yield a better understanding of the genomic differences that define these C. fetus strains, seven isolates were exhaustively characterised phenotypically and genetically and compared with seven previously well characterised isolates. Analysis of these 14 C. fetus samples clearly illustrated that adaption by C. fetus subsp. venerealis to the bovine reproductive tract correlated with increasing genome length and plasticity due to the acquisition and propagation of several mobile elements including prophages, transposons and plasmids harbouring virulence factors. Significant differences in the repertoire of the CRISPR (clustered regularly interspersed short palindromic repeats)-cas system of all C. fetus strains was also found. We therefore suggest that a deficiency in this adaptive immune system may have permitted the emergence of extensive genome plasticity and led to changes in host tropism through gene disruption and/or changes in gene expression. Notable differences in the sub-species complement of DNA adenine methylase genes may also have an impact. These data will facilitate future studies to better understand the precise genetic differences that underlie the phenotypic and virulence differences between these animal pathogens and may identify additional markers useful for diagnosis and sub-typing.