Lactic acid bacteria (LAB) dominate human vaginal microbiota and inhibit pathogen proliferation. In other mammals, LAB do not dominate vaginal microbiota, however shifts of dominant microorganisms occur during ovarian cycle. The study objectives were to characterize equine vaginal microbiota in mares by culture-dependent and independent methods and to describe its variation in estrus and diestrus. Vaginal swabs from 8 healthy adult Arabian mares were obtained in estrus and diestrus. For culture-dependent processing, bacteria were isolated on Columbia blood agar (BA) and Man Rogosa Sharpe (MRS) agar. LAB comprised only 2% of total bacterial isolates and were not related to ovarian phases. For culture-independent processing, V3/V4 variable regions of the 16S ribosomal RNA gene were amplified and sequenced using Illumina Miseq. The diversity and composition of the vaginal microbiota did not change during the estrous cycle. Core equine vaginal microbiome consisted of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria at the phylum level. At the genus level it was defined by Porphyromonas, Campylobacter, Arcanobacterium, Corynebacterium, Streptococcus, Fusobacterium, uncultured Kiritimatiaellae and Akkermansia. Lactobacillus comprised only 0.18% of the taxonomic composition in estrus and 0.37% in diestrus. No differences in the relative abundance of the most abundant phylum or genera were observed between estrus and diestrus samples.

Sheep estrous synchronization is mainly based on progestagen-impregnated sponges which could cause vaginitis. Several species of Lactobacillus used as probiotics are commonly used in the treatment or prevention of urogenital infections in humans. However, no studies have been performed to analyze the potential use of probiotics to prevent urogenital infections in sheep. A randomized controlled clinical trial was conducted with 21 one-year-old ewes to develop a model of probiotic infusion in vaginal sponges in order to study their influence in ewe’s vaginal microbiota, general health status, fertility and prolificity. Synchronization of estrus was based on intravaginal sponges for 14 days. Bacterial communities (Enterobacteriaceae and lactic acid bacteria) were highly fluctuating over time and between animals. The safety of probiotic infusion (mix of Lactobacillus spp. 60% L. crispatus, 20% L. brevis and 20% L. gasseri) in the vagina of healthy ewes was firstly confirmed. Neutrophils were observed in 80% (8/10) of the control ewes compared to 36% (4/11) of the ewes in the probiotic group 2 days after sponge removal (p = 0.056). Fertility in the control and probiotic groups was 60% (6/10) and 91% (10/11), respectively p = 0.097. These results suggest that Lactobacillus spp. infusion in the ewe’s vagina does not affect general health status or fertility.

Mycoplasma bovis is an important bovine pathogen causing pneumonia, mastitis, and arthritis and is responsible for major economic losses worldwide. In the absence of an efficient vaccine, control of M. bovis infections mainly relies on antimicrobial treatments, but resistance is reported in an increasing number of countries. To address the situation in Spain, M. bovis was searched in 436 samples collected from beef and dairy cattle (2016–2019) and 28% were positive. Single-locus typing using polC sequences further revealed that two subtypes ST2 and ST3, circulate in Spain both in beef and dairy cattle, regardless of the regions or the clinical signs. Monitoring of ST2 and ST3 isolates minimum inhibitory concentration (MIC) to a panel of antimicrobials revealed one major difference when using fluoroquinolones (FQL): ST2 is more susceptible than ST3. Accordingly, whole-genome sequencing (WGS) further identified mutations in the gyrA and parC regions, encoding quinolone resistance-determining regions (QRDR) only in ST3 isolates. This situation shows the capacity of ST3 to accumulate mutations in QRDR and might reflect the selective pressure imposed by the extensive use of these antimicrobials. MIC values and detection of mutations by WGS also showed that most Spanish isolates are resistant to macrolides, lincosamides, and tetracyclines. Valnemulin was the only one effective, at least in vitro, against both STs.

Bovine respiratory disease (BRD) is an important viral and/or bacterial disease that mainly affects feedlot calves. The involvement of Mycoplasma bovis in BRD can lead to chronic pneumonia poorly responsive to antimicrobial treatment. Caseonecrotic bronchopneumonia is a pulmonary lesion typically associated with M. bovis. In Spain, M. bovis is widely distributed in the feedlots and circulating isolates are resistant to most antimicrobials in vitro. However, the role of this species in clinical respiratory disease of feedlot calves remains unknown. Furthermore, available data are relative to a fixed panel of antimicrobials commonly used to treat BRD, but not to the specific set of antimicrobials that have been used for treating each animal. This study examined 23 feedlot calves raised in southeast Spain (2016–2019) with clinical signs of respiratory disease unresponsive to treatment. The presence of M. bovis was investigated through bacteriology (culture and subsequent PCR), histopathology and immunohistochemistry. The pathogen was found in 86.9% (20/23) of the calves, mainly in the lungs (78.26%; 18/23). Immunohistochemistry revealed M. bovis antigens in 73.9% (17/23) of the calves in which caseonecrotic bronchopneumonia was the most frequent lesion (16/17). Minimum inhibitory concentration assays confirmed the resistance of a selection of 12 isolates to most of the antimicrobials specifically used for treating the animals in vivo. These results stress the importance of M. bovis in the BRD affecting feedlot calves in Spain.

The vaginal microbiota plays a key role in animals’ health. Understanding its diversity and composition and associated changes occurring through the reproductive cycle represents valuable knowledge to disclose the mechanisms leading to dysbiosis and eventually to infection. Even if the human vaginal microbiota has been thoroughly studied, scarce research has been conducted on the vaginal microbiota of livestock. In this study, 16S rRNA gene-based sequencing was performed on vaginal samples of ten nulliparous ewes at three different sampling points: before the estrus synchronization protocol (T0), at the time of estrus before mating (Testrus), and the day of the pregnancy diagnosis (Tpreg). Preputial samples from the three males collected pre and post-mating were also analyzed. Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria were the most abundant phyla in vaginal samples. The most abundant genera were Porphyromonas, Anaerococcus, and Peptinophilius. Vaginal microbiota biodiversity decreased during pregnancy. Tenericutes (Ureaplasma spp.) increased significantly at Tpreg in both pregnant and non-pregnant ewes. Differences were observed between pregnant and non-pregnant ewes at Tpreg where pregnant ewes had a significantly higher abundance of Actinobacillus spp. and Ureaplasma spp. Ewes that were diagnosed with pregnancy at Tpreg showed a decreased abundance of gram-negative bacteria such as Bacteroidales, Campylobacterales, and Enterobacteriales. In addition, a significant decrease in the relative abundances of genera within Firmicutes, such as Alloicoccus (Lactobacillales), Atopostipes (Lactobacillales), and an uncultured bacteria W5053 from Family XI (Firmicutes, Clostridiales) was observed in non-pregnant ewes at Tpreg. The four most abundant phyla in the rams’ prepuce were the same as in the ewes’ vagina. The most abundant genus was Corynebacterium. No major differences were observed in the ram’s preputial microbiota between pre and post-mating samples. Nevertheless, the differences in the taxonomic composition of ewes’ vaginal microbiota between Testrus and Tpreg could be explained by the exposure to the preputial microbiota. This study offers new insights into the effects of several key steps of the ewe’s reproductive cycle such as estrus-synchronization protocol, mating, and pregnancy on ovine vaginal microbiota. The knowledge of the microbiota dynamics during the reproductive cycle can help improve the reproductive outcomes of dams by identifying biomarkers and putative probiotics.