In Vitro Interaction between Mycoplasma agalactiae and Small Ruminants' Endogenous Bacterial Strains of Enterococcus spp. and Coagulase-Negative Staphylococcus.

Recently, an antimicrobial effect on Mycoplasma agalactiae (Ma), the main etiological agent of contagious agalactia (CA), was reported in vitro with strains of Enterococcus spp. from ovine and caprine milk. The aim of this work was to evaluate the interaction of Ma with the same Enterococcus spp. isolated from other anatomical locations (vagina) and other bacterial populations present in milk, such as coagulase-negative staphylococci (CNS). The vaginal Enterococcus strains and the raw milk CNS were isolated from sheep and goats. Experimental in vitro conditions were prepared to assess the growth of Ma with and without the presence of these strains. The selected vaginal strains were identified as Enterococcus (E.) hirae and E. mundtii, and the strains of CNS were identified as Staphylococcus petrasii. Different interactions of Ma with ovine and caprine wild vaginal strains of Enterococcus and dairy strains of CNS are described for the first time: Ma can grow exponentially during 15 h with the selected strains, although with certain strains, its optimal growth can be negatively affected (p < 0.05). The colonization and/or excretion of Ma could, therefore, be influenced by certain endogenous bacterial strains. Our results increase the knowledge about possible bacterial ecology dynamics surrounding CA.

Antibacterial potential of commercial and wild lactic acid bacteria strains isolated from ovine and caprine raw milk against Mycoplasma agalactiae.

Introduction: The complexity of fighting contagious agalactia (CA) has raised the necessity of alternative antimicrobial therapies, such as probiotics. Lactic acid bacteria (LAB) are present in the mammary gland of small ruminants and their antimicrobial effect have been previously described against species like Mycoplasma bovis but never against Mycoplasma agalactiae (Ma). This in vitro study aims to evaluate the antimicrobial activity against Ma of ovine and caprine LAB strains and a human commercial probiotic (L2) of Lactobacillus spp. Methods: A total of 63 possible LAB strains were isolated from nine ovine and caprine farms in Spain, three isolates (33B, 248D, and 120B) from the 63 strains were selected, based on their capacity to grow in a specific medium in vitro, for an in vitro experiment to assess their antimicrobial activity against Ma in Ultra High Temperature (UHT) processed goat milk (GM). A women commercial vaginal probiotic was also included in the study. The inoculum of L2 was prepared at a concentration of 3.24 × 108 CFU/mL and the average concentration of the inoculum of the wild LAB varied from 7.9 × 107 to 8.4 × 108 CFU/mL. Results: The commercial probiotic L2 significantly reduced the concentration of Ma to 0.000 log CFU/mL (p < 0.001), strain 33B reduced it from 7.185 to 1.279 log CFU/mL (p < 0.001), and 120B from 6.825 to 6.466 log CFU/mL (p < 0.05). Strain 248D presented a bacteriostatic effect in GM. Moreover, the three wild strains and the commercial probiotic produced a significative reduction of the pH (p < 0.001). Discussion: This is the first in vivo report of the antimicrobial potential of LAB strains against Ma and its interaction. Our results support possible future alternative strategies to antibiotic therapy, previously not contemplated, to fight CA in small ruminants. Further studies are necessary to elucidate the action mechanisms through which these LAB are able to inhibit Ma and to assess the safety of using these strains in possible in vivo studies.

Description of the vaginal microbiota in nulliparous ewes during natural mating and pregnancy: preliminary signs of the male preputial microbiota modulation.

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.

Search of antimicrobial lactic acid bacteria from Salmonella-negative dogs.

BACKGROUND: Salmonellosis is one of the most important food-borne zoonotic disease affecting both animals and humans. The objective of the present study was to identify gastrointestinal (GI) lactic acid bacteria (LAB) of canine-origin from Salmonella-negative dogs' faeces able to inhibit monophasic Salmonella Typhimurium previously isolated from dogs' faeces, in order to be used as a potential probiotic in pet nutrition. RESULTS: Accordingly, 37 LAB were isolated from Salmonella-negative dogs' faeces and tested against monophasic S. Typhimurium using the spot on lawn method out of which 7 strains showed an inhibition halo higher than 2.5 cm. These 7 strains were also tested with the co-culture method and one showed the greatest inhibition value (p < 0.05). Subsequently, the isolate was identified through 16S rRNA sequencing and sequence homology and designated as Ligilactobacillus salivarius (L. salivarius). LAB from Salmonella-positive dogs were also identified and none was the selected strain. Finally, to identify the mechanism of inhibition of L. salivarius, the supernatant was analyzed, and a dose response effect was observed. CONCLUSIONS: It is concluded that the canine-origin L. salivarius, could possess some in vitro functional attributes of a candidate probiotic and could prevent monophasic S. Typhimurium colonization or inhibit its activity if the infection occurs.

Review of the bacterial composition of healthy milk, mastitis milk and colostrum in small ruminants.

Bacterial infections are the cause of many reproductive disorders of economic importance, such as mastitis, in livestock. Unfortunately, very little is known about the microbiota and the changes occurring during an infection state in small ruminants. The sequencing of regions of the 16S rRNA gene, is the useful tool to describe the whole dairy microbiome. Using this technique, studies have identified various phyla such as Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, Fusobacteria and Cyanobacteria; and also several genera from raw milk of small ruminants. Nevertheless, there does not seem to be a consensus on the predominant genera nor phyla, even within the same breed. There is a lack of information about the mammary microflora in meat-oriented breeds, and about the microflora of colostrum and mastitis milk. Further studies comparing the microbiota between artificial and natural lactations and between healthy and mastitis milk are necessary. Considering the concerns arising from the use overuse of antibiotic therapy in Veterinary Medicine, it would be interesting to develop alternative strategies for the control of mastitis. Probiotics, such as lactic acid bacteria (LAB), have proven to be an interesting antibiotic-free strategy. Therefore, their presence in the dairy microflora of small ruminants and their interactions with other bacteria, such as mastitis-causing pathogens, should be scrutinized, given that the efficacy of probiotics increase when the bacterial strains used are specific to their host.