Microbial composition of goat buck's ejaculates is modified by the process of preparing and storing refrigerated semen doses.

Ejaculates present their own microbiota, and a link between ejaculates' microbiota and sperm quality and fertility exists. With the development of artificial insemination in animal breeding, ejaculates must be manipulated by diluting them with extenders and storing them at temperatures below body temperature. The effects that these processes have on the original semen microbiota have never been studied. This study explores the effects of the protocol for preparing refrigerated goat buck semen doses and storing on seminal microbiota. Semen from six adult goat bucks of the Murciano-Granadina breed (24 ejaculates) was used, cooled to 4 °C in a skimmed milk-based extender, and stored at this temperature for 24 h. Samples were taken in different steps: in the raw ejaculates (ejaculates), after dilution with the refrigeration extender (diluted), immediately after reaching 4 °C (chilled 0 h) and the samples refrigerated at 4 °C and stored at this temperature for 24 h (chilled 24 h). Sperm quality (motility and integrity of plasma and acrosomal membrane, and mitochondrial functionality) was also evaluated. Bacterial 16S rRNA sequencing was used to study the seminal microbiota. Our results indicated that both refrigeration and storage at 4 °C negatively affected sperm quality parameters. Preparing semen doses and their subsequent conservation caused a significant change in the bacterial community structure. Raw ejaculates showed a lower Pielou's evenness index than the other samples (diluted, chilled 0 h and chilled 24 h). Ejaculates also had a lower Shannon's diversity index (3.44) than the diluted semen (4.17) and the semen chilled for 24 h (4.43). Regarding beta diversity, significant differences were detected between ejaculates and the other treatments. Differences were also found in unweighted UniFrac distances between the semen chilled for 0 h and that chilled for 24 h. At the genus level, marked effects of preparing doses and their subsequent conservation were also evident: 199 genera that were absent in ejaculates were found in the semen chilled and stored for 24 h; 177 genera that were present in ejaculates disappeared after 24-h refrigeration. In conclusion, the extender and protocol for preparing refrigerated goat buck semen doses considerably modify microbial ejaculate composition.

Effect of selection for growth rate on the rabbit (Oryctolagus cuniculus) immune system and its response after experimental Staphylococcus aureus infection.

The aim of the work was to evaluate if genetic selection for daily gain may affect the immune system. Two experiments were performed. The first one involved 80 rabbit females and their first two litters to explore the effect of selection on the ability of animals to maintain immune competence. Two generations from a line selected for average daily gain (ADG) were evaluated (VR19 generation 19th, n = 43; VR37 generation 37th, n = 37). In females, the effect of selection and its interaction with physiological state were not significant for any trait. In litters, the selection criterion increased the granulocyte to lymphocyte ratio. The second experiment involved 73 19-week-old females (VR19, n = 39; VR37, n = 34) to explore the effect of genetic selection on immune response after S. aureus infection. The VR37 rabbit females had lower counts for total lymphocytes, CD5+, CD4+, CD8+, CD25+, monocytes, the CD4+/CD8+ ratio and platelets than those of VR19 (-14, -21, -25, -15, -33, -18, -11 and -11%, respectively; P < 0.05). VR37 had less erythema (-8.4 percentage points; P < 0.05), fewer nodules (-6.5 percentage points; P < 0.05) and a smaller nodule size (-0.65 cm3 on 7 day post-inoculation; P < 0.05) compared to VR19. Our study suggests that genetic selection for average daily gain does not negatively affect the maintenance of a competent immune system or the ability to establish immune response. It seems that such selection may improve the response to S. aureus infections.

Microbiota in Goat Buck Ejaculates Differs Between Breeding and Non-breeding Seasons.

Changes in semen microbiota are associated with alterations to sperm quality and fertility. However, the microbiota from most livestock species has not yet been studied. Goats are seasonal breeders, but semen microbiota has never been described in this species, and it is unknown how seasonality affects it. Our study objective is 2-fold: to describe the microbiota in goat buck ejaculates and to determine if it differs between breeding and non-breeding seasons. Semen from six males of the Murciano-Granadina breed was collected during both seasons. Two replicates were performed per male and season on different days. The microbiota was characterized by genomic sequencing technology. Sperm quality was also evaluated. Repetition was not significant for the studied variables. Sperm velocities were higher for the breeding than for the non-breeding season. The ejaculates from both seasons also differed in the proportion of apoptotic spermatozoa. The five dominant phyla were Firmicutes, Proteobacteria, Fusobacteria, Actinobacteria, and Bacteroidetes during the breeding season and Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Cyanobacteria during the non-breeding season. The dominant genus during both seasons was Ureaplasma. Differences in microbial community structure (the beta diversity) were found. A decrease in the relative abundance of the genus Faecalibacterium and an increase in the genera Sphingomonas and Halomonas were observed in the ejaculates collected during the breeding season. Sphingomonas and Faecalibacterium abundance favorably and unfavorably correlated with sperm quality, respectively. In conclusion, the semen microbiota from goat bucks varies between breeding and non-breeding seasons, and the microbiota remains stable for 7 days within a season. In addition, the genera Sphingomonas and Faecalibacterium could be possible biomarkers of semen quality in goat bucks. These results contribute to an in-depth understanding of the effects of reproductive seasonality on goat buck ejaculates.

Marked Presence of Methicillin-Resistant Staphylococcus aureus in Wild Lagomorphs in Valencia, Spain.

The appearance of methicillin-resistant strains of Staphylococcus aureus (MRSA) in several animal species (including rabbits) has set off alarms for their capacity to act as reservoirs for this bacterium. This is especially important in wild animals given its epidemiological implications. The objectives of this study were to identify and characterize S. aureus, specifically MRSA, strains in wild lagomorph high-density areas. Ten hares and 353 wild rabbits from 14 towns with a high rabbit density in the Valencian region (eastern Spanish coast) were sampled. Swabs from the nasal cavity, ears, perineum and lesions (when present) were taken for microbiological studies. The detection of different genes and antibiotic susceptibility studies were also carried out. Of all the animals, 41.3% were positive for S. aureus, of which 63.3% were MRSA. Ears were the anatomical location with more S. aureus and MRSA strains. The more frequently identified MLST type was ST1945 (97.1%, 136/140). The mecA gene was found only in one sample. The rest (n = 139) carried the mecC gene and were included in CC130, except one. Penicillin resistance was detected in 28 mec-negative isolates and, in one case, bacitracin resistance. mecA isolate presented resistance to enrofloxacin and tetracycline, and 10 mecC isolates also showed bacitracin resistance. No MRSA isolate was positive for genes chp, sea, tst and PVL. Two ST1945 isolates contained IEC type E (comprising genes scn and sak). mecA-isolate was positive for blaZ. Of the 28 MSSA strains showing resistance to penicillin, 22 carried the blaZ gene. These surprising results highlight the marked presence of MRSA strains in wild rabbits in high-density areas.

Pathogenesis of Intradermal Staphylococcal Infections: Rabbit Experimental Approach to Natural Staphylococcus aureus Skin Infections.

Despite the enormous efforts made to achieve effective tools that fight against Staphylococcus aureus, the results have not been successful. This failure may be due to the absence of truly representative experimental models. To overcome this deficiency, the present work describes and immunologically characterizes the infection for 28 days, in an experimental low-dose (300 colony-forming units) intradermal model of infection in rabbits, which reproduces the characteristic staphylococcal abscess. Surprisingly, when mutant strains in the genes involved in virulence (JΔagr, JΔcoaΔvwb, JΔhla, and JΔpsmα) were inoculated, no strong effect on the severity of lesions was observed, unlike other models that use high doses of bacteria. The inoculation of a human rabbitized (FdltBr) strain demonstrated its capacity to generate a similar inflammatory response to a wild-type rabbit strain and, therefore, validated this model for conducting these experimental studies with human strains. To conclude, this model proved reproducible and may be an option of choice to check both wild-type and mutant strains of different origins.

Differences in virulence between the two more prevalent Staphylococcus aureus clonal complexes in rabbitries (CC121 and CC96) using an experimental model of mammary gland infection.

Staphylococcal mastitis is a major health problem in humans and livestock that leads to economic loss running in millions. This process is currently one of the main reasons for culling adult rabbit does. Surprisingly, the two most prevalent S. aureus lineages isolated from non-differentiable natural clinical mastitis in rabbits (ST121 and ST96) generate different immune responses. This study aimed to genetically compare both types of strains to search for possible dissimilarities to explain differences in immune response, and to check whether they showed similar virulence in in vitro tests as in experimental intramammary in vivo infection. The main differences were observed in the enterotoxin gene cluster (egc) and the immune-evasion-cluster (IEC) genes. While isolate ST121 harboured all six egc cluster members (seg, sei, selm, seln, selo, selu), isolate ST96 lacked the egc cluster. Strain ST96 carried a phage integrase Sa3 (Sa3int), compatible with a phage integrated into the hlb gene (ß-haemolysin-converting bacteriophages) with IEC type F, while isolate ST121 lacked IEC genes and the hlb gene was intact. Moreover, the in vitro tests confirmed a different virulence capacity between strains as ST121 showed greater cytotoxicity for erythrocytes, polymorphonuclear leukocytes and macrophages than strain ST96. Differences were also found 7 days after experimental intramammary infection with 100 colony-forming units. The animals inoculated with strain ST121 developed more severe gross and histological mastitis, higher counts of macrophages in tissue and of all the cell populations in peripheral blood, and a significantly larger total number of bacteria than those infected by strain ST96.

Characterization of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates Obtained From Commercial Rabbitries Located in the Iberian Peninsula.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have been a growing problem in human medicine since the 1960s, and more recently in veterinary medicine with the appearance of livestock-associated MRSA (LA-MRSA). Nevertheless, information about the presence of MRSA in rabbits is quite scarce since only one LA-MRSA identification has been previously reported. The present study aimed to determine genotypic characterization by verifying the presence of resistance determinants, virulence, and toxin genes of different S. aureus strains that cause lesions in rabbits, and their phenotypic traits based on the antimicrobial susceptibility profile. The analysis of 240 S. aureus isolates obtained from different lesion types collected from 89 Spanish and Portuguese rabbit commercial farms in the last 4 years (2014-2017) was performed. The methicillin-resistant gene mecA was found in 11.25% of the studied isolates (27 of 240) from 19 farms (13 Spanish and 6 Portuguese). Staphylococcal cassette chromosome mec (SCCmec) typing predominantly revealed type III (n = 15). Additionally, three MRSA isolates carrying the mecC gen were detected in samples from three different farms (two Spanish and one Portuguese). None of the 30 MRSA isolates was PVL-positive or tst-positive. After the multilocus sequence typing (MLST) procedure, 16 belonged to ST2855, 6 to ST146, 6 to ST398, and 2 ST4774. No ST121 isolate was mec-positive. ST398 and ST4774 isolates lacked the immune-evasion-cluster (IEC) genes. ST2855 strains were associated with the presence only of the sak gene, and ST146 isolates were ascribed to IEC type E. Therefore, this is the first description of LA-MRSA from rabbits belonging to ST2855. Interestingly, one ST2855 and two ST4774 isolates were mecC-positive, which could act as a mecC-MRSA reservoir. More studies are needed to further characterize these isolates and their relationship with humans and other animal species.