Production of recombinant proteins including the B-cell epitopes of autolysin A of Staphylococcus aureus isolated from clinical sheep mastitis and their potential for vaccine development.

Staphylococcus aureus is the most common clinical mastitis-associated pathogen in sheep which contributes to reduced welfare of affected animals and, therefore, compromises the quality and quantity of milk production. To prevent mastitis and its spread, it is essential to guarantee adequate breeding conditions and animal health, through the adoption of good farm management practices and the application of suitable biosecurity measures. Vaccination can play a strategic role in prevention, control, and eradication of diseases. The identification of secreted and cellular antigens of the predominant sheep-CC130/ST700/t1773 lineage would assist in the design of effective vaccine against mammary infections caused by S. aureus. In the current study, we carried out a 3D structural prediction analysis with the identification of the best B cell epitopes of the whole and secreted portion of S. aureus AtlA. Fragments of atlA, containing the main predicted epitopes, were amplified, cloned, and expressed in Escherichia coli for recombinant protein production. Two selected clones produced recombinant proteins (rAtl4 and rAtl8) showing strong reactivity with a hyperimmune serum against the native AtlA and with blood sera collected from sheep with clinical S. aureus mastitis. These may represent potential candidate protein-based vaccines able to elicit a protective immune response to be evaluated by vaccination and subsequent challenge of the vaccinated sheep.

Comparative profiling of agr locus, virulence, and biofilm-production genes of human and ovine non-aureus staphylococci.

BACKGROUND: In a collaboration between animal and human health care professionals, we assessed the genetic characteristics shared by non-aureus staphylococci (NAS) infecting humans and dairy ewes to investigate their relatedness in a region concentrating half of the total National sheep stock. We examined by PCR 125 ovine and 70 human NAS for biofilm production, pyrogenic toxins, adhesins, autolysins genes, and accessory gene regulator (agr) locus. The microtiter plate assay (MPA) was used for the phenotypic screening of biofilm production. Ovine NAS included S. epidermidis, S. chromogenes, S. haemolyticus, S. simulans, S. caprae, S. warneri, S. saprophyticus, S. intermedius, and S. muscae. Human NAS included S. haemolyticus, S. epidermidis, S. hominis, S. lugdunensis, S. capitis, S. warneri, S. xylosus, S. pasteuri, and S. saprophyticus subsp. bovis. RESULTS: Phenotypically, 41 (32.8%) ovine and 24 (34.3%) human isolates were characterized as biofilm producers. Of the ovine isolates, 12 were classified as biofilm-producing while the remaining 29 as weak biofilm-producing. All 24 human isolates were considered weak biofilm-producing. Few S. epidermidis isolates harbored the icaA/D genes coding for the polysaccharide intercellular adhesin (PIA), while the bhp, aap, and embp genes coding biofilm accumulation proteins were present in both non-producing and biofilm-producing isolates. Fifty-nine sheep NAS (all S. epidermidis, 1 S. chromogenes, and 1 S. haemolyticus) and 27 human NAS (all S. epidermidis and 1 S. warneri) were positive for the agr locus: agr-3se (57.8%) followed by agr-1se (36.8%) predominated in sheep, while agr-1se (65.4%), followed by agr-2se (34.6%) predominated in humans. Concerning virulence genes, 40, 39.2, 47.2%, 52.8, 80 and 43.2% of the sheep isolates carried atlE, aae, sdrF, sdrG, eno and epbS respectively, against 37.1, 42.8, 32.8, 60, 100 and 100% of human isolates. Enterotoxins and tsst were not detected. CONCLUSIONS: Considerable variation in biofilm formation ability was observed among NAS isolates from ovine and human samples. S. epidermidis was the best biofilm producer with the highest prevalence of adhesin-encoding genes.

Molecular Typing and Antimicrobial Susceptibility Profiles of Streptococcus uberis Isolated from Sheep Milk.

Intramammary infections are a major problem for dairy sheep farms, and Streptococcus uberis is one of the main etiological agents of ovine mastitis. Surveys on antimicrobial resistance are still limited in sheep and characterization of isolates is important for acquiring information on resistance and for optimizing therapy. In this study, a sampling of 124 S. uberis isolates collected in Sardinia (Italy) from sheep milk was analyzed by multilocus-sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) for genetic relatedness. All isolates were also subjected to antimicrobial susceptibility analysis by the disk diffusion test using a panel of 14 antimicrobials. Resistance genes were detected by PCR assays. MLST analysis revealed that the isolates were grouped into 86 sequence types (STs), of which 73 were new genotypes, indicating a highly diverse population of S. uberis. The most frequently detected lineage was the clonal complex (CC)143, although representing only 13.7% of all characterized isolates. A high level of heterogeneity was also observed among the SmaI PFGE profiles, with 121 unique patterns. Almost all (96.8%) isolates were resistant to at least one antimicrobial, while all exhibited phenotypic susceptibility to oxacillin, amoxicillin-clavulanic acid and ceftiofur. Of the antimicrobials tested, the highest resistance rate was found against streptomycin (93.5%), kanamycin (79.8%) and gentamicin (64.5%), followed by novobiocin (25%) and tetracycline-TE (19.3%). Seventy-four (59.7%) isolates were simultaneously resistant to all aminoglycosides tested. Seventeen isolates (13.7%) exhibited multidrug resistance. All aminoglycosides-resistant isolates were PCR negative for aad-6 and aphA-3' genes. Among the TE-resistant isolates, the tetM gene was predominant, indicating that the resistance mechanism is mainly mediated by the protection of ribosomes and not through the efflux pump. Three isolates were resistant to erythromycin, and two of them harbored the ermB gene. This is the first study reporting a detailed characterization of the S. uberis strains circulating in Sardinian sheep. Further investigations will be needed to understand the relationships between S. uberis genotypes, mastitis severity, and intra-mammary infection dynamics in the flock, as well as to monitor the evolution of antimicrobial resistance.

Identification of secreted and cellular antigens of Staphylococcus aureus causing dairy sheep mastitis and their potential for vaccine development.

Staphylococcus aureus is the leading cause of clinical mastitis and is associated with persistent subclinical infections in ewes, significantly compromising the quality and quantity of milk productions. To date, vaccines intended for use in sheep have been mainly focused on biofilm production traits, but many S. aureus pathogenic isolates do not produce biofilm, including those circulating in Sardinia, one of the leading sheep milk producers in Europe. The aim of this work was to identify suitable immunodominant, alternative candidates to biofilm components for vaccine and diagnostic development. An immunoproteomics study was carried out by testing sera from naturally infected sheep with a prevalent S. aureus lineage against cellular and secreted antigens, followed by tandem mass spectrometry identification of the most prominent immunogens. Four cellular and three secreted S. aureus antigens elicited a strong humoral host immune response. The four cellular antigens were the housekeeping proteins pyruvate kinase, elongation Factor Tu, dihydrolipoyl dehydrogenase, and alpha-keto acid dehydrogenase. The three secreted antigens were the bifunctional autolysin (Atl) and the two components of the Panton-Valentine leukocidin, lukF-PV/lukM, demonstrating the carriage of prophage phiPV83 in a sheep isolate and the strong response of the sheep host against them. In consideration of the key role played by these secreted proteins in S. aureus replication and immune evasion, these antigens may represent suitable candidates for developing vaccines eliciting a more successful immunological protection in areas where non-biofilm forming Staphylococcus spp. are the most widespread intramammary pathogens.

Isolation of Leptospira interrogans from a Bottlenose Dolphin (Tursiops truncatus) in the Mediterranean Sea.

The pathogenic Leptospira species are very widespread in nature, persisting in the renal tubules of many domestic and wild animal reservoirs. We report the isolation of Leptospira interrogans serovar Pomona in a bottlenose dolphin (Tursiops truncatus) stranded along the coast of Sardinia, Italy, in 2016.

Discrimination between Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capricolum using PCR-RFLP and PCR.

In this study, the dihydrolipoyl dehydrogenase (lpdA) gene was used to distinguish Mycoplasma mycoides subsp. capri (Mmc) from Mycoplasma capricolum subsp. capricolum (Mcc), two of four Mycoplasma species that cause contagious agalactia in sheep and goats. After alignment of nucleotide sequences of both species, specific primer sets were designed from unchanging and variable gene segments. The first primer set LPD-C1-F/LPD-C1-R was used to amplify a 911 bp fragment that was subsequently co-digested with FastDigest PstI, SspI, EcoRI and ClaI enzymes. The PCR-RFLP profiles differentiated the two mycoplasma species. The second primer set was used to distinguish Mmc from Mcc by single tube PCR. Both methods were further applied to identify 54 isolates collected from dairy herds from different provinces in Sardinia. The results of this study showed that PCR-RFLP and PCR could be used in routine diagnosis for rapid and specific simultaneous discrimination of Mmc and Mcc.

Development of a sensitive and specific enzyme-linked immunosorbent assay based on recombinant antigens for rapid detection of antibodies against Mycoplasma agalactiae in sheep.

We developed a new recombinant enzyme-linked immunosorbent assay (rELISA) for serodiagnosis of contagious agalactia (CA), a disease caused by Mycoplasma agalactiae in sheep and goats. The assay is based on two M. agalactiae surface proteins, namely, P80 and P55. Identification of these immunodominant and common antigens was accomplished by examining the antibody response elicited in sheep during experimental infection and comparing it to the protein expression profiles of 75 M. agalactiae field strains. Our rELISA was tested with 343 sera, collected from sheep with a laboratory-confirmed diagnosis of CA (n = 223) and from healthy animals (n = 120). All sera had previously been tested by Western blotting (WB) for reactivity against M. agalactiae. In addition, our rELISA was compared with a commercial routine ELISA based on inactivated antigens (CHEKiT). Among the 223 samples that were WB positive for M. agalactiae, 209 (93.7%) tested positive for rP80-P55 with our ELISA, whereas only 164 (73.8%) tested positive with the CHEKiT ELISA. Among the 120 samples tested that were WB negative for M. agalactiae, 96.7% were confirmed as negative with our rELISA, while only 75.8% were confirmed as negative with the CHEKiT ELISA. A comparison of the results with receiver operating characteristic curves indicated that the differences observed between our rELISA and the CHEKiT ELISA are statistically significant. The use of recombinant peptides instead of inactivated antigens could significantly improve the discrimination of positive and negative animals, bringing significant advantages in controlling the import/export of live animals and helping in eradication of this economically detrimental disease.