Development of a specific immunomagnetic capture-PCR for rapid detection of viable <i>Mycoplasma agalactiae</i> in sheep milk samples

Aims: To develop an immunomagnetic capture (IMC) to detect viable Mycoplasma agalactiae in routine ovine milk samples. Methods and Results: Polyclonal antibodies against two M. agalactiae membrane surface proteins (P80 and P55) were covalently conjugated to magnetic beads (MBs) to form MB-Ab80 and MB-Ab55. Mycoplasma agalactiae cells were captured by a specific antigen–antibody reaction and magnetic separation. Immunomagnetic capture (IMC) was used to isolate and concentrate M. agalactiae in serial decimal dilutions and in artificially contaminated milk to facilitate subsequent detection by PCR. A 375-bp fragment of M. agalactiae was amplified using a pair of M. agalactiae-specific primers in PCR. The limit of detection of IMC-PCR method ranged from 10 to 102 CCU ml -1 when mycoplasmas were resuspended in PBS and from 102 to 103 CCU ml-1 when mycoplasmas were resuspended in uncontaminated ovine milk. This study also describes the application of IMC-PCR method to test for M. agalactiae in 516 milk samples collected from sheep with suspected contagious agalactia. Its performance was evaluated relative to culture. Conclusions: This report has demonstrated for the first time, the effective use of rapid and reliable IMC combined with PCR assay for the detection of viable M. agalactiae. Significance and Impact of the Study: The method IMC-PCR provides an alternative to conventional microbiological detection, method and it could be applied to quick detection of M. agalactiae in routine sheep milk samples.</br

Detection and characterization of Rickettsial strains in ticks from Sardinia, Italy

The aim of this study was, firstly, to detect the presence of Rickettsial DNA by PCR and, then, to identify the Rickettsiae species using restriction endonuclease fragment length polymorphism (RFLP) on two amplified genes

Detection and characterization of Rickettsial strains in ticks from Sardinia, Italy

The aim of this study was, firstly, to detect the presence of Rickettsial DNA by PCR and, then, to identify the Rickettsiae species using restriction endonuclease fragment length polymorphism (RFLP) on two amplified genes

Species identification by MALDI-TOF MS and gap PCR–RFLP of non-aureus Staphylococcus, Mammaliicoccus, and Streptococcus spp. associated with sheep and goat mastitis

Staphylococci and streptococci are common causes of intramammary infection in small ruminants, and reliable species identification is crucial for understanding epidemiology and impact on animal health and welfare. We applied MALDI-TOF&nbsp;MS and gap PCR-RFLP to 204 non-aureus staphylococci (NAS) and mammaliicocci (NASM) and to 57 streptococci isolated from the milk of sheep and goats with mastitis. The top identified NAS was Staphylococcus epidermidis (28.9%) followed by Staph. chromogenes (27.9%), haemolyticus (15.7%), caprae, and simulans (6.4% each), according to both methods (agreement rate, AR, 100%). By MALDI-TOF MS, 13.2% were Staph. microti (2.9%), xylosus (2.0%), equorum, petrasii and warneri (1.5% each), Staph. sciuri (now Mammaliicoccus sciuri, 1.0%), arlettae, capitis, cohnii, lentus (now M. lentus), pseudintermedius, succinus (0.5% each), and 3 isolates (1.5%) were not identified. PCR-RFLP showed 100% AR for Staph. equorum, warneri, arlettae, capitis, and pseudintermedius, 50% for Staph. xylosus, and 0% for the remaining NASM. The top identified streptococcus was Streptococcus uberis (89.5%), followed by Strep. dysgalactiae and parauberis (3.5% each) and by Strep. gallolyticus (1.8%) according to both methods (AR 100%). Only one isolate was identified as a different species by MALDI-TOF MS and PCR-RFLP. In conclusion, MALDI-TOF MS and PCR-RFLP showed a high level of agreement in the identification of the most prevalent NAS and streptococci causing small ruminant mastitis. Therefore, gap PCR-RFLP can represent a good identification alternative when MALDI-TOF MS is not available. Nevertheless, some issues remain for Staph. haemolyticus, minor NAS species including Staph. microti, and species of the novel genus Mammaliicoccus

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

Investigation into Cryptosporidium and Giardia in bivalve mollusks farmed in Sardinia region and destined for human consumption

Cryptosporidium and Giardia are protozoan parasites transmitted by fecal-oral ingestion of (oo)cysts, and are responsible for enteritis in several animal species and humans worldwide. These (oo)cysts can survive for over a year in aquatic environments and can accumulate in bivalve mollusks, which filter large volumes of water. The aim of this study is to evaluate the natural occurrence of Cryptosporidium and Giardia contamination in different specimens of edible bivalves mollusks from farming sites of the western and north-eastern coasts of Sardinia. From April 2011 to February 2012, 1095 specimens of Mytilus galloprovincialis and 240 of Crassostrea gigas were sampled from Olbia and Oristano gulf and San Teodoro pond. Hepatopancreas and gills, including the labial palp, were examined for oocysts and cysts after pooling and homogenisation using different techniques: i) staining for light and fluorescence microscopy; ii) direct immunofluorescence (IF) Merifluor® test Cryptosporidium/ Giardia (Meridian Bioscience Inc., Cincinnati, OH, USA); and iii) molecular procedures. However, in the context under study, all mollusks examined with the three main diagnostic techniques were negative for both parasites pointing out the hypothetically low zoonotic risk related to Cryptosporidium and Giardia in bivalves, especially Mytilus galloprovincialis and Crassostrea gigas

Staphylococcus aureus and methicillin-resistant staphylococci and mammaliicocci in the bulk tank milk of dairy cows from a livestock-dense area in northern Italy

Staphylococcus aureus is the main etiologic agent of contagious dairy cow mastitis, while non-aureus staphylococci and mammaliicocci (NASM) are the bacteria most frequently isolated from milk. Beyond their impact on animal health, NASM can harbor antimicrobial resistance (AMR) genes with potential for bidirectional transfer with S. aureus, and methicillin-resistant (MR) staphylococci (MRS) can raise significant One Health concerns. In our study, we evaluated the prevalence and characteristics of MRS in the bulk tank milk (BTM) of 88 dairy farms in the livestock-dense province of Lodi, Lombardy, northern Italy. S. aureus was isolated from 32.95 % of BTM samples, with the Ribosomal Spacer PCR (RS-PCR) genotype B being the most prevalent, identified in 37.93 % of S. aureus positive farms. All isolates carried the ica genes (icaA, icaB, icaC, icaD) indicating the potential to produce biofilm. MRS were isolated in 56.81 % of farms. According to MALDI-TOF MS analysis, the most prevalent MR species included S. epidermidis (MRSE, 35.59 %) followed by S. aureus (MRSA, 18.64 %), M. sciuri (15.25 %), S. saprophyticus (11.86 %), S. borealis (6.78 %), S. haemolyticus (5.08 %), M. fleurettii, (3.39 %), S. cohnii, and S. pettenkoferi (1.70 % each). Most MR isolates carried the mecA gene, while none carried mecC. The staphylococcal cassette chromosome mec (SCCmec) was predominantly type V in MRSA (45.45 %) and type IV in MRSE (61.90 %). Given their relevance to One Health, monitoring AMR in all staphylococci and mammaliicocci isolated from milk is essential for understanding the prevalence, characteristics, and transmission dynamics of MR gene pools within dairy herds