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

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

Antimicrobial susceptibilities and population structure of <i>Staphylococcus epidermidis</i> associated with ovine mastitis

Intramammary infections are a serious problem for dairy sheep farms, and Staphylococcus epidermidis is one of the main etiological agents of ovine mastitis. In this work, 131 S. epidermidis isolates, collected from 2201 dairy Sarda sheep belonging to 14 flocks with high somatic cell count scores, were studied. The flocks were located in diverse geographical areas of Sardinia, Italy. The aim of study was to assess the susceptibility of isolates to 13 antimicrobial agents, many of which are frequently used in mastitis therapy. Oxacillin was used for detecting methicillin-resistant S. epidermidis (MRSE) by disk diffusion test. Thirty-eight percent of the isolates (n = 50) were resistant to penicillin, 7.6% (n = 10) were resistant to tetracycline, and 2.3% (n = 3) were resistant to both penicillin and tetracycline (PTRSE). Two isolates were resistant to five antimicrobials including methicillin. Analysis of staphylococcal cassette chromosome mec (SCCmec) elements showed that both MRSE isolates harbored SCCmec type IVa. Based on pulsed-field gel electrophoresis (PFGE) typing by SmaI macrorestriction, S. epidermidis isolates were grouped into four clusters at 75% similarity level. The two multi-drug resistant MRSE isolates displayed distinct PFGE patters. This study indicates that S. epidermidis isolates from sheep milk samples may accumulate resistance markers for different antimicrobial agents. Furthermore, the occurrence of PTRSE and MRSE suggests to adopt adequate hygienic measures when handling animals with intramammary infections, in order to prevent spreading PTRSE and MRSE strains to humans through direct contact and/or consumption of contaminated food

Clinical findings in sheep farms affected by recurrent bacterial mastitis

This study was aimed to investigate the relationships existing between clinical findings and bacterial entities isolated from milk of dairy sheep affected by mastitis. The influence of other parameters on the clinical picture, such as age, nutritional state, breeding conditions, and milking techniques, was also evaluated. All sheep belonged to flocks suffering from serious and repeated outbreaks of infectious mastitis. A total of 2198 Sarda dairy sheep were subjected to a detailed clinical examination, and at least one clinical sign of mastitis was detected in 1666 sheep (75%). Bacteriological examination of milk samples collected from all animals produced 1093 positive results (49.7%). Of bacterial species identified, three accounted for 55.3% of all isolates: Streptococcus uberis (25.6% of positives and 12.7% of total), Staphylococcus epidermidis (16.2% of positives and 8% of total), and Staphylococcus aureus (13.5% of positives and 6.7% of total). Upon investigation of correlations existing among clinical signs and bacterial species responsible for the outbreak, S. uberis showed a statistically significant correlation with serous appearance of milk, presence of clots in secretions, and reactivity of supramammary lymph nodes (p &lt; 0.05); S. epidermidis showed a statistically significant correlation with presence of pustules and ulcers (p &lt; 0.05); and S. aureus showed a statistically significant correlation with clinical signs of chronic mastitis: nodules, abscesses, and atrophy (p &lt; 0.05%). Manual milking techniques were more associated to udder infections than mechanical milking. However, an interesting correlation emerged between presence of S. uberis and mechanical milking with small portable devices. In conclusion, this study revealed interesting and unprecedented correlations among clinical signs, bacterial species isolated from infected milk, and farm management techniques. The results reported here emphasize the primary role played by clinical practice in managing infectious ovine mastitis outbreaks, and strengthen its relevance for recovery of affected flocks

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

Comparazione fra quadri clinici, rilievi microbiologici nel latte e altri parametri di allevamento in greggi caprine con problemi di mastite infettiva

La ricerca è stata condotta su 1388 capre appartenenti a 31 allevamenti distribuiti su tutto il territorio della regione Sardegna. Nell’isola si alleva circa un quarto dell’intero patrimonio caprino dell’Italia. Il lavoro ha previsto la registrazione di dati e informazioni legate ai singoli allevamenti, una visita clinica dettagliata della mammella degli animali in lattazione con registrazione di presenzaassenza dei segni clinici ed esami microbiologici dei campioni di latte. I dati raccolti sono stati sottoposti ad analisi statistica per evidenziare eventuali correlazioni esistenti