Purpose. Alternatives to antibiotic therapy for mastitis in ruminants are needed. We present an e... more Purpose. Alternatives to antibiotic therapy for mastitis in ruminants are needed. We present an evaluation, in two trials, of the efficacy of an intramammary infusion of a live culture of Lactococcus lactis for the treatment of subclinical and clinical mastitis in ewes. Methodology. In total, 67 animals were enrolled: 19 lactating ewes (study 1), including healthy (N=6) and coagulase-negative staphylococci (CNS)-infected ewes (N=13); and 48 lactating ewes (study 2) with either CNS mastitis (N=32), or Staphylococcus aureus mastitis (N=16), for a total of 123 mammary glands. Intramammary infusions were performed with either L. lactis or PBS for 3 (study 1) or 7 (study 2) consecutive days. Antibiotic-treated and untreated control glands were included. Milk samples for microbiology, somatic cell analysis and milk production were collected before and after treatment. Results/Key findings. L. lactis rapidly activated the mammary glands' innate immune response and initiated an inflammatory response as evidenced by the recruitment of polymorphonuclear neutrophils and increased somatic cell counts. But while leading to a transient clearance of CNS in the gland, this response caused mild to moderate clinical cases of mastitis characterized by abnormal milk secretions and udder inflammation. Moreover, S. aureus infections did not improve, and CNS infections tended to relapse. Under our experimental conditions, the L. lactis treatment led to a transient clearance of the pathogen in the gland, but also caused mild to moderate clinical cases of mastitis. We believe it is still early to implement bacterial formulations as alternatives in treating mastitis in ruminants and further experimentation is needed.
The intramammary infusion of lactic acid bacteria has recently emerged as a potential new alterna... more The intramammary infusion of lactic acid bacteria has recently emerged as a potential new alternative to antibiotics for preventing and treating bovine mastitis. in this study we first assessed in vitro the probiotic potential of Lactococcus lactis LMG 7930. We then evaluated, in two trials, the efficacy and safety of the intramammary infusion of a live overnight culture of L. lactis for the treatment of subclinical and clinical mastitis in lactating ewes. Finally, we in-depth investigated the mammary gland immune response induced by L. lactis intramammary inoculum using the mouse mastitis model. We found that L. lactis showed in vitro probiotic properties. In vivo, L. lactis rapidly activated the mammary innate immune system. This led to a transient clearance of the pathogen in the gland, but also caused both mild and moderate inflammations in ewes. in the mouse mammary gland, L. lactis inoculum caused variable histological features, ranging from undamaged tissue to severe inflammation. in conclusion, our results suggest that L. lactis can act as mastitis-causing pathogens when inoculated at high density into the mammary glands of mammals. The use of bacterial formulations as alternatives for the treatment of mastitis in ruminants remains in need of further experimentation.
The study summarises the results obtained over the period 2002-2013 by the Italian IT-Enter-Vet n... more The study summarises the results obtained over the period 2002-2013 by the Italian IT-Enter-Vet network, aimed at collecting data on Salmonella isolates from non-human sources. A total of 42,491 Salmonella isolates were reported with a progressive decrease over the years. S. Typhimurium was the most frequent serovar up to 2011, but then, it was overtaken by S. 4,[5],12,:i:-, S. Derby, S. Livingstone and S. Enteritidis alternated as the third most commonly isolated serovars. With regard to the sources of isolation, S. Typhimurium was distributed ubiquitously among the animal species. On the contrary, S. 4,[5],12,:i:-and S. Derby were strictly associated with pigs, whereas S. Livingstone, S. Enteritidis and S. Infantis were clearly related to poultry. Intriguingly, when the frequency of serovar distribution along the food chain was considered, it was evident that S. Typhimurium and S. Derby tended to persist along the chain, as they were isolated even more frequently from foods than from animals. A similar distribution was found for S. Enteritidis and S. Hadar. Despite limitations related to nonmandatory participation of laboratories in the network, the data presented are valuable to obtain a picture of the evolution of Salmonella from non-human sources over time in Italy.
In this study, a bank of 195 Streptococcus isolates collected in Sardinia (Italy) from sheep and ... more In this study, a bank of 195 Streptococcus isolates collected in Sardinia (Italy) from sheep and goat milk samples was analysed. The purpose of this research was to compare the accuracy of different methods of identifying Streptococcus at the species level: API® 20 Strep, PCR-RFLP and MALDI-TOF MS. For PCR-RFLP analysis, the housekeeping gap gene was used as target gene. After alignment of nucleotide sequences of different Streptococcus species, a primer set was designed to amplify a 945-bp DNA fragment. Amplicons of gap gene were submitted to restriction fragment length polymorphism analysis with the enzyme AluI. When PCR-RFLP patterns were different from those of their reference strains, gap gene products were sequenced for definitive identification. Most of the isolates displayed the same PCR-RFLP profile of Streptococcus uberis type strain (124, corresponding to 63.6%), followed by S. dysgalactiae (18, corresponding to 9.2%) and S. suis (14, corresponding to 7.2%). Overall, in comparison with PCR-RFLP and sequence analysis results, 161 over 195 Streptococcus isolates were identified to the same species levels by means API® 20 Strep (82.6% agreement, CI95 = 77.2-87.9) versus 166/195 (85.1% agreement, CI95 = 80.1-90.1) using MALDI-TOF. In conclusion, the PCR-RFLP is the most accurate and reproducible method for the identification of streptococci collected from milk samples, although high cost and technical difficulty hinder its usage in routine diagnosis. In our study, the accuracy of MALDI-TOF MS in detecting streptococci at the species level has not been perfect; however we believe that MALDI-TOF has the advantage in terms of its rapidity, reliability and low cost.
The aim of this study was to evaluate the prevalence of Salmonella spp. in the Sardinian pig prod... more The aim of this study was to evaluate the prevalence of Salmonella spp. in the Sardinian pig production chain in order to establish the incidence of monophasic serovariant of Salmonella Typhimurium on isolates with molecular methods (real-time PCR and multiplex PCR). Samples were collected in three EC slaughterhouses, four small slaughterhouses annexed to farmhouses, one meat distribution center, four meat cutting laboratories and four sausage processing plants. A total of 166 samples were collected and analyzed: 46 environmental samples, 48 finishing pigs, 16 piglets, 24 samples of non-processed meat, 28 meat preparations and 4 meat products. All samples were processed with an initial screening using the real-time PCR MicroSEQ® Salmonella spp detection Kit (Applied biosystems, life technologies) and with the TaqMan® Real-time PCR to confirm the kit results. Samples that tested positive for Salmonella spp were confirmed with cultural method using the standard ISO 6579. Positive samples were submitted to phenotypic identification. One colony from each positive sample was serotyped with multiplex PCR method. Salmonella spp was isolated in 7 on 166 samples (4.22 %). Among the positive samples, two came from finishing pigs, two belonged to the category meat preparations, two to meat products, one was an environmental sample. Multiplex PCR confirmed that the collected strains belonged to the species Salmonella Typhimurium (1), Salmonella derby (3) and monophasic serovariant of Salmonella Typhimurium (3).
Latvian dairy goat breeds as Saanen, Latvian Native and crossed breed suitability for cheese prod... more Latvian dairy goat breeds as Saanen, Latvian Native and crossed breed suitability for cheese production.
Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacteriu... more Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. To accelerate the detection of viable pathogen, a conventional (peptide mediated magnetic separation: PMS) and novel (phagebead qPCR: PBQ) phage based assay was optimized. A superior limit of detection (LOD) of 10 MAP per 10 mL milk was suggested for PBQ compared to 100 cells/10 mL for PMS-phage assay. Via PBQ, viable MAP was found in 48.78% out 41 unpasteurized sheep and goat milk samples. Sheep milk samples (n = 29) that were tested by PMS-phage assay contained no viable MAP. The absence of viable MAP in milk collected from 21 of the recent sheep animals was also confirmed by PBQ after a 2-week gap. Although, the two phage assays comparably detected no viable MAP in the milk samples, MAP DNA and antibodies against MAP were recognized in milk and sera of some of these animals within two instances of sampling representing that some sheep animals were MAP shedders. In conclusion, PBQ and PMS-phage could be promising methods for the assessment of MAP viability in milk samples. However, PBQ was privileged over the PMS-phage assay due to the lower LOD, rapidity, higher sensitivity, lack of need to M. smegmatis and consequent virucidal treatment that are essential in PMS-phage assay for making lawn and inactivation of exogenous mycobacteriophages respectively. Paratuberculosis or Johne's disease (JD) is a chronic gastrointestinal condition among ruminants 1 that is triggered by Mycobacterium avium subsp. paratuberculosis (MAP) 2 , an acid fast bacillus with complicated growth requirements 3 . MAP-infected animals frequently excrete the pathogen into milk or the environment via feces and put the susceptible animals at risk of the disease 4,5 . Detection of viable MAP could impede the circulation of the pathogen to new recipients remarkably. Many studies have consensus on the strength of culture as a confirmatory assay for the diagnosis of MAP viability in clinical specimens such as milk, blood, and feces. However, MAP has a lengthy generation time (more than 24 h) that elongates the isolation of bacterium to 7-16 weeks . Recently, the selective capture of viable bacteria in milk and other matrixes has been facilitated via biotechnological approaches such as magnetic separation (MS) through purifying the target bacteria and reducing the background signals . Up to now, MS underwent modifications and the capture efficiency of magnetic beads was improved through the application of MAP-specific ligands such as antibodies (monoclonal/polyclonal) and peptides . MAP-complementary peptides of aMp3 and aMptD are specific ligands that routinely used in MS for assessment of MAP viability in various samples. aMp3-and aMptD-mediated magnetic beads could retrieve MAP in specimens containing 10 4 to 10 3 cfu/mL of the bacterium by 85-100% reducing the rate of cross-reactivity with close mycobacterial species to less than 1% 13 . The functionality of peptide mediated magnetic bead separation (PMS), in isolation and integrated with other confirmatory diagnostic methods such as culture, qPCR IS900 (Insertion sequence IS900 is a conserved region that has 16-22 copies in whole MAP genome and is usually targeted in molecular diagnosis of MAP ), immunoassays (e.g. antigen detection immunoassay), and phage assay 13 have been evaluated in few studies . Currently, PMS-phage assay introduced a remarkable velocity to the MAP-viability assessments reducing the diagnostic time to only 48 h 23 . D29 is a tailed-lytic mycobacteriophage that is frequently applied in PMS-phage assay and it has various mycobacterial hosts such as M.
Abstract: An LC–Orbitrap–HRMS method was developed and validated for the simultaneous
determinati... more Abstract: An LC–Orbitrap–HRMS method was developed and validated for the simultaneous determination of four tetracyclines—oxytetracycline (OTC), tetracycline (TC), doxycycline (DC), and chlortetracycline (CTC)—in milk. This method involves sample extraction with McIlvaine– EDTA buffer solution (pH 4) and solid-phase extraction (SPE) with Oasis HLB cartridges, followed by the evaporation of the extract and its reconstitution with a 14% methanol aqueous solution before injection into the instrumental system. This method has been validated in terms of linearity, sensitivity, selectivity, precision, and accuracy, in accordance with Commission Decision 2002/657/EC requirements. Compared to existing methods, this approach optimally combines a quantitative procedure for extracting analytes from the milk of different species, including sheep, bovines, and goats, with a very short LC–Orbitrap–HRMS instrumental analysis time (only 8 min), simultaneously ensuring high precision, sensitivity, and applicability as a rapid confirmation method in official food control laboratories. The proposed method was applied to determine the concentration levels of OTC in milk samples derived from healthy Sardinian sheep and those naturally infected with Streptococcus uberis, after the intramuscular administration of an antibiotic, in order to evaluate how much of the drug was “subtracted” during penetration from blood into milk, with a potential effect of reducing its therapeutic efficacy.
Premessa. Assistiamo con incredulità al diffondersi di convinzioni pseudoscientifiche, reso ancor... more Premessa. Assistiamo con incredulità al diffondersi di convinzioni pseudoscientifiche, reso ancora più acuto dalla pandemia Covid-19 e da certe sue interpretazioni sconfinanti nell’irrazionale. Intendiamo riferirci a quelle convinzioni non confortate da prove evidenti e accettabili e per le quali non sono immaginabili conferme o verifiche che ne possano mettere in crisi le affermazioni1. Il presente contributo esamina e intende fornire possibili interpretazioni al fenomeno delle false credenze, oggi amplificato dalla rete e dai social, e si interroga su che cosa porti al rifiuto delle evidenze scientifiche e a idee cospirative, rivolgendo l’attenzione in particolar modo ai fattori culturali. L’articolo tratta principalmente delle credenze arrivate alla ribalta mediatica con la pandemia Covid-19, come la sua negazione, le teorie complottiste sorte in risposta e la negazione dell’efficacia dei vaccini. Proprio quest’ultima convinzione, scelta come esempio, viene esaminata con maggiore attenzione, in quanto legata a un milieu più complesso di ragioni culturali, ideologiche e sociali, seppure spesso professata da soggetti che accettano anche altre credenze2. Le conclusioni proposte potranno comunque essere generalizzate.
Nel presente studio è stata valutata l'utilità della diagnosi immunoistochimica su encefali d... more Nel presente studio è stata valutata l'utilità della diagnosi immunoistochimica su encefali di ovini e caprini con lesioni istologiche riferibili a Listeria monocytogenes , correlandole, ove disponibile, con l'esame batteriologico; è inoltre stata valutata l'efficacia di alcuni trattamenti di smascheramento antigenico, già risultati efficaci nell'aumentare la sensibilità tecnica quando applicati nella identificazione di altri antigeni localizzati a livello encefalico.
Le mastiti rappresentano la patologia più diffusa nel settore ovino e caprino da latte e sono res... more Le mastiti rappresentano la patologia più diffusa nel settore ovino e caprino da latte e sono responsabili d'importanti perdite economiche dovute alla riduzione e scadimento della qualità delle produzioni, alla perdita di animali e ai costi sanitari. La conoscenza dell'eziologia e della reale diffusione della malattia nel territorio costituisce uno strumento basilare per elaborare e verificare l'efficacia di piani di controllo e risanamento delle patologie mammarie; rappresenta, inoltre, un elemento utile per indirizzare gli investimenti nell'ambito della programmazione sanitaria. Differenti, infatti, sono le strategie da adottare e i risultati attesi nella lotta alle mastiti in rapporto al microrganismo coinvolto che può essere responsabile di mastiti contagiose o ambientali.
In Italy Bovine Tubercolosis (TB) eradication program has been run since 1995, it is based on a s... more In Italy Bovine Tubercolosis (TB) eradication program has been run since 1995, it is based on a single intradermal skin test, slaughter of positive animals and post-mortem inspection. Sardinia, Italy´s second largest island in the Mediterranean Sea, was officially declared Tubercolosis-free in 1998, according to a National law (DM 592/95). Although this programme fulfilled our law requirements, it failed to eradicate TB: in fact, a few outbreaks of the disease still occur. The spread of TB infection occurred in 2007 in extensive cattle herds located in a defined area of Sardinia, named Goceano. In this area, on the basis of the skin test and/or gamma interferon positivity, 700 animals, belonging to 75 affected herds, have been slaughtered during three years. Moreover, the presence of TB infection was monitored in wildlife. We randomly checked wild boar population by inspection of head lymph nodes and, in addition, we collected several samples during two hunting seasons (2007/2008 an...
In this study, we examined in Sardinia the brain of 555 autochthonous sheep, 50 goats, and 4 mouf... more In this study, we examined in Sardinia the brain of 555 autochthonous sheep, 50 goats, and 4 mouflons which were found affected by neurological signs. We found 6 goats and one mouflon with meningoencephalitis caused by Cryptococcus sp. There was no evidence of cryptococcal infections in any of the examined sheep. MLST genotyping on Cryptococcus sp. isolates identified Cryptococcus gatti genotype AFLP4/VGI and Cryptococcus neoformans var. neoformans genotype AFLP2/VNIV. Phylogenetically, all Cryptococcus gattii isolates fell within the autochthonous animal, human and environmental Mediterranean isolate cluster, forming a distinct branch along with environmental strains from Alicante, in the southern Mediterranean coast of Spain.
Counting 3,300,000 heads and over 15,000 herds dairy sheep farming is the main agricultural busin... more Counting 3,300,000 heads and over 15,000 herds dairy sheep farming is the main agricultural business in Sardinia (Italy). Mastitis represents the most widespread pathology in dairy sheep and it is responsible for significant economic losses due to the decrease of milk quality and yield, animal replacement and health care costs. Knowing about aetiology and the rates of mastitis sprerad can help to develop disease control strategies based on characteristics of heach bacterial species involved.
In the present paper, a Quality Systems Theory is presented. Certifiable Quality Systems are trea... more In the present paper, a Quality Systems Theory is presented. Certifiable Quality Systems are treated and interpreted in accordance with a Thermodynamics-based approach. Analysis is also conducted on the relationship between Quality Management Systems (QMSs) and systems theories. A measure of entropy is proposed for QMSs, including a virtual document entropy and an entropy linked to processes and organisation. QMSs are also interpreted in light of Cybernetics, and interrelations between Information Theory and quality are also highlighted. A measure for the information content of quality documents is proposed. Such parameters can be used as adequacy indices for QMSs. From the discussed approach, suggestions for organising QMSs are also derived. Further interpretive thermodynamic-based criteria for QMSs are also proposed. The work represents the first attempt to treat quality organisational systems according to a thermodynamics-related approach. At this stage, no data are available to compare statements in the paper.
The identification of coagulase-negative staphylococci (CNS) causing ovine infections remains pro... more The identification of coagulase-negative staphylococci (CNS) causing ovine infections remains problematic, although these bacteria are considered the main etiologic agents of subclinical mastitis in sheep and goats. In this study, 226 CNS isolates were collected from 2201 milking sarda sheep belonging to 15 flocks with high somatic cell count scores. All isolates were subjected to identification with the API Staph ID test, and then to the amplification of staphylococcal 16S rRNA and gap genes by PCR assays. The gap gene was subjected to restriction fragment length polymorphism analysis with the restriction endonuclease AluI, whereas the 16S rRNA gene was subjected to ribosomal fingerprinting with the restriction endonucleases RsaI, PstI and AluI. When PCR–RFLP patterns of CNS isolates were different from those of their reference strains, gap gene amplicons were sequenced for definitive identification. The API Staph ID test, in alternative to the genotypic identification method, produced considerably different results in terms of species identified within each group. Using the PCR–RFLP assay, most of the isolates clustered together with the Staphylococcus epidermidis type strain (131, corresponding to 57.9%), followed by S. caprae (34, corresponding to 15%) and S. chromogenes (30, corresponding to 13.2%). In conclusion, the PCR–RFLP assay of 16S rRNA and gap genes is a more reliable and reproducible method than the API Staph ID test for the identification of CNS causing sheep mastitis.
This study was aimed to investigate the relationships existing between clinical findings and bact... more 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 < 0.05); S. epidermidis showed a statistically significant correlation with presence of pustules and ulcers (p < 0.05); and S. aureus showed a statistically significant correlation with clinical signs of chronic mastitis: nodules, abscesses, and atrophy (p < 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.
Antibiograms of selected mastitis pathogens, performed during the decade 1995-2004, were retrospe... more Antibiograms of selected mastitis pathogens, performed during the decade 1995-2004, were retrospectively analysed in order to evaluate antimicrobial resistance and determine whether resistance changed over time. Results of 2763 strains of Staphylococcus aureus, coagulase negative staphylococci, Streptococcus uberis and Escherichia coli, against penicillin, ampicillin, oxytetracycline, kanamycin and streptomycin are discussed. Strains were isolated in clinical milk samples from sheep suspected of mastitis. The evolution over time of resistance was evaluated by means of logistic regression analysis. The resistance of staphylococci to penicillin appeared to be lower than those usually reported (4.1% of resistant strains in S. aureus; 15.3% for CNS). Higher rates of resistance were observed for aminoglycosides, relevant for S. uberis (84.5% for kanamycin and 92.5% for streptomycin) and S. aureus (14.6% for kanamycin and 63.3% for streptomycin). Overall resistance appeared to confirm the lower resistance in ovine pathogens than in bovine ones. Logistic regression highlighted no trends to increase for resistance over time.
Purpose. Alternatives to antibiotic therapy for mastitis in ruminants are needed. We present an e... more Purpose. Alternatives to antibiotic therapy for mastitis in ruminants are needed. We present an evaluation, in two trials, of the efficacy of an intramammary infusion of a live culture of Lactococcus lactis for the treatment of subclinical and clinical mastitis in ewes. Methodology. In total, 67 animals were enrolled: 19 lactating ewes (study 1), including healthy (N=6) and coagulase-negative staphylococci (CNS)-infected ewes (N=13); and 48 lactating ewes (study 2) with either CNS mastitis (N=32), or Staphylococcus aureus mastitis (N=16), for a total of 123 mammary glands. Intramammary infusions were performed with either L. lactis or PBS for 3 (study 1) or 7 (study 2) consecutive days. Antibiotic-treated and untreated control glands were included. Milk samples for microbiology, somatic cell analysis and milk production were collected before and after treatment. Results/Key findings. L. lactis rapidly activated the mammary glands' innate immune response and initiated an inflammatory response as evidenced by the recruitment of polymorphonuclear neutrophils and increased somatic cell counts. But while leading to a transient clearance of CNS in the gland, this response caused mild to moderate clinical cases of mastitis characterized by abnormal milk secretions and udder inflammation. Moreover, S. aureus infections did not improve, and CNS infections tended to relapse. Under our experimental conditions, the L. lactis treatment led to a transient clearance of the pathogen in the gland, but also caused mild to moderate clinical cases of mastitis. We believe it is still early to implement bacterial formulations as alternatives in treating mastitis in ruminants and further experimentation is needed.
The intramammary infusion of lactic acid bacteria has recently emerged as a potential new alterna... more The intramammary infusion of lactic acid bacteria has recently emerged as a potential new alternative to antibiotics for preventing and treating bovine mastitis. in this study we first assessed in vitro the probiotic potential of Lactococcus lactis LMG 7930. We then evaluated, in two trials, the efficacy and safety of the intramammary infusion of a live overnight culture of L. lactis for the treatment of subclinical and clinical mastitis in lactating ewes. Finally, we in-depth investigated the mammary gland immune response induced by L. lactis intramammary inoculum using the mouse mastitis model. We found that L. lactis showed in vitro probiotic properties. In vivo, L. lactis rapidly activated the mammary innate immune system. This led to a transient clearance of the pathogen in the gland, but also caused both mild and moderate inflammations in ewes. in the mouse mammary gland, L. lactis inoculum caused variable histological features, ranging from undamaged tissue to severe inflammation. in conclusion, our results suggest that L. lactis can act as mastitis-causing pathogens when inoculated at high density into the mammary glands of mammals. The use of bacterial formulations as alternatives for the treatment of mastitis in ruminants remains in need of further experimentation.
The study summarises the results obtained over the period 2002-2013 by the Italian IT-Enter-Vet n... more The study summarises the results obtained over the period 2002-2013 by the Italian IT-Enter-Vet network, aimed at collecting data on Salmonella isolates from non-human sources. A total of 42,491 Salmonella isolates were reported with a progressive decrease over the years. S. Typhimurium was the most frequent serovar up to 2011, but then, it was overtaken by S. 4,[5],12,:i:-, S. Derby, S. Livingstone and S. Enteritidis alternated as the third most commonly isolated serovars. With regard to the sources of isolation, S. Typhimurium was distributed ubiquitously among the animal species. On the contrary, S. 4,[5],12,:i:-and S. Derby were strictly associated with pigs, whereas S. Livingstone, S. Enteritidis and S. Infantis were clearly related to poultry. Intriguingly, when the frequency of serovar distribution along the food chain was considered, it was evident that S. Typhimurium and S. Derby tended to persist along the chain, as they were isolated even more frequently from foods than from animals. A similar distribution was found for S. Enteritidis and S. Hadar. Despite limitations related to nonmandatory participation of laboratories in the network, the data presented are valuable to obtain a picture of the evolution of Salmonella from non-human sources over time in Italy.
In this study, a bank of 195 Streptococcus isolates collected in Sardinia (Italy) from sheep and ... more In this study, a bank of 195 Streptococcus isolates collected in Sardinia (Italy) from sheep and goat milk samples was analysed. The purpose of this research was to compare the accuracy of different methods of identifying Streptococcus at the species level: API® 20 Strep, PCR-RFLP and MALDI-TOF MS. For PCR-RFLP analysis, the housekeeping gap gene was used as target gene. After alignment of nucleotide sequences of different Streptococcus species, a primer set was designed to amplify a 945-bp DNA fragment. Amplicons of gap gene were submitted to restriction fragment length polymorphism analysis with the enzyme AluI. When PCR-RFLP patterns were different from those of their reference strains, gap gene products were sequenced for definitive identification. Most of the isolates displayed the same PCR-RFLP profile of Streptococcus uberis type strain (124, corresponding to 63.6%), followed by S. dysgalactiae (18, corresponding to 9.2%) and S. suis (14, corresponding to 7.2%). Overall, in comparison with PCR-RFLP and sequence analysis results, 161 over 195 Streptococcus isolates were identified to the same species levels by means API® 20 Strep (82.6% agreement, CI95 = 77.2-87.9) versus 166/195 (85.1% agreement, CI95 = 80.1-90.1) using MALDI-TOF. In conclusion, the PCR-RFLP is the most accurate and reproducible method for the identification of streptococci collected from milk samples, although high cost and technical difficulty hinder its usage in routine diagnosis. In our study, the accuracy of MALDI-TOF MS in detecting streptococci at the species level has not been perfect; however we believe that MALDI-TOF has the advantage in terms of its rapidity, reliability and low cost.
The aim of this study was to evaluate the prevalence of Salmonella spp. in the Sardinian pig prod... more The aim of this study was to evaluate the prevalence of Salmonella spp. in the Sardinian pig production chain in order to establish the incidence of monophasic serovariant of Salmonella Typhimurium on isolates with molecular methods (real-time PCR and multiplex PCR). Samples were collected in three EC slaughterhouses, four small slaughterhouses annexed to farmhouses, one meat distribution center, four meat cutting laboratories and four sausage processing plants. A total of 166 samples were collected and analyzed: 46 environmental samples, 48 finishing pigs, 16 piglets, 24 samples of non-processed meat, 28 meat preparations and 4 meat products. All samples were processed with an initial screening using the real-time PCR MicroSEQ® Salmonella spp detection Kit (Applied biosystems, life technologies) and with the TaqMan® Real-time PCR to confirm the kit results. Samples that tested positive for Salmonella spp were confirmed with cultural method using the standard ISO 6579. Positive samples were submitted to phenotypic identification. One colony from each positive sample was serotyped with multiplex PCR method. Salmonella spp was isolated in 7 on 166 samples (4.22 %). Among the positive samples, two came from finishing pigs, two belonged to the category meat preparations, two to meat products, one was an environmental sample. Multiplex PCR confirmed that the collected strains belonged to the species Salmonella Typhimurium (1), Salmonella derby (3) and monophasic serovariant of Salmonella Typhimurium (3).
Latvian dairy goat breeds as Saanen, Latvian Native and crossed breed suitability for cheese prod... more Latvian dairy goat breeds as Saanen, Latvian Native and crossed breed suitability for cheese production.
Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacteriu... more Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. To accelerate the detection of viable pathogen, a conventional (peptide mediated magnetic separation: PMS) and novel (phagebead qPCR: PBQ) phage based assay was optimized. A superior limit of detection (LOD) of 10 MAP per 10 mL milk was suggested for PBQ compared to 100 cells/10 mL for PMS-phage assay. Via PBQ, viable MAP was found in 48.78% out 41 unpasteurized sheep and goat milk samples. Sheep milk samples (n = 29) that were tested by PMS-phage assay contained no viable MAP. The absence of viable MAP in milk collected from 21 of the recent sheep animals was also confirmed by PBQ after a 2-week gap. Although, the two phage assays comparably detected no viable MAP in the milk samples, MAP DNA and antibodies against MAP were recognized in milk and sera of some of these animals within two instances of sampling representing that some sheep animals were MAP shedders. In conclusion, PBQ and PMS-phage could be promising methods for the assessment of MAP viability in milk samples. However, PBQ was privileged over the PMS-phage assay due to the lower LOD, rapidity, higher sensitivity, lack of need to M. smegmatis and consequent virucidal treatment that are essential in PMS-phage assay for making lawn and inactivation of exogenous mycobacteriophages respectively. Paratuberculosis or Johne's disease (JD) is a chronic gastrointestinal condition among ruminants 1 that is triggered by Mycobacterium avium subsp. paratuberculosis (MAP) 2 , an acid fast bacillus with complicated growth requirements 3 . MAP-infected animals frequently excrete the pathogen into milk or the environment via feces and put the susceptible animals at risk of the disease 4,5 . Detection of viable MAP could impede the circulation of the pathogen to new recipients remarkably. Many studies have consensus on the strength of culture as a confirmatory assay for the diagnosis of MAP viability in clinical specimens such as milk, blood, and feces. However, MAP has a lengthy generation time (more than 24 h) that elongates the isolation of bacterium to 7-16 weeks . Recently, the selective capture of viable bacteria in milk and other matrixes has been facilitated via biotechnological approaches such as magnetic separation (MS) through purifying the target bacteria and reducing the background signals . Up to now, MS underwent modifications and the capture efficiency of magnetic beads was improved through the application of MAP-specific ligands such as antibodies (monoclonal/polyclonal) and peptides . MAP-complementary peptides of aMp3 and aMptD are specific ligands that routinely used in MS for assessment of MAP viability in various samples. aMp3-and aMptD-mediated magnetic beads could retrieve MAP in specimens containing 10 4 to 10 3 cfu/mL of the bacterium by 85-100% reducing the rate of cross-reactivity with close mycobacterial species to less than 1% 13 . The functionality of peptide mediated magnetic bead separation (PMS), in isolation and integrated with other confirmatory diagnostic methods such as culture, qPCR IS900 (Insertion sequence IS900 is a conserved region that has 16-22 copies in whole MAP genome and is usually targeted in molecular diagnosis of MAP ), immunoassays (e.g. antigen detection immunoassay), and phage assay 13 have been evaluated in few studies . Currently, PMS-phage assay introduced a remarkable velocity to the MAP-viability assessments reducing the diagnostic time to only 48 h 23 . D29 is a tailed-lytic mycobacteriophage that is frequently applied in PMS-phage assay and it has various mycobacterial hosts such as M.
Abstract: An LC–Orbitrap–HRMS method was developed and validated for the simultaneous
determinati... more Abstract: An LC–Orbitrap–HRMS method was developed and validated for the simultaneous determination of four tetracyclines—oxytetracycline (OTC), tetracycline (TC), doxycycline (DC), and chlortetracycline (CTC)—in milk. This method involves sample extraction with McIlvaine– EDTA buffer solution (pH 4) and solid-phase extraction (SPE) with Oasis HLB cartridges, followed by the evaporation of the extract and its reconstitution with a 14% methanol aqueous solution before injection into the instrumental system. This method has been validated in terms of linearity, sensitivity, selectivity, precision, and accuracy, in accordance with Commission Decision 2002/657/EC requirements. Compared to existing methods, this approach optimally combines a quantitative procedure for extracting analytes from the milk of different species, including sheep, bovines, and goats, with a very short LC–Orbitrap–HRMS instrumental analysis time (only 8 min), simultaneously ensuring high precision, sensitivity, and applicability as a rapid confirmation method in official food control laboratories. The proposed method was applied to determine the concentration levels of OTC in milk samples derived from healthy Sardinian sheep and those naturally infected with Streptococcus uberis, after the intramuscular administration of an antibiotic, in order to evaluate how much of the drug was “subtracted” during penetration from blood into milk, with a potential effect of reducing its therapeutic efficacy.
Premessa. Assistiamo con incredulità al diffondersi di convinzioni pseudoscientifiche, reso ancor... more Premessa. Assistiamo con incredulità al diffondersi di convinzioni pseudoscientifiche, reso ancora più acuto dalla pandemia Covid-19 e da certe sue interpretazioni sconfinanti nell’irrazionale. Intendiamo riferirci a quelle convinzioni non confortate da prove evidenti e accettabili e per le quali non sono immaginabili conferme o verifiche che ne possano mettere in crisi le affermazioni1. Il presente contributo esamina e intende fornire possibili interpretazioni al fenomeno delle false credenze, oggi amplificato dalla rete e dai social, e si interroga su che cosa porti al rifiuto delle evidenze scientifiche e a idee cospirative, rivolgendo l’attenzione in particolar modo ai fattori culturali. L’articolo tratta principalmente delle credenze arrivate alla ribalta mediatica con la pandemia Covid-19, come la sua negazione, le teorie complottiste sorte in risposta e la negazione dell’efficacia dei vaccini. Proprio quest’ultima convinzione, scelta come esempio, viene esaminata con maggiore attenzione, in quanto legata a un milieu più complesso di ragioni culturali, ideologiche e sociali, seppure spesso professata da soggetti che accettano anche altre credenze2. Le conclusioni proposte potranno comunque essere generalizzate.
Nel presente studio è stata valutata l'utilità della diagnosi immunoistochimica su encefali d... more Nel presente studio è stata valutata l'utilità della diagnosi immunoistochimica su encefali di ovini e caprini con lesioni istologiche riferibili a Listeria monocytogenes , correlandole, ove disponibile, con l'esame batteriologico; è inoltre stata valutata l'efficacia di alcuni trattamenti di smascheramento antigenico, già risultati efficaci nell'aumentare la sensibilità tecnica quando applicati nella identificazione di altri antigeni localizzati a livello encefalico.
Le mastiti rappresentano la patologia più diffusa nel settore ovino e caprino da latte e sono res... more Le mastiti rappresentano la patologia più diffusa nel settore ovino e caprino da latte e sono responsabili d'importanti perdite economiche dovute alla riduzione e scadimento della qualità delle produzioni, alla perdita di animali e ai costi sanitari. La conoscenza dell'eziologia e della reale diffusione della malattia nel territorio costituisce uno strumento basilare per elaborare e verificare l'efficacia di piani di controllo e risanamento delle patologie mammarie; rappresenta, inoltre, un elemento utile per indirizzare gli investimenti nell'ambito della programmazione sanitaria. Differenti, infatti, sono le strategie da adottare e i risultati attesi nella lotta alle mastiti in rapporto al microrganismo coinvolto che può essere responsabile di mastiti contagiose o ambientali.
In Italy Bovine Tubercolosis (TB) eradication program has been run since 1995, it is based on a s... more In Italy Bovine Tubercolosis (TB) eradication program has been run since 1995, it is based on a single intradermal skin test, slaughter of positive animals and post-mortem inspection. Sardinia, Italy´s second largest island in the Mediterranean Sea, was officially declared Tubercolosis-free in 1998, according to a National law (DM 592/95). Although this programme fulfilled our law requirements, it failed to eradicate TB: in fact, a few outbreaks of the disease still occur. The spread of TB infection occurred in 2007 in extensive cattle herds located in a defined area of Sardinia, named Goceano. In this area, on the basis of the skin test and/or gamma interferon positivity, 700 animals, belonging to 75 affected herds, have been slaughtered during three years. Moreover, the presence of TB infection was monitored in wildlife. We randomly checked wild boar population by inspection of head lymph nodes and, in addition, we collected several samples during two hunting seasons (2007/2008 an...
In this study, we examined in Sardinia the brain of 555 autochthonous sheep, 50 goats, and 4 mouf... more In this study, we examined in Sardinia the brain of 555 autochthonous sheep, 50 goats, and 4 mouflons which were found affected by neurological signs. We found 6 goats and one mouflon with meningoencephalitis caused by Cryptococcus sp. There was no evidence of cryptococcal infections in any of the examined sheep. MLST genotyping on Cryptococcus sp. isolates identified Cryptococcus gatti genotype AFLP4/VGI and Cryptococcus neoformans var. neoformans genotype AFLP2/VNIV. Phylogenetically, all Cryptococcus gattii isolates fell within the autochthonous animal, human and environmental Mediterranean isolate cluster, forming a distinct branch along with environmental strains from Alicante, in the southern Mediterranean coast of Spain.
Counting 3,300,000 heads and over 15,000 herds dairy sheep farming is the main agricultural busin... more Counting 3,300,000 heads and over 15,000 herds dairy sheep farming is the main agricultural business in Sardinia (Italy). Mastitis represents the most widespread pathology in dairy sheep and it is responsible for significant economic losses due to the decrease of milk quality and yield, animal replacement and health care costs. Knowing about aetiology and the rates of mastitis sprerad can help to develop disease control strategies based on characteristics of heach bacterial species involved.
In the present paper, a Quality Systems Theory is presented. Certifiable Quality Systems are trea... more In the present paper, a Quality Systems Theory is presented. Certifiable Quality Systems are treated and interpreted in accordance with a Thermodynamics-based approach. Analysis is also conducted on the relationship between Quality Management Systems (QMSs) and systems theories. A measure of entropy is proposed for QMSs, including a virtual document entropy and an entropy linked to processes and organisation. QMSs are also interpreted in light of Cybernetics, and interrelations between Information Theory and quality are also highlighted. A measure for the information content of quality documents is proposed. Such parameters can be used as adequacy indices for QMSs. From the discussed approach, suggestions for organising QMSs are also derived. Further interpretive thermodynamic-based criteria for QMSs are also proposed. The work represents the first attempt to treat quality organisational systems according to a thermodynamics-related approach. At this stage, no data are available to compare statements in the paper.
The identification of coagulase-negative staphylococci (CNS) causing ovine infections remains pro... more The identification of coagulase-negative staphylococci (CNS) causing ovine infections remains problematic, although these bacteria are considered the main etiologic agents of subclinical mastitis in sheep and goats. In this study, 226 CNS isolates were collected from 2201 milking sarda sheep belonging to 15 flocks with high somatic cell count scores. All isolates were subjected to identification with the API Staph ID test, and then to the amplification of staphylococcal 16S rRNA and gap genes by PCR assays. The gap gene was subjected to restriction fragment length polymorphism analysis with the restriction endonuclease AluI, whereas the 16S rRNA gene was subjected to ribosomal fingerprinting with the restriction endonucleases RsaI, PstI and AluI. When PCR–RFLP patterns of CNS isolates were different from those of their reference strains, gap gene amplicons were sequenced for definitive identification. The API Staph ID test, in alternative to the genotypic identification method, produced considerably different results in terms of species identified within each group. Using the PCR–RFLP assay, most of the isolates clustered together with the Staphylococcus epidermidis type strain (131, corresponding to 57.9%), followed by S. caprae (34, corresponding to 15%) and S. chromogenes (30, corresponding to 13.2%). In conclusion, the PCR–RFLP assay of 16S rRNA and gap genes is a more reliable and reproducible method than the API Staph ID test for the identification of CNS causing sheep mastitis.
This study was aimed to investigate the relationships existing between clinical findings and bact... more 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 < 0.05); S. epidermidis showed a statistically significant correlation with presence of pustules and ulcers (p < 0.05); and S. aureus showed a statistically significant correlation with clinical signs of chronic mastitis: nodules, abscesses, and atrophy (p < 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.
Antibiograms of selected mastitis pathogens, performed during the decade 1995-2004, were retrospe... more Antibiograms of selected mastitis pathogens, performed during the decade 1995-2004, were retrospectively analysed in order to evaluate antimicrobial resistance and determine whether resistance changed over time. Results of 2763 strains of Staphylococcus aureus, coagulase negative staphylococci, Streptococcus uberis and Escherichia coli, against penicillin, ampicillin, oxytetracycline, kanamycin and streptomycin are discussed. Strains were isolated in clinical milk samples from sheep suspected of mastitis. The evolution over time of resistance was evaluated by means of logistic regression analysis. The resistance of staphylococci to penicillin appeared to be lower than those usually reported (4.1% of resistant strains in S. aureus; 15.3% for CNS). Higher rates of resistance were observed for aminoglycosides, relevant for S. uberis (84.5% for kanamycin and 92.5% for streptomycin) and S. aureus (14.6% for kanamycin and 63.3% for streptomycin). Overall resistance appeared to confirm the lower resistance in ovine pathogens than in bovine ones. Logistic regression highlighted no trends to increase for resistance over time.
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Papers by Stefano Lollai
determination of four tetracyclines—oxytetracycline (OTC), tetracycline (TC), doxycycline (DC),
and chlortetracycline (CTC)—in milk. This method involves sample extraction with McIlvaine–
EDTA buffer solution (pH 4) and solid-phase extraction (SPE) with Oasis HLB cartridges, followed
by the evaporation of the extract and its reconstitution with a 14% methanol aqueous solution
before injection into the instrumental system. This method has been validated in terms of linearity,
sensitivity, selectivity, precision, and accuracy, in accordance with Commission Decision 2002/657/EC
requirements. Compared to existing methods, this approach optimally combines a quantitative
procedure for extracting analytes from the milk of different species, including sheep, bovines, and
goats, with a very short LC–Orbitrap–HRMS instrumental analysis time (only 8 min), simultaneously
ensuring high precision, sensitivity, and applicability as a rapid confirmation method in official food
control laboratories. The proposed method was applied to determine the concentration levels of OTC
in milk samples derived from healthy Sardinian sheep and those naturally infected with Streptococcus
uberis, after the intramuscular administration of an antibiotic, in order to evaluate how much of the
drug was “subtracted” during penetration from blood into milk, with a potential effect of reducing
its therapeutic efficacy.
determination of four tetracyclines—oxytetracycline (OTC), tetracycline (TC), doxycycline (DC),
and chlortetracycline (CTC)—in milk. This method involves sample extraction with McIlvaine–
EDTA buffer solution (pH 4) and solid-phase extraction (SPE) with Oasis HLB cartridges, followed
by the evaporation of the extract and its reconstitution with a 14% methanol aqueous solution
before injection into the instrumental system. This method has been validated in terms of linearity,
sensitivity, selectivity, precision, and accuracy, in accordance with Commission Decision 2002/657/EC
requirements. Compared to existing methods, this approach optimally combines a quantitative
procedure for extracting analytes from the milk of different species, including sheep, bovines, and
goats, with a very short LC–Orbitrap–HRMS instrumental analysis time (only 8 min), simultaneously
ensuring high precision, sensitivity, and applicability as a rapid confirmation method in official food
control laboratories. The proposed method was applied to determine the concentration levels of OTC
in milk samples derived from healthy Sardinian sheep and those naturally infected with Streptococcus
uberis, after the intramuscular administration of an antibiotic, in order to evaluate how much of the
drug was “subtracted” during penetration from blood into milk, with a potential effect of reducing
its therapeutic efficacy.