Impiego di un programma integrato di bioattivazione per il controllo della polmonite micoplasmica del suino

Introduzione. La qualit\ue0 dell\u2019aria nelle strutture di allevamento dei suini dipende soprattutto dalla concentrazione dei biogas, derivati dall\u2019azione della flora microbica fecale sull\u2019urea contenuta nelle urine; il peggioramento della qualit\ue0 ambientale \ue8 alla base di molti problemi sanitari, basati in primo luogo sull\u2019impossibilit\ue0 degli animali a mantenere un bilancio organico corretto. Una soluzione possibile e innovativa \ue8 rappresentata dalla bioattivazione con miscele batterico-enzimatiche; per questa via, si pu\uf2 ottenere una riduzione delle emanazioni, controllando anche la formazione di sostanze dannose per l\u2019ambiente. Materiali e metodi. \uc8 stato programmato un intervento in due aziende suinicole (il reparto di magronaggio-ingrasso di un ciclo chiuso completo e un allevamento di solo ingrasso), per controllare il danno indotto dall\u2019infezione micoplasmica nelle fasi di produzione a flusso continuo; sono stati messi a confronto gruppi trattati e non trattati, diversi tipi di pavimentazione (grigliato completo, concreto completo e misto grigliato-concreto) e pi\uf9 schemi d\u2019intervento, utilizzando, da soli o in combinazione, bioattivatori in polvere e liquidi. Il sistema di bioattivazione impiegato comprende due bioattivatori (in polvere e soluzione) e un prodotto naturale di origine vegetale da aggiungere alla razione alimentare. La polvere \ue8 stata distribuita sulle pavimentazioni, in ragione di 0,5 Kg per 100 mq ogni 15 giorni mediante spargimento manuale, mentre la soluzione \ue8 stato aerosolizzata nell\u2019ambiente di stabulazione, mediante impianto computerizzato, in ragione di 1 litro ogni 100 mq al mese; il prodotto vegetale, per la sua composizione in grado di riequilibrare la flora intestinale, \ue8 stato invece aggiunto quotidianamente alla razione, in ragione di 500 g/t di mangime. I rilievi e le osservazioni hanno riguardato la concentrazione di azoto ammoniacale (rilevata con metodo chimico), la presenza di anticorpi specifici per M. hyopneumoniae prima e dopo il periodo di trattamento (valutata con metodo ELISA), i punteggi polmonari al macello (valutati con metodo quantitativo) e il peso medio di partita. Risultati. In tutti i gruppi trattati, la sieroprevalenza per M. hyopneumoniae \ue8 stata elevata, a dimostrazione della circolazione dell\u2019infezione nel periodo di osservazione, ma la diminuzione della concentrazione ambientale dell\u2019ammoniaca, ottenuta grazie all\u2019impiego dei bioattivatori) ha permesso di ottenere benefici evidenti sul punteggio polmonare medio, confermati dalla significativit\ue0 statistica delle differenze, e sul peso medio di partita. In rapporto ai tipi di pavimentazione, la bioattivazione ha fornito i risultati migliori combinando polvere e liquido sul grigliato completo. Conclusioni. I risultati ottenuti dimostrano che nelle situazioni di allevamento, la stabilizzazione del bilancio organico degli animali attraverso una gestione ambientale che ne garantisca anche il benessere \ue8 una soluzione attuabile, in una logica di depotenziamento delle infezioni; essi sono la conseguenza dell\u2019accresciuta reattivit\ue0 degli animali, correlata ad un miglior bilancio organico, piuttosto che l\u2019effetto di un\u2019azione diretta sui patogeni; \ue8 in corso la valutazione della reattivit\ue0 immunitaria aspecifica dei suini sottoposti a trattamento, per correlare stato sanitario e produttivit\ue0 al miglioramento della qualit\ue0 ambientale

Antimicrobial Effects of Conditioned Medium From Amniotic Progenitor Cells in vitro and in vivo : toward Tissue Regenerative Therapies for Bovine Mastitis

There is increasing evidence to suggest that, in addition to their regenerative effect, mesenchymal stromal cells (MSCs), and their secretome have an anti-inflammatory and antimicrobial role in the innate immune response in conditions such as sepsis. However, there is no published information on the effect of MSCs in bovine mastitis. Mastitis often results in extensive tissue damage due to multi-microorganism co-infection. This study investigated the ability of amniotic-derived conditioned medium (CM), in vitro and in vivo, to counteract microbial action and restore healthy tissue capable of milk production. Following determination of a dose\u2013response curve, 10,000 colony-forming units (CFU) of Staphylococcus aureus (S. aureus) were inoculated into bovine mammary epithelial cell culture with and without 10% CM (supplemented either at the time of bacteria inoculation or after 4 h). Acridine orange staining was used to assess cell viability/apoptosis. Additionally, an in vivo study was performed using 48 dairy cows with acute and chronic mastitis, treated with CM (treated group) or antibiotics (control group). In vitro results showed that CM can attenuate bacterial growth, as evaluated by the number of CFU. After 24 h of culture with S. aureus, 89.67% of mammary epithelial cells treated with CM were still alive, whereas all cells cultured without CM were dead. Rates of epithelial cell survival (60.67%) were similar when CM was added 4 h after bacteria inoculation. There was no difference in somatic cell count between cases of acute mastitis in the CM-treated or control group in the in vivo study. However, relapses in chronic mastitis were less common in the group receiving CM. Our results show that CM is able to mitigate bacterial growth in vitro and may be particularly useful in the treatment of chronic mastitis, aiding restoration of milk production in cows that would otherwise be removed from the production cycle

Enhanced diagnostic protocol to identify E.coli VTEC from milk filters

The EHEC (enterohemorrhagic E. coli) are a subgroup of VTEC with strong pathogenicity. The most well-known EHEC serotype is E. coli O157:H7, which has been implicated in many large outbreaks of deadly human diseases. However, EHEC strains of other serotypes have increasingly been implicated in sporadic cases and outbreaks of serious illness in humans, e.g., serotypes O26, O45, O103, O111, O121 and O145. Epidemics studied from 1982 to date have shown that ruminants, and in particular bovine, appear almost always involved in the transmission of these bacteria to humans through direct or indirect fecal contamination of foods. Unpasteurized milk and milk products are considered minor, but important sources of infection. The possible ways to the entrance of VTEC in milk are fecal contamination and mammary excretion during E.coli mastitis. Between the two ways, the first is considered much more frequent in practice, but it cannot be excluded that a small part of EHEC found in milk resulting from mammary gland, as reported. Previous investigation suggested that milk filters used in milking machines could be a useful control point to identify the presence of EHEC in dairy herds. However, conventional methods to identify the presence of EHEC have a poor sensitivity due the high content of fecal bacteria of these filters. In order to set up a monitoring scheme to identify herds at risk, we developed and tested a diagnostic protocol involving VIDAS\uae UP E.coli serogroups (ESPT) which is a method using phage recombinant proteins for the immuno-concentration (IC) of E.coli serogroups O157, O26, O103, O111, O145, O45 and O121 from food, multiplex PCR and high resolution melting analysis (HRMA). Practically, bulk tank milk or washing solution obtained from milk filters after stomacher mixing were analyzed by Vidas ESPT. After incubation, the solution obtained was analyzed by multiplex PCR based on serotype-specific primers coding for O-antigen regions of the seven major VTEC serogroups available in literature. If PCR was positive for any of the seven serogroups, a HRMA-based protocol to detect virulence-predictive SNPs, as discovered by Norman et al., 2012, was applied to confirm the presence of a EHEC strain. The protocol was preliminary validated by inoculation of milk and milk filters with a known concentration of the seven EHEC serotypes (O26, O45, O103, O111, O121, O145, O157). The results confirmed that this protocol was able to identify as low as 101 UFC in both milk and milk filters. The protocol applied to milk and milk filters obtained from 70 dairy herds allow to identify 2 EHEC from milk and 17 from milk filters for one or more of the EHEC serogroups considered. The proposed protocol confirmed to be useful in detecting the presence of EHEC and that milk filters are an important critical control point to identify herd at risk

A new integrated approach to analyze bulk tank milk and raw milk filters for the presence of the E. coli serogroups frequently associated with VTEC status

We optimized a combination of microbiological and molecular methods to quickly identify the presence of the O157 and the six non-O157 serogroups (O26, O45, O103, O111, O121 and O145) most frequently associated with VTEC status, at herd level. The lower detection limit of this methodology is 101 CFU/ml for each of the serogroups tested. We tested 67 bulk tank milk (BTM) and raw milk filters (RMF) derived from dairy herds located in Lombardy and Trentino Alto Adige. We identified 3 positive samples and 20 positive samples out of 67 respectively in the BTM and RMF. Interestingly, several samples showed positivity for more than one serogroups at the same time. We also identified the presence of E. coli O45 and O121 for the first time in raw milk and raw milk filters. Once screened the seven serogroups of interest in our samples, we evaluated the real pathogenicity of our positive, non-O157 samples through two parallel molecular biology methods: virulence gene research by PCR, and HRMA and sequencing. The most frequently isolated serogroups in milk were O157 (2.64%), O103 (2.11%), and O145 (1.06%), while in RMF the frequencies were, respectively 14.92%, 4.48%, and 2.98%. Moreover, this is the first published report in Italy of positive recovery of O45 and O121 serogroups in milk and milk filters. The new diagnostic approach proposed investigate the presence of the O157 and big six non-O157 serogroups at farm level and not to identify VTEC hazard only once the product is processed and/or is ready to be consumed

Evaluation of virulence factors profiles and antimicrobials resistance of Escherichia coli isolated from bulk tank milk and raw milk filters

Data on the presence of pathogenic Escherichia coli in bulk tank milk (BTM) and raw milk filters (RMF) are not available in Italy and there are few studies worldwide. Therefore, a study under field condition was conducted to assess the presence of E.coli pathogenic and commensal (CoEC) strains in BTM and RMF samples and their associated AMR pattern. One hundred forty-nine E.coli isolates were characterized. Among all the isolates, 53 (35.6%) were classified as pathogenic while the other ones were classified as CoEC. Among the pathogenic ones, 23 (54.7%) were classified as enterotoxigenic E.coli (ETEC), 6 (11.3%) as enteroinvasive E.coli (EIEC), 2 (3.8%) as enteroaggregative E.coli (EAEC), 12 (22.6%) harboured virulence factors (VF) common to ETEC+EIEC, and 2 (3.8%) common to ETEC+EAEC. To our knowledge, it is the first time that ETEC isolates harboring VF associated with EAEC or EIEC are observed in raw milk. These data support the presence of transmission of VFs genes among isolates. None of the isolates showed resistance to three or more antimicrobials. The CoEC role as a vector of AMR was confirmed by the presence of 18% ampicillin- and cephalexin-resistant isolates. The presence of AMR in CoEC supports the role of these bacteria as source of resistance genes. Monitoring raw milk by either BTM or RMF analysis, and the relatively cheap procedure applied to identify E.coli pathotypes can be useful to identify hazards related to the spread of enteric diseases and antimicrobial resistance

TECNICHE DIAGNOSTICHE APPLICATE ALL’EPIDEMIOLOGIA AZIENDALE DELLE INFEZIONI DA STREPTOCOCCUS SUIS NEL CICLO PRODUTTIVO DEL SUINO TRADIZIONALE ITALIANO

IN ORDER TO CHECK THE EPIDEMIOLOGY OF STREPTOCOCCUS SUIS INFECTIONS IN FARM, A DIAGNOSTIC PROTOCOL HAS BEEN APPLIED. THE RESULTS WERE THE ISOLATION OF 119 STRAINS OF S. SUIS FROM 187 VAGINAL AND TONSILS SWABS CARRIED ON SOWS AND RELATED LITTERS. ANIMALS WERE FOLLOWED UP TO 160 DAYS OF AGE AND THE S. SUIS STRAINS WERE CAPSULAR TYPED WITH A CO- AGGLUTINATION TEST. THE IMPORTANCE OF SOWS IN THE DISTRIBUTION OF THE INFECTION AND THE NEGATIVE EFFECT OF THE WEANING WAS CONFIRMED. PERCENTAGE OF INSULATION AROUND 40% WERE ALSO FOUND IN FATTENING UNIT. CO- AGGLUTINATION TEST CONFIRMED THE PRESENCE OF MORE THAN ONE capsular type IN THE SAME HERD AND EVEN IN THE SAME ANIMAL