Cytokine mRNA expression in the bronchoalveolar lavage cells from horses affected by different equine asthma subtypes.

Equine asthma (EA) is a respiratory syndrome associated with the increase of different leukocyte populations in the bronchoalveolar lavage fluid (BALF). Its pathogenetic mechanisms remain unclear. This study aimed to evaluate the associations between the mRNA expression of different cytokines in the BALF, different EA subtypes and lung function. Fifteen horses underwent physical examination, airway endoscopy, BALF cytology and lung function testing (8/15). One horse did not have evidence of EA and was used as healthy reference, while the others were classified as affected by neutrophilic or mixed granulocytic EA. Cells isolated from the residual BALF were used for IL-1ß, IL-2, IFN-γ, IL-4, IL-17A genes expression by quantitative RT-PCR., Cytokine expression was compared between groups, and their correlations with BALF leukocyte and lung function were evaluated. IL-1ß expression was positively correlated with BALF neutrophils count (p=0.038, r=0.56) and with increased expiratory resistance (p=0.047, r=0.76). IFN-γ was correlated with BALF mast cells (p=0.029, r=0.58). IL-4 was higher in horses with mixed granulocytic EA than neutrophilic (p=0.008), positively correlated with BALF mast cells (p=0.028, r=0.59) and inversely with whole-breath (p=0.046, r=-0.76) and expiratory reactance (p=0.003, r=-0.93). Finally, IL-17A was inversely correlated with expiratory reactance (p=0.009, r=-0.92). These results support that multiple immune responses are involved in EA pathogenesis; innate, Th2, and Th17 responses. Innate immunity appeared associated with neutrophilic inflammation, and Th2 response with increased mast cells. The role of Th1 response in EA remains questionable.

Non-aureus staphylococci and mammaliicocci isolated from bovine milk in Italian dairy farms: a retrospective investigation.

Non-aureus staphylococci and mammaliicocci (NASM) are associated with bovine mastitis and increased milk somatic cell count (SCC) but their relationships with mammary gland health at the species level are not clearly defined. Regional differences have also been reported in their specific prevalence. The implementation of MALDI-TOF MS in milk microbiology is generating large and dependable datasets with the potential of providing useful epidemiological information. We present the retrospective analysis of 17,213 milk samples sent to our laboratory in 2021-2022, including 13,146 quarter samples from cows with subclinical (SCM) or clinical mastitis (CM) from 104 farms, and 4,067 composite herd survey (HS) samples from 21 farms. NASM were isolated from 21.12% of SCM, 11.49% of CM, and 15.59% of HS milk samples. The three most frequently identified NASM in SCM milk were Staphylococcus chromogenes (33.33%), S. haemolyticus (26.07%), and S. epidermidis (10.65%); together with S. microti and S. hyicus, these species were significantly more prevalent in quarters with SCM (p < 0.05). The three most frequently identified NASM in CM milk were S. chromogenes (31.69%), S. haemolyticus (21.42%), and Mammaliicoccus sciuri (18.38%), although no significant associations were found between these NASM species and CM. The three most frequently identified NASM in HS milk were S. chromogenes (44.49%), S. epidermidis (17.84%), and S. haemolyticus (17.23%), with S. chromogenes being isolated in all the farms sending HS milk (100%). In conclusion, this retrospective study provides the first information on the NASM species isolated from cow milk in Italy, expanding our knowledge on the epidemiology of NASM at the species level and providing further insights into their relationships with mammary gland health in modern dairy farms.

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-response 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.

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.

The effect of anaerobic digestion and storage on indicator microorganisms in swine and dairy manure.

The aim of this experimental study was to evaluate the influence of anaerobic digestion and storage on indicator microorganisms in swine and dairy excreta. Samples were collected every 90 days for 15 months at eight farms, four pig, and four dairy farms, four of them having a biogas plant. Moreover, to evaluate storage effects on samples, 20 l of manure and slurry taken at each farm (digested manure only in farms with a biogas plant) were stored in a controlled climatic chamber at 18 °C, for 6 months. The bacterial load and the chemical-physical characteristics of excreta were evaluated at each sampling time, stored slurry, and manure were sampled and analyzed every 2 months. A high variability of the concentration of bacteria in the different excreta types was observed during the experiment, mainly depending on the type and time of treatment. No sample revealed either the presence of Escherichia coli O157:H7 or of Salmonella, usually linked to the temporary rearing of infected animals in facilities. Anaerobic digestion and storage affected in a significant way the reduction of indicator bacteria like lactobacilli, coliforms, and streptococci. Anaerobic digestion lowered coliforms in pig slurry (- 2.80 log, P < 0.05), streptococci in dairy manure (- 2.44 log, P < 0.001) and in pig slurry (- 1.43 log, P < 0.05), and lactobacilli in pig slurry (- 3.03 log, P < 0.05). Storage lowered coliforms and the other indicators counts, in particular in fresh wastes, while clostridia did not show a reduction in concentration.

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 101CFU/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.

Effects of disinfectant fogging procedure on dust, ammonia concentration, aerobic bacteria and fungal spores in a farrowing-weaning room.

INTRODUCTION AND OBJECTIVE: In the last decades, large-scale swine production has led to intensive rearing systems in which air quality can be easily degraded by aerial contaminants that can pose a health risk to the pigs and farm workers. This study evaluated the effects of fogging disinfectant procedure on productive performance, ammonia and dust concentration, aerobic bacteria and fungal spores spreading in the farrowing-weaning room. MATERIALS AND METHOD: This trial was conducted in 2 identical farrowing-weaning rooms of a piggery. In both rooms, 30 pregnant sows were lodged in individual cages. At 75 days of age, the piglets were moved to the fattening room. In the treated room, with the birth of the first suckling-pig, the fogging disinfection with diluted Virkon S was applied once a day in the experimental room per 15 minutes at 11:00. The fogging disinfectant treatment was switched between rooms at the end of the first trial period. Temperature, relative humidity, dust (TSP-RF fractions and number of particles), ammonia concentration and aerial contaminants (enterococci, Micrococcaeae and fungal spores) were monitored in both rooms. RESULTS: Ammonia concentration reduction induced by fogging disinfection was estimated 18%, total suspended particles and the respirable fraction were significantly lower in the experimental room. Fungal spores resulted in a significant reduction by the fogging procedure, together with dust respirable fraction and fine particulate matter abatement. CONCLUSIONS: The fogging disinfection procedure improved air quality in the piggery, thereby enhancing workers and animals health.