IDENTIFICATION OF BACTERIOCIN-PRODUCING LACTIC ACID BACTERIA AND IN VITRO EVALUATION OF THEIR POTENTIAL ROLE IN MASTITIS CONTROL

Bovine mastitis is one of the most significant causes of economic losses for the dairy industry. On the other hand, public health authorities advise prudent use of antibiotics because they could promote bacterial resistance and leave residues in food chain. The dairy industry could benefit from the development of safe antimicrobial agents and bacteriocins could be attractive alternatives to antibiotics. Due to the safety of Lactic acid bacteria (LAB), their bacteriocins have the potential to be used as antimicrobials in veterinary clinical application. We analyzed the efficacy of antibacterial substances produced by bacteriocinogenic Lactococcus lactis subsp. lactis strains against contagious and environmental mastitis pathogens. Thereafter, we investigated how lactococcal strains or their bacteriocins could influence mammary gland innate immune response in vitro. Out of 65 LAB strains tested, 3 were active against mastitis pathogens: 2 strains produced Nisin, one Lacticin 481 and in addition a novel molecule with likely antibacterial activity. To analyze the immune response of mammary epithelial cells when stimulated with lactococcal strains or bacteriocins, a stabilized epithelial cell line, BME-UV1, was used. Both lactococcal live cultures and their antibacterial products were shown to modulate the non-specific immune response of BME-UV1 cells: Lysozyme and N-acetil-\u3b2-D-glucosaminidase excretion were overall enhanced by bacteriocins and live-culture treatments, while intracellular amounts were unaffected by treatments. Proinflammatory cytokine expression of treated BME-UV1 was similar to that observed in control cells, except for Lactococcus lactis subsp. lactis SL153. Such strain induced a significant reduction of TNF\u3b1 transcriptional level. The stimulation of enzyme secretion due to the administration of lactococci or of their antibacterial products, with potential enhancement of pathogens cleaning, can be of interest for the prevention of intra mammary infections. In addition, Lactococcus lactis subsp. lactis SL153 strain could be advantageous for its potential anti-inflammatory properties and could be of interest for the development of intra-mammary probiotic treatments

Combinatorial Discriminant Analysis Applied to RNAseq Data Reveals a Set of 10 Transcripts as Signatures of Exposure of Cattle to Mycobacterium avium subsp. paratuberculosis

Paratuberculosis or Johne's disease in cattle is a chronic granulomatous gastroenteritis caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). Paratuberculosis is not treatable; therefore, the early identification and isolation of infected animals is a key point to reduce its incidence. In this paper, we analyse RNAseq experimental data of 5 ELISA-negative cattle exposed to MAP in a positive herd, compared to 5 negative-unexposed controls. The purpose was to find a small set of differentially expressed genes able to discriminate between exposed animals in a preclinical phase from non-exposed controls. Our results identified 10 transcripts that differentiate between ELISA-negative, clinically healthy, and exposed animals belonging to paratuberculosis-positive herds and negative-unexposed animals. Of the 10 transcripts, five (TRPV4, RIC8B, IL5RA, ERF, CDC40) showed significant differential expression between the three groups while the remaining 5 (RDM1, EPHX1, STAU1, TLE1, ASB8) did not show a significant difference in at least one of the pairwise comparisons. When tested in a larger cohort, these findings may contribute to the development of a new diagnostic test for paratuberculosis based on a gene expression signature. Such a diagnostic tool could allow early interventions to reduce the risk of the infection spreading

Antenatal vitamin D status is not associated with standard neurodevelopmental assessments at age 5 Years in a well-characterized prospective maternal-infant cohort

Background: Although animal studies show evidence for a role of vitamin D during brain development, data from human studies show conflicting signals. Objective: We aimed to explore associations between maternal and neonatal vitamin D status with childhood neurodevelopmental outcomes. Methods: Comprehensive clinical, demographic, and lifestyle data were collected prospectively in 734 maternal-infant dyads from the Cork BASELINE Birth Cohort Study. Serum 25-hydroxyvitamin D [25(OH)D] concentrations were quantified at 15 weeks of gestation and in umbilical cord sera at birth via a CDC-accredited liquid chromatography-tandem mass spectrometry method. Children were assessed at age 5 y through the use of the Kaufman Brief Intelligence Test (2nd Edition, KBIT-2) and the Child Behaviour Checklist (CBCL). Linear regression was used to explore associations between 25(OH)D and neurodevelopmental outcomes. Results: 25(OH)D concentrations were <30 nmol/L in 15% of maternal and 45% of umbilical cord sera and <50 nmol/L in 42% of mothers and 80% of cords. At age 5 y, the mean ± SD KBIT-2 intelligence quotient (IQ) composite score was 104.6 ± 8.6; scores were 107.2 ± 10.0 in verbal and 99.8 ± 8.8 in nonverbal tasks. Developmental delay (scores <85) was seen in <3% of children across all domains. The mean ± SD CBCL total problem score was 21.3 ± 17.5; scores in the abnormal/clinical range for internal, external, and total problem scales were present in 12%, 4%, and 6% of participants, respectively. KBIT-2 and CBCL subscale scores at 5 y were not different between children exposed to low antenatal vitamin D status, either at 30 or 50 nmol/L 25(OH)D thresholds. Neither maternal nor cord 25(OH)D (per 10 nmol/L) were associated with KBIT-2 IQ composite scores [adjusted β (95% CI): maternal –0.01 (−0.03, 0.02); cord 0.01 (−0.03, 0.04] or CBCL total problem scores [maternal 0.01 (−0.04, 0.05); cord 0.01 (−0.07, 0.09)]. Conclusion: In this well-characterized prospective maternal-infant cohort, we found no evidence that antenatal 25(OH)D concentrations are associated with neurodevelopmental outcomes at 5 y. The BASELINE Study was registered at www.clinicaltrials.gov as NCT01498965; the SCOPE Study was registered at http://www.anzctr.org.au as ACTRN1260700055149

Assessment of antibacterial activity of donkey milk lysozyme : safety and hygiene issues

Donkey milk contains as much lysozyme (LZ) as equine milk (6000 times higher than bovine milk). LZ shows antibacte- rial activity against Gram-positive and some Gram-negative bacteria. LZ activity in donkey milk is very high and some authors supposed that donkey resistance to mammary infec- tion is probably due to high amount of LZ. There is scarce literature on this topic; the aim of the present study is thus to evaluate the antibacterial activity of 3 different donkey bulk milk (DBM) samples, on a total of 42 bacterial strains. The microorganisms were isolated from donkey and bovine milk. Lysozyme activity was evaluated by EnzCheck Ly- sozyme Assay Kit; the milk antibacterial activity was tested by Minimal Inhibitory Concentration (MIC) method, in micro- titer plates. The first milk sample inhibited only one Pseudo- monas sp strain (MIC of LZ = 5800 U/ml); the second sample had no antibacterial effect (MIC > 2740 U/ml), whereas the third sample prevented the growth of 50% of Staphylococcus aureus strains from DBM (MIC = 3400 U/ml). LZ showed no activity against bovine strains. Finally, the authors underline the importance of further studies for a deeper understanding of LZ antimicrobial activity, both for veterinary health purpo- ses and to protect a vulnerable group of consumers, such as in- fants affected by intolerance to some milk proteins

Methicillin-resistant Staphylococcus pseudintermedius as causative agent of dairy cow mastitis.

Staphylococcus pseudintermedius is a coagulase-positive specie similar to Staphylococcus intermedius, frequently associated with pyoderma, otitis and urinary tract infections of dogs and cats (van Duijkeren and others 2011). No information about bovine mastitis caused by S pseudintermedius is available in the literature. Antimicrobial resistance among S pseudintermedius strains is increasing: in the past, susceptibility to most antibiotics was common (Bond and Loeffler 2012), but in the last few years methicillin-resistant S pseudintermedius (MRSP) strains have emerged as a significant animal health problem in veterinary medicine (Schwarz and others 2008, van Duijkeren and others 2008, Weese and van Duijkeren 2010). The methicillin-resistance of MRSP is mediated by the mecA gene, such as in Staphylococcus aureus (MRSA). The gene is located on a chromosomal mobile element (staphylococcal chromosomal cassette, SCCmec), which can be horizontally transferred (Weese and others 2010), raising concern about spreading of resistance among species and hosts (Bond and Loeffler, 2012). Therefore, the aim of this study was to report the occurrence of bovine mastitis by MRSP in a dairy herd and to investigate the characteristics of the isolates collected during a control programme for contagious pathogens

Preliminary results of antimicrobial activity of bacteriocins secreted by lactic acid bacteria against mastitis pathogens

In the last thirty years antimicrobial peptides known as bacteriocins, that are secreted by strains of lactic acid bacteria (LAB), have been studied mainly in the dairy industry, for their antibacterial activity. Recently, their prophylactic and therapeutic efficacy against bacteria agents of dairy cow mastitis has been also demonstrated. Few studies have been performed about the efficacy of different bacteriocins in clinical and subclinical mastitis and as teat sealant. We tested 18 LAB strains for production of antibacterial metabolites after different culture times and with different extraction methods. Minimum inhibitory concentration (MIC) test of culture supernatants was performed using the microdilution broth method. Two strains (SC20 and LL11) showed antibacterial activity against different mastitis pathogens (S. agalactiae, S. uberis, S. aureus, E. faecalis and L. monocytogenes), but the efficacy changed in relation to the bacteria tested. The highest MIC values were achieved using LL11, after extraction of bacteriocins at ph 2. The most sensitive strains were S. agalactiae, which were inhibited at a dilution of 1:380, while all the other isolates showed variable sensitivity. Our results represent the starting point for further studies aimed to the development of non-antibiotic formulations as prophylactic and therapeutic tools in dairy cow mastitis. Antibacterial substances produced by LAB have the potential to reduce the use of antibiotics in the dairy farm, with a consequent reduction of the risk of residues for the consumer

Evaluation of biofilm formation using milk in a flow cell model and microarray characterization of Staphylococcus aureus strains from bovine mastitis

It was hypothesized that biofilm could play an important role in the establishment of chronic . Staphylococcus aureus bovine mastitis. The . in vitro evaluation of biofilm formation can be performed either in closed/static or in flow-based systems. Efforts have been made to characterize the biofilm-forming ability of . S. aureus mastitis isolates, however most authors used static systems and matrices other than UHT milk. It is not clear whether such results could be extrapolated to the mammary gland environment. Therefore, the present study aimed to investigate the biofilm-forming ability of . S. aureus strains from subclinical bovine mastitis using the static method and a flow-based one. One hundred and twelve strains were tested by the classic tissue culture plate assay (TCP) and 30 out of them were also tested by a dynamic semi-quantitative assay using commercial UHT milk as culture medium (Milk Flow Culture, MFC) or Tryptic Soy Broth as control medium (TS Flow Culture, TSFC). Only 6 (20%) strains formed biofilm in milk under flow conditions, while 36.6% were considered biofilm-producers in TCP, and 93.3% produced biofilm in TSFC. No agreement was found between TCP, MFC and TSFC results. The association between strain genetic profile, determined by microarray, and biofilm-forming ability in milk was evaluated. Biofilm formation in MFC was significantly associated with the presence of those genes commonly found in bovine-associated strains, assigned to clonal complexes typically detected in mastitis. Based on our results, biofilm-forming potential of bovine strains should be critically analysed and tested applying conditions similar to mammary environment

Duplex real-time PCR assay for rapid identification of Staphylococcus aureus isolates from dairy cow milk

Staphylococcus aureus isolates from dairy cow mastitis are not always consistent with the characteristic morphology described, and molecular investigation is often needed. The aim of the study was to develop a duplex real-time PCR assay for rapid identification of S. aureus isolates, targeting both nuc and Sa442. Overall, 140 isolates collected from dairy cow mastitis in 90 different herds, were tested. All strains had been identified using morphological and biochemical characteristics. DNA from each strain was amplified in real-time PCR assay, to detect nuc or Sa442. Thereafter, a duplex real-time PCR assay was performed, and specificity of the amplified products was assessed by high resolution melting curve analysis. Out of 124 S. aureus isolates, 33 did not show the typical morphology or enzymatic activity; in 118 strains, the two melt-curve peaks consistent with nuc and Sa442 were revealed, while 2 isolates showed only the peak consistent with Sa442. Four isolates bacteriologically identified as S. aureus, were PCR-negative and were further identified as S. pseudintermedius by sequencing. Staphylococcus pseudintermedius and coagulase-negative staphylococci did not carry nuc or Sa442. The results showed the correct identification of all isolates, comprehending also coagulase- or nuc-negative S. aureus, while other coagulase-positive Staphylococci were correctly identified as non-S. aureus. Both sensitivity and specificity were 100%. High resolution melting analysis allowed easy detection of unspecific products. Finally, the duplex real-time PCR was applied directly to 40 milk samples, to detect infected mammary quarters. The assay confirmed the results of bacteriological analysis, on S. aureus-positive or -negative samples. Therefore, the proposed duplex real-time PCR could be used in laboratory routine as a cost-effective and powerful tool for high-throughput identification of atypical S. aureus isolates causing dairy cow mastitis. Also, it could be applied directly to milk samples, to detect S. aureus mammary infections avoiding bacteriological analysis