Efficacy of a teat dip containing the bacteriocin lacticin 3147 to eliminate Gram-positive pathogens associated with bovine mastitis.

On most dairy farms teat dips are applied to the teats of cows either before or after milking in order to prevent pathogens from gaining access to the mammary gland via the teat canal. In the present experiments, a natural teat dip was developed using a fermentate containing the live bacterium Lactococcus lactis DPC 3251. This bacterium produces lacticin 3147, a two-component lantibiotic which was previously shown to effectively kill Gram-positive mastitis pathogens. Lacticin 3147 activity in the fermentate was retained at 53% of its original level following storage for 3 weeks at 4 degrees C. In the initial experiments in vitro, 105 colony-forming units/ml (cfu/ml) of either Staphylococcus aureus, Streptococcus dysgalactiae or Streptococcus uberis were introduced into the lacticin-containing fermentate. Neither Staph. aureus nor Str. dysgalactiae could be detected after 30 min or 15 min, respectively, while Str. uberis was reduced approximately 100-fold after 15 min. Following these trials, preliminary experiments were performed in vivo on teats of lactating dairy cows. In these experiments, teats were coated with each of the challenge organisms and then dipped with the lacticin-containing fermented teat dip. Following a dip contact time of 10 min, staphylococci were reduced by 80% when compared with the undipped control teat. Streptococcal challenges were reduced by 97% for Str. dysgalactiae and by 90% for Str. uberis. These trials showed that the teat dip is able to reduce mastitis pathogens on the teats of lactating cows.

Administration of a live culture of Lactococcus lactis DPC 3147 into the bovine mammary gland stimulates the local host immune response, particularly IL-1beta and IL-8 gene expression.

Mastitis is one of the most costly diseases to the dairy farming industry. Conventional antibiotic therapy is often unsatisfactory for successful treatment of mastitis and alternative treatments are continually under investigation. We have previously demonstrated, in two separate field trials, that a probiotic culture, Lactococcus lactis DPC 3147, was comparable to antibiotic therapy to treat bovine mastitis. To understand the mode of action of this therapeutic, we looked at the detailed immune response of the host to delivery of this live strain directly into the mammary gland of six healthy dairy cows. All animals elicited signs of udder inflammation 7 h post infusion. At this time, clots were visible in the milk of all animals in the investigation. The most pronounced increase in immune gene expression was observed in Interleukin (IL)-1beta and IL-8, with highest expression corresponding to peaks in somatic cell count. Infusion with a live culture of a Lc. lactis leads to a rapid and considerable innate immune response.

Occurrence of ssl genes in isolates of Staphylococcus aureus from animal infection.

The occurrence of 7 of the 11 known ssl genes that are found within the vSaalpha genomic island of Staphylococcus aureus and encode the novel Ssl family of exoproteins was examined in isolates from cows (42 isolates), goats (4 isolates), sheep (1 isolate), rabbits (3 isolates) and chickens (2 isolates). Based on seven S. aureus genome sequences for human strains NCTC 8325, N315, Mu50, COL, MRSA 252, MW2 and MSSA-476, and bovine strain RF122, along with the ssl reference gene sequences from strains NCTC 6571, FRI326 and NCTC 8325, ClustalW-generated alignments were used to design PCR primers for unique regions of the ssl genes that are present in the allelic variants of each, except for the ssl4 gene for which specific primers for the set2 and set9 allelic variants were designed individually. The genotypes of isolates were determined using random amplified polymorphic DNA (RAPD) typing. All of the animal-associated S. aureus isolates contained an ssl locus, but there were minor variations in the number of ssl genes present. Forty-nine of the animal isolates possessed a vSaalpha genomic island containing the ssl3 (set8), ssl5 (set3/set10), ssl7 (set1/set11), ssl8 (set12), ssl9 (set5/set13) and ssl10 (set4/set14) genes. One bovine and one goat isolate lacked the ssl3 gene. The ssl9 gene was absent in one bovine isolate. The goat isolate lacking the ssl3 gene was the only animal isolate that possessed the set2 allele of the ssl4 gene. PCR for the set9 allele of the ssl4 gene was inconclusive. Isolates that showed identical RAPD fingerprints had the same complement of ssl genes, but the ssl gene pattern was not RAPD-type specific. Southern blot hybridization showed similar ssl gene RFLPs in isolates of the same RAPD type.

Interdependence and distribution of subclinical mastitis and intramammary infection among udder quarters in dairy cattle.

The objective of the present study was to determine the distribution of subclincial mastitis and intramammary infection (IMI) across udder quarters and to quantify, if any, the level of interdependence between quarters. Subclinical mastitis was defined as an udder quarter somatic cell count of > or =250,000. Intramammary infection was defined as the presence of pathogens in an udder quarter. The data used in the analyses consisted 6577 test-day records from 2034 lactations with observations on subclinical mastitis and 8428 test-day records from 2575 lactations with observations on IMI; each test-day record consisted data on all four udder quarters. Records were from animals on three research herds in southern Ireland. Observed distributions of IMI were compared to expected distributions, estimated using a binomial probability distribution, assuming independence among quarters. Generally, the observed frequencies deviated significantly from the expected frequencies, indicating some level of interdependence between quarters. Intraclass correlations were estimated from a mixed model including cow and test-day record nested within cow as random effects; fixed effects in the model included herd, year of calving, month of calving, parity, days in milk at sampling and interactions. Intraclass correlations within test-day record ranged from 0.14 to 0.50 for subclinical mastitis and from 0.02 to 0.40 for IMI. Thus, substantial interdependence between quarters exists which should be accounted for when analysing data from experiments across udder quarters. Failure to do so may result in incorrect inferences from statistical tests.

Superantigen genes encoded by the egc cluster and SaPIbov are predominant among Staphylococcus aureus isolates from cows, goats, sheep, rabbits and poultry.

In recent years several new staphylococcal enterotoxins (SEs) have been described, which currently have largely unknown frequencies of occurrence and roles in human or animal disease. One hundred and ninety-one Staphylococcus aureus isolates from cows (99), goats (39), sheep (23), rabbits (15), chickens (15) and a cat (1) were screened for SE genes sea-see, seg-seo and seq and for the tst gene encoding staphylococcal toxic shock syndrome toxin-1 using multiplex PCRs and individual PCRs for the seb and sek genes. One hundred and ten isolates tested positive for at least one of these 16 superantigen (SAg)-encoding genes. There were statistically significant differences in the frequencies of some of these SAg genes between isolates from different animals. No strain possessed either the sea or see gene. The sec gene was present in 51 isolates, the sed gene in eight and the seb gene in one. The seh gene was found in four strains and the sek and seq genes together in one isolate. The most common combinations of genes were the egc cluster, bearing the seg, sei, sem, sen and seo genes, in 47 isolates, the sec, sel and tst gene combination typical of the SaPIbov pathogenicity island in 44 isolates, the egc cluster lacking the seg gene in 11 isolates, the sed and sej genes in nine isolates, and the sec and tst genes without the sel gene in seven isolates. The higher frequencies of the sec and tst genes together and the lower frequencies of the egc gene cluster among the SAg gene-positive sheep or goat isolates compared to bovine isolates were statistically significant. Of 36 bovine isolates that were mitogenic for human T lymphocytes, four were negative for the 16 SAg genes tested for, while a further 14 gave borderline results in the mitogenicity assay, 12 of which were SAg gene-negative. Twenty-nine strains lacking all the SAg genes did not induce T-cell proliferation. This survey indicates that novel SE genes seg, sei, sel, sem, sen and seo along with the sec and tst genes predominate in S. aureus from animal hosts. The mitogenicity assays indicate that further uncharacterized SAgs may be present in bovine isolates.

Update on the development of a novel dry cow therapy using a bismuth-based intramammary teat seal in combination with the bacteriocin lacticin 3147.

: Public concerns over the widespread prophylactic use of antibiotics have led to a search for alternatives to dry cow therapy for the prevention of intramammary infections. A popular alternative is to infuse a teat seal at drying-off. The teat seal is a viscous non-antibiotic formulation and when it is infused into the teat canal and the teat sinus it forms an internal seal that provides a physical barrier to invasion by mastitis-causing pathogens. Enhancement of teat seal formulations may be achieved using non-antibiotic additives such as bacteriocins, potent proteins produced by some bacteria that have the ability to kill other microorganisms. This paper traces the history of investigations at Moorepark Research Centre into the efficacy of teat seal plus lacticin 3147, a bacteriocin produced by Lactococcus lactis DPC3147, in the prevention of intramammary infections in dry cows. Indications from on-going investigations are that a dry cow formulation combining the two products has considerable potential as a non-antibiotic prophylactic product.

Dry cow therapy with a non-antibiotic intramammary teat seal - a review.

: Dry cow antibiotic therapy is used to eliminate existing intramammary infections and to prevent new infections in the dry period. It is implemented as part of a total management system known as the 'Five-Point Plan' for mastitis control. Recent public concerns over the widespread prophylactic use of antibiotics, coupled with an increasing interest in organic farming, have lead to a re-evaluation of the treatment of cows at drying-off. As a result, attention has focussed on the use of novel alternatives to antibiotic therapy at the end of lactation. One such therapy involves the application of a non-antibiotic bismuth-based intramammary teat seal designed for use in cows with low cell counts at the end of lactation. Like the keratin plug that forms naturally in teats of cows that have been dried-off, teat seal forms a physical barrier to invading pathogens. To date, a number of independent studies have shown that teat seal is as effective as traditional dry cow antibiotic products in preventing the occurrence of new infection during the dry period in cows with somatic cell counts of ≤200,000 cells ml-1 at drying-off. This paper reviews the efficacy of teat seal in preventing dry period mastitis in both conventional and organic dairying systems.

Global Immunization Vision and Strategy (GIVS): a mid-term analysis of progress in 50 countries.

Within the overall framework set out in the Global Immunization Vision and Strategy (GIVS) for the period 2006-2015, over 70 countries had developed comprehensive Multi-Year Plans (cMYPs) by 2008, outlining their plans for implementing the GIVS strategies and for attaining the GIVS Goals at the midpoint in 2010 or earlier. These goals are to: (1) reach ≥90% and ≥80% vaccination coverage at national and district level, respectively; and (2) reduce measles-related mortality by 90% compared with the 2000 level. Fifty cMYPs were analysed along the four strategic areas of the GIVS: (1) protecting more people in a changing world; (2) introducing new vaccines and technologies; (3) integrating immunization, other health interventions and surveillance in the health system context; and (4) immunizing in the context of global interdependence. By 2010, all 50 countries planned to have introduced hepatitis B (HepB) vaccine, 48 the Haemophilus influenzae type B (Hib) vaccine and only a few countries had firm plans to introduce pneumococcal or rotavirus vaccines. Countries seem to be inadequately prepared in terms of cold-chain requirements to deal with the expected increases in storage that will be required for vaccines, and in making provisions to establish a corresponding surveillance system for planned new vaccine introductions. Immunization contacts are used to deliver other health interventions, especially in the countries in the World Health Organization (WHO) Africa Region. The cost for the planned immunization activities will double to U$27 per infant, of which U$5 per infant is the expected shortfall. Global Alliance for Vaccines and Immunization (GAVI) funding is becoming the largest contributor to immunization programmes.

Molecular genetic typing reveals further insights into the diversity of animal-associated Staphylococcus aureus.

Staphylococcus aureus is an important pathogen of man, but is also able to colonize and cause disease in a wide variety of mammals and birds. An extended multilocus sequencing approach, involving multilocus sequence typing (MLST), sas typing, spa typing and agr typing, was used to examine the molecular diversity of 118 S. aureus isolates recovered from a range of host species and to compare these data with the known diversity of human-derived isolates. MLST revealed that the commonest animal-associated MLST types were ST133, ST5, ST71, ST97, ST126 and ST151. ST133 appears to be an ungulate-animal-specific genotype, as no evidence of ST133 associating with humans has yet been found in the literature. Novel and unique sas alleles were identified in the animal-associated strains that may represent animal-associated sas alleles. However, sas typing exhibited a lower typeability than MLST for the animal strains (91.3 %). Phylogenetic analyses using neighbour-joining and maximum-parsimony trees localized ruminant-associated MLST lineages to both previously identified S. aureus subspecies aureus subgroups, thus explaining the finding of all four agr types within the ruminant-associated strains. S. aureus isolates recovered from chickens and rabbits were genotypically more similar to known human genotypes than the ruminant-associated lineages.

Intramammary infusion of a live culture of Lactococcus lactis for treatment of bovine mastitis: comparison with antibiotic treatment in field trials.

A treatment containing a live food-grade organism, Lactococcus lactis DPC3147, was compared with conventional antibiotic therapy for its potential to treat bovine chronic subclinical or clinical mastitis in two separate field trials. Effects on disease symptoms and bacteriology were monitored in response to infusion with the culture in each trial. In the first trial, the live culture treatment was compared with an intramammary antibiotic (n=11 quarters for each treatment). Results from this small trial demonstrated that the live culture had potential to be as effective at eliminating chronic subclinical infections as an antibiotic treatment. By day 12, 7 of the 11 quarters treated with the live culture were pathogen-free compared with 5 of the 11 antibiotic-treated infected quarters. Somatic cell counts (SCC) remained relatively unchanged regardless of treatment: average log SCC pre- and post-treatment in the lactococci-treated group were 6.33+/-0.41 (day 0) and 6.27+/-0.43 cells/ml (day 12) and average log SCC pre- and post-treatment in the antibiotic-treated group were 6.34+/-0.37 and 6.22+/-0.46 cells/ml on day 0 and on day 12, respectively. In the second trial, the live culture was compared with an intramammary antibiotic for the treatment of naturally occurring clinical mastitis cases (n=25 quarters for each treatment). Following a 14-d experimental period, similar bacteriological responses were observed in 7 out of 25 live culture treated quarters and 9 out of 25 antibiotic-treated quarters. Additionally, 15 of 25 cases treated with the culture and 18 of 25 cases treated with the antibiotic did not exhibit clinical signs of the disease following treatment. The results of these trials suggest that live culture treatment with Lc. lactis DPC3147 may be as efficacious as common antibiotic treatments in some instances.

Intramammary infusion of a live culture for treatment of bovine mastitis: effect of live lactococci on the mammary immune response.

In the accompanying article, we demonstrated that a live culture of Lactococcus lactis compares favourably with antibiotics for treatment of bovine mastitis in two initial field trials. In an effort to explain the mechanism involved, this study investigated the effect of culture administration on the local immune response. In this respect we initially observed that infusion of the live culture Lactococcus lactis stimulated substantial recruitment of polymorphonucleocytes (PMN) and lymphocytes to the udder. For instance, in one assay, quarters infused with the probiotic experienced a dramatic increase (approximately 20,000-fold) in neutrophils over the first 48-h period from an average value of 83.6 cells/ml pre-treatment to 1.78 x 106 cells/ml 48 h post-infusion. Levels of the acute phase proteins haptaglobin and milk amyloid A were also elevated significantly in comparison with controls following infusion of the culture. The results of flow cytometric assays also demonstrated that while infusion of a live lactococcal culture led to an enhanced recruitment of PMN to the udder (from 1.85 x 104 cells/ml pre-infusion to 1.45 x 106 cells/ml 24 h post-infusion) cell-free supernatant from the same culture was not able to do so, indicating that live Lc. lactis can specifically trigger the mammary immune response to elicit PMN accumulation. These results suggest that the mechanism responsible for this probiotic treatment of mastitis is associated with stimulation of the host intramammary immune system.

The lantibiotic lacticin 3147 produced in a milk-based medium improves the efficacy of a bismuth-based teat seal in cattle deliberately infected with Staphylococcus aureus.

A preparation of the bacteriocin lacticin 3147 (prepared from a demineralized whey protein fermentation liquor) was combined as a powder with a bismuth-based intramammary teat seal and evaluated for its potential as an antimicrobial in non-lactating cows. The lacticin/teat seal formulation enabled significant bacteriocin release from the seal without the requirement for a surfactant. Studies in vivo in lactating cows demonstrated that this formulation was effective in reducing bacterial recoveries (approximately 20-fold) from teats deliberately inoculated with Staphylococcus aureus after infusion. Moreover, this formulation also significantly reduced the numbers of Staph. aureus recovered from teats that were exposed to the challenge bacterium before the infusion of the teat seal preparation. The powdered preparation of lacticin 3147 did, however, cause some teat irritation as evidenced by associated rises in somatic cell count (SCC). However, this effect was short-lived and when the mean SCC readings pre-infusion and the final two readings post-infusion were compared, there was no significant difference in the immunological acceptance between treatments.

Effect of frequency of milking on yield, composition and processing quality of milk.

The objective was to determine the effect of once-daily milking (ODM) and omitting one evening milking each week (13TWM), in late lactation on milk production, composition and processability. Seventy-two cows were assigned to three treatments (ODM, 13TWM and twice-daily milking [TDM]) from 4 October to 12 December. Cows were on average 218 d into lactation at the start of the trial, and all cows were managed similarly throughout the trial. Milk yields and gross milk composition of cows on all treatments were measured, and milk samples for detailed compositional and processability analysis were collected from TDM and ODM treatments at two consecutive milkings and at one milking each week, respectively. Milk yield was significantly reduced (P < 0.001) and milk fat and protein concentrations were increased (P < 0.01) with ODM compared with TDM. Milk yield and fat and protein concentrations of milk from TDM and 13TWM herds were similar. Casein concentrations in ODM and TDM milks were similar, but ODM milk had a higher (P < 0.05) whey protein content. Somatic cell count of ODM and TDM milks was similar. Rennet coagulation time (RCT) and curd firmness (A60) of milk were not affected by milking frequency. However, rate of curd aggregation (K20) of ODM milk was reduced (P < 0.05) compared with that of TDM milk. Plasmin activity in ODM milk was numerically higher than in TDM milk, but the effect was not significant. ODM milk had higher NAGase activity than TDM milk (P < 0.01). In conclusion, once daily milking reduced milk yield by 29% and did not adversely affect the processability of milk. Moreover, one evening milking per week could be eliminated without adverse effects on milk yield or composition.

Heterogeneity of proteolytic enzyme activities in milk samples of different somatic cell count.

Milk contains the alkaline proteinase plasmin and lysosomal proteinases; the significance of the latter is ill-defined. The objective of this study was to investigate composition and activities of several different proteolytic enzymes in milk samples of varying somatic cell count (SCC). Increasing milk SCC was correlated with increased plasmin, cathepsin D and cysteine protease activities, with concomitant increases in proteolysis in milk. Addition of plasmin inhibitors confirmed the heterogeneity of proteinase activities in milk, as urea-PAGE analysis of milk samples showed casein hydrolysis in milk after 7 d storage even in samples with inhibitors added; extent and heterogeneity of proteolysis was correlated with milk SCC. Rennet coagulation properties were not significantly correlated with SCC, or activities of measured enzymes. Milk of increasing SCC also exhibited decreased physical stability during incubation of milk at 37 degrees C. Pasteurized milk was more stable than raw milk, suggesting that the enzyme(s) or mechanisms leading to such instability are impaired by pasteurization. Overall, milk has a very heterogeneous proteolytic enzyme population, with a higher significance of non-plasmin enzymes, such as cathepsin D and cysteine proteinases, than perhaps previously recognised.