Temporal trends in bulk tank somatic cell count and total bacterial count in Irish dairy herds during the past decade.

The objective of this study was to document temporal trends in bulk tank somatic cell count (SCC) and total bacterial counts (TBC) in Irish dairy herds during the years 1994 to 2004. Three milk processors participated in the study, providing data on 2,754,270 individual bulk tank SCC and 2,056,992 individual bulk tank TBC records from 9,113 herds. Somatic cell counts decreased during the years 1994 to 2000, followed by an annual increase thereafter of more than 2,000 cells/mL. A tendency existed for TBC to decrease over time. Across all years, bulk tank SCC were the lowest in April and highest in November; TBC were the lowest in May and highest in December. The significant seasonal pattern observed in herd SCC and TBC was an artifact of seasonal calving in Ireland. In general, herds selling more milk had lower bulk tank SCC and TBC. Herds having the highest SCC (i.e., > 450,000 cells/mL) and the lowest SCC (i.e., < or = 150,000 cells/mL) both contributed substantially to the mean SCC of the milk pool collected by the milk processors. Derived transition matrices showed that between adjacent years, herds had the greatest probability of remaining in the same annual mean SCC or TBC category.

Isolation and characterization of two anti-staphylococcal bacteriophages specific for pathogenic Staphylococcus aureus associated with bovine infections.

AIMS: The aim of this study was to isolate and characterize bacteriophages against bovine Staphylococcus aureus associated with mastitis. METHODS AND RESULTS: We describe the isolation of two anti-staphylococcal phages namely DW2 and CS1 from farmyard slurry. Both phages were characterized by electron microscopy and restriction analysis and shown to belong to the Siphoviridae family. CS1 and DW2 were lytic for representatives of all three clonal groups of Irish mastitis-associated staphylococci. These phages were compared with the previously characterized Myoviridae phage K. Infusion of a cocktail of all three phages at 10(8) PFU ml(-1) into live cow teats resulted in no detectable increase in somatic cell counts in milks indicating that the phages did not irritate the animal. CONCLUSION: Two new anti-staphylococcal phages CS1 and DW2 were isolated and characterized and tested for immunogenicity in animal teats. SIGNIFICANCE AND IMPACT OF THE STUDY: The phages isolated in this study are active against pathogenic S. aureus and may be incorporated into teat-dips or teat-washes as a non-antibiotic prophylaxis against staphylococcal bovine mastitis.

The recombinant phage lysin LysK has a broad spectrum of lytic activity against clinically relevant staphylococci, including methicillin-resistant Staphylococcus aureus.

This study concerns the cloning, characterization, and expression of the lysin (LysK) from staphylococcal phage K in Lactococcus lactis. Lactococcal lysates containing recombinant LysK were found to inhibit a range of different species of staphylococci isolated from bovine and human infection sources, including methicillin-resistant Staphylococcus aureus. LysK thus has potential as an antimicrobial for applications in the prevention and/or treatment of infections caused by staphylococci.

Inhibition of bacteriophage K proliferation on Staphylococcus aureus in raw bovine milk.

AIMS: To assess the ability of staphylococcal bacteriophage K to inhibit Staphylococcus aureus in raw milk. METHODS AND RESULTS: The ability of bacteriophage (phage) to replicate in milk is important in situations where phage might be used as a therapeutic for bovine mastitis. Phage K was able to replicate normally, leading to elimination of the host culture in milk, which had been previously heat-treated. When raw milk was used under identical conditions, the phages were unable to replicate. Phage adsorption assays were performed and these demonstrated that adsorption of phage was significantly reduced in the raw milk while it was restored in the heat-treated sample (86.50% compared with 99.96% adsorption respectively). When confocal microscopy with a Live/Dead Bac light staining system was employed, it was observed that in raw milk S. aureus formed clusters associated with fat globules, while in heat-treated milk, bacterial agglutination had not occurred. CONCLUSIONS: Raw milk inhibits staphylococcal phage K proliferation. SIGNIFICANCE AND IMPACT OF THE STUDY: This observation has implications for the exploitation of staphylococcal therapeutic phage in milk.

Potential of the polyvalent anti-Staphylococcus bacteriophage K for control of antibiotic-resistant staphylococci from hospitals.

The increasing prevalence of antibiotic-resistant staphylococci has prompted the need for antibacterial controls other than antibiotics. In this study, a lytic bacteriophage (phage K) was assessed in vitro for its ability to inhibit emerging drug-resistant Staphylococcus aureus strains from hospitals and other species of Staphylococcus isolated from bovine infections. In in vitro inhibitory assays, phage K lysed a range of clinically isolated methicillin-resistant S. aureus (MRSA) strains, S. aureus with heterogeneous vancomycin resistance and vancomycin resistance, and teicoplanin-resistant strains. In these assays, 14 of the MRSA strains were initially only weakly sensitive to this phage. However, propagation of phage K on these less-sensitive strains resulted in all 14 being sensitive to the modified phages. The results enforce the principle that, while certain target bacteria may be relatively insensitive to lytic phage, this can be overcome by obtaining modified phage variants from passage of the phage through the insensitive strains. Model in situ hand wash studies using a phage-enriched wash solution resulted in a 100-fold reduction in staphylococcal numbers on human skin by comparison with numbers remaining after washing in phage-free solution. Infusion of the phage into a nonimmunogenic bismuth-based cream resulted in strong anti-Staphylococcus activity from the cream on plates and in broth.

Genome of staphylococcal phage K: a new lineage of Myoviridae infecting gram-positive bacteria with a low G+C content.

Phage K is a polyvalent phage of the Myoviridae family which is active against a wide range of staphylococci. Phage genome sequencing revealed a linear DNA genome of 127,395 bp, which carries 118 putative open reading frames. The genome is organized in a modular form, encoding modules for lysis, structural proteins, DNA replication, and transcription. Interestingly, the structural module shows high homology to the structural module from Listeria phage A511, suggesting intergenus horizontal transfer. In addition, phage K exhibits the potential to encode proteins necessary for its own replisome, including DNA ligase, primase, helicase, polymerase, RNase H, and DNA binding proteins. Phage K has a complete absence of GATC sites, making it insensitive to restriction enzymes which cleave this sequence. Three introns (lys-I1, pol-I2, and pol-I3) encoding putative endonucleases were located in the genome. Two of these (pol-I2 and pol-I3) were found to interrupt the DNA polymerase gene, while the other (lys-I1) interrupts the lysin gene. Two of the introns encode putative proteins with homology to HNH endonucleases, whereas the other encodes a 270-amino-acid protein which contains two zinc fingers (CX(2)CX(22)CX(2)C and CX(2)CX(23)CX(2)C). The availability of the genome of this highly virulent phage, which is active against infective staphylococci, should provide new insights into the biology and evolution of large broad-spectrum polyvalent phages.

Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens.

Previous studies have demonstrated that a proportion of Staphylococcus aureus isolates from bovine mastitis coproduce toxic shock syndrome toxin (TSST) and staphylococcal enterotoxin C (SEC). In this study, molecular genetic analysis of one such strain, RF122, revealed the presence of a 15,891-bp putative pathogenicity island (SaPIbov) encoding the genes for TSST (tst), the SEC bovine variant (sec-bovine), and a gene (sel) which encodes an enterotoxin-like protein. The island contains 21 open reading frames specifying hypothetical proteins longer than 60 amino acids including an integrase-like gene. The element is bordered by 74-bp direct repeats at the left and right junctions, and the integration site lies adjacent to the 3' end of the GMP synthase gene (gmps) in the S. aureus chromosome. SaPIbov contains a central region of sequence identity with the previously characterized tst pathogenicity island SaPI1 (J. A. Lindsay et al., Mol. Microbiol. 29:527-543, 1998). A closely related strain, RF120, of the same multilocus enzyme electrophoretic type, random amplified polymorphic DNA type, and ribotype, does not contain the island, implying that the element is mobile and that a recent insertion/deletion event has taken place. TSST and TSST/SEC-deficient mutants of S. aureus strain RF122 were constructed by allele replacement. In vitro bovine Vbeta-specific lymphocyte expansion analysis by culture supernatants of wild-type strains and of tst and sec-bovine allele replacement mutants revealed that TSST stimulates BTB13-specific T cells whereas SEC-bovine stimulates BTB93-specific T cells. This suggests that the presence of SaPIbov may contribute to modulation of the bovine immune response.

Protection against Staphylococcus aureus mastitis in dairy cows using a bismuth-based teat seal containing the bacteriocin, lacticin 3147.

We assessed the effectiveness of a novel dry cow treatment containing lacticin 3147 using deliberate challenge studies in lactating cows. Infection-free quarters of lactating cows were infused with Teat seal (Cross Vetpharm Group, Ltd., Dublin, Ireland) combined with the food-grade bacteriocin, lacticin 3147. Natural infection of the teat was simulated by deliberately introducing Staphylococcus aureus into the teat duct and teat sinus. Relative to control quarters, teat seal plus lacticin 3147 reduced the number of teats shedding viable cells when an inoculum of either approximately 1.7 x 10(3) or approximately 6.8 x 10(3) cfu per teat was used. In addition, the numbers of challenge organisms in those teats from which S. aureus was subsequently recovered were also reduced. However, when the concentration of bacteriocin in the teat seal formulation was reduced by approximately 50%, the number of teats shedding S. aureus cells was not reduced. These data indicate the potential for lacticin 3147 to prevent staphylococcal mastitis infections when a sufficient concentration of the bacteriocin is present. This study also highlights the application of a lactating-cow model to assess the effectiveness of antimicrobial intramammary products on mastitic cell populations.

Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection.

Staphylococcus aureus isolates from cows in Ireland (n = 102) and the USA (n = 42) were characterized by RAPD-PCR and analysed for the production of a number of putative virulence factors. Of these strains 63 representative isolates were screened for the corresponding virulence factor genes by PCR or Southern hybridization or both. The isolates were divided into 12 distinct clonal types on the basis of their RAPD fingerprint profiles. Of the isolates, 107 (74.3%) tested positive for clumping factor in a slide agglutination test, all 24 RAPD type 7 isolates being negative for clumping factor. PCR analysis of region R, a repeat region of the clfA gene, revealed eight region-R sizes. There was a strong association between RAPD type and the clfA region-R genotype among Irish isolates. Of the RAPD type 7 isolates, 21 (87.5%) coproduced toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin C (SEC). Over 90% of isolates demonstrated haemolytic activity on sheep or rabbit red blood cells and all isolates harboured the gamma-haemolysin (hlg) locus. Of the Irish isolates, all those of RAPD type 7 were sensitive to penicillin G, whereas 86% of RAPD types 4 and 5 strains were resistant. Furthermore, RAPD types 5 and 7 were more likely to be associated with clinical mastitis whereas RAPD type 4 isolates were more often associated with a latent infection. The current study identifies some of the putative virulence factors produced by the predominant clonal types of bovine Staph. aureus that may be considered as components of a vaccine.

The natural food grade inhibitor, lacticin 3147, reduced the incidence of mastitis after experimental challenge with Streptococcus dysgalactiae in nonlactating dairy cows.

Lacticin 3147 is a broad-spectrum bacteriocin produced by the food-grade organism Lactococcus lactis. Lacticin 3147 is active at a neutral pH and has been shown to be bactericidal to streptococci and staphylococci in vitro. The effectiveness of an intramammary teat seal formulation, and a teat seal containing lacticin 3147 was evaluated at drying off in 68 uninfected quarters of 18 cows. Following infusion of either teat seal or lacticin 3147 combined with teat seal, a deliberate infection challenge of Streptococcus dysgalactiae (approximately equal to 1.5 x 10(4) cfu per teat) was administered by direct inoculation into the teat sinus. During an 8-d experimental period following inoculation, 61% of control quarters and 6% of the treatment quarters either developed clinical mastitis or were shedding the challenge organism. Randomly amplified polymorphic DNA polymerase chain reaction genetic typing was used to confirm that both the new infections and the bacteria surviving in the teats at the end of the experiment were the challenge strain. The combination of teat seal and lacticin 3147 was well tolerated within the udder and elicited only a temporary increase in somatic cell count to 5.7 x 10(5)/ml (88 h after infusion) in a previously uninfected lactating udder quarter. Therefore, we concluded that this nonantibiotic approach to mastitis prevention may contribute to a reduction in the routine application of antibiotics at drying off in the future.

Developing applications for lactococcal bacteriocins.

While much of the applied research carried out to date with bacteriocins has concerned nisin, lactococci produce other bacteriocins with economic potential. An example is the two component bacteriocin lacticin 3147, which is active over a wide pH range and has a broad spectrum of activity against gram-positive bacteria. Since the genetic determinants for lacticin 3147 are encoded on a large self-transmissible plasmid, the bacteriocin genes may be conveniently transferred to different lactococcal starters. The resulting food-grade strains can then be used to make a significant impact on the safety and quality of a variety of fermented foods, through the inhibition of undesirable microflora. The bacteriocin is heat stable so it can also be used as an ingredient in a powdered form such as a spray-dried fermentate. Given the observation that lacticin 3147 is effective at physiological pH, there is also considerable potential for biomedical applications. Field trials have demonstrated its efficacy in the prevention of mastitis infections in dairy cows. In contrast to lacticin 3147, the lactococcin bacteriocins A, B and M have a narrow spectrum of activity limited to lactococci. Strains which produce these inhibitors can be exploited in the acceleration of cheese ripening by assisting the premature lysis of starter cultures.

The natural food grade inhibitor, lacticin 3147, reduced the incidence of mastitis after experimental challenge with Streptococcus dysgalactiae in nonlactating dairy cows.

Lacticin 3147 is a broad-spectrum bacteriocin produced by the food-grade organism Lactococcus lactis. Lacticin 3147 is active at a neutral pH and has been shown to be bactericidal to streptococci and staphylococci in vitro. The effectiveness of an intramammary teat seal formulation, and a teat seal containing lacticin 3147 was evaluated at drying off in 68 uninfected quarters of 18 cows. Following infusion of either teat seal or lacticin 3147 combined with teat seal, a deliberate infection challenge of Streptococcus dysgalactiae (approximately equal to 1.5 x 10(4) cfu per teat) was administered by direct inoculation into the teat sinus. During an 8-d experimental period following inoculation, 61% of control quarters and 6% of the treatment quarters either developed clinical mastitis or were shedding the challenge organism. Randomly amplified polymorphic DNA polymerase chain reaction genetic typing was used to confirm that both the new infections and the bacteria surviving in the teats at the end of the experiment were the challenge strain. The combination of teat seal and lacticin 3147 was well tolerated within the udder and elicited only a temporary increase in somatic cell count to 5.7 x 10(5)/ml (88 h after infusion) in a previously uninfected lactating udder quarter. Therefore, we concluded that this nonantibiotic approach to mastitis prevention may contribute to a reduction in the routine application of antibiotics at drying off in the future.

Effect of decreased milking frequency of cows in late lactation on milk somatic cell count, polymorphonuclear leucocyte numbers, composition and proteolytic activity.

The effects of reducing the frequency of milking of cows in late lactation on milk somatic cell count (SCC), polymorphonuclear leucocyte (PMN) content, chemical composition and proteolytic activity were investigated. Intermittent milking is frequently practised by Irish farmers in late lactation, and the objective of this study was to determine whether this procedure could be linked to altered quality of milk. Seventeen Holstein Friesian cows in late lactation (> 215 d in milk) were assigned to two treatment groups, and were either milked twice a day until drying-off (control group) or milked intermittently as the yield fell (test group). Milk composition and enzymic characteristics were measured on two occasions. At the first sampling, day 7, test cows were on once daily milking and at the second, day 15, the test cows were being milked every second day. Milk yields were significantly lower in test than control animals and decreased between days 7 and 15 in both groups. Milk SCC and PMN levels were increased on reducing milking frequency and, at day 15, the increase was not linked to decreased milk yield. Milk lactose levels were significantly decreased and pH, alpha-lactalbumin levels, plasmin activity and plasminogen activity significantly increased by reducing milking frequency. In conclusion, reduced frequency of milking in late lactation leads to the production of milk that is abnormal in character and this may be linked to reduced quality of dairy products manufactured from such milk.

Evaluation of lacticin 3147 and a teat seal containing this bacteriocin for inhibition of mastitis pathogens.

Lacticin 3147 is a broad-spectrum bacteriocin produced by Lactococcus lactis subsp. lactis DPC3147 which is bactericidal against a range of mastitis-causing streptococci and staphylococci. In this study, both lacticin 3147 and the lantibiotic nisin were separately incorporated into an intramammary teat seal product. The seal containing lacticin 3147 exhibited excellent antimicrobial activity and might form the basis of an improved treatment for the prevention of mastitis in dry cows.

Fine-structure molecular epidemiological analysis of Staphylococcus aureus recovered from cows.

Sixty-three Staphylococcus aureus isolates recovered from bovine sources in the USA and the Republic of Ireland were characterized by multilocus enzyme electrophoresis (MLEE), ribotyping, and random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) typing at two separate laboratories. The S. aureus isolates were assigned by MLEE to 10 electrophoretic types (ETs) (Index of Discrimination, D = 0.779). In contrast, the same isolates were assigned to 13 ribotypes (D = 0.888), and to 12 RAPD types (D = 0.898). A common clone, ET3, of worldwide distribution, was represented by six distinct combinations of ribotypes and RAPD types. S. aureus clones recovered from cows in Ireland were also associated with mastitis in dairy cows in the USA. These findings are consistent with the hypothesis that only a few specialized clones of S. aureus are responsible for the majority of cases of bovine mastitis, and that these clones have a broad geographic distribution.

A systematic method for evaluating the potential viability of local vaccine producers.

Vaccine production exists in > 55 countries, but many production facilities cannot assure a reliable supply of existing or new vaccines. By analysing the characteristics of successful producers, we have identified seven critical elements for viability, each defined by several indicators. Each indicated weakness implies an investment to correct it. Thirty-one manufacturers were assessed based on these viability indicators and the implied investment costs. Three general groupings were found. 'Viable' producers scored well in all seven categories. Those with a 'low probability' of viability are weak in all areas. Facilities regarded as 'potentially viable' may produce sufficient vaccine to meet national needs, but must develop appropriate structures to effectively manage change. This analysis provides a logical system for governments and donors to evaluate the potential effectiveness of further investment in local vaccine production.

Persistence of detectable residues of penicillin and cloxacillin in normal and mastitic quarters following intramammary infusion.

The excretion rates of sodium penicillin and sodium cloxacillin from treated and untreated quarters of normal and mastitic cows were studied. Penicillin was detected in normal and infected quarters for 72 hours after treatment. Cloxacillin was detected in normal and infected quarters for 64 and 48 hours, respectively, after treatment. Differences in the excretion rates of both antibiotics from normal and infected treated quarters were not significant. Penicillin was detected in the untreated quarters of both normal and mastitic cows but cloxacillin was only detected in the untreated quarters of mastitic cows.