James Flynn

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.

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.

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

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.

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.

Pathogen profile of clinical mastitis in Irish milk-recording herds reveals a complex aetiology

Effective mastitis control requires knowledge of the predominant pathogen challenges on the farm. In order to quantify this challenge, the aetiological agents associated with clinical mastitis in 30 milk-recording dairy herds in Ireland over a complete lactation were investigated. Standard bacteriology was performed on 630 pretreatment quarter milk samples, of which 56 per cent were culture-positive, 42 per cent culture-negative and 2 per cent contaminated. Two micro-organisms were isolated from almost 5 per cent of the culture-positive samples. The bacteria isolated were Staphylococcus aureus (23 per cent), Streptococcus uberis (17 per cent), Escherichia coli (9 per cent), Streptococcus species (6 per cent), coagulase-negative Staphylococci (4 per cent) and other species (1 per cent). A wide variety of bacterial species were associated with clinical mastitis, with S aureus the most prevalent pathogen overall, followed by S uberis. However, the bacterial challenges varied widely from farm to farm. In comparison with previous reports, in the present study, the contagious pathogens S aureus and Streptococcus agalactiae were less commonly associated with clinical mastitis, whereas, the environmental pathogens S uberis and E coli were found more commonly associated with clinical mastitis. While S aureus remains the pathogen most commonly associated with intramammary infection in these herds, environmental pathogens, such as S uberis and E coli also present a considerable challenge.

Increased detection of mastitis pathogens by real-time PCR compared to bacterial culture

Rapid and accurate identification of mastitis pathogens is important for disease control. Bacterial culture and isolate identification is considered the gold standard in mastitis diagnosis but is time consuming and results in many culture-negative samples. Identification of mastitis pathogens by PCR has been proposed as a fast and sensitive alternative to bacterial culture. The results of bacterial culture and PCR for the identification of the aetiological agent of clinical mastitis were compared. The pathogen identified by traditional culture methods was also detected by PCR in 98 per cent of cases indicating good agreement between the positive results of bacterial culture and PCR. A mastitis pathogen could not be recovered from approximately 30 per cent of samples by bacterial culture, however, an aetiological agent was identified by PCR in 79 per cent of these samples. Therefore, a mastitis pathogen was detected in significantly more milk samples by PCR than by bacterial culture (92 per cent and 70 per cent, respectively) although the clinical relevance of PCR-positive culture-negative results remains controversial. A mixed infection of two or more mastitis pathogens was also detected more commonly by PCR. Culture-negative samples due to undetected Staphylococcus aureus infections were rare. The use of PCR technology may assist in rapid mastitis diagnosis, however, accurate interpretation of PCR results in the absence of bacterial culture remains problematic.

Characterization of the bovine innate immune response in milk somatic cells following intramammary infection with Streptococcus dysgalactiae subspecies dysgalactiae

The innate immune response of milk somatic cells in cows to Streptococcus dysgalactiae ssp. dysgalactiae was investigated by deliberate intramammary challenge. Cows were challenged with 2,500 colony-forming units of Strep. dysgalactiae DPC 5435, previously isolated from a clinical mastitis case. Eight of the 9 cows treated showed clinical signs of mastitis (swollen udders, increased somatic cell score, and clotted milk) within 1 wk of challenge. Messenger RNA levels of IL-1β and toll-like receptor 4 (TLR4) in milk somatic cells increased approximately 40 fold within 48 h of infusion, whereas tumor necrosis factor α increased 16 fold within the same time frame. Interestingly, cows homozygous for the G allele of the C-X-C chemokine receptor type 1 (CXCR1)-777 polymorphism had higher IL-8 and CXCR1 transcript abundance at 24h postinfusion compared with cows homozygous for the C allele. The difference in expression of these genes at this critical time point may influence the severity of disease within different genotypes.

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.