Cholera outbreaks are proposed to propagate in explosive cycles powered by hyperinfectious Vibrio cholerae and quenched by lytic vibriophage. However, studies to elucidate how these factors affect transmission are lacking because the field experiments are almost intractable. One reason for this is that V. cholerae loses the ability to culture upon transfer to pond water. This phenotype is called the active but non-culturable state (ABNC; an alternative term is viable but non-culturable) because these cells maintain the capacity for metabolic activity. ABNC bacteria may serve as the environmental reservoir for outbreaks but rigorous animal studies to test this hypothesis have not been conducted. In this project, we wanted to determine the relevance of ABNC cells to transmission as well as the impact lytic phage have on V. cholerae as the bacteria enter the ABNC state. Rice-water stool that naturally harbored lytic phage or in vitro derived V. cholerae were incubated in a pond microcosm, and the culturability, infectious dose, and transcriptome were assayed over 24 h. The data show that the major contributors to infection are culturable V. cholerae and not ABNC cells. Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low. However, V. cholerae failed to colonize the small intestine after 24 h of incubation in pond water—the point when the phage and ABNC cell titers were highest. The transcriptional analysis traced the transformation into the non-infectious ABNC state and supports models for the adaptation to nutrient poor aquatic environments. Phage had an undetectable impact on this adaptation. Taken together, the rise of ABNC cells and lytic phage blocked transmission. Thus, there is a fitness advantage if V. cholerae can make a rapid transfer to the next host before these negative selective pressures compound in the aquatic environment.

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

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.

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.

Effective mastitis control requires knowledge of the predominant pathogen challenges on 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 pre-treatment quarter milk samples, of which 56% were culture-positive, 42% culture-negative and 2% contaminated. Two microorganisms were isolated from almost 5% of the culturepositive samples. The bacteria isolated were Staphylococcus aureus (23%), Streptococcus uberis (17%), Escherichia coli (9%), Streptococcus spp (6%), coagulase negative staphylococci (4%) and other species (1%). 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 S. 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.