J.H. Williamson

Application of Streptococcus uberis multilocus sequence typing: analysis of the population structure detected among environmental and bovine isolates from New Zealand and the United Kingdom.

ABSTRACT We recently developed a multilocus sequence typing (MLST) scheme to differentiate S. uberis isolates and facilitate an understanding of the population biology of this pathogen. The scheme was initially used to study a collection of 160 bovine milk isolates from the United Kingdom and showed that the majority of isolates were from one clonal complex (designated the ST-5 complex). Here we describe the MLST analysis of a collection of New Zealand isolates. These were obtained from diverse sources, including bovine milk, other bovine anatomical sites, and environmental sources. The complete allelic profiles of 253 isolates were determined. The collection was highly diverse and included 131 different sequence types (STs). The New Zealand and United Kingdom populations were distinct, since none of the 131 STs were represented within the previously studied collection of 160 United Kingdom S. uberis isolates. However, seven of the STs were members of the ST-5 clonal complex, the major complex within the United Kingdom collection. Two new clonal complexes were identified: ST-143 and ST-86. All three major complexes were isolated from milk, other bovine sites, and the environment. Carriage of the hasA gene, which is necessary for capsule formation, correlated with clonal complex and isolation from clinical cases of mastitis.

Streptococcus uberis -specific T cells are present in mammary gland secretions of cows and can be activated to kill S. uberis

The presence, phenotype and function of Streptococcus uberis-specific T cells in the mammary gland secretion (MGS) and blood of cows exposed to S. uberis were assessed. MGS T cells in the udder were purified and incubated with autologous blood monocytes as antigen-presenting cells (APC). Most cows, irrespective of prior S. uberis infection status and lactation status, were shown to have S. uberis-specific T cells both in MGS and in the blood. When cells from a subgroup of cows were studied, it was found that the S. uberis-specific T cells produced high levels of interferon-gamma (IFN-γ), but low levels of interleukin-10 (IL-10). A high percentage of responding T cells were of the CD8+ memory (CD45RO) subset. T cells from the MGS specific for S. uberis were propagated from animals during the drying off period and expanded in vitro using interleukin-2 (IL-2) and S. uberis antigens. This led to the accumulation of T cells of the CD8+ subset bearing memory cell markers (CD45A−, CD45RO+), which released high levels of IFN-γ. Four of the five T cell lines derived from the MGS of three animals had substantial direct killing activity towards S. uberis in vitro. It is concluded that there is an emergence of S. uberis-specific bactericidal T cells in the MGS of cows after infection or environmental exposure to S. uberis. Vaccines aimed at activating and expanding this T cell population in the mammary glands of cattle may offer an avenue for the prevention of mastitis caused by S. uberis.

Experimentally induced intramammary infection with multiple strains of Streptococcus uberis.

The effect of infusing a mixture of 5 Streptococcus uberis strains into mammary quarters of 10 lactating cows was investigated. All 5 strains, which included 2 originally isolated from the dairy environment and 3 from clinical cases of mastitis, were capable of establishing an intramammary infection when infused individually. However, when the 5 strains were infused together, a single strain predominated in 7 out of 10 quarters. One strain in particular prevailed in 4 mammary quarters and was also found to inhibit the growth of the other 4 strains with deferred antagonism on esculin blood agar. The genes required for the production of bacteriocins nisin U and uberolysin were identified in this strain, whereas the other 4 strains contained only uberolysin genes. Direct competition may have occurred between strains within the mammary gland but competition was not apparent when cultured together in UHT milk, where no strain predominated. Although the mechanism is unknown, these results imply that a selection process can occur within the mammary gland, leading to a single strain that is detected upon diagnosis of mastitis.

Peripartum infection with Streptococcus uberis but not coagulase-negative staphylococci reduced milk production in primiparous cows

The effect of an intramammary infection (IMI) at calving on the milk yield of heifers during their first 200 d in milk (DIM) was estimated by comparing monozygotic twins, where one member had a naturally occurring IMI detected at the first milking after calving and the other twin did not. Data collected weekly over a full lactation for 29 twin pairs were used to estimate the effects of a peri-calving Streptococcus uberis IMI on milk yield and composition. Data for 19 twin pairs were used to estimate the effects of pericalving coagulase-negative staphylococci (CNS) IMI. A heifer with a Strep. uberis IMI produced 200 kg (7%) less milk during the first 200 d of lactation compared with her uninfected twin, with significant differences evident throughout the 200-d period. Similar milk losses were recorded for heifers that developed CM or remained subclinical. An elevated milk SCC for infected heifers was only apparent for the first month (d 2-30), although SCC tended to remain high during the second (d 31-60) and third (d 61-90) months. Milk protein concentrations were greater in the Strep. uberis-infected twin over the 200-d period, whereas fat and lactose concentrations showed little change. An IMI caused by Strep. uberis was associated with a lower milk yield, whereas an IMI by CNS was not, despite CNS-infected twins having a higher SCC than their uninfected twin for the first 30 d of lactation.

Heifer teats sprayed in the dry period with an iodine teat sanitizer have reduced Streptococcus uberis teat-end contamination and less Streptococcus uberis intra-mammary infections at calving

Heifers managed under pastoral conditions are at risk from Streptococcus uberis mastitis infections at calving. A total of 397 heifers from six farms around New Zealand were enrolled in a study to identify and enumerate S. uberis on teat-ends of heifers in the peri-partum period, and to understand the effect of teat-spraying in the pre-calving period on the prevalence and incidence of S. uberis mastitis post-calving. Heifers were randomly assigned to Control or Sprayed groups. Sprayed heifers were teat-sprayed once, three times a week (Monday, Wednesday and Friday) with a commercial iodine-based teat sanitizer, starting at 3 weeks prior to calving and ending at day of calving. Across three farms, all glands of cows in both groups were sampled at calving to determine S. uberis intra-mammary infection (IMI) prevalence. For all farms, clinical mastitis (CM) cases detected during the week after calving were sampled and submitted for bacteriological analysis. Swabbing of teat-ends of 54 heifers from one farm showed that heifers had a pre-existing S. uberis contamination averaging 610 colony-forming units per swab (cfu/swab), at 3 weeks prior to calving. At calving, teat-end contamination was 560 cfu/swab for Sprayed heifers and 1775 cfu/swab for Control heifers. Two weeks after calving, teat-end contamination was similar between both groups, at 30 cfu/swab. The prevalence of S. uberis IMI was significantly lower in the Sprayed (3.5% glands) vs. the Control (7.4%) heifers in the first week after calving. There was a trend for Sprayed heifers (3.6% heifers) to have a lower incidence of S. uberis CM compared with Control heifers (7.4% heifers). It is concluded that teat-spraying in the dry period is a management option that could contribute to controlling heifer S. uberis mastitis in the transition period.

Dairy cows produce cytokine and cytotoxic T cell responses following vaccination with an antigenic fraction from Streptococcus uberis

Streptococcus uberis is a major cause of mastitis in dairy cows worldwide and currently, there is no vaccine commercially available against this form of mastitis. In the current study, cell-free extracts (CFE) were prepared from each of three different S. uberis strains, designated as #3, #24 and #363 representative of the three main sequence types of S. uberis that cause mastitis in New Zealand. These proteins were formulated into vaccines with Emulsigen-D and the immunogenicity of the vaccines was determined in both calves and dairy cows. Two groups of calves (n=5/group) were vaccinated subcutaneously with CFE from strain #24 or strains #3, #24 and #363 formulated with Emulsigen-D, respectively. A third group (n=5) was vaccinated with CFE from the three strains formulated with Emulsigen-D and also containing recombinant bovine granulocyte macrophage colony-stimulating factor while, a control group (n=5) was not vaccinated. Vaccinated animals produced strong antibody responses to the S. uberis antigens and an antigen-specific cytotoxic effect against blood monocytes/macrophages that had phagocytosed S. uberis, with no significant differences in responses observed between the three vaccinated groups. In a second trial, the safety and immunogenicity of the vaccine containing CFE from all three strains of S. uberis and Emulsigen-D was determined in dairy cows. A group of six cows were vaccinated subcutaneously at 3 and 1 week prior to dry off and revaccinated 2-3 weeks before calving. Immune responses in blood and mammary gland secretions (MGS) were monitored during the dry period and in the subsequent lactation. The vaccine was well tolerated with no adverse effect from vaccination observed in any of the cows. Vaccination induced an antigen-specific cytotoxic effect against blood monocytes/macrophages that had phagocytosed S. uberis, moderate antigen-specific IFN-γ responses in blood and strong antibody responses in both blood and MGS. In conclusion, the results suggest vaccination of cattle with S. uberis CFE by the subcutaneous route can induce both cellular and humoral responses.