Molecular Genetic Typing of Staphylococcus aureus from Cows, Goats, Sheep, Rabbits and Chickens

End of project reportsS. aureus can also cause a number of infections in animals such as tick-associated pyaemia in lambs, staphylococcosis in rabbits, septicaemia, abscesses and chondronecrosis in chickens and pneumonia and osteomyelitis complex in turkeys. S. aureus is the most frequent cause of bovine mastitis, a disease that is of economic importance worldwide (Beck et al., 1992). Typically staphylococcal mastitis is chronic in nature, with subclinical mastitis being the most common form

A study of the somatic cell count (SCC) of Irish milk from herd management and environmental perspectives

End of project reportThe objective of this study was to investigate the herd management practices associated with somatic cell count (SCC) and total bacteria count (TBC), to geographically analyse SCC on a national basis, to investigate cow factors associated with SCC and to estimate the milk loss associated with high SCC across parities. From the 400 farms surveyed during farm visits throughout spring and winter, a profile of herd management was developed and the associations between management practices and milk SCC and TBC were established. Management practices associated with low SCC included the use of dry cow therapy, participation in a milk recording scheme, the use of teat disinfection post-milking, a higher frequency of cleaning and increased farm hygiene. Management practices associated with low TBC included the use of heated water in the milking parlour, participation in a milk recording scheme, tail clipping of cows at a frequency greater than once per year and increased farm hygiene. The spatial analysis showed that the south of the country had the greatest density of milk-recording herds. Approximately 60% of all herds in the study were from four counties (Cork, Kerry, Limerick and Tipperary). Average bulk tank SCC increased from 110,264 cells/mL in 2003 to 118,782 cells/mL in 2005, followed by a decrease to 108,454 cells/mL in 2007. Spatial clustering of high SCC scores was not observed (i.e., SCC on one farm was not related to SCC on other farms), which is consistent with mastitis being a herd problem as opposed to an area-based problem. SCC increased with parity from 97,000 cells/mL in parity 1 to 199,000 cell/mL in parity 6. SCC decreased between the period 5 to 35 days in milk (DIM) and 36 to 65 DIM, and increased thereafter. Cows calving in the months of January and September were associated with lower average 305 day SCC. The rate of increase in SCC from mid to late lactation was greatest in older parity animals. There was a test day milk loss of 1.43, 2.08, 2.59, 2.56 and 2.62 litres (parities 1 to 5, respectively) associated with an increase of SCC category from 400,000 cells/mL. When SCC was adjusted (test day SCC/dilution estimate, and test day SCC + (-ß)(test day milk yield)) to account for milk yield, similar trends in milk loss were observed. Alternatively, adjusting SCC (SCC*test day milk yield/mean test day milk yield) to account for milk yield showed an increase in test day milk with increasing SCC category. The results from this study highlight that adherence to best milking/farming practice will help reduce SCC and TBC on farms. The results contribute to the knowledge relating to SCC through increasing the accuracy of milk loss due to SCC and management practices associated with SCC. The results in the study can also be used in the development of strategies to reduce SCC on farms

Molecular Characterisation of Bacteriophage K Towards Applications for the Biocontrol of Pathogenic Staphylococci

End of project reportThe aim of this work was to characterise staphylococcal bacteriophage (a bacterial virus) and to assess their potential as therapeutic agents against pathogenic strains of Staphylococcus aureus, particularly mastitis-causing strains. The project included the use of two newly isolated phage CS1 and DW2, and an existing polyvalent phage. The new phage were isolated from the farmyard and characterised by electron microscopy and restriction analysis. Both phage were shown to belong to the Siphoviridae family and were lytic for representatives of all three clonal groups of Irish mastitis-associated staphylococci. A cocktail of three phage (CS1, DW2 and K) at 108 (plaque forming units) PFU/ml was infused into cows teats in animal trials. The lack of an increase in somatic cell counts in milks indicated strongly that the phage did not irritate the animal. In addition, the most potent phage used in this study, phage K, was further studied by genome sequencing, which revealed a linear DNA genome of 127,395 base pairs, which encodes 118 putative ORFs (open reading frames)

Population and Virulence Factor Analysis of Staphylococcus aureus from Bovine Mastitis.

End of Project ReportsStaphylococcus aureus is a major cause of bovine mastitis and the disease is responsible for substantial economic losses in the dairy industry world-wide. A large number of commonly accepted virulence factors are associated with S. aureus but it is yet to be elucidated which of these are important for infection of the bovine udder. A rational and effective strategy for the control of intramammary infections may need to be directed against clones of S. aureus that commonly cause disease. The objective of this study was to characterise the genetic variance of S. aureus isolate populations from infected udders in Ireland using RAPD-PCR, ribotyping and multilocus enzyme electrophoresis (MLEE). Similar S. aureus isolates collected in the USA were also typed in order to compare strain differences in staphylococcal populations in a different environment. Phenotypic diversity based on a number of presumed virulence factors together with antibiotic sensitivity was examined and correlations between phenotype and genotype were identified, if present. In addition, a pathogenicity island encoding multiple superantigens was completely sequenced and characterised. Knockout mutants of these superantigens were also constructed and in vitro functional analysis performed. Laboratory animal experiments (mice and rabbits) were used to study the relative pathogenicity of individual staphylococcal strains (mice) and also to measure the immunological responses after prolonged exposure to the predominant strains (rabbits)

Quality and safety of milk from farm to dairy product

End of Project ReportNeutrophils (PMN cells) constitute one of the main cell types in milk. Increased PMN level is an indication of mastitis. An ELISA method has been developed to determine PMN levels in milk. This may allow (in addition to somatic cell count [SCC]) selection of infected quarters at drying off, thereby allowing antibiotic therapy to be limited to those quarters. PMN counts may also be used to select milk for processing. Little information is available on the contribution of different somatic cells in milk to cheese-making efficiency. The overall objective of this study was to establish the influence of the quality of raw milk, as determined by somatic cell level and type, on milk biochemistry and cheese quality. The work firstly included modification to a method for an enzyme immunoassay, which could enumerate milk PMN. Subsequently, the impact of somatic cell and PMN content on biochemistry of individual udder quarter milks and simulated bulk cow milks, and quality of cheese manufactured from such milks was investigated. The somatic cell and PMN content of bulk herd milks was also investigated. The modification to the test of O’Sullivan et al (1992) allowed the accurate measurement of PMN levels in milk. The strong relationship or correlation between SCC and PMN of 92% in the individual quarter milks has confirmed previous preliminary data. This is important since PMN in conjunction with SCC may now provide a more reliable method of selecting milks for processing. The reduction in casein at elevated SCC and PMN levels may have resulted in the trend towards deteriorated milk coagulation properties. A very heterogeneous selection of proteolysis patterns was observed in the miniature cheeses. This substantial difference in proteolytic activity in milk from different quarters had not been observed previously. Enzymes associated with the cells in high SCC milk were retained in the cheese curd and thus, contributed to proteolysis during ripening. Addition of low volumes of high SCC milk had an obvious impact on proteolysis patterns and cheese ripening. However, such trends were generally less clear with increasing PMN milk than those observed for addition of high SCC milk. The poor correlation between SCC and PMN obtained in both cow and herd bulk milks, compared to the correlation in quarter milks was probably due to the mixing of high and low SCC milks from either quarters or cows. Thus, the true effect of PMN may not be observed in bulk herd milk but may still have an adverse effect on milk quality. Whether elevated bulk milk SCC and PMN level is due to milk from a smaller number of cows with extremely high SCC/PMN being included with milk from a predominantly healthy herd, or, to large numbers of cows with sub-clinical infections, probably contributes to variation in the effects of SCC/PMN on dairy products

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

peer-reviewedPublic 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

Association of childhood trauma with cognitive function in healthy adults: a pilot study

BACKGROUND: Animal and human studies suggest that stress experienced early in life has detrimental consequences on brain development, including brain regions involved in cognitive function. Cognitive changes are cardinal features of depression and posttraumatic stress disorder. Early-life trauma is a major risk factor for these disorders. Only few studies have measured the long-term consequences of childhood trauma on cognitive function in healthy adults. METHODS: In this pilot study, we investigated the relationship between childhood trauma exposure and cognitive function in 47 healthy adults, who were identified as part of a larger study from the general population in Wichita, KS. We used the Cambridge Neuropsychological Test Automated Battery (CANTAB) and the Wide-Range-Achievement-Test (WRAT-3) to examine cognitive function and individual achievement. Type and severity of childhood trauma was assessed by the Childhood Trauma Questionnaire (CTQ). Data were analyzed using multiple linear regression on CANTAB measures with primary predictors (CTQ scales) and potential confounders (age, sex, education, income). RESULTS: Specific CTQ scales were significantly associated with measures of cognitive function. Emotional abuse was associated with impaired spatial working memory performance. Physical neglect correlated with impaired spatial working memory and pattern recognition memory. Sexual abuse and physical neglect were negatively associated with WRAT-3 scores. However, the association did not reach the significance level of p < 0.01. CONCLUSIONS: Our results suggest that physical neglect and emotional abuse might be associated with memory deficits in adulthood, which in turn might pose a risk factor for the development of psychopathology

Current trends in oxysterol research

In this short review we provide a synopsis of recent developments in oxysterol research highlighting topics of current interest to the community. These include the involvement of oxysterols in neuronal development and survival, their participation in the immune system, particularly with respect to bacterial and viral infection and to Th17-cell development, and the role of oxysterols in breast cancer. We also discuss the value of oxysterol analysis in the diagnosis of disease

Reduced Plasma Levels of 25-Hydroxycholesterol and Increased Cerebrospinal Fluid Levels of Bile Acid Precursors in Multiple Sclerosis Patients

Multiple sclerosis (MS) is an autoimmune, inflammatory disease of the central nervous system (CNS). We have measured the levels of over 20 non-esterified sterols in plasma and cerebrospinal fluid (CSF) from patients suffering from MS, inflammatory CNS disease, neurodegenerative disease and control patients. Analysis was performed following enzyme-assisted derivatisation by liquid chromatography-mass spectrometry (LC-MS) exploiting multistage fragmentation (MS n ). We found increased concentrations of bile acid precursors in CSF from each of the disease states and that patients with inflammatory CNS disease classified as suspected autoimmune disease or of unknown aetiology also showed elevated concentrations of 25-hydroxycholestertol (25-HC, P &#60; 0.05) in CSF. Cholesterol concentrations in CSF were not changed except for patients diagnosed with amyotrophic lateral sclerosis (P &#60; 0.01) or pathogen-based infections of the CNS (P &#60; 0.05) where they were elevated. In plasma, we found that 25-HC (P &#60; 0.01), (25R)26-hydroxycholesterol ((25R)26-HC, P &#60; 0.05) and 7α-hydroxy-3-oxocholest-4-enoic acid (7αH,3O-CA, P &#60; 0.05) were reduced in relapsing-remitting MS (RRMS) patients compared to controls. The pattern of reduced plasma levels of 25-HC, (25R)26-HC and 7αH,3O-CA was unique to RRMS. In summary, in plasma, we find that the concentration of 25-HC in RRMS patients is significantly lower than in controls. This is consistent with the hypothesis that a lower propensity of macrophages to synthesise 25-HC will result in reduced negative feedback by 25-HC on IL-1 family cytokine production and exacerbated MS. In CSF, we find that the dominating metabolites reflect the acidic pathway of bile acid biosynthesis and the elevated levels of these in CNS disease is likely to reflect cholesterol release as a result of demyelination or neuronal death. 25-HC is elevated in patients with inflammatory CNS disease probably as a consequence of up-regulation of the type 1 interferon-stimulated gene cholesterol 25-hydroxylase in macrophage