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Project 1: Evaluate and optimize parameters for the use of electrolyzed oxidizing (EO) water as a cleaning and sanitizing agent for on-farm pipeline milking systems. <P>
Project 2: A) Optimize oPSU broth for detection of Listeria monocytogenes in dairy processing environments; B) Use oPSU broth to detect and isolate a large number of strains of L. monocytogenes from dairy processing environments and dairy products; C) Determine the molecular subtypes of isolates from objective B using various DNA fingerprinting methods; and D) Use the above information to track L. monocytogenes contamination in dairy processing plants and establish Good Manufacturing Practices (GMPs) and/or Critical Control Points (CCPs) that prevent post-pasteurization contamination. <P>Project 3: Develop a PC-based, robust quantitative Risk Assessment and Evaluation predictive model for Listeria monocytogenes. Specific objectives are: A) To experimentally measure the spatial pH and water activity distributions during the ripening phase. B) To develop diffusion theory-based mathematical model for predicting pH and water activity as parameter. C) To develop a PC-environment, user-friendly and interactive computational model. D) To use the experimental data to calculate material parameters. E) To measure the survival and growth of L. monocytogenes during the ripening of soft cheese and validate the PC-based model. <P>Project 4: This study is based on the observation that certain genotypes of S. aureus are isolated more frequently from field cases of bovine mastitis than others. Recent studies in our laboratory also have shown that the most prevalent genotypes of S. aureus differentially express factors that may allow them to overcome or suppress essential host defense mechanisms and successfully colonize mammary parenchyma when compared to the rare variants. A lactose-specific component of the phosphotransferase system (lactose-specific enzyme II) was recently identified as a differentially expressed transcript in high prevalent strains of S. aureus using differential display. However, the role of the lactose-specific enzyme II (EII) in the pathogenesis of bovine mastitis is unknown at this time. We hypothesize that the enhanced expression of the EII gene by predominant mastitis-causing strains of S. aureus enables these pathogens to metabolize lactose as a primary carbohydrate source and preferentially grow in milk. The specific aims are: a) determine if alterations in the catabolite repression system of the lactose operon can modify the growth of S. aureus in a lactose-rich microenvironment; b) determine if inactivation of the EII gene results in loss of growth potential of S. aureus in a lactose-rich microenvironment; and c) determine if the growth of S. aureus in a lactose-rich microenvironment can be modified with EII-specific antibodies. <P>
Project 5: This project seeks to map genes and quantitative trait loci (QTL) for clinical mastitis (CM), to determine the genetic and molecular basis of variation for CM, and to use this information in the dairy industry to breed dairy cattle that are genetically resistant to CM.
NON-TECHNICAL SUMMARY: Milk can be contaminated with bacteria that cause human disease from bacteria within the mammary gland, from the milking environment and the processing environment. All three areas need to be better understood before we can reduce milk-related disease. These include reductions in mastitis caused by staphylococcus better dairy sanitation practices that will reduce bacterial contamination of milk, and better processing plant sanitation practices that will prevent post farm-gate contamination. On the farm, we can produce genetically improved cows that are resistant to Staphylococcus. This could be supplemented with an improved vaccine that will further reduce the content of bacteria in fluid milk. On farm milk handling and later processing can be improved by tracking sources of contamination from the environment using advanced biotechnology-based tools. This knowledge of sources of contamination can result in further improvements in milk quality.
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APPROACH: Project 1: Effectiveness of EO water will be determined for inactivating microorganisms on various materials used in milking systems. A laboratory scale milk pipeline system for evaluation of EO water will be constructed, and tested with EO water as a cleaning and sanitizing agent. EO water technology will also be compared with traditional cleaning and sanitizing procedures. <P>Project 2: A) Further optimize oPSU broth for detecting L. monocytogenes in dairy processing environments. B) Use oPSU broth to isolateL. monocytogenes from a wide variety of dairy foods and processing environments. C) Use various DNA fingerprinting methods to subtype the various isolates from objective B. D) Use that information to track L. monocytogenes contamination in dairy processing plants and develop GMPs and HACCP systems that will prevent post-pasteurization contamination, and then share this information with the dairy industry. <P>Project 3: Camembert, a soft cheese, will be used to measure the pH and water activity at select time interval during ripening. The time-domain diffusion model will be used for spatial pH changes. Experimental data will be used to deduce material parameters. A finite element PC-based computational model will be developed and verified. An integrated approach for dynamic growth and finite element models, developed and validated by the PI's group, will form the basis to develop a predictive model. The calculated pH and water activity coefficients will be used to determine the survival and growth of L. monocytogenes during cheese ripening. <P>Project 4: Establish EII as a critical rate limiting factor responsible for the efficient growth of S. aureus in a lactose-rich microenvironment, such as milk. The ability to control S. aureus growth rates by modifying the expression of the EII gene and/or the biological activity of the gene product will form the foundation for subsequent studies aimed at establishing EII as a virulence factor associated with bovine mastitis. The potential development of a subunit mastitis vaccine which incorporates only relevant antigenic determinants has not been investigated. Experiments outlined in this proposal will establish EII as a putative S. aureus virulence factor that may contribute to the pathogenesis of mastitis and could potentially be used to formulate an efficacious mastitis vaccine. <P>Project 5: We will perform a genome scan for QTL affecting CM using a 20 cM marker density. We will study five paternal half-sib Holstein families in U.S. commercial dairy herds by collecting data on CM and on traits related to CM from 600 daughters per sire for a total of 3000 daughters. We will genotype 160 DNA markers on five selected sires, 900 daughters defined by selective genotyping, and 40 quality controls for a total of 945 samples. We will carry out multipoint linkage analysis for the traits studied. These analyses will allow us to identify promising chromosomal regions for follow up using a 5-10 cM marker density and genotyping all family members.
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PROGRESS: 2001/09 TO 2004/08<BR>
1: Response surface models were developed for materials used in milking systems to demonstrate the effectiveness of electrolyzed oxidizing (EO) water on sanitation and cleanliness. EO water was evaluated on a pilot scale milking system by using 5, 7.5, and 10-min treatment times at 60C and on the milking system for longterm by using 7.5-min treatment time at 60C and compared with the conventional method. Results indicate that EO water has the potential to be used as a cleaning and sanitizing agent for CIP cleaning of on-farm milking systems. <BR> <BR> 2: L. monocytogenes is a bacterial pathogen that can contaminate a variety of dairy products & cause a rare severe foodborne disease, listeriosis. A multi-virulence-locus sequence typing (MVLST) method was developed for molecular tracking of L. monocytogenes in dairy processing plants by analyzing 6 virulence genes. Results showed that MVLST provided higher discriminatory power than ribotyping & pulsed-field gel electrophoresis analyses of L. monocytogenes. A multiplex PCR assay was developed to further reduce the time & cost of MVLST and to provide a simultaneous serotype identification of L. monocytogenes. <BR> <BR> 3: Detailed spatial distributions of pH & moisture content (mc) during the ripening phase of Camembert cheese were measured. The pH values increased as the mc values decreased. Results showed that L. innocua could be used as an indicator for replacing L. monocytogenes. A semi-empirical dimensionless model was developed and verified for pH evolution. A diffusion theory based moisture content and dimensionless pH models have been partially integrated with a predictive model for the population of L. monocytogenes during ripening. <BR> <BR> 4: A lactose-specific component of the phosphotransferase system was identified as a unique transcript in high prevalent strains of S. aureus using differential display. The results of screening predominant and rare type S. aureus strains by quantitiative RT-PCR indicated that there was a correlation between the status of S. aureus strains and the average EII transcript expression levels at early-exponential growth phase when bacterial metabolic machineries are geared towards fast growth.<BR> <BR> 5: Developed a mouse model of Escherichia coli induced mastitis and performed gene expression studies using the GeneChip Mouse Expression Set 430 which allows evaluating of mouse genes. Identified genes that are differentially expressed in diseased and healthy mammary tissue. We tested mRNA sampled at 24 & 48hr post-injection with E. coli or saline solution. We performed a linear mixed effect ANOVA analysis on the expression data and identified 1,575 differentially expressed genes. The distribution of these genes with known biological function relevant to host immune-defense-inflammatory response was as follows: apoptosis 35%, innate immunity 17%, humoral immunity 12%, chemotaxis 10%, lymphocyte activation 6% and reactive oxygen species 6%. Most of the genes had unknown function. We are using the power of cluster analysis in this group of genes with unknown function to uncover novel genes and regulatory elements that might be biologically relevant to host inflammatory and immunity response.
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IMPACT: 2001/09 TO 2004/08<BR>
1: Demonstrated that EO water is an effective cleaning agent for the CIP of on-farm pipeline milking systems and can eliminate the need to purchase, transport, handle, and dispose of the concentrated chemicals that are traditionally used to clean milking systems. EO water can be manufactured on-site using salt water and electricity and will be a safer and cheaper alternative to the current cleaning agents. <BR> <BR> 2: A DNA sequence-based subtyping method MLVST, was developed for tracking of pathogenic L. monocytogenes in dairy processing plants. Can be used to identify and help food processors to develop effective intervention strategies to control and prevent its contamination in the finished products.<BR> <BR> 3: An interactive computational model for quantification of microbial hazard associated with microorganisms has been partially developed to be an alternative tool for quantitative Risk Assessment and Evaluation. A non-pathogen, L. innocua, can be used by the dairy industry as a suitable indicator organism for L. monocytogenes in plant studies to eliminate hazard associated with the pathogen.<BR> <BR> 4: Identified important virulence factors involved in the pathogenesis of S. aureus mastitis in dairy cattle that can be used to generate a vaccine for the control of S. mastitis.<BR> <BR> 5: Potential to identify DNA markers and gene sequence variants that affects vulnerability to CM that can be used by the dairy industry to develop dairy cows that are less vulnerable to CM, improve animal health, reduce the use of antibiotics and drugs in dairy farms, and improve quality and safety of dairy food.