Progress 09/01/01 to 08/31/04
Outputs
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. 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. 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. 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. 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.
Impacts
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. 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. 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. 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. 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.
Publications
- Walker, S. P., Demirci, A., Graves, R. E., and Spencer, S. B. 2002. Efficacy of electrolyzed oxidizing water to clean milking system materials. ASAE Paper No. NABEC 02-029. American Society of Agricultural Engineers. St. Joseph, MI. 10 pp.
- Walker, S. P., Demirci, A., Graves, R. E., Spencer, S. B. and Roberts, R. F. 2003. CIP cleaning of a pipeline milking system using electrolyzed oxidizing water. ASAE Paper No. 03-6181. American Society of Agricultural Engineers. St. Joseph, MI. 11 pp.
- Walker, S. P., Demirci, A., Graves, R. E., and Spencer, S. B. 2003. Response surface modeling for cleaning and disinfecting materials used in milking systems with electrolyzed oxidizing water. The Institute of Biological Engineering Annual International Meeting. Athens, GA. Abstract 3-5.
- Walker, S. P., Demirci, A., Graves, R. E., Spencer, S. B. and Roberts, R. F. 2003. Response surface modeling for cleaning and disinfecting materials used in milking systems with electrolyzed oxidizing water. ASAE Paper No. 03-7018. American Society of Agricultural Engineers. St. Joseph, MI. 10 pp.
- Walker, S. P. 2003. Cleaning milking systems using electrolyzed oxidizing water. Thesis (M.S.). Pennsylvania State University. 124 pp.
- Liu, S., Puri, V. M., and Demirci, A. 2004. Fate of Listeria innocua during manufacturing and ripening of Camembert cheese. NABEC Paper No. 040024. ASAE, St. Joseph, MI. pp 14.
- Liu, S., Puri, V. M. and Demirci, A. 2004. Survival and growth of Listeria monocytogenes during manufacturing and ripening of Camembert cheese. ASAE Paper No. 046050. ASAE, St. Joseph, MI. pp 15.
- Liu, S., Puri, V. M. and Demirci, A. 2004. Comparison and evaluation of Listeria innocua as a suitable indicator for replacing Listeria monocytogenes during ripening of Camembert cheese. ASAE Paper No. 046025. ASAE, St. Joseph, MI. pp 13.
- Zhang, W., Jayarao, B. M., and Knabel, S. J. 2004. Multi-virulence-locus sequence typing of Listeria monocytogenes. Appl. Environ. Microbiol. 70: 913-920
- Zhang, W., Hughes, A., Wilt, G., and Knabel, S. J. 2004. The BAX PCR for screening Listeria monocytogenes targets a partial putative gene lmo2234. J. Food Prot. 67: 1507-1511
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Progress 01/01/03 to 12/31/03
Outputs
Project 1: In 2003, construction of a pilot scale milking system was completed and the effectiveness of electrolyzed oxidizing (EO) water was evaluated on the milking system by using 5, 7.5, and 10-min treatment times at 60oC as suggested by the developed surface response model and compared with the conventional method in terms of microbial reduction and cleanliness. Project 2: A multi-virulence-locus sequence typing (MVLST) method was developed for subtyping Listeria monocytogenes by analyzing six virulence or virulence-associated genes (prfA, inlB, inlC, dal, lisR, and clpP). This method was optimized by developing a multiplex PCR assay. Comparison of MVLST to pulsed-field gel electrophoresis (PFGE), ribotyping (RT) and multilocus sequence typing (MLST) methods demonstrated that MVLST provided the highest discriminatory power for differentiating L. monocytogenes strains. Project 3: Experimental data for pH and water activity (aw) during the ripening phase of
Camembert showed that: 1) pH values increased gradually and throughout from 4.5 to 7.0, 2) aw values decreased from 0.6 to 0.4. These experimental data are being used to develop and verify mathematical models for pH and aw. Five locations in three sections 120 degrees apart were used to measure the survival and growth of L. innocua during the ripening phase. Similar data for L. monocytogenes are being measured. A finite element formulation for Camembert cheese has been completed, which is being used to develop and verify a MATLAB-based computational model. Project 4: A lactose-specific component of the phosphotransferase system, lactose-specific enzyme II (EII), 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 (predominant vs rare) and the average EII
transcript expression levels at early-exponential growth phase when bacterial metabolic machineries are geared towards fast growth. Since lactose is the major carbohydrate in milk, the ability to uptake and utilize lactose as the energy source may have important impact on S. aureus growth in the mammary gland in general. Project 5: We use positional cloning and functional genomics to identify genetic risk factors for clinical mastitis (CM). Simulation studies indicate that a linkage genome scan that includes 50 half-sib families with 30 daughters per family provides greater than or equal to 0.91 statistical power to detect genes for CM. We plan to identify genes that are differentially expressed in mouse mammary tissue injected with E. coli and saline suspension using GeneChip(R) Mouse Expression Set 430 (Affymetrix, CA). To test CM candidate genes, we developed a case-control sample which includes data on CM traits and DNA for 1,031 dairy cows (895 CM cases and 136 healthy
controls).
Impacts
1: EO water is an effective cleaning agent for cleaning on-farm pipeline milking systems. Using EO water to clean milking systems would eliminate the need to purchase, transport, handle, and dispose of concentrated chemicals. EO water may be manufactured onsite (using salt water & electricity) and could be a safer/cheaper alternative. 2: Provides an effective molecular tool to track pathogenic L. monocytogenes strains in the food supply. It will help dairy processors identify and integrate effective intervention strategies to reduce L. monocytogenes contamination of pasteurized milk & other dairy products and prevent costly recalls & outbreaks. 3: A user-friendly/interactive computational model for quantification of microbial hazard associated with microorganisms will be available as an effective tool for quantitative Risk Assessment & Evaluation. Dairy producers, processors and researchers will have an assessment & evaluation tool for growth & survival of
microorganisms in soft cheeses. 4: Will identify important virulence factors involved in the pathogenesis of S. aureus mastitis in dairy cattle. These virulence factors may be used to generate a vaccine for the control of S. mastitis. 5: Mastitis is a common disease in US dairy herds with a cost of ~$2 billion/annually. Potentially identify DNA markers & genetic risk factors for CM susceptibility. This info can be used to develop CM resistant dairy cattle. This may help to improve animal health, reduce the use of antibiotics & drugs in dairy farms, improve quality-safety of dairy food, & increase farmers profitability.
Publications
- Liu, S. and Puri, V. M. 2003. Measurement of pH and water activity values during ripening of Camembert cheese. ASAE Paper No. 036108. ASAE, St. Joseph, MI. pp 20.
- Vallejo, R. L. 2003. Power to detect loci linked to common diseases of dairy cattle using identical-by-descent based methods of half-sib pair linkage analysis. J. Anim. Sci. 81:160 (Suppl. 1).
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Progress 01/01/02 to 12/31/02
Outputs
Project 1: Response surface models were developed for materials used in milking systems to demonstrate the effectiveness of electrolyzed oxidizing (EO) water and characterize the effect of treatment time and temperature on bacterial reduction and also cleanliness by ATP bioluminescence technique. A pilot scale milking system was also constructed for use in full-scale testing to be completed in 2003. Project 2: Preliminary results indicated that further optimization of oPSU broth is needed to inhibit the growth of background microflora in raw milk. Two novel DNA fingerprinting approaches - 16S-23S rRNA Internal Transcribed Spacer PCR (ITS-PCR) and Multi-locus Sequence Typing (MLST) were introduced in this study. ITS-PCR could not differentiate `Listeria' isolates; however, MLST showed strong discriminatory power. MLST is being optimized for molecular tracking and will be compared with CDC-standardized PFGE to determine which is better suited for molecular tracking of
`L. monocytogenes' in dairy processing environments. Project 3: Measurements of water activity and pH during the ripening phase of Camembert have been initiated. For precise space-time distribution maps, the data are being measured at 3 cross-sections, 120 degrees apart, of cylinder-shaped (diameter, 108 mm x height, 32 mm) Camembert cheese blocks. To obtain detailed information, 16 measurement locations are used in each cross-section. Initial results show that: 1) pH values increase, and 2) water activity values decrease, continually and throughout during ripening. A computational model for predicting pH and water activity during the ripening phase is being developed and verified. Project 4: This study determined the mechanisms which predominant `Staphylococcus aureus' genotypes are able to resist mammary gland defense mechanisms. A lactose-specific component (EII) of the phosphotransferase system was identified as a unique transcript in high prevalent strains of `S. aureus' using
differential display. Screening predominant and rare type `S. aureus' strains by quantitative RT-PCR indicated that there was a correlation between the prevalence status of `S. aureus' strains and the average EII transcript expression levels at early-exponential growth phase. Since lactose is the major carbohydrate in milk, the ability to uptake and utilize lactose as the energy source may have impact on `S. aureus' growth in the mammary. Project 5: A study design of 10 half-sib (HS) families with 60 clinical mastitis (CM) affected and 20 CM unaffected daughters per family provides a statistical power >/= 0.96 to detect loci linked to CM. We identified 10 highly heterozygous Holstein bulls that are as least related as possible and widely used in the US dairy industry. These bulls will be used as parental sires to develop informative HS families. We are developing a case-control sample for CM. This sample will be used in association tests to identify gene variants for CM. We are
collecting blood/milk samples and CM data for cows from PA dairy farms.
Impacts
The use of EO water for the clean-in-place process of cleaning on-farm pipeline milking systems would eliminate the need to purchase, transport, handle, and dispose of the concentrated chemicals that are traditionally used to clean milking systems. `Listeria monocytogenes' is a potentially lethal foodborne pathogen often found in food processing plants. Some strains are known to contaminate ready-to-eat foods (including dairy products) after pasteurization, resulting in foodborne illness and/or costly recalls. We are optimizing a DNA fingerprinting method, Multilocus Sequence Typing, permitting tracking of these `L. monocytogenes' strains in dairy processing plants and leading to reduced contamination of dairy products. An interactive computational model for quantification of microbial hazard associated with microorganisms will be validated, leading to an effective alternative tool for quantitative Risk Assessment and Evaluation and possible implementation in HACCP.
Dairy producers, processors and researchers will have a rapid assessment and evaluation tool for growth and survival of microorganisms in soft cheeses. Mastitis is a common disease in U.S. dairy herds with an annual cost of $2 billion. Identification of virulence factors involved in the pathogenesis of `Staphylococcus aureus' mastitis in dairy cattle may be used to generate efficacious vaccines. We will identify mastitis susceptibility genes and enable the dairy industry to develop mastitis resistant dairy cattle, leading to reduced antibiotic use on dairy farms and enhanced quality/safety of dairy food.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Assembly of pilot-scale milking system is underway. Methods for contaminating materials and evaluating effectiveness have been established. Preliminary experiments have shown significant bacterial reduction on surfaces various milking equipment materials contaminated with raw milk and cleaned with EO water. We are optimizing the EO water treatment for highest inactivation. The oPSU broth for enrichment and presumptive detection of Listeria monocytogenes (LM) in farm milk samples was optimized. Quarter milk (n=300) and bulk tank milk samples (n=248) were tested for the presence of L. monocytogenes using oPSU one-tube test. L. monocytogenes was isolated from 11 of 248 (4.6%) bulk tank milk samples; none of the quarter milk samples (n=300) were positive for L. monocytogenes. We are optimizing oPSU one-tube test for isolation of L. monocytogenes from environmental samples taken from a dairy processing plant. Camembert cheese shelf-life is currently limited to 28 days. To
extend shelf-life, experiments were done to observe the effect of starter culture, cut size of curd pieces, and drainage. An optimum starter culture dose range has been determined. Texture parameters needed to characterize the cheese were identified and are being evaluated. Precision water activity and pH sensors are being procured. Feasibility of an incubator is being evaluated. Initial steps for modeling of pH and water activity changes during ripening are being studied. Certain genotypes of Staphylococcus aureus are isolated more frequently from field cases of bovine mastitis. The most prevalent are more virulent based on their ability to resist neutrophil phagocytosis and killing compared to the rare variants. The predominant genotypes differentially express gene transcripts that may allow them to overcome or suppress essential host defense mechanisms and successfully colonize mammary parenchyma. A lactose-specific component of the phosphotransferase system, lactose-specific
enzyme II (EII), was identified as a unique transcript in high prevalence strains of S. aureus using differential display. Screening of predominant and rare type S. aureus strains by quantitiative RT-PCR showed 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. Ability to uptake and utilize lactose as the energy source may impact S. aureus growth in the mammary gland. Studies showed that varying growth media lactose content affects the growth curves of S. aureus. Equipment for processing and long-term storage of perishable blood, milk, and DNA samples was installed. Laboratory protocols were refined for high throughput isolation of genomic DNA from milk and blood samples of dairy cattle. A study based on computer simulations indicate that a design of 10 half-sib (HS) families with 80 mastitis-affected daughters per family (total of
800 daughters) would provide a statistical power of >0.70 to detect mastitis susceptibility genes. Collection of blood and milk samples for HS families with mastitis-affected cows from U.S. dairy farms is in progress.
Impacts
This research hopes to show that electrolyzed oxidizing (EO) water is an effective cleaning agent for the clean-in-place process of cleaning on-farm pipeline milking systems. The use of EO water to clean milking systems would eliminate the need to purchase, transport, handle, and dispose of the concentrated chemicals that are traditionally used to clean milking systems. Because EO water may be manufactured on-site using only salt water and electricity, it would be a safer and perhaps cheaper alternative to the currently available cleaning methods. A PC-based, user-friendly and interactive computer model for quantification of microbial hazard associated with L. monocytogenes would be available. The validated computer model will be an effective alternative tool for quantitative Risk Assessment and Evaluation, and possible implementation of HACCP. The dairy producers, processors and researchers will have a rapid assessment and evaluation tool for growth and survival of
L. monocytogenes in soft cheeses. This research will identify important virulence factors involved in the pathogenesis of Staphylococcus aureus mastitis in dairy cattle. These putative virulence factors may be used to generate and efficacious vaccine for the control of staphylococcal mastitis. Mastitis is a common disease in U.S. dairy herds with an annual cost of $2 billion. This study will identify mastitis susceptibility genes and enable the dairy industry to develop mastitis resistant dairy cattle. This will reduce the use of antibiotics/drugs in dairy farms and enhance the quality/safety of dairy food.
Publications
- No publications reported this period
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