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Preharvest Food Safety: Reducing the Risk of Preharvest Pathogens, Antibiotic Residues and Antibiotic Resistance in Cows

Investigators
Sears, Philip
Institutions
Michigan State University
Start date
2007
End date
2011
Objective
  1. Determine animal safety of Nisin in the mammary gland An antimicrobial protein (Nisin) has demonstrated bacteriocidal activity toward staphylococcal and streptococcal intramammary infections. A purified Nisin compound has been prepared from commercial available nisaplin. This preparation of Nisin will be tested for purity and affect on bovine gland tissue following intramammary infusion. Batches of Nisin can vary and can produce an elevated milk somatic cell count when infused into the mammary gland, however this variation does not affect bactericidal activity. Five fractions of the Nisaplin preparations have been identified, but the specific component(s) contributing to the adverse cellular response have not been determined. Nisin peptide interferes with the Limulus Assay for endotoxin testing, thus the most sensitive test available is in the mammary gland.
  2. Determine formulation efficacy of Nisin towards mastitis pathogens in lactating animals. To confirm the optimal effective intramammary dose of Nisin and in the treatment of clinical and subclinical mastitis in lactating dairy cows caused by Streptococcus agalactiae, Staphylococcus aureus, E. coli, and Streptococcus spp. (non-agalactiae) under variable field conditions.
  3. Determination of the formulation efficacy of Nisin towards mastitis pathogens in the non-lactating period Establish efficacy for pathogen reduction in non-lactation by treating cows at dry off. Two hundred thirty one multiparous Holstein cows near the end of their lactation cycle will be selected for participation in the dry cow treatment trial. Elevated somatic cell counts and two cultures will be a prerequisite for entering the study. Cultures may be positive for staphylococci (including but not limited to S. aureus), Streptococcus agalactiae, Streptococcus dysagalactiae, or Streptococcus uberis. In these studies, cases of Gram-negative mastitis will be excluded. There is preliminary evidence that Nisin, in formulation will be effective against mastitis caused by Gram-positive bacteria. Infected animals will be randomly assigned to one of three treatment groups to be given either 50 mg of Nisin (Test treatment), 50 mg of Dry-Clox (Positive Control; Fort Dodge, cloxacillin benzathine), or aqueous solution control (Negative Control) as an intramammary infusion of 10 ml in the infected gland. We calculate that this will result in at least 76 infected quarters in each treatment group. Each infected gland will receive a single intamammary infusion immediately after the last regular milking at dry off. Based on previous experiments, we anticipate a 10-20% spontaneous cure rate (negative control) and up to a 60% cure rate for Dry-Clox (positive control).
More information
NON-TECHNICAL SUMMARY: Objective 1. To demonstrate animal safety for the antimicriobial protein, nisin. Objective 2. To demonstrate the bacteriocidal activity of nisin towards Gram-positive pathogens and the reduction of pathogens in lactating cows with subclinical mastitis. Objective 3. To demonstrate nisin's efficacy by the reduction of intramammary infections in the non-lactating (dry) period. Bovine mastitis is a major disease effecting dairy cattle that requires the use of antibiotic therapy. The use of antibiotic in the treatment of mastitis pathogens increases the risk of antibiotic residues in milk and the development of antibiotic resistance on dairy farms. The use of non-antibiotic antimicrobial proteins can reduce the risk of antibiotics in food products and lower the risk of drug resistance in preharvest pathogens. Our studies clearly demonstrates that nisin is a safe antimicrobial protein, that is effective against staphylococcal and streptococcal pathogens including antibiotic resistant pathogens. The field studies for clinical mastitis have been submitted to FDA for product approval. Treatment in the non-lactating (dry) period is in progress. In addition to developing an effective treatment that may result in a zero day withdraw, on-farm mastitis diagnostic testing has been initiated to reduce the antibiotic use on farms, thus reducing the risk of antibiotic residues and reducing the risk of antibiotic resistance. On farm studies have shown that through the use of this on-farm pathogen testing many farms can reduce their antibiotic use over 50%.

APPROACH: Research Protocol Objective 1: A representative sample will be collected from the total milk of each individual quarter. Samples taken prior to or following milking are not as accurate and less repeatable. In this study, effects between quarters within the same cow are not significant at this level of cellular responses as determined in previous testing. A minimum of four cows will be used in each trial to obtain accurate sampling and observation for each of the treatments. Four trials will be needed to collect the 50-quarter tests necessary to evaluate all components. Other component testing will be added if needed to determine which components are producing cellular changes. Additional testing will be addressed if formulation is changed or modified based on the dosage trials in Objective 2. Objective 2: General descriptive data for each participating herd will be recorded, and for each cow that qualifies for the study, an individual production/udder health profile will also be submitted. All clinical observations will be performed and recorded by the same individual (herd manager) under the supervision of the investigator. Treatment will be administered by the herd manager based on a predetermined treatment schedule. Treatments will be assigned using a random number generating computer program. Two post-treatment milk samples will be collected by the herd manager at 14 and 28 days following treatment. The investigator or trained technician authorized by the investigator will monitor the participation of the herd manager and will transport milk samples back to the laboratory on a weekly or as needed basis. Objective 3: Bacterial evaluation. FDA Guideline 49 requires that a minimum of two microbiology test samples be analyzed during the assessment period following treatment. These milk samples will be collected on day 3 and 5 post-calving. The samples will be plated on BHI agar and colonies identified and counted. Clinical evaluation. Other health measurements will be recorded for each animal before and after treatment including rectal temperature and udder physical health (tenderness & swelling). Cytological evaluation. Quarter milk will be collected prior to dry off and then on Day 3 and 5. Somatic Cell Counts will be analyzed through the DHIA laboratory for each treatment.

PROGRESS: 2007/01 TO 2007/12
OUTPUTS: Objective 1. To demonstrate the bacteriocidal activity of nisin towards Gram-positive pathogens and the reduction of pathogens in lactating cows with subclinical mastitis. Objective 2. To demonstrate systemic and intramammary efficacy by the reduction of intramammary infections in the non-lactating (dry) period. Objective 3. To demonstrate systemic and intramammary efficacy by the reduction of intramammary infections in the prepartum heifers at calving. Bovine mastitis is a major disease effecting dairy cattle that requires the use of antibiotic therapy. The use of antibiotic in the treatment of mastitis pathogens increases the risk of antibiotic residues in milk and the development of antibiotic resistance on dairy farms. The use of non-antibiotic antimicrobial proteins can reduce the risk of antibiotics in food products and lower the risk of drug resistance in preharvest pathogens. Our studies clearly demonstrates that nisin is a safe antimicrobial protein, that is effective against staphylococcal and streptococcal pathogens including antibiotic resistant pathogens. The field studies for clinical mastitis have been submitted to FDA for product approval. Treatment in the non-lactating (dry) period has been completed. Treament in prepartum heifers is in progress. In addition to developing an effective treatment that may result in a zero day withdraw, on-farm mastitis diagnostic testing has been initiated to reduce the antibiotic use on farms, thus reducing the risk of antibiotic residues and reducing the risk of antibiotic resistance. On farm studies have shown that through the use of this on-farm pathogen testing many farms can reduce their antibiotic use over 50%. Demonstration farms have been established to measure the economic impact.
PARTICIPANTS: Two large commerical farms of 2000 cows, 3200 cows respecively were enrolled in the on farm diagnostic and therapy testing. Herd managers were trained to perform on-farm milk culturing to accompany treatment trial.
TARGET AUDIENCES: The target audience is commerical dairy farms targeting their use of antibiotic treatment to reduce mastitis and low milk somatic cell counts. This project is aimed at reducing the amount of antibiotic used on dairy farms and reduce the risk of antibiotic residues.

IMPACT: 2007/01 TO 2007/12
Bovine mastitis is a major disease effecting dairy cattle that requires the use of antibiotic therapy. The use of antibiotic in the treatment of mastitis pathogens increases the risk of antibiotic residues in milk and the development of antibiotic resistance on dairy farms. The use of non-antibiotic antimicrobial proteins can reduce the risk of antibiotics in food products and lower the risk of drug resistance in preharvest pathogens. Our studies clearly demonstrates that nisin is a safe antimicrobial protein, that is effective against staphylococcal and streptococcal pathogens including antibiotic resistant pathogens. The field studies for clinical mastitis have been submitted to FDA for product approval. Treatment in the non-lactating (dry) period is completed and prepartum heifer trials are in progress. On farm studies have shown that through the use of this on-farm pathogen testing many farms can reduce their antibiotic use over 50%. Our studies, demonstrate that dairy farms can improve there diagnostic efforts in identifying pathogen by "on-farm diagnostic" or through their local veterinarian and establish protocols that will lead to targeted treatments and reduction in antibiotic use.

PROGRESS: 2006/01/01 TO 2006/12/31
There are 3 objectives that are being addressed in the preharvest pathogen studies. Objective 1. To demonstrate animal safety for the antimicriobial protein, nisin. Objective 2. To demonstrate the bacteriocidal activity of nisin towards Gram-positive pathogens and the reduction of pathogens in lactating cows with subclinical mastitis. Objective 3. To demonstrate nisin's efficacy by the reduction of intramammary infections in the non-lactating (dry) period. Early data indicate good efficacy consistent with those studies seen in preliminary investigation on the university herd. Adverse reaction to the product has not been reported confirming data reported in Objective 1. Nisin has been initiated in commercial clinical trial and evaluation of clinical data from the lactation studies indicate value as a commercial product to treat clinical mastitis. Further investigations have been iniated to meet FDA protocols for lactating products. Objective 3 has been formulated and the dry cow (non-lactation) therapy studies will be initiated. In addition to the efficacy studies, on-farm diagnostic testing has been initiated in study herds. The cause of mastitis can be identified on the farm and treatment targeted at specific causes. Several farms have been identified for on-farm studies to evaluate both efficacy of treating based on farm diagnostic testing and the economic gains by implementing pathogen identification prior to therapy invention. This diagnostic protocol has been provided in extension meetings, producer workshops and is available on-line at our extension website (www.cvm.msu.edu/extension dairy programs).

IMPACT: 2006/01/01 TO 2006/12/31
Bovine mastitis is a major disease effecting dairy cattle that requires the use of antibiotic therapy. The use of antibiotic in the treatment of mastitis pathogens increases the risk of antibiotic residues in milk and the development of antibiotic resistance on dairy farms. The use of non-antibiotic antimicrobial proteins can reduce the risk of antibiotics in food products and lower the risk of drug resistance in preharvest pathogens. Our studies clearly demonstrates that nisin is a safe antimicrobial protein, that is effective against staphylococcal and streptococcal pathogens including antibiotic resistant pathogens. The field studies for clinical mastitis have been submitted to FDA for product approval. Treatment in the non-lactating (dry) period is in progress. In addition to developing an effective treatment that may result in a zero day withdraw, on-farm mastitis diagnostic testing has been initiated to reduce the antibiotic use on farms, thus reducing the risk of antibiotic residues and reducing the risk of antibiotic resistance. On farm studies have shown that through the use of this on-farm pathogen testing many farms can reduce their antibiotic use over 50%. Our studies, demonstrate that dairy farms can improve there diagnostic efforts in identifying pathogen by "on-farm diagnostic" or through their local veterinarian and establish protocols that will lead to targeted treatments and reduction in antibiotic use.

PROGRESS: 2005/01/01 TO 2005/12/31
There are 4 objectives that are being addressed in the preharvest pathogen studies. Objective 1. To demonstrate animal safety for the antimicriobial protein, nisin. Objective 2. To reduce clinical signs and demonstrate the bacteriocidal activity of nisin towards Gram-positive pathogens in lactating cows. Objective 3. To demonstrate the bacteriocidal activity of nisin towards Gram-positive pathogens and the reduction of pathogens in lactating cows with subclinical mastitis. Objective 4. To demonstrate nisin efficacy by the reduction of intramammary infections in the non-lactating (dry) period. Over the last year, field studies have been implemented for subclinical and clinical mastitis (Objective 2). Early data indicate good efficacy consistent with those studies seen in preliminary investigation on the university herd. Adverse reaction to the product has not been reported confirming data reported in Objective 1. Upon evaluation of clinical data from the lactation studies, dry cow (non-lactation) therapy studies will be initiated. In addition to the efficacy studies, on-farm diagnostic testing has been initiated in study herds. The cause of mastitis can be identified on the farm and treatment targeted at specific causes. This diagnostic protocol has been provided in extension meetings, producer workshops and is available on-line at our extension website (www.cvm.msu.edu/extension dairy programs).

IMPACT: 2005/01/01 TO 2005/12/31
Bovine mastitis is a major disease effecting dairy cattle that requires the use of antibiotic therapy. The use of antibiotic in the treatment of mastitis pathogens increases the risk of antibiotic residues in milk and the development of antibiotic resistance on dairy farms. The use of non-antibiotic antimicrobial proteins can reduce the risk of antibiotics in food products and lower the risk of drug resistance in preharvest pathogens. Our studies clearly demonstrates that nisin is a safe antimicrobial protein, that is effective against staphylococcal and streptococcal pathogens including antibiotic resistant pathogens, and it is safe as an intramammary infusion for the treatment of mastitis in dairy cattle. All adverse reactions have been identified and addressed in our recent studies so the present product (nisin) can be studied in the field in a NDA studies (FDA approval). This product has received with very short milk withdrawal (24 hours) period. The field studies are in progress on several Michigan dairy farms. Preliminary data is very promising and will be reviewed and reported upon in the coming year. In addition to developing an effective treatment that may result in a zero day with-draw, on-farm mastitis diagnostic testing has been initiated to reduce the antibiotic use on farms, thus reducing the risk of antibiotic residues and reducing the risk of antibiotic resistance. On farm studies have shown that through the use of this on-farm testing many farms can reduce their antibiotic use over 50 percent.

PROGRESS: 2004/01/01 TO 2004/12/31
There are 4 objectives that are being addressed in the preharvest pathogen studies. Objective 1. To demonstrate animal safety for immune modulators-ID-1, a USDA approved bovine IgG and an antimicriobial protein, nisin. Objective 2. To reduce clinical signs and demonstrate the bacteriocidal activity of nisin towards Gram-positive pathogens in lactating cows. Objective 3. To demonstrate the bacteriocidal activity of nisin towards Gram-positive pathogens and the reduction of pathogens in lactating cows with subclinical mastitis. Objective 4. To demonstrate nisin efficacy by the reduction of intramammary infections in the non-lactating (dry) period. This year, objective 2 was completed with the acceptance of a nisin compound accepted by FDA for a new drug approval application so it can be used in field trials to determine efficacy in lactating and dry cows, objective 3 & 4. FDA has requested additional drug safety testing of new product to meet the Good Manufacturing Practices requirements. Nisin is to be prepared and will be tested on commerical farms in 2005 on mastitis in lactating and non-lactating cows (field trials). Preliminary studies show good bacteriocidal active toward streptococcal and staphylococcal mastitis pathogens without elevating milk somatic cell counts or producing any adverse physiological responses in the glands. This product will be studied in lactating and non-lactating cows to provide data for the FDA new drug approval clinical and subclinical efficacy trials (objectives 3). A second immune modulator-ID-1, a bovine IgG product was studied this year looking for it's safety when used in the lactating mammary gland. It is believed that the local immune response can be modulated to aid in the elimination of intramammary infection. Preliminary data has been collected and submitted to the USDA for review.

IMPACT: 2004/01/01 TO 2004/12/31
Bovine mastitis is a major disease effecting dairy cattle that requires the use of antibiotic therapy. The use of antibiotic in the treatment of mastitis pathogens increases the risk of antibiotic residues in milk and the development of antibiotic resistance on dairy farms. The use of non-antibiotic antimicrobial proteins can reduce the risk of antibiotics in food products and lower the risk of drug resistance in preharvest pathogens. Our preliminary studies clearly demonstrates that nisin, a very safe antimicrobial protein, is effective against staphylococcal and streptococcal pathogens including antibiotic resistant pathogens, and it is safe as an intramammary infusion for the treatment of mastitis in dairy cattle. All adverse reactions have been identified and addressed in our recent studies so the present product (nisin) can be used in commercial studies (FDA approval). This product has received with very short milk withdrawal (24 hours) period. This will make commercial field studies practical and data can be collected in a timely fashion. Our preliminary studies have provided sufficient information required by the FDA and provides the basis for future field studies. A immune modulator, bovine IgG has also been studied this year. This product may serve as an aid in the treating of clinical and subclinical mastitis and reduce the levels of antibiotic needed for the treatment of this disease in dairy cattle.

PROGRESS: 2003/01/01 TO 2003/12/31
There are 4 objectives that are being addressed in the preharvest pathogen studies. Objective 1. To demonstrate animal safety for the antimicriobial protein, nisin. Objective 2. To reduce clinical signs and demonstrate the bacteriocidal activity of nisin towards Gram-positive pathogens in lactating cows. Objective 3. To demonstrate the bacteriocidal activity of nisin towards Gram-positive pathogens and the reduction of pathogens in lactating cows with subclinical mastitis. Objective 4. To demonstrate nisin efficacy by the reduction of intramammary infections in the non-lactating (dry) period. Over the last year, trials testing the product(nisin)were completed by examining production consistency and testing nisin in non-infected mammary gland. These studies demonstrated that nisin can be produced in a consistent form that is effective in killing bacteria without causing inflammation of the mammary gland or producing adverse reaction when infused into the mammary gland. This data has been presented and accepted by the FDA for drug safety approval. Preliminary studies of intramammary infusions of the refined product has demonstrated bacteriocidal active toward streptococcal and staphylococcal pathogens without elevating milk somatic cell counts or producing any adverse physiological responses in the glands. Upon FDA new drug approval clinical and subclinical efficacy trials (Objectives 2-3) will be initiated in commercial herds with cases assigned for randomized drug testing.

IMPACT: 2003/01/01 TO 2003/12/31
Bovine mastitis is a major disease effecting dairy cattle that requires the use of antibiotic therapy. The use of antibiotic in the treatment of mastitis pathogens increases the risk of antibiotic residues in milk and the development of antibiotic resistance on dairy farms. The use of non-antibiotic antimicrobial proteins can reduce the risk of antibiotics in food products and lower the risk of drug resistance in preharvest pathogens. Our preliminary studies clearly demonstrates that nisin, a very safe antimicrobial protein, is effective against staphylococcal and streptococcal pathogens including antibiotic resistant pathogens, and it is safe as an intramammary infusion for the treatment of mastitis in dairy cattle. All adverse reactions have been identified and addressed so the present product (nisin) can be used in commercial studies (FDA approval). This product has received a very short milk withdrawal (24 hours) period from the FDA. This will make commercial field studies practical and data can be collected in a timely fashion. Our preliminary studies have provided sufficient information required by the FDA and provides the basis for future field studies.

PROGRESS: 2002/01/01 TO 2002/12/31
Objective 1. Through fecal sample testing at slaughter, it is evident the further the animal is shipped to slaughter and the greater the length of time combined with mixing of animals, the more likely E. coli and Salmonella pathogens are present in the fecal samples at slaughter and the greater the number of organisms being shed. The greatest influence was obsereved in the summer months when heat stress also increases the shedding for both pathogens. Seasonal effects was greatest for Salmonella shedding, particularly for S. typhimurium. The isolation increased from 4.79% in winter to 13.86% in summer of the cases for all Salmonella, but specificly from <1% to over 30%, respectively for S. typhimurium. Thus, there is a real need for preharvest HACCP program to reduce the risk of pathogens at slaughter. Objective 2. . We have three parts of this objective that includes: a) the demonstrating animal safety for Nisin; b) demonstrating Nisin efficacy towards mastitis infected lactating cows; c) demonstrating Nisin efficacy towards infected cow treated in the non-lactating (dry) period. The isolation and extraction of Nisin by column separation produce a compound that did not produce elevate milk somatic cell counts when infused into the mammary gland. The infusion of nisin into the mammary glands of lactating cows confirmed that nisin that can be produced in a manner that eliminates other inflammatory contaminates and does not cause adverse reaction when infused into non-infected glands. These findings have provided needed information and safety assurances necessary to proceed in looking at the efficacy of Nisin towards mastitis pathogens in dairy cows (Objective 2b and 2c). In vitro studies indicate a high efficacy towards most Gram-positive pathogens, specifically streptococccal and staphylococcal pathogens. Some of the preliminary studies using Nisin against these pathogens in the mammary gland has been very effective in reducing bacteria numbers without elevating milk somatic cell count or producing adverse physiological responses in the treated glands. Objective 2 b will be initiated first as product receives FDA new drug approval and herds are assigned for randomized testing.

IMPACT: 2002/01/01 TO 2002/12/31
1. Pathogen reduction in dairy cattle at slaughter is important in order to reduce the risk contamination during slaughter and meat processing. It is evident that the level of pathogens can increase by methods of shipment and by season of the year. Meat processed from cattle slaughter during the summer months is at a higher risk than in winter months. Therefore, more care must be taken when slaughter animals during these higher stress period. There are real needs for preharvest HACCP programs to reduce risk of pathogens at slaughter.
2. Bovine mastitis is still a major disease on dairy farms that require the use of antibiotic therapy. The use of non-antibiotic antimicrobial proteins can reduce the risk of drugs in food and reduce the risk of antibiotic resistance in mastitis pathogens on dairy farms. Our preliminary studies indicates that Nisin is an effective treatment against streptococcal and staphylococcal mastitis pathogens and Nisin use does not cause any adverse reaction in the gland or adverse effects on the cow. The safety Nisin in the cow has provided needed information necessary to proceed with the efficacy trials on clinical and subclinical mastitis.

Funding Source
Nat'l. Inst. of Food and Agriculture
Project source
View this project
Project number
MICL02016
Accession number
191577
Categories
Chemical Contaminants
Natural Toxins
Risk Assessment, Management, and Communication