Dairy/Milk Testing Report Detecting Elevated Levels of Bacteria in Milk-On-Site Direct- From-The-Cow Within Minutes as Indicator of Mastitis EnZtek Diagnostics Incorporated has investigated and successfully conducted on-the-farm testing of milk samples to detect elevated levels of bacteria present as a means of quickly identifying cases of Mastitis due to bacterial infection. The most common test used to check for Mastitis is a Somatic Cell Count (SCC) test which tests each of the four quarters of the cow s udders. Somatic cells are composed of white blood cells and occasional sloughed epithelial cells. The Somatic Cell test performed is the CMT, or California Mastitis Test. The CMT reagent is simply a detergent plus bromcresol purple (used as an indicator of ph). The degree of reaction between the detergent and the DNA of cell nuclei is a measure of the number of somatic cells in milk. The relationship between SCC (Somatic Cell Count) values and CMT is not precise because of the high degree of variability in SCC values of each CMT score. (1) Additionally, the test must be read in a matter of seconds as the reaction disappears within 20 seconds. The sensitivity of the CMT is reported to be 66.7% and specificity of 54.8%. When the CMT was used for selective dry cow therapy the CMT test correctly identified 75%-80% of infected cows depending upon the study and type of pathogens present. However, it was concluded that a large percentage of uninfected quarter would receive dry cow therapy because 23% - 40% of animals without infections with major pathogens were detected by CMT.(2) The focus of this study however, is to actually detect bacteria. Disease causing bacteria are called pathogens. The most common mastitis pathogens are found in the udder tissues, spreading from cow-to-cow (contagious pathogens) or in the herd s surroundings (environmental pathogens), such as bedding materials, manure and soil. This distinction may be important when assessing the challenges present in a herd and the measures which may be taken to reduce or treat mastitis.(3)
The chart below describes environmental mastitis pathogens, and contagious pathogens: Classification Bacteria Contagious or Source Spread Control Treatment* Staphylococcus spp. Staph. aureus Contagious Infected udders, hands of milkers Milking time Post-dip, DCT 1, segregation and cull if necessary Label recommendations for broad-spectrum antibiotics, if early lactation 5-7 d pirlimycin, do not treat chronic infections Coagulase (-) staph. & S. hyicus Neither Skin flora & occasionally environment Infect teat canal from skin sources Post-dip, DCT Treat clinical cases (broad spectrum), DCT Strep. agalactiae Contagious Infected udders Milking time Milking time hygiene, postdip, DCT Label recommendations for broad-spectrum antibiotics Streptococcus spp. and Enterococcus spp. Strep. dysgalactiae Strep. uberis strep & Enterococcus spp. Contagious and environmental Infected udders and environment Environment early dry period Environment Milking time & environmental New IMI 2 during early dry period Milking time hygiene, pre- & post-dip, DCT, teat seal Milking time hygiene, pre- & post-dip, DCT, teat seal Milking time hygiene, pre- & post-dip, DCT, teat seal Label recommendations for broad-spectrum antibiotics Label recommendations for broad-spectrum antibiotics and consider IMM 3 therapy 4-5 d penicillin systemically (3.5 cc/100 lb body weight)** Continues Next Page
Escherichia coli Bedding, manure, soil Klebsiella spp. Organic bedding Enterobacter spp. Bedding, manure, soil Cows clean & dry, use of sand bedding, pre-dip, a J5 vaccine Avoid sawdust & recycled manure, predip, J5 vaccine Cows clean & dry, use of sand bedding, pre-dip, a J5 vaccine Do not treat local cases. Systemic cases 2-3 L hypertonic saline IV, followed by oral fluid therapy, NSAID*** and injectable antibiotics Gram negatives Serratia spp. Soil and plants Cows clean & dry, pre-dip (no chlorhexidine products) Pseudomonas spp. Water & wet bedding No water use in parlor, no cooling ponds, sand bedding, a J5 vaccine 180-300 ml hypertonic saline IMM infusion Proteus spp. Bedding, feed & water Not much known, use of sand bedding, a J5 vaccine Pasteurella spp. Probably contagious Upper respiratory tract of mammals and birds Unknown likely cow to cow Prevent teat injuries, remove affected cows from herd Do not respond to IMM treatment Continues Next Page
Yeast & mold Soil, plants, water Dirty infusions Aseptic infusions No treatment Corynebacterium bovis & other coryneforms Contagious Infected udders Cow to cow Post-dip Treat clinical cases and DCT Other Prototheca Soil, plants, water Dirty infusions, infected udders Aseptic infusions, eliminate infected cow No treatment cull cow Bacillus spp. Soil, water, air Dirty infusions Arcanobacterium pyogenes Contagious/Environm ental Aseptic infusions Teat injuries Flies Fly control Broad-spectrum antibiotic Kill affected quarter or remove from herd Information obtained from NMC Laboratory Handbook on Bovine Mastitis and veterinary consultation for treatment recommendations). *These are general treatment recommendations; actual recommendations may vary from herd to herd. Please consult your veterinarian. **Extra label usage; please consult your veterinarian before starting this protocol. ***Nonsteroidal anti-inflammatory drugs. 1 DCT, dry cow therapy; 2 IMI, intramammary infection; 3 IMM, intramammary. As indicated by the chart above, the primary area of concern is Gram Positive bacteria such as Staphylococcus aureas and Streptococcus.(4) The EnZtek assays for Total Aerobic Organisms (detects gram positive and gram negative) and the Gram Negative Aerobic Bacteria (detects gram negative) can be utilized for this purpose. Additionally, the EnZtek assay for Staphylococcus aureus can be used to identify the presence of that specific gram positive bacteria. The EnZtek assays are designed to be used On-The-Farm and can provide results within 30 minutes from sample collection directly from the cow. There have been other on-the-farm products available, such as the 3M Petrifim on-the-farm culture system (POFCS). However, this system will not provide results until after a 24 hour incubation. While the 3M product tested with accuracy of 93.8% in their laboratory trials, in the hands of the producers it was reduced to and overall sensitivity and specificity of 63.8% and 48.4%, respectively. (5) References: (1) Milk Quality and Mastitis Tests, Pamela L. Ruegg, DVM, MPVM, Douglas J. Reinemann, PhD University of Wisconsin, Madison, 2002 (2) Milk Quality and Mastitis Tests, Pamela L. Ruegg, DVM, MPVM, Douglas J. Reinemann, PhD University of Wisconsin, Madison, 2002 (3) dairy.co.org.uk,2012 (4) On-Farm Culture Use Experience and Impact on Antimicrobial Use, Greg Keefe, Kimberley MacDonald and Marguerite Cameron, Maritime Quality Milk, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE CANADA, 2012 (5) On-Farm Culture Use Experience and Impact on Antimicrobial Use, Greg Keefe, Kimberley MacDonald and Marguerite Cameron, Maritime Quality Milk, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE CANADA, 2012
EnZtek tests detect the enzymes produced by bacteria, and a healthy bacteria can produce thousands of molecules of the target enzyme. Most other bacteria detection tests need to find an actual bacteria cell, which requires the bacteria to grow, and this takes time, usually 24 hours or more of incubation. Even PCR bacteria tests must grow the bacteria before enough DNA can be obtained and replicated. Bacterial Enzymes (light green) EnZtek develops substrates which identify the target enzyme produced by the target bacteria, and a match is made, resulting in a detectable reaction. This is depicted in the Lock and Key Analogy diagram. This reaction can be seen with an easy to see color reaction or fluorescence, or it can be seen as a numerical reading on a fluorometer.
During on-site testing, tests were conducted on seven suspect milk samples (6031, 1979, 2367, 1925, 6947, 2638, 1301), using the EnZquik handheld fluorometer and EnZquik Fluorescent Assays. The fluorometer results are in RFU or Relative Fluorescent Units. The seven milk samples were tested following the EnZquik procedures for testing at a dairy by comparing each suspect milk sample to a known healthy cow milk sample. A healthy cow milk sample was used as a control. The testing was intended as a presumptive screen to detect elevated levels of bacteria which would be an indication of Mastitis. The tests performed were for Total Aerobic Organisms, Gram Negative Aerobic Bacteria, E.coli and Staphylococcus aureus. Since many substances fluoresce, a positive result from reactions between the substrate and the bacterial enzyme is indicated when the sample has a higher RFU reading than the control. As both the control and sample will have some natural hydrolysis, the cut-off value is usually a difference of 1,000 RFU or more for a positive result. All readings are recorded after 10 minutes of reaction time. This allows for On-The-Spot results.
These next pages provide the results for the milk samples: Total Aerobic Organisms difference Samples 10 minute Readings sample less control result Control 5121 6031 32648 27527 high positive Control 4739 1979 19469 14730 medium positive Control 5042 2367 7980 2938 low positive Control 5502 1925 7900 2398 low positive Control 5120 6947 21155 16035 medium positive Control 4713 2638 6457 1744 low positive Control 5610 1301 48996 43386 high positive Gram Negative Aerobic Bacteria difference Samples 10 minute Readings sample less control result Control 16247 6031 22158 5911 medium positive Control 19567 1979 16407-3160 negative Control 19023 2367 25551 6528 medium positive Control 20598 1925 35746 15148 high positive Control 19695 6947 4870-14825 negative Control 18822 2638 25405 6583 medium positive Control 22878 1301 32084 9206 medium positive The control RFU value is subtracted from the sample RFU. If the difference is 1,000 RFU or higher, the result is positive. All of the milk samples tested positive for Total Aerobic Organisms using the handheld fluorometer and fluorescent assays. The control RFU value is subtracted from the sample RFU. If the difference is 1,000 RFU or higher, the result is positive. Milk samples 6031, 2367, 1925, 2638, and 1301 tested positive for Gram Negative Aerobic Bacteria using the handheld fluorometer and fluorescent assays. Milk samples 1979 and 6947 tested negative for Gram Negative Aerobic Bacteria using the handheld fluorometer and fluorescent assays.
E.coli difference Samples 10 minute Readings sample less control result Control 1548 6031 2522 974 negative Control 1713 1979 2497 784 negative Control 1283 2367 1246-37 negative Control 1720 1925 2760 1040 low positive Control 1163 6947 4801 3638 high positive Control 1650 2638 2103 453 negative Control 1727 1301 3987 2260 medium positive Staphyloc occus aureus difference Samples 10 minute Readings sample less control result Control 3333 6031 60141 56808 high positive Control 3783 1979 21246 17463 low positive Control 3336 2367 9514 6178 low positive Control 3455 1925 8915 5460 low positive Control 3534 6947 40232 36698 medium positive Control 3757 2638 4632 875 negative Control 3828 1301 78775 74947 high positive The control RFU value is subtracted from the sample RFU. If the difference is 1,00 RFU or higher, the result is positive. Milk samples 1925, 6947, and 1301 tested positive for E.coli using the handheld fluorometer and fluorescent assays. Milk samples 6031, 1979, 2367 and 2638 tested negative for E.coli using the handheld fluorometer and fluorescent assays. The control RFU value is subtracted from the sample RFU. If the difference is 1,000 RFU or higher, the result is positive. Milk samples 6031, 1979. 2367, 1925, 6947, and 1301 tested positive for Staphylococcis aureus using the handheld fluorometer and fluorescent assays. Milk samples 2638 tested negative for Staphylococcus aureus using the handheld fluorometer and fluorescent assays.
Below is an overview of all of the fluorescent results: Sample Total Organisms Gram Negative Total Coliforms E.coli Staphylococcus aureus 6031 xxx xx neg xxx 1979 xx neg neg x 2367 x xx neg x 1925 x xxx x x 6947 xx neg xxx xx 2638 x xx neg neg 1301 xxx xx xx xxx x=low xx=med xxx=high
Conclusion: A thorough examination of the seven milk samples was conducted using the EnZquik handheld fluormeter and EnZquik Fluorescent Assays. Positive results were successfully obtained on site at the dairy providing results within minutes. Performing the test including the 10 minute reaction time, yields results in less than 30 minutes. This conclusion reconfirms EnZtek s goal for the EnZquik Fluorescent Assays and handheld fluorometer to be used On-Site and provide test results within minutes of sample collection to identify elevated levels present as an indicator for Mastitis. Sample Total Organisms Gram Negative Total Coliforms E.coli Staphylococcus aureus 6031 xxx xx neg xxx 1979 xx neg neg x 2367 x xx neg x 1925 x xxx x x 6947 xx neg xxx xx 2638 x xx neg neg 1301 xxx xx xx xxx x=low xx=med xxx=high