Written in English — pages. Subjects Mastitis , Laboratory manuals , Handbooks, manuals. Laboratory handbook on bovine mastitis , National Mastitis Council. Libraries near you: WorldCat. Laboratory and field handbook on bovine mastitis.
Laboratory and field handbook on bovine mastitis First published in Subjects Mastitis , Laboratory manuals , Handbooks, manuals. Edition Notes Spiral bound. Includes bibliographical references.
Genre Laboratory manuals. Table 1a. An alternative method of calculating milk loss is to assign a 1. For example, if SCS increases from 2 to 5, multiply 3 units x 1. This number times the price of milk gives an estimate of the dollar value of milk lost per day.
Determining the exact amount of milk lost at a specific SCC or SCS is not possible, but the relationships are real and these estimates can be a valuable tool for encouraging producers to improve mastitis control and for evaluating the response to improvements made. Samples taken over consecutive days or weeks are most useful. Extreme caution should be taken when interpreting results from a single BTM sample.
Interpreting Bulk Tank Milk Cultures Mastitis pathogens Bulk tank milk analysis can be useful in determining if mastitis pathogens such as Staphylococcus aureus, Streptococcus agalactiae, or Mycoplasma spp. Presence of these pathogens in BTM almost always indicates the presence of infected quarters in the herd.
However, negative culture results do not necessarily mean that the herd is negative for infections caused by these pathogens. Raw milk quality Bulk tank analysis may also be used to determine raw milk quality based on quantitation of certain bacterial groups. Enzymes, such as lipases and proteases, secreted by bacterial species in raw milk can break down milk fats and proteins, respectively, resulting in reduced milk quality and possibly reduced shelf life.
Theoretically, any bacterial isolate from BTM could arise from an intramammary infection, but elevated counts of certain bacterial groups may also result from milking wet udders, organic soil in milk lines, cracked inflations, inadequately heated wash water, and inadequate cooling of milk. Laboratory pasteurization count LPC measures the number of thermoduric bacteria, species able to survive pasteurization, in BTM. Preliminary incubation count PIC measures the number of psychrotrophic bacteria, bacteria able to grow at cold temperatures.
Bulk tank milk cultures can be valuable supplements to quarter- or cow-level milk samples, but never a substitute for determining infection incidence and prevalence based on quarter- or cow-level milk samples. Interpretive criteria pertaining to raw milk quality suggested here are based on data collected throughout Pennsylvania only and are simply meant to provide helpful guidelines for bulk tank monitoring and troubleshooting.
Ideal counts given in this publication are not meant to be substituted for government standards. Continue to spread until the milk has been completely absorbed into the agar, taking care not to gouge the agar.
Never obtain samples from a bulk tank's milk release valve. If a plate has too many colonies to count. If analyzing more than one sample from the same bulk tank, use the mean value for each bacterial count when interpreting results Table Unfortunately, the practice of submitting clinical mastitis milk samples to laboratories for microbial culture has not been routinely utilized because of extended turnaround times 36 to 48 hours , cost, and inconvenience.
This has resulted, historically, in the non-selective treatment of mastitis cases. However, rapid on-farm culture systems OFCS can be used to direct mastitis treatment decisions, resulting in reduced antibiotic usage, without changing the probability of achieving treatment success. In instances where selective dry cow therapy programs are utilized, OFCS may be valuable in making decisions about which mammary quarters to treat.
However, the success of an o n f- arm culture OFC program will be depend on adequate training and experience of those individuals collecting milk samples, plating the milk, interpreting culture results, the presence of adequate laboratory conditions and quality assurance programs, and the existence of logical treatment protocols, as developed with the herd veterinarian.
Ultimately, the development of an OFCS can hasten the diagnostic process and have the potential to economically return a result in a timely manner to dairy managers. Gram-negative bacteria Minnesota Easy Culture System, However, simple techniques and tools are available that can be used in an on-farm laboratory or in a local veterinary clinic.
Most OFCS make use of one or more types of selective media designed to promote bacterial growth of only certain classes of organisms. Paul, MN , which offers two different types of selective culture media systems. The Bi-Plate system is a plate with two different types of agar, MacConkey agar on one-half of the plate selectively grows Gram-negative organisms, whereas Factor agar on the other half of the plate selectively grows Gram-positive organisms e.
The Tri-Plate system is a plate with three different types of agar. In addition to including MacConkey agar Gram-negative growth and Factor agar Gram-positive growth , it also includes a section of MTKT agar, which is selective for streptococci or streptococcal-like organisms Figure In both cases, producers dip a sterile cotton swab into the milk sample and then apply it over the media surface estimate 0.
If no growth is observed, plates are rechecked after 48 hours and then discarded Flowchart 2. No growth at 48 hours yields a presumptive diagnosis of culture negative. Hence, this OFCS can be reliable and reasonably accurate in the hands of trained farm personnel for diagnosing broad groups of mastitis pathogens to facilitate treatment decisions. Minnesota Easy Culture System, Paul, MN has also been used for on-farm diagnosis of mastitis pathogens. This system is very similar to the University of Minnesota agar plate system in terms of using selective and differential media.
If growth is detected on the Staph Express count plate, then confirmation of S. The STX disk contains deoxyribonuclease and a dye that reacts to produce a distinct pink zone around S.
The need to dilute the milk is an extra step that requires aseptic technique and could increase the error rate. Both systems can be used to identify S. In general, neither system is designed to identify pathogens to the species level. Another shortcoming is that neither system can effectively recognize pathogens that are outside the common mastitis pathogens discussed. For example, a sample of milk from a quarter with an inrramammary infection caused by Mycoplasma spp.
Alternatively, Prorocheca spp. Count Plate Petrifilm System. Count Plate Petrifilrn System. Hence, lactating cow therapy for clinical mastitis is generally aimed at and most effective against Gram-positive Staphylococcus spp. Producers can use OFCS to decide which mammary quarters are likely to respond to antibiotic treatment. Selective lactating cow therapy has significant advantages. This program will save on the cost of antibiotic, cost of labor and management involved in treatment, decrease the amount of discarded milk, and potentially reduce the risk of an antibiotic residue violation.
The major negative factor of OFCS may be the time needed to determine the outcome of the milk culture, which is generally 24 hours. Culture time can delay treatment, which may reduce the success of therapy of sensitive pathogens or may prolong the cow's duration of infection.
However, the consensus is that OFCS program positives outweigh the negatives, with respect to targeted therapy. Also, recent data suggest that a hour delay in treatment does not appreciably impact overall outcomes in udder health. Selective dry cow therapy Globally, there is interest in moving away from blanket dry cow therapy approaches, where all mammary quarters of all cows receive intramammary treatment just before dry-off, to selective programs that only treat subclinically infected mammary quarters.
Diagnosis of an infection can be done indirectly by monitoring signs of inflammation e. The advantage of using OFCS prior to dry-off would be to identify the specific pathogen groups Gram-positive versus Gram-negative or genera to make informed treatment decisions in consultation with the herd veterinarian.
Mammary quarters yielding no growth may be protected from new intramammary infections during the dry period by use of an internal or external teat sealant. A more detailed description on collecting high quality milk samples, setting up and operating an on-farm culture laboratory, and interpreting on-farm culture results are provided in the Minnesota Easy Culture System User's Guide However, a summary of these requirements is provided below.
Laboratory space Producers will need a clean, heated, designated space, free of barn traffic manure, dust, etc. The lab area should be insulated against large fluctuations in room temperature and free of drafts. The workspace countertop must be clear, free of food or drink, and easily disinfected.
The lab worker s should always wear clean disposable gloves when working with mastitic milk samples or culture plates. Incubator setup and operation Laboratory incubators are available from a variety of laboratory supply sources, with many options size and price to choose from. Even low-cost egg incubators can be made to work on smaller dairies to incubate a small number of plates. It should have a thermometer that is regularly checked, plus a dish of water inside to maintain constant humidity.
Temperature and water level in the incubator should be checked daily, as variation can result in erroneous culture results, including "no bacterial growth. Stored media should be refrigerated, not frozen.
Farm staff must be trained in aseptic milk sample collection techniques, as described elsewhere in this handbook. If a sample is frozen, allow it to thaw completely in the refrigerator before plating. Mix the sample well by gently inverting the vial several times before plating.
Plating procedures may differ among different OFCS. As such, users must refer to manufacturer guidelines for the specific OFCS in place on the farm. The following is an example for the Minnesota Easy Culture system.
After washing hands and putting on new disposable gloves, turn the culture plate upside down and label the bottom of the plate cow ID, quarter, date. Use a new sterile disposable cotton swab, place the swab into the milk sample for approximately 10 seconds or until it is saturated with milk Figure Streak the milk over the entire surface of the culture media.
Re-dip the swab in the milk sample between each different section of the culture plate. Once finished swabbing, immediately place the lid back onto the media plate and reseal the lid on the milk sample. Freeze the milk sample immediately in case it is needed for confirmatory testing at a later date. Place the plate in the incubator upside down i.
Once plating is completed, dispose of any garbage e. After 18 to 24 hours. For interpretation, refer to the guidelines recommended by the manufacturer of the culture system being used. A record keeping system should be in place to record, at a minimum, the sample date, cow ID, quarter affected. Culture results may later be transferred into an on-farm computerized record keeping system e. Such records are necessary to monitor the types of organisms causing mastitis on a given dairy and to evaluate treatment success.
If OFCS results are suspicious, not easily interpreted, or there is routine failure of mastitis cases to respond to appropriate therapy, then frozen stored milk samples should be submitted to a commercial mastitis laboratory for routine culture and verification of OFCS results. No growth results If no bacterial growth is evident after 48 hours, then the sample is considered a "No Growth," as shown in Figure Explanations for this could include: 1.
The quarter sampled was not infected true negative. The quarter sampled was infected, but the cow's immune system had already eliminated the bacteria before the sample was collected true negative. This is believed to be common in Gram-negative infections. Equipment failure or errors in sample collection, storage, or culture technique resulted in a false-negative result. The type of bacteria or other organism causing the mastitis does not grow under the conditions of the culture system false negative; e.
Contaminated samples A milk sample is considered "Contaminated" when three or more types of bacteria are identified on the Minnesota Easy Culture System media Figure Typically, when individual quarter milk samples are collected properly, only a single mastitis pathogen is identified. When a milk sample is "Contaminated," it is uncertain which, if any, of the bacteria found are causing disease and which are merely environmental bacteria that contaminated the milk sample during the collection process.
Consideration must be given to the types of bacteria isolated, the number of colonies appearing on the plate, and the stage of infection. Frozen milk samples may also be submitted to a microbiology laboratory for confirmatory testing. Veterinarians or other qualifiec professionals should visit the dairy regularly and work with farm staff i. Milk sample collection and storage i. Storage of new plates i.
Labeling of culture plates i. Plating technique i. Placing of plates in the incubator i. Time plates spend in the incubator. Reading of plates from that day with on-farm culture technician. Used plates should be stored in a refrigerator different from the one where storing new plates to: a. Evaluate agreement with the on-farm culture technician results. Additional testing e.
Evaluate plating technique. Identify contaminated samples. Observe hygiene practices e. Records evaluation: a. Accurate entering of culture results. Matching between culture results and treatment decisions. Discarding of plates i.
It is recommended that farm staff working with OFCS periodically verify their culture results by regularly reviewing and interpreting plates with the regular herd veterinarian or by sending frozen milk samples to a local diagnostic laboratory for culture, and then compare culture results with those obtained on farm. Frequent verification should be used. Any growth present? Mix thoroughly and heat with frequent agitation; boil for 1 minute to completely dissolve the agar.
Dispense into ml aliquots, using ml flasks with vented stoppers or aluminum foil. Mix well and pour into Petri plates, using aliquots of 12 to 14 ml for each x mrn plate. Use within 2 weeks because plates become dehydrated and do not support growth of some microorganisms. Advantages Use of blood agar plates permits growth of most microorganisms that are isolated from milk samples. Limitations Blood agar plates do not detect esculin reactions or support the growth of mycoplasmas.
Quality control of Dehydrated media should be purchased in sufficient quantities so that media preparation one lot can be used over a long period of time 6 to 12 months. Each lot should be checked for its ability to produce expected reactions, according to the American Public Health Association's Standard Methods for the Examination of Dairy Products. Each bottle of dehydrated media should be labeled, indicating the date received and opened, and stored in a cool, dry location protected from light, or in a refrigerator or freezer, if recommended by the manufacturer.
Any expired media should be discarded or if any change in color or texture is noted. Complete mixing of media is necessary prior to autoclaving. Quality control Each lot of newly prepared blood agar plates should be tested with blood agar plates quality control microorganisms to ensure that the media will support growth and provide correct differential reactions for hemolysis. Suggested quality control microorganisms: Streptococcus dysgalactiae ATCC to ensure proper growth of fastidious streptococci.
Staphylococcus aureus ATCC , alpha and beta toxin positive to ensure correct hemolytic patterns. Note: Blood agar plates can be prepared from small batches of agar, as described above or from large batches of media that are prepared in commercial agar sterilizers and with automated plate pourers, according to the manufacturer's instructions. Mix thoroughly and heat with frequent agitation; boil for 1 minute co completely dissolve the agar powder.
Dispense into ml aliquots into ml flasks with vented stoppers or aluminum foil. Mix well and pour into Petri plates using aliquots of 12 to 14 ml for each x mm plate. Limitations Alpha hemolysis of some Streptococcus agalactiae isolates may be confused with esculin splitting. Blood-esculin agar plates are available commercially from some vendors.
Each lot should be checked for its ability to produce expected reactions according to the American Public Health Association's Standard Methods for the Examination of Dairy Products. Each bottle of dehydrated media should be labeled indicating the date received and opened, and stored in a cool, dry location protected from light, or in a refrigerator or freezer if recommended by the manufacturer.
Expired media should be discarded or if any change in color or texture is noted. Quality control of Each lot of newly prepared blood-esculin agar plates should be tested blood-esculin agar plates with quality control microorganisms to ensure that the media will support growth and provide correct differential reactions for hemolysis and utilization of esculin.
Streptococcus uberis ATCC , esculin positive to ensure proper esculin reaction. Note: Blood-esculin agar plates can be prepared from small batches of agar as described above or from large batches of media that are prepared in commercial agar sterilizers and with automated plate pourers, according to the manufacturer's instructions. Allow the hemolysin to dry before streaking samples. Swab method Saturate a sterile swab with beta-hemolysin and streak a single line across the diameter of blood agar plate.
If the plate is to contain four milk samples, streak a second line perpendicular to the first. Drop method With a sterile syringe and small gauge needle, place 6 drops of beta-hemolysin across the diameter of a blood agar plate. If four samples are to be streaked on the plate, add another 6 drops on the perpendicular axis.
Chloroform method Two methods are available to prepare beta-hemolysin and are described below. Ingredients and supplies Staphylococcus aureus colonies showing beta-hemolysin production. Blood agar plates. Brain Heart Infusion or tryptose broth. Transfer an isolated S. Transfer one typical colony to broth. The volume of broth depends on the quantity of beta-hemolysin desired.
Add 1 ml of chloroform per 10 ml broth culture this will kill the S. Incubate at room temperature for 18 to 24 hours. Store at room temperature or refrigerate for up to 6 weeks. Chloroform will be the bottom layer. After each use, swirl the chloroform throughout the hemolysin to remove contaminants, which may have entered the supply during use.
Filtration method Reagents and equipment Staphylococcus aureus colonies showing beta-hemolysin production. Filters with 0. Vacuum or air supply. Procedures 1. Transfer an S.
Transfer one colony to broth. Centrifuge at 1, x g for 30 minutes. Filter supernatant through sterilizing filters with the aid of a vacuum or air supply. Dispense in small quantities for use. Store under refrigeration. The preparation should be stable for several months. I Oxoid Boil to dissolve the agar and distribute ml volumes into flasks ml large enough to hold additives. Complete working agar 1. Ready to use when agar surface is dry.
Pass through a sterile, disposable, 0. After cooling, centrifuge at 1, x g for 45 minutes. Remove the supernatant fluid and filter several times through decreasing pore size on filters 1.
Penicillin Dissolve 5 million IU of potassium penicillin Gin 25 ml of deionized distilled water. DNA solution Dissolve 0. Dextrose solution Dissolve 50 g of dextrose in ml of deionized distilled water.
Break clots and separate serum by centrifugation at 2, x g. Gently heat solution by immersing the tube in boiling water until digitonin powder is completely dissolved. Prepare a broth culture 4-day culture of the isolate to test, or use a sample of fresh milk with the infectious mycoplasma isolate to test.
Allow the surface to dry. Place a digitonin disc on the surface of agar. Measure the zone of inhibition from the edge of the disc to the edge of the clear zone mm around the disc under the stereomicroscope. Store nisin stock solution 5. This stock solutio; can be used for up to 24 months. Make a IO-fold dilution of nisin from stock solution with 10 mM citrate pH 3. The two types of disc will contain Place undiluted and diluted nisin discs on the surface of agar.
Measure the zone of inhibition from the edge of the disc to the edge of the clear zone mm around the disc under the stereornicroscope. Mix solution and heat to dissolve.
Pour 13 to 15 ml per plate. Store inverted in a refrigerator. Dispense 5 ml per tube tube size 16 mm ID. Cool in a slanted position to obtain deep butt. Store refrigerated. OOC Cool in a slanted position. Mix and dissolve MTA in distilled water. Add TTC solution. Dispense 5 ml into x mm tubes. Akfl'-esculin Plates Ingredients Trypticase soy agar Mix and dissolve trypticase soy agar and esculin in distilled water.
Pour 12 to 15 ml per plate. Ingredients Phenol red broth base Distilled water Procedmes 1. Mix and dissolve ingredients. Dispense 4 ml into x mm screw-top tubes. Add peptone, yeast extract, neutral red, and agar to distilled water. Add filter to sterilized propylene glycol and filter to sterilized galactopyranoside. Store inverted in refrigerator. Media should be used within 5 days.
Trypticase Soy Broth Ingredients Trypticase soy broth powder IO g NaCl Dissolve broth base and sodium hippurate in 1, ml distilled water. Mark level of medium in a tube before incubating. Store broth solution refrigerated. Store ferric chloride solution at room temperature. Mix solution, heat to dissolve, and adjust pH to 5. Suspend potassium hydrogen phthalate and sodium hydroxide in 1, ml distilled water. Add remaining ingredients, except 5-fluorocytosine, heat to dissolve, and adjust pH to 5.
Add filter to sterilized 5-fluorocytosine. Mix ingredients except sterile blood and heat to dissolve. Add sterile blood and swirl gently. Mix solution, except for potassium tellurite, and heat to dissolve.
Add sterile potassium tellurite solution. Streak streptococcal cultures perpendicular to and within 1 to 2 mm of the S. Ten isolates may be tested per plate, with five on a side. Positive esculin hydrolysis Browning around organism growth e. Carbohydrate Fermentation for identifying streptococci and coliform bacteria Inoculation 1.
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Scope of laboratory field handbook on mastitis in the individual animals for herds characterized primarily by the risk. Washed and aetiology of laboratory and field handbook on bovine subclinical in scc. Mastitis -- Handbooks, manuals, etc. Mastite -- Guides, manuels, etc. View all subjects More like this Similar Items. Allow this favorite library to be seen by others Keep this favorite library private. Save Cancel. Find a copy in the library Finding libraries that hold this item Find more information about: Joseph S Hogan.
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