PEER REVIEWED

Developing a practice-based microbiological laboratory to guide Inastitis treatinent on dairy farins

James W. Bennett, DVM; Joanna L. Gardner, DVM Northern Valley Dairy Production Medicine Center, 900 N. Wabasha, Plainview, MN 55964 Corresponding author: Dr. James Bennett, email: [email protected]

Abstract Introduction

Culture-based treatment ofmastitis has been ad­ Microbiological identification of masti tis pathogens vocated to reduce expenses of treatment and discarded has typically been performed retrospectively to identify milk and to improve treatment results. Ideally, identi­ pathogens to guide treatment of subsequent cases, or fication of mastitis pathogens should be performed in a to obtain an antibiogram ofpathogens.37 More recently, laboratory accessible to dairy farms within a reasonable culture-based treatment has been advocated to reduce amount of time. Results can usually be made available drug use, reduce treatment costs or increase treatment to farms the following day, before the time of day that success. 8,9,13,16,17 ,18,20,21,24,25,21,31,32,42,43 treatments are normally administered. Mastitis patho­ On-farm culture has been successful on some gens can be successfully identified in a practice-based farms, although its use is often limited because of laboratory using standardized methods. Examples of questionable results or discontinuation of the practice.42 specific, step-by-step methods, including needed materi­ Additionally, bi-plate or tri-plate media and the 3M Pet­ als, are described to aid in developing and operating a rifilm systems available for use by on-farm laboratories mastitis laboratory. A novel method ofreporting results are designed to identify only the most common bovine 8 13 22 utilizing Google Docs is described. mastitis pathogens. • • As a result, their use on farms without use of mic;roscopic evaluation and other tests Key words: dairy, mastitis, laboratory, treatment may result in misdiagnosis, particularly if the mastitis is caused by less common mastitis pathogens such as Resume yeasts, Arcanobacterium, Corynebacterium, Pseudomo­ nas, Serratia, Prototheca, and others. 42 Le traitement de la mammite par l'entremise des Delay of treatment for 24 hours to seven days is 30 31 cultures bacteriologiques a ete mis de l'avant afin de not detrimental to treatment success, • thus a 24- to reduire les depenses reliees au traitement et a la perte 48-hour interval between first diagnosis of clinical mas­ de lait et pour ameliorer les resultats du traitement. titis and treatment, typically achievable in a veterinary Idealement, !'identification des agents pathogenes asso­ practice-based lab, should not reduce treatment success. cies a la mammite devrait etre faite dans un laboratoire While methods to identify mastitis patho­ capable de fournir aux fermes laitieres des resultats gens utilizing microbiological techniques have been dans un delai de temps raisonnable. Les resultats sont described, 9,10,15,22,29,35,36,37,38,39,44 few resources that offer habituellement disponibles le jour suivant avant que detailed, complete, step-by-step methodology needed ne debute les traitements administres a la ferme. Les to properly identify mastitis pathogens are available to agents pathogenes associes a la mammite peuvent etre the veterinary practitioner desiring to design a mastitis identifies avec succes dans un laboratoire de pratique laboratory. This article will list and explain detailed veterinaire utilisant des methodes standardisees. Des methods utilized in the authors' laboratory to assist oth­ exemples de methodes specifiques decrites pas a pas, er practitioners wishing to develop a laboratory or those incluant le materiel necessaire, sont presentes afin wishing to refine processes in an existing laboratory. d'assister au developpement et a !'operation d'un labo­ This paper is not meant to represent the only or best ratoire devolu a la mammite. Une nouvelle methode de methods available for use in a practice laboratory, nor presentation des resultats par l'intermediaire de Google is it suggested that identification techniques elucidated Docs est presentee. in this paper are new or novel. Furthermore, it should

18 THE BOVINE PRACTITIONER-VOL. 46, NOd be stressed that often a diagnosis can be made without preliminary results are obtained (usually 18 to 24 hours completing the entire diagnostic scheme outlined for any after the samples arrive), a notation of"preliminary" is particular organism in this paper. In general, simpler made on the form and results are reported to the farm. and faster is better, but at times additional testing will An example form is shown in Figure 1. Final results be needed to correctly identify a pathogen. For this should be reported on standardized, easily readable reason, a relatively thorough methodology is described. forms which can be developed using a variety of soft­ In the authors' practice, dairy producers typically ware programs. treat animals based on protocols developed by the farm veterinarian following delivery of laboratory results Materials Needed for the Typical Practice-Based to the farm. Often at least 50% of dairy farm mastitis Mastitis Laboratory samples produce growth of a coliform bacteria or no growth at all. 8,16,19,21,25,26,33 Common treatment protocols Media for these cases suggest no antibiotic treatment unless Washed bovine blood plates 37 the animal is ill,8,13,16,17,18,24•25•27•32•33• thus antibiotic us­ Citrate media in tubes age for mastitis is reduced. Since more than 50% of Factor plates antibiotic treatments used on a typical dairy farm are MacConkey plates for mastitis, use of culture-based treatments can sub­ Modified Hayflick plates (for Mycoplasma) 12 stantially reduce the overall use of antibiotics. ,28,31 In Motility media with color indicator (or Motility/ addition, more appropriate treatment protocols, either Indole/Ornithase media) in tubes in choice of antibiotic or duration of treatment, may be Optional selective media, for example, potato used when pathogens are identified, such as extended­ dextrose agar for yeast and Prototheca duration therapy for treatment of mastitis caused by Modified TKT (MTKT) plates . Staphylococcus aureus. The combined practice-based Candle jar (for Mycoplasma) laboratory and on-farm treatment protocols have been Glass rod well received by clients in the authors' practice. Nearly Dissecting microscope (for Mycoplasma) all clinical cases of mastitis are cultured on many farms, Filter paper and cows are treated based on culture results. Freezer Gram stain kit Sampling Hydrogen peroxide, 3% Incubator Proper sample collection is critical for obtaining Microscope, slides, cover slips, and microscopy oil correct, meaningful results. Sampling methods have National Mastitis Council (NMC) Laboratory been well described by others.9,29,37 Handbook on Bovine Mastitis, 1999 Oxidase reagent Timing of Sampling Potassium hydroxide PYR test kit Mastitis is most often detected at milking time, Refrigerator which is a convenient time to collect samples. Samples Rabbit plasma may also be taken from fresh cows when mastitis is Staphylococcus aureus cultures suspected during colostrum collection. Samples should Sterile, 0.1 mL disposable inoculating loops be refrigerated or immediately frozen if they cannot be Urease reagent delivered to the laboratory within 24 hours. Samples Kovac's reagent (for use with MIO tubes) should be delivered to the laboratory under refrigera­ 6.5% NaCl broth with BromCresol indicator tion. Factor and MTKT agar are available from The Recording Samples Laboratory for Udder Health.a Other supplies may be obtained from Hardy Diagnosticsh or other sources. When samples arrive at the laboratory, a record The actual list of materials used in any practice­ should be created with a chronological listing of ac­ based lab will vary. Each practitioner should choose cessions. 3,34 This log should include the farm name, appropriate tests for his/her own laboratory. test(s) requested, date completed, technician's name or initials, and an area for notation that results have been Procedures for Identifying Mastitis Pathogens reported. A form to record test results should also be created for each sample at this time. This form will be A variety of techniques have been described for updated as microbial identification is completed. After identifying pathogens. 9,10,15,22,29,35,36,37,38,39,44 An excellent

SPRING 2012 19 Individual Culture ID Sheet (Lab Use Only)

Client: Date samples plated: Date samples submitted: Date results reported:

Quarter Sampled N STAPH STREP COLIFORM Identification Tests Cow ID OTHER LIL IR IR I G SIC E I KI E coag catal oxid KOH ! camp gram FIRIFIRIC A IN C 1 L 1 N I I I I

24-Hour Reading DVM Initials: _____ 48-Hour Reading DVM Initials: _____

Figure 1. Example form to record milk sample information and laboratory results.

resource is the Laboratory Handbook on Bovine Masti­ diagnosis was corrected. For example, misidentifying an tis, 15 published by the National Mastitis Council. Most Enterobacter species as Escherichia coli will likely not organisms can be identified in a practice-based labora­ change the recommended treatment, but misidentifying tory by culturing samples on selective media, and follow­ yeast as a coagulase-negative Staphylococcus species ing with one or more simple laboratory tests. Reading will more likely change the treatment decision. Thus, cultures on colony morphology alone often results in a reasonable goal is to promptly produce correct results misdiagnosis. 41 Reading cultures using morphology, whenever possible, and to not misdiagnose organisms microscopic examination, and supplemental testing where the choice of treatment would be changed. may still occasionally result in an incorrect diagnosis. 14 In consideration of this goal, there is no need to This can occur because of variable colony morphology or perform unnecessary procedures and tests to achieve a variable response to supplemental testing. In addition, diagnosis with a reasonable level of confidence. Those results of biochemical and other supplemental tests are considering or actively building a mastitis laboratory sometimes subjective. should understand that a diagnosis is often simple, and A practice-based laboratory designed to enable that the sum of the tests and procedures in this docu­ culture-based treatment of mastitis does not need to ment are not needed in most cases. Two useful resources produce absolutely correct results 100% of the time. for ensuring laboratory quality are the Laboratory Pro­ However, it is important for the diagnosis to be correct ficiency Testing Program offered by Cornell University's when the recommended treatment would change if the Quality Milk Production Services,1 and known standard

20 THE BOVINE PRACTITIONER-VOL. 46, NO. 1 cultures which can be plated and run through the diag­ patterns of growth on different media often make the nostic process in the laboratory. Cultures are available diagnosis difficult without microscopy. from a number of sources, including Hardy Diagnostics. h Standardized procedures are necessary for quality Simple Tests Useful for Identification ofMastitis results in the mastitis laboratory. Procedures used in the Pathogens and Their Uses authors' practice laboratory are as follows: Milk is plated with a 0.01 mL loop on each of the • CAMP test: this test is used to differentiate be­ following plates: washed bovine blood agar, Modified tween CAMP-positive Streptococci (Strep. agalac­ TKT agar, MacConkey agar, and Factor agar. Up to tiae, some human isolates) and CAMP-negative four samples can be inoculated on each plate by divid­ Streptococci (primarily Strep. dysgalactiae ). ing plates into sections. Plates can typically be read 18 • Catalase test: commonly used to differentiate hours post-incubation. An attempt is made to generate between staphyloccal (catalase-positive) and strep­ preliminary results and deliver them to the farm by tococcal (catalase-negative) organisms. It may also phone, fax, or email prior to the time of day the producer be used to differentiate between Arcanobacterium typically administers intramammary treatments. The pyogenes (catalase-negative) and Corynebacterium particular time of day varies from farm to farm, therefore bovis (catalase-positive). Serratia and Proteus are polling individual producers regarding daily treatment vigorous, rapid catalase reactors, while most other schedules is important. If samples are delivered to gram-negative mastitis pathogens are slow, weakly the laboratory on the day of collection, producers can positive or negative reactors. 4,4o · generally administer treatments after receiving culture • Citrate test: this test is used for differentiation be­ results the following morning. tween citrate-negative (E. coli) and citrate-positive The first step in evaluating plates is to determine (Klebsiella, Enterobacter), gram-negative organisms. upon which plates colonies are present. MTKT agar is • Coagulase test: used to confirm coagulase-positive selective for Streptococcus and Enterococcus; Factor agar (Staph. aureus) organisms. selects for gram-positive organisms; and MacConkey • Gram stain: commonly used to confirm the identi­ agar selects for gram-negative organisms,22 but there fication of yeast and Prototheca and to aid in identi­ are exceptions. For example, Pasteurella multocida, fication of less common mastitis pathogens.· a gram-negative organism, only grows on blood agar, • Motility media: used to distinguish motile (Entero­ while Mannheimia haemolytica typically grows on blood, bacter, E. coli) from non-motile organisms (Klebsiella) Factor, and sometimes MacConkey agar. A variety of • MIO (motility, indole, ornithase) media: this less common pathogens, including yeasts, fungi, and the test is used to indicate motility, production of in­ algae Prototheca, will grow inconsistently on a variety of dole, and metabolism of ornithine. It is useful for plates. Yeasts typically grow on blood, Factor, occasion­ differentiation oflactose-fermenting gram-negative ally MTKT, and rarely MacConkey. Prototheca typically pathogens, including E. coli, Enterobacter, Citrobac­ grows on all four plates, but is most recognizable on ter, Klebsiella, and some Serratia isolates. blood agar due to more rapid and robust growth. With • Oxidase test: this test distinguishes Pseudomonas the exceptions noted, a presumptive diagnosis can often (positive) from non-pigmented Serratia (negative). be quickly made by examining the patterns of growth • Potassium hydroxide (KOH) test: this test dis­ on the agar plates. tinguishes between gram-negative (KOH-positive) When a definitive diagnosis can be made by visual and gram-positive (KOH-negative) organisms grow­ examination only, no further testing is necessary and the ing on blood agar. It is also useful to confirm that a plates are returned to the incubator for 24 or more hours. Mannheimici or Pasteurellµ, (gram-negative, KOH­ For example, E. coli is often fairly easy to identify on positive) is growing on a Factor plate. MacConkey agar by its characteristic dry, flat, pink colo­ • PYR test: used to distinguish gram-positive, nies with surrounding pink precipitate. Typically, how­ esculin-positive, PYR (pyrrolidonyl arylamidase)~ ever, performing one or more simple tests are required positive (Enterococcus) organisms from PYR-nega­ to confirm the identification of organisms. 9,10,15,36,37,38,39,44 tive (Streptococcus uberis) organisms. If a clear diagnosis is not reached by visual ex­ • 6.5% NaCl broth (with color indicator): used to amination, a variety of simple tests can be performed distinguish gram-positive, esculin-positive, organ­ in the laboratory, including gram staining. The gram isms capable of growing in 6.5% NaCl (Enterococ­ stain should be performed on any colonies that cannot be cus) from those incapable of growth (Streptococcus definitively identified. Visual examination under high uberis). power will often lead to a diagnosis. For example, yeasts • Urease test: this test distinguishes urease-positive usually are easy to recognize following gram staining (Proteus, Pseudomonas) from urease-negative (Ser­ with a microscope, but variable colony morphology and ratia) organisms.

SPRING 2012 21 Further information regarding specific tests and ty-positive organisms will show fuzziness radiating test procedures is available from a variety of other out from the stab line and the media may appear sources. 7,10,1s,29,36,37,3s,39,44 cloudy. Ornithase-positive organisms will keep the media purple, while ornithase-negative organisms Testing Methods will turn the media yellow. Indole-positive organ­ isms will show a red line at the top of the media • CAMP test: streak known Staph. aureus colonies in after adding two drops of Kovac's reagent. a straight line down the center of a blood agar plate. • Oxidase test: place a circle of filter paper on top Streak the suspected Strep. agalactiae in a line of cardboard to protect the countertop. Place a drop perpendicular to the Staph. aureus so that the two of oxidase reagent onto the filter paper. Pick up a lines come within 1-2mm of each other. Formation colony with a loop, then drag the loop across the of a clear, arrowhead-shaped zone ofbeta-hemolysis wet area of the filter paper. A positive reaction has after 24 hours of incubation is a positive reaction. occurred when the filter paper immediately turns • Catalase test: place a drop of 3% hydrogen perox­ purplish-black where it came in contact with the ide onto a microscope slide. Emulsify a colony into loop. No color change, a slight, or pink color change, the drop of peroxide, being careful not to gouge the or a color change occurring after ten or more seconds agar (red blood cells will create a false-positive test). is negative. Bubbling is positive; no bubbling is negative. • PYR test: moisten the paper disk slightly with dis­ • Citrate: inoculate the tube with a few colonies. Posi­ tilled water. Pick two to three well isolated colonies tive isolates will turn the green media blue within from the blood plate and rub into a small area of 24 hours. the PYR disk so that there is a visible paste. Allow • Coagulase test: thaw a 5 mL tube of frozen rabbit to react for two minutes, then add one drop of PYR plasma. Using a loop, transfer several colonies into reagent. A bright pink or cherry red color will ap­ the tube and mix thoroughly. Incubate and examine pear within one minute for a positive test. No color at 1, 2, 4, and 24 hours after inoculation. Semi-solid change, or orange, salmon, or yellow color should be to solid gelling is a positive reaction. If no gelling interpreted as a negative result. has occurred at any time and the plasma is liquid • 6.5% NaCl broth (with color indicator): inocu­ at 24 hours, the test is negative. late the broth with a few colonies. Positive isolates • Gram stain: using a loop, mix a colony into a drop will turn the broth blue within 24 hours. of sterile water on a glass microscope slide. Air • Urease test: moisten filter paper with a few drops of dry and fix the slide by passing over a flame. Ap­ 10% urea agar base concentrate. Rub some culture ply crystal violet to the slide, wait 30-60 seconds, onto the paper with a glass rod. Positive isolates then drain. Apply Grams Iodine to the slide, wait will show a pink or red streak on the paper within 30-60 seconds, then drain. Decolorize with decolor­ two minutes. izer (75% acetone/25% isopropyl alcohol) by gently rinsing until no color is visible in the runoff. Rinse Procedures for Identification Using a Plate­ with tap water. Apply safranin and wait 60 seconds. Based Identification Flow Rinse with tap water, blot dry with bibulous paper, and examine under the microscope with oil. Note One method to identify organisms is to serially size, shape, arrangement, and color of the cells. examine all of the plates for growth as follows: • KOH test: place a small drop of KOH onto a plastic cover slip. Use a loop to transfer several colonies 1. Remove plates from incubator and let stand for into the drop. Thoroughly mix the colonies into the 20 minutes. This allows the esculin reaction to drop with the loop, then gently raise the loop while take place on the MTKT plates. looking closely for any evidence of a tiny string be­ 2. Examine the blood agar plate. tween the drop and the loop. Stringing is a positive a. No growth~ fewer than three identical colo­ (gram-negative organism) reaction. A false negative nies (one or more Staph. aureus or Strep. ag. may occur ifthere is too much KOH, too few colonies, colonies are considered positive growth) are or,inadequate mixing. present. • Motility media with color indicator: inoculate b. Arcanobacterium pyogenes - white, pinpoint the tube with a deep stab into the media. Positive beta-hemolytic colonies, which typically isolates will show reddish lines radiating from the do not appear until after 30 to 48 hours of stabbed area after 24 hours of incubation. incubation, are present. Also A pyogenes • MIO media: inoculate the tube with a deep stab grows on the Factor plate and sometimes is into the media. After 24 hours of incubation, motili- visible on the MTKT plate. Confirm with a

22 THE BOVINE PRACTITIONER-VOL. 46, NO. 1 catalase test (negative) and gram stain (tiny k. Prototheca - small, dry, gray, flat colonies purple rods). that are often difficult to notice at 24 hours, c. Bacillus - large, flat colonies, which can be are present. Prototheca grows on all four rough or moist, dry or slimy, and sometimes types of agar, but grows fastest on blood asterisk-shaped, are present. Sometimes agar. Gram stain to confirm the identity of beta-hemolysis is present. Consider Bacillus the organism (large cells, 10 to 20 times the a contaminant unless no other organisms size of bacteria, five times the size of yeast, are present and there are three or more variable in color, variable in shape and size, colonies. Bacillus also grows on the Factor often can see daughter cells pinching off of plate. parent cells). d. Corynebacterium bovis - tiny, white or gray 1. Staphylococcus - large, 2-5 mm, smooth, non-hemolytic colonies, which do not appear white, creamy, yellow, gold, or grayish-white until after 30 to 48 hours of incubation, are colonies are present. They may or may not present. They should also grow on Factor be hemolytic; any degree ofbeta-hemolysis plate. C. bovis may be confused with Pro­ is suggestive of Staph. aureus (use coagulase totheca. Confirm the diagnosis with a gram test to confirm). Grows on Factor, but not stain (tiny, pleomorphic rods) and catalase on MTKT plates. test (positive). m. Streptococcus and Enterococcus - small, 1-3 e. E. coli - colonies are 3-5mm in size, gray, mm, smooth, translucent, convex colonies moist, and have a fecal odor. Less than 15% are present. No hemolysis, alpha- or beta­ are hemolytic. They do not grow on Factor hemolysis may be present. Examine MTKT or MTKT plates. Refer to MacConkey plate plate. to differentiate. n. Serratia - small, 2-3 mm in diameter, white, f. Enterobacter and Citrobacter - appear simi­ yellow, or gray colonies are present, which lar to E. coli, are non-hemolytic, and have may resemble Staphylococci. Seratia may be a fecal odor. The organisms do not grow on hemolytic, and may develop bright red color Factor or MTKT plates. Proceed to MacCo­ at room temperature. nkey plate. o. Yeast - white, waxy-textured, or sometimes g. Klebsiella - appear similar to E. coli, are of­ gray and raised colonies are present. Yeast ten mucoid, and are non-hemolytic. They do also grow on Factor, possibly MTKT, and not grow on Factor or MTKT plates. Proceed rarely MacConkey plates. Gram stain to to MacConkey plate. confirm. They will be about five times the h. Pasteurella and Mannheimia - colonies size of bacteria, usually purple, but variable often look like spilled milk (wet, confluent, in color, fairly consistently sized oval-shaped or lobed, white or grayish). Examine for cells; one can often see evidence of budding; growth on MacConkey and Factor plates fungal hyphae may be seen. (Mannheimia), Factor but not MacConkey p. Contaminated sample - if the sainple is plates (Pasteurella or Mannheimia species), from a single quarter, and more than two or blood agar only (Pasteurella multocida). distinct colony morphologies are present Confirm with a KOH test (positive) and (not including Bacillus), the sample is con­ gram stain (tiny, pink, bipolar staining rods side-red contaminated. Ifthe sample is from that may look like two cocci stuck together). more·than one quarter, report all organisms i. Proteus - colonies are gray, slimy, and with more than two colonies (one or more for swarming with a putrid odor. Colonies may Staph. aureus and Strep. agalactiae) on the quickly cover the entire plate. Proteus also plate. grows on MacConkey agar. Agram stain may 3. Examine the Factor plate. show long, filament-like "swarm cells".29 a. If no growth, proceed to the MacConkey Proteus is urease-positive and oxidase­ plate. negative. b. Bacillus, Mannheimia, Pasteurella, Proto­ j. Pseudomonas - large, 3-4mm, flat, grayish­ theca, Arcanobacterium, and Corynebacte­ blue colonies are present. Beta-hemolysis rium - see blood plate above. Mannheimia is usually present. Occasionally the colo­ and some Pasteurella species will grow on nies can be rough or dry. A grape-like odor Factor agar; Pasteurella multocida will not. typically is noticeable. Pseudomonads are Mannheimia may grow, sometimes with urease- and oxidase-positive. pinpoint red colonies, 29 on MacConkey agar.

SPRING 2012 23 Mannheimia haemolytica will show beta­ the agar around the colonies turns pink). hemolysis on blood agar; most Pasteurella E. coli are citrate-negative. species will not. ii. Enterobacter - typically pink, dry c. Beta-hemolytic colonies - set up a coagulase colonies, but sometimes wet and mucoid test to confirm Staph. aureus (positive). If colonies are present. There is no bile this test is negative, report as coagulase­ precipitate. Organisms are citrate-pos­ negative Staph. itive, motility-positive, and ornithase­ d. Non-hemolytic, or alpha-hemolytic colonies positive, but indole-negative. (MIO +-+) - check to see if they are growing on MTKT m . Klebsiella - mucoid, wet-looking, pink plate. colonies with a cream-colored center. 1. Growth on MTKT - proceed to MTKT Precipitated bile salts are not present. flow. Klebsiella are citrate-positive, motility­ 11. No growth on MTKT-two possibilities negative, and ornithase-negative. Most remain: Klebsiella are indole-negative (MIO ---), 1. Yeast - white, waxy-textured, or but Klebsiella oxytoca is indole-positive sometimes gray and raised colonies (MIO-+-). are typical. See blood agar above. 1v. Serratia rubidaea - this species of Serra­ 2. Coagulase-negative Staph - colonies tia ferments lactose, and is an unusual are typically white, yellow, or gray, cause of bovine mastitis. Colonies are and shiny. pink and wet-looking. Sixty percent 4. Examine the MTKT plate. of isolates will have red pigment and a. Prototheca - see blood agar plate above. appear as bright red colonies. Pigment b. Esculin-positive colonies (darkened or black is more likely to be produced at room agar surrounding the colonies) temperature. Organisms are motility­ i. Strep. uberis - colonies appear black or positive, indole-negative, ornithase­ gray. The organism will not grow in 6.5% negative, and citrate-positive (MIO+--). NaCl broth, and is PYR-negative. Serratia are vigorous catalase reactors, 11. Enterococcus - colonies appear black, while other lactose-fermenters are slow 4 4 gray, greenish-gray, or brown. Confirm reactors, negative, or weakly positive. , o with PYR test (positive) or NaCl broth v. Citrobacter - wet-looking colonies are (positive). present. Citrobacter are motility-, in­ c. Esculin-negative colonies dole-, ornithase-, and citrate-positive 1. Strep. agalactiae-a zone ofbeta- (clear) (MIO+++). hemolysis will be present around colo­ vi. Mannheimia haemolytica - pinpoint nies. Perform CAMP test (positive). If reddish colonies may be present due negative, report as Strep. dysgalactiae. to fermentation of lactose. Look for It may be necessary to submit CAMP­ growth on Factor plate; note "spilled positive samples to another lab, or use milk" morphology on blood agar. Gram an API systemc for confirmation. stain to confirm. Mannheimia is nega­ ii. Strep. dysgalactiae - non-hemolytic, tive for motility, indole, and ornithase, esculin-negative colonies of variable size (MIO---), citrate-negative, and oxidase­ and color are present. negative or weakly positive. d. Opportunities for occasional misdiagnosis vii. Special note for diagnosis of lactose­ using this scheme include the isolation of positive organisms: often a diagnosis of an occasional esculin-positive Strep. dysga­ E. coli, Klebsiella, and Enterobacter can lactia, or isolation of other streptococcal spe­ be made by colony morphology alone, but cies that are less common causes of bovine the possibility exists that misdiagnosis mastitis.15 More definitive diagnoses can be will occasionally occur. For example, made with an API, or similar system. Enterobacter are typically dry-looking, 5. Examine the MacConkey plate. pink colonies with no bile precipitate. a. Prototheca - see blood agar above. However, some Enterobacter will be b. Lactose-fermenting colonies (pink and mucoid, and thus may be confused with opaque) Klebsiella. Non-pigmented Serratia i. E. coli - dry, pink colonies are surround­ rubidaea may also be confused with ed by precipitated bile salts (surface of Klebsiella. E. coli can occasionally ere-

24 THE BOVINE PRACTITIONER-VOL. 46, NO. 1 ate mucoid colonies. Citrobacter is an not result in a diagnosis. Alternatively, an uncommon cause of bovine mastitis. API 20E systemc or other similar system can MIO and citrate testing can definitively be used to definitively identify pathogens in diagnose most lactose-fermenting organ­ such circumstances. Typically, however, such isms. systems are more costly than simple tests c. Non-lactose fermenting colonies (translu­ that can be run in a practice laboratory. cent, gray, tan, or yellow): 6. Special considerations for Nocardia and Myco­ 1. Pseudomonas - colonies are tan, trans­ plasma lucent, and may have a grape-like odor. a. Nocardia - this gram-positive organism is Confirm with the oxidase (positive) test. seldom seen on mastitis cultures. Infection If oxidase-negative, report as Serratia. is uncommon, and refrigeration and freez­ 11. Serratia - most Serratia isolates are ing of milk samples make isolation difficult. non-pigmented, and will appear very They are gram-positive, irregular rods with similar to Pseudomonas. The remainder filamentous branching, have no spores, and produce a red pigment, especially if left are catalase-positive. Airborne hyphae out for 20 minutes at room temperature. are potentially pathogenic to humans, so Perform an oxidase test to confirm (neg­ plates with suspected or confirmed Nocardia ative). Serratia are vigorous catalase should be handled carefully. reactors. 4·40 b. Mycoplasma species require microaerophilic m. Proteus - swarming, slimy colonies often conditions for growth. This can be accom­ cover the entire plate. A putrid odor may plished with a carbon dioxide incubator or a be present. Proteus are vigorous catalase candle jar. Plates are examined at 24 to 48 reactors.4·40 Proteus are urease-positive, hours after plating for growth. Samples are while Serratia are urease-negative. not considered negative unless no growth is d. Differentiation between gram-negative present at seven days. Specific procedures organisms, and particularly the Enterobac­ for culture and examination are found else­ teraciae, may be difficult in some situations. where.15·29 If Mycoplasma culture is to be Table 1 and Table 2 show typical reactions performed in a practice-based laboratory, of gram-negative organisms to a variety one should follow the procedures described of tests. These tables have been compiled above for other organisms, and then supple­ by the authors from a variety of sourc­ ment results with Mycoplasma culture es.10·15·29·39·44 Tables are provided as an aid in results, which are typically available seven diagnosis when less thorough testing does days after plating.

Table 1. Differentiation of gram-negative mastitis pathogens with biochemical tests.

Organism Oxidase Motility Lysine Ornithine Urease Citrate Indole Lactose

Enterobacter + + + + + aerogenes Enterobacter + + +/- + + cloacae Klebsiella + + + + Klebsiella + + + + + oxytoca E.coli (+)most (+)most +/- + + Citrobacter (+)most (+)most +/­ (+)most + + Serratia + + + (-)most + marcescens Serratia (+)most +/- + + rubidaea Pseudomonas + +(top) + +/- Pasteurella weak+ + + (-)most Mannheimia +/- (+)most Proteus + +/- + +/- +/-

SPRING 2012 25 Table 2. Motility/Indole/Ornithase (MIO) test inter­ Reporting Results pretation. Cows are often treated at a particular time of day Motility Indole Ornithase on many dairy farms. This might, for example, be dur­ ing milking, or following a particular milking shift. It is Enterobacter + + often possible to provide preliminary results to the farm Klebsiella pneumoniae on the day after plating so that treatment is not delayed. Klebsiella oxytoca + Cellular phones make dairy farm personnel nearly al­ E.coli (+)most + +/- Citrobacter (+)most + (+)most ways available, and many farms have fax machines for Serratia marcescens + + reporting results. Email is also very commonly used Serratia rubidaea (+)most on farms; however, a farm computer or smart phone is required to receive results. Recently a commercial soft­ ware program has been utilized to electronically report Procedures for Identification of Mastitis results. 23 A combination of reporting methods may be Pathogens Using Flow Charts used, but it is advisable to find the preferred method for each farm and then use that method to consistently Flow charts are an alternative way to represent report results. processes of identification of mastitis pathogens. Figures A relatively new method combines email with 2 through 4 are flow charts representing the decision "cloud" storage. An example of such a system is Google process used to identify pathogens in the authors' labo­ Docs. A document can be created in word processing, ratory. Note that Pasteurella multocida grows only on spreadsheet, or presentation format from the Google blood agar, and Prototheca typically grows on all four Docs website. Alternatively, a document using one of agar types. It should again be stressed that these flow these formats can be uploaded to Google Docs. Once a charts are examples and are not meant to represent the document is created or uploaded, the user can designate best, or only available, diagnostic methods. individuals with which to share the document. Sharing can mean viewing, editing, or both, depending on the Susceptibility Testing designation used by the owner of the document. When an individual is allowed to share a document, an email Susceptibility testing has been advocated as a ba­ notification is sent, which allows the person to click on sis for therapy selection for nearly 50 years. However, a portion of the message and be forwarded to the online the disk-diffusion method typically used has several document for viewing. The editing function can be useful limitations. 2•4•5•6•7•11 In the authors' opinion, routine sus­ for veterinarians to add comments, such as treatment ceptibility testing of bovine mastitis isolates is seldom recommendations. indicated for formulating routine treatment decisions. Google Docs is used by the authors' laboratory and Typical sensitivity patterns ofmastitis pathogens using has been very useful. As soon as results are available, minimum inhibitory concentration data are available, an Excel spreadsheet containing the results is created by and can be used to formulate treatment protocols. the laboratory technician using a report template. The document is then uploaded to Google Docs, with the farm Veterinarian Involvement veterinarian and the appropriate farm personnel listed as viewers. The farm veterinarian is also designated Having both a veterinarian and a veterinary tech­ as an editor. Both the veterinarian and the producer nician examine results and plates is advisable and serves receive email notification of completed results, allowing several purposes. 3 First, it allows for another opinion. them to see the document with one click. The veterinar­ On occasion some judgment is required to formulate ian can add a notation, or send an email notation to the final results, and thus another trained individual is of producer at the time of viewing. When a notation is value. Second, veterinarians can soon become profi­ made, the producer will receive another email indicating cient in identification procedures, and can be utilized a document is ready to be viewed. This system offers to determine results when a technician is absent, or on several advantages. First, it can be done quickly and the weekends. Third, having the veterinarian sign-off on ev­ document is available almost instantaneously. Second, ery report can help maintain a standard of quality in the there is no need for a hard copy of the document to find laboratory. In the authors' laboratory, both preliminary its way into a veterinarian's inbox, or on a desk, or in a and final results are examined by a practice-member pile of documents somewhere in the office. Third, the veterinarian. The veterinarian may agree, disagree, or document is available for viewing from virtually any request further testing. The tracking form is initialed by internet-connected device at any time. Unlike email the veterinarian both for preliminary and final results. attachments, for example, it is not necessary to locate

26 THE BOVINE PRACTITIONER-VOL. 46, NO. 1 the computer in which an electronic copy of a docu­ Treatment of Cows ment is stored. A producer or veterinarian can access the information from a smart phone, tablet computer, Use of standardized treatment protocols on dairy or similar device while on the farm. Fourth, it is easy farms has been advocated for a variety of reasons. 24,41 for the farm veterinarian to add comments. There are Following notification of mastitis culture results, farm other similar services available, such as Windows Live, staff can administer treatments according to existing that are available for free on the internet. farm-specific treatment protocols. Typically, contact

Growth on Factor Plate Gram+, Pasteurella, Mannheimia,• yeast, Prototheca

Growth on MTKT + Growth on MTKT - + Go to MTKT flow chart l White, creamy, White, gray, or tram, Large, flat, White or gray or yellow lucent, lobed, or rough or moist pinpoint colonies colonies confluent colonies colonies visible at 30-48 hours + of incubation Mannheimia, Bacillus• Pasteurella Catalase + +Catalase - ( Gram stain to confirm)

Hemolysis + Hemolysis - Corynebacterium Arcanobacterium bovis pyogenes

Staph.+ Coagulase-negative+ Staph.

Coagulase + Coagulase -

Staph. aureus+ Coagulase-+ negative Staph.

Note: Colony morphology and gram stain can be used to identify yeast, Mannheimia, Pasteurella, Bacillus, Arcanobacterium, Corynebacterium, or Prototheca. Prototheca grows on MacConkey, blood agar, Factor, and MTKT plates.

Figure 2. Flow chart used to identify pathogens on Factor agar plates.

SP.RING 2012 27 with the farm veterinarian is not needed. On farms with outlined above. Whole-herd culture for contagious patho­ employees, employees will usually be able to administer gens can be performed in a practice-based laboratory. the designated treatments without contacting the farm Bulk-tank milk culture and differentiation, and culture owner or manager. Treatment protocols need to specify of bedding, towels, calf milk, and colostrum all are rela­ the pathogen isolated for this system to work properly. tively easy to perform once the laboratory materials and Protocols should identify the drug to use, the duration procedures are in place. and frequency of treatment, and the proper milk and On-farm pasteurizers can be monitored by cultur­ meat withholding periods. Protocols may be updated ing pre- and post-pasteurized milk, using techniques based on the perceived treatment response, product very similar to culturing of bulk-tank milk. preference, cost, availability, ease of administration, and other factors. It is the veterinarian's responsibility Conclusions to develop specific treatment protocols for any farm. A discussion of specific treatments is beyond the scope of A practice-based mastitis laboratory can provide this paper. valuable service to dairy farms by providing pathogen identification in a timely manner. Isolation and iden­ Other Procedures tification methods are not difficult to learn or perform. By using available technologies it is possible to report The techniques developed and materials available results quickly and efficiently, allowing dairy producers in the mastitis laboratory can be utilized for other test­ to treat mastitis cases without excessive delay. This ing. Cultures can be performed on cows with suspected paper describes methods used in the authors' labora­ subclinical or chronic mastitis by using the procedures tory, in both text and flow chart form, and is meant to

Growth on MTKT Plate

Probable Strep., Enterococcus,+ Prototheca, yeast l

Esculin + Esculin -

PYR+ PYR­ Beta-hemolysis No hemolysis NaCl+ NaCl-

CrP + i CAMP _ Enterococcus Strep. uberis l Presumptive LStrep . dysgalactiae Strep. agalactiae ( Confirm at outside lab)

Note: Prototheca grows on blood agar, MacConkey, Factor, and MTKT plates. Yeast may not always grow on MTKT agar. Gram stain should be used to confirm Prototheca or yeast.

Figure 3. Flow chart used to identify pathogens on MTKT agar plates.

28 THE BOVINE PRACTITIONER-VOL. 46, NO. 1 provide an example of a relatively complete "how-to" hHardy Diagnostics, Santa Maria, CA, guide that other practitioners might find useful when www.HardyDiagnostics.com developing a mastitis laboratory. The methods described cbioMerieux, Inc, Durham, NC are not meant to represent the only, or necessarily, best methods available. References

Endnotes 1. Animal Health Diagnostic Center, Cornell University College of Veterinary Medicine, Ithaca, NY. http://ahdc.vet.cornell.edu/sects/ QMPS/Programs/proficiency.cfm. Accessed November 2011. aThe Laboratory for Udder Health, MVDL, University of Minnesota, St. Paul, MN, [email protected]

Growth on MacConkey Plate t Gram-, Prototheca

Lactose fermentation + Lactose fermentation - (pink, opaque colonies) (translucent, not pink)

Wet-looking Dry-looking Pinpoint red colonies colonies• colonies + MIO Bile Bile Mannheimia +++ +-+ +-- -( +/-)- precipitate + precipitate - citrate - MIO --- + Factor growth + Citrob:cter l Klebsiella t ( Gram stain to E. coli Enterobacter confirm) Enterobacter citrate• - citrate+ ~, MIO++(+/-) MIO+-+ Serratia rubidaea (may be red-colored)

Tan, translucent, Swarming, slimy or red colonies · colonies, putrid odor

Oxidase + Oxidase -

Proteus oxidase - Pseudomonas Serratia urease + (grape odor) (maybe red) urease + urease - (most)

Note: Prototheca grows on MacConkey, blood agar, Factor, and MTKT plates.

Figure 4. Flow chart used to identify pathogens on MacConkey agar plates.

SP,RING 2012 29 2. Apparo M, Ruegg P, Lago A, Godden S, Bey R, Leslie K. Relationship 21. McCarron JL, Keefe GP, McKenna SL, Dohoo IR, Poole DE. between in-vitro susceptibility test results and treatment outcomes for Laboratory evaluation of 3M Petrifilms and University of Minnesota gram-positive mastitis pathogens following treatment with cephapirin Biplates as potential on-farm tests for detection of clinical mastitis. J sodium. J Dairy Sci 2009;92:2589-2597. Dairy Sci 2009;92:2297-2305. 3. Blackmer PE. A practitioner's micro lab and on-farm culturing, 22. Minnesota Easy Culture System IL Quality Milk Production Quality Milk Seminar JC, session handout. Am Assoc of Bov Pract Services, Animal Diagnostic Laboratory, University of Minnesota, Conf, Saint Paul, MN, 2006. St. Paul, MN. http://www.ahdc.vet.cornell.edu/sects/QMPS/Services/ 4. Chester B, Moskowitz LB. Rapid catalase test for identification of minnesota culturemanual.pdf. 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