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Mastitis Isolation

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					Major
Mastitis Pathogens
Isolation
           Selective Media


Philip Sears, DVM, PhD
Department of Large Animal Clinical Science



                   Veterinary Extension
                   Michigan state University
                   517-355-9610
                   www.cvm.msu.edu (extension)
                                   Mastitis Screening
                                           California Mastitis Test

The California Mastitis Test (CMT) is an indirect test used to determine the somatic cell count in milk. Milk
from quarters that react to the CMT indicates intramammary infection.

Procedure:
 Clean teat and remove a single steam of milk from each quarter
 Collect milk (about 2 ml/1 tsp) in appropriate well: A, right fore; B, right rear; C, left fore D, left rear
 Add equal amount (2 ml/1 tsp) CMT solution
 Rotate mixture gently for 20 seconds
 Determine agglutination score:
               Score          Milk gelling Somatic Cell Count (cell/ml)           Milk Quality
                Neg              none            <10,000-200,000                    Good
               Trace            slight            150,000-500,000                   Good
                 1               mild             400,000-1,500,000               In-between
                 2             moderate           800,000-5,000,000                  Bad
                 3              strong             over 5,000,000                    Bad
 Record each quarter reaction score
 Quarters with mild to strong gel should be considered infected and cultured for mastitis management




                                                CMT Scores and Production
                                                CMT Score               Production Lost
             N                 1                                      Cows         Quarters
                                                 Neg/Trace             6%           9.0%
                                                    1                 10%          19.5%
                  2                3                2                 16%          31.8%
                                                    3                 24%           43.4%
                                                                                                Schalm, 1971
                                                                                                   Sears, 2002
                                      Major Mastitis Pathogen Isolation
                                                   Philip Sears, DVM, PhD
Milk samples must be taken using aseptic technique and placed on ice or refrigerated immediately. Contaminated
samples or mishandling of the sample can lead to misdiagnosis and confusion.
Sample consideration:
 Individual:   Composite samples used to screen herd for infected cows.
               Quarter samples when evaluating individual or clinical cows.
 Bulk Tank:     Used to screen or monitor herd for contagious mastitis (Strep ag, Staph aureus, Mycoplasma)
 Sampling:      Clean teats (predip and dry) before sampling.
                Clean teat end with 70% alcohol using cotton balls, pledgets or wipes.
                Teats must be dry before collection sample. Use a tight sealing vial or tube.
                Discard first milk and collect (midstream -45o angle) into a sterile vial or tube (5 to 10 ml).
                Store sample on ice or refrigerate until plating. (Freeze, if greater than 48 hours).
Plating Selective Agar:
                 Plate milk sample as soon after collecting as possible. Fresh is best.
                 Single plate media (such as blood agar plates) can be quartered for a cow or half for composites.
                 Selective agar tri-plate (Blood agar, TKT agar, MacConkey agar): plate clinical quarter per plate.
                 Read selective media first:
                   • MacConkey agar selective for coliforms
                   • TKT agar selective for streptococcal organisms
                   • Blood agar will grow all organisms
                 Gram Stain and other tests are helpful in identifying oragans


                                                               Selective Media

Organisms       Test             Blood Agar                        TKT Agar                          MacConkey Agar

E. coli          KOH +           growth                            no growth                         growth
                 Gram -          large grey colonies                                                 pink colonies/dry
                                 mostly non-hemolytic                                                pink zone ppt in agar
Klebsiella sp    KOH +           growth                            no growth                         growth
                 Gram -          large mucoid colonies                                               pink-yellow colonies
                                                                                                      mucoid
                                                                                                      “fish eye” appearance
Strep            Gram +          growth                            growth                            no growth
 agalactiae      H2O2 -          small translucent colonies        esculin negative
                 CAMP +          beta-hemolytic (clear zone)       clear zone

Strep sp         Gram +          growth                            growth                            no growth
                                 small translucent colonies        esculin positive
  except;                                                           (black colonies)
  (dysagalactiae) CAMP -                                           esculine negative
Staph aureus      Gram +         growth                            no growth                         no growth
                  H2O2 +         large opaque colonies
                  Coagulase +    (creamy-golden-yellow)
                                 double zone hemolysis
                                 (complete & partial)
Staph sp         Gram +          growth                            no growth                         no growth
                 H2O2 +          opaque colonies
                 Coagulase -     white to yellow color
                                 non-hemolytic or complete                                                   Sears, 2003
             Major Mastitis Pathogen Isolation
      Selective Media – Blood agar/TKT agar/MacConkey agar
Streptococcal Isolation

               growth                                              Streptococus agalactiae

                                                                   Gram positive small colonies
                                                                   catalase neg  esculin neg
                 BA                                                CAMP positive -hemolytic
             TKT MAC         No
                           growth
                                         -hemolytic colonies



                                                        Strep ag   Strep ag produce clearing (arrow-shape)
                                                                   other strep sp: esculin +, CAMP neg
                                          Strep sp


                                             CAMP Test
                 BA                                                 Streptococus species
             TKT MAC
                                                                    Gram positive small colonies
                          No                                        catalase neg  esculin positive
                        growth
                                                                    CAMP negative non-hemolytic


                                           esculin positive

 Staphylococcal Isolation
                                                                    Staphylococcus aureus

                                                                    large colonies (cream-tan color)
                                                                    Gram positive
                                                                    catalase positive (H2O2 +)
                                             Staph aureus           coagulase positive
                   BA                                               -hemolytic (double zone)
             TKT    MAC
                                                +
      No                  No
    growth              growth                      -
                                              Coagulase Test
                                                                    Staphylococcus species

                                                                    large colonies (white to yellow)
                                                                    Gram positive
             Veterinary Extension                                   catalase positive (H2O2 +)
             Michigan state University                              coagulase negative
             Philip Sears, DVM, PhD                                 no hemolysis or complete hemolysis
             517-355-9610                       Staph sp
                                                                                              Sears, 2003
                Coliforms

                                                                                   E. coli

                                                                                   Blood agar:
                                                                                    large colonies (grey, moist)
                                                                                    Gram negative rods
                         BA
                                                                                    not hemolytic
                   TKT    MAC                                                      MacConkey agar:
                                                                                    lactatose positive(pink, dry, flat colonies)
                                                                                     pink zone of ppt in agar




                                                                                    Kkebsiella sp
                                                pink zone around colonies
                                                                                    Blood agar:
                                                                                     large colonies (grey, mucoid )
                     BA
                                                                                     Gram negative rods
                   TKT MAC                                                           not hemolytic
                                                                                    MacConkey agar:
                                                                                     lactatose positive(pink, mucoid)
                                                                                     pink center “fish eye” appearance


                                                 pink center “fish eye”

Organisms           Test           Blood Agar                         TKT Agar                            MacConkey Agar
E. coli              KOH +         growth                             no growth                           growth
                     Gram -        large grey colonies                                                    pink colonies/dry
                                   mostly non-hemolytic                                                   pink zone ppt in agar
Klebsiella sp        KOH +         growth                             no growth                           growth
                     Gram -        large mucoid colonies                                                  pink-yellow colonies
                                                                                                           mucoid
                                                                                                          “fish eye” appearance
Strep                Gram +        growth                             growth                              no growth
 agalactiae          H2O2 -        small translucent colonies         esculin negative
                     CAMP +        beta-hemolytic (clear zone)        clear zone
Strep sp             Gram +        growth                             growth                              no growth
                                   small translucent colonies         esculin positive
 except;                                                               (black colonies)
 (dysagalactiae)     CAMP -                                           esculine negative
Staph aureus         Gram +        growth                             no growth                           no growth
                     H2O2 +        large opaque colonies
                     Coagulase +   (creamy-golden-yellow)
                                   double zone hemolysis
                                   (complete & partial)
Staph sp             Gram +        growth                             no growth                           no growth
                     H2O2 +        opaque colonies
                     Coagulase -   white to yellow color
                                   non-hemolytic or complete                                                      Sears, 2003
                                                                 Adapted from NMC Laboratory Handbook on Bovine Mastitits
   Biplates using blood agar with 1% esculin (BA) and MacConkey agar (MC). MacConkey agar is used to
   determine the presence of Escherichia coli or Klegsiella (A,B). Streptococci is differentiated using esculin
   hydrolysis and placing esculine-negative streptococci on a CAMP test. Streptococcus agalactiae (C) will be
   esculin-negative and positive. All other streptococcci can be listed as Streptococcus spp (D). The hemolysis
   patterns around the colonies identify Staphylococcus aureus (E) from other staphylococcci (F).


      MC




                     A    B                                                                 E    F
                                                       C         D



      BA

                    Coliforms                       Streptococci                        Staphylococci

  Coliforms
                                                           E. coli                        Klebsiella sp




           A                                 A                                   B
           BA: large greyish colonies      MC: pink zone around colonies         MC: pink center “fish eye”

 Streptococci
                     Strep ag                          Strep sp



                                                                                                      Strep ag

                                                                                      Strep sp
                                    C                                  D

              beta-hemolytic colonies               esculin positive                     CAMP Test
Staphylococci
                     Staph aureus                                                          Staph sp



                                                         +
                                                             -

              E                                                                                            F
                                                                                             Staph sp
                   hemolysis                       Coagulase Test
                                       Table 1. Major Mastitis Pathogen Isolation
Milk samples must be taken using aseptic technique and placed on ice or refrigerated immediately. Contaminated
samples or mishandling of the sample can lead to misdiagnosis and confusion.
Sample consideration:
 Individual:       Composite samples used to screen herd for infected cows.
                   Quarter samples when evaluating individual or clinical cows.
 Bulk Tank:        Used to screen or monitor herd for contagious mastitis (Strep ag, Staph aureus, Mycoplasma)

 Sampling:       Clean teats (predip and dry) before sampling.
                 Clean teat end with 70% alcohol using cotton balls, pledgets or wipes.
                 Teats must be dry before collection sample. Use a tight sealing vial or tube.
                 Discard first milk and collect (midstream -45o angle) into a sterile vial or tube (5 to 10 ml).
                 Store sample on ice or refrigerate until plating. (Freeze, if greater than 48 hours).
Plating Selective Agar:
                 Plate milk sample as soon after collecting as possible. Fresh is best.
                 Single plate media (such as blood agar plates) can be quartered for a cow or half for composites.
                 Selective agar biplate (Blood agar w/1%esculin, MacConkey agar): plate clinical quarter per plate.
                 Read selective media first:
                   • MacConkey agar selective for coliforms
                   • Esculin in agar to identify streptococcal organisms
                   • Blood agar will grow all organisms
                 Gram Stain and other tests are helpful in identifying oraganisms.

                        Bacterial growth on biplates selective for the identification of mastitis pathogens
                                                               Selective Media-Biplates

     Organisms          Test             Blood Agar                                          MacConkey Agar

     E. coli             KOH +           growth                                              growth
                         Gram -          large grey colonies                                 pink colonies/dry
                                         mostly non-hemolytic                                pink zone ppt in agar
     Klebsiella sp       KOH +           growth                                              growth
                         Gram -          large mucoid colonies                               pink-yellow colonies
                                                                                              mucoid
                                                                                              “fish eye” appearance
     Strep               Gram +          growth                                              no growth
      agalactiae         H2O2 -          small translucent colonies, esculin negative
                         CAMP +          beta-hemolytic (clear zone)

     Strep sp            Gram +          growth                                              no growth
                                         small translucent colonies
                                         esculin positive, (black colonies)
       except;
       (dysagalactiae) CAMP -            esculine negative
     Staph aureus      Gram +            growth                                              no growth
                       H2O2 +            large opaque colonies
                       Coagulase +       (creamy-golden-yellow)
                                         double zone hemolysis
                                         (complete & partial)
     Staph sp            Gram +          growth                                              no growth
                         H2O2 +          opaque colonies
                         Coagulase -     white to yellow color
                                         non-hemolytic or complete
Other Mastitis Pathogens (less common)


                   Pseudomonas spp.
                   Pseudomonas sp are environmental pathogens.
                   Associated with contaminated water or teat dip.

                   Gram-negative rods

                   MacConkey agar:translucent, shiny colonies




                    A. pyogenes
                    “Summer mastitis”result of wounds, injuries
                    and forms incurable abscess in the udder.

                    Grows slow on blood agar (similar to C. bovis)
                    Hemolytic on blood agar
                    Gram-positive rods (pleomorphic, clubshape)




                     Pasteurella spp.
                     Uncommon pathogen, from respiratory tract

                     Colonies are grey, very mucoid and confluent
                     Gram-negative rods, bipolar staining
                     Do not grow on MacConkey agar




                                                          Sears, 2003
Yeast
Contaminated or excessive use of
intramammary treatment infusions

Dry yeast (top) White and dry-smooth or
wrinkled

Moist yeast (middle) White to creamy
color mucoid or waxy.

Gram-postive: ovid or elliptical usually
exibit budding. Two to three times the
size of staphylococci. (bottom)




Serratia spp.
Environmental and water contamination

Yellow to grey colonies on blood agar,
but can develop a red pigment when
incubated at room temp (top)

Gram-negative rod

MacConkey agar: translucent colonies,
often with red pigment (bottom)




                                   Sears, 2003
                              Mastitis Pathogen Identification
                                                                    Mastitis
                                                                     cow



                                                                                                                           CMT Score
   Clinical mastitis Score                                                                                             N    No gelling
1 abnormal-milk                                                                                                        T   Slight gelling
                                                  Clinical                              Subclinical
2 Abnormal-milk, udder                                             Physical                                            1   Mild gel
3 Abnormal-milk, udder, cow                                         Exam                                               2   Moderate gel
                                                                                                                       3   Thick gel


                 TKT agar                                                                                                  MacConkey agar
              TKT agar selects                                                                                          Coliform selective agar
              for Streptococcial                                                                                              Lactose rxn:
                                                                     Culture
                    growth                                                                                                 Coliforms produce
               Esculin rxn : (+)                                                                                        lactose +, pink colonies
                 CAMP Testg

                                                                                                                   Lact (+)                      Lact (-)

   CAMP (+)
                               CAMP (-)
                                                                                                   Klebsiella sp                E. coli              Other Gram-neg
                                                                  Blood Agar
                                                                                                                                                       Lactose negative
                                                                                                    Lactose positive        Lactose positive
                                                                 All bacteria grow                                                                    Pseudomonas spp
                                                                                     Gram neg       Pink- "fish eye"        Pink-dry colonies
                                                                  on blood agar                                                                          Proteus spp
                                                                                                    mucoid colonies           Bile salt ppt -
                                                                                                                                                         Serratia spp
                                                                                                                           cloudy pink in agar
                                                                                                                                                        Pasturella spp
                                                                     Gram pos
  Strep ag                  Strep sp.
                                                                Gram pos. cocci
Gram-pos cocci            Gram-pos cocci
  CAMP (+)                    CAMP (-)                               H202 rxn                                          Staphylococci
                                                   H2o2 (-)                                     H202 (+)
  Esculin (-)           Strep dysagalactiae
                                                                                                                       Coagulase Test
                             esculin (-)                      Strep - negative
                       Strep sp. - esculine (+)               Staph - positive
                                                                                                                 Coag (+)             Coag (-)


                                                                                                      Staph aureus                          Staph sp
                                                                                                     Gram-pos cocci                       Gram-pos cocci
                                                                                                   Coagulase- positive                    Coagulase-neg
                                                                                                  double zone hemolysis                 sometime hemoylitic




 Proceedures:
 1. Physical Exam - Clinical severity or CMT Score
 2 Culture milk sample on:
               Tri-plates (Blood agar/TKF agar/MacConkey agar)
               Bi-plate (Blood agar+1% esculin/MacConkey agar)
 3. Identify mastitis pathogens:
    Step 1: MacConkey agar: Identify all coliforms and other Gram-negative organisms
    Step 2: TKT agar: Identify streptococci and differentiate using esculin rxn and CAMP Test
    Step 3: Blood agar: Grow all pathogens, but identify staphylococci using other agar elimination.
               Use Gram Stain and hydrogen peroxide to differentiate from coliforms and streptococci
               Use coagulase test to differentiate Staph aureus from other Staph sp
                                                                                                                                                            Sears, 2003
                      Culture Media Sources
Hardy Diagnostic Headquarters                       Troy Biological/
1430 West McCoy lane, Santa Maria CA 9345           1238 Rankin
Phone: 805-346-2760, 800-266-2222                   Troy, MI 48083
www.Hardy Diagnostic. Com                           800-521-0445 Fax 248-585-2490
   Esculin SBA plates - cat no A189                 email:support@troybio.com
   Mycoplasma plates - cat no. G109                 www.troybio.com



University of Minnesota Veterinary Medical Center
                                                    VWR International
1365 Gortner Avenue
                                                    1451 Raddant Rd.
St. Paul MN 55108
                                                    Batavia, IL 60510
612-625-1919
                                                    800-932-5000
    Bi-plates (blood agar/MacConkey agar
                                                       Bi-plates; BB4321290
    Tri-plate (Blood agar/TKT/MacConkey agar



Veterinary Medicine Teaching and Research
University of California - Davis
18830 RD. 112
Tulare, CA 93274
559-688-1731
www.umbms.usdavis.edu
    Mycoplasma plates - Item 1559
    Mycoplasma broth - Item 1558




                                  Incubator
 NASCO.Fort Atkinson
 901 Janesville Ave
 Fort Atkinson, WI 53538
 414-563-2446, Fax 414-563-8296
 www.nascofa.com
    Incubator, SAo1623N - $255.00

				
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