Udder Health Systems

Coagulase Tube Test

Purpose

The presence of coagulase enzyme in Staphylococcus organisms is considered a key pathogenic feature for the diagnosis of Staphylococcus aureus. Testing for the presence of this enzyme in isolated Staphylococcus colonies is a standard method for differentiating the pathogenic Staph. aureus strain from other pathogenic and non pathogenic staphylococcal strains.

Description

Animal strains of Staph. aureus from cows and dogs will frequently produce a large partial hemolytic zone on blood agar culture indicating its ability to produce Beta toxin. In Bovine Mastitis microbiology, the presence of this “Beta Zone” is considered diagnostic for Staph. aureus. In addition to these Beta toxin producing strains, cultures from Staph. aureus mastitis cows will also show that not all Staph aureus strains are Beta producing strains. Some Staph. aureus strains only produce an alpha hemolysin that causes a small clear hemolytic zone on blood agar and some are non hemolytic. Coagulase testing is the recommended method of identifying these non-Beta producing strains. An isolated colony of coagulase producing Staph. aureus may produce the gelling reaction in as little as 4 hours. The coagulase enzyme will cause the partial or complete gelling of blood plasma. In the picture above the top tube shows that the plasma is still liquid, indicating a negative result. The bottom tube shows a positive reaction. The plasma in the bottom tube is coagulated and remains adhered to the bottom of the tube when it is tilted.

 

The Udder Health Systems coagulase plasma is shipped in the liquid or frozen state and is ready to use. It is typically dispensed in approximately 1 ml aliquots to perform the test. Unlike lyophilized rabbit plasma, UHS coagulase plasma does not need to be reconstituted and is typically lower in test cost than rabbit plasma. It is good for several years in the frozen state and remains effective for at least 12 weeks at refrigerated temperatures.

 

Refer to table on back for expected test responses.
Udder Health Systems

Coagulase Test

Culture Response

Organism	Growth	Reaction
Strep. agalactiae	Excellent
Staph. aureus *	Excellent	Coagulation
Mycoplasma	Not Applicable
Strep. dysgalactiae	Excellent
Strep. uberis	Excellent
E- strep	Excellent
Staph. species *	Excellent
E. Coli †	Excellent	False Positive
Klebsiella pneumoniae	Excellent
Klebsiella species	Excellent
Pseudomonas species	Excellent
Pseudomonas aeruginosa	Excellent
Pasteurella	Excellent
Proteus	Excellent
Serratia	Excellent
Bacillus	Excellent
Yeast	Excellent
Mold	Excellent
Nocardia	Excellent
Prototheca	Excellent
Arcanobacterium pyogenes	Excellent
C. bovis	Excellent

        All of these organisms will grow in the coagulase broth, this growth will produce a cloudy appearance. This is not to be interpreted as a positive reaction, only the coagulation reaction is a positive result. The coagulation can be detected in as little as 4 hrs. Typically the reaction is still readable in 24 hrs. Some false negatives will occur when coagulation occurs but the clot will lyse again by 24 hrs.

        * All Staph species will grow in the coagulase broth, but only Staph. aureus will generate a positive coagulation reaction. The coagulation can be detected in as little as 4 hrs. Typically the reaction is still readable in 24 hrs. Some false negatives will occur when coagulation occurs but the clot will lyse again by 24 hrs.

      ^† Some strains of E. coli. can produce the coagulase reaction. A false positive result can occur if the analyst mistakenly tests an E. coli colony. It is the responsibility of the analyst to only perform this test only on Staphylococcus genus isolates.  

 

 

 

 

 

Udder Health Systems

Mycoplasma Agar

Purpose

 This is the standard isolation agar used at Udder Health Systems Laboratory for cultivating mycoplasma organisms from individual cow and bulk tank milk samples.

Description

This light to medium amber agar is prepared from a purified agar base with the addition of PPLO broth. This agar has been formulated with inhibitors to discourage a variety of non-target organisms.  The agar is especially formulated to inhibit slow growing contaminating organisms that show up in 10 day incubation.   The agar has been tested against University of California Davis mycoplasma agar, with side by side comparisons of field isolates.  The agar demonstrated an equivalent or greater recovery of mycoplasma.

Refer to table on back for expected culture responses.

Udder Health Systems

Mycoplasma Agar

Culture Response

Organism	Growth	Reaction
Strep. agalactiae	Inhibited
Staph. aureus	Inhibited
Mycoplasma	Excellent	Fried-Egg colonies
Strep. dysgalactiae	Inhibited
Strep. uberis	Inhibited
E- strep	Inhibited
Staph. species	Inhibited
E. Coli	Inhibited
Klebsiella pneumoniae	Inhibited
Klebsiella species	Inhibited
Pseudomonas species	Inhibited
Pseudomonas aeruginosa	Inhibited
Pasteurella	Inhibited
Proteus	Inhibited
Serratia	Inhibited
Bacillus	Inhibited
Yeast	Inhibited
Mold	Inhibited
Nocardia	Inhibited
Prototheca	Inhibited
Arcanobacterium pyogenes	Inhibited
C. bovis	Inhibited

 

 

 

 

 

 

Udder Health Systems

MP2 Agar

Purpose

 This is a selective isolation agar used at Udder Health Systems Laboratory for cultivating gram positive mastitis organisms from individual cow milk samples.

Description

This cherry red, agar is prepared from a modified Columbia agar base with the addition of 5% washed bovine erythrocytes and full strength esculin.  This selective agar has been formulated to enhance the growth of gram-positive staphylococci, streptococci and enterococci bacteria while inhibiting the growth of all gram- negative Enterobacteriaceae and Pseudomonas organisms. The addition of esculin enhances the capability of differentiating Streptococci.  Certain Streptococci will hydrolyze esculin, causing a mild background blackening for E-strep and a mild greening or no background coloration for suspect Streptococcus agalactiae and Streptococcus dysgalactiae organisms.  All suspect Streptococcus agalactiae organisms should be moved to our special Camp media for confirmation. 

Refer to table on back for expected culture responses.

 

Udder Health Systems

MP2 Agar

Culture Response

Updated: 1/6/05

Organism	Growth	Reaction
Strep. agalactiae	Excellent	Esculin negative
Staph. aureus	Excellent	Hemolytic (beta)
Mycoplasma	Not Applicable
Strep. dysgalactiae	Excellent	Esculin variable
Strep. uberis	Excellent	Esculin positive
E- strep	Excellent	Esculin positive
Staph. species	Excellent	Hemolytic variable
E. Coli	Inhibited
Klebsiella pneumoniae	Inhibited
Klebsiella species	Inhibited
Pseudomonas species	Inhibited
Pseudomonas aeruginosa	Inhibited
Pasteurella	Fair
Proteus	Inhibited
Serratia	Inhibited
Bacillus	Ínhbited
Yeast	n/a
Mold	n/a
Nocardia	n/a
Prototheca	Inhibited
Arcanobacterium pyogenes	Excellent	24-48 hour
C. bovis	n/a

 

 

 

 

 

 

Udder Health Systems

TNT Biplates

Purpose

 This biplate is used at Udder Health Systems Laboratory for selection and identification of both gram-positive and gram-negative organisms.

Description

·        The bright red agar portion of the biplate is prepared from a modified Columbia base agar with the addition of 5% washed bovine blood. This selective agar has been formulated to enhance the growth of gram-positive staphylococci, streptococci and enterococci bacteria while inhibiting the growth of all gram-negative Enterobacteriaceae and Pseudomonas organisms. 

·        The pink agar portion of the biplate is specialized media for the detection of gram-negative organisms. Since the antimicrobial agents in this agar inhibit the growth of gram-positive bacteria, the media focuses on the organisms’ ability to ferment lactose. This fermentation causes the bacterial colony to change to a pinkish color.  Non-lactose fermenting colonies will remain colorless and opaque.

Refer to table on back for expected culture responses.

Udder Health Systems

TNT Biplate

Culture Response

Updated: 1/6/05

	GP Agar		MacConkey Agar
Organism	Growth	Reaction	Growth	Reaction
Strep. agalactiae	Growth		Inhibited
Staph. aureus	Growth	hemolysis	Inhibited
Mycoplasma	Not Applicable		Not Applicable
Strep. dysgalactiae	Growth		Inhibited
Strep. uberis	Growth		Inhibited
E- strep	Growth		Inhibited
Staph. species	Growth	Hemolytic variable	Inhibited
E. Coli	Inhibited		Excellent	Pink colonies
Klebsiella pneumoniae	Inhibited		Excellent	Pink mucoid colonies
Klebsiella species	Inhibited		Excellent	Pink mucoid colonies
Pseudomonas species	Inhibited		Excellent	Colorless colonies
Pseudomonas aeruginosa	Inhibited		Excellent	Green/colorless colonies
Pasteurella	Weak		No Growth
Proteus	Ínhibited		No Growth
Serratia	Inhibited		Excellent	Pinkish/red pigment
Bacillus	Inhibited		Inhibited
Yeast	Not Applicable		No Growth
Mold	Not Applicable		Not Applicable
Nocardia	Not Applicable		Not Applicable
Prototheca	Not Applicable		Not Applicable
A. pyogenes	Growth	Small hemolytic	Inhibited
C. bovis	Not Applicable		Not Applicable

 

 

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A BULK TANK CULTURING PROGRAM FOR

MONITORING MILK QUALITY AND UDDER HEALTH

A. M. Britten¹, and T. Emerson²

¹Udder Health Systems, Inc.

Bellingham, Washington

²Darigold Farms

Seattle, Washington

A bulk tank culturing service has been available to producers of Darigold Farms in Washington, Oregon, Idaho, and northern California since 1986². The goal of  the culture is to identify the specific types of bacteria that make up the total raw bacteria count. It is a voluntary program and has grown in popularity over the years. At this point in time over 600 of the 1100 producers in the cooperative use this service each year.

Sample Procedure

The culture test is performed on a well mixed sample of bulk tank milk from the herd. Typically this sample is taken by the milk hauler and placed is a specially marked 4 oz. sample cup at the time of milk pick up. On some dairies the sample is pulled on a sporadic basis at the request of the producer. A large portion of the producers have arranged with the field personnel and the hauler for samples to be automatically taken each month. The samples are frozen and transported to the testing laboratory usually within 48 hrs.

Testing method

In the laboratory the sample is thawed and plated on four different solid media. Inoculums of .01 mls. of milk are plated onto a whole plate of washed cow blood agar, and  a portion of mycoplasma agar and a selective strep. agar plates. Also inoculums of .1 mls. are plated onto portions of selective staph. agar plates. The non-selective washed cow blood agar is used for general  identification and counting of a wide variety of bacteria groups. Bacteria are identified according to standard methods³.

The selective  staph agar is similar in composition to the washed cow blood agar but has 7.5% sodium chloride added. Hemolytic staph colonies from this agar that are positive to the coagulase test are reported as Staph aureus. The selective strep agar is a modified Edwards⁴ media with added esculin, and Staph aureus beta hemolysin. Catalase negative, esculin negative colonies with CAMP like hemolytic zones around them are reported as Strep. ag. The selectivity of these media and the larger inoculum size allow us to attain  greatly improved detection efficiency over the whole blood agar alone¹. The mycoplasma agar is used as a screening test on all bulk tanks  for organisms that will grow on this media. A separate fluorescent antibody test is recommended for confirmation of all mycoplasma like colonies grown on this media.

Results

A report is produced and mailed to the producer and the field personnel which shows the identification and total count of the various bacteria found in his bulk tank sample. With the aid of the fieldman and veterinary consultants, the producer can use the report as an aid in analyzing various milk quality and udder health problems in the herd. High somatic cell count tank milk  by definition comes from herds with significant mastitis problems. The bulk tank culture can frequently reveal the likely pathogenic causes for these  high cell counts. High bacteria counts in bulk tank milk may result from mastitic milk or from contamination, cleaning or cooling problems. On many occasions identifying the organism causing the high count may help guide the producer to more specific and appropriate correction efforts.  When a major mastitis pathogen such as Strep ag. or Staph. aureus is causing a high bacteria count, we know disease control measures and abnormal milk diversion is the way to solve the problem. Conversely if organisms in the pseudomonas or bacillus group are too numerous then mastitis is probably not the culprit and attention should be turned toward environmental sanitation.

A special effort has been made to optimize the detection for the major mastitis pathogens Streptococcus agalactiae, Staphylococcus aureus, and Mycoplasma sp. Staph. aureus infection in herds is common and some producers just hope to keep the bulk tank counts for this pathogen low. Many progressive producers strive to keep the bulk tank counts for all three of these mastitis pathogens at zero. The table below shows pathogen detection statistics for a twelve month period ending in 1995.

Table 1. Pathogen Detection from Bulk Tank Culture Program

	Herds Positive	% Herds Positive
Strep ag.	31	5
Staph aureus	479	81
Mycoplasma sp.	34	6
Total herds tested:	589

References

1.  Britten, A.M., 1996. Use of a selective agar for improving streptococcus agalactiae detection. Proc. 35 Annual Meeting of the National Mastitis Council.

2.  Emerson, T., 1989. Bulk milk bacterial culturing - an aid to quality milk production. Proc. 28 Annual Meeting of the National Mastitis Council, pp 49-53.

3.  National Mastitis Council. Laboratory and Field Handbook on Bovine Mastitis. Atkinson, WI: W.D. Hoard and Sons Co.

4.  Schalm, O.W., Carrol, E.J., Jain, N.C., 1971. Bovine Mastitis. Philadelphia, PA: Lea & Febiger. pp 171-173.

 

 

 

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USE OF A SELECTIVE AGAR FOR IMPROVING

STREPTOCOCCUS AGALACTIAE DETECTION

A. M. Britten

Udder Health Systems, Inc.

Bellingham, Washington

Streptococcus agalactiae is widely recognized as a very dangerous mastitis pathogen. Dairy managers rely on sensitive tests to detect this organism in prevention (bulk tank monitoring) and intervention (test and treat) programs. In bulk tank culture, because the sample is always a mixed culture, there is concern that the small Strep. ag. colonies may be missed because of competing bacteria. The common practice in herd culture procedures is to collect a single composite sample of the four quarters from each cow.  This also leads to a situation where one is likely to obtain a mixed culture. Add to this the possibility of organisms from the skin contaminating the culture and again we have the concern that the Strep. ag. colonies may be missed. The use of selective streptococcus agar such as Edward’s² media has been advocated as a means of aiding the diagnosis of Strep. ag.  Two investigations are reported here evaluating the hypothesis that using a selective strep agar for detection of Strep ag. on bulk tank samples and on individual cow composite cultures would, improve detection over conventional blood agar.

Materials and Methods

The two media used for comparison are a washed cow blood agar plate with added esculin (BA) and a selective strep agar plate (E). The E agar is a modified Edward’s media with added esculin, and Staph aureus beta hemolysin. The 5074 bulk tank samples used in the study were submitted to the laboratory from 589 herds whose tanks were sampled from 1 to 57 times during a one year period. The 2120 individual cow composite samples tested in the study came from three herds with known Strep ag. histories.

For the bulk tank study, an inoculum of .01 mls. from a pipetter was placed onto a whole plate of BA, and onto half an E agar plate. The plates were incubated and read at 40-48 hours. For the individual cow composite samples a 3 mm platinum loop was used to streak the specimen onto a quarter section of the BA and E plates. These plates were incubated and read in 24 hours. The milk samples were incubated for 4-6 hours. For those specimens that had no growth at 24 hours, the plate was re-streaked with the incubated milk.

Streptococcus agalactiae identification was made according to standard methods¹. From the BA plate non-esculin strep. candidates were selected for testing for CAMP reaction on a separate CAMP plate. From the E plate, catalase negative, esculin negative colonies with large hemolytic zones around them were selected for re-testing for CAMP reaction on a separate plate.  The presence of at least a single CAMP positive colony resulted in that sample being reported as Strep. ag. positive.

Results

Of the 5074 bulk tank samples tested, 94 tanks tested positive for Strep. ag.when the E plate was used to detect the pathogen, versus only 43 tanks testing positive on the BA plate. (Table 1.). A similar increase in detection rate was seen when this data was analyzed by herd. Of the 589 herds tested, 31 herds tested positive for Strep. ag. on the E plates, versus only 15 on the BA plates (Table 2.). For the tank samples tested, the E plate always detected the Strep. ag. samples that were detected by the BA plate. An even more dramatic difference is seen on the composite cow samples tested (Table 3.). Of the 2120 composite samples tested, 220 tested positive for Strep. ag. on the E plates, versus only 49 on BA plates. On these samples the E plate detected all but three of the samples testing positive on the BA plate. Although the degree of difference for composite samples was strongly influenced by one herd, the difference was significant in all three herds. Although the incubation procedure increased the positive detection rates for both the E and BA plates it was only significant for the E plate.

Table 1. Test results for detection of Streptococcus agalactiae comparing a strep selective agar E versus washed cow blood agar BA from 5074 bulk tank samples.

	E+	E-	Totals
BA+	43	0	43
BA-	51	4980	5031
Totals	94	4980

Table 2. Test results for detection of Streptococcus agalactiae comparing a strep selective agar E versus washed cow blood agar BA from samples shown in Table. 1. when analyzed by herd.

	E+	E-	Totals
BA+	15	0	15
BA-	16	558	574
Totals	31	558

Table 3. Test results for detection of Streptococcus agalactiae comparing a strep selective agar E versus washed cow blood agar BA from 2120 cow composite samples from three  problem herds.

	E+	E-	Totals
BA+	46	3	49
BA-	174	1897	2071
Totals	220	1900

References

1.  National Mastitis Council. Laboratory and Field Handbook on Bovine Mastitis. Atkinson, WI: W.D. Hoard and Sons Co.

2.  Schalm, O.W., Carrol, E.J., Jain, N.C., 1971. Bovine Mastitis. Philadelphia, PA: Lea & Febiger. pp 171-173.

 

 

 

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Udder Health Systems Technical Report

Inulin Agar Evaluation Study

This study was conducted to test the Udder Health Systems Inulin Agar for its suitability in identification of Streptococcus uberis organisms from mastitis isolates. Inulin agar is a special formulation that contains two principal systems that are required to make this media useful for identification of Strep uberis and differentiating this species from other e-strep mastitis isolates in cow milk samples. There is an enrichment nutrient system in the agar that allows for the growth requirements of these streptococci. The agar uses the sugar inulin as the single source of carbohydrate. When organisms that ferment inulin are grown on the agar, there is an indicator system which shows a distinct yellowing of the agar to show that the organism has metabolized this sugar. Strep uberis is one of the streptococci that will ferment inulin. It is suggested by Udder Health Systems that this agar be used in a multi-step secondary screening procedure to identify this pathogen from cow milk samples. The test for inulin fermentation capability should only be done on esculin positive isolates from cow milk in the proposed scheme. In this scheme if an esculin positive cow milk isolate is Inulin Agar test positive (ferments inulin) then it is Strep uberis.  The goal of the investigation was to estimate the sensitivity or specificity of this detection scheme. Sensitivity (the ability of the test to detect Strep uberis) was simply determined by comparing the number of isolates that were test positive to the total number of Strep uberis tested. The Specificity (the ability of the test to correctly identify non-strep uberis e-streps) was demonstrated by comparing the number of non-strep uberis organisms that were test negative to the total number of non-strep uberis tested.

The table below summarizes the data from the study in the form of a truth table.

	API RESULTS
	Strep. uberis	Non- Strep uberis
INULIN +	93	8	101
INULIN -	6	95	101
TOTAL	99	103	202
	Sensitivity	Specificity
	94%	92%

A total of 202 isolates of e-streps isolated from mastitic cow milk were included in the study. All the e- streps were speciated using the API rapid strep identification system. Of the 99 Strep uberis isolated tested, 93 of them were inulin test positive on this agar, resulting in a 94% Sensitivity. Of the 103 non-strep uberis organisms, 95 of them were test negative resulting in a 92% Specificity. In this study, the 8 organisms that were False Positive (inulin positive non-strep uberis) included 6 Strep bovis, 1 Ent faecium, 1 Aerococcus sp. The 6 False Negative organisms were by definition inulin negative Strep uberis.

Udder Health Systems Technical Report

2004 New Mycoplasma Agar Comparison Study

This study was conducted to assess the suitability of Udder Health System’s new ultra clear Mycoplasma agar in isolating mycoplasma organisms from bovine milk. The two agars in the study were the new ultra clear Mycoplasma agar plates manufactured by Udder Health Systems, Inc. Bellingham, Washington and the comparison mastitis mycoplasma agar plates manufactured by the University of California at Davis.

Mycoplasma isolation agar is a special formulation that contains two principal systems that are required to make this media useful for detection of mastitis causing mycoplasma from milk samples. There is an enrichment nutrient system, typically including serum, which allows for the special growth requirements of these fastidious, slow growing organisms. There are also inhibitor systems, which include antibiotics, to discourage non-mycoplasma growth. Typically non-target organisms may be present in individual cow milk samples and will always be present in bulk tank samples. To be successful in detecting mycoplasma organisms, these non-target species must be inhibited. If they do grow on the mycoplasma agar, they may overwhelm and obscure the mycoplasma growth and prevent their detection. 

The goal of this study was to determine if there were any differences in the sensitivity or specificity of the two agars. Sensitivity (the ability to detect the mycoplasma in the specimen) was simply determined by comparing the organism counts based on plating the same specimen on a whole plate of each agar. The Specificity of the agar (the ability to only grow mycoplasma colonies and not grow competing bacteria) was demonstrated by the number of plates showing contaminating growth on each agar.

The following pages contain the raw data from the study. The samples used had been previously identified as containing mycoplasma, and had been stored frozen for up to one year before the study was initiated. Samples were identified by Sample number and Source (cow/tank). Each sample was plated in duplicate on UHS Media agar and UC Davis Media agar. The total number of mycoplasma colonies growing on each plate is shown in the column totals for each agar. It is clear from the raw data that the UHS Media is as sensitive or more sensitive than the UC Davis media is.

We also looked at natural contaminants that grew on the two agars from these field specimens. These are shown in the columns labeled Cont. In these data an advantage is shown in the inhibitor system in the UHS media, which we believe is particularly important for laboratories using a 10-day incubation (which UHS recommends). Contaminants showed in five of the UC Davis plates but only three of the UHS plates. Contaminant inhibition is important in selective agars like mycoplasma agar. Non-mycoplasma growth may interfere with the detection of low numbers of mycoplasma that may show up between day 5 and 10 of incubation.