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Department of Dairy Science, University of Wisconsin, Madison 53706
Corresponding author: Pamela L. Ruegg; e-mail: plruegg{at}wisc.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: mastitis • culture • Petrifilm • microbiology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Negative results of bacteriological culture of mastitis cases can result from spontaneous clearance of pathogens (Eberhart et al., 1979; Smith et al., 1985), cyclical shedding of chronic Staphylococcus aureus infections (Sears et al., 1990), or the presence of few bacterial colonies in milk (Sears et al., 1990). Milk samples from infected quarters may also be negative if bacteria have been engulfed by phagocytes (Newbould and Neave, 1965; Hill et al., 1978). The occurrence of false negative results of milk samples submitted for culture can result in maintenance of infected animals within a herd and may contribute to the failure of programs designed to control contagious mastitis. It is important to balance the use of sensitive and costly laboratory procedures against the cost of other interventions. The choice to use a more rigorous diagnostic method should be determined for each herd based on the herd goals and the manager’s ability to use the resulting data.
Several methods have been used to enhance recovery of pathogens from milk samples. The use of larger inocula increased the relative sensitivity for Staph. aureus from 78 (0.01 mL) to 90% (0.1 mL) (Lam et al., 1996). Incubation of milk samples for 4 to 18 h before culture increased the recovery rate of bacterial pathogens compared with the use of standard microbiological methods (Dinsmore et al., 1992). Freezing and subsequent thawing of milk samples before inoculation increased the recovery by 2.5 and 1.5 times for Streptococcus agalactiae and Staph. aureus respectively (Villanueva et al., 1991). Centrifugation of milk samples and subsequent culture of sediment increased the recovery of Staph. aureus by 86% (Zecconi et al., 1997). The use of various combinations of these methods has also been reported (Dinsmore et al., 1992; Sol et al., 2002).
Petrifilm plates (3M, Minneapolis, MN) are sample-ready selective culture systems that are marketed for rapid bacteriological isolation and enumeration of bacteria from food products. The Petrifilm Staph Express Count plate contains chromogenic, modified Baird-Parker medium that is selective and differential for Staphylococcus spp. Confirmation of Staph. aureus is performed using a disk that contains deoxyribonuclease and a dye that reacts to produce a distinct pink zone around Staph. aureus colonies.
A series of 4 trials were performed to evaluate the use of Petriflim Staph Express Count plate used for isolation of Staph. aureus from milk samples. The objective of experiment 1 was to compare microbiological results of Petrifilm to standard and augmented culture techniques for diagnosis of mastitis caused by Staph. aureus. The objective of experiment 2 was to compare results of Petrifilm using composite and quarter milk samples. The objective of experiment 3 was to determine the specificity of Petrifilm based on different interpretative criteria. The objective of experiment 4 was to determine the repeatability of Petrifilm among several individuals that read the plates.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Thawed milk samples were vortexed and streaked onto one quarter of blood agar plates using 0.01-mL disposable plastic loops, and plates were incubated at 37°C for 24 h (standard method). A 1-mL aliquot of each sample was plated on Petrifilm Staph Express plates and incubated at 37°C for 24 h following the manufacturer’s instructions (Petrifilm method). A 5-mL aliquot of each sample was placed in a separate vial and centrifuged at 2000 x g for 15 min. The supernatant was then discarded and a 0.01-mL aliquot of the sediment was spread using a loop on a 5% blood agar plate and incubated at 37°C for 24 h (centrifugation method). The remaining aliquot of the original sample was incubated for 18 h at 37°C in the original plastic vial. When no bacterial growth was observed using the standard method, 0.01 mL of the incubated sample was plated on blood agar and incubated at 37°C for 24 h (incubation method).
All bacterial growth was identified and recorded after 24 and 48 h of incubation. For the standard, centrifugation, and incubation methods, Staph. aureus were identified by hemolytic pattern, gram-staining characteristics, positive catalase reaction, positive mannitol agar reaction, and positive tube coagulase test (National Mastitis Council, 1999). Other bacteriological results were recorded as coliforms, Streptococcus sp., CNS, Corynebacterium spp., Bacillus spp., and others as defined by the National Mastitis Council (1999).
Samples were considered contaminated if 3 or more dissimilar colony types were found in the same sample. To meet the objective of this project, cultures were considered negative when <3 colonies were seen on the plate, except for Staph. aureus, when the presence of 
1 colony was considered a positive sample.
For the Petrifilm method, Staph. aureus were initially identified following manufacturer’s instructions for the Staph Express Count plate and the Staph Express disk. The disk contains a dye and deoxyribonuclease. Staphylococcus aureus produces DNA, which reacts with the dye to form a pink zone around colonies. The disk was applied to all Staph Express plates with any evidence of bacterial growth after 24 h of incubation. Positive Petrifilm tests were recorded when a colony was associated with a distinct pink zone after 2 more hours of incubation. When the type of colony growth was diffuse and without apparent individual colonies, a 1:10 dilution was made using sterile distilled water and inoculated on Petrifilm. Results of the diluted sample were used in the analysis. When growth occurred only on Petrifilm, colonies were picked, regrown, and confirmed using standard methods. The Petrifilm results were considered false positives when the standard methods did not result in confirmation of Staph. aureus. Final identification of these colonies was performed using a commercial microbial identification system (BBL Crystal ID; Becton Dickinson, Franklin Lakes, NJ).
Experiment 2
Samples were collected from Farm A. Composite (n = 100) and quarter (n = 386) milk samples were collected by university personnel from cows (n = 100) with high SCC (>200,000 cells/mL) on the current monthly DHI test. Quarter milk samples were collected as described in experiment 1. For composite sampling, a 50-mL graduated vial was used and approximately 5 to 10 mL of milk was collected from each quarter. Samples were cooled immediately and delivered within 24 h to the laboratory. All samples were frozen and analyzed within 15 d of collection. Sample handling and microbiological techniques were the same as experiment 1 except that the centrifugation method was not used.
Experiment 3
Quarter milk samples (n = 332) were obtained from all lactating cows (n = 88) located on a commercial farm (Farm B) that used a robotic milking system. The herd was selected because of a suspected problem with subclinical mastitis caused by Staph. aureus. Cows were restrained in headlocks and milk samples were collected by university personnel using aseptic procedures as recommended by the National Mastitis Council (1999). Immediately after collection, a 1-mL aliquot of each milk sample was spread on a Petrifilm Staph Express Count plate following manufacturer’s directions. Samples were incubated at 37°C for 24 h. Staphylococcus aureus were identified following manufacturer’s instructions for the 3M Staph Express Count plate and the 3M Staph Express disk. The disk was applied to all Staph Express plates with evidence of bacterial growth after 24 h of incubation (n = 320). Petrifilm plates with evidence of zones associated with colonies (n = 127) were recorded (weak or distinct) after 2 more hours of incubation. All colonies that formed weak or distinct pink zones on Petrifilm were picked and regrown on blood agar plate. Staphylococcus aureus were identified by hemolytic pattern, gram-staining characteristics, positive catalase reaction, growth on mannitol, and positive tube coagulase test. Final identification of CNS was performed using a commercial microbial identification system (BBL Crystal ID).
Experiment 4
Composite milk samples (n = 240) were collected from all lactating cows on a commercial farm (Farm C) in Wisconsin. Milk samples were collected in the milking parlor by university personnel after premilking cow preparation and before attachment of the milking unit using aseptic procedures as recommended by the National Mastitis Council (1999). Samples were cooled immediately and delivered within 8 h to the laboratory. A 1-mL aliquot of each milk sample was spread on each of a Petrifilm Staph Express Count plate and a Petrifilm Aerobic count plate following manufacturer’s directions (3M). Petrifilm Staph Express plates were initially read after incubation at 37°C for 24 h by 3 readers with varying levels of experience. Readers A and B had some previous experience with interpretation of Petrifilm, whereas reader C had no experience with the product. Plates were read independently without consultation between readers. Readers recorded the number of colonies (0, 1 to 50, 51 to 150, >150), colony color (red-violet, black, blue-green, other), and colony size (tiny, normal, large, irregular). After the initial reading of the plates, the disk was applied to all Petrifilm Staph Express plates that contained evidence of bacterial growth (1 cfu/ mL) and plates were incubated at 37°C for a further 2 h. Plates with disks were independently read by the same 3 readers. Readers recorded the intensity of pink zones surrounding colonies (none, weak, distinct). When 2 of 3 readers agreed that colonies formed a weak or distinct pink zone on Petrifilm, the suspect colonies were picked and regrown on blood agar plate. Staphylococcus aureus were identified by hemolytic pattern, gram-staining characteristics, positive catalase reaction, growth on mannitol, and positive tube coagulase test. Final identification of CNS was performed using a commercial microbial identification system (BBL Crystal ID).
Colonies from Petrifilm Aerobic Count plates that contained >25 cfu/mL were picked and regrown on blood agar and MacConkey agar. Identification of bacteria was performed using microbiological procedures as outlined by the National Mastitis Council, (1999). The morphology and hemolytic patterns of bacterial colonies were determined and organisms were differentiated using standard microbiologic methods. Staphylococcus aureus were identified using mannitol and coagulase reactions, Streptococci were differentiated using the CAMP test, esculin reactions and agglutination, and gram-negative bacteria were identified using MacConkey agar, motility, indole and ornithine reactions, and triple sugar iron slants.
Statistical Analyses
Statistical analyses for experiments 1 and 2 were performed using the McNemar test for paired data using SAS statistical package (SAS Institute, 1999). Statistical significance was defined at P 
0.05. In experiment 1, the relative sensitivity was defined as the probability of isolating Staph. aureus using a single technique compared with the probability of isolation using the 4 microbiological techniques (gold standard). Two samples with false-positive Petrifilm results (based on standard microbiological methods) were excluded from the data to compare prevalence of Staph. aureus.
In experiment 2, the prevalence of Staph. aureus results was compared on a cow basis and quarter basis. When quarter samples were used, cows were considered positive if Staph. aureus was isolated from any quarter. The cow status for composite sampling was determined using results of the composite sampling. The gold standard was defined based on the parallel Staph. aureus isolation of the 3 methods together. Four cows with false-positive results on the Petrifilm method were excluded from the calculations of prevalence.
In experiment 3, specificity and predictive values were estimated using epidemiological software (Win Episcope 2.0; available at http://www.clive.ed.ac.uk) based on formulas derived from standard epidemiological methods (Thrushfield, 1995; Martin et al., 1987). Three records with incomplete laboratory results were excluded from statistical analysis. False positives were defined as colonies that exhibited a weak or distinct pink zone on Petrifilm (after application of the disk) but were identified as other pathogens using standard microbiological methods. The association between zone intensity and accuracy of identification of Staph. aureus was determined using 
2 analysis (Win-Episcope 2.0).
In experiment 4, specificity and the association between zone intensity and accuracy of identification of Staph. aureus was estimated as in experiment 3. The association among readers and results of Petrifilm was evaluated using 2 analysis (WinEpiscope 2.0).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). The sensitivity of the standard method was significantly lower than that of the incubation and Petrifilm methods (P < 0.05).
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). There was no significant difference in the rate of contamination among methods, and the prevalence of isolation of pathogens was highest for incubated samples. Centrifugation resulted in increased recovery of Bacillus compared with the standard method, whereas incubation increased the recovery of all microorganisms except for Staph. aureus and Corynebacterium sp.
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). The use of Petrifilm resulted in a 62.5% increase in positive Staph. aureus samples compared with the standard method (P = 0.03) (Table 2).
Experiment 2
The estimated prevalence of Staph. aureus was 86% higher for quarter samples processed using Petrifilm compared with composite samples processed using the standard method (Table 3). Although the prevalence of Staph. aureus was numerically higher for quarter samples, there was no significant difference in estimated prevalence of Staph. aureus between quarter and composite samples for samples processed using any method (Table 3). There was a tendency for both quarter samples (P = 0.06) and composite samples (P = 0.07) processed using the standard method to have lower prevalence compared with the gold standard.
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). In this high prevalence population, the occurrence of a distinct pink zone surrounding a colony was highly specific for Staph. aureus and had a specificity of 98.5% (Table 4). The use of a weak pink zone to diagnose Staph. aureus resulted in a high rate of false-positive results (22.4%). The intensity of the pink zone was strongly associated with the odds of confirming Staph. aureus (P < 0.001). Colonies exhibiting distinct pink zones were 120 times more likely to be confirmed as Staph. aureus compared with colonies that exhibited weak pink zones. A variety of staphylococci was associated with the development of weak pink zones. Staphylococcus haemolyticus (n = 16) and Staph. simulans (n = 7) were the most commonly isolated staphylococci.
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). The relative specificities were 87.2 and 96.0% for weak and distinct pink zones, respectively. The positive predictive value was less than experiment 3 because of the lower prevalence of infection with Staph. aureus. Interpretation of Petrifilm Staph Express count plates was influenced by reader (Table 6). Readers varied in their assessment of number of colonies, colony size and presence or intensity of pink zones. There was no significant association among readers and identification of colony color.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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The increased sensitivity of Petrifilm for isolation of Staph. aureus may be a result of the larger inoculum volume used with that method. In this study, the detection limit was decreased from 100 to 1 cfu/mL. The low numbers of microorganisms shed by infected mammary glands and the intermittent shedding pattern of Staph. aureus in milk supports the use of lower detection limits for Staph. aureus (Sears et al., 1990). Increased sensitivity for Staph. aureus can be useful in segregation programs because of more efficient detection of infected cows. Conversely, the risk of false-positive results is increased when lower detection limits are used. The use of aseptic sampling technique and proper handling and storage of samples are essential. Diagnostic results of multiple samples obtained from the same quarter are more reliable than diagnosis based on a single sample and could be used in situations where larger economic values are involved (such as in culling of high genetic merit animals). The significance of isolation of a single colony of Staph. aureus from a 1-mL inoculation is unknown and in some cases will not reflect infection status of the gland. It is important to have additional information (such as monthly SCC values and the individual cow history of clinical mastitis) to make informed decisions regarding the significance of isolation of such few colonies of a suspected pathogen.
No significant difference was observed between results of samples processed using centrifugation or standard methodology. This finding is in agreement with those of Zecconi et al. (1997) when comparing Staph. aureus results in 3 of the 6 herds studied. Zecconi et al. (1997) inoculated the whole sediment from a 10-mL milk sample centrifugation and in this study, only 0.01 mL from a 5-mL sample sediment was plated; therefore, detection limits were different. Although centrifugation does enhance recovery of Staph. aureus, the use of this technique is extremely labor intensive and may not be practical for many laboratories.
In this study, using our definition of contamination, preincubation increased the recovery of major mastitis pathogens without increasing the number of contaminated samples. Sol et al. (2002) also reported improvement in isolation of major mastitis pathogens when using a similar method. The relative sensitivity of Petrifilm plates was similar to the incubation method. The use of either method would result in increased isolation of Staph. aureus but the incubation method requires a longer period until diagnosis. The use of incubation did result in isolation of more minor pathogens.
The use of composite milk samples is often preferred by farmers to reduce the cost of diagnosis (Ruegg and Reinemann, 2002). The relative sensitivity of a single composite milk sample used to detect Staph. aureus has been estimated to be 63% (Lam et al., 1996). In this experiment, analyzing composite milk samples with the standard method resulted in the lowest sensitivity, and this method should be avoided in situations when false-negative results are undesirable (such as when introducing new animals to a herd or in segregation programs). Lam et al. (1996) improved the relative sensitivity of testing composite milk samples by using multiple samples and by utilizing a greater inoculum volume.
In this study, the sensitivity of Petrifilm Staph Express was equivalent to traditional techniques. Petrifilm may be preferred in situations where rapid decisions are required or when a sample-ready system is preferable (such as on-farm culture programs). In herds that have segregation programs to control Staph. aureus, evaluation of milk samples obtained from postpartum cows is often recommended. The use of Petrifilm in this situation may result in quicker diagnosis and allow better implementation of segregation plans.
The manufacturer’s interpretive criteria for use of Petrifilm Staph Express suggest that the appearance of red-violet colonies on the initial incubation is presumptive evidence for diagnosis of Staph. aureus. Results of experiment 4 do not support this recommendation for milk samples and demonstrate the necessity of using the Staph Express disk for confirmation even when red-violet colonies are the only colonies present. Of plates classified as containing red-violet colonies (n = 35), only 8 were confirmed as Staph. aureus. Therefore, assessment of results of Petrifilm Staph Express should include the use of the confirmatory disk.
Diagnosis of Staph. aureus using Petrifilm Staph Express is dependent upon the ability of individuals to observe variations in colony color after the secondary incubation. Different intensities of pink zones appear around colonies after incubation with the Staph Express disk. In this study, no attempt was made to train individuals or standardize interpretive criteria of the zones. Results from this study demonstrate that the ability to accurately and carefully read zone intensity highly influences the specificity of this test. Thus, it is important that people reading the plates have excellent visual abilities and the ability to discern colors. In cases where different individuals will be reading plates, it is important that adequate training be performed to standardize the interpretation of color associated with the confirmatory test. Training should include observation of Petrifilm Staph Express plates (with and without the confirmatory disks) that have been inoculated with milk known to contain both Staph. aureus and CNS. In circumstances where false-positive results are highly undesirable, results of positive Petrifilm tests should be confirmed using traditional laboratory methods.
The use of Petrifilm products for on-farm culture programs is appealing because of the simplicity of use compared with standard microbiological methods. Variability observed in reading the Petrifilm plates demonstrates that standardization and training of personnel reading the plates is fundamental for achieving reliable results. Periodic assessment of accuracy of on-farm methods by submission of duplicate samples to specialized microbiology laboratories is recommended to minimize errors. Under these circumstances, the use of the Petrifilm method on farms would reduce the time between obtaining the milk sampling and obtaining results, and would allow farmers to use the results in control and treatment programs in herds with mastitis suspected to be caused by Staph. aureus.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Received for publication November 24, 2004. Accepted for publication April 22, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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