PCR as a Diagnostic Tool for Mastitis: Weighing the Options

PCR can be a useful diagnostic aid when combined with other currently available tools and herd data.
PCR as a Diagnostic Tool for Mastitis: Weighing the Options - Articles

With advances in research and improvements in the interpretation of assay results, PCR and other molecular biology techniques are likely to gain a more prominent place in mastitis diagnostics in the future. Producers should weigh their options and expectations along with consideration of the means by which the results will be utilized when determining whether to opt for PCR as a diagnostic tool for mastitis on their farm.

Molecular biology techniques have staked a claim in the realm of diagnostic testing related to milk quality and herd health issues. Polymerase chain reaction (PCR) -based assays continue to gain a share in the marketplace with the availability of commercial kits. Some testing services have added this diagnostic tool to their repertoire of resources. This technique relies on deoxyribonucleic acid (DNA) sequences found within target microorganisms to help with their identification. Very short pieces of DNA with specific sequences called primers that are unique to that organism are targeted. If these sequences are found within the sample, then the fragment is replicated many times through repetition of temperature change cycles resulting in an exponential increase in copies of the target DNA sequence. A positive result occurs if the target is detected. Real-time PCR (qPCR) assays allow rapid interpretation of results as a signal with increasing intensity is produced, correlating to a rapid increase in the number of copies of the specific target DNA sequence. As dairy producers consider utilizing PCR and other molecular diagnostic tools, there are some aspects that they should weigh in making their decision.

Advantages

  • PCR-based assays can be viewed as more expedient than some of the lengthy protocols that are used to identify and confirm organisms via culture-based methods. The more rapid availability of results could allow for swifter treatment of infections or yield savings in refraining from administering treatment unnecessarily.
  • A high throughput is possible as a large number of samples can be processed at one time, which may be a consideration when conducting a herd survey.
  • With advances in technology, the testing process using PCR has become more automated.
  • This method can facilitate the pooling of samples, which may be more cost effective when testing larger herds for contagious mastitis pathogens such as Mycoplasma bovis, Staphylococcus aureus, and Streptococcus agalactiae (Murai et al., 2014).
  • Specific microorganisms can be targeted as long as the primer for that target is included in the assay and the assay has been validated. In one study directly comparing a PCR kit to culture results, a number of clinical samples were positive using PCR but negative via culture. This was interpreted as an exhibition of greater sensitivity using the PCR assay (Koskinen et al., 2010).
  • Similarly, the detection of mixed culture positives through multiplex PCR assays, which include primers for multiple microorganisms, is possible.
  • Some testing services will collect samples to be used with PCR assays as part of their routine sampling requiring no additional effort on the part of the producer beyond that associated with their regular testing program.

Limitations

  • Non-viable bacteria can be detected and amplified as reported in a recent study of Streptococcus agalactiae using real-time PCR (de Carvalho et al., 2015). Non-viable bacteria may be present in the sample, but not a cause of infection.
  • Along the same lines, clinical relevance of positives should be considered. A recent study by Hiitiö et al. (2015) demonstrated that a real-time PCR assay using a commercially available kit was useful in identifying Staphylococcus aureus as a causative agent, but additional diagnostic information was needed in identifying other Staphylococcal organisms.
  • Additional studies are needed to aid in the interpretation of results of positive mixed cultures (Keane et al., 2013). Treatment options to deal with mixed culture positive results also warrant further research. Prudent and judicious use of antibiotics is at the forefront requiring a logical process for prioritizing treatment, which may require the consideration of multiple sources of information.
  • The assay will only provide results for the specific target organisms. Some cases of mastitis may be caused by less common agents that may not be part of a routine PCR assay.
  • Sampling techniques have raised some concern of possible carry over from previous samples as demonstrated by a recent study that employed qPCR to diagnose infections caused by Streptococcus agalactiae (Mahmmod et al., 2014). Additional research to determine the cycling threshold (Ct) values used to determine a positive result is warranted. This is key for minimizing false positives that may lead to unnecessary treatment and service costs.
  • Conventional culture is viewed as providing additional information about the microorganism that can be considered in determining treatment.

PCR can be a useful diagnostic aid when combined with other currently available tools and herd data. With advances in research and improvements in the interpretation of assay results, PCR and other molecular biology techniques are likely to gain a more prominent place in mastitis diagnostics in the future. Producers should weigh their options and expectations along with consideration of the means by which the results will be utilized when determining whether to opt for PCR as a diagnostic tool for mastitis on their farm. Producers should consult their regional animal diagnostic laboratory or their county extension educator on the choice of test for identification of mastitis-causing organisms.

References

  • de Carvalho, N. L., J. L. Gonçalves, B. G. Botaro, L. F. de Prada e Silva, and M. V. dos Santos. 2015. Detection and enumeration of Streptococcus agalactiae from bovine milk samples by real-time polymerase chain reaction. Curr. Microbiol. 71:363-372.
  • Hiitiö, H., R. Riva, T. Autio, T. Pohjanvirta, J. Holopainen, S. Pyörälä, and S. Pelkonen. 2015. Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture. J. Dairy Res. 82:200-208.
  • Keane, O. M., K. E. Budd, J. Flynn, and F. McCoy. 2013. Increased detection of mastitis pathogens by real-time PCR compared to bacterial culture. Vet. Rec. Sept. 21, 2013.
  • Koskinen, M. T., G. J. Wellenberg, O. C. Sampimon, J. Holopainen, A. Rothkamp, L. Salmikivi, L., W. A. van Haeringen, T. J. Lam,. J. and S. Pyörälä. 2010. Field comparison of real-time polymerase chain reaction and bacterial culture for identification of bovine mastitis bacteria. J. Dairy Sci. 93:5707-5715.
  • Muhmmod, Y. S., M. M. Mweu, S. S. Nielsen, J. Katholm, and I. C. Klaas. 2014. Effect of carryover and presampling procedures on the results of real-time PCR used for diagnosis of bovine intramammary infections with Streptococcus agalactiae at routine milk recordings. Prev. Vet. Med. 113:512-521.
  • Murai, K., T. W. Lehenbauer, J. D. Champagne, K. Glenn, and S. S. Aly. 2014. Cost-effectiveness of diagnostic strategies using quantitative real-time PCR and bacterial culture to identify contagious mastitis cases in large dairy herds. Prev. Vet. Med. 113:522-535.

Authors

Diagnostic Microbiology Veterinary Public Health Food Safety Epidemiology New and Emerging Infectious Diseases

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