Mycoplasma hyopneumoniae, the etiologic agent of porcine enzootic pneumonia, continues to be one of the main bacterial respiratory pathogens in modern porcine production. Strategies to diagnose, control and eliminate infections are being widely implemented in the field and thus, a constant scrutiny and refinement of the practices put in place is recommended.

Different diagnostic approaches have been described for M. hyopneumoniae, and they can be applied depending on the information required at a given moment. Most commonly, veterinarians may seek to determine the presence or absence of the pathogen in a clinical sample, the degree of infection of an exposed pig or the duration of bacterial shedding, especially at the late stages of the disease. For all these scenarios, and because of the intrinsic difficulty of M. hyopneumoniae to be cultivated in the laboratory, the main practical tool that can be relied on for detection is PCR, which targets and amplifies DNA present in a sample, and can give a relative approximation of bacterial load.

In general, diagnostic techniques based on PCR are widely used in animal disease diagnostics, mainly due to their high sensitivity and specificity and their cost-benefit advantage over other tests. Accordingly, research has focused on maximizing the accuracy of M. hyopneumoniae PCR testing, for example by comparing the performance of different sample types for antemortem sampling (i.e., laryngeal secretions vs. deep tracheal secretions)¹ or by investigating the effect that sample pooling has on sensitivity and diagnostic costs². Nevertheless, current PCR testing for M. hyopneumoniae can be challenging to interpret, as PCR detection in clinical samples is based on targeting bacterial DNA and current assays do not differentiate between viable and non-viable bacteria.

Moreover, DNA molecules are extremely stable in the environment and resistant to degradation even in extreme conditions. Examples of the resistance of DNA molecules can be observed in archeological discoveries involving ancient DNA in buried bones for thousands of years, or the use of DNA in forensic investigations in long-term archived samples in police departments. Thus, pathogen DNA can be present in a sample long after it has been inactivated and consequently, there is no direct relationship between viability of bacteria (and by extension, potential infectivity) and their detection by PCR.

Although the inability to differentiate between viable and non-viable M. hyopneumoniae might not be a problem in some scenarios, such as when detection is coupled with the presence of the typical clinical signs in acute phases of the disease. Knowing the viability of the detected pathogen is essential when evaluating and monitoring the efficacy of control measures, such as antibiotic treatments or eradication programs.

For this reason, our research group has worked on developing a novel PCR assay³ for the detection of viable M. hyopneumoniae by taking advantage of the fantastic properties that PCR assays have in terms of sensitivity and specificity³, while circumventing their main pitfall, the inability to determine viability. The process of development and validation of this new viability PCR followed the guidelines suggested by the Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians.

The results from that meticulous validation process showed that the newly developed viability PCR is highly sensitive³. It is also highly specific³, as it does not detect the genetic material of swine pathogens other than M. hyopneumoniae, and is highly repeatable³. Additionally, detection of viable cells was obtained from various tested clinical specimen matrices from pigs experimentally infected with M. hyopneumoniae, while detection was negative in non-infected pigs³.

Once the validation process was completed, side-by-side comparisons between the new viability PCR and the current PCR assays were performed to evaluate the detection of viable M. hyopneumoniae or DNA, respectively, using inactivated bacterial cultures. While M. hyopneumoniae DNA detection by the current PCR assay was constant irrespective of the viability status, viable M. hyopneumoniae was only detected for a very short period, immediately after inactivation³. Thus, the newly developed assay was able to differentiate between viable (or very recently inactivated) and non-viable M. hyopneumoniae cells³.

In summary, a culture-independent method to assess M. hyopneumoniae viability, both in vitro and in clinical specimens has been developed and validated. While it is not yet available for routine diagnostic purposes, this quantitative, rapid and cost-efficient assay can help porcine practitioners and diagnosticians better understand the clearance of M. hyopneumoniae from the respiratory tract. It can also help elucidate the bacterial detection post-treatment and shedding dynamics in the chronic phase of infection, particularly at the end of elimination programs. The newly developed PCR test will shed light in the challenging interpretation of Mycoplasma diagnostics and will support disease control and eradication efforts.

Acknowledgements
This research work has been supported by Zoetis.

References

Source: Albert Canturri and Maria Peters, who are solely responsible for the information provided, and wholly owns the information. Informa Business Media and all its subsidiaries are not responsible for any of the content contained in this information asset.