Characterizing the persistence of inactivated Mycoplasma hyopneumoniae DNA detection in the respiratory tract of pigs

umnswinenews

2 years ago

The detection of Mycoplasma hyopneumoniae in clinical samples is usually determined by PCR targeting bacterial DNA. However, a positive M. hyopneumoniae PCR result may eventually represent the detection of non-viable bacteria, complicating the diagnostic interpretation. The objective of this study was to evaluate the PCR detection of non-viable M. hyopneumoniae and its residual cell-free DNA in live pigs.

MATERIALS AND METHODS

A lung homogenate containing 1×105.5CCU/mL of M. hyopneumoniae strain 232 and Friis medium was administered to pigs, either in an active or an inactivated form.
The active inoculum, containing viable bacteria, was maintained at -80ºC and prepared just minutes prior to inoculation. The inactive inoculum, containing non-viable M. hyopneumoniae, was inactivated via autoclaving at 121ºC and 15 psi for 40 minutes.
Sixteen, three-week-old pigs were intratracheally inoculated with 10 mL of either the active or inactive inoculum (n=8/experimental group) and ante-mortem and post-mortem samples were collected at various times post-inoculation (Figure 1).
All samples were tested via a commercial M. hyopneumoniae species-specific real-time PCR assay (VetMAX™, Thermo Fisher Scientific)

**Figure 1.** Experimental design and sample collection scheme. Eight pigs were inoculated with inactivated *Mycoplasma hyopneumoniae*, while eight pigs were inoculated with the viable bacterium. Two pigs were humanely euthanized at every time point (one pig per experimental group).

RESULTS

In pigs that received the active inoculum, M. hyopneumoniae was detected by PCR starting at 2 dpi in tracheal secretions and BALF, and at 3 dpi in bronchial secretions, tracheal scrapings and thoracic lymph nodes (Figures 2-3).

Detection of M. hyopneumoniae DNA by PCR was not obtained at any timepoint post-inoculation in pigs receiving the inactivated inoculum (Figures 2-3).

**Figure 2.** *Ante-mortem* detection of *Mycoplasma hyopneumoniae* by PCR in tracheal secretion samples. Each row represents an individual pig. A dash represents target not detected, while numbers represent Ct values. Gray shaded cells represent samples not collected (pigs euthanized at a previous timepoint).

**Figure 3.** *Post-mortem* detection of *Mycoplasma hyopneumoniae* by PCR in various sample types. Results are expressed as Ct values. Gray dots: Active inoculum. Black dots: Inactivated inoculum.

CONCLUSIONS AND DISCUSSION

Results obtained in this study indicated that, in pigs with non-compromised mucociliary apparatuses and immune systems, the DNA of non-viable M. hyopneumoniae was not detected.
In healthy pigs, a positive PCR result for M. hyopneumoniae may not be affected by the residual presence of genetic material from of non-viable cells, suggesting true colonization at the time of sampling.
Coinfections with other pathogens and certain environmental conditions that could influence the clearance of genetic material from the respiratory tract were not evaluated in this study and thus, further research in field conditions is warranted.

Read the open-access short report here: https://doi.org/10.1186/s13567-024-01273-2

MATERIALS AND METHODS

RESULTS

CONCLUSIONS AND DISCUSSION

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