By Karine Ludwig Takeuti, College of Veterinary Medicine, University of Minnesota; David E. S. N. de Barcellos, College of Veterinary Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil  and Maria Pieters, College of Veterinary Medicine, University of Minnesota

Mycoplasma hyopneumoniae is one of the most important pathogens affecting the respiratory tract of pigs. This bacterium causes Enzootic pneumonia, a chronic condition that is clinically evident in grow-finishers, even though pigs in all phases of production may be colonized with M. hyopneumoniae. At the barn level, Enzootic pneumonia is characterized by a dry cough and growth retardation, subsequently, at the industry level, these infections lead to significant economic losses, especially due to respiratory co-infections with other bacterial and viral pathogens, decreased performance, and increased costs associated with disease control.

Diagnosis of M. hyopneumoniae infections can be attempted using several methods. For example, bacterial isolation, identification of a serological response against the agent and detection of the bacterium’s genetic material. However, the bacterium is extremely difficult to grow in laboratory conditions, requires a specialized medium and grows very slowly, and for those reasons, growing M. hyopneumoniae in vitro is not usually considered an option for simple diagnostic purposes.  Antibody detection, although very common, brings various limitations for interpretation. Current ELISA assays used to detect M. hyopneumoniae antibodies cannot differentiate the response due to natural exposure from the response due to vaccination. In addition, time to seroconversion after exposure can be unusually long.  On the other hand, detection of M. hyopneumoniae genetic material by PCR exhibits a fast turnaround and high accuracy.  Moreover, M. hyopneumoniae infections can be detected in live animals even weeks before the onset of clinical signs and before the development of specific antibodies. Mycoplasma hyopneumoniae detection by PCR can provide information on the infection dynamics and can be performed using samples collected from different body sites along the respiratory tract of the pig, including nasal cavities, tonsils, larynx, trachea, and bronchia, among others. And most commonly, sterile swabs are used to collect these samples.

Several types of swabs can be used to collect clinical samples from pigs. Rayon-bud swabs have been extensively used and reported in the literature to detect M. hyopneumoniae. However, nylon-flocked seem to be minimally used, or reported, for detection of this pathogen, thus its performance to detect M. hyopneumoniae is unknown. Rayon-bud swabs consist of a small wad of rayon wrapped around the end of a rod (Figure 1). The nylon-flocked swabs are made of short nylon fibers working as a brush (Figure 1). Several studies have shown a high diagnostic sensitivity for respiratory epithelial cells or respiratory pathogens detection in humans, such as influenza virus, respiratory syncytial virus and adenovirus. Thus, it can be speculated that nylon-flocked swabs could increase M. hyopneumoniae detection by real-time PCR.

Fig. 1. Swab tips. Top: Rayon-bud. Bottom: Nylon-flocked.

In a study conducted in the Mycoplasma Research Laboratory at the University of Minnesota, we tested the absorption of PBS and lubricant (used as a surrogate for respiratory secretions) using nylon-flocked and rayon-bud swabs. Also, a comparison of M. hyopneumoniae detection by real-time PCR using the same types of swabs was performed. Statistical analysis was performed to compare absorption and Ct values between nylon-flocked and rayon-bud swabs.

No differences in the percent of positive samples detected by each swab type were observed. However, the absorption of PBS and lubricant, and M. hyopneumoniae load detection were significant higher (p<0.05) in nylon-flocked than in rayon-bud swabs, even though mean Ct differences were only 1.1 (Table 1). The results of this study suggest that nylon-flocked swabs are a good alternative for M. hyopneumoniae detection when costs are comparable. Moreover, nylon-flocked swabs could be used to improve M. hyopneumoniae detection when bacterial load is lower, such as in chronic infections. In general, our results suggest that the material of the device that is used for sample collection can significantly influence M. hyopneumoniae detection by PCR.

Table 1. Mean Ct value (and difference) for Mycoplasma hyopneumoniae detection by real-time PCR by type of swab. Different lower case letters denote statistically significant differences between the two swab types (p<0.05).

References:

Calsamiglia, M., Pijoan, C., Bosch, G.J., 1999. Profiling Mycoplasma hyopneumoniae in farms using serology and a nested PCR technique. Swine Health Prod. 7, 263-268.

Daley, P., Castriciano, S., Chernesky, M., Smieja, M., 2006. Comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients. J. Clin. Microbiol. 44, 2265-2267.

Esposito, S., Molteni, C.G., Daleno, C., Valzano, A., Cesati, L., Gualtieri, L., Tagliabue, C., Bosis, S., Principi, N., 2010. Comparison of nasopharyngeal nylon flocked swabs with universal transport medium and rayon-bud swabs with a sponge reservoir of viral transport medium in the diagnosis of paediatric influenza J. Med. Microbiol. 59, 96-99.

Flabet, C., Marois, C., Kobisch, M., Madec, F., Rose, N., 2010. Estimation of the sensitivity of four sampling methods for Mycoplasma hyopneumoniae detection in live pigs using a Bayesian approach. Vet. Microbiol. 143, 238-245.

Friis, N.F., 1975. Some recommendation concerning primary isolation of Mycoplasma suipneumoniae and Mycoplasma flocculare. Nord Vet. Med. 27, 337-339.

Goodwin, R.F.W., Pomeroy, A.P., Whittlestone, P., 1965. Production of enzootic pneumonia in pigs with a mycoplasma. Vet. Rec. 77, 1247–1249.

Hernes, S.S., Quarsten, H., Hagen, E., Lyngroth, A.L., Pripp, A.H., Bjorvatn, B., Bakke, P.S., 2011. Swabbing for respiratory viral infections in older patients: a comparison of rayon and nylon flocked swabs. Eur. J. Microbiol. Infect. Dis. 30, 159-165.

Kurth, K.T., Hsu, T., Snook, E.R., Thacker, E.L., Thacker, B.J., Minion, F.C., 2002. Use of a Mycoplasma hyopneumoniae nested polymerase chain reaction test to determine the optimal sampling sites in swine. J. Vet. Diagn. Invest. 14, 463-469.

Mare, C.J., Switzer, W.P., 1965. New Species: Mycoplasma hyopneumoniae; a causative agent of virus pig pneumonia. Vet. Med. Small Anim. Clin. 60, 841–846.

Pieters, M., Pijoan, C., Fano, E., Dee, S., 2009. An assessment of the duration of Mycoplasma hyopneumoniae infection in an experimentally infected population of pigs. Vet. Microbiol. 134, 261-266.

Pieters, M., Daniels, J., Rovira, A., 2017. Comparison of sample types and diagnostic methods for in vivo detection of Mycoplasma hyopneumoniae during early stages of infection. Vet. Microbiol. 203, 103-109.

Roos, L. R., Fano, E., Homwong, N., Payne, B., Pieters, M., 2016. A model to investigate the optimal seeder-to-naïve ratio for successful natural Mycoplasma hyopneumoniae gilt exposure prior to entering the breeding herd. Vet. Microbiol. 184, 51-58.

Sibila, M., Pieters, M., Molitor, T., Maes, D., Haesebrouck, F., Segalés, J., 2009. Current perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection. Vet. J. 181, 221-231.

Thacker, E.L., Minion, F.C., 2012. Mycoplasmosis, in: Zimmermann, J.J., Karriker, L.A., Ramirez, A., Schwartz, K.J., Stevenson, G.W. (Eds.), Diseases of Swine. Tenth ed. Wiley-Blackwell, Ames, pp.779-797.