Genetic diversity of M. hyopneumoniae isolates of abattoir pigs

Mycoplasma hyopneumoniae is present in the majority of swine herds around the world (Kobisch and Friis, 1996). It is the primary agent involved in porcine enzootic pneumonia (EP). This condition is associated with respiratory disease and reduced productivity in pigs causing severe economic losses to the swine industry. The importance of M. hyopneumoniae is also linked to its ability to increase the severity of infections caused by viruses (Opriessnig et al., 2004), as well as bacteria (Marois et al., 2009; Palzer et al., 2008). When these pathogens are in co-infection with M. hyopneumoniae, the severity of the respiratory lesions is increased. Moreover, M. hyopneumoniae can enhance the quantity and the persistence of PCV2 antigens and can increase the incidence of postweaning multisystemic wasting syndrome (PMWS) in swine (Opriessnig et al., 2004; Thacker et al., 2001).

Isolation of M. hyopneumoniae is known to be fastidious due to the long incubation period needed for its culture (Friis, 1975; Marois et al., 2007) and to the frequent co-isolation of Mycoplasma hyorhinis, a normal flora inhabitant of the upper respiratory tract of young pigs (Kobisch and Friis, 1996). M. hyorhinis has also been involved in a variety of diseases in swine including enzootic pneumonia and respiratory disease in general (Kawashima et al., 1996; Kobisch and Friis, 1996; Lin et al., 2006).

At the genomic level, high heterogeneity has been demonstrated between M. hyopneumoniae isolates throughout the world using various typing techniques such as random amplified polymorphic DNA (RAPD) (Artiushin and Minion, 1996), amplified fragment length polymorphism (AFLP) (Kokotovic et al., 1999) and pulsed-field gel electrophoresis (PFGE) (Stakenborg et al., 2005). However, the RAPD technique and the analysis of polyserine repeats have weak reproducibility rates among different laboratories, and the AFLP and PFGE techniques are considered fastidious. Thus, new techniques based on DNA amplification have been developed in the last few years. The multiple loci variable number of tandem repeats (VNTR) analysis (MLVA) and the PCR combined with restricted fragments length polymorphism (PCR-RFLP) are two methods that can be easily performed, are reproducible and have a high discriminatory power (Marois-Créhan et al., 2012; Stakenborg et al., 2006; Vranckx et al., 2011). Recently, a MLVA assay was described as a tool to differentiate M. hyopneumoniae strains in samples from the respiratory tract without prior cultivation (Vranckx et al., 2011). Previous studies have shown genetic heterogeneity between isolates from different farms (Mayor et al., 2007; Nathues et al., 2011; Stakenborg et al., 2005). However, other reports have shown both genetic heterogeneity and homogeneity between isolates from the same herds (Maes et al., 2008; Marois-Créhan et al., 2012). Field isolates of M. hyopneumoniae have also shown virulence variability (Vicca et al., 2003).

Actually, little is known about M. hyopneumoniae isolates found in Canada. The aim of this study was to evaluate the genetic diversity of M. hyopneumoniae isolated from single or mixed infections from abattoir pigs. A total of 160 swine lungs with lesions suggestive of enzootic pneumonia originating from 48 different farms were recovered from two slaughterhouses and submitted for gross pathology. The pneumonic lesion scores ranged from 2% to 84%. Eighty nine percent of the lungs (143/160) were positive for M. hyopneumoniae by realtime PCR whereas 10% (16/160) and 8.8% (14/160) were positive by PCR for M. hyorhinis and M. flocculare, respectively. By culture, only 6% of the samples were positive for M. hyopneumoniae (10/160). The M. hyopneumoniae isolate and mixed cultures of M. hyorhinis and M. hyopneumoniae showed relatively low minimal inhibitory concentrations against the antibiotics tested (Table 1). No resistance genes or point mutations were found in isolates with slightly higher MICs. This is likely indicative of an absence of acquired antibiotic resistance.

Among the selected M. hyopneumoniae-positive lungs (n = 25), 9 lungs were co-infected with M. hyorhinis, 9 lungs with PCV2, 2 lungs with PRRSV, 12 lungs with S. suis and 10 lungs with P. multocida (Table 2). MLVA and PCRRFLP clustering of M. hyopneumoniae revealed that analyzed strains were distributed among three and five clusters respectively, regardless of severity of lesions, indicating that no cluster is associated with virulence. However, strains missing a specific MLVA locus showed significantly less severe lesions and lower numbers of bacteria. MLVA and PCR-RFLP analyses also showed a high diversity among field isolates of M. hyopneumoniae with a greater homogeneity within the same herd. Almost half of the field isolates presented less than 55% homology with selected vaccine and reference strains. 

This work was supported partly by grants from the "Fédération des Producteurs de Porcs du Québec" (FPPQ), the "Centre de Recherche en Infectiologie Porcine" (CRIP), Zoetis Animal Health and from the Natural Sciences and Engineering Research Council of Canada (M. Archambault, RGPIN-191461). The authors are grateful to Guy Beauchamp for his expertise in statistics. 

Table 1. MICs of M. hyorhinis and M. hyopneumoniae cultures

Table 2. Severity of lesions, quantification of M. hyopneumoniae in lungs with lesions suggestive of EP with or without other pathogens in abattoir pigs