Mycoplasma hyopneumoniae genetic variability within a swine operation.

The objective of our study was to characterize the Mycoplasma hyopneumoniae genetic diversity within a swine operation comingling weaned pigs. Bronchial swabs and tracheal aspirates were collected from 3 nursery-to-finish farms. During the finishing production stages, samples were obtained from mortalities and from live coughing pigs in rooms where mortality was not observed. A total of 105 samples were examined by a M. hyopneumoniae real-time polymerase chain reaction and subjected to genetic typing using a multilocus variable number tandem repeat analysis (MLVA) assay. The MLVA was used to identify genetic variants based on the number of repeats in 2 variable number tandem repeats loci, namely P97 and P146, thought to mediate adherence of M. hyopneumoniae to swine cilia. Four distinguishable M. hyopneumoniae variants were identified: MVLA variants 9-15, 11-21, 9-21, and 7-15. Variant 9-15 was the most prevalent, observed in 79% of rooms, and detected on all 3 farms. Variant 11-21 was present in 37% of the rooms on 2 of the 3 farms. Only one 9-21 variant was identified in 1 farm, and all samples of variant 7-15 were recovered from another farm. Based on the low prevalence and limited geographic distribution of the last 2 variants, it is hypothesized that they might be the result of in-situ recombination. All variants detected in this investigation appeared to belong to 3 clusters. Overall, a limited number of variants and clusters were identified in a system that comingles pigs from different sources, suggesting limited M. hyopneumoniae genetic variation within commercial swine production environments.

Genotyping of Mycoplasma hyorhinis using multiple-locus variable number tandem repeat analysis.

Mycoplasma hyorhinis (M. hyorhinis) has re-emerged as an important swine pathogen in recent years causing significant economic losses in post weaning pigs. Genetic variability of M. hyorhinis has been described based on different molecular methods that have limited resolution and reproducibility. The present study was undertaken to develop a molecular epidemiological typing tool for M. hyorhinis based on multiple loci of variable number of tandem repeats in its genome, termed MLVA. The typing method was designed on the basis of the number of repeats in two hypothetical proteins, MHR_0152 and MHR_0298. A total of 205 samples were analyzed, including field isolates, clinical specimens, and a reference strain. Analysis of the combination of the 2 loci revealed 16 MLVA types in 165 of the 205 samples. In the remaining forty samples only one locus could be amplified. The most frequent types obtained from the set of samples were 8-4 (36.9%), 8-3 (11.5%), 7-4 (11.5%), 9-4 (10.9%) and 10-4 (9.3%). The Simpson's diversity index for the assay was D=0.814 when the 165 samples were taken into account. No clustering was observed based on the geographical location, sample type, or year of isolation or sampling. The MLVA assay developed in this investigation showed to be a reproducible and portable assay which could be easily performed and transferred to other laboratories. The use of this technique will assist in epidemiological investigations and can be used to improve the understanding the molecular biology of M. hyorhinis variants.

Genotype distribution of Mycoplasma hyopneumoniae in swine herds from different geographical regions.

Genetic heterogeneity of Mycoplasma hyopneumoniae in pigs has been reported, however there has been limited reproducibility on the molecular methods employed so far. The aim of this study was to modify and standardize a high-resolution multiple locus variable number tandem repeat analysis (MLVA), to investigate the genetic variability of M. hyopneumoniae circulating in the United States of America (USA), Brazil, Mexico and Spain. The MLVA was standardized on the basis of the number of tandem repeats in two Mycoplasma adhesins, P97 and P146, which are proteins involved in the adherence of the pathogen to cilia. A total of 355 samples obtained from the four countries were analyzed. The Simpson's diversity index for the assay was D=0.976 when samples from all countries were combined. A large number of MLVA types (n=139) were identified, suggesting that multiple M. hyopneumoniae variants are circulating in swine. The locus P97 had 17 different types with 2-18 repeats. The P146 locus showed higher heterogeneity, with 34 different types, ranging from 7 to 48 repeats. MLVA types that presented more than 30 repeats in P146 were found in Spain and Brazil, while shorter repeats were observed in the USA and Mexico. This simplified MLVA method proved to be an efficient tool for typing M. hyopneumoniae with a high degree of stability, repeatability, and discriminatory power. In conclusion, M. hyopneumoniae showed a high variable number tandem repeat heterogeneity and this assay can be applied in molecular epidemiology investigations within farms and productions systems.