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J Clin Microbiol. May 2011; 49(5): 2020–2023.
PMCID: PMC3122671
Notes

Multiple-Locus Variable-Number Tandem-Repeat Analysis Is a Suitable Tool for Differentiation of Mycoplasma hyopneumoniae Strains without Cultivation[down-pointing small open triangle]

Abstract

An assay based on multiple-locus variable-number tandem-repeat analysis allowed differentiating and studying diversity and persistence of Mycoplasma hyopneumoniae strains in pig herds without prior cultivation. The test had a discriminatory index of >0.99 and was applied reliably to porcine bronchoalveolar lavage fluid and tracheal swabs.

TEXT

Mycoplasma hyopneumoniae is the primary agent involved in porcine enzootic pneumonia (EP), a common respiratory disease in pigs characterized by a dry, nonproductive cough and decreased performance. Large differences have been observed between M. hyopneumoniae field strains in terms of virulence (12), proteome (1), and genome (2). Thus, molecular typing methods able to differentiate strains are valuable both for epidemiological studies and experimental trials. Currently, available methods are relatively expensive and labor-intensive, as they require either cultivation of this fastidious organism or sequencing (6, 7, 11). Using MLVA (multiple-locus VNTR [variable-number tandem-repeat] analysis), we aimed to develop a quick and relatively inexpensive assay able to differentiate strains in samples from the respiratory tracts of pigs without prior cultivation.

MLVA assay.

For genotyping of M. hyopneumoniae strains, four loci containing a VNTR were selected (Table 1). The loci were sequenced in 10 field isolates and two reference strains as previously described (11) and were submitted to GenBank under accession no. JF461497 to JF461536. For the MLVA assay, the loci were amplified in a touchdown multiplex reaction with a Mastercycler ep gradient S (Eppendorf, Hamburg, Germany) in a final volume of 20 μl (Table 1). Reaction mixtures contained 1× PCR buffer (20 mM Tris-HCl [pH 8.4], 50 mM KCl), 3 mM MgCl2, 0.2 mM each deoxynucleotide triphosphate, 0.75 U of Platinum Taq DNA polymerase (Invitrogen, Merelbeke, Belgium), 0.1 μM each primer (Table 2) (PET- and NED-labeled primers were from Applied Biosystems, Halle, Belgium; all other primers were from Operon, Huntsville, AL), and 2 μl of template DNA. The cycling conditions were 10 cycles of 30 s at 94°C, 30 s at 63°C (annealing temperature), and 1 min 15 s at 69°C, in which the annealing temperature was incrementally decreased by 1°C per cycle, followed by 40 cycles of 30 s at 94°C, 30 s at 53°C, and 1 min 15 s at 69°C and a final extension step of 5 s at 69°C. Amplicons were kept at 4°C for a maximum of 48 h until further analysis. Two microliters of each sample was mixed with 12 μl of Hi-Di formamide (Applied Biosystems) and 0.7 μl of GeneScan 1200 LIZ size standard (Applied Biosystems). Samples were denatured at 95°C for 5 min and cooled on ice, and electrophoresis was applied on an ABI 3100 genetic analyzer (Applied Biosystems) at 50,000 V for 20 min at 60°C. The peak table containing the length of the fragments obtained with GeneMapper software version 4.0 (Applied Biosystems) was imported into Bionumerics version 4.6 (Applied Maths, Sint-Martens-Latem, Belgium), and a dendrogram was constructed according to the Ward algorithm and by using multistate categorical coefficients with a tolerance level of 1.5 and enabling fuzzy logic. As an estimate for the discriminatory power, the Simpson's index of diversity was calculated according to Hunter and Gaston (5). Multidimensional scaling was used to render a three-dimensional image based on the similarity matrix.

Table 1.
Names of the studied repeats and repeat consensus (amino acid compositions) of the repeat-containing genes, the gene name based on strain USA 232, and oligonucleotide primers used in this study
Table 2.
Average scores for pneumonia lesions and numbers of M. hyopneumoniae-positive pigs per herd and per time point as determined by MLVA on bronchoalveolar lavage fluid (BALF) and/or tracheal swabs

Strains and samples.

A collection of 42 M. hyopneumoniae field isolates (Fig. 1) described previously (10) was included in this study as well as two reference strains, J (ATCC 25934) and USA 232 (8). Furthermore, lungs were collected at six points in time (at least 5 weeks apart) (Table 2) from three Belgian pig herds infected with M. hyopneumoniae and not vaccinated against M. hyopneumoniae. In each herd, one batch of pigs was sampled four times (10, 15, 20, and 26 weeks of age), and two more batches of pigs were sampled once at 26 weeks of age. The lungs were scored for pneumonia lesions (4). Lung tissue (preferably healthy tissue adjacent to a lesion) from the left lung was used for isolation of M. hyopneumoniae (3). Briefly, lung tissue was homogenized and incubated in modified Friis medium. Obtained isolates were then filter cloned on agar plates with Friis medium. A multiplex PCR was performed on the isolates to identify them as M. hyopneumoniae (9). Bronchoalveolar lavage fluid (BALF) and tracheal swabs were obtained from the right lung as previously described (14). Prior to analysis with the MLVA assay, DNA was extracted from all samples (isolates, 200 μl of BALF and tracheal swabs suspended in 200 μl phosphate-buffered saline [PBS]) by using the DNeasy blood and tissue kit (Qiagen) by following the manufacturer's protocol.

Fig. 1.
Dendrogram of a collection of isolates based on the analysis of four variable-number tandem repeats. The dendrogram was constructed according to the Ward algorithm and by using multistate categorical coefficients with a tolerance level of 1.5 and enabling ...

Validation of the MLVA assay.

The detection limit was determined with a series of DNA dilutions in H2O or BALF (negative for M. hyopneumoniae) from a highly virulent strain (F7.2c) (12). The assay was able to detect 4 organisms/μl reaction mixture in H2O and 100 organisms in BALF. The specificity of the assay was examined using a collection of DNA of mycoplasmas and other organisms commonly present in swine (Mycoplasma hyorhinis [ATCC 23234], Mycoplasma flocculare [ATCC 27399], Mycoplasma hyosynoviae [ATCC 25591], Acholeplasma granularum, Pasteurella multocida, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Streptococcus suis, Arcanobacterium pyogenes, Erysipelothrix rhusiopathiae, Staphylococcus aureus, Escherichia coli, Salmonella enterica serovar Typhimurium, Proteus vulgaris, Haemophilus parasuis, Staphylococcus hyicus) as well as porcine and human DNA samples. No cross-reaction was observed. The stability of the assay was determined by analysis of 16 in vitro passages of strain F7.2c. The lengths of the loci remained stable. To detect inhibiting factors, BALF, tracheal, and nasal swabs of animals experimentally infected with strain F7.2c (12) were used. Briefly, an internal control pUC18 plasmid was added containing p97 primer binding sites and a spacer fragment selected from the pBluescript II SK+ plasmid of 166 bp long. M. hyopneumoniae DNA was detected in both BALF and tracheal swabs but only sporadically in nasal swabs. The internal control was visible in BALF and tracheal swabs but could be detected in only a few nasal swabs. The accuracy of the assay was determined by comparing the lengths of the loci obtained with MLVA with the calculated lengths from the available sequences. There was a good correlation between both lengths.

Typing of isolates.

The collection of 42 M. hyopneumoniae field isolates has been typed previously with randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), PCR-restriction fragment length polymorphism (RFLP) of the p146 gene, VNTR analysis of p97 (11), and pulsed-field gel electrophoresis (PFGE) (10) and now also with MLVA (Fig. 1). The discriminatory index for MLVA was >0.99, comparable to or higher than the discriminatory index of the other typing methods. In the different dendrograms of these isolates, only branching at high similarity (>70% for MLVA typing) was the same for all techniques; branching at lower similarities was different. It is therefore difficult to establish a phylogenetic relationship between strains. Two other typing methods not requiring cultivation have been described for M. hyopneumoniae: molecular typing of the p146 gene (sequencing and length of VNTR) (7) and multilocus sequence typing (MLST) (6). The first technique investigates a limited genomic region. A limited change in this region is expected to have a major impact on clustering analysis. MLST is the most robust technique for identifying phylogenetic relationships in highly diverse organisms (6). However, it is an expensive and labor-intensive technique, making it unsuitable for high-throughput screening. Furthermore, it does not allow analysis of samples containing several strains. To test the applicability of the MLVA assay to this type of sample, a dilution series of M. hyopneumoniae strains F1.12 and F13.7 (12) was made. The concentration of F1.12 was kept constant at 100 ng/μl, while the concentration of F13.7 was gradually decreased. Peaks corresponding to both strains were observed in the electropherogram to a concentration of 10 ng/μl for F13.7. Moreover, the proportion of the height of the peaks (F13.7/F1.12) was linear to the proportion of the concentrations (F13.7/F1.12), with R2 values higher than 0.95 for all loci.

Diversity of M. hyopneumoniae strains in three Belgian pig herds.

Analysis of all data obtained in the three herds revealed differences in diversity and persistence of strains between herds. In herd 1, M. hyopneumoniae DNA was detected in the samples at all time points. Clustering analysis (Fig. 2) suggests that one strain with limited clonality persisted in this herd. In herd 2, one strain was detected at the second time point, and three strains were detected from the third time point onward (Fig. 2). Gilts were purchased for this herd more frequently than for the other two herds. New strains might be introduced with these gilts. In half of the sampled pigs in this herd, there was evidence of simultaneous infection with two strains. For some pigs, the strain identified in BALF or tracheal swabs was different from the strain isolated from the lung. For other pigs, double peaks were noticed for several loci in the electropherogram of BALF and/or tracheal swabs. Each of the peaks corresponded with one of the strains present in this herd. Lung scores of these pigs were not statistically different from the scores in other pigs. It should, however, be noted that lung scores were higher from the third time point on. Though in this case the virulence of the strains is unknown, we suspect that simultaneous or subsequent infections with more than one strain might result in more severe lung lesions. As shown by Villarreal et al. (13), pigs inoculated with two different M. hyopneumoniae strains at a 4-week interval developed more severe lung lesions than pigs inoculated with only one strain. The strains identified in herd 3 displayed a peculiar pattern of diversity and persistence. Two strains present at the first three time points could not be distinguished from strains present in herd 2 (Fig. 2). These strains were not observed at the next time point, and the pigs showed very few lesions. A different strain and more lung lesions were observed at the fifth and sixth time points. It seems that some M. hyopneumoniae strains did not persist in this herd. The reason for this finding is not clear. No all-in–all-out system was practiced, and no antimicrobials active against M. hyopneumoniae were used during the trial.

Fig. 2.
Three-dimensional representation created with multidimensional scaling of M. hyopneumoniae DNA detected in three pig herds. In herd 1 (gray), one strain with limited clonality was detected. A higher diversity is seen in herd 2 (black) and herd 3 (white), ...

In conclusion, the MLVA typing developed in the present study is a reliable test for quick and relatively inexpensive differentiation of M. hyopneumoniae strains without prior cultivation or isolation. It has revealed differences in diversity and persistence of M. hyopneumoniae strains between the investigated herds and has also given strong evidence that pigs can be infected with multiple strains of M. hyopneumoniae.

Nucleotide sequence accession numbers.

The VNTR locus sequences determined in the study are available under GenBank accession no. JF461497 to JF461536.

Acknowledgments

The study was financially supported by IWT project number 050642.

We thank Hanne Vereecke and Marleen Foubert for all the help and effort dedicated to the project. The pig producers are acknowledged for their collaboration with this study.

Footnotes

[down-pointing small open triangle]Published ahead of print on 9 March 2011.

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