JCM Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
JCM.02366-07v1
46/8/2491    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Google Scholar
Right arrow Articles by Strait, E. L.
Right arrow Articles by Thacker, E. L.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Strait, E. L.
Right arrow Articles by Thacker, E. L.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, August 2008, p. 2491-2498, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.02366-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Real-Time PCR Assays To Address Genetic Diversity among Strains of Mycoplasma hyopneumoniae{triangledown}

Erin L. Strait,1 Melissa L. Madsen,1 F. Chris Minion,1* Jane Christopher-Hennings,2 Matthew Dammen,2 Katherine R. Jones,1 and Eileen L. Thacker1

Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, Iowa,1 Veterinary Science Department, South Dakota State University, Brookings, South Dakota2

Received 10 December 2007/ Returned for modification 16 February 2008/ Accepted 26 May 2008


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mycoplasma hyopneumoniae is an important cause of pneumonia in pigs around the world, but confirming its presence in (or absence from) pigs can be difficult. Culture for diagnosis is impractical, and seroconversion is often delayed after natural infection, limiting the use of serology. Numerous PCR assays for the detection of M. hyopneumoniae have been developed, targeting several different genes. Recently, genetic diversity among strains of M. hyopneumoniae was demonstrated. The effect of this diversity on the accuracy and sensitivity of the M. hyopneumoniae PCR assays could result in false-negative results in current PCR tests. In this study, a panel of isolates of M. hyopneumoniae, M. flocculare, M. hyorhinis, and M. hyosynoviae were tested with a number of M. hyopneumoniae-specific PCR assays. Some M. hyopneumoniae PCR assays tested did not detect all isolates of M. hyopneumoniae. To increase the efficiency of PCR testing, two new real-time PCR assays that are specific and capable of detecting all of the M. hyopneumoniae isolates used in this study were developed.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia and an integral component of the porcine respiratory disease complex. Considered one of the most important sources of disease-associated losses in swine production, M. hyopneumoniae is also one of the most difficult to detect. This organism is difficult to isolate in pure culture because it is easily overgrown by other contaminating bacteria; therefore, culture is generally not attempted. In addition, seroconversion to M. hyopneumoniae is often slow within a herd and can vary considerably among pigs, making the use of enzyme-linked immunosorbent assays less effective. A number of PCR assays have been developed and reported to be both sensitive and specific for M. hyopneumoniae (4, 6, 11, 19, 20, 22, 24, 26). As a result, this technique is now among the most widely used for detection of M. hyopneumoniae in pigs.

While the development of PCR assays has greatly enhanced our ability to detect M. hyopneumoniae, the genetic variability of the organism (1, 5, 12, 14, 15, 21) can affect detection by PCR. It is not known what effect this heterogeneity has on the sensitivities of the different assays. Real-time PCR has many advantages over traditional PCR assays, including less time to obtain quantitative results and a decrease in environmental contamination. Together, these traits make real-time PCR assays preferred in diagnostic and research settings. A real-time assay that is based on a conserved target common among isolates of M. hyopneumoniae has not yet been described. The two currently published M. hyopneumoniae-specific real-time assays had to be used in combination in order to detect all M. hyopneumoniae field samples in a Swiss collection of isolates (6). In addition, many of the previously published PCR assays were validated using only a small number of mycoplasma isolates. Therefore, the objectives of this study were to measure the ability of several M. hyopneumoniae-specific PCR assays to detect a collection of isolates of M. hyopneumoniae and also to identify new targets for real-time PCR assays for M. hyopneumoniae.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacteria. For this study, four panels of commonly isolated mycoplasmas of swine were assembled, including 36 isolates of M. hyopneumoniae, 12 isolates each of Mycoplasma flocculare and Mycoplasma hyosynoviae, and 10 isolates of Mycoplasma hyorhinis. Also included were single isolates of several infrequently identified mycoplasmas and acholeplasmas of swine and of Mycoplasma bovis from cattle. Clinical samples were obtained from pigs experimentally inoculated with different isolates of M. hyopneumoniae as previously described (11, 28) and from uninfected control pigs. Samples were obtained 28 days postinfection at necropsy (11). Cultures from a number of other swine bacterial pathogens were also evaluated (Table 1). Genomic DNA was obtained from each of the samples by use of the QIAamp DNA minikit (Qiagen, Valencia, CA).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Bacteria used in this study

 
PCR assays. A list of published PCR assays was identified (Table 2). These assays were then used to test DNA from each of the isolates in Table 1. Some assays were performed as previously published (6, 11, 20), while others (4, 19, 22, 23, 26) were modified by substituting MasterAmp E buffer (Epicentre Biotechnologies, Madison, WI) for the published buffer with their published cycling parameters in reaction mixtures consisting of 1x Master Amp E, 0.4 mM each forward and reverse primer, 2.5 U of Taq (New England Biolabs, Inc., Ipswich, ME), template containing 1 to 10 ng of DNA, and a sufficient quantity of PCR-grade water to a final volume of 25 µl. The primer pairs from nested-set PCRs were run as individual assays (not as nested sets) to independently evaluate the ability of each primer pair to form products.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Summary of results for PCR assays tested against a panel of M. hyopneumoniae isolates

 
M. hyopneumoniae mhp165 real-time PCR. The primers and probe for a real-time PCR assay targeting the mhp165 gene of M. hyopneumoniae (GenBank accession no. AE017332, M. hyopneumoniae strain 232 complete genome; bp 195124 to 201267) were designed using PrimerQuest software (Integrated DNA Technologies, Iowa City, IA) (Mhp165 F, Mhp165 R, Mhp165 P) (Table 3). Three isolates of M. hyopneumoniae were sequenced in the region of the real-time product to evaluate sequence conservation in M. hyopneumoniae (Mhp165 seqF and Mhp165 seqR) (Table 3). A panel of forward and reverse primers was then designed (Mhp165 F g-a, Mhp165 R t-c, Mhp165 R t-c g-a, and Mhp165 R g-a) (Table 3) based on the sequence results (Fig. 1). The primers were then tested for their abilities to detect the isolates possessing single-nucleotide polymorphisms (SNPs). One set of forward and reverse primers (Mhp165 F and Mhp165 R) (Table 3) was selected and shown to be unique to M. hyopneumoniae by BLAST analysis. This set was further tested for specificity by use of a SYBR green assay performed as described by the manufacturer (QuantiTect SYBR green PCR kit; Qiagen) with DNA from M. hyopneumoniae, M. flocculare, M. hyorhinis, and M. hyosynoviae. All real-time assays were performed on a Rotor-Gene RG-3000 (Corbett Research, San Francisco, CA). A checkerboard assay of primers at 100 nM, 300 nM, and 900 nM versus probe concentrations of 50 nM, 150 nM, and 250 nM was used to optimize the real-time protocol by use of the universal PCR master mix (Applied Biosystems, Foster City, CA) according to the manufacturer's directions. The following cycling parameters were used: 50°C for 2 min; 95°C for 10 min; and 40 cycles of 95°C, 15 s, and then 60°C for 1 min. Template DNA from four different isolates of M. hyopneumoniae was utilized in replicates of the checkerboard assay. The detection limit of the finalized assay was tested using a dilution series (10 ng/µl to 1 fg/µl) of chromosomal DNA from M. hyopneumoniae strain 232. The assay was also compared to a nested PCR targeting the mhp165 gene that includes an inner set of primers (8) corresponding to a previously published outer set (19) (Mhp165 outerF, Mhp165 outerR; Mhp165 innerF, Mhp165 innerR) (Table 3). By use of the GeneAmp PCR core kit (Applied Biosystems), the following reaction mixture was employed for both the inner and outer assays: 1x PCR buffer II, 2.5 mM MgCl2, 100 µM each deoxynucleoside triphosphate, 0.4 mM each forward and reverse primer, 2.5 U Taq, 2 µl template (PCR product from the outer reaction is the template for the inner "nested" reaction), and a sufficient quantity of PCR-grade water to a final volume of 50 µl. The same cycling parameters were applied to both the outer and inner reaction mixtures as follows: 94°C for 3 min and 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min. Products from these reactions were analyzed on a 1.5% agarose gel.


View this table:
[in this window]
[in a new window]

  		TABLE 3. List of primer and probe sequences used in this study

 

Figure 1
View larger version (37K):
[in this window]
[in a new window]

  		FIG. 1. Sequence alignment of the mhp165 gene real-time PCR target region for isolates of M. hyopneumoniae compared to the consensus sequence from M. hyopneumoniae strain 232 (AE017332). Single underlining indicates the forward primer, dashed underlining the probe, and double underlining the reverse primer, while asterisks indicate sequence homology.

 
M. hyopneumoniae mhp183 real-time PCR. To identify conserved regions for primer and probe design, gene sequences for P97 (mhp183) from 17 different GenBank records were aligned using vector NTI Advance 10 (Invitrogen, Carlsbad, CA) (Fig. 2), and primers and probes were designed with Primer Express version 2.0.0 (Applied Biosystems) (Mhp183 F, Mhp183 R, and Mhp183 P) (Table 3). These primers and probes were shown to be unique to M. hyopneumoniae by a BLAST search. They were further tested for specificity with a SYBR green assay performed as described by the manufacturer (QuantiTect SYBR green PCR kit; Qiagen) with DNA from M. hyopneumoniae, M. flocculare, M. hyorhinis, and M. hyosynoviae. A checkerboard assay of primers versus probe concentrations was performed as described above. Template DNA from four different isolates of M. hyopneumoniae was utilized in replicates of the checkerboard assay. This assay was then tested against each of the bacterial DNA samples listed in Table 1. The detection level of the finalized assay was tested with a dilution series (10 ng/µl to 1 fg/µl) of chromosomal DNA of M. hyopneumoniae.


Figure 2
View larger version (26K):
[in this window]
[in a new window]

  		FIG. 2. Sequence alignment of the mhp183 gene real-time PCR target region for isolates of M. hyopneumoniae compared to the consensus sequence from M. hyopneumoniae strain 232 (AE017332). Single underlining indicates the forward primer, dashed underlining the probe, and double underlining the reverse primer, while asterisks indicate sequence homology.

 
mhp165 and mhp183 multiplex real-time PCR assay. A dilution series of chromosomal DNA of M. hyopneumoniae was tested in the mhp165 and mhp183 real-time PCR assays both individually and multiplexed, and with or without an exogenous internal positive control (EIPC) (Applied Biosystems) (data not shown). The EIPC was used to control for inhibition in the PCR caused by the template sample. Reagents for the EIPC were diluted twofold from the manufacturer's suggestion.

DNA sequencing and analysis. The region of the M. hyopneumoniae genome containing the PCR target of Artiushin et al. (2) and as used by Kurth et al. in the outer pair of a nested PCR (11) was sequenced from strains 232, 95MP1509, 95MP1505, 00MP1502, and 00MP1301. The location of the PCR target was identified by BLAST analysis of the M. hyopneumoniae genome sequence (16) with the sequence from the work of Kurth et al. and was found to include the 5' end of mhp023 and most of mhp024.

Nucleotide sequence accession numbers. The nucleotide sequences in the mhp023 and mhp024 chromosomal regions of strains 95MP1505, 95MP1509, 00MP1301, and 00MP1502 have been submitted to the GenBank database under accession numbers EU658728, EU658729, EU658727, and EU658726, respectively. The nucleotide sequences in the mhp165 chromosomal regions of strains 95MP1509, 96MP0001, and 06MP2501 have been submitted to the GenBank database under accession numbers EU658731, EU658732, and EU658730, respectively.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mycoplasma PCR assay panel. Assays targeting the 16S rRNA gene (4, 20, 24) and the mhp165 gene (8, 19) positively identified all of the M. hyopneumoniae isolates used in this study. However, five of the M. hyopneumoniae-specific PCR assays did not detect every M. hyopneumoniae isolate in our collection (Table 2). These five assays targeted the following three genes: a putative ABC transporter (GenBank accession no. U02537) (6, 26), a repeated element (REP; GenBank accession no. AF004388) (6, 22), and a target that spanned the hypothetical genes mhp023 and mhp024 (GenBank accession no. AE017332; base pairs 27057 to 28020) (11). With the PCR assay used by Kurth et al., some strains of M. hyopneumoniae gave products with the outer primers but failed upon use of the inner primer pair.

Real-time PCR assays. The SYBR green assay using primers for the mhp165 assay was specific only to M. hyopneumoniae, while the primers for the mhp183 assay produced a product for both M. hyopneumoniae and M. flocculare. When analyzed on a 3% agarose gel, the product for M. flocculare was smaller than the amplicon made in M. hyopneumoniae (data not shown), indicating that the targets are not identical in each species.

The most sensitive combination of primers and probes among the M. hyopneumoniae isolates tested for the mhp165 assay was 900 nM forward primer, 900 nM reverse primer, and 50 nM probe. Additionally, Mhp165 probes labeled with Cy3 or Cy5 were found to perform identically regardless of the label used (data not shown). For the mhp183 assay, the most sensitive combination was 50 nM forward primer, 900 nM reverse primer, and 50 nM probe.

Both of the probe-based real-time PCR assays were specific to M. hyopneumoniae, detected all isolates of M. hyopneumoniae in our DNA collection, and detected none of those in the M. flocculare, M. hyorhinis, and M. hyosynoviae panels. In addition, none of the other bacteria tested were positive by either of these assays (100% specificity). Clinical samples taken 28 days postinoculation from M. hyopneumoniae-infected pigs were also tested, including nasal swabs, bronchial swabs, and bronchial alveolar lavage fluid. All samples from all infected pigs were positive by both assays except for one nasal swab tested by the mhp183 assay (Table 4).


View this table:
[in this window]
[in a new window]

  		TABLE 4. Analysis of clinical samples by real-time PCR

 
DNA sequence results. DNA sequence analysis of the PCR target of Kurth et al. was performed due to the failure of the inner product of the nested PCR to detect some strains of M. hyopneumoniae. Sequences from strains 232, 95MP1509, and 95MP1505 are nearly identical (data not shown), but sequences from strains 00MP1502 and 00MP1301 show three regions with only 80 to 87% homology to strain 232 and three regions with no significant homology, including a break in the alignment of strains 00MP1502 and 00MP1301 to strain 232 at the site where the forward inner primer binds. Sequences of the inner-primer binding regions and a graphical representation of the BLAST scores are shown in Fig. 3.


Figure 3
View larger version (9K):
[in this window]
[in a new window]

  		FIG. 3. DNA sequence analysis of the inner PCR product of strains 00MP1502 and 00MP1301 from the work of Kurth et al. (A) DNA sequence alignment of strains 232, 00MP1502, and 00MP1301 in the regions of the inner PCR product primers Mhp3 and Mhp4. Identities are shown by the vertical lines. (B) Graphic representation of the BLAST alignment of the 00MP1502 and 00MP1301 strain inner PCR product sequences with that of strain 232. Gray areas indicate poor or no alignment. White regions indicate homology, with the percent identity indicated below. The region of the inner PCR product is indicated by the double-headed arrow. The outer primers bind outside the indicated region, and DNA sequence was not obtained for that part of the chromosome.

 
Sequence analysis of the 529-bp region containing the primer binding sites for mhp165 for five isolates of M. hyopneumoniae identified SNPs in the reverse and/or forward primers, while the site of probe binding was found to be homologous (Fig. 1). Real-time PCR analysis using DNA templates from isolates 96MP0001, 95MP1509, 06MP2501, and 232 was then performed using primers including each of the identified SNPs (primers for the 132-bp fragment) (Table 3). Every primer pair was able to detect each isolate tested, but variation in the sensitivities for M. hyopneumoniae isolates was observed as a change in the threshold cycle values among the different primer pairs (data not shown).

Detection level. A 10-fold dilution series from 10 ng/µl to 1 fg/µl, including two additional dilutions of 5 fg/µl and 2.5 fg/µl, was made with DNA extracted from M. hyopneumoniae strain 232. One femtogram of chromosomal DNA is considered to be approximately one genome equivalent (11). The nested-set PCR based on the mhp165 gene detected DNA from the 10-ng/µl to 5-fg/µl dilutions. The mhp165 and mhp183 real-time PCR assays each detected DNA from the 10-ng/µl to 2.5-fg/µl dilutions (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Five PCR assays targeting three different genes of M. hyopneumoniae failed to detect all of the M. hyopneumoniae isolates in our collection. The two published real-time assays targeting the ABC and REP genes did not detect every isolate, as had been previously reported (6). Additionally, the standard PCR assays on which these two real-time assays were based failed to detect the same isolates of M. hyopneumoniae in nearly every case (22, 26). Contrary to the findings of Dubosson et al. (6), who used the REP and ABC real-time assays in combination to detect all isolates in their study, there was one isolate (96MP0001 [Table 1]) in the current study that was not detected by either of these PCR assays.

The PCR assay of Kurth et al., a nested assay targeting a unique hypothetical gene of M. hyopneumoniae (11), also failed to detect all isolates. The basis for this failure was genetic variability among the isolates resulting in deletions of and rearrangements in the mhp024 gene. One of the PCR primer binding sites was missing, causing failure in the inner-pair reaction. This demonstrates the importance of evaluating each primer pair individually across multiple isolates in nested-set PCR assays.

Based on these PCR results, we developed two new real-time PCR assays. The nested PCR assay with the mhp165 gene performed well against our panel of isolates, and therefore this gene was selected as a target for one of the new real-time assays. The function of the mhp165 gene has not been identified, but it is a large gene (6,140 bp) containing zinc finger, metallopeptidase, coiled-coiled, transmembrane, secretory signal, and ABC transporter sequence motifs. This information is available in the M. hyopneumoniae database (http://mycoplasma.genome.uab.edu/).

A second real-time PCR assay developed in this study targets the gene for the P97 cilium adhesin (mhp183). The product of this well-characterized gene is important for the adherence of M. hyopneumoniae to ciliated epithelium within the respiratory tract (9, 10, 27). Because it is thought to be necessary for virulence, this gene is likely to be present in all pathogenic isolates of M. hyopneumoniae. Additionally, this gene has been sequenced from a large number of isolates, allowing identification of conserved regions. While a small target was produced with the M. flocculare template using the forward and reverse primer in a SYBR green assay, this product did not hybridize to our M. hyopneumoniae-specific probe. When M. flocculare DNA was added to a dilution series of M. hyopneumoniae DNA, no effect on sensitivity to the real-time assay was found (data not shown).

Due to the restricted parameters used in primer and probe design programs for real-time PCR assays, there is often little choice in the area of the gene that can be targeted. This is particularly true for M. hyopneumoniae, due to its low G+C content (28.6%) (16). Therefore, even though SNPs were identified in the primer binding sites for mhp165, this area was unable to be avoided because of probe design issues; however, the internal probe was conserved between these isolates. In addition to the SNPs identified in the previously sequenced strains 232, J, and 7228, various SNPs were found to be contained in the primer binding regions upon sequencing of three isolates from the M. hyopneumoniae isolate collection. More-extensive sequencing of isolates was not performed due to the fact that all isolates in our panel were detected by this assay. The observed SNPs did have an effect on the sensitivity of the mhp165 assay. This could potentially affect the diagnostic ability of the mhp165 assay in the field. Additionally, this assay would not be as useful for quantitative comparisons of samples from different isolates as the mhp183 assay, the target of which appears to be conserved at the primer and probe binding sites.

Both the mhp165 and mhp183 assays detected M. hyopneumoniae DNA in clinical samples collected from groups of pigs experimentally inoculated with M. hyopneumoniae isolates. One nasal swab sample was found to be negative by the mhp183 assay from a pig challenged with isolate 00MP1301. PCR performed on samples from nasal swabs has previously been shown to have a detection rate lower than that performed on bronchial swab or bronchial alveolar lavage fluid samples (11). The nasal swab sample from the other pig challenged with isolate 00MP1301 and the bronchial swab and bronchial alveolar lavage fluid samples from both pigs were all PCR positive, demonstrating the ability of the mhp183 assay to detect this isolate. The EIPC included in the mhp183 assay gave a positive result for a nasal swab sample that was negative (data not shown), indicating that no sample inhibition occurred and that the negative result was more likely due to the low level of M. hyopneumoniae DNA present in the sample.

The assays targeting the 16S rRNA gene detected all M. hyopneumoniae isolates (4, 20, 24), but many researchers are reluctant to target this gene due to its high level of conservation among bacterial species, which may lead to false-positive results. A number of mycoplasmas and related acholeplasmas that have received little research, such as M. arginini, M. salivarium, M. hyopharyngis, Acholeplasma laidlawii, A. granularum, and others, have been isolated from pigs (3, 7, 17, 18). The prevalence of these organisms is unclear, as are their potentials for eliciting false-positive results in 16S-based PCR assays (or in PCR assays targeting other genes).

Although three strains of M. hyopneumoniae have been sequenced (16, 25), there is little known concerning the extent of genetic variability between M. hyopneumoniae isolates, making it difficult to identify conserved targets for PCR assays. One recent study has shown a considerable amount of genetic diversity among field isolates (12). To improve the diagnostic ability to detect field isolates of M. hyopneumoniae, the two real-time PCR assays developed for mhp165 and mhp183 were multiplexed. An EIPC was included to control for sample inhibition from template contaminants leading to a false-negative result. No decrease in sensitivity was found by comparing the individual assays to the multiplexed assay. Each of the two real-time assays was more sensitive than a nested-set PCR assay to the mhp165 gene (8).

Noteworthy is the fact that the mhp165 and mhp183 assays described here detected all of the isolates tested. This is a significant improvement over previously described real-time PCR assays. Results from this study also support previously reported genetic diversity among isolates of M. hyopneumoniae. The extent of this variability and its effect on currently used diagnostic assays to accurately detect a wide range of M. hyopneumoniae field isolates have yet to be determined. The majority of M. hyopneumoniae isolates used in the present study came from pigs in the United States. A future study comparing these PCR assays against a more diverse international collection of M. hyopneumoniae isolates is under way to further test their sensitivities and specificities.


   ACKNOWLEDGMENTS
 
This study was funded by a grant from the National Pork Board.

We thank Barbara Zimmerman Erickson and Linda Zeller for their assistance in acquiring cultures for this study and Nancy Upchurch for medium preparation. We also thank Michael Carruthers for his technical assistance.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011. Phone: (515) 294-6347. Fax: (515) 294-8500. E-mail: fcminion{at}iastate.edu Back

{triangledown} Published ahead of print on 4 June 2008. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Artiushin, S., and F. C. Minion. 1996. Arbitrarily primed PCR analysis of Mycoplasma hyopneumoniae field isolates demonstrates genetic heterogeneity. Int. J. Syst. Bacteriol. 46:324-328.[Abstract/Free Full Text]
  2. Artiushin, S., L. Stipkovits, and F. C. Minion. 1993. Development of polymerase chain reaction primers to detect Mycoplasma hyopneumoniae. Mol. Cell. Probes 7:381-385.[CrossRef][Medline]
  3. Blank, W. A., B. Z. Erickson, and G. W. Stemke. 1996. Phylogenetic relationships of the porcine mycoplasmas Mycoplasma hyosynoviae and Mycoplasma hyopharyngis. Int. J. Syst. Bacteriol. 46:1181-1182.[Abstract/Free Full Text]
  4. Calsamiglia, M., C. Pijoan, and A. Trigo. 1999. Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs. J. Vet. Diagn. Investig. 11:246-251.[Abstract/Free Full Text]
  5. de Castro, L. A., T. R. Pedroso, S. S. Kuchiishi, M. Ramenzoni, J. D. Kich, A. Zaha, M. H. Vainstein, and H. B. Ferreira. 2006. Variable number of tandem amino acid repeats in adhesion-related CDS products in Mycoplasma hyopneumoniae strains. Vet. Microbiol. 116:258-269.[CrossRef][Medline]
  6. Dubosson, C. R., C. Conzelmann, R. Miserez, P. Boerlin, J. Frey, W. Zimmermann, H. Hani, and P. Kuhnert. 2004. Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples. Vet. Microbiol. 102:55-65.[CrossRef][Medline]
  7. Erickson, B. Z., R. F. Ross, and J. M. Bove. 1988. Isolation of Mycoplasma salivarium from swine. Vet. Microbiol. 16:385-390.[CrossRef][Medline]
  8. Fraser, T., J. Christopher-Hennings, K. Rossow, E. Thacker, P. Ahrens, R. Chapin, A. Wasilk, and E. Nelson. 1999. Detection of Mycoplasma hyopneumoniae by nested PCR: a comparison of specimen type and extraction methods, p.36. Abstr. 42nd Annu. Meet. Am. Assoc. Vet. Lab. Diagn., San Diego, CA.
  9. Hsu, T., S. Artiushin, and F. C. Minion. 1997. Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae. J. Bacteriol. 179:1317-1323.[Abstract/Free Full Text]
  10. Hsu, T., and F. C. Minion. 1998. Identification of the cilium binding epitope of the Mycoplasma hyopneumoniae P97 adhesin. Infect. Immun. 66:4762-4766.[Abstract/Free Full Text]
  11. Kurth, K. T., T. Hsu, E. R. Snook, E. L. Thacker, B. J. Thacker, and F. C. Minion. 2002. Use of a Mycoplasma hyopneumoniae nested polymerase chain reaction test to determine the optimal sampling sites in swine. J. Vet. Diagn. Investig. 14:463-469.[Abstract/Free Full Text]
  12. Madsen, M. L., M. J. Oneal, S. W. Gardner, E. L. Strait, D. Nettleton, E. L. Thacker, and F. C. Minion. 2007. Array-based genomic comparative hybridization analysis of field strains of Mycoplasma hyopneumoniae. J. Bacteriol. 189:7977-7982.[Abstract/Free Full Text]
  13. Mattsson, J. G., K. Bergstrom, P. Wallgren, and K. E. Johansson. 1995. Detection of Mycoplasma hyopneumoniae in nose swabs from pigs by in vitro amplification of the 16S rRNA gene. J. Clin. Microbiol. 33:893-897.[Abstract]
  14. Mayor, D., J. Jores, B. M. Korczak, and P. Kuhnert. 2008. Multilocus sequence typing (MLST) of Mycoplasma hyopneumoniae: a diverse pathogen with limited clonality. Vet. Microbiol. 127:63-72.[CrossRef][Medline]
  15. Mayor, D., F. Zeeh, J. Frey, and P. Kuhnert. 2007. Diversity of Mycoplasma hyopneumoniae in pig farms revealed by direct molecular typing of clinical material. Vet. Res. 38:391-398.[CrossRef][Medline]
  16. Minion, F. C., E. J. Lefkowitz, M. L. Madsen, B. J. Cleary, S. M. Swartzell, and G. G. Mahairas. 2004. The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis. J. Bacteriol. 186:7123-7133.[Abstract/Free Full Text]
  17. Orning, A. P., R. F. Ross, and M. F. Barile. 1978. Isolation of Mycoplasma arginini from swine and from a swine waste disposal system. Am. J. Vet. Res. 39:1169-1174.[Medline]
  18. Roberts, D. H., and T. W. Little. 1976. Isolation of Acholeplasma granularum from porcine faeces. Vet. Rec. 99:13.[Medline]
  19. Sorensen, V., P. Ahrens, K. Barfod, A. A. Feenstra, N. C. Feld, N. F. Friis, V. Bille-Hansen, N. E. Jensen, and M. W. Pedersen. 1997. Mycoplasma hyopneumoniae infection in pigs: duration of the disease and evaluation of four diagnostic assays. Vet. Microbiol. 54:23-34.[CrossRef][Medline]
  20. Stakenborg, T., J. Vicca, P. Butaye, H. Imberechts, J. Peeters, A. De Kruif, F. Haesebrouck, and D. Maes. 2006. A multiplex PCR to identify porcine mycoplasmas present in broth cultures. Vet. Res. Commun. 30:239-247.[Medline]
  21. Stakenborg, T., J. Vicca, D. Maes, J. Peeters, A. de Kruif, F. Haesebrouck, and P. Butaye. 2006. Comparison of molecular techniques for the typing of Mycoplasma hyopneumoniae isolates. J. Microbiol. Methods 66:263-275.[CrossRef][Medline]
  22. Stark, K. D., J. Nicolet, and J. Frey. 1998. Detection of Mycoplasma hyopneumoniae by air sampling with a nested PCR assay. Appl. Environ. Microbiol. 64:543-548.[Abstract/Free Full Text]
  23. Stemke, G. W. 1997. Gene amplification (PCR) to detect and differentiate mycoplasmas in porcine mycoplasmal pneumonia. Lett. Appl. Microbiol. 25:327-330.[CrossRef][Medline]
  24. Stemke, G. W., R. Phan, T. F. Young, and R. F. Ross. 1994. Differentiation of Mycoplasma hyopneumoniae, M flocculare, and M hyorhinis on the basis of amplification of a 16S rRNA gene sequence. Am. J. Vet. Res. 55:81-84.[Medline]
  25. Vasconcelos, A. T., H. B. Ferreira, C. V. Bizarro, S. L. Bonatto, M. O. Carvalho, P. M. Pinto, D. F. Almeida, L. G. Almeida, R. Almeida, L. Alves-Filho, E. N. Assuncao, V. A. Azevedo, M. R. Bogo, M. M. Brigido, M. Brocchi, H. A. Burity, A. A. Camargo, S. S. Camargo, M. S. Carepo, D. M. Carraro, J. C. de Mattos Cascardo, L. A. Castro, G. Cavalcanti, G. Chemale, R. G. Collevatti, C. W. Cunha, B. Dallagiovanna, B. P. Dambros, O. A. Dellagostin, C. Falcao, F. Fantinatti-Garboggini, M. S. Felipe, L. Fiorentin, G. R. Franco, N. S. Freitas, D. Frias, T. B. Grangeiro, E. C. Grisard, C. T. Guimaraes, M. Hungria, S. N. Jardim, M. A. Krieger, J. P. Laurino, L. F. Lima, M. I. Lopes, E. L. Loreto, H. M. Madeira, G. P. Manfio, A. Q. Maranhao, C. T. Martinkovics, S. R. Medeiros, M. A. Moreira, M. Neiva, C. E. Ramalho-Neto, M. F. Nicolas, S. C. Oliveira, R. F. Paixao, F. O. Pedrosa, S. D. Pena, M. Pereira, L. Pereira-Ferrari, I. Piffer, L. S. Pinto, D. P. Potrich, A. C. Salim, F. R. Santos, R. Schmitt, M. P. Schneider, A. Schrank, I. S. Schrank, A. F. Schuck, H. N. Seuanez, D. W. Silva, R. Silva, S. C. Silva, C. M. Soares, K. R. Souza, R. C. Souza, C. C. Staats, M. B. Steffens, S. M. Teixeira, T. P. Urmenyi, M. H. Vainstein, L. W. Zuccherato, A. J. Simpson, and A. Zaha. 2005. Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae. J. Bacteriol. 187:5568-5577.[Abstract/Free Full Text]
  26. Verdin, E., C. Saillard, A. Labbe, J. M. Bove, and M. Kobisch. 2000. A nested PCR assay for the detection of Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs. Vet. Microbiol. 76:31-40.[CrossRef][Medline]
  27. Zhang, Q., T. F. Young, and R. F. Ross. 1995. Identification and characterization of a Mycoplasma hyopneumoniae adhesin. Infect. Immun. 63:1013-1019.[Abstract]
  28. Zielinski, G. C., and R. F. Ross. 1990. Effect of growth in cell cultures and strain on virulence of Mycoplasma hyopneumoniae for swine. Am. J. Vet. Res. 51:344-348.[Medline]

Journal of Clinical Microbiology, August 2008, p. 2491-2498, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.02366-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
JCM.02366-07v1
46/8/2491    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Google Scholar
Right arrow Articles by Strait, E. L.
Right arrow Articles by Thacker, E. L.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Strait, E. L.
Right arrow Articles by Thacker, E. L.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Antimicrob. Agents Chemother. Clin. Microbiol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS