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. 2015 Spring;16(2):176-81.

Isolation and molecular identification of Mycoplasma equigenitalium from equine genital tracts in northern India

K Nehra  1 , R Rana  2 , K N Viswas  3 , T R Arun  1 , V P Singh  4 , A P Singh  5 , S N Prabhu  6
Affiliations

Isolation and molecular identification of Mycoplasma equigenitalium from equine genital tracts in northern India

K Nehra et al. Iran J Vet Res. 2015 Spring.

Abstract

Although Mycoplasma equigenitalium has been implicated in equine reproductive problems, its prevalence is largely unexplored due to the lack of specific diagnostic tests. To address this limitation, the authors developed and optimized species-specific primer pairs that target M. eguigenitalium rpoB (RNA polymerase B subunit) gene sequences. The specificity of the PCR assay developed in this study was determined using 12 field isolates including the type strain of M. equigenitalium and other Mycoplasma species. In the field study, a total of 122 mare and stallion samples comprising of 50 clinical and 72 random samples were subjected to species-specific PCR assay to detect M. equigenitalium in equine genital tracts. Mycoplasma equigenitalium (MEG) species-specific PCR detected 22.13% positive samples; however, only 9.01% of the samples were found to be positive using the conventional culture technique. The PCR established in this study could be used for rapid, specific and accurate diagnosis of M. equigenitalium strains. To the authors' knowledge, this is the first report addressing the development and evaluation of species-specific PCR to detect M. equigenitalium.

Keywords: Diagnosis; Mycoplasma equigenitalium; Polymerase chain reaction; rpoB gene.

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Figures

Fig. 1
Fig. 1
Source and number of clinical and random samples collected from different states of India
Fig. 2
Fig. 2
Alignment report of rpoB genes in different Mycoplasma species
Fig. 3a
Fig. 3a
Species-specific PCR amplification for Mycoplasma equigenitalium. Lane M1 and M2: 100 bp DNA ladder marker, Lane 1-5 and 8-13: DNA of 11 M. equigenitalium isolates from clinical samples, Lane 6: Reference M. equigenitalium DNA, and Lane 7: Negative control
Fig. 3b
Fig. 3b
Specificity of Mycoplasma equigenitalium species specific PCR. Lane M: 100 bp ladder, Lane 1 and 8: Mycoplasma equigenitalium, Lane 2: Acholeplasma equifetale, Lane 3: Acholepasma laidlawi, Lane 4: Mycoplasma bovis, Lane 5: Mycoplasma mycoides subsp. capri, Lane 6: Mycoplasma agalactiae, Lane 7: Mycoplasma bovirhinis, Lane 9: Brucella abortus, Lane 10: E. coli, Lane 11: Pasteurella multocida, Lane 12: Arcobacter butzleri, Lane 13: Listeria monocytogens, and Lane 14: Leptospira interrogans
Fig. 4
Fig. 4
Gel electrophoresis of PCR products amplified from serial dilutions of Mycoplasma equigenitalium DNA. Lane M: 100 bp ladder, Lane 1: 500 ng, Lane 2: 50 ng, Lane 3: 5 ng, Lane 4: 500 pg, Lane 5: 50 pg, Lane 6: 5 pg, and Lane 7: 500 fg
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