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. 2015 Spring;16(2):176–181.

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
PMCID: PMC4827683  PMID: 27175172

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.

Key Words: Diagnosis, Mycoplasma equigenitalium, Polymerase chain reaction, rpoB gene

Introduction

Mycoplasma equigenitalium is associated with infertility, endometritis, vulvitis and abortions in mares, and reduced fertility and balanoposthitis in stallions (Moorthy et al., 1977 ▶; Heitmann et al., 1979 ▶; Kirchhoff et al., 1979 ▶; Spergser et al., 2002 ▶). The impact of M. equigenitalium on equine reproductive health is apparently inconsistent, largely because of strain differences in pathogenic potential, host factors and major roles played by other determinants (Bermúdez et al., 1992 ▶). Nevertheless, in vitro studies of experimental infections have reported M. equigenitalium to cause ciliostasis and cell degeneration in chicken-embryo tracheal explants (Bermúdez et al., 1988 ▶; Miller et al., 1994 ▶).

Identification of M. equigenitalium in clinical samples by standard bacterial culture is time consuming and requires complex media, special handling and further testing for species determination. Confirmed identification of M. equigenitalium at the species level is then achieved by carrying out digitonin sensitivity tests followed by metabolic inhibition tests which cause growth inhibition with species specific anti-sera, (Taylor-Robinson and Furr, 1997 ▶). Presently, only few diagnostic laboratories have specialized facilities and the required expertise to isolate and characterize Mycoplasmas. Therefore, the importance of M. equigenitalium infection in equine genital disorders may be underestimated.

PCR based detection of pathogens has recently gained momentum and emerged as an important tool for disease diagnosis. 16S rRNA based PCR targeting species-specific regions in the 16S rDNA and 16S/23S rDNA intergenic spacer (IGS) sequences were used for mollicute detection (Harasawa, 1999 ▶; Tang et al., 2000 ▶; Kong et al., 2001 ▶). Although 16S rDNA PCR is by far the most widely used technique, it is critically limited as a phylogenetic marker in Mycoplasma, because there are two or more 16S rRNA operons in many Mycoplasma species showing high interspecies variations (Woese et al., 1980 ▶; Pettersson et al., 1996 ▶; Konigsson et al., 2002 ▶). The rpoB gene, or the DNA-dependent RNA polymerase gene, has been proposed as a genome similarity predictor, and an alternative to 16S rDNA gene sequencing for biodiversity studies (Helal et al., 2008 ▶). A high degree of variation in terms of size and sequence was observed among species by the phylogenetic analysis of rpoB sequences of 26 Mycoplasma species. The rpoB gene was described as a useful target for bacterial identification and phylogenetic studies (Mollet et al., 1997 ▶). The extreme sequence diversity of rpoB gene sequence in the Mycoplasma species can be viewed as a good alternative molecular marker and an interesting choice to differentiate or identify wild types of Mycoplasmas (Kim et al., 2003 ▶).

The present study aimed to develop a M. equigenitalium species-specific PCR for the identification of M. equigenitalium directly from clinical samples.

Materials and Methods

Sample collection

Samples were collected from different states in northern parts of India including Rajasthan, Punjab, Haryana, Jammu and Kashmir, Uttar Pradesh and Maharashtra (Fig. 1). Forty eight clinical samples comprising of preputial swabs and washings from stallion and fossa glandis swabs, vaginal discharge/swabs and clitoral fossa swabs were collected from mares subjected to various reproductive disorders, while 9 other samples were collected randomly. Another group of 63 samples (frozen and neat semen) was taken at random from stallions subjected to semen evaluation, while 2 clinical samples were from stallions with balanoposthitis. The swab samples were collected in duplicate, one kept for culture and the other for PCR.

Fig. 1.

Fig. 1

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Source and number of clinical and random samples collected from different states of India

Isolation of M. equigenitalium

All samples were cultured in modified PPLO broth media (Carmichael et al., 1972 ▶). The swabs were squeezed in 2 ml of modified PPLO broth (pH = 7.6-7.8) growth medium containing the following components per liter: 450 ml Hank’s growth medium, 450 ml beef heart infusion, 100 ml horse serum, 2.5 ml (10% w/v) thallous acetate (Sigma) and 5 ml (2,00,000 IU/ml) penicillin (Glaxo Laboratories) and 4 ml 1% (w/v) phenol red. Hanks growth medium was prepared by mixing 225 ml Hank’s buffered salt solution (HBSS), 5.0 g lactalbumin hydrolysate (LAH) and 1.0 g yeast extract (Difco) in a 1 L final volume.

After inoculating the suspected specimen in modified PPLO broth, 10 fold dilutions were made up to 10-4 with PPLO medium. The media were incubated at 37°C for about one week. In case of lack of growth, turbidity or any pH change in liquid media, it was further subcultured and observed for another week to 10 days. Likewise, frozen semen, neat semen and preputial washing samples were diluted and processed in modified PPLO broth (pH = 7.6-7.8). Cultures were observed daily for colour change. Suspected growth in the medium with a faint translucent turbidity was confirmed by transfering about 0.2 ml onto a solid PPLO agar medium and incubation in micro-aerophilic conditions at 37°C for one week to 10 days. The growth of M. equigenitalium isolates was confirmed using digitonin sensitivity and growth inhibition tests conducted with specific standard antiserum, glucose fermentation, phosphatase activity, arginine hydrolysis, liquification of coagulated serum, tetrazolium reduction and film and spot formation tests. Biochemical tests were performed according to the methods described by Aloutto et al. (1970) ▶.

Sample preparation for PCR analysis

Bacterial strains

Genomic DNAs from bacterial cultures were prepared as described by Garcia et al. (1988) ▶. Briefly, cells were suspended in 20 mM Tris-HCl (pH = 8.0) containing 10 mM EDTA, 50 mM glucose, and 2 mg lysozyme per ml. After the addition of sodium dodecyl sulfate (SDS) to a final concentration of 0.5%, the DNA was incubated with 0.1 mg of proteinase-K per ml at 55°C overnight. The DNA was phenol extracted, ethanol precipitated, and suspended in distilled water. Concentrations of genomic DNA isolated from different bacterial strains were measured using a NanoDrop-8000 spectrophotometer (Thermo Fisher scientific, USA) and estimated to be in the range of 1.8-4.6 μg/ml, with the average of 2.56 μg/ml.

Genital swabs

For sample preparation, swabs were suspended in 200 μL sterile phosphate buffered saline (PBS). The 200 μL suspensions and 100 μL semen or pre-ejaculatory fluid samples were centrifuged at 14,000 g for 20 min. Pellets were re-suspended in 385 μL of a lysis buffer containing 20 mM Tris-HCl (pH = 8.0), 1 mM ethylene diamine tetra acetate (EDTA), 30 mM dithiothreitol, 0.5% sodium dodecyl sulphate (SDS) and 0.4 mg proteinase K/ml. The mixture was incubated with agitation overnight at 55°C. DNA was isolated by cetyl trimethyl ammonium bromide (CTAB) extraction as previously described by Kaltenböck et al. (1997) ▶. Phenol-chloroform extraction was followed by precipitation with 3 M sodium acetate and 96% ethanol. The DNA was pelleted, washed with 70% ethanol and re-suspended in 50 μL distilled water.

Primer design

Published rpoB gene sequences of Mollicutes were retrieved from GenBank (NIH, Bethesda, MD), and homology trees were generated using Lasergene® software (DNASTAR, Madison, Wis.). Multiple sequence alignments (MSA) were performed using the Clustal W algorithm. The alignments were visually scanned for unique non-homologous DNA sequences between clusters of highly conserved DNA sequences representing Mycoplasma species (Fig. 2). PCR primers were developed based on these sequences while maximizing established primer design criteria. Potential primer sequences were checked for lack of sequence homology with other prokaryotic and eukaryotic DNA sequences using a BLAST (NCBI, Bethesda, MD) search.

Fig. 2.

Fig. 2

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Alignment report of rpoB genes in different Mycoplasma species

M. equigenitalium species-specific PCR

Specific primers directed against the rpoB gene of M. equigenitalium were designed and used to amplify a 922 bp product. The oligonucleotide sequence for forward primer MEG-(F) was 5´-AGT GAG GGT GAG CTT TCG CTT GG-3´ and the reverse primer was MEG-(R) 5´-ATG GCC CAA TGC TCC GTG CG-3´. The amplification reaction was performed in a 25 μL volume containing 0.2 mM dNTP’s (Fermentas, GmBH, Germany), 15 pmol of each primer (GCC Biotech, India), 1 unit Taq polymerase (Fermentas) and 2 µl of the DNA template. The PCR program was performed for 30 cycles with denaturation (94°C for 1 min), annealing (55°C for 45 s) and extension (72°C for 1 min) in an automated DNA thermal cycler (Eppendorf Mastercycler®, USA). PCR products were analyzed by 1.5% agarose gel electrophoresis. A negative control (the PCR reagents excluding the DNA template) and a positive control, using DNA from M. equigenitalium (NCTC 10176), were also included.

Specificity and sensitivity of primers

To determine primer specificities, strains of Mycoplama species widely associated with equine reproductive tract including M. equigenitalium T37 (NCTC 10176), Acholeplasma laidlawi (NCTC 10171) Acholeplasma equifetale (NCTC 10116), M. subdolum, as well as field isolates of nine M. equigenitalium, six A. laidlawi and two A. equifetale were used in the study. M. agalactiae, M. Capri and M. felis isolates obtained from the Referral Laboratory on Mycoplasma, Division of Bacteriology, Indian Veterinary Research Institute, India, were also used.

Genomic DNA from 12 M. equigenitalium strains and other Mycoplasma species belonging to equine, feline, and ruminant isolates were tested using the M. equigenitalium species specific primer (MESSP) pair. In addition, the specificity of the MESSP pair was tested with 122 clinical swabs. MESSP sensitivity was evaluated by qualitative PCR analysis. The genomic DNA of M. equigenitalium culture was isolated and quantified by NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, USA). Purified DNA was 10 fold serially diluted, each dilution being used as a template and tested by MESSP PCR to determine lower detection limits.

Results

Clinical evaluation

A total of 122 genital sample swabs were cultured to isolate the bacterium of 50 clinical and 72 random samples. The results are summarized in Table 1. M. equigenitalium was isolated from 11 clinical samples, whereas 27 others, including the culture-positive samples, were found to be positive using the direct PCR. The highest percentage of culture positive samples (16.66%) belonged to abortion cases, followed by 15.78%, 13.04%, and 5.55%, from metritis, repeat breeders and random samples respectively. Our results from the direct PCR showed the highest prevalence of M. equigenitalium (47.82%) in the repeat breeders, followed by, 36.84%, 16.66%, 11.11%, from, metritis, abortion and random samples respectively.

Table 1.

Percentage of culture and PCR positive samples and distribution of M. equigenitalium among samples collected from clinically affected and random animals

Sample Sex Clinical condition No of sample Culture positive sample PCR positive sample
Clinical Female Abortion 6 N=1 (% 16.66) N=1 (% 16.66)
Repeat breeder, infertility 23 N=3 (% 13.04) N=11 (% 47.82)
Metritis 19 N=3 (% 15.78) N=7 (% 36.84)
Male Balanoposthitis 2 0 0
Total 50 N=7 (% 14.00) N=19 (% 38.00)
Random Female Apparently normal 9 0 N=4 (% 44.45)
Male Apparently normal 63 N=4 (% 6.35) N=4 (% 6.35)
Total 72 N=4 (% 5.56) N=8 (% 11.11)
Grand total 122 N=11 (% 9.01) N=27 (% 22.13)
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Primer specificity and sensitivity of the PCR assay

Specificities of the selected PCR primers were tested using DNA from one reference strain of M. equigenitalium and 11 M. equigenitalium field isolates. An amplified product of 922 base pairs was generated with all tested isolates (Fig. 3a). The 922 bp rpoB PCR product was sequenced by an automated DNA sequencer (Chromus Biotech, Bangalore), and the gene sequence was submitted to GenBank (GenBank accession No.: KC485074.1). DNA isolated from a variety of other Mycoplasma species (as explained in the previous section) was also subjected to PCR, but none amplified the specific target sequence (Fig. 3b). Lower limits required to detect the M. equigenitalium isolated DNA by PCR were examined. The results showed that 50 pg of purified DNA in the reaction mixture was the minimum needed to obtain a detectable PCR product (Fig. 4).

Fig. 3a.

Fig. 3a

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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

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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

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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

Discussion

Mycoplasmas have been isolated from uterus, cervix and vaginas of mares, the clitoral fossa being most likely the “ecological niche”. In stallions, the preferred isolation site is the fossa glandis, apart from other common sources like urethra, prepuce, penis, and fresh or frozen semen (Lemcke and Kirchhoff, 1979 ▶; Bermúdez et al., 1987 ▶; Spergser et al., 2002 ▶). Hence, clitoral fossa and fossa glandis swabs from mares and stallions were chosen in this study as preferred clinical samples for obvious reasons. The impending pathogenic role of M. equigenitalium in reproductive dysfunctions is yet to be recognized clearly, and further studies are required to ascertain its pathogenic potential (Bermúdez et al., 1988 ▶; Miller et al., 1994 ▶). Information regarding the occurrence and significance of M. equigenitalium in equine reproductive disorders is scarce in scientific veterinary literature, largely because the isolation of the organisms is very tedious, laborious, and time consuming. At the same time, serological tests are not very sensitive and specific. PCR based diagnoses can thus be used as effective tools for detecting pathogens in clinical samples, either directly or in conjunction with culture techniques. The aim of this study was to develop a novel PCR assay specific to M. equigenitalium and to apply it to detect M. equigenitalium DNA taken directly from clinical samples.

In an attempt to develop a species-specific PCR for M. equigenitalium, the rpoB gene was targeted for the construction of specific primers. The PCR reaction using the rpoB gene-based primer set described here was found to be very specific since a precise amplification of 922 bp fragments was obtained using the l2 M. equigenitalium isolates tested. The same test gave negative results with phylogenetically related Mycoplasma species other than M. equigenitalium, some of which are known to be present in the genital tracts of horses.

As evident from the results presented in Table 1, the sensitivity of the PCR assay is superior to that of the conventional Mycoplasma culture technique. However, a combination of culture and PCR is to date the most sensitive approach. Of the tested samples, 9.01% are positive as shown by the conventional culture technique, while 22.13% resulted positively using the M. equigenitalium species specific PCR. The results clearly indicate that a considerable number of horses carrying M. equigenitalium remain unidentified by current culturing techniques, meaning that M. equigenitalium infection is more prevalent and that its importance has probably been underestimated.

Both culture- and PCR-based studies show the maximum number of positive clinical samples to belong to cases of metritis and repeat breeders, highlighting the subclinical involvement of these pathogens. Khurana et al. (2004) ▶ report an occurrence of 15.9% seroprevalence of antibodies to M. equigenitalium amongst repeat breeder and 41.2% seroprevalence in cases of metritis by M. equigenitalium specific ELISA, which is in close agreement with the results of the present study.

A significant number (6.35%) of apparently healthy stallions without any sperm abnormalities were found to be positive for M. equigenitalium, hence its clinical relevance in semen should not be over-interpreted. This may be due to the colonisation of M. equigenitalium in non-testicular tissues, which serves as a source of contamination during ejaculation. This is in agreement with reports on the lack of correlation between sperm abnormalities and isolation of Mycoplasmas or other potentially pathogenic bacteria in stallions and bulls (Malmgren et al., 1998 ▶; Spergser et al., 2002 ▶).

The analysis of the findings from the present study implies that there is indeed a prevalence of M. equigenitalium infections in India. The use of the rpoB gene-based species-specific M. equigenitalium PCR described here can help increase the number of laboratories detecting M. equigenitalium in equine genital infections and enhance current knowledge on the importance and distribution of M. equigenitalium.

Conflict of interest

The authors declare that they do not have any conflict of interest.

Acknowledgements

We sincerely acknowledge the help extended to us by Dr. R. A. J. Nicholas, VLA Weybridge, UK for providing the standard genomic DNA and/or equine origin culture. We also wish to acknowledge the help given to us by Dr. R. K. Singh, Director of the National Research Centre on Equines and VTCC, Hisar, India for his valuable guidance and suggestions. We also thank the Director of the Indian Veterinary Research Institute, Izatnagar, India for financial assistance to carry out this work.

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