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Mycoplasma is an important pathogen of veterinary importance as it causes heavy economic losses by infecting many species of livestock. The major syndrome associated with Mycoplasma infection in small ruminants is pneumonia. The study indicated that PCR can be adapted to screen the field samples for detection of Mycoplasma as it is a rapid and efficient method compared to other methods.

A total number of 20 samples including milk, synovial fluid, conjunctival and nasal swabs from the affected sheep showing symptoms of conjunctivitis, arthritis and mastitis were collected from Nizamabad, Karimnagar and Mahaboobnagar districts of Andhra Pradesh and swabs were immediately placed in PBS and transported to the laboratory on ice.

The Mycoplasma group specific primers which amplify 280 bp fragment of 16S rRNA gene for detection all the species of Mycoplasma genus were used. The sequence of the forward primer was 5’- GGG AGC AAA CAG GAT TAG ATA CCCT-3’ and the reverse primer was 5’-TGC ACC ATC TGT CAC TCT GTT AAC CTC-3’.

The extraction, amplification of DNA and analysis of amplified DNA were performed as per the method described by Van Kuppeveld et al. [1] with slight modifications. Briefly, DNA was isolated from 500 μl of the sample with 10% sodium dodecyl sulfate and 2.5 mg/ml of proteinase K, extracted with Phenol:Chloroform:Isoamyl alcohol mixture (25:24:1), then with chloroform:Isoamyl alcohol mixture (24:1) and precipitated with absolute alcohol in the presence of 3 M sodium acetate. The pellets were washed with 80% alcohol and resuspended in 20 μl of nuclease free water. Positive (Mycoplasma positive cell cultures) and negative controls (Distilled water) were incorporated in the test to rule out false positive and false negative results.

Amplification of DNA was performed using 10 μl of nucleic acid in a 50 μl reaction mixture containing 10X PCR buffer, 25 mM MgCl₂, 25 picomoles of each primer, 10 mM dNTP mix and 1.5 U of TaqDNA polymerase. The thermal profile consisted of initial denaturation at 94°C for 5 min, 40 cycles of denaturation at 94°C for 1 min, primer annealing at 55°C for 1 min and extension at 72°C for 2 min and final extension was done at 72°C for 7 min.

Post PCR analysis was carried out by agarose gel electrophoresis using 1.2% agarose gel stained with ethidium bromide loaded with 15 μl of amplified product using 6X gel loading buffer and 100 bp DNA ladder (GeNei) for comparison.

The positive control and conjunctiva swab samples has shown the band at 280 bp level (Figure 1) indicative of positive for Mycoplasma species [2] whereas the other samples did not show any amplification. The results indicated that the organism may belong to the members of M. conjunctivae , as the DNA from the eye swabs was amplified [3], M. agalactiae when it is correlated with the clinical [4] and M. capricolum subspecies capripneumoniae as reported by the Cetinkaya et al. [2].

Figure 1: Agarose gel electrophoresis of PCR product of 16s rRNA gene of Mycoplasma.

The diagnosis of Mycoplasma infection has not been paid enough attention because of the inadequate laboratory facilities and the causative agent is rather difficult to cultivate in vitro . In the present study, the positive results paved a way to adopt PCR as a diagnostic tool to screen the field samples for Mycoplasma detection, as it is a rapid, specific, and sensitive test when compared to serological and cultural methods which lack specificity and may require several days to establish the presence of the organism. In future, the Mycoplasma species specific primers can be procured and species specific diagnosis can be made.

However, further studies are to be taken up to standardize PCR technique for all the field samples including milk, nasal swabs, vaginal swabs and fecal samples etc. and for all the species of livestock, susceptible for Mycoplasma infection. Moreover, it is necessary to evaluate the best suitable sample for conducting PCR to detect Mycoplasma by testing a number of samples [5].

In this study, an attempt was made to Mycoplasma infection from various field samples such as milk, synovial fluid, swabs from conjunctiva and nasal mucosa. Among the entire samples tested conjunctival swab sample showed positive reaction while rest of others are negative for Mycoplasma species in PCR. The study indicated that PCR can be adapted to screen the field samples for detection of Mycoplasma as it is a rapid and efficient method compared to other methods.