Mycoplasmosis is one of the major diseases of poultry causing significant economic loss worldwide both in commercial sector and backyard system. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the major pathogens of chicken and are considered economically significant among more than 20 Mycoplasma spp. of the class Mollicutes that are known to infect avian hosts (Ferguson and Noormohammadi, 2013). MG is known to cause chronic respiratory disease (CRD) in chickens and infectious sinusitis in turkeys (Osman et al., 2009). MS causes infectious synovitis primarily affecting the synovial membranes of joints. In recent times MS infection is more often associated with air sacculitis, egg peritonitis, eggshell abnormalities, etc., (Feberwee et al., 2009). Mycoplasmosis cause huge economic loss due to mortality, drop in egg production, poor hatchability, poor FCR, reduced growth rate and carcass condemnation in both broilers and layers (Ferguson-Noel and Williams, 2015). Further, secondary bacterial infections, costs involved in vaccination, medication, screening of the flock and depopulation, etc., are the additional burden for the poultry producer.
       
In addition, MG and MS are widespread in many avian species and reported in backyard poultry and in numerous wild bird species (Sawicka et al., 2020; Ayala et al., 2020). Often co-infection and multiple infections with MG/MS complicate the situation. Due to their transmission both by vertical and horizontal mode and persistent infection in the host, mycoplasmosis became quickly endemic in many parts of the world including India. In addition, backyard poultry and wild bird species become infected and remain as reservoirs or carriers for these pathogens posing threat to commercial poultry. High intensity poultry rearing areas multi-age flocks and close contact with backyard poultry and wild birds are the major risk factors for mycoplasmosis.
       
Rapid detection and differentiation of MG and MS is a challenging task. Although culture method is gold standard, due to laboriousness, time-consuming and often contamination with other saprophytic mycoplasmas limits its application for routine screening. Serology does not give an indication of the current infection status in the host. Hence molecular detection methods are widely employed in mycoplasma diagnosis (Wu et al., 2019). Earlier attempts have been made to develop duplex assays. However, the target gene of choice by most of them being 16S-23S rRNA limiting the specificity (Tomar et al., 2017). In the present study, we developed a duplex PCR assay for specific and simultaneous detection of MG and MS in a single tube and validated it with field samples for screening.

Sample collection and DNA extraction
 
A total of 179 backyard poultry flocks located in different states of India viz., Tamil Nadu, Kerala, Telangana, Haryana and Assam were included for screening of MG and MS. Pooled oropharyngeal and choanal swabs from 5 randomly selected birds from each backyard flocks of minimum size 50 were considered as single sample and were included in the study to represent each flock. The birds were either native or local breed /variety or improved backyard variety in the age group of 16-40 weeks. All the swabs were collected aseptically in sterile PBS in container and transported to laboratory under chilled condition. DNA extraction from the samples were performed either within 24 h of collection of samples or stored at -20°C until extraction.
       
The swabs with the PBS were centrifuged at 14,000 rpm for 10 min and 250 ml of supernatant was taken for subsequent extraction. DNA extraction was performed by using Hi PurA® Multi-sample DNA purification kit (Himedia Laboratories Pvt Ltd) following the manufacture’s instruction. The DNA from the samples was stored at -20°C until further use in PCR.
 
Primers design
 
Primers for both MG and MS were designed using NCBI Primer design tool http://www.ncbi.nlm.nih.gov/tools/primer-blast and are presented in Table 1. The mgc2 gene of MG and vlhA (variable lipoprotein hemagglutinin A) gene for MS were selected as target genes for primer design and following criterion were considered while designing and selecting the primer sets. 40-60% GC content, 20-25 bp length, no hairpin loops, Tm° value in the range of 50-55°C and difference between the amplicon size of at least 200 bp between the primer sets.
 

 
Optimization of duplex PCR
 
The DNA extracted from live vaccines of MG and MS (Vaxsafe MG/ MS; Zoetis India Pvt Ltd) were used as template for optimization of duplex PCR assay. Different concentration of primer sets was tested using checkerboard method for getting optimal amplification of both genes from template DNA. The optimized PCR mixture is as follows: 25ml of total PCR reaction in single tube containing 2.5 ml of 10X buffer with MgCl₂, 0.25 ml of Taq polymerase (5U/ml), 0.5 ml of 10 mMol dNTPs, 0.5 ml of MG primer sets, 0.25 ml of MS primer sets, 2 ml of template DNA (50ng/ ml) and nuclease free water. The optimum annealing temperature for PCR was optimized by testing with gradient PCR method. The reaction condition optimized were as follows: initial denaturation at 94°C for 5 min followed by 35 cycles of 94 °C for 1 min, 60°C for 1 min and 72°C for 1 min, with final extension at 72°C for 10 min. The amplified specific gene PCR products were checked in 1.5% agarose gel for 150 and 787 bp respectively.
 
Sensitivity and specificity
 
To determine the sensitivity of duplex PCR primer sets, serial 10-told dilution of positive DNA template with initial concentration of 1 mg/ml was done and each dilution of both MG and MS template DNA was used for duplex PCR up to 10^-12 dilution. The highest dilution of DNA template that gives a visible target gene specific band in PCR was considered as the detection limit or sensitivity. The cross-reactivity of primer sets was tested using DNA isolated from MG and MS live vaccines. The primer set of MG was tested with DNA template from MS positive control and vice versa. The specificity of primer sets was clearly indicated by the absence of amplification in another DNA template. The gene specific products amplified with respective DNA templates were sequenced by Sanger method and obtained sequences were confirmed by BLASTN analysis for the homology.
 
Validation with field samples
 
The DNA extracted from 179 backyard poultry flock samples were subjected to duplex PCR with both MG and MS primer sets in single tube by following the optimized protocol as described earlier. The amplified PCR products were checked in 1.5% agarose gel. Presence of either 150 or 787 bp or both amplicons were considered as positive for MG / MS or both, respectively. Positive control with template DNA from vaccine and negative control were included.
 
Comparison with single PCR for MG and MS
 
The same 179 field samples were subjected to detection of MG and MS by single PCR in separate PCR reaction. Primer sets targeting the 16S rRNA gene for MG (OIE, 2008) and vlhA gene for MS (Jeffery et al., 2007) described earlier were used. The amplification of 185 bp and 350 bp products indicated the samples were positive for MG and MS respectively. The results from single PCR and duplex PCR were compared for agreement between these two assays.

Avian mycoplasmosis by both MG and MS is widely reported from different parts of this country (Yadav et al., 2021). Earlier few studies attempted for duplex PCR assays for MG and MS (Buim et al., 2009; Mettifogo et al., 2015; Malekhoseini et al., 2017; Yadav et al., 2022). However, most of the studies targeted 16S or intergenic spacer region of 16-23 S rRNA genes for the MG attributing to the difficulty in differentiating from other Mycoplasma such as M. imitans. We targeted the mgc2 gene of MG for the development of duplex PCR here, making the assay more specific and avoiding cross amplification with other Mycoplasmas.
       
Screening of field samples with duplex PCR assay showed 133/179 (74.3%) positive for MG and 39/179 (21.78%) positive for MS and 35/179 (19.55%) positive for both MG and MS (Fig 1). Single PCR assay showed 130/179 (72.62%) positive for MG and 37/179 (20.6%) positive for MS and 33/179 (18.43%) positive for both MG and MS. The results of single and duplex PCR assay are presented in Table 2. The sensitivity of duplex PCR was 10^-6 of 1 mg/ml of template DNA. Comparison of the results with single PCR of these samples with duplex PCR showed k value of 0.90 indicating high agreement levels among these assays (Fig 2). The optimized duplex PCR assay in the study showed high sensitivity and specificity, hence ideal assay for rapid surveillance of Mycoplasma in field conditions.
 

 

 

Earlier studies reported 3.5-32% for MG and 6.4-33% prevalence rates for MS in Indian poultry flocks (Ramadass et al., 2006; Tomar et al., 2017; Rajkumar et al., 2018; Bagal et al., 2019; Yadav et al., 2021; Yadav et al., 2022). The wide range due to spatial and temporal variation. The positivity rate in the current study for both MG and MS are very high compared to earlier reports. Most of the earlier reports are from commercial poultry and relatively lower incidence levels may be attributed to the control strategies like vaccination, anti-mycoplasma programs and bio-security employed. However, here the backyard poultry owing to the lack of any control measures and biosecurity high prevalence is observed. Nevertheless, this also indicates that backyard poultry can act as reservoir or carries for Mycoplasma and can potentially contribute spill over to commercial poultry. Emphasis needs to given for the surveillance/ screening of these flocks for the prevalence of Mycoplasma pathogen and devise effective control strategies to minimize the economic loss.

Authors declares that they have no conflict of interest.