Simultaneous Detection of Mycoplasma Infections in Clinically Sick and Apparently Healthy Broilers

Muhammad Asif Zahoor1, Zeeshan Nawaz1, Abu Baker Siddique1, Sajjad ur Rahman2 and Shahid Ali1*

1 Department of Microbiology, Government College University, Faisalabad-Pakistan; 2 Institute of Microbiology, University of Agriculture, Faisalabad-Pakistan.

Abstract | Mycoplasma infections in gallinaceous birds are reported throughout the world and are caused by either Mycoplasma gallisepticum or Mycoplasma synoviae. Hence, in the present study we have detected Mycoplasma spp. from tissue samples collected from clinically sick (60 samples) and apparently healthy broiler birds (30 samples) in a commercial farm, using classical isolation and compared with molecular identification of Mycoplasma infections. A total of 96 tissue samples from trachea, air sacs, lungs and oral swabs along with blood from each bird were collected and initially subjected to isolation of Mycoplasma spp. using brain heart infusion broth supplemented with horse serum. The blood was used for serum plate agglutination assay. Thereafter, the samples were subjected to genomic DNA extraction to detect Mycoplasma spp. using multiplex-PCR. Isolation and identification data showed that a total of 58/96 samples (60.41%) were positive for Mycoplasma infections whereas a total of 61/96 blood samples were also found positive using serum plate agglutination. However, the multiplex-PCR detected Mycoplasma in all of the tissue samples, either one of the species or simultaneous infection. M. gallisepticum was found in 39/96 samples (40.62%) and M. synoviae in 32/96 samples (33.34%), whereas 25/96 samples (26.04%) were detected with simultaneous infection with both species. Altogether, it has been observed that multiplex-PCR is a sensitive tool for early and accurate diagnosis of Mycoplasma infections from samples collected from clinically sick and apparently healthy birds. In this way, current approach could be contributed well towards elimination and reduction of losses due to Mycoplasma infections in commercial poultry.

Editor | Muhammad Abubakar, National Veterinary Laboratories, Park Road, Islamabad, Pakistan.

Received | February 28, 2017; Accepted | April 17, 2017; Published | April 25, 2017

*Correspondence | Shahid Ali, Assistant Professor, Department of Microbiology, Government College University, Faisalabad-Pakistan; Email: shahidali@gcuf.edu.pk

Citation | Zahoor, M.A., Z. Nawaz, A.B. Siddique, S.U. Rahman and S. Ali. 2017. Simultaneous detection of Mycoplasma infections in clinically sick and apparently healthy broilers. Veterinary Sciences: Research and Reviews, 3(2): 34-37.

DOI | http://dx.doi.org/10.17582/journal.vsrr/201 7.3.2.34.37

Key words: Microbiology, Broilers, DNA, Disease, Genome

Introduction

The rearing and housing of gallinaceous birds at commercial levels has emerged as an alternative to beef and mutton throughout the world. These birds are prone to various types of infectious diseases. Among these Mycoplasma infections are of paramount importance causing huge economic losses to commercial poultry. Mycoplasma species are mainly responsible for respiratory distress, air sacculitis and synovitis. Almost 120 species of Mycoplasma have been identified, however only few are known to cause infections in gallinaceous birds. Among these species, M. gallisepticum and M. synoviae are major pathogens associated with respiratory and joint disease and cause huge economic losses worldwide (Bradbury, 2005; Chanie, 2009).

For the diagnosis of Mycoplasma infections; isolation/identification, serological assays and molecular identification have been reported previously (Stanley et al., 2001; Hess et al., 2007; Ehtisham et al., 2011; Siddique et al., 2012). Some of the previous studies descried molecular identification of Mycoplasma spp. with PCR as an efficient and accurate diagnostic tool (Nascimento et al., 1991; Lauerman et al., 1995; García et al., 2005; Hess et al., 2007). Similarly, comparison of different laboratory conditions for optimized protocols of PCR and simultaneous detection of bacterial pathogens has been described using multiplex-PCR (Wang et al., 1997; Hess et al., 2007; Siddique et al., 2012). The prevalence of Mycoplasma infections are reported as 30% in Pakistan (Ehtisham et al., 2011). Therefore, in an extension to the previously published data and research methodologies, we have designed the present study to optimize and validate the multiplex-PCR in our lab conditions to establish an accurate diagnostic technique for Mycoplasma infections.

Materials and Methods

Sample collection and initial processing of samples

A total of 96 tissue samples including trachea (35), air sac exudates (28), lungs (15) and oral swabs (18) along with blood from each bird were collected and labeled properly from clinically sick (60 samples) and apparently healthy broiler birds (30 samples). The birds with tracheal rales, nasal discharge, sneezing and coughing were considered as clinically sick (Siddique et al., 2012). The tissue samples were directly inoculated into the Brain heart infusion broth supplemented with 5% horse serum and incubated at 37oC for 24-72 hours for initial screening and identification of Mycoplasma infections, whereas serum plate agglutination was performed for each blood sample (Branton et al., 1984; Siddique et al., 2012).

Molecular identification of Mycoplasma spp

The genomic DNA from all tissue samples was extracted using phenol chloroform method and the extracted DNA was used in multiplex-PCR. Amplification reaction was carried out in final volume of 50µl reaction mixture using the primers described in Table 1 (Wang et al., 1997; Ehtisham et al., 2011). Multiplex-PCR was performed with an initial de-naturation at 94oC for 5 minutes, followed by 35 cycles at melting temperature 94oC for 1 minute and to annealing temperature of 50oC for 1 minutes, and extension at 72oC for 2 minutes. The sample was then heated at 72oC for 10 min for final extension of the reaction mixture (Wang et al., 1997). The amplified product was visualized under UV light following 1.5% agarose gel electrophoresis.

Table 1: Sequence of primers used for Mycoplasma gallisepticum and Mycoplasma synoviae

Primer | Sequence
M. gallisepticum forward primer | 5’ CTTTCCCATCTCGACCAGGAGAAAA-3
M. gallisepticum reverse primer | 5’ GGATCAATCAGTGAGTAACTGATGA-3’
M. synoviae forward primer | 5’ GTCGTAAATAGTGATATCA-3’
M. synoviae reverse primer | 5’ GAAGCCTCGAAGTTAACAA-3’

Results and Discussion

In the present study, a total of 96 tissue samples were processed for Mycoplasma isolation. Conventional isolation data showed that total of 58/96 samples (60.41%) were positive for Mycoplasma infections whereas 61/96 blood samples were also found positive using serum plate agglutination as shown in Figure 1.

All of the tissue samples (96) were found positive with multiplex-PCR for either one of the species M. gallisepticum or M. synoviae or simultaneous infection with both species. Altogether, all of 96 samples 39 (40.62%) samples were positive for M. gallisepticum, 32 samples (33.34%) were positive for M. synoviae and remaining 25 samples (26.04%) were positive for simultaneous infection as shown in Figure 1 and 2.

Huge economic losses have been reported from almost every part of the world in gallinaceous birds due to Mycoplasma infections which includes reduced egg production (10-20%), increased embryonic and chick mortality (5-10%) and reduced weight gain and feed conversion ratio (10-20%) as described by Kleven, 1990. Mycoplasma infections are caused by either M. gallisepticum or M. synoviae and are characterized by coughing, sneezing, tracheal rales and nasal discharge. M. gallisepticum usually causes subclinical to mild infection whereas the severity of disease increases with simultaneous infection of M. synoviae (Roussan et al., 2008). The diagnosis of Mycoplasma infections are based on serology and isolation/identification of Mycoplasma. Serological identification of Mycoplasma antigen is mainly considered nonspecific due to cross reactivity of Mycoplasma spp. and usually produces false positive results, however it is good screening tool at large scale or commercial poultry farms (Ehtisham et al., 2011, Hutton et al., 2017). Isolation and identification of Mycoplasma is gold standard for diagnosis, however it is time consuming as the micro organism showed slower growth patterns (Siddique et al., 2012). Therefore, in the present study multiplex-PCR was used to simultaneously detect M. gallisepticum and M. synoviae infections from clinically sick and apparently healthy broiler birds. Multiplex-PCR detection has increased diagnostic accuracy and reduced time consumption as compared to serology and isolation/identification of Mycoplasma infections.

In the present study, a total of 96 tissue samples were processed for Mycoplasma isolation. Conventional isolation data showed that 58/96 samples (60.41%) were positive whereas 61/96 blood samples were also found positive using serum plate agglutination. Previously published data also described the isolation/identification and serological assays for Mycoplasma spp. identification (Siddique et al., 2012; Ehtisham et al., 2011). However, in the present study all of the samples were found positive either with M. gallisepticum, M. synoviae or dual infection using multiplex PCR. has detected Mycoplasma.

Further, multiplex-PCR data showed absolute and accurate diagnosis including of simultaneous detection of M. gallisepticum and M. synoviae as compared to isolation/identification and serological data. The highest number of positive samples was M. gallisepticum 39/96 (40.62%), followed by M. synoviae 32/96 (33.34%), whereas simultaneous infection is reported among 25/96 (26.04%). The previously published data also showed a high efficacy and efficiency of molecular identification methods including PCR and multiplex-PCR from respiratory distress cases as compared to isolation/identification and serology (Hess et al., 2007; Siddique et al, 2011).

Conclusion

Finally, based on results of the present study, multiplex-PCR is an ideal approach to detect Mycoplasma infections including M. gallisepticum and M. synoviae or simultaneous infection with both species.

Authors’ Contribution

MAZ and ZN prepared the manuscript, ABS and SA collected the samples and performed the experiments, whereas SUR designed and supervised the study.

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