Detection of Mycoplasma gallisepticum from field samples of Poultry using conventional PCR

Davinder Singh^1*, Nand Kishor Mahajan¹, Aman Kumar², Sushila Maan², Pawan Kumar²

*Corresponding author:drdev18@gmail.com

ARTICLE CITATION: Singh D, Mahajan NK, Kumar A, Maan S and Kumar P (2013). Detection of Mycoplasma gallisepticum from field samples of poultry using conventional PCR. Adv. Anim. Vet. Sci. 1 (1S): 11 – 13.
Received: 2013-08-19, Revised: 2013-08-20, Accepted: 2013-08-21
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ABSTRACT

The present study reports the use of polymerase chain reaction (PCR) for detection of Mycoplasma gallisepticum (MG) infection in poultry using tissues directly. A total of 51 samples (trachea and airsacs) were collected from clinically suspected birds originating from district Hisar, India. PCR was carried out using specie specific primers for MG and identified 18 samples (35.3%). It is concluded that tissues may be used for rapid screening and detection of MG in poultry.

Mycoplasmosis is one of the major respiratory problems in poultry. Avian Mycoplasmosis, caused mainly by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), can cause considerable economic losses in chicken due to chronic respiratory disease, reduced weight gain and meat quality and increased feed conversion ration and mortality in broilers, tremendous drop in egg production in layers and increase in embryonic mortality in breeders (Kleven and Noel, 2008; Ley, 2008). Out of four major pathogenic Mycoplamas, Mycoplasma galliseptcium has been described as the most important species affecting poultry, others include Mycoplasma synoviae (chicken and turkey pathogen), Mycoplasma synoviae (turkey pathogen), and Mycoplasma meleagridis (turkey pathogen) (Mohammed Et al., 1987; Thomas and Sharp, 1990; Ley, 2008). The annual economic impact of MG infections in the United States has been estimated between $118 and $150 million for the layer industry alone in 1994 (Patterson, 1994). In the recent past, the MG outbreaks have caused significant losses to the poultry industry (Ley, 2008).

The flock screening for the pathogen is done by serological assays and the confirmation can be done by cultivation of pathogen or PCR detection (Ramadass Et al., 2006). Isolation of Mycoplasma is very difficult and cumbersome to perform. Diagnosis of Mycoplasma infections by serological procedures is sometimes hampered by interspecies cross–reactions and nonspecific reactions (Hagan Et al., 2004). Therefore, molecular methods for diagnosing the disease may be more effective in comparison to cultural and serological methods. These include a validated PCR assay for MG, MS, and other avian Mycoplasmas based on unique sequences contained in the 16S rRNA gene and nucleic acid probes (Khan and Kleven 1993; Kempf, 1998; Lauerman, 1998; McAuliffe Et al., 2005). The technique has been proven to be very specific and sensitive method even for amplifying low amounts of nucleic acid to a level that cannot be easily detected by other methods.

Therefore, present study was aimed to test feasibility of using tissues directly PCR based identification of Mycoplasma in poultry flocks without any need of enrichment or culture.

Tissues (trachea and airsacs) from poultry flocks (one representative samples was taken from each affected flock) showing typical signs of chronic respiratory disease viz. rales, coughing, nasal discharge, sinusitis, brought for diseases investigation were collected at Disease Investigation Lab., College of Veterinary Sciences, LLR UVAS, Hisar. Broiler flocks around Hisar region of Haryana were included with flock size ranging from 5000 to 10,000 birds. Necropsy of the dead birds revealed pathologic findings such as air sacculitis, tracheitis, and pneumonia. Tissue samples (trachea and airsacs) were collected from such birds in buffered glycerol (50%) and stored at –20^OC till further use.

Tissues were triturated directly without any enrichment in pestle and mortar. Genomic DNA was extracted using DNeasy Blood & Tissue Kit® (Qiagen) following manufacturer’s instructions. The total DNA was measured at 260 nm optical density as per method described by Sambrook and Russel (2001). The extracted DNA was kept at –20^OC till further use.

PCR was carried out by using species–specific primer pair for MG (Kiss et al., 1997) (Table 1). PCR was carried out in 0.5 mL PCR tubes in a final reaction of 25 µL volume. Each reaction mixture contained 2X PCR Master Mix (12.5µL) (TopTaq Master Mix ®, Qiagen), 10 pmol/µL each of MG–F and MG–R primers and 100 ng of the DNA template. The reaction mixtures were adjusted to the final volume by adding Nuclease free water (NFW) and subjected to PCR amplification in MyCycler (Biorad, USA).

The PCR amplified products were separated by agarose gel electrophoresis (2% agarose) in 1X Tris–borate–EDTA buffer (Tris–base10.8g 89 mM, Boric acid 5.5g 2mM, EDTA 4 ml of 0.5 M EDTA pH 8.0). Gel was stained with ethidium bromide (@ 0.5 μg/mL of gel).

Aliquots of 6 µL were applied to the gel. Standard molecular size marker, Gene Ruler 100 bp DNA ladder (Fermentas, USA) was included in gel. Product separation was done at constant voltage of 75V for 1 hour. DNA fragments were observed by ultraviolet transilluminator and photographed in a gel documentation system (Alpha Imager, Germany).

The species specific primers used in the study successfully amplified the portion (713–1243 bp) of 16s RNA gene of MG (530 bp) (Figure 1). Out of 51 samples tested, 18 samples (35. 3%) were found PCR positive for the Mycoplasma gallisepticum. This study showed that MG can be detected from field samples directly without need of culturing this fastidious organism as reported by McAuliffe Et al. (2005). Ramadass Et al., (2006) had documented that tracheal samples gave more isolations than from lung tissues; however the present study reports the use of airsacs along with tracheal tissues for extraction of DNA.

Thus this species specific PCR may be applied for rapid screening and diagnosis of the MG in naturally infected birds before the culture results are obtained as reported by various workers (Lauerman Et al., 1993; Ramadass Et al., 2006).

Mycoplasmas are among those fastidious organisms which are very hard to culture, as they have unique medium requirements. It requires 4–5 days for growth and initial cultures commonly contain other contaminants also (McAuliffe Et al., 2005; Kleven, 2008, Bagheri Et al., 2011). Present study illustrates the combined advantage of simple, cheap and rapid molecular test of PCR, having high sensitivity and specificity, to diagnose MG infection as compared to traditional culture of the organism.

Culture testing can be costly and time–consuming, and can also be inconclusive (Ewing Et al 1998), besides this, culture of Mycoplasma is laborious and can take 3–4 weeks, and even then, the result can be negative or be hampered by mixed infections (Bradbury Et al., 1982; Zain Et al., 1995). As for culture and PCR tests, despite the comparable sensitivity, PCR test lead to rapid results and relatively low costs compared with culture (Lauerman Et al., 1993; Feberwee Et al., 2005; Sakhaei Et al., 2009; Pourbakhsh Et al., 2010). For such reasons, use of rapid and sensitive molecular detection methods, like PCR, can be advantageous (Hyman Et al., 1989; Fernandez Et al., 1993; Kempf Et al., 1993).

In the present study, PCR was used to demonstrate the involvement of Mycoplasma gallisepticum infection in broiler flocks in Haryana, suspected for respiratory disease involvement. Results indicated that M. gallisepticum infection is prevalent in poultry flocks of Haryana as indicated by PCR testing of clinical tissues (trachea along with airsacs). Thus, PCR could be a rapid, effective, sensitive and inexpensive method as compared to the standard cultural technique which is cumbersome, time consuming and laborious, hence PCR directly from tissues can be an alternative and useful method for screening compared to traditional culture for the detection of MG in poultry at various diagnostic laboratories.

In conclusion, PCR application on tissues like trachea and air sacs seems to give encouraging results for detection of Mycoplasma gallisepticum infection in poultry flocks suffering from respiratory disease. Further studies are needed to test the comparative efficacy, sensitivity and specificity of such PCR separately on different tissues, which will allow rapid and specific diagnosis of this infection which would subsequently help in its control.

CONFLICT OF INTEREST

The authors encountered no conflict of interest during the study.

REFERENCES

Bagheri H, Doosti A and Arshi A (2011) Detection of Mycoplasma gallisepticum in Chaharmahal Va Bakhtiari Province Poultry Using PCR. Glob. Vet. 7 (1): 54–59.

Bradbury JM and McLenaghan M (1982). Detection of mixed mycoplasma species. J. Clin. Micro. 16: 314–318.
PMid:6749893 PMCid:PMC272352

Ewing L, Cookson KC, Phillips RA, Turner KR, Kleven SH (1998). Experimental infection and transmissibility of Mycoplasma synoviae with delayed serological response in chickens. Av. Dis. 42: 230–238.
http://dx.doi.org/10.2307/1592472
PMid:9645313

Feberwee A, Mekke DR, de Wit JJ, Hartman EG and Pijpers A (2005) Comparison of culture, PCR, and different serologic tests for detection of Mycoplasma gallisepticum and Mycoplasma synoviae infections. Av. Dis. 49: 260–268.
http://dx.doi.org/10.1637/7274-090804R
PMid:16094832

Fernandez C, Mattson JG, Bolske G, Levisohn S and Johansson KE (1993). Species–specific oligonucleotide probes complementary to 16S rRNA of Mycoplasma gallisepticum and Mycoplasma synoviae. Res. Vet. Sci. 55: 130–136.
http://dx.doi.org/10.1016/0034-5288(93)90047-J

Hagan JC, Ashton NJ, Bradbury JM and Morgan KL (2004). Evaluation of an egg yolk enzyme–linked immunosorbent assay antibody test and its use to assess the prevalence of Mycoplasma synoviae in UK laying hens. Av. Path. 33: 1335–38.

Hyman HC, Levisohn S, Yogev D and Razin S (1989). DNA probes for Mycoplasma gallisepticum and Mycoplasma synoviae: application in experimentally infected chickens. Vet. Micro. 20: 323-337.
http://dx.doi.org/10.1016/0378-1135(89)90057-6

Kempf I, Blanchard A, Gesbert AF, Guittet M and Bennejean G (1993). The polymerase chain reaction for Mycoplasma gallisepticum detection. Av. Path. 22: 739–750.

Kempf I (1998). DNA amplification methods for diagnosis and epidemiological investigations of avian mycoplasmosis. Av. Path., 27: 7–14.

Khan MI and Kleven SH (1993) Detection of Mycoplasma gallisepticum infection in field samples using a species–specific DNA probe. Av. Dis. 37: 880–83.
http://dx.doi.org/10.2307/1592045
PMid:8257385

Kiss MK, Kaszanyitzky E, Chavez Y and Johansson KE (1997). Detection and identification of avian Mycoplasmas by polymerase chain reaction and restriction fragment length polymorphism assay. Vet. Micro. 58: 23–30.
http://dx.doi.org/10.1016/S0378-1135(97)81568-4

Kleven SH and Noel NF (2008). Mycoplasma synoviae infection. In: Diseases of poultry Ames, Iowa State University Press. USA, pp. 845–856.,

Kleven SH (2008). Mycoplasmosis. In: A Laboratory Manual for the Isolation, Identification, and Characterization of Avian Pathogens. L. Dufour–Zavala, D. E. Swayne, J. R. Glisson, J. E. Pearson, W. M. Reed, M. W. Jackwood and P. R. Woolcock, eds. American Association of Avian Pathologists, Athens, GA. pp 59–64.

Lauerman LH, Hoerr FJ, Sharpton AR, Shah SM and Van Santen VL (1993). Development and application of a polymerase chain reaction assay for Mycoplasma synoviae. Av. Dis. 37: 829–34.
http://dx.doi.org/10.2307/1592037
PMid:7504919

Lauerman LH (1998). Mycoplasma PCR Assays. In: Nucleic Amplification Assays for Diagnosis of Animal Diseases, Lauerman L.H., ed. American Association of Veterinary Laboratory Diagnosticians, Auburn, AL, USA, 41–52.

Ley DH (2008). Mycoplasma gallisepticum infection. In: Diseases of poultry, Ames, Iowa State University Press. USA, pp. 805–833

McAuliffe L, Ellis RJ, Lawes JR, Ayling RD and Nicholas RAJ (2005). 16S rDNA PCR and denaturing gradient gel electrophoresis; a single generic test for detecting and differentiating Mycoplasma species. J Med Microbiol. 54: 731–739.
http://dx.doi.org/10.1099/jmm.0.46058-0
PMid:16014426

Mohammed HO, Carpenter TE and Yamamoto R (1987). Economic impact of Mycoplasma gallisepticum and M. synoviae in commercial layer flocks. Av. Dis. 31: 477–482.
http://dx.doi.org/10.2307/1590727
http://dx.doi.org/10.2307/1590726
PMid:3675423

Patterson PH (1994). Coping with Mycoplasma gallisepticum. Internew. 7: 1–3.

Pourbakhsh SA, Shokri1 GR, Banani M, Elhamnia F and Ashtari A (2010). Detection of Mycoplasma synoviae infection in broiler breeder farms of Tehran province using PCR and culture methods. Archi.of Raz. Inst. 65(2): 75–81.

Ramadass P, Ananthi R, Senthilkumar TMA, Venkatesh G and Ramaswamy V (2006). Isolation and characterization of Mycoplasma gallisepticum and Mycoplasma synoviae from poultry. Ind. J. Ani. Sci. 76 (10): 796–798.

Sakhaei D, Pourbakhsh SA, Banani M, Lotfi M, Akhlaghi F and Asli E (2009). Using PCR and culture methods for Mycoplasma testing in poliomyelitis vaccine. Archi.of Raz. Inst. 64(2): 109–114.

Sambrook J and Russel DW (2001) Molecular cloning: A laboratory manual. (3rd edn.) Cold spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Thomas CB and Sharp P (1990). Glycoconjugate heterogeneity among five strains of Mycoplasma gallisepticum. Av. Dis. 34:969–978.
http://dx.doi.org/10.2307/1591391
PMid:2282023

Zain ZM and Bradbury JM (1995). The influence of type of swab and laboratory method on the recovery of Mycoplasma gallisepticum and Mycoplasma synoviae in broth medium. Av. Path. 24: 707–716.