Article history: Received: 05-10-2018, Accepted: 20-02-2019, Published online: 14-04-2019
Corresponding author: Mohamed M. Amer
E-mail: firstname.lastname@example.orgCitation: Amer MM, Mekky HM, Fedawy HS (2019) Molecular identification of Mycoplasma synoviae from breeder chicken flock showing arthritis in Egypt, Veterinary World, 12(4): 535-541.
Aim: Arthritis is one of the most economic problems facing poultry industry worldwide. The study was done to detect possible causes of arthritis in breeder chicken flock with emphasis on molecular identification of Mycoplasma synoviae (MS).
Materials and Methods: This study was carried on chicken from broiler breeder flock of 57 weeks' age in Dakahlia, Egypt, suffered from arthritis with frequently 5-7% decrease in egg production, reduced fertility, and hatchability. Forty blood samples were randomly collected from individual birds in sterile tubes and used for serum separation. Serum samples were tested using serum plate agglutination (SPA) test against colored antigens for Mycoplasma gallisepticum (MG), MS, and Salmonella gallinarum-pullorum (SGP). On the other hand, 24 joint samples were collected. Of those 24 samples, 12 joint samples were subjected to bacteriological examination, while the other 12 were utilized for molecular diagnosis by polymerase chain reaction (PCR) for MS and avian reovirus (ARV).
Results: SPA test results revealed the presence of antibodies against MG, MS, and SGP in tested sera in rates of 14/40 (35%), 35/40 (87.5%), and 9/40 (22.5%), respectively. Furthermore, 19 bacterial isolates were recognized from joint samples and identified as five Staphylococcus spp., nine Escherichia coli, three SGP, one Citrobacter, and one Proteus. The identified Staphylococcal isolates were three coagulase-positive staphylococci (two Staphylococcus aureus and one Staphylococcus hyicus) and two coagulase-negative staphylococci (one Staphylococcus epidermidis and one Staphylococcus lentus), while E. coli isolate serotypes were 1 O11, 2 O55, 3 O78, 1 O124, 1 O125, and 1 untyped. PCR proved that 12/12 (100%) samples were positive for MS variable lipoprotein hemagglutinin A (vlhA) gene, while ARV was not diagnosed in any of the examined samples. Four amplified vlhA gene of MS isolates (named MS-2018D1, MS-2018D2, MS-2018D3, and MS-2018D4) was successfully sequenced.
Analysis of phylogenetic tree revealed the presence of 100% identity between each two sequenced isolates (isolates MS-2018D1 and MS-2018D4 and also isolates 2018D2 and MS-2018D3). However, the nucleotide similarity between four isolates was 88.6%. On the other hand, our field isolates MS-2018D1, MS-2018D4, MS-2018D2, and MS-2018D3 showed nucleotide identity with vaccine strain MS-H 98.4%, 98.4%, 88.1%, and 88.1%, respectively. Furthermore, the nucleotide similarities with field strains from Argentina ranged between 87.8% and 98.6%.
Conclusion: Four field isolates of MS were identified in examined broiler breeder flock. A phylogenetic study of these isolates revealed the variation between isolated MS strains and vaccine strain. Therefore, further studies are required for evaluating the vaccine efficacy against the present field isolates of MS. In addition, application of MS immunization of breeder flocks is necessary for proper control of the disease.
Keywords: arthritis, Mycoplasma synoviae variable lipoprotein hemagglutinin A gene, Mycoplasma synoviae, polymerase chain reaction.
1. Manohar, G.R., Omprakash, A.V. and Kanagaraju, P. (2015) Leg weakness in commercial broiler chicken an overview. Int. J. Sci. Environ. Technol., 4(2): 482-487.
2. Reece, R.L. (1992) The role of infectious agents in leg abnormalities in growing birds. In: Whitehead, C.C., editor. Bone Biology and Skeletal Disorders in Poultry. Carfax Publishing Ltd., Abingdon. p231-263.
3. Jiang, T., Mandal, R.K., Wideman, R.F Jr., Khatiwara, A., Pevzner, I. and Kwon, Y.M. (2015) Molecular survey of bacterial communities associated with bacterial chondronecrosis with osteomyelitis (BCO) in broilers. PLoS One, 10(4): e0124403. [Crossref]
4. Kieronczyk, B., Rawski, M., Jozefiak, D. and Swiatkiewicz, S. (2017) Infectious and non-infectious factors associated with leg disorders in poultry a review. Ann. Anim. Sci., 17(3): 645-669. [Crossref]
5. Wijesurendra, D.S., Chamings, A.N., Bushell, R.N., Rourke, D.O., Stevenson, M., Marenda, M.S., Noormohammadi, A.H. and Stent, A. (2017) Pathological and microbiological investigations into cases of bacterial chondronecrosis and osteomyelitis in broiler poultry. Avian Pathol., 46(6): 683-694. [Crossref] [PubMed]
6. Ferguson-Noel, N. and Noormohammadi, A.H. (2013) Mycoplasma synoviae infection. In: Swayne, D.E., Glisson, J.R., McDougald, L.R., Nolan, L.K., Suarez, D.L. and Nair, V., editors. Diseases of Poultry. 13th ed. Blackwell Publishing, United Status. p900-910.
7. Catania, S., Bilato, D., Gobbo, F., Granato, A., Terregino, C., Iob, L. and Nicholas, R.A. (2010) Treatment of eggshell abnormalities and reduced egg production caused by Mycoplasma synoviae infection. Avian Dis., 54(2): 961-964. [Crossref] [PubMed]
8. Jeon, E.O., Kim, J.N., Lee, H.R., Koo, B.S., Min, K.C., Han, M.S., Lee, S.B., Bae, Y.J., Mo, J.S., Cho, S.H., Lee, C.H. and Mo, I.P. (2014) Eggshell apex abnormalities associated with Mycoplasma synoviae infection in layers. J. Vet. Sci., 15(4): 579-582. [Crossref] [PMC]
9. Sumitha, P. and Sukumar, K. (2017) Incidence of Mycoplasma synoviae induced eggshell apex abnormality in desi chicken. Indian Vet. J., 94(5): 36-37.
10. Morrow, C.J., Bell, I.G., Walker, S.B., Markham, P.F., Thorp, B.H. and Whithear, K.G. (1990) Isolation of Mycoplasma synoviae from infectious synovitis of chickens. Aus. Vet. J., 67(4): 121-124. [Crossref]
11. Landman, W.J.M. and Feberwee, A. (2001) Field studies on the association between amyloid arthropathy and Mycoplasma synoviae infection, and experimental reproduction of the condition in brown layers. Avian Pathol., 30(6): 629-639. [Crossref] [PubMed]
12. Landman, W.J.M. and Feberwee, A. (2012) Longitudinal field study on the occurrence of Mycoplasma synoviae in Dutch Turkey flocks with lameness and experimental induction of the condition. Avian Pathol., 41(2): 141-149. [Crossref] [PubMed]
13. Stanley, W.A., Hofacre, C.L., Speksnijder, G., Kleven, S.H. and Aggrey, S.E. (2001) Monitoring Mycoplasma gallisepticum and Mycoplasma synoviae infection in breeder chickens after treatment with enrofloxacin. Avian Dis., 45(2): 534-539. [Crossref]
14. Uddin, M.I., Abid, M.H., Islam, M.S., Rakib, T.M., Sen, A.B., Chowdhury, S.M.Z., Anwar, M.N. and Kamaruddin, K.M. (2016) Molecular identification of Mycoplasma synoviae from seroprevalent commercial breeder farms at Chittagong district, Bangladesh. Vet. World, 9(10): 1063-1069. [Crossref] [PubMed] [PMC]
15. Malekhoseini, G., Pourbakhsh, S.A., Homayounimehr, A.R., Zolfeghari, M.R., Ashtari, A. and Abtin A.R. (2017) Simultaneous identification of Mycoplasma gallisepticum and Mycoplasma synoviae by duplex PCR assay. Immunol. Case Rep., 1(1): 12-16.
16. Tomar, P., Singh, Y., Mahajan, N.K., Jindal, N. and Singh, M. (2017) Molecular detection of avian mycoplasmas in poultry affected with respiratory infections in Haryana (India). Int. J. Curr. Microbiol. App. Sci., 6(6): 2155-2162. [Crossref]
17. Rachida, A., Omar, B., Elhacene, B., Rachid, K. and Cherif, A.M. (2013) Serological investigation on avian mycoplasmosis in laying hen farms in Eastern Algeria. IJAVMS, 7(5): 170-177. [Crossref]
18. Hong, Y., Garcia, M., Leiting, V., Bencina, D., Dufour-Zavala, L., Zavala, G. and Kleven, S.H. (2004) Specific detection and typing of Mycoplasma synoviae strains in poultry with PCR and DNA sequence analysis targeting the hemagglutinin encoding gene vlhA. Avian Dis., 48(3): 606-616. [Crossref] [PubMed]
19. Senthilnathan, G., Shenbagam, S., Suryanarayana, T. and Thiyageeswaran, M. (2015) Isolation and molecular confirmation of Mycoplasma synoviae infection from broiler breeder farms in Tamilnadu. Indian J. Anim. Res., 49(1): 91-94. [Crossref]
21. Jones, R.C. (2019) Overview of Viral Arthritis in Poultry, Merck Veterinary Manual. Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.
22. Jones, R.C. (2013) Reovirus infections viral arthritis. In: Swayne, D.E., Glisson, J.R., McDougald, L.R., Nolan, L.K., Suarez, D.L. and Nair, V.L., editors. Diseases of Poultry. 13th ed. Iowa State University Press, Ames. p351-373.
23. Wen, C., Zhong, Q., Zhang, J., Lu, J., Zhang, L., Yuan, X., Gan, M., Cai, X. and Zhang, G. (2016) Sequence and phylogenetic analysis of chicken reoviruses in China. J. Integr. Agric., 15(8): 1846-1855. [Crossref]
24. Moreira, F.A., Cardoso, L. and Coelho, A.C. (2017) Review article: Mycoplasma synoviae and Reovirus: Reemerging infectious diseases in broiler breeders. J. Hell. Vet Med. Soc., 68(2): 113-122. [Crossref]
25. Jones, R.C. (2000) Avian reovirus infections. Rev. Sci. Tech., 19(2): 614-625. [Crossref]
26. Bruhn, S., Bruckner, L. and Ottiger, H.P. (2005) Application of rt-PCR for the detection of avian reovirus contamination in avian viral vaccines. J. Virol. Methods, 123(2): 179-186. [Crossref] [PubMed]
27. Teng, L., Xie, Z., Xie, L., Liu, J., Pang, Y., Deng, X., Xie, Z., Fan, Q., Luo, S., Feng, J. and Khan, M.I. (2014) Sequencing and phylogenetic analysis of an avian reovirus genome. Virus Genes, 48(2): 381-386. [Crossref] [PubMed]
28. Hedayati, M., Shoojadost, B., Peighambari, S.M. and Langeroudi, A.G. (2016) Characterization of reoviruses isolated from some broiler breeder flocks in Iran. Arch. Razi Inst., 71(4): 227-234.
29. Ayalew, L.E., Gupta, A., Fricke, J., Ahmed, K.A., Popowich, S., Lockerbie, B., Tikoo, S.K., Ojkic, D. and Gomis, S. (2017) Phenotypic, genotypic and antigenic characterization of emerging avian reoviruses isolated from clinical cases of arthritis in broilers in Saskatchewan, Canada. Sci. Rep., 7(1): 3565. [Crossref]
30. Souza, S.O., De Carli, S., Lunge, V.R., Ikuta, N., Canal, C.W., Pavarini, S.P. and Driemeier, D. (2018) Pathological and molecular findings of avian reoviruses from clinical cases of tenosynovitis in poultry flocks from Brazil. Poult. Sci., 97(10): 3550-3555. [Crossref] [PubMed]
31. National Poultry Improvement Plan and Auxiliary Provisions. (2011) The National Poultry Improvement Plan. United States Department of Agriculture, Animal and Plant Health Inspection Service, Hyattsville, MD.
32. Cruickshank, R., Duguid, P., Marmion, B.D. and Swain, R.H.A. (1975) Medical Microbiology. 12th ed., Vol. 2. Churchill Living-Stone, Edinburgh, London, New York.
33. Edwards, P.R. and Ewing, W.H. (1972) Identification of Enterobacteriaceae. Burgess Publishing Co., Minneapolis, Minnesota.
34. MaCffaddin, J.E. (1980) Biochemical Tests for Identification of Medical Bacteria. 2nd ed. Williams and Wilkins Co., Baltimore, USA.
35. Quinn, P.J., Markey, B.K., Leonard, F.C., FitzPatrick, E.S., Fanning S. and Hartigan, P.J. (2011) Veterinary Microbiology and Microbial Disease. 2nd ed. New Jersey: Wiley-Blackwell.
36. Neville, J. and Brgant, A.F. (1986) Laboratory and Serology. 2nd ed. Saunder Co., Toronto, Canada.
37. Lee, M.D. and Arp. L.H. (1998) Colibacillosis. In: Swayne, D.E., Glisson, J.R., Jackwood, M.W., Pearson, M.W. and Reed, W.M., editors. A Laboratory Manual for the Isolation and Identification of Avian Pathogens. American Association of Avian Pathologists, Kennett Square, PA. p14-16.
38. Ferguson-Noel, N., Laibinis, V.A. and Farrar, M. (2012) Influence of swab material on the detection of Mycoplasma gallisepticum and Mycoplasma synoviae by real-time PCR. Avian Dis., 56(2): 310-314. [Crossref] [PubMed]
39. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipmanl, D.J. (1990) Basic local alignment search tool. J. Mol. Biol., 215(3): 403-410. [Crossref]
40. Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22(22): 4673-4680. [Crossref]
41. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013) MEGA 6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol., 30(12): 2725-2759. [Crossref] [PubMed] [PMC]
42. Nazia, K.K.M, Durrani, N.U., Kamboh, A.A.A., Lakho, S.A., Rind, R.R., Abro, S.H. and Soomro, N.M. (2015) Prevalence of septic arthritis caused by Staphylococcus aureus in poultry birds at Tando Jam, Pakistan. J. Anim. Health Prod., 3(3): 73-77. [Crossref]
43. Amer, M.M. and El-Ghany, W.A.A (2006) Bacterial causes of decrease in performance of the breeder chicken flocks. Beni Suef Vet. Med. J., 16(1): 61-69.
44. Silva, C.B.C., Chagas, W.F.I., Santos, R.F.I., Gomes, L.R.I., Ganda, M.R.I. and Lima, A.M.C. (2015) Seroprevalence of Salmonella and Mycoplasma in commercial broilers, backyard chickens, and spent hens in the region of Triangulo Mineiro, state of Minas Gerais, Brazil. Rev. Bras. Cienc. Avic., 17(1): 57-62. [Crossref]
45. Rasheed, B.Y. (2011) Isolation and identification of bacteria causing arthritis in chickens. Iraqi J. Vet. Sci., 25(2): 93-95. [Crossref]
46. Braga, J.F.V., Chanteloup, N.K., Trotereau, A., Baucheron, S., Guabiraba, R., Ecco, R. and Schouler, C. (2016) Diversity of Escherichia coli strains involved in vertebral osteomyelitis and arthritis in broilers in Brazil. BMC Vet. Res., 12(1): 140. [Crossref]
47. Tawfik, R.G., Khalil, S.A., Ellakany, H.F. and Torky, H.A. (2016) Mycoplasma Synoviae and other associated bacteria causing arthritis in chicken. Alex. J. Vet. Sci., 49(2): 163-169.
48. Moreira, F.A., Cardoso, L. and Coelho, A.C. (2015) Epidemiological survey on Mycoplasma synoviae infection in Portuguese broiler breeder flocks. Vet. Ital., 51(2): 93-98. [PubMed]
49. Rasool, A., Anjum, A.A., Rabbani, M., Lateef, M., Akhter, F., Afroz, H., Muhammad, J. and Nawaz, M. (2018) Molecular characterization and phylogenetic analysis of Mycoplasma synoviae isolated from chicken. J. Anim. Plant Sci., 28(2): 491-497.
50. Mahmoud, H., Armanious, W., Elenbawy, M., Elhariri, M., Elhelw, R. and El-Din, T.S. (2016) The recovery and molecular diagnosis of Mycoplasma gallisepticum infection in commercial poultry flocks in Egypt. Indian J. Sci. Technol., 9(29). Available from: http://www.indjst.org/index.php/indjst/article/view/96001/72080. Last accessed on 01-04-2019.
51. Farouk, H., Nasef, S.A., Erfan, A.M. and El-Enbaawy, M.H. (2017) Molecular detection of Mycoplasma spp. In wild and domestic birds by rapid multiplex real-time PCR. Biosci. Res., 14(4): 1019-1023.52. Reck, C., Menin, A., Canever, M.F. and Miletti, L.C. (2013) Rapid detection of Mycoplasma synoviae and avian reovirus in clinical samples of poultry using multiplex PCR. Avian Dis., 57(2): 220-224. [Crossref] [PubMed]