Early detection and differential serodiagnosis of Mycoplasma hyorhinis and Mycoplasma hyosynoviae infections under experimental conditions

Autoři: Luis G. Giménez-Lirola aff001;  Henrique Meiroz-De-Souza-Almeida aff002;  Ronaldo L. Magtoto aff001;  Aric J. McDaniel aff001;  Maria M. Merodio aff001;  Franco S. Matias Ferreyra aff001;  Korakrit Poonsuk aff001;  Igor R. H. Gatto aff002;  David H. Baum aff001;  Richard F. Ross aff001;  Paulo H. E. Arruda aff001;  Kent J. Schwartz aff001;  Jeffrey J. Zimmerman aff001;  Rachel J. Derscheid aff001;  Bailey L. Arruda aff001
Působiště autorů: College of Veterinary Medicine, Iowa State University, Ames, IA, United States of America aff001;  São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, SP, Brazil aff002
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0223459


Mycoplasma hyorhinis (MHR) and Mycoplasma hyosynoviae (MHS) are common opportunistic pathogens in the upper respiratory tract and tonsils of swine. The identification of the specific species involved in clinical cases using conventional diagnostic methods is challenging. Therefore, a recombinant chimeric polypeptide based on the seven known variable lipoproteins (A-G) specific of MHR and a cocktail of surface proteins detergent-extracted from MHS cultures were generated and their suitability as antemortem biomarkers for serodiagnosis of MHR- and MHS-infection were evaluated by ELISA. M. hyorhinis and MHS ELISA performance, evaluated using serum samples collected over a 56-day observation period from pigs inoculated with MHR, MHS, M. hyopneumoniae, M. flocculare, or Friis medium, varied by assay, targeted antibody isotype, and cutoffs. The progressions of MHR and MHS clinical diseases were evaluated in relation to the kinetics of the isotype-specific antibody response in serum and bacterial shedding in oral fluids during the observation period. In pigs inoculated with MHR, bacterial DNA was detected in one or more of the 5 pens at all sampling points throughout the study, IgA was first detected at DPI 7, one week before the first clinical signs, with both IgA and IgG detected in all samples collected after DPI 14. The peak of MHS shedding (DPI 8) coincided with the onset of the clinical signs, with both IgA and IgG detected in all serum samples collected ≥ DPI 14. This study demonstrated, under experimental conditions, that both ELISAs were suitable for early detection of specific antibodies against MHR or MHS. The diagnostic performance of the MHR and MHS ELISAs varied depending on the selected cutoff and the antibody isotype evaluated. The high diagnostic and analytical specificity of the ELISAs was particularly remarkable. This study also provides insights into the infection dynamics of MHR-associated disease and MHS-associated arthritis not previously described.

Klíčová slova:

Antibody response – Arthritis – DNA extraction – Enzyme-linked immunoassays – Polymerase chain reaction – Respiratory infections – Swine – Mycoplasma


1. Switzer WP. Studies on infectious atrophic rhinitis. IV. Characterization of a pleuropneumonia-like organism isolated from the nasal cavities of swine. American journal of veterinary research. 1955;16: 540–544. 13259032

2. Ross RF, Karmon JA. Heterogeneity among strains of Mycoplasma granularum and identification of Mycoplasma hyosynoviae. Journal of bacteriology. 1970;103: 707–713. 4991614

3. Baseman JB, Tully JG. Mycoplasmas: sophisticated, reemerging, and burdened by their notoriety. Emerging infectious diseases. 1997;3: 21–32. doi: 10.3201/eid0301.970103 9126441

4. Hayflick F., Chanock R.M., 1965. Mycoplasma species of man. Bacteriol. Rev. 29, 185–221. 14304038

5. Hagedorn-Olsen T, Nielsen NC, Friis NF, Nielsen J. Progression of Mycoplasma hyosynoviae infection in three pig herds. Development of tonsillar carrier state, arthritis and antibodies in serum and synovial fluid in pigs from birth to slaughter. Zentralblatt fur Veterinarmedizin. Reihe A. 1999;46: 555–564.

6. Simecka JW, Davis JK, Davidson MK, Ross SE, Städtlander CTKH, Cassell G.H. Mycoplasma diseases of animals. In: McElhaney R.N., Finch L.R., Baseman J.B., editors. Mycoplasma: Molecular biology and pathologenesis. American Society for Microbiology. Washington, DC; 1992. pp. 391–415.

7. Ross RF. Mycoplasmal diseases. In: Straw B, D’Allaire S, Mengeline W, Taylor D, editors. Diseases of Swine. Iowa State University Press, Ames, IA; 1999. pp. 495–510.

8. Clavijo MJ, Oliveira S, Zimmerman J, Rendahl A, Rovira A. Field evaluation of a quantitative polymerase chain reaction assay for Mycoplasma hyorhinis. Journal of Veterinary Diagnostic Investigation. 2014;26: 755–760. doi: 10.1177/1040638714555175 25319032

9. Clavijo MJ, Murray D, Oliveira S, Rovira A. Infection dynamics of Mycoplasma hyorhinis in three commercial pig populations. Vet. Record. doi: 10.1136/vr.104064 28424318

10. Leuwerke B. 2009. Mycoplasma hyorhinis-Field experiences in diagnosis and control.

11. Friis NF, Feenstra AA. Mycoplasma hyorhinis in the etiology of serositis among piglets. Acta Veterinaria Scandinavica. 1994;35: 93–98. 8209825

12. Kobisch M, Friis NF. Swine mycoplasmoses. Revue Scientifique et Technique-Office International des Epizooties. 1996;15:1569–1614.

13. Lin JH, Chen SP, Yeh KS, Weng CN. Mycoplasma hyorhinis in Taiwan: diagnosis and isolation of swine pneumonia pathogen. Veterinary microbiology. 2006;115: 111–116. doi: 10.1016/j.vetmic.2006.02.004 16540266

14. Rovira A. Review of Mycoplsma hyorhinis. Proceedings of the 36th Allen Leman Swine Conference. St. Paul, MN; 2009. pp. 87–88.

15. Gomes-Neto JC, Gauger PC, Strait EL, Boyes N, Madson DM, Schwartz KJ. Mycoplasma-associated arthritis: critical points for diagnosis. J Swine Health Prod. 2012;20: 82–86.

16. Goiš M, ČErný M, Rozkošný V, Sovadina M. Studies on the epizootiological significance of some species of mycoplasma isolated from nasal swabs and lungs of pigs. Zentralblatt für Veterinärmedizin Reihe B. 1969;16: 253–265.

17. Ross RF, Whittlestone P. Recovery of identification of, and serological response to porcine mycoplasmas. Methods in mycoplasmology. 2012;2: 115–127.

18. Friis NF, Ahrens P, Larsen H. Mycoplasma hyosynoviae isolation from the upper respiratory tract and tonsils of pigs. Acta Veterinaria Scandinavica. 1991;32: 425–429. 1818502

19. Makhanon M, Tummaruk P, Thongkamkoon P, Thanawongnuwech R, Prapasarakul N. Comparison of detection procedures of Mycoplasma hyopneumoniae, Mycoplasma hyosynoviae, and Mycoplasma hyorhinis in lungs, tonsils, and synovial fluid of slaughtered pigs and their distributions in Thailand. Tropical animal health and production. 2012;44: 313–318. doi: 10.1007/s11250-011-0022-z 22116666

20. Hariharan H, MacDonald J, Carnat B, Bryenton J, Heaney S. An investigation of bacterial causes of arthritis in slaughter hogs. Journal of veterinary diagnostic investigation. 1992;4: 28–30. doi: 10.1177/104063879200400107 1554766

21. Stemke GW, Phan R, Young TF, Ross RF. Differentiation of Mycoplasma hyopneumoniae, M flocculare, and M hyorhinis on the basis of amplification of a 16S rRNA gene sequence. American journal of veterinary research. 1994;55: 81–84. 8141501

22. Caron J, Ouardani M, Dea S. Diagnosis and differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR amplification of the p36 and p46 genes. Journal of clinical microbiology. 2000;38: 1390–1396. 10747113

23. Timenetsky J, Santos LM, Buzinhani M, Mettifogo E. Detection of multiple mycoplasma infection in cell cultures by PCR. Brazilian journal of medical and biological research. 2006;39: 907–914. doi: 10.1590/s0100-879x2006000700009 16862282

24. Kang I, Kim D, Han K, Seo HW, Oh Y, Park C, et al. Optimized protocol for multiplex nested polymerase chain reaction to detect and differentiate Haemophilus parasuis, Streptococcus suis, and Mycoplasma hyorhinis in formalin-fixed, paraffin-embedded tissues from pigs with polyserositis. Canadian journal of veterinary research. 2012;76: 195–200. 23277698

25. Gomes-Neto JC, Bower L, Erickson BZ, Wang C, Raymond M, Strait EL. Quantitative real-time polymerase chain reaction for detecting Mycoplasma hyosynoviae and Mycoplasma hyorhinis in pen-based oral, tonsillar, and nasal fluids. Journal of veterinary science. 2015;16: 195–201. doi: 10.4142/jvs.2015.16.2.195 25643803

26. Ross RF, Switzer WP. Comparison of isolates of Mycoplasma hyorhinis by indirect hemagglutination. American journal of veterinary research. 1963;24: 622–627. 13975123

27. Goiŝ M, Franz J, Kuksa F, Pokorný J, Ĉerný M. Immune response of gnotobiotic piglets infected with Mycoplasma hyorhinis. Zentralblatt für Veterinärmedizin Reihe B. 1972;19: 379–390.

28. Ross RF, Dale SE, Duncan JR. Experimentally induced Mycoplasma hyorhinis arthritis of swine: immune response to 26th postinoculation week. American journal of veterinary research. 1973;34: 367–372. 4691485

29. Binder A, Likitdecharote B, Kirchhoff H. Fluorescence serological and culture detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis in swine lungs. Berliner und Munchener tierarztliche Wochenschrift. 1989;102: 11–13. 2647076

30. Zimmermann BJ, Ross RF. Modified metabolic-inhibition test for detection of antibodies to Mycoplasma hyosynoviae in swine serum. American journal of veterinary research. 1977;38: 2075–2076. 596706

31. Zimmermann BJ, Ross RF. Antibody response of swine experimentally infected with Mycoplasma hyosynoviae. Veterinary microbiology. 1982;7: 135–146. doi: 10.1016/0378-1135(82)90025-6 7112893

32. Pedersen MW, Webster P, Lind P, Hagedorn-Olsen T, Friis NF. Detection of antibodies against Mycoplasma hyosynoviae using a monoclonal antibody-based ELISA. IOM Lett. 1996;4: 90.

33. Hagedorn-Olsen T, Basse A, Jensen TK, Nielsen NC. Gross and histopathological findings in synovial membranes of pigs with experimentally induced Mycoplasma hyosynoviae arthritis. Apmis. 1999;107: 201–210. 10225318

34. Nielsen EO, Lauritsen KT, Friis NF, Enøe C, Hagedorn-Olsen T, Jungersen G. Use of a novel serum ELISA method and the tonsil-carrier state for evaluation of Mycoplasma hyosynoviae distributions in pig herds with or without clinical arthritis. Veterinary microbiology. 2005;111: 41–50. doi: 10.1016/j.vetmic.2005.08.009 16171955

35. Freeman MJ, Armstrong CH, Sands-Freeman LL, Lopez-Osuna M. Serological cross-reactivity of porcine reference antisera to Mycoplasma hyopneumoniae, M. flocculare, M. hyorhinis and M. hyosynoviae indicated by the enzyme-linked immunosorbent assay, complement fixation and indirect hemagglutination tests. Canadian journal of comparative medicine. 1984;48: 202–207. 6372971

36. Neto JC, Strait EL, Raymond M, Ramirez A, Minion FC. Antibody responses of swine following infection with Mycoplasma hyopneumoniae, M. hyorhinis, M. hyosynoviae and M. flocculare. Veterinary microbiology. 2014;174: 163–171.

37. Kobisch M., 1983. Pathogenicity of Mycoplasma hyorhinis. Yale J. Biol. Med. 56, 922–923.

38. Strasser M, Abiven P, Kobisch M, Nicolet J. Immunological and pathological reactions in piglets experimentally infected with Mycoplasma hyopneumoniae and/or Mycoplasma flocculare. Veterinary immunology and immunopathology. 1992;31: 141–153. doi: 10.1016/0165-2427(92)90092-5 1570675

39. González W, Giménez-Lirola LG, Holmes A, Lizano S, Goodell C, Poonsuk K, et al. Detection of Actinobacillus pleuropneumoniae ApxIV toxin antibody in serum and oral fluid specimens from pigs inoculated under experimental conditions. Journal of veterinary research. 2017;61: 163–171. doi: 10.1515/jvetres-2017-0021 29978069

40. Ross RF, Switzer WP, Duncan JR. Experimental production of Mycoplasma hyosynoviae arthritis in swine. American journal of veterinary research. 1971;32: 1743–1749. 4941436

41. Friis NF. Mycoplasmas cultivated from the respiratory tract of Danish pigs. Acta Veterinaria Scandinavica. 1971;12:69. 5575144

42. Prickett J, Simer R, Christopher-Hennings J, Yoon KJ, Evans RB, Zimmerman JJ. Detection of Porcine reproductive and respiratory syndrome virus infection in porcine oral fluid samples: a longitudinal study under experimental conditions. Journal of Veterinary Diagnostic Investigation. 2008;20: 156–163. doi: 10.1177/104063870802000203 18319427

43. Strait EL, Madsen ML, Minion FC, Christopher-Hennings J, Dammen M, Jones KR, et al. Real-time PCR assays to address genetic diversity among strains of Mycoplasma hyopneumoniae. Journal of clinical microbiology. 2008;46: 2491–2498. doi: 10.1128/JCM.02366-07 18524960

44. Stakenborg T, Vicca J, Butaye P, Imberechts H, Peeters J, De Kruif A, et al. A multiplex PCR to identify porcine mycoplasmas present in broth cultures. Veterinary research communications. 2006;30: 239–247. doi: 10.1007/s11259-006-3226-3 16437299

45. Xiong Qi-Yan Ji Yan, Liu Zhan-Jun Ma Qing-Hong, Feng Zhi-Xin Liu Mao-Jun, et al. Cloning, prokaryotic expression and reactogenicity analysis of a recombinant protein for detecting Mycoplasma hyorhinis. Chinese Journal of Zoonoses. 2013;29: 877–882.

46. Turner GV. A microbiological study of polyarthritis in slaughter pigs. Journal of the South African Veterinary Association. 1982;53: 99–101. 6750122

47. Rosengarten R, Wise KS. Phenotypic switching in mycoplasmas: phase variation of diverse surface lipoproteins. Science. 1990;247: 315–318. doi: 10.1126/science.1688663 1688663

48. Rosengarten RE, Wise KS. The Vlp system of Mycoplasma hyorhinis: combinatorial expression of distinct size variant lipoproteins generating high-frequency surface antigenic variation. Journal of bacteriology. 1991;173: 4782–4793. doi: 10.1128/jb.173.15.4782-4793.1991 1856172

49. Yogev D, Rosengarten R, Watson‐McKown R, Wise KS. Molecular basis of Mycoplasma surface antigenic variation: a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5′ regulatory sequences. The EMBO Journal. 1991;10: 4069–4079. 1721868

50. Cleavinger CM, Kim MF, Wise KS. Processing and surface presentation of the Mycoplasma hyorhinis variant lipoprotein VlpC. Journal of bacteriology. 1994;176: 2463–2467. doi: 10.1128/jb.176.8.2463-2467.1994 7512554

51. Yogev D, Watson-McKown R, Rosengarten R, Im J, Wise KS. Increased structural and combinatorial diversity in an extended family of genes encoding Vlp surface proteins of Mycoplasma hyorhinis. Journal of bacteriology. 1995;177: 5636–5643. doi: 10.1128/jb.177.19.5636-5643.1995 7559353

52. Citti C, Watson-McKown R, Droesse M, Wise KS. Gene families encoding phase-and size-variable surface lipoproteins of Mycoplasma hyorhinis. Journal of Bacteriology. 2000;182: 1356–1363. doi: 10.1128/jb.182.5.1356-1363.2000 10671459

53. Rotolo ML, Giménez-Lirola L, Ji J, Magtoto R, Henao-Díaz YA, Wang C, et al. Detection of porcine reproductive and respiratory syndrome virus (PRRSV)-specific IgM-IgA in oral fluid samples reveals PRRSV infection in the presence of maternal antibody. Veterinary microbiology. 2018;214: 13–20. doi: 10.1016/j.vetmic.2017.11.011 29408024

54. Evans AS. Causation and Disease: The Henle-Koch Postulates Revisited. Yale J Biol Med. 1976;49: 175–195. 782050

55. Friis NF. Mycoplasma suipneumoniae and Mycoplasma flocculare in comparative pathogenicity studies. Acta Veterinaria Scandinavica (Denmark). 1974;15: 507–518.

56. Armstrong CH, Freeman MJ, Sands-Freeman L. Cross-reactions between Mycoplasma hyopneumoniae and Mycoplasma flocculare—practical implications for the serodiagnosis of mycoplasmal pneumonia of swine. Israel journal of medical sciences. 1987;23: 654–656. 3667232

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