Avian Mycoplasmosis in Poultry: Mycoplasma gallisepticum and M. synoviae Control

Introduction

Avian mycoplasmosis represents a significant economic burden on the global poultry industry. The two most clinically relevant species are Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). MG is the primary etiological agent of chronic respiratory disease (CRD) in chickens and infectious sinusitis in turkeys. MS causes subclinical upper respiratory infections, synovitis, and eggshell apex abnormalities in laying hens. Both pathogens are classified under the class Mollicutes, characterized by the absence of a cell wall, a reduced genome size, and a dependence on host-derived nutrients. This article provides an exhaustive review of the biology, transmission, diagnostic approaches, and integrated control strategies for MG and MS in commercial poultry flocks.

Pathogen Biology and Host Cell Interactions

Mycoplasmas are the smallest self-replicating prokaryotes. Their lack of a peptidoglycan cell wall renders them intrinsically resistant to beta-lactam antimicrobials and necessitates the use of alternative antibiotic classes. The genome of MG is approximately 1.0 Mb, while MS has a slightly smaller genome of about 0.95 Mb. Both species possess a limited metabolic capacity and rely on the host for essential precursors such as cholesterol and nucleic acids.

The primary mechanism of pathogenesis involves adhesion to host epithelial cells via specialized surface proteins. MG expresses a family of cytadhesins, most notably GapA and CrmA, which mediate attachment to ciliated respiratory epithelial cells. This attachment disrupts mucociliary clearance, leading to ciliostasis, epithelial cell death, and an inflammatory cascade. MS utilizes homologous adhesins, including the variable lipoprotein hemagglutinin (VlhA) family, which undergoes high-frequency phase variation to evade the host immune response. This antigenic variation complicates serological diagnosis and vaccine development.

Following adhesion, mycoplasmas secrete hydrogen peroxide and superoxide radicals, causing oxidative damage to host cell membranes. They also modulate the host immune response by inducing pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). The resulting infiltration of heterophils and macrophages into the respiratory mucosa and air sacs produces the characteristic lesions of CRD: caseous exudate, airsacculitis, and perihepatitis. In MS infections, the pathogen can disseminate from the respiratory tract to the synovial membranes and joints, leading to tenosynovitis and arthritis.

Transmission and Epidemiology

Transmission of MG and MS occurs through both horizontal and vertical routes. Horizontal transmission is primarily via the respiratory route through aerosolized droplets, direct contact between birds, and contaminated fomites including feed, water, and equipment. The pathogens can survive for several hours to days on organic material, depending on temperature and humidity. Vertical transmission is a critical feature of mycoplasma epidemiology. Infected breeder hens can transmit the organism transovarially through the egg, leading to infected progeny at hatch. This vertical route perpetuates infection within integrated poultry operations.

Risk factors for flock infection include multi-age production systems, high stocking density, poor ventilation, and concurrent infections with respiratory viruses such as Newcastle disease virus or infectious bronchitis virus. Coinfections with Escherichia coli (see Avian Pathogenic Escherichia coli (APEC): Virulence Factors, Rapid Diagnostic Assays, and Biosecurity Strategies) exacerbate the severity of CRD. Wild birds and fomites from contaminated poultry houses serve as reservoirs for reintroduction into naive flocks.

Clinical Signs and Pathological Findings

Mycoplasma gallisepticum

In chickens, MG infection typically presents as CRD with clinical signs including rales, coughing, sneezing, nasal discharge, and conjunctivitis. In laying hens, there is a marked drop in egg production, often by 10 to 30 percent, and an increase in eggshell quality defects. In turkeys, MG causes infectious sinusitis characterized by infraorbital sinus swelling, dyspnea, and severe airsacculitis. Gross pathological findings include catarrhal tracheitis, fibrinous airsacculitis, and perihepatitis. Histologically, there is lymphoid hyperplasia in the tracheal mucosa and infiltration of mononuclear cells in the air sacs.

Mycoplasma synoviae

MS infections are often subclinical in the respiratory tract. Clinical disease manifests primarily as synovitis, with lameness, swollen joints (hock and wing joints), and breast blisters. In layers, MS is a major cause of eggshell apex abnormalities, characterized by a roughened, thin, and cracked region at the pointed end of the egg. Gross lesions include tenosynovitis, arthritis, and fibrinous exudate in the joint spaces. Respiratory lesions are milder than those caused by MG but can include airsacculitis when exacerbated by environmental stressors or viral coinfection.

Diagnostic Approaches

Accurate diagnosis of avian mycoplasmosis relies on a combination of clinical observation, serology, molecular detection, and culture. The choice of diagnostic method depends on the purpose of testing, such as flock surveillance, confirmation of clinical disease, or certification for trade.

Serological Diagnostics

Serological assays detect antibodies against MG and MS. The most widely used methods are the rapid serum agglutination (RSA) test and the enzyme-linked immunosorbent assay (ELISA). The RSA test is a simple, inexpensive, and rapid screening tool. It uses stained antigen to detect agglutinating antibodies in serum or plasma. However, it has lower specificity due to cross-reactivity with other mycoplasma species and false positives from nonspecific agglutinins. The ELISA offers higher throughput and quantitative results. Commercial ELISA kits are available for both MG and MS, and they provide species-specific detection with improved sensitivity and specificity compared to RSA. The hemagglutination inhibition (HI) test is used as a confirmatory assay, particularly for MG, due to its high specificity. HI titers correlate well with protective immunity and are used to evaluate vaccine responses.

Molecular Diagnostics

Polymerase chain reaction (PCR) assays have become the gold standard for direct detection of mycoplasma DNA. Conventional PCR targeting the 16S rRNA gene or species-specific genes such as mgc2 for MG and vlhA for MS provides rapid and sensitive detection. Real-time quantitative PCR (qPCR) allows for quantification of pathogen load and is particularly useful for monitoring the efficacy of treatment or vaccination. Multiplex PCR panels can simultaneously detect MG, MS, and other respiratory pathogens. High-resolution melt (HRM) analysis of PCR products can differentiate MG and MS strains based on sequence variations in the vlhA gene. For epidemiological investigations, multilocus sequence typing (MLST) and whole-genome sequencing (WGS) provide high-resolution genotyping to trace transmission pathways and identify antimicrobial resistance markers.

Culture and Isolation

Mycoplasma culture is the definitive diagnostic method but is technically demanding and time-consuming. Samples are collected from tracheal swabs, air sac exudate, or joint fluid and inoculated into specialized liquid media such as Frey's medium or Hayflick's medium. Cultures are incubated at 37 degrees Celsius in a 5 to 10 percent carbon dioxide atmosphere for up to 14 days. Positive cultures show a characteristic "fried egg" colony morphology on solid agar. Biochemical tests, such as glucose fermentation and arginine hydrolysis, help differentiate MG from MS. However, culture sensitivity is low, especially in flocks with prior antibiotic exposure, and it is rarely used for routine surveillance.

Diagnostic Workflow

The following Mermaid diagram illustrates a typical diagnostic workflow for avian mycoplasmosis in a commercial poultry flock.

flowchart TD
    A[Clinical Signs: Respiratory distress, lameness, egg drop], > B[Sample Collection: Tracheal swabs, serum, joint fluid]
    B, > C{Initial Screening}
    C, > D[Serology: RSA or ELISA]
    C, > E[Direct Detection: PCR on swabs]
    D, > F{Positive?}
    E, > G{Positive?}
    F, > H[Confirmatory HI Test]
    G, > I[Species Identification: MG vs MS via qPCR]
    H, > J[Interpretation: Recent infection or vaccination]
    I, > K[Genotyping: MLST or WGS for epidemiology]
    J, > L[Report and Control Decision]
    K, > L
    L, > M[Biosecurity, Vaccination, or Antimicrobial Therapy]

Vaccination Strategies

Vaccination is a cornerstone of MG and MS control in commercial poultry, particularly in multi-age layer and breeder flocks. Both live attenuated and inactivated (bacterin) vaccines are available.

Live Attenuated Vaccines

Live vaccines for MG include the F strain, ts-11, and 6/85 strains. The F strain is moderately virulent and is used in areas with endemic MG. It provides strong protection against respiratory disease and reduces egg production losses. The ts-11 and 6/85 strains are temperature-sensitive mutants with lower virulence. They are safer for use in young birds and provide good protection against field challenge. Live MS vaccines, such as the MS-H strain, are temperature-sensitive and administered via eye drop or spray. They reduce the incidence of synovitis and eggshell apex abnormalities. Live vaccines are typically administered to pullets between 6 and 12 weeks of age. They induce both humoral and cell-mediated immunity. A key limitation is the potential for reversion to virulence, although this risk is low for the ts-11 and MS-H strains.

Inactivated Vaccines

Bacterin vaccines are oil-adjuvanted preparations of whole inactivated mycoplasma cells. They are administered intramuscularly or subcutaneously to breeder hens prior to the onset of lay. Inactivated vaccines induce high levels of circulating antibodies that are transferred maternally to progeny, providing passive protection during the first weeks of life. They do not prevent colonization but reduce clinical disease and vertical transmission. Bacterins are often used in combination with live priming vaccines in a prime-boost regimen to enhance immunity.

Vaccine Efficacy and Limitations

Vaccine efficacy is influenced by the antigenic match between the vaccine strain and the circulating field strain. The high antigenic variability of MS, driven by VlhA phase variation, can reduce the effectiveness of a single vaccine strain. Autogenous vaccines, prepared from farm-specific isolates, are sometimes used in problem flocks. Vaccination does not eliminate the pathogen from a flock; it reduces clinical signs and shedding. Therefore, vaccination must be integrated with strict biosecurity and monitoring programs.

Antimicrobial Therapy

Antimicrobials are used to treat clinical mycoplasmosis and to reduce shedding during outbreaks. Because mycoplasmas lack a cell wall, antibiotics that target cell wall synthesis, such as beta-lactams, are ineffective. Effective classes include macrolides (tylosin, tilmicosin), tetracyclines (oxytetracycline, doxycycline), pleuromutilins (tiamulin), and fluoroquinolones (enrofloxacin). Tiamulin is particularly potent against both MG and MS. Antimicrobial susceptibility testing is recommended to guide therapy, as resistance to macrolides and tetracyclines has been reported. Treatment is most effective when administered early in the course of infection. In-feed or in-water medication is common for large flocks. However, antimicrobial use is increasingly restricted due to concerns about antimicrobial resistance and food safety. In many regions, prophylactic use of medically important antibiotics is prohibited.

Biosecurity and Management Control

Biosecurity is the most effective long-term strategy for preventing mycoplasma introduction and spread. Key components include:

• All-in/all-out production: Single-age flocks reduce the risk of pathogen carryover between groups.
• Rodent and wild bird control: Rodents and wild birds can mechanically transmit mycoplasmas.
• Personnel and equipment hygiene: Dedicated footwear, coveralls, and disinfection of equipment between houses.
• Water sanitation: Chlorination or acidification of drinking water reduces mycoplasma survival.
• Monitoring and surveillance: Regular serological and PCR testing of sentinel birds or routine flock samples.
• Eradication programs: In breeding stock, eradication through depopulation, cleaning, disinfection, and repopulation with mycoplasma-free stock is the gold standard. This approach is used in primary breeder operations to maintain negative status.

Integrated Control Programs

An integrated control program combines vaccination, biosecurity, monitoring, and targeted antimicrobial use. For MG, a typical program in layer flocks involves vaccination of pullets with a live vaccine, followed by periodic serological monitoring to detect breakthrough infections. For MS, control focuses on maintaining negative breeder flocks through biosecurity and eradication, with vaccination used in endemic areas. The following table summarizes the key control measures for MG and MS.

Conclusion

Avian mycoplasmosis caused by MG and MS remains a persistent challenge in commercial poultry production. The absence of a cell wall, antigenic variability, and the capacity for vertical transmission make these pathogens difficult to control. A multifaceted approach combining accurate molecular and serological diagnostics, strategic vaccination, prudent antimicrobial use, and rigorous biosecurity is essential. Advances in molecular typing and genomic surveillance will continue to refine control strategies, enabling more targeted interventions and ultimately reducing the economic impact of these infections.

References

1. Kleven SH. Mycoplasmosis. In: Swayne DE, editor. Diseases of Poultry. 14th ed. Wiley-Blackwell; 2020.
2. Ley DH, Yoder HW. Mycoplasma gallisepticum infection. In: Saif YM, editor. Diseases of Poultry. 11th ed. Iowa State Press; 2003.
3. Ferguson NM, Leiting VA, Kleven SH. Safety and efficacy of the avirulent Mycoplasma gallisepticum strain K5054 as a live vaccine. Avian Dis. 2004;48(1):91-99.
4. Noormohammadi AH. Role of phenotypic diversity in pathogenesis of avian mycoplasmosis. Avian Pathol. 2007;36(6):439-444.
5. Raviv Z, Callison SA, Ferguson-Noel N, Kleven SH. The Mycoplasma gallisepticum 16S-23S rRNA intergenic spacer region sequence as a novel tool for epizootiological studies. Avian Dis. 2007;51(4):838-845.
6. Whithear KG. Control of avian mycoplasmoses by vaccination. Rev Sci Tech. 1996;15(4):1527-1553.
7. Kleven SH. Mycoplasma synoviae infection. In: Saif YM, editor. Diseases of Poultry. 11th ed. Iowa State Press; 2003.
8. Feberwee A, Mekkes DR, de Wit JJ, Hartman EG, Pijpers A. Comparison of culture, PCR, and different serologic tests for detection of Mycoplasma gallisepticum and Mycoplasma synoviae infections. Avian Dis. 2005;49(2):260-264.
9. Grodio JL, Dhondt KV, O'Connell PH, Schat KA. Detection and quantification of Mycoplasma gallisepticum genome load in conjunctival samples of experimentally infected house finches (Haemorhous mexicanus) using real-time PCR. Avian Dis. 2009;53(4):603-608.
10. Marois C, Dufour-Gesbert F, Kempf I. Detection of Mycoplasma synoviae in poultry environment samples by culture and polymerase chain reaction. Vet Microbiol. 2000;73(4):311-318.
11. Raviv Z, Kleven SH. The development of diagnostic real-time TaqMan PCRs for the four pathogenic avian mycoplasmas. Avian Dis. 2009;53(1):103-111.
12. Bencina D, Bradbury JM. Combination of immunofluorescence and immunoperoxidase techniques for serotyping of Mycoplasma gallisepticum and Mycoplasma synoviae. Avian Pathol. 1992;21(2):295-304.
13. Morrow CJ, Bradbury JM, Gentle MJ. The detection of Mycoplasma gallisepticum and Mycoplasma synoviae in poultry by the polymerase chain reaction. Vet Microbiol. 1990;24(3-4):311-320.
14. Kempf I, Gesbert F, Guittet M. Experimental infection of chickens with an atypical Mycoplasma gallisepticum strain: comparison of diagnostic methods. Res Vet Sci. 1994;57(3):338-343.
15. Kleven SH, King DD, Anderson DP. Airsacculitis in broilers from Mycoplasma synoviae and Escherichia coli. Avian Dis. 1972;16(4):915-924.
16. Yoder HW. A historical account of the diagnosis and control of avian mycoplasma infections. Avian Dis. 1991;35(4):653-660.
17. Evans JD, Leigh SA, Branton SL, Collier SD, Pharr GT, Bearson SM. Mycoplasma gallisepticum: current and developing means to control the avian pathogen. J Appl Poult Res. 2005;14(4):757-763.
18. Ferguson-Noel N, Laibinis VA, Farrar M. Influence of Mycoplasma gallisepticum strain on vaccine efficacy. Avian Dis. 2012;56(4):724-729.
19. Noormohammadi AH, Markham PF, Kanci A, Whithear KG, Browning GF. A novel mechanism for control of antigenic variation in the haemagglutinin gene family of Mycoplasma synoviae. Mol Microbiol. 2000;35(4):911-923.
20. Shahid MA, Markham PF, Markham JF, Marenda MS, Noormohammadi AH. Mutations in the Mycoplasma gallisepticum mgc2 gene are associated with reduced virulence. Infect Immun. 2013;81(6):2100-2108.
21. Ghanem M, Wang L, Zhang Y, et al. Core genome multilocus sequence typing for identification of globally distributed clonal groups of Mycoplasma gallisepticum. Vet Microbiol. 2018;217:94-100.
22. El-Gazzar M, Ghanem M, McDonald K, et al. Development of a multiplex real-time PCR assay for simultaneous detection of Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis. Avian Dis. 2015;59(4):518-523.
23. Feberwee A, de Wit JJ, Landman WJ. Induction of eggshell apex abnormalities in broiler breeder hens following experimental infection with Mycoplasma synoviae. Avian Pathol. 2009;38(1):33-40.
24. Landman WJ, Feberwee A. A longitudinal study on the transmission of Mycoplasma synoviae in broiler breeder flocks. Avian Pathol. 2012;41(3):277-283.
25. Kleven SH. Changing trends in the control of Mycoplasma gallisepticum. World's Poult Sci J. 2008;64(3):297-304.
26. Whithear KG, Soeripto, Harringan KE, Ghiocas E. Safety of temperature sensitive mutant Mycoplasma gallisepticum vaccine. Aust Vet J. 1990;67(5):159-165.
27. Leigh SA, Branton SL, Evans JD, Collier SD. Effects of vaccination with F strain Mycoplasma gallisepticum on egg production and quality in commercial layer hens. J Appl Poult Res. 2006;15(3):387-393.
28. Barbour EK, Hamadeh SK, Eidt A. Infection and immunity in broiler chicken breeders vaccinated with a temperature-sensitive mutant of Mycoplasma gallisepticum and challenged with a virulent strain. Avian Dis. 2000;44(3):580-586.
29. Markham JF, Scott PC, Browning GF. In vitro selection of resistance to tylosin in Mycoplasma gallisepticum. Antimicrob Agents Chemother. 1998;42(9):2382-2385.
30. Gautier-Bouchardon AV, Ferré S, Le Grand D, Paoli A, Gay E, Poumarat F. Overall decrease in the susceptibility of Mycoplasma bovis to antimicrobials over the past 30 years in France. PLoS One. 2014;9(2):e87672.
31. Hannan PC, Windsor GD, de Jong A, Schmeer N, Stegemann M. Comparative susceptibilities of various animal-pathogenic mycoplasmas to fluoroquinolones. Antimicrob Agents Chemother. 1997;41(9):2037-2040.
32. Bencina D, Bradbury JM. Combination of immunofluorescence and immunoperoxidase techniques for serotyping of Mycoplasma gallisepticum and Mycoplasma synoviae. Avian Pathol. 1992;21(2):295-304.
33. Kempf I, Gesbert F, Guittet M. Experimental infection of chickens with an atypical Mycoplasma gallisepticum strain: comparison of diagnostic methods. Res Vet Sci. 1994;57(3):338-343.
34. Kleven SH, King DD, Anderson DP. Airsacculitis in broilers from Mycoplasma synoviae and Escherichia coli. Avian Dis. 1972;16(4):915-924.
35. Yoder HW. A historical account of the diagnosis and control of avian mycoplasma infections. Avian Dis. 1991;35(4):653-660.
36. Evans JD, Leigh SA, Branton SL, Collier SD, Pharr GT, Bearson SM. Mycoplasma gallisepticum: current and developing means to control the avian pathogen. J Appl Poult Res. 2005;14(4):757-763.
37. Ferguson-Noel N, Laibinis VA, Farrar M. Influence of Mycoplasma gallisepticum strain on vaccine efficacy. Avian Dis. 2012;56(4):724-729.
38. Noormohammadi AH, Markham PF, Kanci A, Whithear KG, Browning GF. A novel mechanism for control of antigenic variation in the haemagglutinin gene family of Mycoplasma synoviae. Mol Microbiol. 2000;35(4):911-923.
39. Shahid MA, Markham PF, Markham JF, Marenda MS, Noormohammadi AH. Mutations in the Mycoplasma gallisepticum mgc2 gene are associated with reduced virulence. Infect Immun. 2013;81(6):2100-2108.
40. Ghanem M, Wang L, Zhang Y, et al. Core genome multilocus sequence typing for identification of globally distributed clonal groups of Mycoplasma gallisepticum. Vet Microbiol. 2018;217:94-100.
41. El-Gazzar M, Ghanem M, McDonald K, et al. Development of a multiplex real-time PCR assay for simultaneous detection of Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis. Avian Dis. 2015;59(4):518-523.
42. Feberwee A, de Wit JJ, Landman WJ. Induction of eggshell apex abnormalities in broiler breeder hens following experimental infection with Mycoplasma synoviae. Avian Pathol. 2009;38(1):33-40.
43. Landman WJ, Feberwee A. A longitudinal study on the transmission of Mycoplasma synoviae in broiler breeder flocks. Avian Pathol. 2012;41(3):277-283.
44. Kleven SH. Changing trends in the control of Mycoplasma gallisepticum. World's Poult Sci J. 2008;64(3):297-304.
45. Whithear KG, Soeripto, Harringan KE, Ghiocas E. Safety of temperature sensitive mutant Mycoplasma gallisepticum vaccine. Aust Vet J. 1990;67(5):159-165.
46. Leigh SA, Branton SL, Evans JD, Collier SD. Effects of vaccination with F strain Mycoplasma gallisepticum on egg production and quality in commercial layer hens. J Appl Poult Res. 2006;15(3):387-393.
47. Barbour EK, Hamadeh SK, Eidt A. Infection and immunity in broiler chicken breeders vaccinated with a temperature-sensitive mutant of Mycoplasma gallisepticum and challenged with a virulent strain. Avian Dis. 2000;44(3):580-586.
48. Markham JF, Scott PC, Browning GF. In vitro selection of resistance to tylosin in Mycoplasma gallisepticum. Antimicrob Agents Chemother. 1998;42(9):2382-2385.
49. Gautier-Bouchardon AV, Ferré S, Le Grand D, Paoli A, Gay E, Poumarat F. Overall decrease in the susceptibility of Mycoplasma bovis to antimicrobials over the past 30 years in France. PLoS One. 2014;9(2):e87672.
50. Hannan PC, Windsor GD, de Jong A, Schmeer N, Stegemann M. Comparative susceptibilities of various animal-pathogenic mycoplasmas to fluoroquinolones. Antimicrob Agents Chemother. 1997;41(9):2037-2040.