Avian Mycoplasmosis in Poultry: Diagnostic Approaches and Economic Impact

Introduction

Avian mycoplasmosis represents a group of infectious diseases caused by pathogenic species of the genus Mycoplasma, primarily Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). These organisms are responsible for chronic respiratory disease (CRD), infectious synovitis, and reproductive disorders in commercial poultry, resulting in substantial economic losses worldwide [1, 2]. The disease burden is compounded by frequent co-infections with other respiratory pathogens such as Avian pathogenic Escherichia coli (APEC), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV) [3]. Accurate and timely diagnosis is essential for implementing effective control measures, including biosecurity protocols, vaccination strategies, and antimicrobial therapy. This review provides an exhaustive examination of diagnostic approaches for avian mycoplasmosis and a detailed analysis of its economic impact on poultry production systems.

Etiology and Pathogenesis

MG is the primary etiological agent of CRD in chickens and infectious sinusitis in turkeys [4, 5]. MS causes infectious synovitis and respiratory disease, often presenting as subclinical infections that become apparent under stress or concurrent infections [2, 6]. Both species are classified as notifiable pathogens by the World Organisation for Animal Health (WOAH) [7, 2].

Transmission occurs horizontally via direct contact, aerosol droplets, and contaminated fomites, as well as vertically through the egg [4]. The bacteria colonize the respiratory epithelium, trachea, and air sacs, triggering an inflammatory response characterized by mononuclear cell infiltration, mucosal hyperplasia, and exudation [8, 9]. In MS infections, synovial membranes and joints are also affected, leading to lameness and reduced mobility [2, 6].

Co-infections significantly exacerbate disease severity. Studies have demonstrated that flocks positive for MG or MS frequently harbor concurrent infections with IBV, APEC, or NDV [3]. For example, in a survey of 146 flocks, only 17.24% of mycoplasma-positive flocks had no other detected pathogen, while the remainder showed co-infection with APEC, IBV, or both [3]. Such interactions amplify economic losses through increased mortality, medication costs, and carcass condemnation [3, 9].

Diagnostic Approaches

Serological Methods

Serological testing is widely used for flock-level surveillance. The rapid serum agglutination (RSA) test is a simple, inexpensive screening tool that detects antibodies against MG and MS using stained antigen suspensions [10, 11]. However, RSA has limited sensitivity and specificity, often yielding false positives due to cross-reactions with commensal mycoplasmas [10].

Enzyme-linked immunosorbent assays (ELISA) provide higher throughput and objective quantification. Commercial ELISA kits are available for detection of MG and MS antibodies in serum or egg yolk [7, 12]. A comparative study in Bhaktapur, Nepal, found that dot-ELISA (ImmunoComb) detected 58.7% positivity compared to 32.6% by standard ELISA, indicating that format differences affect sensitivity [10]. Another evaluation of indirect ELISA and DOT blot assays reported comparable performance, with DOT blot offering advantages for field use due to its simplicity [13]. Serological monitoring remains a cornerstone of national surveillance programs, as demonstrated by a 2019 survey in the Russian Federation that identified positive samples for respiratory mycoplasmosis (n=6) and infectious synovitis (n=129) among 2,401 chicken serum samples [7].

Molecular Methods

Polymerase chain reaction (PCR) assays have become the gold standard for specific detection and differentiation of MG and MS. Conventional single (monoplex) PCR targeting the 16S-23S rRNA intergenic spacer region (IGSR) for MG and the hemagglutinin vlhA gene for MS yields amplicons of 812 bp and 1200 bp, respectively [14]. Duplex PCR enables simultaneous detection of both pathogens in a single reaction with high sensitivity and specificity. In a study of 146 flocks, duplex PCR showed 94.44% sensitivity and 100% specificity compared to monoplex PCR, with a Cohen's kappa of 0.97 indicating perfect agreement [14].

Real-time PCR (qPCR) offers quantitative results and reduced turnaround time. A duplex real-time PCR applied to 400 tracheal swabs from commercial and rural laying hens in Sicily detected MG in 12.5% and MS in 23.25% of samples, with higher prevalence in rural flocks [15]. Nested PCR has also been employed to increase sensitivity, particularly when sample quality is compromised [8].

Molecular detection is especially valuable for identifying subclinical infections and differentiating pathogenic from commensal species. In a study from Egypt, PCR confirmed 86 MG and 39 MS isolates from 628 samples, with sequencing revealing genetic diversity among MG strains [16].

Culture and Isolation

Isolation of mycoplasmas requires specialized media, such as pleuropneumonia-like organism (PPLO) broth and agar supplemented with serum and antibiotics (e.g., kanamycin) to suppress contaminants [8, 17]. Colonies exhibit the characteristic "fried-egg" appearance after 3–10 days of incubation under microaerophilic conditions [17]. Although culture provides a definitive diagnosis, it is time-consuming and less sensitive than molecular methods, especially for transport-damaged samples. A study from Bangladesh reported successful isolation from 60% of choanal cleft swabs, with histopathological confirmation of tracheitis, air sacculitis, and pneumonia [9].

Histopathological Examination

Postmortem findings include catarrhal to fibrinous exudate in the trachea and air sacs, pneumonia, and in MS cases, synovial membrane inflammation and joint exudates [9, 18]. Histological examination reveals congestion, mononuclear infiltration, and hyperplasia of lymphoid follicles in the respiratory tract [8]. These changes, while not pathognomonic, support clinical and molecular diagnoses.

Diagnostic Workflow

The following decision tree illustrates a recommended diagnostic approach for avian mycoplasmosis at the flock level.

flowchart TD
    A[Clinical signs: respiratory distress, lameness, egg drop], > B[Pooled sample collection: tracheal swabs, serum, tissues]
    B, > C{Initial screening}
    C, > D[Rapid Serum Agglutination (RSA)]
    C, > E[ELISA for MG/MS antibodies]
    D, > F[Positive RSA or inconclusive?]
    E, > F
    F, >|Yes| G[Confirm by molecular methods]
    F, >|No| H[Consider other pathogens]
    G, > I[Mono/duplex PCR or real-time PCR]
    I, > J[Positive for MG/MS?]
    J, >|Yes| K[Report and implement control measures]
    J, >|No| L[Consider culture or histopathology]
    L, > M[Isolation or histo findings]
    M, > K
    K, > N[Biosecurity, vaccination, treatment]

Economic Impact

Avian mycoplasmosis imposes severe economic burdens on the poultry industry through multiple mechanisms: reduced weight gain, impaired feed conversion efficiency, decreased egg production and hatchability, increased embryo mortality, and higher costs for medication and prophylaxis [2, 12]. In broiler breeders, flocks serologically positive for mycoplasma show lower production indices and higher feed conversion ratios compared to negative flocks [12]. For laying hens, egg production losses can reach 30% during acute outbreaks, as documented in a managed outbreak in Nigeria where treatment restored production from a drop to 80% [17].

The economic consequences extend to processing plants, where carcass condemnation due to airsacculitis and synovitis increases losses. Co-infections, particularly with IBV and APEC, exacerbate these effects, leading to higher mortality and longer recovery times [3]. In Bangladesh, mycoplasmosis prevalence of 26.52% in commercial chickens was associated with significant morbidity, and winter season peaks correlated with higher production losses [9].

Country-specific studies illustrate regional impacts. In Nepal, seroprevalence of 58.7% by dot-ELISA in samples from Bhaktapur highlights widespread infection in the absence of effective biosecurity [10]. In Algeria, isolation rates of 21.67% for MG in broiler and layer chickens underscore the endemic nature of the disease [19]. In the Russian Federation, continuous monitoring from 2015 to 2019 showed a decrease in MG-positive farms but persistent MS circulation, particularly in layer flocks [7].

Vaccination and biosecurity programs represent significant investments but are cost-effective compared to uncontrolled outbreaks. The development of recombinant fowl-pox virus vaccines and live attenuated F-strain vaccines provides options for reducing clinical disease and vertical transmission [20]. Novel therapeutic approaches such as berberine, which inhibits MS by targeting PIK3CA-dependent inflammatory and apoptotic pathways in avian macrophages, may offer alternatives amid rising antimicrobial resistance [6].

Control and Management

Integrated control strategies combine biosecurity, vaccination, antimicrobial therapy, and flock management. Strict all-in-all-out production systems, cleaning and disinfection of facilities (e.g., using peroxygen compounds), and sourcing stock from mycoplasma-free breeders are foundational [17, 21]. Vaccination is recommended for replacement pullets and breeders; both inactivated bacterins and live attenuated vaccines (e.g., F-strain) are available [2, 20]. Recombinant poxvirus-vectored vaccines provide a safer alternative, especially when combined with other poultry vaccines [20].

Antimicrobial treatment can reduce clinical signs and transmission but does not eliminate infection. Tylvalosin tartrate, administered via drinking water, has been effective in restoring egg production during outbreaks [17]. However, the emergence of antimicrobial resistance underscores the need for judicious use and alternative therapies [6]. Berberine, a natural isoquinoline alkaloid, has shown both direct anti-mycoplasmal activity and host-directed effects in vitro [6].

Surveillance programs incorporating regular serological and molecular testing enable early detection and removal of positive flocks. National monitoring, such as that conducted in Russia, helps track trends and evaluate control measures [7]. Flock-level diagnostics should include sampling of multiple birds and, where possible, detection of co-infections that may influence disease expression [3].

Conclusion

Avian mycoplasmosis remains a major constraint to global poultry production. Advances in molecular diagnostics, including duplex PCR and real-time PCR, have improved the speed and accuracy of detection, while serological methods continue to serve as practical screening tools for large-scale surveillance. The economic impact of MG and MS infections is profound, involving losses in production, reproduction, and processing. Effective control requires an integrated approach: rigorous biosecurity, strategic vaccination, targeted antimicrobial use, and continuous monitoring. Future research should focus on affordable point-of-care diagnostics, novel therapeutics to combat antimicrobial resistance, and cost-benefit analyses of vaccination programs in diverse production settings.

References

[1] Umar S, Munir M, ur-Rehman Z, et al. RETRACTED ARTICLE: Mycoplasmosis in poultry: update on diagnosis and preventive measures. Journal. https://www.semanticscholar.org/paper/cda22f74bfdd63b77c7f5c5a78228b3c6429a46d

[2] Yadav J, Tomar P, Singh Y, et al. Insights on Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry: a systematic review. Animal Biotechnology. https://www.semanticscholar.org/paper/b1708d1c5802c0812b783e3f2d42a9980d0782b7

[3] Yadav J, Singh Y, Batra K, et al. Molecular detection of respiratory avian mycoplasmosis associated bacterial and viral concurrent infections in the poultry flocks. Animal Biotechnology. https://www.semanticscholar.org/paper/033cf4d4b0937041b911e33c90e25e17ba866947

[4] Levisohn S, Kleven S. Avian mycoplasmosis (Mycoplasma gallisepticum). Revue scientifique et technique. https://www.semanticscholar.org/paper/e2adbe567ffa0c2a41e2aac47af727394a0197b0

[5] Avian Mycoplasmosis (Mycoplasma gallisepticum, M. synoviae). Journal. https://www.semanticscholar.org/paper/b48e10394ec0e04025949d4c0019ac76b8481021

[6] Sun Y, Hu WJ, Li S, et al. Berberine Inhibits Mycoplasma synoviae Infection by Suppressing PIK3CA-Dependent Inflammatory and Apoptotic Responses in Avian Macrophages. Microbial Pathogenesis. https://www.semanticscholar.org/paper/51656f851cfb8cdf9fb7a975aa3cda819623e1f0

[7] Menshchikova AE, Brundakova TN, Volkov M, et al. Avian mycoplasmosis monitoring in the Russian Federation in 2019. Veterinary Science Today. https://www.semanticscholar.org/paper/aa26a24639cfc86c6cb6d9893e46af719a0c7e4d

[8] Basak P, Banowary B, Arju S, et al. Isolation and molecular detection of avian mycoplasmosis in selected areas of Mymensingh district in Bangladesh. Journal. https://www.semanticscholar.org/paper/6fc747581354eba98e45939a917db796547f71c7

[9] Bepary S, Rahman M, Rahman MS, et al. Macroscopic and microscopic characterization of mycoplasmosis in commercial chickens in Barishal, Bangladesh. Asian-Australasian Journal of Bioscience and Biotechnology. https://www.semanticscholar.org/paper/1469d97da984fcac0e461555a9b610c3b628c5a4

[10] Khanal DR, Ranjit E, Adhikari S, et al. Seroprevalence of mycoplasmosis in poultry of Bhaktapur. Journal. https://www.semanticscholar.org/paper/ed15f5d353766ffcc0c382d372ead5a6c14179da

[11] Sobuj M, Matubber B, Islam S, et al. Seroprevalence and associated risk factors of mycoplasmosis in layer chickens at southern region of Bangladesh. Asian Journal of Medical and Biological Research. https://www.semanticscholar.org/paper/387450777cd39d2f33efdbe02198dacc0c566113

[12] Park DH, Kim KJ, Lim TH, et al. Comparison of Broiler Performance according to Infection Rate of Chicken Mycoplasmosis in Broiler Breeders. Journal. https://www.semanticscholar.org/paper/06eacdcab909f7c8ab85d501bc82eb3088d86a7d

[13] Yadav J, Batra K, Singh Y, et al. Comparative evaluation of indirect-ELISA and DOT blot assay for serodetection of Mycoplasma gallisepticum and Mycoplasma synoviae antibodies in poultry. Journal of Microbiological Methods. https://www.semanticscholar.org/paper/498d1caad126377f4a7a388c9833b27b440c5ce1

[14] Yadav J, Singh Y, Jindal N, et al. Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays. Journal of Microbiological Methods. https://www.semanticscholar.org/paper/60ab48a274ac221c32ac4e002e3b7fdaec20cb42

[15] Galluzzo P, Migliore S, Galuppo L, et al. First Molecular Survey to Detect Mycoplasma gallisepticum and Mycoplasma synoviae in Poultry Farms in a Strategic Production District of Sicily (South-Italy). Animals. https://www.semanticscholar.org/paper/e1dd0cb81d5915eb3d0e6fd6a863ed1aa14a2af4

[16] Marouf S, Moussa I, Salem H, et al. A picture of Mycoplasma gallisepticum and Mycoplasma synoviae in poultry in Egypt: Phenotypic and genotypic characterization. Journal. https://www.semanticscholar.org/paper/5340bfac2f7b4659bc1a7e2f759f02bda1be1781

[17] Bwala DG, Ularamu H, Luka P, et al. Management of mycoplasmosis in a laying flock in Vom, Plateau State, Nigeria. Sokoto Journal of Veterinary Sciences. https://www.semanticscholar.org/paper/792987c76cbfd9198295f55ea93f53881dbd9aef

[18] Nagalakshmi K, Senthilkumar T, Parthiban M, et al. Differential Diagnosis of Avian Mycoplasmosis. Journal. https://www.semanticscholar.org/paper/575ddd88e4f5d9aacd392ebbf45931cee5be5cfd

[19] Heleili N, Mamache B, Chelihi A. Incidence of avian mycoplasmosis in the region of Batna, Eastern Algeria. Journal. https://www.semanticscholar.org/paper/6154279a89c6447a6bc187b4018148ff8498df93

[20] Hashish A, Chaves M, Osemeke O, et al. Evaluation of Vaccination Programs in Layer Pullets Using Recombinant Fowl-Pox Mycoplasma gallisepticum Vaccine in Comparison to Commercially Available F-Strain Live Vaccines. Avian Dis. https://pubmed.ncbi.nlm.nih.gov/41973009/

[21] Kleven S. Current Situation with Avian Mycoplasmosis Prevention and Control. Journal. https://www.semanticscholar.org/paper/5f1623ed903d3926ccb4135c50cdebf2074b68f7

[22] Akade FT, Akade B, Ad AD, et al. Serological Evidence of Avian Mycoplasmosis and Salmonellosis in Different Poultry Birds at the Live Bird Market and Free Range Management System in Taraba State, Nigeria. Journal. https://www.semanticscholar.org/paper/d7ba5219de62d4a9f9ceb7d567f6711c6de96490

[23] Rachida A, Omar B, Elhacène B, et al. Serological investigation on avian mycoplasmosis in laying hen farms in Eastern Algeria. Journal. https://www.semanticscholar.org/paper/84c07579ab84f8ea6f510653ec71ffa9c206dd00

[24] Qasem J, Al-Mouqati SA, AlAli E, et al. Application of Molecular and Serological Methods for Rapid Detection of Mycoplasma gallisepticum Infection (Avian mycoplasmosis). Pakistan Journal of Biological Sciences. https://www.semanticscholar.org/paper/78d94c8459a128776ba8f667698777855fe0b3cb

[25] Venkatesh G. Development Of Recombinant Antigens For Diagnosis Of Avian Mycoplasmosis. Journal. https://www.semanticscholar.org/paper/3d7fa721a54be57453ec3e27bdd7745fa025aec7

[26] Wanasawang W, Nimitkun C, Chansiripornchai N. Current Knowledge of Avian Mycoplasmosis. Journal. https://www.semanticscholar.org/paper/3036e18bb58d33392526a45d6629c82e339eefbd

[27] Rodrigo CH, Kulappu Arachchige SN, Bushell RN, et al. Complex pathogen interactions in upper respiratory tract infections in commercial free-range layers: A cross-sectional study. Vet Microbiol. https://pubmed.ncbi.nlm.nih.gov/42190304/

[28] Zhang Y, Li Y, Wang Z, et al. Cryptosporidium baileyi and Mycoplasma synoviae co-infection enhances M. synoviae colonization and aggravates tissue damage in chickens. Front Microbiol. https://pubmed.ncbi.nlm.nih.gov/42158394/

[29] Adelman JS, Henschen AE, Tillman FE Jr, et al. The devil you know: a longer history of pathogen coevolution predicts higher behavioural tolerance of infection among host populations. Biol Lett. https://pubmed.ncbi.nlm.nih.gov/42124517/

[30] Hartady T, Sugandi SD, Viqih M. A Case of Avian Influenza Co-Infection and Multifactorial Diseases in a Broiler Chicken Farm in Majalengka, West Java, Indonesia. Vet Sci. https://pubmed.ncbi.nlm.nih.gov/42076736/