SPAT, Dot-ELISA or i-ELISA? Choosing the Right Approach for MG and MS Screening

Respiratory disease in a broiler flock can raise concern about infection with Mycoplasma gallisepticum (MG) or Mycoplasma synoviae (MS). Because the two organisms are associated with different clinical manifestations, early detection can help initiate appropriate control measures and strengthen flock surveillance. The choice of serological test also matters, as different methods can produce markedly different levels of apparent seropositivity. 

MG is responsible for chronic respiratory disease (CRD) in chickens, whereas MS is mainly associated with infectious synovitis, airsacculitis, arthritis and mild upper respiratory tract infections, with or without symptoms of septicemia^[1],[2]. 

SPAT: Rapid primary screening 

Serum plate agglutination test (SPAT) is commonly used as a primary screening method for avian mycoplasmosis. It is inexpensive, rapid and sensitive for detecting IgM antibodies. It can also detect seroconversion a few days earlier than haemagglutination inhibition (HI) and ELISA^[1],[3]. 

However, its rapidity does not mean that every positive result should be interpreted in isolation. Differences between antigen sources and batches, poor-quality or frozen sera, antigen preparation and cross-reactions involving MG, MS and Mycoplasma imitans can affect SPAT results^[1]. 

Among 68 serum samples collected from broiler chickens affected with respiratory tract infections in Mathura district, apparent seropositivity by SPAT was 36.76% for MG and 72.06% for MS^[1]. 

i-ELISA: A more sensitive screening approach^[1] 

Indirect ELISA (i-ELISA) is another commonly used serological method. It can screen large numbers of sera, and ELISA is considered more sensitive and specific than other serological tests such as SPAT and HI for detecting avian mycoplasmosis 

In the same group of samples, i-ELISA showed an apparent seropositivity of 85.29% for MG and 80.88% for MS. 

The difference from SPAT is substantial. This illustrates why the diagnostic method should be considered when interpreting flock-level serological findings rather than treating the reported percentage as independent of the assay used. 

Dot-ELISA: A practical alternative 

Dot-ELISA is a solid-phase immunoassay described as simple, reliable and cost-effective for serodetection of important poultry diseases^[1],[4]. A single strip can also be used for serodetection of more than one poultry pathogen, supporting its practical value for screening. 

In these samples, Dot-ELISA detected MG antibodies in 66.18% and MS antibodies in 77.94%. Its relative diagnostic sensitivity compared with i-ELISA was 77.59% for MG and 96.36% for MS, while relative diagnostic specificity was 100% for both. 

Although Dot-ELISA is slightly less sensitive than ELISA, its simplicity, lower cost and ability to provide useful serological information can make it relevant for initial screening^[1]. 

When is further testing needed? 

Serological screening can support early detection, but additional diagnostic methods may be required for confirmation. Pathogen isolation is considered confirmatory, although it is laborious, time-consuming, expensive, and susceptible to overgrowth by secondary bacterial infections^[1]. 

Molecular tests provide rapid and accurate detection of avian mycoplasmas, but their wider use can be limited by the requirement for sophisticated laboratory facilities, skilled manpower and higher costs^[5],[6]. 

Clinical Bottom Line 

SPAT, Dot-ELISA and i-ELISA each have a distinct role in serological screening for MG and MS. SPAT offers rapid primary screening, i-ELISA provides a more sensitive approach for antibody detection, and Dot-ELISA offers a simple and cost-effective alternative. The diagnostic method should therefore be considered alongside the flock's clinical status when interpreting serological findings.

References:

1. Yadav JP, Singh Y, Batra K, Jain U. Evaluation of serological tests for the diagnosis of Mycoplasma gallisepticum and Mycoplasma synoviae infections in broiler chickens with respiratory ailments. https://www.jvas.in/media/articles/pdf/502.pdf
2. Yadav JP, Singh Y, Batra K, Kumar R, Mahajan NK, Jindal N. Isolation, molecular characterization and phylogenetic analysis of Mycoplasma gallisepticum and Mycoplasma synoviae strains recovered from commercial broiler chicken flocks affected with respiratory tract infections. The Microbe. 2025 Jul 26:100485. https://www.sciencedirect.com/science/article/pii/S2950194625002535
3. Wanasawaeng W, Chaichote S, Chansiripornchai N. Development of ELISA and serum plate agglutination for detecting antibodies of Mycoplasma gallisepticum using strain of Thai isolate. The Thai Journal of Veterinary Medicine. 2015;45(4):499-507. https://digital.car.chula.ac.th/cgi/viewcontent.cgi?article=2679&context=tjvm
4. Alam J, Muhammad F, Siddiqui MU, Khan SA, Rehmani S, Ahmad A. Dot-ELISA for Newcastle disease, infectious Bursal disease and mycoplasmosis. Pakistan Journal of Zoology. 2012 Oct 1;44(5). https://www.researchgate.net/profile/Shakeel-Khan-3/publication/236867459
5. Wu Q, Xu X, Chen Q, Zuo K, Zhou Y, Zhang Z, Kan Y, Yao L, Ji J, Bi Y, Xie Q. Rapid and visible detection of Mycoplasma synoviae using a novel polymerase spiral reaction assay. Poultry Science. 2019 Nov 1;98(11):5355-60. https://www.sciencedirect.com/science/article/pii/S0032579119457386
6. Yadav JP, Batra K, Singh Y, Khurana SK, Jindal N. Comparative study on conventional and real-time PCR assays for rapid and specific detection of Mycoplasma gallisepticum infection in poultry. The Microbe. 2024 Jun 1;3:100066. https://www.sciencedirect.com/science/article/pii/S2950194624000335