Rapid Detection of Mycoplasma synoviae and Avian Reovirus in Clinical Samples of Poultry Using Multiplex PCR Carolina Reck, AB A ´ lvaro Menin, BC Marina Feltrin Canever, A and Luiz Claudio Miletti AD A Departamento Produc ¸a ˜o Animal e Alimentos, Universidade do Estado de Santa Catarina-CAV/UDESC, Lages, SC, Brazil, 88520-000 B Instituto de Pesquisa e Diagno ´stico Veterina ´rio–IPDVET, Curitibanos, SC, Brazil, 89520-200 C Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina, Floriano ´polis, SC, Brazil, 88040-970 Received 21 October 2012; Accepted 23 January 2013; Published ahead of print 25 January 2013 SUMMARY. Mycoplasma synoviae and avian reovirus (ARV) are associated with several disease syndromes in poultry and cause notable global economic losses in the poultry industry. Rapid and efficient diagnostics for these avian pathogens are important not only for disease control but also for prevention of clinical disease progression. However, current diagnostic methods used for surveillance of these diseases in poultry flocks are laborious and time-consuming, and they have low sensitivity. The multiplex PCR (mPCR) developed in this study has been proven to be both sensitive and specific for simultaneous M. synoviae and ARV detection and identification in clinical samples. To evaluate the mPCR assay, the diagnostic test was applied to different clinical samples from natural and experimental M. synoviae and ARV-infected poultry. Results were compared with serologic, single PCR, and immunofluorescence analyses. Tibiotarsal articulation could be the best target for simultaneous detection of M. synoviae and ARV infection. The detection limit by visualization of mPCR-amplified products was 100 pg for both pathogens. Overall, the mPCR developed and standardized in this research is a useful tool for diagnosis and screening and for surveillance and control of M. synoviae and ARV infection in poultry flocks. RESUMEN. Deteccio ´n ra ´pida de Mycoplasma synoviae y reovirus aviar mediante PCR mu ´ltiple en muestras clı ´nicas avı ´colas. Mycoplasma synoviae y el reovirus aviar (ARV) esta ´n asociados con varios sı ´ndromes de enfermedades en las aves y causan importantes pe ´rdidas econo ´micas mundiales en la industria avı ´cola. El diagno ´stico ra ´pido y eficaz para estos pato ´genos aviares es importante no so ´lo para el control de las enfermedades, sino tambie ´n para la prevencio ´n de la progresio ´n de la enfermedad clı ´nica. Sin embargo, los me ´todos actuales de diagno ´stico utilizados para la vigilancia de estas enfermedades avı ´colas son laboriosos, consumen mucho tiempo, y tienen baja sensibilidad. El PCR mu ´ltiple (mPCR) desarrollado en este estudio ha demostrado ser ma ´s sensible y especı ´fico para la deteccio ´n e identificacio ´n simulta ´nea de M. synoviae y reovirus en muestras clı ´nicas. Para evaluar el ensayo de PCR mu ´ltiple, la prueba de diagno ´stico se aplico ´ en diferentes muestras clı ´nicas de aves comerciales infectadas de manera natural y experimental con M. synoviae y con reovirus. Los resultados se compararon con ana ´lisis serolo ´gicos, con PCR simples, y con ana ´lisis de inmunofluorescencia. La articulacio ´n tibiotarsal podrı ´a ser la mejor muestra para la deteccio ´n simulta ´nea de la infeccio ´n por M. synoviae y reovirus. El lı ´mite de deteccio ´n por visualizacio ´n de los productos amplificados por PCR mu ´ltiple fue de 100 pg para ambos pato ´genos. En general, el me ´todo de PCR mu ´ltiple desarrollado y estandarizado en esta investigacio ´n es una herramienta u ´til para el diagno ´stico, la deteccio ´n, la vigilancia y el control de la infeccio ´n por M. synoviae y reovirus en las aves comerciales. Key words: multiplex polymerase chain reaction, Mycoplasma synoviae, avian reovirus, poultry disease Abbreviations: ARV 5 avian reovirus; IBDV 5 infectious bursal disease virus; IBV 5 infectious bronchitis virus; IF 5 immuno- fluorescence; Ig 5 immunoglobulin; mPCR 5 multiplex PCR Mycoplasma synoviae and avian reovirus (ARV) are important avian pathogens for poultry and present several disease syndromes that cause large economic losses for the poultry industry (25,30). Mycoplasma synoviae is responsible for infectious synovitis, arthritis, airsacculitis, bursitis, and septicemia in chickens (18,19,30). ARV is an RNA virus in the family Reoviridae, and it is associated with a variety of diseases and conditions in chickens and turkeys, including enteric diseases, chronic respiratory diseases, myocarditis, hepatitis, arthritis/tenosynovitis, and malabsorption syndrome (4,23,25). Mixed infections with M. synoviae and ARV have been known to occur in poultry flocks worldwide, and they also have been associated with severe immunosuppression (27), depression, retarded growth, weight loss, decrease in egg production, and particularly the elimination of lesioned carcasses at the slaughterhouse (15,21,22). These findings suggest simultaneous M. synoviae and ARV infec- tion has a synergistic effect upon clinical symptoms and pathologic changes (14). In this context, efficient diagnostic testing is essential for the surveillance and control of these avian pathogens in poultry flocks. Rapid and early diagnostic detection of M. synoviae and ARV is important to prevent spread of infection and to limit economic losses to the poultry industry. Serologic testing and isolation are methods used for the diagnosis of M. synoviae and ARV (5,29); however, these methods are laborious and time-consuming, and they have low sensitivity. Moreover, serologic analysis is often plagued by nonspecific reactions and problems with reagent cross-reaction (5,29,31). Although, single PCR also has been used to detect these avian pathogens, the technique only allows for the detection of nucleic acids from one specific pathogen per reaction (8,9,31). These limitations can be overcome by using a multiplex PCR (mPCR) assay that incorporates multiple primers to amplify molecular targets from different avian pathogens simultaneously in one reaction (7,20). The benefits of mPCR include cost effectiveness, time efficiency, and potential for use in screening and surveillance of pathogens in commercial poultry flocks (2,10). In this context, the main objective of this research was to develop and optimize an mPCR assay that would allow simultaneous detection and D Corresponding author. E-mail: miletti@cav.udesc.br AVIAN DISEASES 57:220–224, 2013 220