Rapid Detection ofMycoplasma synoviaeand Avian Reovirus in Clinical Samples of Poultry Using Multiplex PCR

Abstract

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.

The development and evaluation of cross-priming amplification for the detection of avian reovirus

“…Descriptions: NC -RNA extracted from noninfected SPF chicken embryo fibroblasts (CEF), avian reovirus -S1133 strain (MSD Animal Health, AA Boxmeer, the Netherlands), REVreticuloendotheliosis virus strain T (REV-T) (Charles river, North Franklin, CT), CAVchicken anaemia virus vaccine strain (CAV-VAC, Intervet, the Netherlands). Liu et al 1999;Bruhn et al 2005;Day et al 2007;Guo et al 2012;Jindal et al 2012;Reck et al 2013;Yun et al 2014). The main drawback of PCR-based methods was the absolute requirement to set up the reactions in a thermal cycler, or PCR system.…”

“…; Reck et al . ). Apart from synovitis , a considerable mortality rate, reaching up to 80%, was observed among affected broiler chickens (Niczyporuk et al .…”

“…Identification of reovirus infection in poultry, on the basis of clinical signs and pathological changes, may be difficult, since they are not observed. In the case of synovitis, clinical signs may be misdiagnosed and mistaken for the Mycoplasma synoviae, Staphylococcus aureus infection, or the consequence of reduced mineral uptake (van Loon et al 2001;Day 2009;Davis et al 2013;Reck et al 2013). Apart from synovitis, a considerable mortality rate, reaching up to 80%, was observed among affected broiler chickens (Niczyporuk et al 2009).…”

“…Other serological methods used in ARV detection include agar gel precipitation assay (AGP) or enzyme-linked immunosorbent assay (ELISA) (Xie et al 2010;Jung et al 2014). Recently, an increase in the application of reverse transcription PCR (RT-PCR) and real-time RT-PCR in ARV detection has been observed (Liu et al 1999;Bruhn et al 2005;Day et al 2007;Guo et al 2012;Jindal et al 2012;Reck et al 2013;Yun et al 2014). These methods are targeted to specifically detect rC proteins, encoded by the S1 segment, or rNS encoded by the S4 segment of the ARV genome.…”

Avian rotavirus enteritis – an updated review

“…A diverse number of AvRVs can be detected by employing such assay targeting NSP3 of virus in fecal samples (Mijatovic-Rustempasic et al 2013). Some workers have developed a multiplex (m) RT-PCR assay, which could differentially detect presence of AsTVs (turkey astrovirus-2) and AvRVs in feces of birds Jindal et al 2012;Day and Zsak 2013;Reck et al 2013). Otto et al (2012) detected avian RVs of group A (58.8%) and group D (65.9%) using a more sensitive and group specific real-time-PCR.…”