Rapid Detection of Mycoplasma synoviae and Avian Reovirus in Clinical Samples of Poultry Using Multiplex PCR Author(s): Carolina Reck, Álvaro Menin, Marina Feltrin Canever, and Luiz Claudio Miletti Source: Avian Diseases, 57(2):220-224. 2013. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10425-101712-Reg.1 URL: http://www.bioone.org/doi/full/10.1637/10425-101712-Reg.1

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.

BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

AVIAN DISEASES 57:220–224, 2013

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 MilettiAD 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 immunofluorescence; 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 infection has a synergistic effect upon clinical symptoms and pathologic changes (14). In this context, efficient diagnostic testing is essential D

Corresponding author. E-mail: [email protected]

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

220

221

Mycoplasma synoviae and avian reovirus using multiplex PCR

Table 1.

Oligonucleotide primer sequence used in single PCR and multiplex polymerase chain reactions.

Avian pathogen

ARV M. synoviae

Primer

MK87F MK88R MSF MSR

Sequence(59-39)

59-GGTGCGACTGCTGTATTTGGTAAC-39 (location 55–77) 59-AATGGAACGATAGCGTGTGGG-39 (location 568–587) 59-GAAGCAAAATAGTGATATCA-39 (location 1194–1213) 59-GTCGTCTCCGAAGTTAACAA-39 (location 1381–1400)

differentiation of M. synoviae and ARV in one reaction. Our results provide insight on the use of mPCR for detection as well as surveillance of two important avian pathogens in commercial poultry flocks. MATERIALS AND METHODS Field samples and experimental infection. For this study, 212 (42day-old) broiler chickens with lesions of infectious arthritis in tibiotarsal articulations were obtained from 21 commercial flocks from the state of Santa Catarina, Brazil. During slaughter, 10–12 tibiotarsal joints with arthritic injury were collected from each flock. Broiler chickens were not vaccinated for M. synoviae, ARV, or both and were not previously identified for infection by these pathogens. Tissue samples including synovial membrane and digital flexor tendons were collected in the slaughterhouse and conserved in liquid nitrogen (2196 C) for nucleic acid extraction. Samples were embedded in Tissue-TekH OCTTM compound (Sakura Finetek USA, Torrance, CA) for immunofluorescence (IF) analysis and formalin fixed for histopathologic examination. For experimental infection with M. synoviae and ARV, we used 16 female broiler chicks with 10-day-old (lineage Cobb). The animals were maintained in the experimental station of the Centro de Cieˆncias Agroveterina´rias/Universidade do Estado de Santa Catarina (CAV/ UDESC), Lages, Brazil, under standard conditions and in accordance with guidelines established by the current laws of animal protection in Brazil (3). All procedures were evaluated previously and approved by the Ethics Committee on Animal Experiments, CAV/UDESC, under protocol 1.31.11. At 15 days old, chicks were simultaneously inoculated with 8 3 105 colony-forming units/ml M. synoviae, MS-H strain (Merial, Campinas, Brazil) through aerosol route and footpad injection, and a suspension of ARV S1133 strain (Biovet, Vargem Grande Paulista, Brazil) containing 104.8 median tissue culture infective dose/0.05 ml, through oral route and footpad injection. Thirty days after infection, euthanasia and necropsy of the poultry were performed. Trachea, lung, air sacs, liver, spleen, tibiotarsal articulation, and blood samples were collected and conserved in liquid nitrogen (2196 C) for nucleic acid extraction, embedded in Tissue-Tek OCT compound (Sakura Finetek USA) for IF analysis, and formalin fixed for histopathologic examination. Experimentally infected chickens were tested before and after inoculation. Serologic analyses to detect antibodies against M. synoviae and ARV were performed by ELISA using the IDEXX MS Ab TestH and IDEXX REO Ab TestH (IDEXX Laboratories, Westbrook, ME), following manufacturer’s instructions. Nucleic acid extraction. The nucleic acid extraction protocol was adopted for the simultaneous extraction of total DNA (M. synoviae) and RNA (ARV). The procedures were carried out according to the TRIzolH (Invitrogen, Carlsbad, CA) manufacturer’s protocol and as described previously (11,28). In brief, after homogenization with TRIzol and addition of chloroform, the tissue sample was centrifuged to separate the phases. The mixture separated into a lower, red, phenol-chloroform phase (DNA); an interphase; and a colorless, upper aqueous phase (RNA). RNA was precipitated by adding isopropyl alcohol, and then DNA was precipitated by the addition of ethanol. Positive controls used were pure cultures of M. synoviae MS-H strain (Merial) and ARV S1133 strain (Biovet). Reverse transcription reaction and cDNA synthesis. The total RNA (ARV) extracted was used for the reverse transcription-PCR and preparation of the first-strand complementary DNA synthesis using

Amplicon size (bp)

Reference

532

25

207

12

ProtoScriptH M-MuLV First Strand cDNA Synthesis kit (New England Biolabs, Ipswich, MA), following the manufacturer’s instructions. In brief, the first-strand cDNA synthesis was initially performed in a volume of 8 ml, in which the reaction mixture contained 6 ml of viral RNA (100 ng) and 2 ml of random primer mix. RNA was denatured for 5 min at 70 C. Next, 10 ml of Moloney murine leukemia virus (MMuLV) reaction mix and 2 ml of M-MuLV enzyme mix were added to the solution, for a final volume of 20 ml. Reverse transcription was carried out in the thermal cycler for one cycle at 25 C for 5 min and at 42 C for 60 min. The enzyme was then inactivated at 80 C for 5 min. Reaction was diluted to 50 ml with 30 ml of sterile deionized water. The cDNA product was stored at 220 C. Single PCR for M. synoviae and ARV detection. Single PCR for M. synoviae and ARV was performed to detect single avian pathogen in each reaction. Oligonucleotide primer sets that specifically amplify the target sequence of the S1 gene from ARV (31) and MS-16S rRNA sequence from M. synoviae (17) are described in Table 1. All sets of oligonucleotide primers were synthesized by Invitrogen. The addition of both extracted DNA and synthesized cDNA provided templates for the amplification of M. synoviae and ARV, respectively. The conditions for single PCR for M. synoviae and ARV detection were performed as described previously (17,31). mPCR for simultaneous detections M. synoviae and ARV. In the mPCR assay, M. synoviae and ARV were detected using oligonucleotide primers (Table 1) described previously (17,31). Detection involved optimizing several PCR parameters, including testing different primers concentrations, concentrations of MgCl2, and annealing temperatures to achieve optimum conditions for the simultaneous amplification of target genomic sequences from M. synoviae and ARV. The mPCR reactions were performed in a 20-ml volume. Extracted DNA and synthesized cDNA were mixed in equal proportions of each (100 ng), thereby providing the template for M. synoviae and ARV detection, respectively. The mPCR reactions were carried out using optimized MgCl2, 103 PCR buffer II (500 mM KCl and 100 mM Tris HCl, pH 8.3; Invitrogen), 200 mM of each dNTP (Invitrogen), 20 pmol of ARV primers MK87R and MK88F, 20 pmol of M. synoviae primers MSR and MSF (Invitrogen), and 5 units of Taq DNA polymerase (Invitrogen). Various concentrations of MgCl2 (2.5, 3.0, 3.5, and 4.0 mM) were tested as well. All reactions were carried out using a gradient thermal cycler (Axygen, Inc., Union City, CA). The cycling protocol consisted of an initial denaturation at 94 C for 5 min and then 35 cycles of denaturation at 94 C for 1 min, annealing for 1 min; various temperatures were tested (48.5, 49.8, 51.5, 52.6, and 54.0 C) and extension was at 72 C for 1 min. The sample was then heated at 72 C for 10 min for a final extension. Each mPCR run included positive and negative controls for PCR reagents and sample extractions. The negative control did not contain template DNA and consisted of PCR master mix, all four sets of primers, and deionized water. The mPCR amplification products were analyzed by electrophoresis in 1.5% (w/v) agarose gel and TBE buffer (89 mM Tris, 89 mM borate, and 2 mM EDTA). Ten-microliter volumes of single PCR and mPCR products were separated in agarose horizontal gel by electrophoresis at 80 V. A 100-bp DNA ladder was used as the molecular-size marker. Gels were stained with ethidium bromide solution (0.5 mg/ml) for 10 min, visualized by ultraviolet light, and photographed on a MiniBIS ProH digital transilluminator (BioAmerica Biotech, Miami, FL). mPCR sensitivity and specificity. Sensitivity of the mPCR was determined by performing 10-fold serial dilutions (100 ng) of the DNA extracted from M. synoviae (MS-H strain) and RNA extracted from ARV

222

C. Reck et al.

Fig. 2. Sensitivity of mPCR. Detection limit of mPCR for M. synoviae and ARV to different concentrations of DNA and RNA (used for cDNA synthesis). Lane M, 100-bp DNA ladder; lane 1, only M. synoviae (100 ng); lane 2, only ARV (100 ng); lane 3, 100 ng; lane 4, 50 ng; lane 5, 10 ng; lane 6, 1 ng; lane 7, 0.5 ng; lane 8, 100 pg; lane 9, 10 pg; lane 10, negative control (agarose gel).

Fig. 1. Optimization of mPCR assay developed for the detection of M. synoviae and ARV. (A) Agarose gel showing simultaneous mPCR amplification of target M. synoviae/ARV with different annealing temperatures. Lane M, 100-bp DNA ladder; lane 1, only ARV; lane 2, only M. synoviae; lanes 3–7, different annealing temperatures mPCR: lane 3, 51.5 C; lane 4, 49.8 C; lane 5, 52.6 C; lane 6, 48.5 C; lane 7, 54 C. (B) mPCR assay optimized with 52.6 C temperature of annealing. Lane M, 100-bp DNA ladder; lane 1, only M. synoviae; lane 2, only ARV; lanes 3 and 4, M. synoviae + ARV. (S1133 strain). The different extracted ARV RNA dilutions were used for cDNA synthesis as described previously (2,12). Extracted DNA and cDNA of each dilution were used as the template for mPCR. The DNA and RNA were quantified spectrophotometrically (BioPhotometerH, Eppendorf AG, Hamburg, Germany). Determination of the mPCR specificity was carried out by examining the ability of the test to detect and differentiate only M. synoviae and ARV. The mPCR was tested against other agents found in broilers, such as Mycoplasma gallisepticum, Escherichia coli, Pasteurella multocida, Staphylococcus aureus, Enterococcus faecalis, infectious bursal disease virus (IBDV), and infectious bronchitis virus (IBV). In addition, this assay was used to test articulations of 15 uninfected chickens to examine the specificity. The age of the healthy chickens varied from 35 to 47 days. For further confirmation, amplified DNA bands were excised and purified using QIAquick Gel Extraction (QIAGEN, Valencia, CA). Sequencing of mPCR amplicons that amplified 207- and 532-bp products of the 16S rRNA M. synoviae gene and ARV S1 gene, respectively, were cloned in a pGEMH-T Easy vector (Promega, Madison, WI) following the manufacturer’s instructions. Competent DH5a E. coli cells were used for chemical transformation. Transformed clones were selected in Luria-Bertani medium supplemented with ampicillin (100 mg/ml). Confirmation of the insert presence and size was performed by direct PCR amplification from the bacterial colony using primers specific for the pGEM-T Easy vector (pGEM-F and EXCELR). Colonies positive by PCR for insert presence were selected for

sequencing. Sequencing of clones was carried out using MegaBaceH 1000 DNA Analysis System (GE Healthcare, Little Chalfont, U.K.). The sequencing reactions were prepared using the DYEnamic ET Dye TerminatorH kit (GE Healthcare), following the manufacturer’s guidelines. The sequences were analyzed and compared with those from the international gene bank at the National Center for Biotechnology Information (Bethesda, MD) using the BLAST program. Immunofluorescence assay and histopathologic analysis. IF assays were performed on tissues (articular capsule/synovial membrane, tendon, lung, and liver) embedded in Tissue-Tek OCT compound. Four-micrometer sections were fixed with cold acetone in slides pretreated with poly-L-lysine (QIAGEN). The blocking of tissue sections was performed using skim milk solution (1%) for 60 min. Then, the sections were rinsed with phosphate-buffered saline (PBS) and incubated overnight at 4 C with the primary antibodies mouse anti-ARV antibody (Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil) or rabbit anti-M. synoviae antibody (Bcell, RS, Brazil), diluted 1:200 in PBS. Sections were rinsed with PBS and then incubated for 60 min with the secondary antibody AlexaFluorH 488 anti-rabbit immunoglobulin (Ig)G (Invitrogen) or fluorescein isothiocyanate antimouse IgG (2 mg/ml; Invitrogen), diluted 1:400 in PBS. After mounting, slides were analyzed in a fluorescence microscope (Axioplan; Carl Zeiss, Jena, Germany) and photographed with aDP72H/chassrgecoupled device system (Olympus America, Center Valley, PA). For the histopathologic analysis, tissues samples were fixed in 10% neutral buffered formalin and dehydrated in ethanol solutions. After dehydration, samples were paraffin embedded, sectioned (4 mm), and stained by hematoxylin and eosin. The histologic lesions suggestive of M. synoviae and ARV were defined as described previously (1,19,23).

RESULTS

The optimization of mPCR parameters, including annealing temperature, extension time, cycle quantity, and primer concentrations, was performed to achieve efficient conditions for simultaneous M. synoviae and ARV detection. Optimal mPCR conditions for detection of these two important avian pathogens were observed when using the concentration of 3.5 mM MgCl2 and a primer annealing temperature of 52.6 C (Fig. 1). Detection by visualization of PCR-amplified DNA products was 100 pg for M. synoviae and 100 pg of RNA used for cDNA synthesis for ARV (Fig. 2). The primer specificities were confirmed by sequencing the mPCR products. Sequencing analysis demonstrated the sequences of mPCR amplicons were identical to the sequences of respective ARV (MK87R/MK88F; 100%) and M. synoviae (MSR/ MSF; 100%) templates. The mPCR was found to be specific assay for M. synoviae and ARV, with no amplification of nucleic acids from M. gallisepticum,

223 76.88 (163/212) NT NA NT NA NT B

A

MS 5 M. synoviae. NA 5 not applicable. C NT 5 not tested.

25.47 (54/212) 58.01 (131/212) 25.47 (54/212) 58.01 (131/212) 19.33 (41/212) 51.41 (109/212) NT NT NT NT NT NT Field samples (n 5 212) Tibiotarsal articulation Serum

100 (16/16) 100 (16/16) 100 (16/16) NT NA NA NA 100 (16/16) NAB NA NA 100 (16/16) 100 (16/16) 100 (16/16) 0 NT 100 (16/16) 0 100 (16/16) NT 100 (16/16) 100 (16/16) 0 NT 100 (16/16) 0 100 (16/16) NT 100 (16/16) 100 (16/16) 0 NT Experimentally infected broilers chickens (n 5 16) Tibiotarsal articulation 100 (16/16) Lung/airsacs 0 Liver 100 (16/16) Serum NTC

MS

Serology (ELISA)

ARV MS

IF positive, % (no./total)

ARV MS

mPCR positive, % (no./total)

ARV MSA

The mPCR developed in this research was efficient in the simultaneous detection of M. synoviae and ARV infection in tissue samples from naturally and experimentally infected broiler chickens. These important avian pathogens cause chronic infectious disease and are transmitted rapidly among broiler chickens or poultry flocks (4,8,13). Despite the importance of these avian pathogens, systematic data on infection in Brazilian commercial flocks are scarce. Therefore, rapid diagnosis of these pathogens is an important tool for surveillance programs and for control of the spread of infection. Molecular biology has been used widely for the detection of various infectious agents (9), including several avian pathogens (2,10). The optimization of mPCR involves important steps such as testing the best annealing temperature, determining the optimal concentration of MgCl2 and Taq DNA polymerases, establishing efficient methods for DNA and RNA extraction, calibrating equipment (20). Low annealing temperature cycles can lead to nonspecific amplification, and high annealing temperatures can reduce primer pairing and the PCR amplification yield (6,26). Diagnosis of ARV is dependent on virus detection; however, the presence of the virus cannot necessarily be confirmed as the cause of the disease, except when ARV is detected in the affected joint tissue (13). In addition, in arthritis/synovitis cases caused by M. synoviae infection, high bacterial load can be observed within the joint tissue affected (22). Similarly, these studies have shown tibiotarsal articulation can be a major target for simultaneous detection by mPCR of M. synoviae and ARV infection in broiler chickens. The difference between the M. synoviae/ARV (mPCR/IF) detection data

ARV

DISCUSSION

Single PCR positive, % (no./total)

E. coli, P. multocida, S. aureus, E. faecalis, IBDV, and IBV. In addition, mPCR analysis of 212 lesions of arthritis from broiler chickens (Fig. 3) showed positive amplification for M. synoviae in 58.01% (131/212) of samples, ARV in 25.47% (54/212) of samples, and both M. synoviae and ARV in 18.86% (40/212) of samples (Table 2). Histopathologic analyses of broilers field samples showed suggestive lesions of M. synoviae, ARV infection, or both in 76.88% (163/212) of samples. The broiler chickens presented primarily diffuse lymphohistiocytic inflammatory infiltrate with accumulation of heterophils in the synovial capsule and digital flexor tendon. Furthermore, we observed hyperplasia and hypertrophy of synovial cells.

Clinical sample

Fig. 3. Clinical application of the mPCR for the simultaneous detection of M. synoviae and ARV. Lane M, 100-bp DNA ladder; lanes 1–4, field samples positive for M. synoviae and ARV; lanes 5, 7, and 8, field samples positive for M. synoviae; lane 6, field samples positive for ARV (50-ng concentration of DNA and cDNA template, agarose gel).

Table 2. Detection frequency of M. synoviae and ARV by single PCR, mPCR, IF, ELISA, and histopathology in experimentally and naturally infected clinical samples.

Histopathologic analysis

Mycoplasma synoviae and avian reovirus using multiplex PCR

224

C. Reck et al.

and histopathologic analysis possibly is due to infection by M. synoviae, ARV, or both not having a lesional pathognomonic standard (13,24), and lesions also can be associated with other infectious agents (13,16). The mPCR developed in this research has been proven to be sensitive and specific for detection and able to identify M. synoviae and ARV simultaneously in one reaction. Nevertheless, the sensitivity of mPCR assay depends on reaction condition optimization, purity of DNA and RNA, DNase and RNase activity, and the presence of inhibitors (20). This mPCR is useful for diagnosis, screening, and epidemiologic studies that require fast processing with large numbers of samples, as well as for surveillance of poultry flocks for early detection of pathogens and avoidance of economic losses. REFERENCES 1. Bradbur, J. M. Y., and A. Garuti. Dual infection with Mycoplasma synoviae and a tenosynovitis-inducing reovirus in chickens. Avian Pathol. 7:407–419. 1978. 2. Caterina, K. M., S. Frasca Jr, T. Girshick, and M. I. Khan. Development of a multiplex PCR for detection of avian adenovirus, avian reovirus, infectious bursal disease virus, and chicken anemia virus. Mol. Cell. Probes 18:293–298. 2004. 3. [COBEA] Cole´gio Brasileiro de Experimentac¸a˜o Animal. Princı´pios e´ticos da experimentac¸a˜o animal. Cole´gio Brasileiro de Experimentac¸a˜o Animal, Sa˜o Paulo, Brazil. 2005. 4. Dobson, K. N., and J. R. Glisson. Economic impact of a documented case of reovirus infection in broiler breeders. Avian Dis. 36:788–791. 1992. 5. Doman´ska-Blicharz, K., G. Tomczyk, and Z. Minta. Comparison of different molecular methods for detection of Mycoplasma synoviae. Bull. Vet. Inst. Puławy 53:357–360. 2009. 6. Edwards, M. C., and R. A. Gibbs. Multiplex PCR: advantages, development, and applications. Genome Res. 3:65–75. 1994. 7. Elnifro, E. M., A. M. Ashshi, R. J. Cooper, and P. E. Klapper. Multiplex PCR: optimization and application in diagnostic virology. Clin. Microbiol. Rev. 13:559–570. 2000. 8. Feberwee, A., D. R. Mekkes, J. J. Wit, E. G. Hartman, and A. Pijpers. Comparison of culture, PCR, and different serologic tests for detection of Mycoplasma gallisepticum and Mycoplasma synoviae infections. Avian Dis. 49:260–268. 2005. 9. Fiorentin, L., M. A. Z. Mores, I. M. Trevisol, S. C. Antunes, J. L. A. Costa, R. A. Soncini, and N. D. Vieira. Test Profiles of broiler breeder flocks housed in farms with endemic Mycoplasma synoviae infection. Rev. Bras. Cienc. Avic. 5:37–43. 2003. 10. Henegariu, O., N. A. Heerema, G. H. Vance, and P. H. Vogt. Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23:504–511. 1997. 11. Hummon, A. B., S. R. Lim, M. J. Difilippantonio, and T. Ried. Isolation and solubilization of proteins after TRIZOLH extraction of RNA and DNA from patient material following prolonged storage. Biotechniques 42:467–472. 2007. 12. Jindal, N., Y. Chander, D. P. Patnayak, S. K. Mor, A. F. Ziegler, and S. M. Goyal. A multiplex RT-PCR for the detection of astrovirus, rotavirus, and reovirus in turkeys. Avian Dis. 56:592–596. 2012. 13. Jones, R. C. Avian reovirus infections. Rev. Sci. Tech. 19:614–625. 2000. 14. Kibenge, F. S. B., R. C. Jones, and C. E. Savage. Effects of experimental immunosuppression on reovirus-induced tenosynovitis in light-hybrid chickens. Avian Pathol. 16:73–92. 1987.

15. Landman, W. J. M., and A. Feberwee. Aerosol-induced Mycoplasma synoviae arthritis: the synergistic effect of infectious bronchitis virus infection. Avian Pathol. 33:591–598. 2004. 16. Landman, W. J. M., D. R. Mekkes, R. Chamanza, P. Doornenbal, and E. Gruys. Arthropathic and amyloidogenic Enterococcus faecalis infections in brown layers: a study on infection routes. Avian Pathol. 28:545–557. 1999. 17. Lauerman, L. H. Mycoplasma PCR assays. In: Nucleic acid amplification assays for diagnosis of animal diseases. L. H. Laureman, ed. American Association of Veterinary Laboratory Diagnostics, Auburn, AL. pp. 41–52. 1998. 18. Lockaby, S. B., and F. J. Hoerr. Virulence of Mycoplasma synoviae in poultry: a review. Worlds Poult. Sci. J. 55:175–185. 1999. 19. Lockaby, S. B., E. J. Hoerr, L. H. Lauerman, and S. H. Kleven. Pathogenicity of Mycoplasma synoviae in broiler chickens. Vet. Pathol. 35:178–190. 1998. 20. Markoulatos, P., N. Siafakas, and M. Moncany. Multiplex polymerase chain reaction: a practical approach. J. Clin. Lab. Anal. 16:47–51. 2002. 21. Mohammed, H. O., T. E. Carpenter, and R. Yamamoto. Economic impact of Mycoplasma gallisepticum and Mycoplasma synoviae in commercial layer flocks. Avian Dis. 31:477–482. 1987. 22. Morrow, C. J., I. G. Bell, S. B. Walker, P. F. Markham, B. H. Thorp, and K. G. Whithear. Isolation of Mycoplasma synoviae from infectious synovitis of chickens. Aust. Vet. J. 67:121–124. 1990. 23. Ni, Y., and M. A. Kemp. A comparative study avian reovirus pathogenicity: virus spread and replication and induction lesions. Avian Dis. 39:554–566. 1995. 24. Reck, C., A. Menin, C. Pilati, and L. C. Miletti. Clinical and histologic lesions of mixed infection with Avian orthoreovirus and Mycoplasma synoviae in broilers. Pesq. Vet. Bras. 32:687–691. 2012. 25. Rosenberger, J. K., and N. O. Olson. Viral arthritis. In: Diseases of poultry, 10th ed. B. W. Calnek, H. J. Barnes, C. W. Beard, L. R. McDougald, and Y. M. Saif, eds. Iowa State University Press, Ames, IA. pp. 711–719. 1997. 26. Sambrook, J., and D. W. Russell. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring, NY. 2001. 27. Sent Es-Cue, G., H. L. Shivaprasad, and R. P. Chin. Systemic Mycoplasma synoviae infection in broilers chickens. Avian Pathol. 34: 137–142. 2005. 28. Triant, D. A., and A. Whitehead. Simultaneous extraction of highquality RNA and DNA from small tissue samples. J. Hered. 100:246–250. 2009. 29. Yin, H. S., and L. H. Lee. Development and characterization of a nucleic acid probe for avian reoviruses. Avian Pathol. 27:423–426. 1998. 30. Yoder, H. W. Jr Mycoplasmosis. In: Diseases of poultry, 9th ed. B. W. Calnek, J. J. Barnes, C. W. Beard, W. M. Reid, and H. W. Yoder Jr, eds. Iowa State University Press, Ames, IA. pp. 198–212. 1991. 31. Xie, Z., A. A. Fadl, T. Girshick, and M. I. Khan. Amplification of avian reovirus RNA using the reverse transcriptase-polymerase chain reaction. Avian Dis. 41:654–660. 1997.

ACKNOWLEDGMENTS The authors are indebted to Dr. Edmundo C. Grisard Lab of Universidade Federal de Santa Catarina for sequencing. This study was funded by Fundac¸a˜o de Amparo a Pesquisa e Inovac¸a˜o do Estado de Santa Catarina (FAPESC) and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES).

Rapid detection of Mycoplasma synoviae and avian reovirus in clinical samples of poultry using multiplex PCR.

Mycoplasma synoviae and avian reovirus (ARV) are associated with several disease syndromes in poultry and cause notable global economic losses in the ...
227KB Sizes 1 Downloads 4 Views