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A Multiplex RT-PCR for the Detection of Astrovirus, Rotavirus, and Reovirus inTurkeysAuthor(s): Naresh Jindal, Yogesh Chander, Devi P. Patnayak, Sunil K. Mor, Andre F. Ziegler, and SagarM. GoyalSource: Avian Diseases, 56(3):592-596. 2012.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/9958-100911-ResNote.1URL: http://www.bioone.org/doi/full/10.1637/9958-100911-ResNote.1

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Research Note—

A Multiplex RT-PCR for the Detection of Astrovirus, Rotavirus, andReovirus in Turkeys

Naresh Jindal,AB Yogesh Chander,A Devi P. Patnayak,A Sunil K. Mor,A Andre F. Ziegler,A and Sagar M. GoyalAC

ADepartment of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, 1333 Gortner Avenue,Saint Paul, MN 55108

BDepartment of Veterinary Public Health and Epidemiology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and AnimalSciences, Hisar 125 004, Haryana, India

Received 12 December 2011; Accepted 1 May 2012; Published ahead of print 2 May 2012

SUMMARY. This study was undertaken to develop and validate a multiplex reverse transcription-polymerase chain reaction(mRT-PCR) for simultaneous detection of avian rotavirus, turkey astrovirus-2 (TAstV-2), and avian reovirus. Primers targeting theconserved regions of NSP4 gene of avian rotavirus, polymerase gene of TAstV-2, and S4 gene of avian reovirus were used. Theposition of bands at 630, 802, and 1120 base pairs on agarose gel confirmed the presence of rotavirus, TAstV-2, and reovirus,respectively. This mRT-PCR was found to be specific as no amplification was observed with avian influenza virus, Newcastledisease virus, turkey coronavirus, avian metapneumovirus, and intestinal contents of uninfected turkey poults. Intestinal contents ofpoults from flocks suspected of exhibiting ‘‘poult enteritis syndrome’’ were pooled and tested. Of the 120 pooled samples tested,70% were positive for TAstV-2, 45% for avian rotavirus, and 18% for avian reovirus. These three viruses were detected alone or indifferent combinations. Of the samples tested, 20% were negative for these three viruses, 38% were positive for a single virus(TAstV or rotavirus or reovirus), and 42% were positive for two or three viruses. This single-tube mRT-PCR assay has the potentialto serve as a rapid diagnostic method for the simultaneous detection of the three enteric viruses in turkeys.

RESUMEN. Nota de Investigacion—Un metodo de RT-PCR multiple para la deteccion de astrovirus, rotavirus y reovirus enpavos.

Este estudio se llevo a cabo para desarrollar y validar un metodo de transcripcion reversa y reaccion en cadena de la polimerasamultiple (mRT-PCR) para la deteccion simultanea de rotavirus aviar, astrovirus de los pavos 2 (TAstV-2), y reovirus aviar. Losiniciadores estuvieron dirigidos a las regiones conservadas del gene NSP4 de los rotavirus aviares, al gene de la polimerasa delTAstV-2, y para el gen S4 del reovirus aviar. La posicion de las bandas de ADN con longitudes de 630, 802 y 1120 pares de basesen geles de agarosa confirmo la presencia de rotavirus, TAstV-2, y reovirus, respectivamente. Este metodo de RT-PCR multipleresulto ser mas especıfico, no se observo amplificacion con el virus de la influenza aviar, con el virus de la enfermedad de Newcastle,con el coronavirus de los pavos, con metapneumovirus aviar, y o con el contenido intestinal de pavos no infectados. Los contenidosintestinales de los pavipollos de parvadas sospechosas de presentar el ‘‘sındrome de la enteritis de los pavipollos’’ fueron agrupados yse analizaron. De las 120 muestras agrupadas analizadas, el 70% fueron positivas para TAstV-2, el 45% fue positivo para elrotavirus aviar, y el 18% para reovirus aviar. Estos tres virus se detectaron solos o en combinaciones diferentes. De las muestrasanalizadas, el 20% fueron negativos para estos tres virus, el 38% fueron positivos para un solo virus (rotavirus o TAstV o reovirus), yel 42% fueron positivos para dos o tres virus. Este metodo de PCR multiple tiene el potencial para servir como un metodo rapidode diagnostico para la deteccion simultanea de los tres virus entericos en los pavos.

Key words: turkey astrovirus, avian rotavirus, avian reovirus, multiplex reverse transcription-polymerase chain reaction, poultenteritis syndrome

Abbreviations: AIV 5 avian influenza virus; AMPV 5 avian metapneumovirus; bp 5 base pairs; mRT-PCR 5 multiplex reversetranscription-polymerase chain reaction; MVDL 5 Minnesota Veterinary Diagnostic Laboratory; NDV 5 Newcastle disease virus;PEC 5 poult enteritis complex; PEMS 5 poult enteritis and mortality syndrome; PES 5 poult enteritis syndrome; RT-PCR 5 reverse transcription-polymerase chain reaction; TAstV 5 turkey astrovirus; TAstV-1 5 turkey astrovirus-1; TAstV-2 5 turkey astrovirus-2

Poult enteritis complex (PEC) is of paramount importance inpoultry industry because of losses associated with enteritis,retardation of growth, and mortality. The etiology of PEC iscomplex and may involve infection with one or more pathogensincluding viruses (avian rotavirus, enterovirus, turkey astrovirus,turkey coronavirus, and avian reovirus), bacteria (Salmonella sp.,Escherichia coli, Enterococcus sp.), and protozoa (Eimeria, Cryptospo-ridium) (1). In recent years, a new disease called ‘‘poult enteritissyndrome’’ (PES) has been detected in Minnesota turkeys, which is

characterized by depression, retarded growth, and fluid-filledintestines in turkey poults (7). We reproduced PES by inoculatingturkey poults orally with intestinal contents from PES-affected birds;the intestinal contents contained rotavirus, turkey astrovirus, andSalmonella sp. (8).

Enteric viruses play an important role in causing enteritis inanimals and birds. Rotavirus is an important pathogen causingdiarrhea in humans and animals and has also been detected indomestic poultry and wild birds (3,5,11,12,25,27) includingdiarrheic turkeys (8,17,19) and broiler chickens with runting andstunting syndrome (15). Astrovirus is an important cause of enteritisin humans and animals. Turkey astrovirus (TAstV) was firstCCorresponding author. E-mail: goyal001@umn.edu

AVIAN DISEASES 56:592–596, 2012

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identified in 1980 in the United Kingdom from diarrheic turkeypoults (13) and was subsequently reported from the United States(where it was named TAstV-1) (18,19). Later, another turkeyastrovirus (TAstV-2), genetically distinct from TAstV-1, wasidentified and characterized (10,20). Astroviruses have also beenassociated with ‘‘poult enteritis and mortality syndrome’’ (PEMS)(10,28). Although both TAstV-1 and TAstV-2 have been detected inthe United States (16,17), the occurrence of TAstV-2 is much higherthan that of TAstV-1 (17). TAstV-2 has also been detected in poultsaffected with PES (6). Avian reovirus has been isolated from bothPEMS and PEC and has been associated with a wide range of diseasepresentations in avian species (4,14,24). This virus has also beendetected in PES-affected poults (6).

Rapid detection of enteric viruses has important economical andclinical implications. Reverse transcription-polymerase chain reac-tion (RT-PCR) assays for the detection of individual enteric viruses(rotavirus, TAstV, reovirus, or coronavirus) have been developed(9,16,17,26). Such assays can detect a single virus at a time but donot detect infections involving more than one virus. Multiplex RT-PCR (mRT-PCR) assays have been developed for the detection ofenteric viruses (2,21,23), but none of these methods detect avianrotavirus, TAstV, and avian reovirus simultaneously in a singlereaction. We report the development of such an assay for thesimultaneous detection of avian rotavirus, TAstV, and avian reovirusbecause these viruses have been detected in various combinations incases of PES in Minnesota (6).

MATERIALS AND METHODS

Viruses. Known avian rotavirus and TAstV (grown in embryonatedeggs) were kindly provided by Prof. Y. M. Saif (Wooster, OH). RNAfrom avian reovirus (SEP108) was obtained from Dr. J. M. Day(Southeast Poultry Research Laboratory, Athens, GA). Total RNA wasextracted from avian rotavirus and turkey astrovirus using the Trizol LSreagent (Invitrogen, Carlsbad, CA). The RNA from these three viruseswas used as positive control.

Multiplex RT-PCR. Primers used for the development of mRT-PCRare listed in Table 1. Amplification was carried out using a OneStep RT-PCR kit (Qiagen,Valencia, CA). The reaction mix consisted of 13 RT-PCR reaction buffer, 320 mM each dNTP, 0.6 mM forward and reverseprimer of each virus, 2 ml of enzyme blend, 1.5 mM of MgCl2, 3 ml ofextracted RNA, and nuclease-free water to make a total volume of 50 ml.Amplification steps consisted of reverse transcription at 50 C for 30 min,initial denaturation at 94 C for 15 min, 35 cycles of denaturation at 94 Cfor 1 min, annealing at 53 C for 2 min, extension at 72 C for 2 min, anda step of final extension at 72 C for 10 min. The PCR products wereseparated by electrophoresis on 1.2% agarose gel in Tris-acetate-EDTAbuffer. A 100 base pair (bp) DNA ladder was used as a molecular weightmarker and bands at 630, 802, and 1120 bp positions on agarose gelconfirmed the presence of rotavirus, TAstV-2, and reovirus, respectively.For further confirmation, amplified DNA bands were excised, purifiedusing Qiagen gel purification kit (Qiagen, Valencia, CA) and sequencedin both directions (forward and reverse) at the Biomedical GenomicCenter, University of Minnesota, St. Paul. The nucleotide sequences

thus obtained were aligned with the existing database using BLASTsearch tool available online (www.ncbi.nlm.nih.gov) to confirm theiridentity.

Specificity and sensitivity of the mRT-PCR. To test the specificityof the mRT-PCR assay, RNA extracted from isolates of Newcastledisease virus (NDV), avian influenza virus (AIV), turkey coronavirus,and avian metapneumovirus (AMPV) were used. Known strains ofNDV, AIV, and AMPV were retrieved from the repository of theMinnesota Veterinary Diagnostic Laboratory (MVDL), St. Paul, MN,and tested by this mRT-PCR. However, known strain of turkeycoronavirus NC95 was kindly provided by Prof. James Guy (NorthCarolina State University, NC). In addition to these four viruses, thisassay was also employed to test intestinal contents of 10 uninfectedturkey poults to test the specificity; the age of the healthy poults variedfrom day 1 to 20 days. Sensitivity of the assay was determined using5-fold dilutions (1:5–1:625) of RNA extracts from known isolates ofavian rotavirus, TAstV-2, and reovirus. The RNA of these three viruseswas quantified spectrophotometrically.

Screening of field samples by mRT-PCR. Intestinal contents fromPES-affected cases (n 5 120) were screened by this mRT-PCR for thedetection of enteric viruses. Intestinal contents of birds (n 5 5) from asingle flock/case with PES were pooled to make one pooled sample. Atotal of 120 such pooled samples from different turkey flocks werecollected. A 10% suspension of the pooled samples was made inphosphate-buffered saline (pH 7.4). The suspension was homogenizedfollowed by centrifugation at 784 3 g for 20 min. The supernatant wascollected and processed further for the extraction of total RNA using theTRIzol LS reagent (Invitrogen, Carlsbad, CA) followed by testing withthe mRT-PCR.

RESULTS

Initially the reaction conditions were optimized with known avianrotavirus, TAstV, and avian reovirus using primers for all threeviruses in a single reaction. Using these primers, all three viruses wereamplified, and gel electrophoresis of the PCR products revealedbands of sizes 630, 802, and 1120 bp specific for avian rotavirus,TAstV-2, and avian reovirus, respectively (Fig. 1). Each band wasexcised from the agarose gel, purified, and sequenced. BLASTanalysis of aligned sequences confirmed that each distinct bandidentified the appropriate virus. The BLAST analysis of TAstVsequence gave match only with TAstV-2.

Specificity of this assay was found to be high for the targetedviruses as no specific bands at these positions were observed whenthis multiplex assay was tested with AIV, NDV, AMPV, and turkeycoronavirus. In addition, intestinal contents of all 10 uninfectedturkey poults were also found negative for these three viruses as nospecific bands were observed. For sensitivity testing, fivefold serialdilutions of targeted viruses were tested by this mRT-PCR, and allthree viruses could be detected up to the last dilution (1:625) tested.Total RNA concentrations at the last dilution were TAstV-2(110 ng), avian reovirus (27 ng), and rotavirus (257 ng).

Of the 120 pooled samples tested, 70% were positive for TAstVfollowed by avian rotavirus (45%) and avian reovirus (18.3%). A

Table 1. Primers used in this study for multiplex RT-PCR assay.

Virus Primer sequence Gene Reference

Avian rotavirus F: 59-GGGCGTGCGGAAAGATGGAGAAC-39 NSP4 17R: 59-GGGGTTGGGGTACCAGGGATTAA-39

Turkey astrovirus -2 F: 59-TGGACCGACCCRRTTTTYACCA-39 Polymerase 9R: 59-GGCCCGACYTCAGGMAGTTGT-39

Avian reovirus F: 59-GTGCGTGTTGGAGTTTCCCG-39 S4 16R: 59-TACGCCATCCTAGCTGGA-39

mRT-PCR for avian enteric viruses 593

total of 38% samples were positive for a single virus (TAstV orrotavirus or reovirus), while 42% were positive for two or threeviruses. The most predominant combination was TAstV and avianrotavirus (Fig. 2). The remaining 20% samples were negative for allthree viruses (Fig. 2).

DISCUSSION

Enteric diseases in poultry have been recognized for decades and areresponsible for decreased growth and mortality in poults leading toconsiderable economic losses. Various enteric syndromes such ascoronaviral enteritis of turkeys, poult malabsorption syndrome, runtingand stunting syndrome of turkeys, maldigestion syndrome, PEMS,spiking mortality of turkeys, and turkey viral enteritis have beenreported in turkeys and are a part of the PEC complex (1). A number ofenteric viruses have been identified from these enteric syndromes withrotavirus, TAstV, enterovirus, reovirus, and turkey coronavirus beingthe predominant viruses detected. Because of their adverse effects inturkeys, it is important to detect these viruses in a flock as early aspossible so that effective control strategies can be initiated.

Several RT-PCR methods have been developed during the last fewyears to detect enteric viruses (rotavirus, astrovirus, reovirus,coronavirus, and adenovirus) individually in turkeys (9,16,17,26).The mRT-PCR assays have also been described for simultaneousdetection of some of these viruses (2,21,23). For example, Sellers etal. (21) developed an mRT-PCR assay that detects turkey astrovirusand turkey coronavirus. The real-time mRT-PCR method describedby Spackman et al. (23) detects TAstV, turkey coronavirus, andturkey-origin reovirus in a single reaction. The mRT-PCR methoddescribed by Day et al. (2) identifies TAstV types 1 and 2, avianrotavirus, and avian nephritis virus in a single reaction. None ofthese methods detects avian rotavirus, TAstV, and avian reovirus in asingle reaction. The present study was therefore undertaken todevelop and evaluate an mRT-PCR assay for the simultaneousdetection of these three viruses in a single-tube system. We chosethese three viruses for mRT-PCR because these three viruses havebeen detected frequently in PES cases from Minnesota (6).

Specificity of the newly developed mRT-PCR assay was evidentbecause of lack of amplification with AIV, NDV, AMPV, and turkeycoronavirus. Fivefold serial dilutions of RNA extracts were tested bythis assay to determine the sensitivity of the method, and all three

Fig. 1. PCR amplification of avian rotavirus, turkey astrovirus, and avian reovirus in a multiplex RT-PCR assay. Lanes: M 5 molecular marker;1 5 positive control for rotavirus; 2 5 positive control for turkey astrovirus; 3 5 positive control for reovirus; 4–6 5 field samples positive forrotavirus and turkey astrovirus; 7 5 field sample positive for turkey astrovirus and reovirus; 8–11 5 field samples positive for rotavirus, turkeyastrovirus, and reovirus; 12–14 5 negative controls (turkey coronavirus, avian influenza virus, Newcastle disease virus).

Fig. 2. Pie chart showing poult enteritis syndrome cases tested for enteric viruses by multiplex RT-PCR. ARV 5 avian reovirus, RV 5 rotavirus,TAstV5 turkey astrovirus.

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viruses could be detected up to the last dilution tested (1:625). Theamount of viral RNA in the specimen is probably lower than thereported amount, so the sensitivity will actually be better thanreported since the calculation will include nonviral RNA. Theamplified products of the last dilution were clearly visible on 1.2%agarose gel. For determining test sensitivity, RNA extracts fromembryo-passaged intestinal material were used. The sensitivity of theassay may be lower when performed on clinical samples because ofthe potential presence of PCR inhibitors. This study indicated thatthe mRT-PCR assay developed was sensitive and specific for thetargeted viruses. Furthermore, BLAST analysis of nucleotidesequences obtained from purified bands gave match with the specificviruses thereby confirming their identity.

The PES, characterized by depression, retarded growth, and fluid-filled intestines in turkey poults has been observed in Minnesotaturkeys. It is an infectious, multifactorial disease of young turkeysusually identified between 1 day and 7 wk of age. Analysis of data of151 PES flocks from 2002 to 2007 revealed that 73.5%, 23.2%, and3.3% flocks had poults less than 3 wk, 3–6 wk, and 6–9 wk of age,respectively. We also found significantly more occurrence of PES inpoults less than 3 wk of age as compared to poults more than 3 wk ofage (7). We detected viruses (rotavirus, small round virus), bacteria(Salmonella, nonhemolytic Escherichia coli, Enterococcus), andprotozoa (Eimeria spp.) alone or in different combinations fromPES cases with Salmonella and rotavirus being the most commonlyidentified pathogens. The viruses in these cases were detected only byelectron microscopy (7). Subsequently we detected rotavirus, TAstV-2, and avian reovirus from PES cases by RT-PCR (6); the viruseswere detected by individual RT-PCR for each virus.

To validate the use of this mRT-PCR assay, 120 pooled samplesfrom PES cases were tested. Of the 120 samples, 43 samples werecollected in 2007–2008 (6), and the remaining 77 were collected in2009–2010. Samples positive for all three viruses gave clear bands onagarose gel; these bands were distinguishable from each otherwithout any ambiguity. Twenty percent of the pools were negativefor all three viruses. This may be due to very low quantity of entericviruses (rotavirus, TAstV, and reovirus) in the intestinal contents.Second reason could be the involvement of enteric pathogens otherthan these three viruses in PES cases. In a retrospective study on PESfrom 2002–2007, we have observed the involvement of Salmonella,E. coli, and Eimeria spp. in PES cases (7). Third reason could bethe noninfectious etiology of enteritis. The role of nutritional ormanagement factors in causing enteritis under field conditionscannot be ruled out (7). Another possibility could be the role ofinhibitors present in fecal samples that interfere in the PCR. It mayalso be possible that the cases presented to the MVDL were in thestage at which they revealed the disease and pathology related toPES; however, the infection had already been cleared. Spackman etal. (22) experimentally infected turkeys with rotavirus or TAstV-2alone or in combination and reported huddling and diarrhea 4–11 days post-infection in poults of all groups. However, rotaviruswas shed only by 0%–25% birds at 7 days post-infection in differentgroups. It indicated that virus(es) induced clinical findings, andpathology may persist in affected birds for a longer duration and theinfection leaves the host, thereby leading to nondetection of virus(es)from such cases. Under field conditions, it is possible that the casesbrought for disease diagnosis at a later stage may lead tononidentification of the virus.

In this study 70% samples were positive for turkey astrovirusfollowed by rotavirus (45%) and reovirus (18.3%). In a survey,Pantin-Jackwood et al. (17) tested 96 samples from turkeys in eightcommercial operations and a research facility and reported 89.5%

samples to be positive for astrovirus and 67.7% for rotavirus. All flocksin their study at all time points were negative for turkey coronavirus,reovirus, and group 1 adenovirus. In another study, Pantin-Jackwoodet al. (16) examined intestinal contents from 33 commercial turkeyflocks for enteric viruses by RT-PCR and reported the presence ofTAstV-1 and TAstV-2 in 15.4% and 100% of the turkey flocks,respectively. Rotaviruses and reoviruses were present in 69.7% and45.5% of the turkey flocks tested. Such a variation in prevalence overdifferent geographical regions can be expected.

Simultaneous detection of three viruses by this assay shouldreduce the time and expense in evaluating clinical samples and maybe of use in veterinary diagnostic laboratories where large numbers ofsamples are tested on a routine basis for these enteric viruses. Earlydetection of these viruses in PES cases may lead to early initiation ofcontrol measures at the farm level. Since these viruses have also beendetected in apparently healthy flocks (5,17), it would be interestingto use this mRT-PCR to further understand the epidemiology ofthese viruses in such flocks. In summary, this single-tube mRT-PCRassay holds potential as a rapid diagnostic method for thesimultaneous detection of three avian enteric viruses in turkeys.

REFERENCES

1. Barnes, H. J., J. S. Guy, and J. P. Vaillancourt. Poult enteritiscomplex. Rev. Sci. Tech. Off. Int. Epiz. 19:565–588. 2000.

2. Day, J. M., E. Spackman, and M. Pantin-Jackwood. A multiplex RT-PCR test for the differential identification of turkey astrovirus type 1, turkeyastrovirus type 2, chicken astrovirus, avian nephritis virus, and avianrotavirus. Avian Dis. 51:681–684. 2007.

3. Gough, R. E., W. J. Cox, and J. Devoy. Isolation and identificationof rotavirus from racing pigeons. Vet. Rec. 130:273. 1992.

4. Heggen-Peay, C. L., M. A. Qureshi, F. W. Edens, B. Sherry, P. S.Wakenell, P. H. O’Connell, and K. A. Schat. Isolation of a reovirus frompoult enteritis and mortality syndrome and its pathogenicity in turkeypoults. Avian Dis. 46:32–47. 2002.

5. Jindal, N., D. P. Patnayak, Y. Chander, A. F. Ziegler, and S. M.Goyal. Detection and molecular characterization of enteric viruses in breederturkeys. Avian Pathol. 39:53–61. 2010.

6. Jindal, N., D. P. Patnayak, Y. Chander, A. F. Ziegler, and S. M.Goyal. Detection and molecular characterization of enteric viruses frompoult enteritis syndrome in turkeys. Poult. Sci. 89:217–226. 2010.

7. Jindal, N., D. P. Patnayak, A. F. Ziegler, A. Lago, and S. M. Goyal. Aretrospective study on poult enteritis syndrome in Minnesota. Avian Dis.53:268–275. 2009.

8. Jindal, N., D. P. Patnayak, A. F. Ziegler, A. Lago, and S. M. Goyal.Experimental reproduction of poult enteritis syndrome: clinical findings,growth response and microbiology. Poult. Sci. 88:949–958. 2009.

9. Koci, M. D., B. S. Seal, and S. Schultz-Cherry. Development of an RT-PCR diagnostic test for an avian astrovirus. J. Virol. Methods 90:79–83. 2000.

10. Koci, M. D., B. S. Seal, and S. Schultz-Cherry. Molecularcharacterization of an avian astrovirus. J. Virol. 74:6173–6177. 2000.

11. Legrottaglie, R., V. Rizzi, and P. Agrimi. Isolation and identificationof avian rotavirus from pheasant chicks with signs of clinical enteritis. Comp.Immunol. Microbiol. Infect. Dis. 20:205–210. 1997.

12. McNulty, M. S., G. M. Allan, and J. C. Stuart. Rotavirus infection inavian species. Vet. Rec. 103:319–320. 1978.

13. McNulty, M. S., W. L. Curran, and J. B. McFerran. Detection ofastroviruses in turkey faeces by direct electron microscopy. Vet. Rec.106:561. 1980.

14. Nibert, M. L., and L. A. Schiff. Reoviruses and their replication. In:Field’s virology, 4th ed. D. M. Knipe and P. M. Howley, eds. LippincottWilliams and Wilkins, Philadelphia. pp. 1679–1728. 2001.

15. Otto, P., E. M. Liebler-Tenorio, M. Elschner, J. Reetz, U. Lohren,and R. Diller. Detection of rotaviruses and intestinal lesions in broiler chicksfrom flocks with runting and stunting syndrome (RSS). Avian Dis.50:411–418. 2006.

mRT-PCR for avian enteric viruses 595

16. Pantin-Jackwood, M. J., J. M. Day, M. W. Jackwood, and E.Spackman. Enteric viruses detected by molecular methods in commercialchicken and turkey flocks in the United States between 2005 and 2006.Avian Dis. 52:235–244. 2008.

17. Pantin-Jackwood, M. J., E. Spackman, J. M. Day, and D. Rives.Periodic monitoring of commercial turkeys for enteric viruses indicatescontinuous presence of astrovirus and rotavirus on the farms. Avian Dis.51:674–680. 2007.

18. Reynolds, D. L., and Y. M. Saif. Astrovirus: a cause of an entericdisease in turkey poults. Avian Dis. 30:728–735. 1986.

19. Saif, L. J., Y. M. Saif, and K. W. Theil. Enteric viruses in diarrheicturkey poults. Avian Dis. 29:798–811. 1985.

20. Schultz-Cherry, S., D. R. Kapczynski, V. M. Simmons, M. D. Koci,C. Browns, and H. J. Barnes. Identifying agent(s) associated with poultenteritis mortality syndrome: importance of the thymus. Avian Dis.44:256–265. 2000.

21. Sellers, H. S., M. D. Koci, E. Linnemann, L. A. Kelly, and S. Schultz-Cherry. Development of a multiplex reverse transcription-polymerase chainreaction diagnostic test specific for turkey astrovirus and coronavirus. AvianDis. 48:531–539. 2004.

22. Spackman, E., J. M. Day, and M. J. Pantin-Jackwood. Astrovirus,reovirus, and rotavirus concomitant infection causes decreased weight gain inbroad-breasted white poults. Avian Dis. 54:16–21. 2010.

23. Spackman, E., D. Kapczynski, and H. Sellers. Multiplex real-timereverse transcription-polymerase chain reaction for the detection of three

viruses associated with poult enteritis complex: turkey astrovirus, turkeycoronavirus, and turkey reovirus. Avian Dis. 49:86–91. 2005.

24. Spackman, E., M. Pantin-Jackwood, J. M. Day, and H. Sellers. Thepathogenesis of turkey origin reoviruses in turkeys and chickens. AvianPathol. 34:291–296. 2005.

25. Takehara, K., H. Kiuchi, M. Kuwahara, F. Yanagisawa, M.Mizukami, H. Matsuda, and M. Yoshimura. Identification and character-ization of a plaque forming avian rotavirus isolated from a wild bird inJapan. J. Vet. Med. Sci. 53:479–486. 1991.

26. Tang, Y., M. M. Ismail, and Y. M. Saif. Development of antigencapture enzyme-linked immunosorbent assay and RT-PCR for detection ofturkey astroviruses. Avian Dis. 49:182–188. 2005.

27. Theil, K. W., and Y. M. Saif. Age-related infections with rotavirus,rotaviruslike virus, and atypical rotavirus in turkey flocks. J. Clin. Microbiol.25:333–337. 1987.

28. Yu, M., M. M. Ismail, M. A. Qureshi, R. N. Dearth, H. J. Barnes,and Y. M. Saif. Viral agents associated with poult enteritis and mortalitysyndrome: the role of a small round virus and a turkey coronavirus. AvianDis. 44:297–304. 2000.

ACKNOWLEDGMENT

This study was funded in part by a grant from the Rapid AgriculturalResponse Fund, University of Minnesota.

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