Pesq. Vet. Bras. 36(7):595-599, julho 2016 DOI: 10.1590/S0100-736X2016000700006 Detection of enteric viruses in pancreas and spleen of broilers with runting-stunting syndrome (RSS)1 2 2 2, 3 2,4 5 Luis Fabian N. Nuñez , Silvana H. Santander Parra , Claudete3* S. Astolfi-Ferreira Claudia Carranza , David I.D. De La Torre , Antonio C. Pedroso ABSTRACT.- and Antonio J. Piantino Ferreira - Detection of enteric viruses in pancreas and spleen of broilers Nuñez with L.F.N., runting-stunting Santander Parra S.H.,syndrome Astolfi-Ferreira (RSS). Pesquisa C.S., Carranza Veterinária C., De Brasilei La Tor- rare D.I.D.,36(5):595-599. Pedroso A.C. Departamento & Ferreira A.J.P. de Medicina 2016. Veterinária Preventiva e Saúde Animal, Facul- dade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando alterations,Marques de elevatedPaiva 87, feedSão Paulo,conversions SP 05508-270, an Brazil. E-mail: [email protected] virusesEnteric disease is a multifactorial problem in chickens, which causes gastrointestinal d impairment. In the last years, several enteric- were implicated in enteric disease; case reports have shown their presence alone or in concomitant infections during outbreaks and have suggested that they might be de termining factors in the aetiology of enteric disease. This study shows high detection rates of enteric viruses in the pancreas and spleen in samples from an outbreak of enteritis and malabsorption in 16 chicken flocks (n=80 broilers). Avian nephritis virus (ANV) was the most ubiquitous virus, present in 75% of the flocks followed by avian rotavirus group A (ART-A) with 68.75%, and by chicken astrovirus (CAstV) and chicken parvovirus (ChPV) in 43.75% of samples. Viruses were present in the pancreas of positive flocks at extremely high rates: 100% for ART-A, 91.7% for ANV, 100% for CAstV and 57.14% for ChPV. By- contrast, only 16.7% and 57.14% of intestine samples were positive for ANV and CAstV, resultrespectively. of viremia Avian reovirus (AReo) and avian adenovirus group 1 (FAdV-1) were not de tected. These results suggest that high viral detection rates in pancreas samples may be a during enteric disease, with subsequent damage of the exocrine pancreas, leading to runting-stunting syndrome (RSS). RESUMO.- [DetecçãoINDEX TERMS:de vírus Broiler, entéricos enteric em viruses, pâncreas detection, e pancreas,Nos últimos spleen, anos,runting-stunting os vírus entéricossyndrome, foramRSS. associados à baço de frangos com a síndrome de nanismo e raqui- doença entérica; casos reportados mostraram a infecção tismo (RSS).] A doença entérica é um problema multifa- de um único vírus e também infecções concomitantes du- torial em galinhas que causa alterações gastrointestinais, rante os surtos sugerindo a presença de múltiplos fatores alta taxa de detecção dos vírus entéricos em amostras de conversão1 alimentar elevada e deficiência de crescimento. pâncreasetiológicos e baçonas doenças de um surto entéricas. de enterite Este estudo e má-absorção mostra uma em - 2 Received on July 27, 2015. Departamento de Patologia, Faculdade de Medicina Veterinária e Zoo- ria (ANV) foi o vírus mais detectado, estando presente em tecniaAccepted (FMVZ), for Universidade publication on de April São 19,Paulo 2016. (USP), São Paulo, SP 05508- 75%16 lotes dos de lotes frangos seguido (n=80 pelo frangos). rotavírus O aviário vírus de grupo nefrite A (ART aviá- 3 Departamento de Medicina Veterinária Preventiva e Saúde Animal -A) em 68,75% dos casos, e pelo astrovirus (CAstV) e par- 270, Brazil. - thor: (VPS),4 FMVZ-USP, São Paulo, SP 05508-270, Brazil. *Corresponding au vovírus aviários (ChPV), ambos em 43,75% das amostras. [email protected] em percentuais elevados: 100% para ART-A e CAstV; 91,7% Os vírus estavam presentes no pâncreas dos lotes positivos 5 FaculdadDepartamento de Medicina de Medicina Veterinaria Veterinária, y Zootecnia, Faculdade Universidad de Medicina Central Veteri del- náriaEcuador, e Zootecnia, Quito, Ecuador. Universidade Federal da Fronteira Sul (UFFS), BR-182 16,7% e 57,14%, em amostras de intestino, foram positi- para ANV, e em 57,14% para ChPV. Em contraste, somente Km 466, Realeza, PR 85770-000, Brazil. vos para ANV e CAstV, respectivamente. Reovírus aviário 595 596 Luis Fabian N. Nuñez et al. (AReo) e o adenovírus do grupo 1 (FAdV-1) não foram de- Sampling - tuais de vírus detectados em amostras de pâncreas podem . Five broilers aged 8 to 28 days randomly, chosen, estartectados. associados Estes resultados à viremia sugerem durante quea doença os elevados entérica, percen com from each affected flock described above, were submitted to the Avian Diseases Laboratory at the FMVZ-USP, São Paulo/SP, Brazil subsequente lesão no pâncreas exócrino das aves levando giving a total of 80 birds analyzed. A separate pool of each organ (pool)(gastrointestinal of intestinal tract, tract, spleen 16 samplesor pancreas) (pool) was of obtained pancreas from and the10 - samplesfive sampled (pool) birds of spleen, from each giving flock, a total so wereof 42 collected samples, 16all samples ao desenvolvimento da síndrome de nanismo e raquitismo. TERMOS DE INDEXAÇÃO: Frangos, vírus entéricos, detecção, pân creas, baço, síndrome de nanismo e raquitismo, RSS. wereNucleic submitted acid to extraction molecular for analyses viral detection for the presence. Tissues of from enteric the INTRODUCTION viruses. intestine, pancreas or spleen of five (05) birds of each flock were Enteric disease in poultry is a multifactorial problem- pooled separately and homogenized; pooled samples from each that is not fully understood. This disorder (also known tissue type and from each flock were homogenized separately and as malabsorption syndrome and as runting-stunting syn diluted in 1.5-mL microcentrifuge tubes containing 0.1 M sterile drome or RSS) is a multifactorial disease of chickens which PBS, pH 7.4, at a 1:1 proportion. These diluted samples were then- results in gastrointestinal alterations along with elevated frozen at -20°C for 10 minutes and thawed at 56°C for 1 minute, and this process was repeated three times, including homogeniz feed conversion and decreased body weight (Rebel et al.- ation between freeze-thaw cycles and vortexing for 20 seconds.- 2006, Saif 2013, Nuñez et al. 2016a, Nuñez et al. 2016b). For DNA extraction, the samples were centrifuged at 12.000xg for Bacterial, protozoal and viral agents are known to be in 20 minutes at 4°C, and for RNA extraction, the samples were cent volved in the development of outbreaks, which are also rifuged for 30 minutes under the same conditions. ®The (Invitrogen, resulting related to flock management and to the immunological- supernatants were stored at -20°C until analysis. DNA and RNA status of flocks (Nuñez & Ferreira 2013, Moura-Alvarez et werePCR extracted and RT-PCR from the detection supernatants of viral using agents TRIzol. The screen- al. 2013, Moura-Alvarez et al. 2014). In the last years, en ingValencia, procedures CA, USA) performed according to to detectthe manufacturer’s the most common instructions. enteric teric viruses, such as chicken astrovirus (CAstV), chicken- parvovirus (ChPV), avian rotavirus (ART), fowl adenovirus viruses in Brazilian flocks (Moura-Alvarez et al. 2013) included type I (FAdV I) and avian reovirus (AReo) have been in polymerase chain reaction (PCR) assays for the detection of creasingly implicated. This report is showing its presence avian adenovirus group 1 (FAdV-1) (Meulemans et al. 2001) and alone or in concomitant infections during outbreaks and chicken parvovirus (ChPV) (Zsak et al. 2009) and RT-PCR analysis havesuggesting led to that the they development play an important of experimental role in the models aetiology to for chicken astrovirus (CAstV), avian nephritis virus (ANV), avian of enteric disease (Zsak et al. 2013). These recent reports- rotavirus group A (ART-A) (Day et al. 2007) and Avian reovirus (AReo) (Pantin-Jackwood et al. 2008). Target genes, primers and- recreate the disease and to try to determine its etiopatho amplicon sizes are listed in Table 1. ChPV, FAdV-1, CAstV and ANV genicity (Zsak et al. 2013, Decaesstecker et al. 1989, Nuñez strains previously isolated from Brazilian flocks and whose iden et al. 2015a). However, due to the complex nature of this- tity was confirmed by sequencing were used as positive controls disease, the exact aetiology remains unknown. in the molecular assays. Additionally, Nebraska calf diarrhoea virus and the S1133 strain were used as controls for rotavirus and The objective of this study was to detect viruses asso reovirus, respectively. RESULTS ciated with RSSMATERIALS in Brazilian ANDcommercial METHODS chicken flocks. Description of the problem. - ing undigested feed and aqueous feces In late 2012, 16 broiler flocks Table 2 shows the detection rates of the screened viruses. from the state of Minas Gerais, Brazil, were reported to be excret- All flocks that presented enteric problems were positive for and to be exhibiting delayed at least one of these pathogens. Avian nephritis virus (ANV) duodenitisgrowth and increased mortality, at variable ( rates. Necropsy re and avian rotavirus group A (ART-A) were present in more vealed severe congestion of the duodenal walls compatible with than 60% of the flocks (75% and 68.75%, respectively). Both , and Tablerunting-stunting 1. Primer sequences,syndrome RSS) amplicon was mooted. sizes for RT-PCRchicken and PCR astrovirus assays performed(CAstV) and to detectchicken enteric parvovirus (ChPV) viruses in chickens with RSS in Brazil VirusAvian rotavirus group A TargetNSP4 gene PrimerNSP4-F30 name GGG CGTPrimer GCG sequence GAA AGA (5’-3’) TGG AGA AC Amplicon630 size (bp) NSP4-R660 GGG GTT GGG GTA CCA GGG ATT AA ANV pol 1R CRT TTG CCC KRT ART CTT TRT Avian nephritis virus Polymerase ANV pol 1F GYT GGG CGC YTC YTT TGA YAC 473 CAS pol 1R TCA GTG GAA GTG GGK ART CTA C Chicken astrovirus Polymerase CAS pol 1F GAY CAR CGA ATG CGR AGR TTG 362 PVR1 TTT GCG CTT GCG GTG AAG TCT GGC TCG AvianChicken reovirus parvovirus NSS4 S4-F13PVF1 GTGTTC TAACGT TAAGTT CGAGGA TATGTT CAC TCC TCACG AGT TTC 1120561 S4-R1133 TAC GCC ATC CTA GCT GGA Avian adenovirus Hexon Hexon A CAA RTT CAG RCA GAC GGT 897 group 1 Hexon B TAG TGA TGM CGS GAC ATC AT Pesq.