Viral metagenomic analysis of chickens with runting-stunting syndrome in the Republic of Korea

Abstract

Background and aims: Runting-stunting syndrome (RSS) in chickens, also known as malabsorption syndrome, which is characterized by mild to severe enteritis and diagnosed through typical histopathologic examination as well as clinical signs, results in considerable economic losses. Despite the many studies carried out over decades to determine the etiologic agents of RSS involved in the disease, several outbreaks remained without the elucidation of, potentially multiple, etiologies involved.

Methods: We performed comparative analysis of viral metagenomes from four chicken flocks affected with RSS using next-generation sequencing. Primers for the detection of chicken enteric viruses were designed from the sequencing data obtained with metagenomics. Multiplex reverse transcription-polymerase chain reaction (PCR) and PCR were performed to detect a variety of etiological agents previously described in natural cases of RSS.

Results: The most abundant viral families identified in this study were Astroviridae, Picornaviridae, Parvoviridae, Caliciviridae, Reoviridae and Picobirnaviridae. Chicken astrovirus sequences were present in all four samples, suggesting an association between chicken astrovirus and RSS and chicken astrovirus as a candidate pathogen responsible for RSS. Picobirnavirus and the newly identified chapparvovirus were found in chickens in the Republic of Korea for the first time, and the genetic diversity of enteric viruses and viral communities was showed.

Conclusions: Chicken astrovirus was consistently detected in broilers affected with RSS and the result of this study may contribute to knowledge of enteric diseases and viruses in chickens.

Keywords: Metagenomics; RNA virus; Runting-stunting syndrome; Viral enteritis.

Fig. 1

Diagnosis of runting-stunting syndrome using (a) histopathological lesions showing distension of the crypts in the small intestine lined with a flattened epithelium, exfoliated cells in the crypts and inflammatory cell infiltration in the adjacent lamina propria (100×). The black arrow indicates that the crypt contains necrotic cellular debris observed at higher magnification (right, 400×). Bar indicates 25 μm. b Negative contrast electron micrograph of feces and intestine homogenates of chickens with viral enteritis

Fig. 2

Phylogenetic analysis of viruses among (a) Astroviridae, (b) Picornaviridae, (c) Parvoviridae, (d) Caliciviridae, and (e) Picobirnaviridae identified from chickens with runt-stunting syndrome. A phylogenetic tree was generated using the neighbor-joining method and used to compare individual sequence reads to corresponding sequences from known viruses. Bootstrap values were deduced from 1000 replicates. The viruses that were identified in this study are highlighted with asterisk