Molecular Characterization and Determination of Relative Cytokine Expression in Naturally Infected Day-Old Chicks with Chicken Astrovirus Associated to White Chick Syndrome

Abstract

White chick syndrome (WCS) is an emergent disease that affects hatchability and hatched chicks, resulting in high mortality and economic losses, and is related to chicken astrovirus (CAstV). This syndrome has been reported in several countries worldwide, and groups A iii and B vi of CAstV have been determined; however, in Brazil, the virus has not been genotyped. The innate immunity of chicks affected by WCS or any CAstV is poorly understood and studied, and it is important to determine whether relative cytokine expression occurs during the early stages of the life of chicks. The aim of the present investigation is to detect and molecularly characterize CAstV associated with WCS, examine the macroscopic and microscopic lesions in the jejunum and spleen, and determine cytokine expression in the jejunum, liver, spleen and thymus of chicks naturally infected with WCS. To do so, we applied a pathological and molecular approach for CAstV detection and characterization, as well as the quantification of the relative mRNA expression of several cytokine genes. The phylogenetic analyses of the sequences obtained herein classified CAstV as uniquely belonging to group B iv, showing a high similarity of nucleotides (NT) (75.7–80.6%) and amino acids (AA) (84.2–89.9%) with the members of group B and a low similarity of NT (46.7–47.9%) and AA (37.8–38.9%) with the virus belonging in group A. CAstV was also detected and quantified in the serum, spleen, thymus and jejunum, the latter being the organ where CAstV had the highest viral concentration. However, this organ did not present any microscopical alterations. In contrast, we observed necrotic hepatitis in the liver of the affected subjects. On the other hand, we observed the activation of several T helper 1 (Th1)- and T helper 2 (Th2)-cytokines (IFN-γ, IL-2, IL-8, IL-12p40, IL-15, TGF-β4, TNF-SF-15 and t-BET), without being able to control the viral replication due to the high concentration of viral particles in some organs, principally in the gut. One possible role of these cytokines is contributing to the control of inflammation and cell protection of intestinal cells, principally during the early activation of immune responses. However, the fact that these responses are not mature enough to control the viral infection means that more studies need to be carried out to elucidate this topic.

Experimental co-infection of variant infectious bursal disease virus and fowl adenovirus serotype 4 increases mortality and reduces immune response in chickens

“…Approximately 500 ng of RNA was reverse-transcribed with PrimeScript™ RT Master Mix (Takara, Beijing, China) at 37 °C for 15 min, 85 °C for 5 s in accordance with the manufacturer’s instructions. To evaluate the cytokine gene expression, primers for chicken β-actin, interleukin (IL)-6, IL-1β, interferon (IFN)-α, and IFN-γ (Table 1 ) described in previous studies were used [ 22 – 24 ]. RT-qPCR for each gene was performed in a 20-µL volume of mixture composed of 2 × M5 HiPer SYBR Premix EsTaq (with Tli RNaseH) (Mei5bio, Beijing, China), primers, cDNA, and nuclease-free water.…”

An Insight into the Molecular Characteristics and Associated Pathology of Chicken Astroviruses

“…Presently, three to four types of CAstV-related illnesses or conditions exist based on infectious virus strains [4]: RSS or uneven growth [13,25], enteric and locomotion disorders [55], kidney disease with visceral gout [16], and white chick hatchery disease [17] or white chick syndrome (WCS) [2,67,68].…”

“…For over 40 years, CAstV has been associated with a hatchability condition referred to as "white chick" [20,68]. The condition, which is mostly observed in 1-day-old broiler chicks, is typified with overall pale plumage, irregular curly brown feathers on the neck region extending to the head, and green to bronze discolored liver lesions [2,17,20,53,67,68]. Additionally, the observable signs and lesions in white chick syndrome (WCS) are similar to those in RSS, including abnormal feathering, runting, underperformance, wet cloaca, weakness, and mortality within the first few hours and up to 3-5 days after hatching [2,17,20,68].…”

Transcriptome Sequencing of the Spleen Reveals Antiviral Response Genes in Chickens Infected with CAstV

“…White chicks syndrome (WCS) has recently been associated with CAstV. Ongoing outbreaks of WCS have been reported in Canada, Brazil, and some countries in Europe, including Poland [16,18,46,47]. Studies have described an increased embryo mortality in hatcheries.…”

“…Only a few studies on the immunology of TAstV (turkey astrovirus) infection in turkeys [54,55] and cytokine expression in day-old chicks with WCS have been published [46]. Here, to further facilitate investigations into CAstV pathogenesis and chickens' response to infection, we performed a spleen transcriptome analysis in uninfected chickens and in chickens 4 dpi following infection with a WCS CAstV strain.…”