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. 2019 Nov 27;30(4):551–561. doi: 10.1007/s13337-019-00554-1

Molecular characterization of chicken astroviruses in gout-affected commercial broiler chickens in Haryana, India

Sumitra Panigrahi 1, Naresh Jindal 1,✉, Pawan Kumar 2, Sanjay Barua 3, Naveen Kumar 3, Thachamvally Riyesh 3, Yogesh Chander 3
PMCID: PMC6917690  PMID: 31897418

Abstract

Chicken astroviruses (CAstVs) infect young chicks and are associated with gastroenteritis, stunted growth or visceral gout (gout). True incidence and distribution of CAstVs as well as virus variants circulating in India is not well understood. In this study, 80 gout-affected broiler chicken flocks from Haryana, a north-western state of India, were tested for the presence of astroviruses by targeting the polymerase gene of both CAstV and avian nephritis virus (ANV) and capsid gene of CAstV. Of these, 22 (27.5%) flocks were found positive for CAstV, 7(8.75%) for ANV and 2 (2.5%) for both CAstV and ANV genome by reverse-transcription-polymerase chain reaction. CAstV was isolated by inoculating tissue (kidney) homogenate from gout-affected birds into specific-pathogen free embryonated chicken eggs where the infected embryos showed stunted growth with necrosis of liver and enlarged kidney with urate deposits. Capsid gene-based phylogenetic analysis revealed the clustering of CAstV strains from this study with Indian strains of serogroup Biii suggesting their antigenic relatedness. Thus the present study reveals the presence of chicken astroviruses in broiler chickens affected with gout.

Keywords: Gout, Chicken astrovirus, Avian nephritis virus, Commercial broiler chicken

Introduction

Astroviruses infect a wide variety on mammalian and avian species. Most common astrovirus infections are characterized by gastroenteritis [18]. Two astroviruses species namely; chicken astrovirus (CAstV) and avian nephritis virus (ANV) infect chickens and both are associated with stunted growth, enteritis and kidney lesions in young chickens [1, 3, 22]. More recently CAstV has also been implicated in severe kidney disease of young broilers with visceral gout (gout) and the ‘White Chicks’ hatchery disease [3, 13, 25, 28]. CAstVs are one of the earliest viruses that infect young chicks, when immunity is least developed. CAstVs can cause varying degree of growth retardation viz; runting stunting syndrome (RSS) in broiler chickens [7, 12]. The isolation and characterization of CAstV has been reported from broilers exhibiting RSS and dead-in-shell chicks having hatchability problems [1, 29]. Few recent reports also suggest CAstVs infection in poultry in India [3, 11, 23].

ANV was originally regarded to be a picornavirus but later (year 2000) characterized as an avian astrovirus on the basis of gene sequence similarity [9]. The true incidence and distribution of the disease due to ANV is not well known due to subclinical nature of the infection and difficulties with virus isolation. Astroviruses have been detected from poultry flocks and other domestic birds from various parts of the world [8, 10, 15, 19]. In a previous study conducted in our laboratory, we detected the presence of CAstV and ANV in enteritis-affected broiler chicken flocks by virus specific reverse-transcription-polymerase chain reaction (RT-PCR) [11].

Astroviruses are positive-sense single stranded non-enveloped RNA viruses of 25–35 nm diameter belonging to the family Astroviridae [16, 24]. The genome contains 6800–7900 nucleotides (nt) with three open reading frames (ORFs) where ORF1a encodes non-structural polyprotein (NSP); highly conserved, ORF1b encodes RNA dependent RNA polymerase and highly variable ORF2 encodes capsid protein which is a major determinant of immune response [27]. Two distinct serogroups (serogroup A and B) of CAstV are known that have minimal degree of cross reactivity with heterologous antisera. This serogrouping of the CAstV is also supported by genotyping studies that showed clustering of astrovirus strains into two groups due to lower level of shared amino acid (aa) identity [26].

Commercial broiler sector is one of the fastest growing poultry sectors in India. However, infectious and non-infectious diseases hamper the production potential to be achieved to its maximum. Gout is not a single disease entity, rather the result of kidney damage due to several factors. Among the infectious causes, viruses such as infectious bronchitis virus (IBV), ANV and CAstV have been reported to cause gout in different avian species [2, 3, 31]. CAstV infections usually occur within the first few days or week of life where the infected baby chicks suffer from diarrhoea, retarded growth or RSS. In India, very little attempts have been made to study CAstV infection in poultry [3, 11, 23]. The nature of antigenic variants (serogroups) circulating in the country is not known. Moreover, there are limited reports on isolation of CAstV from poultry. Furthermore, CAstV prototypes that can be used as reference strain for molecular epidemiological studies, for preparation of vaccines and diagnostics are largely unavailable. Therefore, in this study, besides evaluating the status of astrovirus infection in poultry in Haryana (India), we isolated and characterized CAstV strains circulating in the region.

Materials and methods

Collection of samples

During disease investigation, some of the broiler chicken flocks revealed clinical findings such as dullness, depression, chalky white faeces and mortality along with evidence of deposition of urates in visceral organs (liver, kidney and heart) at necropsy (Fig. 1a, b). Such flocks were diagnosed as suffering from gout and detailed history of these flocks was collected. Kidney specimens (n = 80) were collected in sterile containers from gout-affected flocks. A single specimen represented pooled kidney samples from 4 to 5 dead birds from each flock. Kidney samples (n = 10) were also collected from apparently healthy broiler chicken flocks. The specimens were stored at − 20 °C until processed. A part of the sample was triturated using PBS with antibiotics-antimycotic solution (5:1) to make 10% suspension followed by two freeze–thaw cycles at − 80 °C for 10 min. The suspension was clarified by centrifugation at 4000 rpm for 10 min and the resulting supernatant was used for virological and molecular assays. The research work was duly approved by the Institutional Animal Ethics Committee of the University.

Fig. 1.

Fig. 1

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a Urate deposits on pericardium, proventriculus and intestine of a bird affected with gout. b Swollen kidneys and prominent ureters with urate deposition in a broiler chick affected with visceral gout

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted from supernatant using Trizol (Invitrogen, USA) as per the manufacturer’s instructions and reverse transcribed to cDNA using random hexamer as per standard procedures of First Strand cDNA Synthesis kit (Thermo Scientific). A set of published primer was used to specifically amplify the polymerase gene of CAstV (forward primer-5′-GAYCARCGAATGCGRAGRTTG-3′ and reverse primer -5′ TCAGTGGAAGTGGGKARTCTA-3′) and polymerase gene of ANV (forward primer-5′-GYTGGGCGCYTCYTTTGAYAC-3′and reverse primer 5′-CRTTTGCCCKRTARTCTTTRT-3′) [5]. The capsid gene of CAstV was amplified using a set of self-designed primer pair (forward primer 5′-ATCTGGTAGAGGGATGGACCGA-3′ and reverse primer 5′-GCMCTTYTCAAAGCGGACRAG-3′). The PCR amplification was carried out in 20 µl reaction mixture containing 10 µl DreamTaq Green PCR Master Mix (2×), 1 µl (10 pmol/μl) each of the forward and reverse primers and 2 µl of template cDNA. Thermal cycler conditions were: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 53 °C (CAstV) and 56 °C (ANV) for 30 s, extension at 72 °C for 30 s with a step of final extension at 72 °C for 5 min for polymerase gene. Amplification of capsid gene of CAstV was carried out with initial denaturation at 95 °C for 5 min followed by 35 cycles of denaturation at 95 °C for 40 s, annealing at 53 °C for 40 s and extension at 72 °C for 110 s. The PCR amplifications of CAstV polymerase gene and capsid gene were expected to produce 362 bp and 2200 bp fragments, respectively (Fig. 2a, b) whereas for ANV polymerase gene it was 473 bp (Fig. 2c).

Fig. 2.

Fig. 2

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a 362 bp PCR products of field samples using polymerase gene specific primers of chicken astrovirus on 1.5% agarose gel. Lane M = 100 bp marker; Lane NC = Negative control; Lanes 1–8 = Field samples positive for CAstV; Lane PC = Positive control. b 2.2 kbp PCR products of kidney samples using capsid gene specific primers of chicken astrovirus on 1% agarose gel. Lane M = 1 kb marker; Lane NC = Negative control; PC = Positive control; Lanes 1-7 = Field isolates of CAstV. c 473 bp PCR products of kidney samples using polymerase gene specific primers of avian nephritis virus on 1% agarose gel. Lane M = 100 bp marker; Lane PC = Positive control; Lanes 1–3 = Field samples positive for ANV; Lane NC = Negative control

Nucleotide sequencing and phylogenetic analysis

PCR products were purified using QIAquick PCR purification kit (Qiagen, Valencia) and directly sequenced in both directions using forward and reverse PCR primers at the DNA Sequencing Facility, Department of Animal Biotechnology, College of Veterinary Sciences, LUVAS, Hisar (India). DNA Baser (version 4.39) software programme was employed for trimming off the low-quality end sequences and generation of final contig sequence which was subjected to BLAST (NCBI) analysis to confirm their identity. The nucleotide sequences were then aligned using MEGA6.0 software [30] by ClustalW method. The evolutionary distances were computed by pairwise distance method using the maximum composite likelihood model. A phylogenetic tree of aligned sequences was constructed by Neighbor-joining method (1000 replicates for bootstrap). A conservation plot was constructed using Bioedit software program to determine variations at the nucleotide (nt) level among different strains of the present study and earlier reported sequences from India and abroad. Further, the nt sequences were translated to deduced amino acid (aa) sequences, which were also compared using conservation plot to determine differences at the aa level, if any. The sequences were submitted to the GenBank under the following accession number; CAstV polymerase gene (MF416951-MF416961), CAstV capsid gene (MF405733-MF405737) and ANV polymerase gene (MF380438-MF380440).

Virus isolation

The specimens that were found positive for CAstV or ANV viral genome in PCR were subjected to virus isolation by inoculating 0.2 ml of the tissue homogenate into 9–10 days old specific-pathogen free (SPF) embryonated chicken eggs (ECE) via allantoic route. Negative control(s) were inoculated with 0.2 ml phosphate buffer saline (PBS). The eggs were then incubated at 37 °C in an egg incubator. Mortality occurring within 24 h was considered as non-specific. The eggs were candled twice daily for 5 days after which kept at 4 °C overnight and the allantoic fluid was harvested. Embryos were examined for any gross pathological lesions in liver, kidney and spleen. A piece of these organs was collected in a sterile container and stored at -80 °C. Additionally, organs were collected in 10% formalin for histopathological studies. Formalin fixed tissues were processed and 5–6 µm thick sections were stained with routine haematoxylin and eosin stain [14]. The evidence of the virus replication/adaptation in eggs was confirmed by amplification of virus-specific gene segments in embryonic tissues collected at each passage up to 4th egg passage.

Results

Chicken astrovirus in clinical samples

Of the 80 gout-affected specimens tested for the presence of astrovirus-specific genome, 22 (27.5%) were positive for CAstV alone, 7 (8.75%) for ANV alone and 2 (2.5%) for both CAstV and ANV genome by RT-PCR. Overall a total of 31 (38.75%) kidney specimens were found positive for chicken astroviruses. All kidney samples from apparently healthy birds were found negative for both CAstV and ANV genome by RT-PCR. Analysis of data revealed that percent morbidity (10.63%) and mortality (7.41%) were higher in gout-affected flocks positive for astroviruses as compared to the flocks negative for these viruses (5.67% and 3.22% respectively). Likewise, case fatality rate was 63.77% in CAstV- or ANV-positive gout-affected flocks and 56.77% in these viruses negative flocks (Table 1).

Table 1.

Epidemiological indices in gout-affected commercial broiler flocks

Epidemiological indices Gout affected flocks negative for CAstVs (n = 49) Gout affected flocks positive for CAstVs (n = 31)
Morbidity (%) 5.67 10.63
Mortality (%) 3.22 7.41
Case fatality rate (CFR; %) 56.77 63.77
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Virus isolation

CAstV or ANV singly infected (PCR positive) specimens were inoculated into 10-days-old SPF embryonated chicken eggs. At 5 days post inoculation, CAstV-infected embryos revealed stunting growth (Fig. 3a), liver necrosis (Fig. 3b) and enlargement of the kidneys with deposition of urate crystals (Fig. 3c). Out of the 22 CAstV-positive samples by RT-PCR, virus isolation was successful in 8 specimens. Virus isolation was unsuccessful in all the ANV-positive samples.

Fig. 3.

Fig. 3

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a Chicken embryos at 5 days post-inoculation with kidney homogenate positive for chicken astrovirus. b Embryos showing necrosis of liver after inoculation with chicken astrovirus. c Embryo showing enlarged kidney and ureters with urate deposition after inoculation with chicken astrovirus

Histopathology of infected embryos

Hematoxylin and eosin (H& E) staining of kidney, liver and heart sections was performed from dead embryos at 5 days post-infection. Heart sections revealed severe congestion (Fig. 4a), liver showed necrotic foci and different degrees of degenerative changes and deposition of urate crystals around the central vein (Fig. 4b) and mild to moderate interstitial nephrosis in kidneys (Fig. 4c).

Fig. 4.

Fig. 4

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a Section of heart showing severe congestion in 15 days old embryo infected with chicken astrovirus (H&E × 400). b Section of liver showing focal necrosis around central vein in 15 days old embryo infected with chicken astrovirus (H&E × 400). c Section of kidney showing interstitial nephritis in 15 days old embryo infected with chicken astrovirus (H&E staining × 400)

Sequence analysis

Nucleotide sequences of CAstV polymerase gene corresponding to nucleotide positions 766-1086 (321 nt) were compared with other published sequences originated either from India or rest part of the world. CAstV strains from India showed 79.06–100% sequence identities among themselves and 77.78–100% identity with strains from abroad. The aa sequences of CAstV (corresponding to aa positions from 256 to 362 of ORF1b of protein ID APR72389.1) showed 92.40–100% identity among themselves, 91.30–100% to that of other Indian strains and 89.20–100% with CAstV strains from abroad.

Capsid gene of CAstV (858 bp corresponding to nucleotide positions 1243-2100) showed 67.70–99.80%, 63.20–98.90% and 46.20–85.20% identity, respectively within themselves, with other Indian strains and with strains from abroad. At amino acid level the respective identities were 66.80–99.60%, 64.40–99.60%, 48.40–90.50%, respectively. Changes at the amino acid level in various CAstV strains of this study are depicted in Table 2. One of the strains [CAstV/50/16/HR(Ind)/Chicken] showed change at position 260 (E260 K). Six of the 11 CAstV strains of this study revealed S279A mutation. G334R change observed in a CAstV strain [CAstV/52/16/HR(Ind)/Chicken] of this study was unique, as this was not observed in any of the CAstV strains used for comparison.

Table 2.

Changes at the amino acid level in CAstV

Gene Mutation as compared to reference strain No. of strains showing the change Name of the strain
Polymerase gene E260K 1/11 CAstV/50/16/HR(Ind)/Chicken
A266D 1/11 CAstV/50/16/HR(Ind)/Chicken
S279A 6/11 CAstV/40/14/HR(Ind)/Chicken
CAstV/41/15/HR(Ind)/Chicken
CAstV/50/16/HR(Ind)/Chicken
CAstV/52/16/HR(Ind)/Chicken
CAstV/63/16/HR(Ind)/Chicken
CAstV/D1/17/HR(Ind)/Chicken
A299E 1/11 CAstV/50/16/HR(Ind)/Chicken
A300E 1/11 CAstV/50/16/HR(Ind)/Chicken
V316I 6/11 CAstV/40/14/HR(Ind)/Chicken
CAstV/41/15/HR(Ind)/Chicken
CAstV/50/16/HR(Ind)/Chicken
CAstV/52/16/HR(Ind)/Chicken
CAstV/63/16/HR(Ind)/Chicken
CAstV/D1/17/HR(Ind)/Chicken
I327V 1/11 CAstV/50/16/HR(Ind)/Chicken
G334R 1/11 CAstV/52/16/HR(Ind)/Chicken
S355A 1/11 CAstV/50/16/HR(Ind)/Chicken
Capsid gene G584V 1/5 CAstV/21/16/HR(Ind)/Chicken
A585V 2/5 CAstV/G2/16/HR(Ind)/Chicken
CAstV/20/16/HR(Ind)/Chicken
T635Y 1/5 CAstV/39/14/HR(Ind)/Chicken
R638Y 1/5 CAstV/39/14/HR(Ind)/Chicken
Y640C 1/5 CAstV/39/14/HR(Ind)/Chicken
E649D 2/5 CAstV/52/16/HR(Ind)/Chicken
CAstV/D1/17/HR(Ind)/Chicken
P667S 1/5 CAstV/39/14/HR(Ind)/Chicken
F693I 1/5 CAstV/20/16/HR(Ind)/Chicken
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Partial gene sequence analysis (384 bp corresponding to nucleotide position 676-1059) of ANV polymerase gene revealed nt identity of 96.53–100%, 96.53–100%, 86.13–96.50% within themselves, with the Indian strains and with other strains from abroad, respectively. At amino acid level the respective identities were 99.20–100%, 91.30–100%, 89.20–100%.

Phylogenetic analysis

For phylogenetic analysis, eight isolates of CAstV and three CAstV strains along with previously published polymerase gene sequences viz; nine of Indian origin and 20 from abroad were used for comparison (Fig. 5a). The isolates of this study were found in two different groups. One isolate (CAstV/50/16/HR(Ind)/Chicken) was found to cluster in a distinct group and this group did not have any CAstV strain from India used for comparison. Remaining all isolates and strains of this study formed a single cluster and were grouped with previously reported CAstVs strains from India. The outgrouping viruses such as turkey astrovirus, duck astrovirus and ANV were in a different clade in polymerase gene phylogeny (Fig. 5a).

Fig. 5.

Fig. 5

Fig. 5

Fig. 5

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a Phylogenetic tree on polymerase gene of chicken astrovirus. Tree was constructed in MEGA 6.06 using neighbor-joining method and 1000 bootstrap replicates. The strains with diamond in front are the strains of this study. b Phylogenetic tree on capsid gene of chicken astrovirus. Tree was constructed in MEGA 6.06 using neighbor-joining method and 1000 bootstrap replicates. c Phylogenetic tree on polymerase gene of avian nephritis virus. Tree was constructed in MEGA 6.06 using neighbor-joining method and 1000 bootstrap replicates. The strains with diamond in front are the strains of this study

Phylogenetic analysis based on CAstV partial capsid gene (15 previously published sequences of Indian origin and 13 from abroad) showed grouping of all the Indian CAstV strains (of gout origin) into single cluster (formed by CAstV serogroup Biii isolates) (Fig. 5b).

All three ANV strains of present study were analyzed for their phylogenetic relationship and were found close to previously reported Indian strains forming a common phylogenetic group (Fig. 5c). The outgrouping viruses such as turkey astrovirus, duck astrovirus and CAstV were in a different clade in polymerase gene phylogeny of ANV.

Discussion

The present study was undertaken to detect and characterize astroviruses from gout cases in broiler flocks. In poultry the CAstV has been reported to cause enteritis along with growth depression and higher mortality [4]. Due to growth depression, the economic losses to the poultry farmers are considerable. This virus has been detected from RSS in chickens [7, 12] and ‘white chick’ in day-old chicks [13, 25, 28]. Strict biosecurity measures remain the only available option for their control in the absence of a vaccine for commercial broiler chickens [26].

In the present study, 38.75% specimens from gout-affected poultry flocks were positive either for CAstV or ANV or in combination by RT-PCR. In our study CAstV was detected more (27.5%) as compared to ANV (8.75%). Most of the earlier studies have detected chicken astrovirus alone or in combination with other viruses from enteritis cases in chicken/turkeys [6, 10, 11, 17, 21]. However, a few studies indicate the detection of these two viruses in gout in poultry [3]. Percent morbidity, mortality and case fatality rate were higher in astrovirus positive gout-affected flocks as compared to flocks that were astrovirus negative. Analysis of data also revealed that all the gout-affected flocks positive for astroviruses (n = 31) in this study had birds between one to two weeks of age indicating that this virus affects mostly the young birds. CAstV infections usually occur very early and are either transmitted horizontally, or some CAstV strains can also be vertically transmitted, and such chicks may hatch shedding high levels of CAstV [26]. Testing of hatched chicks before placement may throw light on this aspect. If a flock is positive, follow-up may help us to understand whether that flock develops gout or not.

In the present study, only two gout-affected flocks revealed the presence of both viruses (CAstV and ANV). It may be possible that presence of more than one virus in gout-affected flocks may cause more severe adverse effects than produced by an individual virus. An interpretation of the role of each agent involved in gout still remains a challenge. Studying complete virome of gout-affected birds and other animals (at next generation sequencing platform) would provide more insights for better understanding the dynamics of mixed infections. Furthermore, experimental studies are needed to appraise the synergism between these two viruses and other nephropathogenic viruses. In our study, 51 samples were negative for the tested viruses. There are a number of factors (nutritional, management, viruses etc.) other than the tested viruses that can cause gout in broiler chicks. The possibility of involvement of these factors in causation of gout cannot be ruled out.

There are a few published reports on isolation of astroviruses from embryonated chicken eggs (ECE) [3, 20, 25]. Out of the 22 samples that were positive for CAstV by RT-PCR, this virus could be isolated in eight samples. Infected embryos were found to be haemorrhagic and had stunted growth. Similar findings in embryos have been reported earlier [3, 20]. While the embryonic lesions are not pathognomonic for the detection and confirmation of CAstV, haemorrhagic lesions and dwarfism can be suggestive of CAstV [20]. For further confirmation, three additional passages were made in this study; presence of CAstV-specific genome in kidneys at 4th passage confirmed successful adaptation of the virus in the eggs. Deposition of urates in kidneys of embryos in this study also suggests CAstV to be the probable cause of gout in birds. In the present study, we could not isolate ANV in SPF eggs. It might be due to low viral load in the samples or inability of the virus to adapt in SPF eggs.

The CAstVs of this study revealed changes at amino acid level in both polymerase and capsid genes. Like other RNA viruses, CAstVs are also subjected to genetic mutation and recombination which is reflected by variability of CAstV strains. Though polymerase gene is considered as quite conserved, mutation observed might be due to positive selection because of synonymous to nonsynonymous nucleotide substitution [21]. The serogrouping of the CAstV (serogroup A and B) is also supported by genotyping studies that showed clustering of astrovirus strains into two groups due to lower level of shared amino acid identity [26]. CAstV capsid gene is largely responsible for antigenic variation and altered pathogenicity [22, 27]. In our study, capsid gene-based phylogenetic analysis revealed clustering of all the Indian CAstV strains of gout origin into single group (which is formed by CAstV serogroup Biii viruses). This suggest that the CAstV strains from north India (reported under this study) are likely to be antigenically similar to those reported previously from southern part of the country [3] and that virus prototypes reported in this study may be used for development of common prophylactic and diagnostic reagents.

In conclusion, detection and isolation of astroviruses from gout cases suggests their possible role in disease. An interpretation of the role of each agent involved in gout remains a challenge for future studies. It seems that there are a number of factors that may interact for causation of gout and/or increasing the severity of the gout in broiler chicks. The present study was conducted in commercial broiler chickens; further studies are needed to ascertain the status of astroviruses in breeder and layer flocks.

Acknowledgements

The authors are thankful to National Research Centre on Equines, National Centre for Veterinary Type Culture Collection, Hisar, Haryana (India) for their support during the entire research.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no any conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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