Molecular Detection and Phylogenetic Analysis of Chicken Astrovirus Associated with Poultry Enteritis

Absorption and assimilation of nutrients depends on the health of gastrointestinal tract (GIT). Any abnormality in the functioning of GIT results in decreased growth, feed conversion ratio (FCR) and production losses (Devaney et al., 2016 and Lobani et al., 2016). Enteric viruses such as CAstV, Avian coronavirus (ACV), Avian rotavirus (ARV), Avian reovirus (AReoV), Fowl adenovirus-I (FAdV-I) and Chicken parvovirus (ChPV), have been detected in chickens with diarrhoea and other signs of Runting and Stunting Syndrome (RSS) (Kaithal et al., 2016; Nunez et al., 2016 and Mettifogo et al., 2014). CAstV is recognized as the causal agent of enteritis in chicken primarily in young birds (Mettifogo et al., 2014 and Pantin-Jackwood et al., 2011). Involvement of CAstV with an unusual condition in chicks known as “white chicks” has been reported from Brazil (Nuñez et al., 2016b).
       
CAstV is a small, round, non-enveloped, positive-sense single-stranded RNA virus having 5 or 6-pointed star-like surface projections. It belongs to the genus Avastrovirus of the Astroviridae family (Nuñez and Ferreira, 2013). Its genome is approximately 7 kb in length having three ORFs, it begins with a 5’ untranslated region (UTR) followed by three Open Reading Frames (ORF); ORF1a, ORF1b and ORF2 (Nuñez and Ferreira, 2013). CAstV has been detected from poultry enteritis and RSS from UK, USA, India, Netherland, Korea and Brazil (Day et al., 2007, Pantin-Jackwood et al., 2007, Kaithal et al., 2016; Saraswat et al., 2021; Todd et al., 2009; Smyth et al., 2009, Kang et al., 2012, Koo et al., 2013 and Nuñez et al., 2016b).
       
Present study was aimed to find out the occurrence of CAstV in poultry enteritis cases from unexplored areas of Rajasthan state of India, describe the partial molecular characterization of CAstV and observe gross and microscopic changes in the intestine and liver of the affected birds.

The present study was conducted at Department of Veterinary Microbiology, College of Veterinary and Animal Science, Navania, Udaipur, Rajasthan. To investigate theproblem of poultry enteritis associated with CAstV, in commercial poultry farms in four districts viz. Ajmer, Dungarpur, Sirohi and Pratapgarh of Southern Rajasthan during the period of January 2019 to February 2020. A total of 604 dead birds had signs of enteritis, rough feather and poor weight gain, were included in this study. The birds were subjected to post-mortem and molecular screening for CAstV. Intestine and liver were subjected to histopathological examination.
 
Post-mortem examination
 
All visceral organs were examined for gross lesions. Intestine of four birds of same age group were pooled to make one biological sample for molecular testing, while a piece of intestine and liver were collected in 10% buffered formalin for histopathology.
 
Molecular detection
 
Pooled Intestinal samples were subjected to molecular detection of the CAstV using RT-PCR. Tissue samples were processed as described (Saraswat, 2019). The supernatants were treated with TRIzol™ reagent (Sigma life science technologies) for the extraction of RNA according to the manufacturer’s instruction. For RT-PCR nuclease-free water was used as a negative control. While the CAstV positive samples stored in our laboratory was used as a positive control. 
       
The RNA obtained was subjected to a reverse transcription reaction to obtain complementary DNA (cDNA) as described (Saraswat, 2019), cDNA obtained was submitted to PCR.
 
Polymerase chain reaction (PCR)
 
ORF -1b gene specific primers has been used in this study as described (Day et al., 2007). With forward and reverse sequences 5¢-GAYCARCGAATGCGRAGRTTG-3¢ and 5¢-TCAGTGGAAGTGGGKARTCTAC-3¢ respectively. The PCR reaction mix of 20 μl contained the following components: 0.5 μl of each forward and reverse primers (10 pmol), 10 μl of 2x PCR master mix (Thermo Scientific), 2 μl of cDNA as template and 7 μ nuclease free water. PCR amplification was performed under the following conditions a cycle of 95°C for 5 min as initial denaturation, 35 cycles at 95°C for 30 sec, 51°C (annealing temperature) for 1min and 72^°C for 1 min and a cycle of 72°C for 10 min as final extension. The amplified product (362 bp) was submitted to electrophoresis in 1.5% agarose gel.
 
DNA sequencing and phylogenetic analysis
 
The amplified products of the ORF1b gene of CAstV from 2 positive samples were purified using Sure Trap Gel Extraction Kit as described by the manufacturer (Genetix biotech Asia Pvt. Ltd.). Each purified product was sequenced in forward and reverse direction using Applied Biosystem by life science technology 3130 XL sequencer (at the Department of Biochemistry, University of Delhi, South Campus, New Delhi).
 
Nucleotide sequences were edited in Bio-Edit software. The aligned sequences were analysed on the NCBI website http://www.ncbi.nlm.gov using BLAST to confirm their identity. The nucleotide sequences were then aligned using MEGAX version 5 software by CLUSTAL W method, using partial sequences of 16 astroviruses downloaded from the NCBI. The nucleotide phylogenetic tree was inferred using the neighbour-joining method with 1,000 bootstrap replicates that were integrated into MEGA X version 5 software (Saitou and Nei, 1987 and Kumar et al., 2018).
 
GenBank accession numbers
 
The sequence data were submitted to the GenBank database and were assigned accession numbers MT263725 and MT263726 (Table 3). Table 1 shows the Gene Bank accession numbers of sequences used for molecular analysis in the present work.
 
 

 

 
Histopathological examinations
 
From CAstV positive case, intestine and liver samples preserved in buffered formalin were subjected to histopathological examination by paraffin embedding using acetone and benzene technique (Lillie, 1965 and Luna et al., 1968). Further, the histopathological lesions were analysed using a research microscope at 10x and 40x magnifications.

Samples
 
A total of 151 pooled Samples  from 604 birds were collected from affected flocks of four districts of southern Rajasthan (Table 2).
 

 
Molecular detection
 
All the collected intestinal samples were subjected to molecular detection, by PCR which amplified the ORF1b gene of CAstV and obtained an amplicon of 362 bp in agarose gel electrophoreses as shown in Fig 1. CAstV was detected in 20.52% (31/151) of pooled samples. The highest CAstV occurrence in poultry enteritis was recorded from Ajmer district 37.83% followed by Sirohi 20.83%, Pratapgarh 17.64% and minimum occurrence was recorded as 7.69% in Dungarpur district (Table 2). This variation in prevalence among these districts of Rajasthan may be due to size of chicken flocks reared and number of samples collected. Most of the samples from Ajmer district were collected from commercial poultry farms, containing large number of chickens reared in a close confined area. While from other three districts most of the samples were from small flocks. So the close contact is relatively less and probably low chance of horizontal transmission. In our study, high prevalence of enteric CAstV infection was observed in 0-1 week of age group birds as 19/71 (26.76%) whereas the prevalence among 2-12 weeks of age birds was 12/80 (15%).
 

       
Saraswat (2019) reported 3.33% a lower prevalence of CAstV in and around Udaipur district of Rajasthan during 2018-19. High occurrence of CAstV (87.69%) in Haryana state has been reported by Kaithel et al., (2016). The variation among various state of same country has also been reported by DelaTorre et al., (2018) 12 states of Brazil and found CAstV in only two state this shows that occurrence of the virus can vary between regions (DelaTorre et al., 2018). Globally various researchers also reported the prevalence of CAstV i.e. DelaTorre et al., (2018) from Brazil 8.1%, Lobani et al., (2016) from Jordan 29.7%, Mettifogo et al., (2014) from Brazil 21.1%, Koo et al., (2013) from Korea 38.2%. Roussan et al., (2012) 38.6% and Pantin-Jackwood et al., (2008) reported 86% prevalence of CAstV. Low prevalence in our study may also be due to low titres ofvirus in samples, although to increase the titre of virus in one biological sample we have pooled four birds from same age group and same flocks.

The relation of CAstV and poultry enteritis has been shown by Baxendale and Mebatsion, who conducted an experimental study in which they inoculated orally in day-old specific pathogen free (SPF) white leghorn chicks with the CAstV. The inoculated chicks were developed clinical signs within 4 days such as mild diarrhoea with moderately digested food in faeces and distension of small intestine (Baxendale and Mebatsion, 2004).
 
DNA sequencing and phylogenetic analysis of enteric CAstV
 
The use of a particle-associated nucleic acid sequencing technique led to the identification and sequencing of CAstV, showing the ORF 1b gene to be the most conserved region. The sequences of CAstV from enteritis-affected birds were edited and compared with other sequences from genBank using the BLAST tool. The results showed high similarity with other CAstV. A fragment of 362 bp from sequences of CAstV from the intestine of the affected bird in the present work was compared with other sequences. The Indian sequences of CAstV obtained here revealed a high similarity to the nucleotide (nt) (97%-92%) and amino acid (aa) of (100%-98%) between the sequences. Comparison with sequences from other countries also showed a high similarity of nucleotide ( Table 1 and Fig 2). Various authors (Kaithel et al., 2016; DelaTorre et al., 2018; Nunez et al., 2016b; Koo et al., 2013 and Pantin-Jackwood et al., 2006) had also reported similar findings. In the present study, a part of polymerase gene was amplified; determination of the complete genome therefore needs to be considered for genotyping of CAstVs. Though the present study was conducted in a specific region, screening of suspected enteritis cases in large geographical region may help to uncover CAstVs infection circulating in poultry. Further, phylogenetic tree was constructed using neighbour-joining method algorithm, which is a distance based method. The evolutionary relationship among 18 sequences weredepicted and analysed. It divided majorly in two branches I and II which further subdivided in four clusters namely A, B, C and D. Branch I, which having cluster A and B contains all Indian variants of CAstV including recently submitted sequences. Where, branch II which comprises with cluster C and D having variants from South Korea, Poland, USA and Brazil. These finding suggest the closest phylogenetic relationship of CAstV sequences (MT263726 and MT263725) with sequence (MF491634) submitted from Hyderabad, Telangana, India and other Indian sequences found in cluster A. However, cluster B comprises with single sequence found distantly related with recently submitted sequences, in spite of country-wise origin. They share a common ancestry with other Indian sequences as well sequences found in different countries.
 

 
Pathological finding of intestine and liver affected from poultry enteritis
 
Gross examination of intestine and liver
 
On gross examination of intestine showed severe congestion with watery fluid and undigested material in the lumen. Some part of intestine showed distension and ballooning with thin transparent in appearance and in liver hepatomegaly, severe congestion with pinpoint necrotic patches. asess andend severe congestion with pinpoint necrotic pathesin transparent appearence (Fig 3a and b).
 

 
Histopathological examination of intestine and liver
 
Histopathological examination of affected intestine reveled mild degenerative changes and necrosis of the mucosal villi with severe infiltration of mononuclear cells into mucosa and submucosa. Submucosal glandes were atrophied and some become cystic. Mild congestion was observed in the muscularis layer (Fig 3c). Liver showed mild to moderate degenerative changes, severe congestion and hemorrhages along with infiltration of inflammatory cells especially mononuclear cells infiltration (Fig 3d). These observations are well in accordance with those described by Rosenberger and Direction (2012); Qamar et al., (2013) and Hauck et al., (2016).

Authors thankfully acknowledged Prof. Vishnu Sharma, Hon’ble Vice Chancellor RAJUVAS Bikaner and Prof. R.K. Joshi, Dean, CVAS, Navania, Vallabhnagar, Udaipur for providing necessary facilities.