Detection of variant infectious bronchitis viruses in broiler flocks in Libya

Peer review under responsibility of Faculty of Veterinary Medicine, Cairo University.

Abstract

A number of broiler flocks with respiratory disease and high mortality in five broiler farms in Libya were sampled for detection of infectious bronchitis virus (IBV). Twelve IBV strains from these farms were detected by reverse transcription polymerase chain reaction (RT-PCR) and differentiated by nucleotide sequencing of the hypervariable region of the S1 gene. A pair-wise comparison of the sequences showed two distinctive patterns. Those from farms 1, 2, 4 and 5, formed a separate cluster with 94–99% relatedness to the Egyptian IBV strains CK/Eg/BSU-2/2011, CK/Eg/BSU-3/2011 and Eg/1212B. Sequences from the farm 3 formed another cluster with 100% relatedness to Eg/CLEVB-2/IBV/012 and IS/1494/06. This appears to be the first report on the co-circulation these variant IBVs in Libya.

Keywords:

• Chicken
• Libya
• Variant infectious bronchitis virus

Abbreviations:

• EXO, Exonuclease
• FTA, Flinders Technology Associates
• IBD, infectious bursal disease
• IBV, infectious bronchitis virus
• ND, Newcastle disease
• OP, oropharyngeal swab
• RNA, ribonucleic acid
• RT-PCR, reverse transcription-polymerase chain reaction
• SAP, shrimp alkaline phosphatase

1 Introduction

Avian infectious bronchitis virus (IBV) causes a highly contagious disease in chicken. It mainly affects the respiratory tract, and frequently causes damage to the kidneys and reproductive systems [1]. Although vaccination is commonly adopted, outbreaks continue to occur worldwide with significant economic consequences due to a substantial decrease in production performances [1,2]. Different genotypes of IBV have been identified worldwide, and new variants continue to emerge [3]. A number of IBV variant genotypes have been reported in the Middle East, including Iran/793B/19/08, Iraq/Sul/01/09, Israel/720/99, Israel/885/00, IS/1494/06, Egypt/Beni-Seuf/01, Egypt/F/03, Egypt/D/89, CK/CH/LDL/97I, and CK/CH/SCYA/10I [4–9]. Some of these genotypes in particular IS/885/00 and IS/1494/06, have become dominant in the majority of farms in the Middle East countries, causing respiratory and renal diseases [4,10,11]. To date, there is no information available on the circulation of variant IBVs in Libya. In the Middle East, the vaccination against IBV is performed with vaccines that contain live-attenuated or killed viruses belonging to the Massachusetts serotype [10]. In the past few years, vaccine strains belonging to 793B and D274 serotypes are also widely used. In spite of this, IBV infection is considered endemic and widely spread both in vaccinated and unvaccinated poultry farms generally associated with kidney damages [4]. The aim of this study is to provide information on the molecular characteristic and the phylogenetic relationship of strains in Libya in comparison to other strains reported in the Middle East.

2 Materials and methods

2.1 Case history and clinical samples

In July 2012, a number of broiler flocks in five different farms with respiratory disease and high mortality at East Libya were visited. The flocks had no vaccination against IBV but were vaccinated against Newcastle disease (ND) and infectious bursal disease (IBD) (Table 1). All flocks showed clinical signs of respiratory distress, manifested by sneezing, tracheal râles, gasping, nasal discharge, head swelling, conjunctival congestion and frothy eyes. Post-mortem examination revealed lesions of inflamed trachea, cheesy exudate in airsacs and swelling of the kidneys. Mortality on the day of sampling ranged from 1.4% to 3.7% (Table 1).

Table 1 Flock details, RT-PCR and genotype results.

Farm	Number of day-old chicks placed	Age (days)	Vaccination programme	Mortality on the day of sampling (%)	RT-PCR (Laboratory sequence No., Genebank Accession No.)
					OP	Turbinates	Trachea	Lungs	Kidneys
1	6000	32	None	2	–	–	+(01, KF007922)^⁎	+(02, KF007923)	+(03, KF007924)
2	5000	22	IBD (day 14)	3	+(04, KF007924)	–	–	–	–
3	9000	40	NDV (day 7) IBD (day 14)	3.7	–	–	+(05, KF007926)	–	+(06, KF007927)
4	10,000	26	IBD (day 14)	2	–	–	–	–	+(07, KF007928)
5	7500	16	NDV (day 7)	1.4	+(08, KF007929)	+(09, KF007930)	+(10, KF007931)	+(11, KF007932)	+(12, KF007933)

⁎ For details, see Fig. 1.

From each of the farms, oropharyngeal swabs (OP) were collected from a total of 40 chicks. These swabs were divided into sets of 10 and were dipped into bijou tubes containing 2 ml of sterile water. After vigorous shaking, 100 μl of the mixture was spotted onto the Flinders Technology Associates (FTA) cards. Ten to twenty diseased birds per farm were killed and tissues of turbinates, trachea, lungs and kidneys were collected. The like-tissues were rubbed gently onto matrix areas of the FTA cards. These cards were air-dried and transported to the poultry virology laboratory at the University of Liverpool for analysis.

2.2 RNA extraction

The FTA cards were processed as described by the manufacturer with some modification. Briefly, the spotted or imprint area of the FTA card were cut using sterile scissors and forceps, each sample was placed into bijou tubes containing 2 ml of guanidinium thiocyanate and stored at −20 °C until required. RNA was extracted using guanidinium thiocyanate–phenol chloroform method as described [12]. Three hundred microliters of the mixtures above were placed in 1.5 ml eppendorf tube containing 300 μl guanidinium thiocyanate and stored at −20 °C for few hours. After thawing, this mixture was transferred into eppendorf tube and 50 μl of 2 M sodium acetate and 650 μl of phenol–chloroform were added. The suspensions were vortexed and centrifuged at 13,000g for 5 min. The aqueous phase containing the RNA was mixed with 500 μl isopropanol and stored at −20 °C overnight for precipitation of the RNA. The supernatant was carefully removed, and the precipitated RNA was pelleted at 13,000g for 15 min and washed twice with 100% ethanol. The pellet was dried and resuspended in 30 μl of treated water and used for RT-PCR.

2.3 RT-PCR and DNA sequencing

Procedures for the IBV RT-PCR have been described by [13]. Briefly, detection of the IBV genome and molecular characterization were achieved by identifying (380) base pairs of the S1 region of the S protein gene. The RT-PCR procedure including the primers used were as described [14]. For sequencing, the nested-PCR products corresponding to the spike glycoprotein were purified with 0.15 μl Exonuclease 1 (EXO) and 0.99 μl shrimp alkaline phosphatase (SAP) at 37 °C for 30 min, followed by 80 °C for 10 min to remove any extraneous material. The purified product, together with positive sense primer (forward direction using primer SX3+), were submitted to external laboratory for analysis of the partial S1 gene sequences.

2.4 Phylogenetic analysis and nucleotide comparison

Multiple sequence alignments were carried out with Clustal W [15], and phylogenic tree was constructed with MEGA 5 software [16], using the Neighbor-joining tree method with 1000 bootstrap replicates to assign confidence levels to branches. The IBV sequences were aligned and compared with reference and vaccine strains that were found or used in the Middle East. The sequences were retrieved from GenBank (National Centre of Biotechnology Information) and BLAST search was carried out. The other S1 gene sequences used for comparison or phylogenetic analysis were CK/Eg/BSU-2/2011 (JX174185), CK/Eg/BSU-3/2011 (JX174186), Eg/1212B (JQ839287), Eg/CLEVB-2/IBV/012 (JX173488), IS/885/00 (AY279533) and IS/1494/06 (EU780077), M41 (GQ219712), H120 (GU393335), D274 (X15832), 4/91 (JN600614), CR88 (JN542567) and 793B (Z83979).

3 Results and discussion

The daily mortality on the day sampling is given in the Table 1. At necropsy, the main lesions found were tracheitis, lung congestion, air-sacculitis and enlarged kidneys. IBV was detected in samples obtained from all the farms. The nucleotide sequences of these IBVs were submitted to the GenBank and the assigned accession numbers are shown in the Table 1. Fig. 1 and Table 2 shows the relatedness between the Libyan IBV sequences in comparison to those found in the Middle East and the reference IBVs. A pair-wise comparison of the IBVs, showed two distinctive patterns. The Libyan IBVs, from farm 1, 2, 4, and 5, formed a separate cluster, with 94–99% homology to CK/Eg/BSU-2/2011, CK/Eg/BSU-3/2011 and Eg/1212B. The percent similarity to another regionally important IBV (IS/885/00), which was first detected in Israel ranged from 85–89% (Table 2). The Egyptian IBV strains CK/Eg/BSU-2(also 3)/2011 were associated with high mortality, respiratory and renal pathology [17]. The IS/885/00 strain was isolated in 2000 from broiler chickens in Israel. This strain was reported to cause acute renal disease, severe morbidity and high mortality ranging from 15% to 25% [4].

Fig. 1 Phylogenetic tree based on a partial sequence of the S1 gene, showing the relationship between the Libyan and other IBV strains.

Table 2 Nucleotide and amino acid identity of the part-S1 glycoprotein gene of the Libyan in comparison to other IBV strains.

Those IBVs detected in farm 3 (IBV/Chicken/Libya/05/2012 and IBV/ Chicken/Libya/06/2012) formed another cluster, with 100% relatedness to Eg/CLEVB-2/IBV/012 and IS/1494/06 (Table 2). The IS/1494/06 was first identified in Israel in 2006, was recognised as a nephropathogenic IBV, and later classified as variant 2. It has been reported that birds vaccinated with H120 were poorly protected when challenged with these strains [4,9].

These findings show both, IS/885/00-like and IS/1494/06-like IBVs, are now circulating in Libya. Not much is known about the mode of IBV spread between the countries in the Middle East, however, cross-border movements of poultry and poultry-related products are likely an important factor. These finding shows that high mortality and severe respiratory diseases in Libyan chicken farms is likely contributed by these variant IBVs. The role of other co-infections and other exacerbating factors (e.g. immunosuppression, poor ventilation, stocking density, poor management) need to be examined. In North Africa, both classical and variant IBVs have been reported in Egypt, Tunisia and Morocco [6,17–19]. In this study, even though only small numbers of farms were sampled, our findings have highlighted the circulation of variant IBVs in Libya for the first time. Further studies should include sero-surveillance, isolation and serotyping/genotyping of IBVs in the region.

Acknowledgement

The authors wish to thanks Anne Forrester and Emma Newsham for technical assistance and help with the molecular analysis respectively.

Notes

Peer review under responsibility of Faculty of Veterinary Medicine, Cairo University.