Trop anim health production journal oumed

Trop Anim Health Prod (2014) 46:1297–1301 DOI 10.1007/s11250-014-0643-0

REGULAR ARTICLES

Identification and molecular analysis of infectious bursal disease in broiler farms in the Kurdistan Regional Government of Iraq Oumed Gerjis M. Amin & Daral J. Jackwood

Accepted: 6 July 2014 / Published online: 16 July 2014 # Springer Science+Business Media Dordrecht 2014

Abstract The present study was undertaken to characterize field isolates of infectious bursal disease virus (IBDV). The identification was done using reverse transcriptionpolymerase chain reaction (RT-PCR) and partial sequencing of the VP2 gene. Pooled bursal samples were collected from commercial broiler farms located in the Kurdistan Regional Government (KRG) of Iraq. The genetic material of the IBDV was detected in 10 out of 29 field samples. Sequences of the hypervariable VP2 region were determined for 10 of these viruses. Molecular analysis of the VP2 gene of five IBDVs showed amino acid sequences consistent with the very virulent (vv) IBDV. Two samples were identified as classic vaccine viruses, and three samples were classic vaccine viruses that appear to have mutated during replication in the field.

Phylogenetic analysis showed that all five field IBDV strains of the present study were closely related to each other. On the basis of nucleotide sequencing and phylogenetic analysis, it is very likely that IBD-causing viruses in this part of Iraq are of the very virulent type. These IBDVs appear to be evolving relative to their type strains.

Keywords Infectious bursal disease . RT-PCR . Molecular characterization . Broiler farms . Kurdistan Regional Government of Iraq

O. G. M. Amin (*) Animal Health Department, General Directorate of Animal Wealth and Veterinary, Ministry of Agriculture and Water Resources, Kurdistan Regional Government, Erbil, Iraq e-mail: oumedgergis6@yahoo.com D. J. Jackwood Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, 25 Edgington Hall, 1680 Madison Ave., Wooster, OH 44691, USA

Introduction Infectious bursal disease virus (IBDV) is the etiological agent of infectious bursal disease (IBD), which is a highly contagious, immunosuppressive disease of young chickens. IBDV destroys premature B lymphocytes in the bursa of Fabricius in young chickens, causing an acute infection and immunosuppression (Lasher and Shane 1994). The disease was first discovered in the USA in 1962 (Cosgrove 1962) and later spread worldwide. Currently, IBD is endemic in poultry farms in Iraq. The first report of an outbreak of IBD in Iraq was published by Al-Sheikhly et al. (1978).

IBDV is classified as a member of the family Birnaviridae and belongs to the genus Avibirnavirus. They are nonenveloped, double-stranded bi-segmented (segments A and B) RNA viruses (Eterradossi and Saif 2008). Two serotypes (1 and 2) of IBDV have been identified; only serotype 1 viruses are pathogenic to chickens. Based on virulence, classical virulent, subclinical variant, and vvIBDV strains exist (van den Berg et al. 2004).

The large genome segment A encodes four structural viral proteins: VP5 and a polyprotein that is self-cleaved into VP2VP4-VP3. The smaller segment B encodes the VP1 RNA polymerase protein (Becht 1981). Because conventional serological tests do not differentiate between vvIBDV and classical virulent IBDV, molecular techniques such as reverse transcriptionpolymerase chain reaction (RT-PCR) have been used to rapidly identify IBDV in clinical samples (Lee et al. 1992).

Within VP2 of IBDV, serotype-specific antigenic determinant binding sites are located that are responsible for induction of the neutralizing antibody against IBDV (Synder et al.

1988). The variable domain of VP2 is very prone to high mutation rates which may be associated with alteration of key amino acids responsible for the molecular basis of antigenic variation. Therefore, molecular characterization of IBDV has been focused primarily on the study of the VP2

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gene, which contains a hypervariable region (hvVP2) (Bayliss et al.1990). The hvVP2 region of IBDV can be utilized for the molecular differentiation of IBD strains (Jackwood 2004). A hypervariable region within the VP2 gene especially between nucleotides 762 to 1151 can be utilized to differentiate classical, variant, and very virulent IBDV pathotypes (Heine et al.

1991; Brown et al. 1994).

Isolates of vvIBDV reported from different countries are genetically similar and have three unique amino acid (aa) residues located at positions A222, I256, and I294. Initial studies indicated that a newly isolated virus can be classified as vvIBDV if these three aa residues are detected (Hoque et al.

2001; Banda and Villegas 2004). Subsequent studies have shown that the vvIBDV phenotype is also dependent on the VP1 gene in genome segment B. Identification of this genome segment is also necessary for a vvIBDV diagnosis (Le Nouën et al. 2006; Hon et al. 2006).

Since vvIBDV has not been reported before in Iraq, the objective of this work was to investigate the possibility of circulation of vvIBDV strains in broiler flocks in the Kurdistan Regional Government (KRG) of Iraq.

Conventional RT-PCR was conducted. The GenAmp RNA PCR kit (PerkinElmer, Roche Molecular Systems, Branchburg, NJ, USA) was used for the RT and PCR assays.

Primers 743–1: 5′-GCCCAGAGTCTACACCAT-3′ and 743– 2: 5′-CCCGGATTATGTCTTTGA-3′ were used to amplify a 743-bp region of the hypervariable sequence in the VP2 gene (hvVP2). The RT was conducted at 42 °C for 60 min and was followed by 35 cycles of PCR at 95 °C for 1.0 min, 53 °C for 1.5 min, and 72 °C for 1.0 min. Following PCR, a 7.0-min extension at 72 °C was included. A Wizard SV Gel and PCR Clean-up System (Promega Corp., Madison, WI, USA) was used to purify the RT-PCR products according to the manufacturer’s instructions. Purified samples were sent for nucleotide sequencing to the University of Wisconsin Biotechnology Center DNA sequence facility (Madison, WI, USA). The results were downloaded using Chromas (Technelysium Pty. Ltd., Queensland, Australia) and analyzed using Accelrys Gene v2.5 (Accelrys, San Diego, CA, USA). The sequence data of KRG field IBDV isolates were submitted to GenBank and acquired accession numbers: KC352659.1, KC352660.1, KC352661.1, KC352662.1, KC352663.1, KC352664.1, KC352666.1, KC352667.1, KC352668.1, and KC352669.1.

Materials and methods

Comparison of aligned sequences

Bursal sample collection

To detect similarities and differences in sequences among strains, nucleotide and predicated sequences of Kurdistan IBDV isolates and published IBDV strains were aligned and compared using the BioEdit program. VP2 sequences from 11 strains in the GenBank database, including classical, attenuated, vvIBDV, and variant viruses, were used in the phylogenetic analysis (Table 1). vvIBDV from neighboring countries were also included in order to examine the potential for a genetic relationship. A phylogenetic tree was constructed from IBDV genome sequences using the MEGA 5.1 software, and the tree was calculated with 1,000 bootstrap replicates based on the VP2 hypervariable region (Tamura et al. 2011).

During the period between September and November 2012, 29 pooled bursal samples were collected from the 29 commercial broiler flock farms in Erbil and Dohuk cities (KRG, Iraq). These broiler flocks had several clinical signs related to respiratory diseases and a mortality rate up to 19 %. Postmortem examination of dead and euthanized birds showed variable degrees of airsacculitis and atrophied hemorrhagic bursae.

Molecular detection and sequencing The viral genomic RNA was extracted from FTA cards (Whatman, Inc., Piscataway, NJ, USA). A 4-mm circular section of the FTA card containing the sample was placed in an Eppendorf tube containing 300 μl TNE buffer (10 mM Tris– HCl, pH 8.0; 100 mM NaCl; 1 mm ethylenediaminetetraacetic acid). After a 10-min incubation at room temperature, a 900-μl volume of TRIzol LS Reagent (Life Technologies, Invitrogen Corp., Carlsbad, CA, USA) was added and incubation continued for 5 min at room temperature. A 200-μl volume of chloroform was then added, and the sample was shaken to mix the reagents.

Following centrifugation at 12,000×g for 15 min, the aqueous phase was collected and added to a 500-μl volume of isopropyl alcohol. The RNA precipitate was collected by centrifugation (12,000×g) and then suspended in 50 μl of a 90 % solution of DMSO and sterile water. A 1.0-μl volume was used for each RTPCR assay.

Results Sequence analysis A comparison of the deduced aa sequences showed that five of Kurdistan sequences Iraq 4, 5, 16, 26, and 27 were similar to the following vvIBDV strains: EUvvIBDV, Turkish vvIBDV, Israel vvIBV, Shiraz vvIBDV, and UPM92-04 vvIBDV (Fig. 1). All these viruses contained the markers of virulence associated with vvIBDV genome segment A (residues A222, I242, I256, I294, and S299).

IBDV strain D78, which is a vaccine strain, was included in the analysis as some of the flocks were vaccinated with this

Trop Anim Health Prod (2014) 46:1297–1301 Table 1 IBDV virus strains used in this study for comparison

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Virus ID

GenBank accession no.

Strain

Country

52/70 UK661 Shiraz UPM92-04 Israel99ISR30

D00869 AJ878898 JX983160 AF262030 DQ916203

Classical virulent Very virulent Very virulent Very virulent Very virulent

UK France Iran Malaysia Israel

EL2004-1/TR_VP2 Var-E T1 Mexico04M84 Colombia04C81 D78 vaccine

EF043078 AY819703 AF281238 AF498619 DQ916185 Y14962

Very virulent Variant Variant Variant Variant Attenuated

Turkey USA USA Mexico Colombia Netherlands

vaccine at 12 days of age. The Kurdistan isolates, Iraq 14 and Iraq 14.2, were similar to the classical vaccine strain D78 (and had amino acids 253H and 284 T typically associated with attenuation). Compared to the D78 vaccine, VP2 of the Iraq 10, Iraq 11, and Iraq 21 isolates was similar to the classic D78 vaccine viruses; however, they were different from D78 by having a 253Q residue similar to the classical virulent strain 52/70. Amino acid sequence comparisons between the vvIBDV and classical attenuated IBD vaccine strain (D78) showed no similarities through segment A.

viruses (Fig. 2). All the Kurdistani vvIBDVs and published vvIBDVs were placed in one clade which was different from the clade of classical and variant IBDVs. Samples Iraq 14 No. 2, 14, 10, 11, and 21 were placed in a separate clad together with the attenuated D78 vaccine strain. As expected, variant viruses branched in a distinct cluster from other strains.

Discussion Phylogenetic analysis In 2012, 10 IBD virus isolates were recovered from broiler flocks in the KRG. Phylogenetic analysis was conducted on the predicated amino acid sequences of VP2 on genome segment A; included were reference IBDV strains (classical viruses 52/70; vvIBDV strains UK661, UPM92-04, EL20041/TR, Israel99ISR30, Shiraz; variant viruses Var-E, T1, Colombia04C81, and Mexico04M84) and all 10 Kurdistan aa sequences.

The phylogenetic tree revealed four distinct groups consisting of vvIBDVs and classical, attenuated, and variant Fig. 1 Alignment of deduced amino acid sequences of Iraqi IBDV isolates and published isolates

Since 1978, when the first detection of virulent IBD was reported in Iraq (Al-Sheikhly et al. 1978), the IBDVs have become widespread and established in the poultry industry in the KRG of Iraq. However, genetic information on the IBDV field strains that circulate in the KRG and Iraq poultry flocks has remained unknown.

Due to a lack of local production, Iraq, including the KRG, has extensively imported day-old chicks, feed, drugs, and vaccines from abroad. Beginning in 1999, several outbreaks of IBD occurred that caused 50 and 20 % mortality in layer pullet and broiler farms, respectively. These IBD outbreaks were diagnosed based on clinical signs and postmortem

1300

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Fig. 2 Phylogenetic tree constructed by neighbor-joining method. The Kurdistan vvIBDV strains in this study are marked with squares, and the D78 vaccine strain is marked with a triangle

vvIBDV strains

Classical virulent strains

Antigenic variant strains

Classical attenuated strains

changes; however, no attempts were made to genetically characterize the field IBDV strains circulating in the KRG. We investigated the National Center for Biotechnology Information (NCBI) and found no report of the occurrence of vvIBDVs in the KRG and Iraq. However, circulation of vvIBDV strains has been reported regularly in Middle East countries such as Israel and in countries neighboring the Kurdistan Regional Government such as Turkey and Iran.

In this study and through RT-PCR followed by molecular sequencing, we detected amino acid residues A222, I256, I294, and S299 in the hypervariable region of VP2 of five Kurdistani IBDV (Iraq 4, 5, 16, 26, and 27)-positive samples. These aa residues are conserved genetic markers and are considered unique to most typical vvIBDV strains (Eterradossi et al. 1999).

Although a pathogenicity study was not carried out to evaluate the phenotype of the vvIBDV viruses under our study, the high mortality observed in the commercial flocks of origin also suggests that the viruses are vvIBDV. To confirm our data, genome segment B needs to be identified or the viruses should be used to inoculate specific pathogen-free chicks (van den Berg et al. 1991).

Phylogenetic data revealed that all the Kurdistani vvIBDV field isolates were grouped in the same cluster with vvIBDV from Iranian, Turkish, Israeli, European, and Malaysian isolates, which may indicate their ancestral relationships. It is possible that the vvIBDV strains were introduced into Iraq from neighboring countries or European countries because there is limited restriction on the movement of poultry products from Turkey and Iran to the KRG. In addition, it is also possible that the vvIBDV came from a more distance source

due to the annual importation of huge numbers of chicks from all over the world to the KRG and Iraq.

Because the D78 IBD vaccine strain is widely used in the poultry industry in the KRG and Iraq, sequences belonging to the D78 vaccine virus were obtained from GenBank and included in the molecular comparison.

A multiple comparison of the aa positions at the VP2 hypervariable domain from positions 222 to 300 in Kurdistani field isolates and published IBDV isolates showed that the Kurdistani isolates Iraq 14 No. 2 and Iraq 14 encoded threonine at position 284 T and glutamine at position 253H, which is typically found in vaccine strains (Brandt et al. 2001).

The Kurdistani isolates (Iraq 10, 11, 21) had the aa residue 284 T, but instead of 253H, they had the aa residue glutamine at position 253Q (which is usually associated with pathogenic viruses). The phylogenetic analysis confirmed this relationship as it placed Iraq 14, 14 No. 2, 10, 11, and 21 isolates with the attenuated D78 vaccine strain.

Our study revealed that the three IBD isolates Iraq 10, Iraq 11, and Iraq 21 were probably classic vaccine viruses (D78) that have replicated in the field and mutated, as this is consistent with a report from Jackwood et al. (2008) who reported that reversion from vaccine to virulent strain can occur following a point mutation at position 253.

We obtained variant strain sequences belonging to North and South America from GenBank for comparison.

Interestingly, our molecular analysis data revealed that KRG IBD field isolates did not have amino acid similarity with variant strains and the variant strains distinctly clustered into a separate branch. This data suggests that the variant IBDV

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strains may not be circulating in KRG poultry farms. A more comprehensive survey of KRG flocks is needed to confirm this supposition.

In conclusion, molecular investigation by RT-PR followed

by DNA sequencing and phylogenetic comparison is a powerful tool in the detection and characterization of IBDV strains circulating in poultry farms.

Based on partial sequencing of the hypervariable region of the VP2 gene in segment A of the IBDV genome, the present study demonstrated molecular evidence of vvIBDV strains in broiler flocks in the KRG of Iraq. To the best of our knowledge, this is the first molecular investigation published on the characterization of vvIBDVs in KRG and Iraq. Further molecular studies on circulating field strains of vvIBDVand other IBDV strains are needed to reveal possible mutations that may change their virulence or antigenicity.

Acknowledgments We thank the Ohio Agricultural Research and Development Center, Ohio University, USA, for partly funding this study.

We also would like to thank the Barash Company in Erbil, KRG, for helping collect the samples from the broiler farms.

Conflict of interest The authors declare that they have no conflict of interest.

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