Seroprevalence and molecular characterisation of infectious bronchitis virus (IBV) in broiler farms in Sabah, Malaysia

Abstract

Background: Infectious bronchitis virus (IBV) is classified as a highly contagious viral agent that causes acute respiratory, reproductive and renal system pathology in affected poultry farms. Molecular and serological investigations are crucial for the accurate diagnosis and management of IBV.

Objectives: The purpose of this study was to determine the seroprevalence of IBV and to characterise the circulating IBV in poultry farms in Sabah Province, Malaysia.

Methods: To determine IBV antibodies, a total of 138 blood samples and 50 organ samples were collected from 10 commercial broiler flocks in 3 different farms by using the enzyme-linked immunosorbent assay (ELISA) (IDEXX Kit) and reverse transcription-polymerase chain reaction (RT-PCR) followed by sequencing.

Results: A total of 94.2% (130/138) of the samples were seropositive for IBV in the vaccinated flock, and 38% (52/138) of the birds was the IBV titre for infection. The selected seropositive samples for IBV were confirmed by RT-PCR, with 22% (11/50) being IBV positive amplified and sequenced by targeted highly conserved partial nucleocapsid (N) genes. Subsequently, phylogenetic analysis constructed using amplified sequences again exposed the presence of Connecticut, Massachusetts, and Chinese QX variants circulating in poultry farms in Sabah, Malaysia.

Conclusions: The unexpectedly increasing mean titres in serology indicated that post infection of IBV and highly prevalent IBV in selected farms in this study. The sequencing and phylogenetic analysis revealed the presence of multiple IBV variants circulating in Malaysian chicken farms in Sabah, which further monitoring of genetic variation are needed to better understand the genetic diversity.

Keywords: ELISA; evolution; infectious bronchitis virus; nuclocapsid protein; qRT-PCR; seroprevalence.

FIGURE 1

(a) Sample collection area in different regions of Sabah. (b) Screening results of all IBV isolates in different flocks of selected farms in Sabah state, Malaysia. NP: total 10 flock – TU: Tuaran farm (3 flocks); TA: Tawau farm (4 flocks); PA: Papar farm (3 flocks).

FIGURE 2

Gross pathology of suspected IBV infection. (a) Tracheal haemorrhage, (b–d) cystic oviduct distention and yellowish fluid accumulation, and (e) swollen and congested kidney.

FIGURE 3

IBV‐positive samples showed stunting, curling, twisted and shrunken embryos whereas IBV‐negative samples exhibited normal embryos as a control in 13‐day‐old embryo.

FIGURE 4

Prevalence of IB individual titre in 10 flocks of three different farm with vaccination program (Tuaran, Tawau and Papar) of Sabah, Malaysia.

FIGURE 5

(a) Gel electrophoresis (1% agarose gel) results of orf gene of IBV detection by universal primers compared with other Non‐IBV. Lane 1: Hyperladder 100 bp (Transgenbiotech, China). Lane 2: Positive control (Ma5 IBV vaccine strain) showed the clear band. Lane 3: positive PCR band of orf gene at 143 bp. Lane 4–12 = NDV (Newcastle virus); IBD (Infectious bursal disease); AI (Avian influenza); E coli (Escherichia coli); MG (Mycoplasma gallisepticum); IC (Infectious coryza); ILT (Infectious laryngotracheitis virus), respectively. (b) Universal detection by the orf gene from all farms is isolated. Lane 1: Hyper ladder 1 kb (Transgenbiotech, China). Lane 3: Positive PCR band of orf gene at 143 bp from 11 samples of farm isolate. (c) Electrophoresis results of N gene of IBV detection from positive samples by specific primers in Lanes 4 −12: Positive PCR band of partial N gene at 406 bp.

FIGURE 6

The phylogenetic trees show the possible evolutionary relationship of IBV isolated in this study with other selected IBVs. The maximum likelihood (ML) trees were constructed by using partial nucleotide sequences of the IBV N gene. Nucleotide sequences were aligned with MAFTT (version 7.450) in Geneious (version 10.2.2, Biomatters, Ltd., Auckland, New Zealand). The ML trees were constructed under the GTR substitution model, and 1000 bootstrap re‐samplings using tools available in CLC Genomics Workbench (version 9.5.4). The numbers on the left show bootstrap values as percentages and the labels at branch tips refer to GenBank accession numbers followed by isolates and country of origin. The clade and subclade related to this study are highlighted in different colours, and IBV isolates sequenced in this study are shown in the pink colour.