Avian Infectious Bronchitis Virus: Molecular Detection in Southwestern Ethiopia Chickens

Infectious bronchitis virus (IBV) is a significant threat to poultry worldwide, but its status in Ethiopia remains understudied. Thus, this study aimed to detect the virus and associated risk factors in South West Ethiopia. Ninety oropharyngeal swab samples were purposively collected from symptomatic chickens located in Jimma town, Seqa Chekorsa, and Tiro Afeta woredas of the Jimma zone between November 2021 and April 2022 to detect IBV virus by using RT-PCR. A side-by-side questionnaire was administered to assess risk factors. Total RNA was extracted, reverse transcription polymerase chain reaction (RT-PCR) was conducted, and products were visualized under UV light. The overall proportion of IBV was 16.6% (15/90). No statistical association was observed between any of the animal risk factors and the detection of the virus (P=0.57, 0.586, and 1). However, the proportion of birds infected by the virus was higher in males, exotic breeds, and adults compared to females, local breeds, and young birds. Similarly, none of the management risk factors had a significantly different effect on virus detection (P=0.25, 0.09, 0.088, and 0.726). However, improper carcass disposal (OR = 0.43, 95% CI: 0.13–1.4), lack of veterinary services (OR = 2.7, 95% CI: 0.8–8.3), and the presence of wild birds/rodents (OR = 4.4, 95% CI: 0.88-22.3) were associated with increased IBV risk but not cleaning of feeders/drinkers (OR = 1.1, 95% CI: 0.2–4.8). These findings underscore the need for enhanced biosecurity practices and further research to implement informed IBV control strategies in Ethiopia.


Introduction
Infectious bronchitis virus (IBV), classifed within the genus Gammacoronavirus of the Coronaviridae family, is an archetypal avian coronavirus that infects domestic chickens (Gallus gallus).It causes infectious bronchitis, an acute, highly contagious respiratory disease in poultry, characterized by depression, snicking, coughing, head-shaking, and nasal and ocular discharges.Although primarily a respiratory disease, IBV also afects the female reproductive organs, leading to weak, cracked, or misshapen-shelled eggs with watery albumen.Some strains target the kidneys, causing nephritis and signifcant mortality [1].
Infections can be diagnosed by detecting IBV or specifc antibody responses [2].Reverse-transcription polymerase chain reaction (RT-PCR) targeting the 3′ untranslated region (UTR), N gene, and S1 and S2 parts of the IBV genome is commonly used for viral detection [3].
Since 2017, IBV has been reported in Ethiopia, detected through molecular methods [17,18] and serology [19][20][21].Despite its widespread presence and early discovery, IBV remains understudied, particularly in Ethiopia.Tis study aimed to determine the occurrence of infectious bronchitis virus (IBV) in diseased poultry and identify associated risk factors in southwestern Ethiopia using RT-PCR.

Sample Collection, Study Design, and Risk Assessment.
Ninety swab samples were purposively collected from symptomatic, IBV-unvaccinated, backyard, and small-scale chickens between November 2021 and April 2022.Te sampled birds were brought to veterinary clinics in the study sites due to respiratory distress, rufed feathers, or oral/ocular discharge.Oropharyngeal swabs were collected by inserting sterile, cotton-tipped swabs (Miraclean Technologies, China) into the oropharynx of the birds and gently rotating them against the walls.Te swabs were stored in labeled, sterile universal bottles containing virus transport media (VTM).All collected swabs were transported to the National Agricultural Biotechnology Research Center Animal Biotechnology Research facility, Holeta, via cold chain, and preserved at −80 °C until further processing.A semistructured questionnaire was administered to bird owners to assess associated animal and management risk factors (Appendix 1).Questions focused on fock and disease management practices.Outcomes were recorded and correlated with the virus occurrence rate.A total of 58 farmers were interviewed.

RNA Extraction and Reverse Transcription Polymerase
Chain Reaction.Te preserved samples were prepared for extraction after removal of gross contaminants and debris.Cryovials containing swab samples were thawed and centrifuged at 10,000 × g for 5 minutes.Te supernatant was collected in a separately labeled 1.5 ml Eppendorf tube.Total RNA was extracted from 100 μl of each sample using the DaAn Gene RNA Purifcation Kit (DaAn Gene Co., Ltd., China), according to the manufacturer's instructions.Te extracted RNA was stored at −80 °C for further analysis.
Nucleotide sequences for a set of primers with an amplicon product size of 149 bp were obtained from Rashid et al. [23], and a pair of primers was synthesized accordingly (Sigma-Aldrich, USA).Te forward primer was 5′-GCT TTT GAG CCT AGC GTT-3′ and the reverse primer was 5′-GCC ATG TTG TCA CTG TCT ATT-3′.Te annealing temperature and cycling conditions for the primers were optimized using the VaxSafe ® IB Ingham Strain as the positive control.
Finally, reverse transcription and the polymerase chain reaction were conducted in a single reaction tube using the AccuPower Dual-HotStart RT-PCR Kit (Bioneer, Korea).Briefy, 1 µl of each of the forward and reverse primers, 2 µl of the extracted RNA as a template, and 16 µl of nucleasefree water which added up to 20 µl were dispensed into the master mix pellet and vortexed to create a homogenous mixture.Ten, the mixture was placed in a PCR machine (Mastercycler ® PCR thermal cycler, Germany), and the machine was set for thermal conditions of 51 °C for 1 h for reverse transcription, followed by 95 °C for 3 min, initial denaturation, and 36 cycles of 94 °C for 20 s, 54 °C for 20 s, and 72 °C for 20 s, with a fnal extension step at 72 °C for 5 min.
Finally, the products were visualized on a 1% agarose gel stained with ethidium bromide using a gel documentation system (BioDoc-It, USA) to detect the presence of the expected amplicon product in comparison with the reference 100 bp ladder (Bio Basic Inc., Canada).

Statistical Analysis.
Te data collected from the molecular tests were dichotomous.Te responses recorded from the interviews were organized and trimmed for extreme values or homogenous responses.Finally, all adjusted data were analyzed for descriptive statistics, cross-tabulation (chi-square), and likelihood ratio analysis using SPSS V.26.
None of the management risk factors signifcantly affected virus detection (P � 0.25, 0.09, 0.088, and 0.726).However, birds from households that disposed of carcasses in open felds were twice as likely to be infected (22.2%) compared to those where carcasses were fed to pets (11.1%).Te odds of infection were almost double in the frst group (OR � 0.4; 95% CI: 0.13-1.4).Birds without treatment for various conditions were more than twice as likely to be infected (OR � 2.7; 95% CI: 0.8-8.3)compared to those receiving treatment.Te presence of wild birds and rodents increased the risk of infection by more than fourfold (OR � 4.4; 95% CI: 0.88-22.3).However, cleaning poultry feeders or drinkers did not signifcantly afect virus distribution (OR � 1.1; 95% CI: 0.2-4.8).

2
International Journal of Microbiology

Discussion
Te proportion of IBV-positive samples detected by RT-PCR was 16.6% (15/90).Te distribution of IBV under Ethiopian conditions has been understudied, emerging as a concern within the past few years.In 2017, IBV isolates belonging to a vaccinal genotype were identifed through sequencing from a government-owned breeder farm in Bishoftu, likely the frst report in the country [17].
Subsequent detection and genetic characterization were performed in 2022 in the southwest region, revealing isolates with a genetic composition similar to the frst [18].Recent studies in the Central Gondar Zone [24] and two districts of the East Shewa Zone [21] detected IBV using RT-PCR in swab samples.Despite molecular detection, seropositive birds were found in the Ada'a District, Bishoftu, Bonga, Hawass, Kality, Holeta [17,[19][20][21]25], and areas in Gondar and West Gojjam [24,26].Tese seroprevalence and viral detections across diferent geographical areas indicate the virus' expansion in a country with limited vaccination practices.We found no signifcant diference in susceptibility to the virus between male and female birds.An experimental study comparing pathogenesis and host immune responses in one-week-old chicks showed no signifcant diference, except for minor disease severity in males [27].Similarly, a Nigerian study reported nonsignifcant diferences in seroprevalence between sexes [28].Terefore, despite physiological diferences, both sexes appear equally susceptible to IBV.
It was found that susceptibility to the virus was not agedependent, with similar virus prevalence in both age categories.A study experimentally infecting 3-and 10-week-old chicks revealed less prominent nephritogenic lesions in younger birds [29].Another study reported longer viral clearance from various organs in older birds compared to younger birds, except for the trachea [30].However, younger chickens have been reported to be more susceptible to oviduct-afecting strains [31].Tese conficting fndings suggest that the most susceptible age might depend on viral tropism, pathogenesis (respiratory vs. nephropathic), and sample type (respiratory swab vs. kidney or oviduct tissue).
We observed no signifcant diference in virus distribution between exotic and local birds.In contrast, studies comparing inbred white Leghorn lines [32] and brown and white Leghorn lines [33] reported signifcant diferences in susceptibility and mortality rates, respectively.Da Silva described the involvement of MHC alleles B2 and B18 in breed-related susceptibility to IBV [34].Without evaluating these chromosomes and loci in our study breeds, resistance assessment can only be based on phenotypic performance.
Management risk factors such as carcass disposal, access to veterinary services, exposure to living fomites or carriers, and cleaning equipment practices could afect virus transmission.Birds whose owners disposed of carcasses in open felds were twice as likely to be infected (22.2%) compared to those where carcasses were fed to pets (11.1%).Te importance of proper carcass disposal in preventing pathogen spread was highlighted by Blake during the 2002 Virginia outbreak of low pathogenic avian infuenza, where infected carcass movement was implicated in virus dissemination [35,36].Open carcass disposal can disrupt disease control and create infection cycles.
Access to veterinary services, including deworming, antibiotic treatment, and micronutrient supplementation, indirectly impacts poultry health.Birds receiving treatment for various conditions were less likely to be infected compared to those without treatment.Deworming [37,38], antibacterial treatment [39] and micronutrient supplementation [40] have been reported to enhance viral immunity against NDV and IBV.Terefore, the lower IBV prevalence among treated birds is unsurprising.
Chicken residing in premises accessible to wild birds and rodents exhibited higher susceptibility to IBV compared to those in isolated environments.Te detection of IBV antibodies in free-living pigeons and Japanese quails in Nigeria and the detection of wild-type strains in Brazil indicate their potential as viral reservoirs [41,42].Rodents are well-known vectors for various pathogens [43], and their role in disease transmission could extend beyond currently recognized pathogens.
While enveloped viruses, including coronaviruses, are generally susceptible to detergents [44], the efectiveness of cleaning practices can vary based on the cleaning agent and frequency.Daily cleaning of feeders with diluted bleach solution has shown to reduce parasite burden in fnches [45].However, aerosol infection and environmental contamination might override the impact of cleaning feeders and drinkers in viral disease control.

Conclusion
Although limited studies have been conducted, the detection of IBV in various regions of Ethiopia, including the southwest, confrms the virus' widespread distribution.Serological fndings further support the expansion of IBV within the country.Tese results emphasize the urgent need for enhanced biosecurity measures and comprehensive research to develop efective IBV control strategies in Ethiopia.

Figure 1 :Figure 2 :
Figure 1: Map showing the study area.

Table 1 :
Association of risk factors with detection of the virus.