Molecular Biology and Pathological Process of an Infectious Bronchitis Virus with Enteric Tropism in Commercial Broilers

Abstract

Infectious bronchitis virus (IBV) induces respiratory and urogenital disease in chickens. Although IBV replicates in the gastrointestinal tract, enteric lesions are uncommon. We have reported a case of runting-stunting syndrome in commercial broilers from which an IBV variant was isolated from the intestines. The isolate, CalEnt, demonstrated an enteric tissue tropism in chicken embryos and SPF chickens experimentally. Here, we determined the full genome of CalEnt and compared it to other IBV strains, in addition to comparing the pathobiology of CalEnt and M41 in commercial broilers. Despite the high whole-genome identity to other IBV strains, CalEnt is rather unique in its nucleotide composition. The S gene phylogenetic analyses showed great similarity between CalEnt and Cal 99. Clinically, vent staining was slightly more frequent in CalEnt-infected birds than those challenged with M41. Furthermore, IBV IHC detection was more evident and the viral shedding in feces was overall higher with the CalEnt challenge compared with M41. Despite underlying intestinal lesions caused by coccidiosis and salmonellosis vaccination, microscopic lesions in CalEnt-infected chickens were more severe than in M41-infected chickens or controls, supporting the enteric tropism of CalEnt. Further studies in SPF chickens are needed to determine the pathogenesis of the virus, its molecular mechanisms for the enteric tropism, and its influence in intestinal health.

Keywords: IBV; enteric tropism; infectious bronchitis; runting-stunting syndrome; variants; whole-genome sequencing.

Figure 1

Phylogenetic analyses of the whole genome (A), the full S gene (B) and the hypervariable region of the S gene (C) of infectious bronchitis virus isolates. The maximum likelihood method with 1000 bootstrap replicates was used. The IBV CalEnt sequence is in red. G = genotype, L = lineage.

Figure 2

Similarity plot comparing the S gene of CalEnt (reference) to the S gene of Cal99, GA/1476/15, and IBS037A/14. The y axis represents the percent similarity to CalEnt, and the x axis represents the nucleotide position of the S gene. The vertical red lines represent the nucleotide position in which there is a sequence crossover.

Figure 3

Respiratory sign scores of commercial broilers challenged with either IBV M41 or CalEnt oculonasally or via crop gavage. Different superscripts represent statistical significance (p < 0.05). Same letters represent no statistical difference (p > 0.05). Groups were compared using Kruskal–Wallis (for non-parametric data) followed by Dunn’s multiple comparison tests.

Figure 4

Percentage of commercial broilers presenting vent staining after a challenge with either IBV M41 or CalEnt oculonasally or via crop gavage. Different superscripts represent statistical significance (p < 0.05). Same letters represent no statistical difference (p > 0.05). Groups were compared using Kruskal–Wallis (for non-parametric data) followed by Dunn’s multiple comparison tests.

Figure 5

Difference between viral load in tears and viral load in cloacal swabs collected from commercial broilers challenged with either IBV M41 or CalEnt oculonasally or via crop gavage. Numbers above zero represent a higher viral load in tears. Numbers below zero represent a higher viral load in cloacal swabs. Group 5 (unchallenged controls) is not represented because no IBV amplification was observed in any sample at any timepoint.

Figure 6

Infectious bronchitis virus antigen detection in intestinal sections of broilers challenged with CalEnt by immunohistochemistry (IHC): (A) IBV IHC staining in the cytoplasm of enterocytes from CalEnt-infected chickens at 4 dpi via oculonasal route. Scale bar = 40 µm; (B) IBV IHC staining in the cytoplasm of lymphocytes of the intestinal lamina propria from CalEnt-infected chickens at 14 dpi via oculonasal route. Scale bar = 20 µm.