Currently, QX is a major genotype of IBV in poultry flocks in Asia, Europe and other parts of the world, causing tremendous economic losses to the poultry industry [21, 22]. Several reports have illustrated the pathogenicity of different QX-type isolates in specific organs of young chickens [23, 24]. However, alterations of the oviduct after viral infection in laying phase are still unknown. In this study, we pioneered the analysis of the pathological effects of QX-type IBV on different segments of the layers’ oviduct and compared them to the Mass-type reference strain M41.
Binding to the host cell is the first crucial step in a virus replication cycle [25]. A previous study confirmed that the QX strain could infect epithelial cells of the pullet’s oviduct in vitro [26]. In this study, we found that both the QXL and M41 strains could infect the epithelial cells of the layers’ oviduct; however, the QXL strain also infected the tubular glands on a large scale. This result suggests that the QXL strain can easily be released from the mucosal epithelium to the lamina propria, possibly via an unknown mechanism mediated by the QXL strain and not by the M41 strain. Previous reports have shown that QX-type IBV caused characteristic dilatation and serum-like fluid accumulation in the oviduct of young chickens, although few alterations were observed microscopically in this organ [11]. In the present study, the QXL strain caused severe injury to different segments of the layers’ oviduct, including the expansion of tubular glands in the infundibulum and magnum, inflammatory cell infiltration in the infundibulum and isthmus, and interstitial dilation of the lamina propria in the uterus. This discrepancy might be caused by the different ages of the birds or by experimental conditions. In the M41 group, only desquamation of epithelial cells was observed in the isthmus and uterus. This result is consistent with a previous study showing that Mass-type IBV causes degeneration and desquamation of epithelial cells in oviduct-organ culture [27].
Generally, the host defends against intracellular viruses via classical inflammatory responses and antiviral immune responses, which are mediated by interferons (IFNs). IFN-γ is a key cytokine for Th1-controlled responses, which is essential for controlling intracellular pathogens [28]. IFN-γ can also cooperate with proinflammatory cytokines, such as IL-1β and IL-6, in the inflammatory response. Previous data demonstrated that infection with attenuated IBV upregulated the expression of proinflammatory cytokines, including IL-1β, IL-6, and IFN-γ [18]. The current results showed that in the QXL group, expression of IL-1β, IL-2, and IL-6 was increased approximately 2-30-fold above normal values, whereas IFN-γ demonstrated a much stronger increase, in some cases more than 500-fold above normal. In this study, macrophages were observed in the infundibulum of the oviduct in the QXL group, where the expression of IFN-γ was increased nearly 500-fold. Type I interferon is a hallmark of innate immune responses to pathogens, and can directly or indirectly act on natural kill (NK) cells, CD8+ cytotoxic T lymphocytes (CTLs), dendritic cells (DCs), and B cells to regulate adaptive immune responses [29]. The current results demonstrated that in the QXL group, the expression of IFN-α was elevated in the infundibulum and magnum. Furthermore, we also observed that the levels of granzyme and perforin were significantly increased in the oviduct of the QXL group, except for the isthmus. These data indicate that the QXL strain induces cell-mediated inflammatory responses driven by Th1 cells, subsequently recruiting macrophages, NK cells and other innate immune cells to secrete type I IFN and cytotoxic molecules for virus suppression. Excessive immune responses can also cause oviduct lesions. In the isthmus of the QXL group, however, neither IFN-α nor cytotoxic molecules were detected, and IHC results showed that the viral antigens were most widely distributed in the isthmus. These results suggest that the isthmus is more susceptible to the QXL strain than other segments of the oviduct are. In the M41 strain, expression of proinflammatory cytokines and antiviral molecules was not significantly altered. Thus, we speculate that the ability to induce innate immune responses may be related to viral pathogenicity. This was in agreement with a previous study [30].
Infection with IBV can affect the quality of eggs, such as soft shell eggs and sand shell eggs. Therefore, we evaluated the expression of genes involved in eggshell and eggshell membranes formation. Type I collagen is expressed in the isthmus, and the eggshell membrane forms during the passage of eggs through the oviduct [31]. The current results showed that the expression of type Ⅰ collagen was downregulated in the isthmus 8 days after challenge in the QXL group. In the avian eggshell gland, calcium-binding protein (calbindin, CaBP) exists as a high-molecular- weight protein of 28 kDa (CaBP-D28k) and plays a role in the transportation of Ca2+ for shell formation [32]. Our results demonstrated that the expression of CaBP-D28k was significantly lower in the QXL group than in the control group at both 4 and 8 days after viral challenge. The decline in the synthesis of type Ⅰ collagen and CaBP-D28K in the isthmus and uterus might be associated with the elevation of immune-related molecules. Therefore, the QXL strain may affect the formation of shell membranes and eggshells by inhibiting the synthesis of type Ⅰ collagen and CaBP-D28k. These results also support the findings of Nii et al. [18], who showed that attenuated IBV could decrease the expression of type Ⅰ collagen and CaBP-D28k.