Mallards and blue-winged teals are important reservoir hosts for avian influenza viruses (3, 24, 25); they are both widely distributed waterfowl species and commonly infected with both LPAIV and HPAIV. Our study documents both within and between-species variation in viral shedding as well as for SAα2,3Gal, the viral receptor for many LPAIVs. In mallards, but not teals, we found viral shedding was related to percentage of SAα2,3Gal. While we expected to see positive linear relationships between virus titers and SAα2,3Gal in all tissues and cell types, the mallard ileum was the most predictive of virus titers, with a positive relationship between virus titers and SAα2,3Gal in ileum villi enterocytes, and a negative relationship between virus titers and SAα2,3Gal in the ileum brush border. Despite the lack of relationship between viral shedding and SAα2,3Gal in teals, we observed significantly higher viral shedding by teals, and a higher percentage of SAα2,3Gal compared to mallards.
As the direction (positive or negative) of the correlation between SAα2,3Gal and virus titer varied across mallard tissue locations, our data highlight the importance of understanding tissue-specific tropism as it relates to cell surface SAα2,3Gal distribution. Within mallards, the positive relationship between virus titer and SAα2,3Gal in the ileum villi enterocytes was expected given that LPAIV replicates in intestinal enterocytes by binding SAα2,3Gal on the surface of the cell for cell entry (27). A reason why ileum villi enterocytes were most correlated with viral titer compared to ileum crypt enterocytes may be that the villi have closer direct contact with digesta and as a result, closer direct contact with virus passing through the gut. For example, previous studies have found LPAIV antigen via immunohistochemistry more consistently in mallard villi enterocytes compared to the crypts (12, 13). Surprisingly, however, the other three intestinal tissue types: proximal, cecum, and colon, were not associated with virus titers in the MLR models.
The lack of a statistically significant relationship between SAα2,3Gal and virus titer in the mallard colon was unexpected, given numerous studies have indicated the colon as a site for high LPAIV replication (11–13, 28). The lack of a statistically significant relationship between colon SAα2,3Gal and viral shedding may be due to the statistical approach we used. The MLR method was designed to identify the tissue or tissues which most contributed to the variation observed in virus titers while eliminating issues from the model that exhibited multicollinearity. Since SAα2,3Gal in the ileum and colon were 63% correlated with each other (Additional File 11), the colon could also have a contributing effect to viral load, but not as strongly as the ileum.
The statistically insignificant relationship between SAα2,3Gal in the mallard proximal intestine and virus titers may be explained by the low percentage of SAα2,3Gal in the proximal intestine compared to the ileum, cecum, and colon. Previous studies have assessed the presence of SAα2,3Gal in villi enterocytes and goblet (mucus producing) cells of the duodenum and jejunum, but with conflicting results. Costa et. al found high expression of SAα2,3Gal in the epithelial cells of the duodenum and jejunum of mallards but not in goblet cells (17), while Franca et. al only found SAα2,3Gal in the goblet cells of the duodenum and jejunum of mallards, but not the enterocytes (13, 18). These differences could be a result of the microscopic imaging techniques used, where Costa et. al used light microscopy and Franca et. al used florescence microscopy. Our analysis purposefully excluded positive staining goblet cells since they produce mucins which also express SAα2,3Gal and may inhibit cell entry and viral replication (29, 30). Although the consensus is not clear concerning the presence of SAα2,3Gal in the mallard proximal intestine; previous findings indicate that positive viral antigen via immunohistochemistry is more commonly found in the ileum, cecum, and colon when cloacal swab virus titers are high (12, 13), which would indicate that the proximal intestine is not a main site of LPAIV replication. Therefore, we suggest that the lower percentage of SAα2,3Gal in the proximal enterocytes could explain the lack of a relationship to shedding of LPAIV.
Two hypotheses could explain the negative relationship between SAα2,3Gal in the ileum brush border and virus titer. Initially, we expected to see a positive relationship between SAα2,3Gal in the brush border of all intestinal tissues and virus titers since the receptors are located on the surface of the cell and more likely to be exposed to virus (31). However, as a virion attaches to a receptor, the virion along with the receptor becomes engulfed by the cell for replication, therefore removing the receptor from the surface of the cell (32). This idea is also consistent with the differences observed in percentage of SAα2,3Gal between infected and control mallards, where control mallards had higher SAα2,3Gal in the ileum and colon brush border compared to infected birds. Secondly, mucus is also found along the brush border and LPAIV has been found to bind SAα2,3Gal in mucus, which would prohibit the virus from reaching the enterocyte for virus replication (15, 30, 33); thereby reducing the quantity of virus shed. Up-regulation of mucins have also been observed in response to other viruses which bind sialic acid receptors (29), such as human rotavirus infections (34). To investigate the true explanation for the negative relationship between percentage of SAα2,3Gal in the ileum brush border and virus titers, further experimental research is warranted.
The bursa epithelial cells are also considered to be an important site of replication for LPAIV in waterfowl, including mallards (12, 13). However, given autolysis of tissue samples we were unable to analyze the relationship between SAα2,3Gal in the bursa and viral shedding in mallards. In teals, lectin staining was very high in the bursa; however, it was not significantly related to viral shedding. Lack of a significant relationship to viral titer in teals could be attributed to the lack of individual variation in SAα2,3Gal expression in the bursa or to a sporadic correlation between bursa and cloacal swab virus quantity.
The premise of our study was to determine if the percentage of SAα2,3Gal in the intestines and bursa may be associated with cloacal shedding; hence, we predicted the variation of SAα2,3Gal in control and infected birds would not differ. Our data suggest this is not the case. In the cecum, the percentage of SAα2,3Gal was higher in the crypts of infected mallards compared to their conspecific controls. Similarly, in teals the percentage of SAα2,3Gal was higher in the cecum villi and brush border of infected birds. The ceca have a unique role in the functioning of the vertebrate immune system. The cecal tonsils, a major lymphoid tissue in the cecum, enlarges during gut infections due to infiltration of immune cells, which includes macrophages (35). Macrophages express Gal-specific receptors (36), which could explain the higher abundance of SAα2,3Gal in the cecum of infected birds relative to controls. White leghorn chickens have a greater abundance of sialic acid receptors than silky fowl and this corresponded with a higher number of immune cells in the cecum in the leghorns (37). The cecum has a unique response to LPAIV infection compared to other intestinal tissues, which warrants further analysis of SAα2,3Gal in this tissue.
Contrary to differences in SAα2,3Gal expression between LPAIV-infected and control birds in the cecum, control mallards expressed more SAα2,3Gal in the ileum and colon brush border than infected mallards. Franca et. al (13) found that SAα2,3Gal was lower in the cecum, colon, and bursa of infected birds compared to control birds. Their hypothesis indicated that the SAα2,3Gal expression level may decrease after infection because the neuraminidase function of the virus allows cleaving of the receptor releasing virions from the cell (38). When the receptor is cleaved, it is no longer present on the cell surface which would reduce lectin binding. While Franca et. al (13) did not specify whether the decrease in lectin staining was on the surface of the enterocyte, we found mallards to have a higher percentage of SAα2,3Gal only in the brush border. Our results indicate the importance of assessing the specific location of SAα2,3Gal in determining their function in influenza studies.
An interesting finding was the species-specific differences between mallards and teals in variation and viral shedding. The variation observed in mallards in our study is consistent with what has been observed in other experimental infection studies (10, 39). To our knowledge this is the first experimental infection of blue-winged teals with LPAIV; hence we do not have any other study against which to compare our results. We found that teals shed significantly more virus and had a higher percentage of SAα2,3Gal than mallards but expressed less variation in both measurements. Jankowski et al. (21) also found species-based variation in sialic acid receptor expression. In their study, which analyzed the variation of sialic acid receptors expressed by erythrocytes in various avian species, it was found that approximately 20% of the species expressed 80% of the overall sialic acid receptor quantity in all species studied. Although teals were not included in the Jankowski et. al. (21) study, mallards and three other Anas species (A. acuta, A. Americana, and A. crecca) were among the species assessed. Interestingly, mallards had the lowest quantity of sialic acid receptors on erythrocytes compared to the other three Anas species. In our study, the higher shedding of LPAIV and SAα2,3Gal identified in teals is important because teals have the highest LPAIV prevalence rates during winter months in Texas (26, 40), implicating them as important hosts for overwintering LPAIV in the U.S. Our results and the results of Jankowski et. al suggest that species variation is an important factor to consider for sialic acid receptor variation. Since the quantity of virus shed can directly impact transmission dynamics and is an important parameter for predicting disease risk in a population (41), if certain species can be identified as more infectious than other species, species-specific parameters could improve our ability to predict and mitigate disease. Hence, knowing which species are likely to contribute more to environmental contamination is key for determining the transmission risk of LPAIV.
Since both virus titers and the percentage of SAα2,3Gal were significantly higher in teals compared to mallards, it may be hypothesized that the higher teal virus titers were a result of the higher SAα2,3Gal. It has already been shown that teals have higher binding affinity to MAL I lectin than mallards (18). Additionally, different lectins vary in their affinity for SAα2,3Gal with slightly different molecular structure, such that MAL I preferentially binds SAα2,3Gal with a β1-4Glc(NAc) linkage (42), while MAL II preferentially binds SAα2,3Gal with a β1-3Gal(NAc) linkage (43). Different LPAIV strains also vary in binding affinity to SAα2,3Gal with different molecular structures (44). Although, LPAIV H5N9 (Ratite/New York/12716/94) has similar affinity for the receptors targeted by each lectin (43, 44), we did not test the specific receptor affinity of the LPAIV H5N9 (A/northern pintail/California/44221-761/2006) used in this study. If LPAIV H5N9 (A/northern pintail/California/44221-761/2006) does have a higher affinity for SAα2,3Gal with a β1-4Glc(NAc) linkage, which is the preferred binding affinity of MAL I, then further evidence would be provided to explain the higher LPAIV H5N9 virus titers in teals.
Sex was not a significant factor in viral titers nor SAα2,3Gal when examined separately; yet, the MLR analysis did show that when SAα2,3Gal in the ileum villi enterocytes and brush border are held constant, there was a positive relationship between virus titer and SAα2,3Gal in male mallards. We are unsure why this was the case and likely warrants a study with larger samples sizes for both sexes to draw conclusions about the effect of sex on expression of SAα2,3Gal and LPAIV shedding. Few experimental AIV infection studies have investigated sex-based differences in viral shedding (but see Pepin et al. 2012 (45)). Pepin et al. (45) observed significantly higher cloacal shedding of LPAIV in males than females but did not draw a conclusion about the mechanism underlying this difference. There is more evidence for sex-based differences in infectiousness for other host-pathogen systems (46); however, much of the difference is related to a bird’s reproductive state and/or hormone levels (47, 48). The birds in our study were only 6–12 weeks old and not reproductively mature; hence, any sex-related differences are unlikely due to variation in hormone production.
The identified positive relationships between viral RNA in cloacal swabs, ileum tissue, and bursa tissue further supports the importance of the ileum and bursa for cloacal shedding of LPAIV. Prior to this study, it was well known that LPAIV replicates in duck intestines and the bursa of Fabricius (11–13). While testing for virus in cloacal swabs is the standard method for determining AIV fecal shedding (49, 50), the direct relationship between tissue replication and virus shed by the cloaca was unknown. Through quantifying viral RNA via RT-qPCR in ileum and bursa tissue, significant positive relationships were found between virus titers in cloacal swabs, ileum tissue, and bursa tissue, indicating the contribution of these tissues to the cloacal virus shed. The positive relationship between virus titers in the ileum and cloacal swabs provides additional evidence to support our conclusion that ileum SAα2,3Gal was associated with virus titer. Lastly, these positive relationships add validity to collecting cloacal swabs as an indicator of virus titer in the ileum and bursa and perhaps the infection status of the bird as a whole.