- Email: [email protected]
Lower expression of sialic acid receptors in the cecum of silky fowl (Gallus gallus domesticus Brisson) compared to white leghorn
Lower expression of sialic acid receptors in the cecum of silky fowl (Gallus gallus domesticus Brisson) compared to white leghorn Deping Han,∗ Yanxin Hu,† Kedao Teng,† and Xuemei Deng∗,1 ∗
National Engineering Laboratory for Animal Breeding and Key Laboratory of Animal Genetics, Breeding, and Reproduction of the Ministry of Agriculture, China Agricultural University, Beijing 100193, China; and † College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China
Key words: Silky Fowl, influenza virus, sialic acid-α-2,3-galactose receptor, sialic acid-α6-galactose receptor 2016 Poultry Science 0:1–6 http://dx.doi.org/10.3382/ps/pew065 2,3-GAL) receptor and sialic acid-α-2, 6-galactose (SA-α-2, 6-GAL) receptors (Suzuki 2005; Stevens et al., 2006). The avian influenza virus primarily binds the SA-α-2,3-GAL receptor that is predominately expressed on avian respiratory epithelial cells, and, thus, infect birds but not humans (Perkins and Swayne 2003; Liu et al., 2007). Conversely, the epithelial cells of the human respiratory tract mostly express the SA-α-2, 6-GAL receptor and are primarily infected by the human influenza virus (Shinya et al., 2006; Chua and Chai 2012). Recent studies have shown that SA-α-2,3-GAL and SA-α-2, 6-GAL receptors are co-expressed in the respiratory epithelia of the pig and tiger (Ito et al., 1998; Keawcharoen et al., 2004), suggesting that interspecies transmission could occur if avian and human influenza viruses recombine while co-infecting the same intermediate host animal. Although avian influenza virus infect birds by binding specific receptors, during a pandemic, large numbers of birds become infected that display only mild pathological changes suggestive of widely varying susceptibilities to the virus (Zhou et al., 1999; Kim et al., 2009). Previous studies have found that AIV infection of chickens accelerated influenza virus evolution and resulted in high mortalities, but infection of ducks resulted in no obvious signs of influenza virus infection (Huang et al., 2013). Thus, the respiratory and gastrointestinal tracts of different species may have varied susceptibilities to AIV infection. Different susceptibilities to AIV have also been found among different breeds of chicken (van Riel et al., 2007; Wang et al., 2014).
INTRODUCTION As an anthropozoonosis, researches on avian influenza virus (AIV) have received a great deal of scientific attention for many years. AIV has a broad tissue tropism and can infect many kinds of cells and organs, but susceptibilities to infection vary between different organs and different breeds (van Riel et al., 2007; Wang et al., 2014). It is well known that trachea and lung are the primary target tissues of AIV under natural infection conditions, and AIV attacks the epithelial cells and macrophages in the airway resulting in acute lung injury and acute respiratory distress syndrome (Xu et al., 2006; Hu et al., 2012; Jang et al., 2015). The proximal cause of acute lung injury and mortality in both humans and animals is the uncontrolled release of cytokines by hosts in response to viral infection, often called “cytokine storm” (Lipatov et al., 2005; Burggraaf et al., 2014). The gastrointestinal tract is an additional key route for oral infection where the macrophages provide an ideal microenvironment for influenza virus replication (Ito et al., 2000). Many studies have found that sialic acid receptors expressed on the cells of susceptible tissues are key determinants for influenza virus infection in both birds and humans (Suzuki 2005; Stevens et al., 2006). The cell surface glycan moieties recognized by influenza viruses are sialic acid-α-2,3-galactose (SA-α C 2016 Poultry Science Association Inc. Received October 22, 2015. Accepted January 14, 2016. 1 Corresponding author: [email protected]
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Leghorn chickens were compared. The results showed that sialic acid-α-2,3-galactose receptors and sialic acidα6-galactose receptors were both observed in Silky Fowl and White Leghorn, but fewer positive cells were detected in Silky Fowl with significant difference in the cecum. The lower abundance of sialic acid receptors likely results from the lower abundance of CD3 and F4/80 immune cells in the cecum of Silky Fowl.
ABSTRACT Avian influenza virus has received increasing attention in recent years because of the potential for recombination with the human virus. Distributions of sialic acid receptors on target cells are determinants of the susceptibilities of different species to influenza virus infection. In this study, the distribution of sialic acid receptors in the respiratory and gastrointestinal tracts of Silky Fowl and White
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MATERIALS AND METHODS Animals Ten SF and 10 WL chicks, aged 3 wk old, evenly distributed with regard to sex, were obtained from the experimental farm of China Agricultural University. Chickens were anesthetized with pentobarbital sodium and then sacrificed by severing their jugular veins. The tissues were sampled in duplicate; one was fixed in 4% paraformaldehyde solution for paraffin sectioning and the other was stored frozen in liquid nitrogen for crystal section. Animal use and animal trials in this study were approved by The Beijing Municipal Committee of Animal Management and The Ethics Committee of China Agricultural University.
Hematoxylin and Eosin Stain (H&E) Samples were allowed to fixation in 4% paraformaldehyde solution for a minimum period of 24 h before use. Tissues were trimmed, dehydrated by alcohols, and embedded in paraffin. After that, 5 μm serial sections were made. The sections were dewaxed, rehydrated, and incubated in hematoxylin (Zhongshan GoldenBridge Biotechnology Co. Ltd., Beijing, China) for 5 min. After being rinsed with distilled water, the sections were rinsed with 1% hydrochloric acid in 75% alcohol for 20 s, and then incubated in phosphate buffered saline (PBS) for 5 min. The sections were incubated sequentially in 70% and 80% alcohol for 2 min each, and then were stained with Eosin (Zhongshan GoldenBridge Biotechnology Co. Ltd.) for 30 s. After destaining in 95% al-
cohol for 1 min, the sections were dehydrated in 100% alcohol and xylene, and then were mounted for observation under light microscope. The histological distributions of melanin in SF were observed and pictured using a Zeiss camera system (Carl Zeiss AG, Oberkochen, Germany).
Immunofluorescence Frozen tissue samples were trimmed and embedded in OCT (Leica Camera AG, Wetzlar, Germany), and then were made for 7 μm serial frozen sections. After dried at room temperature, the sections were fixed in 4% paraformaldehyde solution for 10 min. Rinsed with distilled water, the sections were incubated in 3% hydrogen peroxide solution for 15 min. Then the sections were washed with the distilled water and blocked with 1% bovine serum albumin (BSA) for 20 min. All the above operations were performed at room temperature. The sections were incubated with FITC labeled MAA (1:500, Vector Labs, Burlingame, CA) or SNA (1:500, Vector Labs) for overnight at 4◦ C in a humidified chamber. After being rinsed with PBS, the sections were mounted for observation under a fluorescence microscope.
Immunohistochemistry Frozen sections were washed with distilled water and incubated in 3% hydrogen peroxide solution for 15 min at room temperature. Then the sections were washed with the distilled water and blocked with 1% BSA for 20 min at room temperature. The sections were incubated with primary antibody mouse anti-chicken Bu-1b (1:100, Santa Cruz Biotechnology, Inc., Dallas, TX), mouse anti-chicken CD3 (1:100, Southern Biotech, Birmingham, AL) and rat anti-chicken F4/80 (1:100, Santa Cruz Biotechnology, Inc.) overnight at 4◦ C. After rinsing with PBS, sections were incubated with the cognate secondary antibodies (goat anti-mouse IgG or goat anti-rat IgG) conjugated with horseradish peroxidase (Zymed Laboratories Inc., Beijing, China) at room temperature for 1 h. After reacted with diaminobenzidine as chromogen (Zymed Laboratories Inc.) for 10 min at room temperature in dark, the sections were counterstained with hematoxylin. For the negative control, the primary antibody was replaced with PBS. The number of CD3, Bu-1b and F4/80 cells were estimated by counting the cells in 10 independent microscopic fields at 200× magnification and the mean numbers of positive cells were calculated. Samples were coded and examined by a single investigator to eliminate sampling bias.
Statistical Analysis Data were expressed as means ± standard errors (SE). The significance of the variability among different groups was determined by one-way analysis of
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This study compared and contrasted the expression and distribution of sialic acid receptors expression in the airways and gastrointestinal tracts of two chicken breeds (Silky Fowl [SF] and White Leghorn [WL]) using the lectins (Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA)) specific for SA-α2,3-GAL and SA-α-2, 6-GAL receptors, respectively. SF has a prominent feature of the wide distribution of melanocytes shown as hyperpigmentation in the inner organs. Melanocyte is a class of dendritic cells. Many studies have found that melanocytes play important roles in some biological processes. Melanocytes in the epidermis can protect the skin from sunburn by interaction with keratinocytes (Bellono et al., 2013), and macrophage-like melanocytes in the inner ear help maintain the integrity of the blood barrier in combination with the endothelial cells. Depletion of these cells leads to increased permeability and intravascular substance leakage leading to hearing loss (Zhang et al., 2012). The correlation of the distribution of different sialic acids in epithelial cells of the respiratory and gastrointestinal tracts in the two breeds of chicken may shed light on the possible mechanism for different susceptibilities observed between breeds.
SIALIC ACID RECEPTORS IN TWO BREEDS OF CHICKEN
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variance (ANOVA) contained in the SPSS software suite (version 12.0; SPSS Taiwan Corp., Taiwan), and P < 0.05 was considered statistically significant.
RESULTS AND DISCUSSION The specificity of infection of AIV for different tissues and breeds of chicken is related to the interaction between virus and the receptors found on the surface of host cells. Immunohistochemical staining of tissues indicated the presence of MAA and SNA positive cells in the lungs and gastrointestinal tracts of both SF and WL, suggesting the presence of both SA-α-2,3-GAL and SA-α-2, 6-GAL receptors. This indicates that both avian and human influenza viruses could potentially infect SF and WL chickens. In a previous study, only SA-α-2, 6-GAL was detected in the trachea and lung of chicken but not in duck (Gambaryan et al., 2003), suggesting that differences of influenza virus susceptibilities
exist in different species. In this study, a few MAApositive cells but no SNA-positive cells were detected in the mucosa and lamina propria of the trachea in SF. Nevertheless, both MAA- and SNA-positive cells were observed in the trachea of WL, suggesting that WL may provide more opportunities than SF for infection by human influenza virus in these two breeds. Besides the respiratory tract, the gastrointestinal tract is another key route for influenza virus infection. Both MAA- and SNA-positive cells were observed in the mucosal epithelial cells, glandular cells, and cells in the lamina propria of the stomach and intestines of both breeds (Figures 1A and 2A). These results indicated that the gastrointestinal tracts of SF and WL could potentially support infection by both avian and human influenza viruses. Significantly fewer SA-α-2, 3-GAL– and SA-α-2, 6-GAL–positive cells were observed in the cecum of SF than were found in WL (P < 0.01, Figure 1B; P < 0.05, Figure 2B). So, we conclude that different from WL, there is lower
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Figure 1. MAA expression in the lung and cecum of Silky Fowl and White Leghorn by immunofluorescence (A). Bar = 100 μ m. MAA in the cecum of SF is significantly lower than in that of WL (∗∗ P < 0.01) (B).
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susceptibility in SF during influenza virus infection by gastrointestinal tract, especially the cloaca. As a peripheral immune organs in birds, the cecum contains a large number of immune cells. In order to investigate whether expression of the different influenza virus receptors was associated with the distribution of immune cells in the cecum, we stained and calculated the number of CD3-, Bu-1b– and F4/80-positive cells in the cecum of SF and WL. As shown in Figure 3A, significantly lower number of CD3+ cells were observed in the lamina propria of the cecum of SF than in WL (Figure 3B, P < 0.01). Bu-1b+ cells were mostly detected in the lymphoid nodule in the lamina propria of the cecum (Figure 3A) and their numbers were similar between SF and WL (Figure 3B). Significantly more F4/80-positive cells were observed in the cecum of WL than in that of SF (Figure 3, P < 0.01). Influenza virus receptors have been detected on macrophages, and direct infection of macrophages by influenza virus was reported (Ibricevic
et al., 2006; van Riel et al., 2007). So the significant differences of sialic acid receptors expressions between SF and WL might be related to the aberrant immune-cell development in SF (Han et al., 2015; Li et al., 2014). Compared with WL, fewer SNA-positive cells were detected in the cecum of SF, and the most SNA-positive cells were observed in the lamina propria, not in the mucosa and intestinal gland, which was likely a result in there being fewer numbers of macrophages. In addition, fewer numbers of MAA-positive cells were detected in SF, which also may be correlated with a lower number of immune cells. As reported in our previous work (Han et al., 2015), SF had widely distributed melanocytes in the trachea, lung, glandular stomach, muscular stomach and intestines. No melanocytes were observed in the corresponding tissues of WL. In this study, the differences of sialic acid receptor expression were only detected in the cecum between SF and WL, but there were no
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Figure 2. SNA expression in the lung and cecum of Silky Fowl and White Leghorn by immunofluorescence (A). Bar = 100 μ m. Obvious lower SNA expression in the cecum of SF than in that of WL (∗ P < 0.05) (B).
SIALIC ACID RECEPTORS IN TWO BREEDS OF CHICKEN
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Figure 3. Immune cells in the cecum of Silky Fowl and White Leghorn. Immunohistochemistry of Bu-1b, F4/80 and CD3 cells (A). Bar = 100 μ m. Numbers of Bu-1b, F4/80 and CD3 cells in the cecum of SF and WL (∗∗ P < 0.01) (B).
differences of melanocyte distribution in the duodenum, jejunum, ileum, cecum, and colon of SF. Moreover, the melanocytes embedded in organs of SF did not stain positively for either sialic acid receptor. So, melanocytes may not serve directly as host cells for the virus. In our work, the melanocytes in the air-
ways and gastrointestinal tracts of SF were mainly located around blood vessels, similar to mast cells (Han et al., 2015); thus, we predicted that melanocytes may not only act as constitutive cells, but also have some role in the immune response to certain diseases.
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Taken together, these results suggest that the lower quantity of sialic acid receptors in the cecum of SF is a reflection of a lower abundance of immune cells. Given that influenza virus infection can cause an exaggerated inflammatory response, an increase in the number of immune cells could increase the abundance of sialic acid receptors and cooperatively increase the likelihood of virus infection.
ACKNOWLEDGMENTS
REFERENCES Bellono, N. W., L. G. Kammel, A. L. Zimmerman, and E. Oancea. 2013. UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes. Proc. Natl. Acad. Sci. U S A. 110:2383–2388. Burggraaf, S., A. J. Karpala, J. Bingham, S. Lowther, P. Selleck, W. Kimpton, and A. G. Bean. 2014. H5N1 infection causes rapid mortality and high cytokine levels in chickens compared to ducks. Virus Res. 185:23–31. Chua, K. H., and H. C. Chai. 2012. Hemagglutinin protein of Asian strains of human influenza virus A H1N1 binds to sialic acid—a major component of human airway receptors. Genet. Mol. Res. 11:636–643. Gambaryan, A. S., A. B. Tuzikov, N. V. Bovin, S. S. Yamnikova, D. K. Lvov, R. G. Webster, and M. N. Matrosovich. 2003. Differences between influenza virus receptors on target cells of duck and chicken and receptor specificity of the 1997 H5N1 chicken and human influenza viruses from Hong Kong. Avian Dis. 47:1154– 1160. Han, D., S. Wang, Y. Hu, Y. Zhang, X. Dong, Z. Yang, J. Wang, J. Li, and X. Deng. 2015. Hyperpigmentation results in aberrant immune development in Silky Fowl (Gallus gallus domesticus Brisson). PLoS One. 10:e0125686. Hu, Y., Y. Jin, D. Han, G. Zhang, S. Cao, J. Xie, J. Xue, Y. Li, D. Meng, X. Fan, L. Q. Sun, and M. Wang. 2012. Mast cell-induced lung injury in mice infected with H5N1 influenza virus. J. Virol. 86:3347–3356. Huang, Y., Y. Li, D. W. Burt, H. Chen, Y. Zhang, W. Qian, H. Kim, S. Gan, Y. Zhao, J. Li, K. Yi, H. Feng, P. Zhu, B. Li, Q. Liu, S. Fairley, K. E. Magor, Z. Du, X. Hu, L. Goodman, H. Tafer, A. Vignal, T. Lee, K. W. Kim, Z. Sheng, Y. An, S. Searle, J. Herrero, M. A. Groenen, R. P. Crooijmans, T. Faraut, Q. Cai, R. G. Webster, J. R. Aldridge, W. C. Warren, S. Bartschat, S. Kehr, M. Marz, P. F. Stadler, J. Smith, R. H. Kraus, Y. Zhao, L. Ren, J. Fei, M. Morisson, P. Kaiser, D. K. Griffin, M. Rao, F. Pitel, J. Wang, and N. Li. 2013. The duck genome and transcriptome provide insight into an avian influenza virus reservoir species. Nat. Genetics. 45:776–783. Ibricevic, A., A. Pekosz, M. J. Walter, C. Newby, J. T. Battaile, E. G. Brown, M. J. Holtzman, and S. L. Brody. 2006. Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells. J. Virol. 80:7469–7480. Ito, T., J. N. Couceiro, S. Kelm, L. G. Baum, S. Krauss, M. R. Castrucci, I. Donatelli, H. Kida, J. C. Paulson, R. G. Webster, and Y. Kawaoka. 1998. Molecular basis for the generation in pigs
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This work was supported by the Natural Science Foundation of China (31472082), Chinese University Scientific Fund (2014BH003), Program for Changjiang Scholar and Innovation Research Team in University (IRT1191), and National System for Layer Production Technology (CARS-41).
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National Engineering Laboratory for Animal Breeding and Key Laboratory of Animal Genetics, Breeding, and Reproduction of the Ministry of Agriculture, China Agricultural University, Beijing 100193, China; and † College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China
Key words: Silky Fowl, influenza virus, sialic acid-α-2,3-galactose receptor, sialic acid-α6-galactose receptor 2016 Poultry Science 0:1–6 http://dx.doi.org/10.3382/ps/pew065 2,3-GAL) receptor and sialic acid-α-2, 6-galactose (SA-α-2, 6-GAL) receptors (Suzuki 2005; Stevens et al., 2006). The avian influenza virus primarily binds the SA-α-2,3-GAL receptor that is predominately expressed on avian respiratory epithelial cells, and, thus, infect birds but not humans (Perkins and Swayne 2003; Liu et al., 2007). Conversely, the epithelial cells of the human respiratory tract mostly express the SA-α-2, 6-GAL receptor and are primarily infected by the human influenza virus (Shinya et al., 2006; Chua and Chai 2012). Recent studies have shown that SA-α-2,3-GAL and SA-α-2, 6-GAL receptors are co-expressed in the respiratory epithelia of the pig and tiger (Ito et al., 1998; Keawcharoen et al., 2004), suggesting that interspecies transmission could occur if avian and human influenza viruses recombine while co-infecting the same intermediate host animal. Although avian influenza virus infect birds by binding specific receptors, during a pandemic, large numbers of birds become infected that display only mild pathological changes suggestive of widely varying susceptibilities to the virus (Zhou et al., 1999; Kim et al., 2009). Previous studies have found that AIV infection of chickens accelerated influenza virus evolution and resulted in high mortalities, but infection of ducks resulted in no obvious signs of influenza virus infection (Huang et al., 2013). Thus, the respiratory and gastrointestinal tracts of different species may have varied susceptibilities to AIV infection. Different susceptibilities to AIV have also been found among different breeds of chicken (van Riel et al., 2007; Wang et al., 2014).
INTRODUCTION As an anthropozoonosis, researches on avian influenza virus (AIV) have received a great deal of scientific attention for many years. AIV has a broad tissue tropism and can infect many kinds of cells and organs, but susceptibilities to infection vary between different organs and different breeds (van Riel et al., 2007; Wang et al., 2014). It is well known that trachea and lung are the primary target tissues of AIV under natural infection conditions, and AIV attacks the epithelial cells and macrophages in the airway resulting in acute lung injury and acute respiratory distress syndrome (Xu et al., 2006; Hu et al., 2012; Jang et al., 2015). The proximal cause of acute lung injury and mortality in both humans and animals is the uncontrolled release of cytokines by hosts in response to viral infection, often called “cytokine storm” (Lipatov et al., 2005; Burggraaf et al., 2014). The gastrointestinal tract is an additional key route for oral infection where the macrophages provide an ideal microenvironment for influenza virus replication (Ito et al., 2000). Many studies have found that sialic acid receptors expressed on the cells of susceptible tissues are key determinants for influenza virus infection in both birds and humans (Suzuki 2005; Stevens et al., 2006). The cell surface glycan moieties recognized by influenza viruses are sialic acid-α-2,3-galactose (SA-α C 2016 Poultry Science Association Inc. Received October 22, 2015. Accepted January 14, 2016. 1 Corresponding author: [email protected]
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Downloaded from http://ps.oxfordjournals.org/ at George Mason University on March 25, 2016
Leghorn chickens were compared. The results showed that sialic acid-α-2,3-galactose receptors and sialic acidα6-galactose receptors were both observed in Silky Fowl and White Leghorn, but fewer positive cells were detected in Silky Fowl with significant difference in the cecum. The lower abundance of sialic acid receptors likely results from the lower abundance of CD3 and F4/80 immune cells in the cecum of Silky Fowl.
ABSTRACT Avian influenza virus has received increasing attention in recent years because of the potential for recombination with the human virus. Distributions of sialic acid receptors on target cells are determinants of the susceptibilities of different species to influenza virus infection. In this study, the distribution of sialic acid receptors in the respiratory and gastrointestinal tracts of Silky Fowl and White
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HAN ET AL.
MATERIALS AND METHODS Animals Ten SF and 10 WL chicks, aged 3 wk old, evenly distributed with regard to sex, were obtained from the experimental farm of China Agricultural University. Chickens were anesthetized with pentobarbital sodium and then sacrificed by severing their jugular veins. The tissues were sampled in duplicate; one was fixed in 4% paraformaldehyde solution for paraffin sectioning and the other was stored frozen in liquid nitrogen for crystal section. Animal use and animal trials in this study were approved by The Beijing Municipal Committee of Animal Management and The Ethics Committee of China Agricultural University.
Hematoxylin and Eosin Stain (H&E) Samples were allowed to fixation in 4% paraformaldehyde solution for a minimum period of 24 h before use. Tissues were trimmed, dehydrated by alcohols, and embedded in paraffin. After that, 5 μm serial sections were made. The sections were dewaxed, rehydrated, and incubated in hematoxylin (Zhongshan GoldenBridge Biotechnology Co. Ltd., Beijing, China) for 5 min. After being rinsed with distilled water, the sections were rinsed with 1% hydrochloric acid in 75% alcohol for 20 s, and then incubated in phosphate buffered saline (PBS) for 5 min. The sections were incubated sequentially in 70% and 80% alcohol for 2 min each, and then were stained with Eosin (Zhongshan GoldenBridge Biotechnology Co. Ltd.) for 30 s. After destaining in 95% al-
cohol for 1 min, the sections were dehydrated in 100% alcohol and xylene, and then were mounted for observation under light microscope. The histological distributions of melanin in SF were observed and pictured using a Zeiss camera system (Carl Zeiss AG, Oberkochen, Germany).
Immunofluorescence Frozen tissue samples were trimmed and embedded in OCT (Leica Camera AG, Wetzlar, Germany), and then were made for 7 μm serial frozen sections. After dried at room temperature, the sections were fixed in 4% paraformaldehyde solution for 10 min. Rinsed with distilled water, the sections were incubated in 3% hydrogen peroxide solution for 15 min. Then the sections were washed with the distilled water and blocked with 1% bovine serum albumin (BSA) for 20 min. All the above operations were performed at room temperature. The sections were incubated with FITC labeled MAA (1:500, Vector Labs, Burlingame, CA) or SNA (1:500, Vector Labs) for overnight at 4◦ C in a humidified chamber. After being rinsed with PBS, the sections were mounted for observation under a fluorescence microscope.
Immunohistochemistry Frozen sections were washed with distilled water and incubated in 3% hydrogen peroxide solution for 15 min at room temperature. Then the sections were washed with the distilled water and blocked with 1% BSA for 20 min at room temperature. The sections were incubated with primary antibody mouse anti-chicken Bu-1b (1:100, Santa Cruz Biotechnology, Inc., Dallas, TX), mouse anti-chicken CD3 (1:100, Southern Biotech, Birmingham, AL) and rat anti-chicken F4/80 (1:100, Santa Cruz Biotechnology, Inc.) overnight at 4◦ C. After rinsing with PBS, sections were incubated with the cognate secondary antibodies (goat anti-mouse IgG or goat anti-rat IgG) conjugated with horseradish peroxidase (Zymed Laboratories Inc., Beijing, China) at room temperature for 1 h. After reacted with diaminobenzidine as chromogen (Zymed Laboratories Inc.) for 10 min at room temperature in dark, the sections were counterstained with hematoxylin. For the negative control, the primary antibody was replaced with PBS. The number of CD3, Bu-1b and F4/80 cells were estimated by counting the cells in 10 independent microscopic fields at 200× magnification and the mean numbers of positive cells were calculated. Samples were coded and examined by a single investigator to eliminate sampling bias.
Statistical Analysis Data were expressed as means ± standard errors (SE). The significance of the variability among different groups was determined by one-way analysis of
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This study compared and contrasted the expression and distribution of sialic acid receptors expression in the airways and gastrointestinal tracts of two chicken breeds (Silky Fowl [SF] and White Leghorn [WL]) using the lectins (Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA)) specific for SA-α2,3-GAL and SA-α-2, 6-GAL receptors, respectively. SF has a prominent feature of the wide distribution of melanocytes shown as hyperpigmentation in the inner organs. Melanocyte is a class of dendritic cells. Many studies have found that melanocytes play important roles in some biological processes. Melanocytes in the epidermis can protect the skin from sunburn by interaction with keratinocytes (Bellono et al., 2013), and macrophage-like melanocytes in the inner ear help maintain the integrity of the blood barrier in combination with the endothelial cells. Depletion of these cells leads to increased permeability and intravascular substance leakage leading to hearing loss (Zhang et al., 2012). The correlation of the distribution of different sialic acids in epithelial cells of the respiratory and gastrointestinal tracts in the two breeds of chicken may shed light on the possible mechanism for different susceptibilities observed between breeds.
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variance (ANOVA) contained in the SPSS software suite (version 12.0; SPSS Taiwan Corp., Taiwan), and P < 0.05 was considered statistically significant.
RESULTS AND DISCUSSION The specificity of infection of AIV for different tissues and breeds of chicken is related to the interaction between virus and the receptors found on the surface of host cells. Immunohistochemical staining of tissues indicated the presence of MAA and SNA positive cells in the lungs and gastrointestinal tracts of both SF and WL, suggesting the presence of both SA-α-2,3-GAL and SA-α-2, 6-GAL receptors. This indicates that both avian and human influenza viruses could potentially infect SF and WL chickens. In a previous study, only SA-α-2, 6-GAL was detected in the trachea and lung of chicken but not in duck (Gambaryan et al., 2003), suggesting that differences of influenza virus susceptibilities
exist in different species. In this study, a few MAApositive cells but no SNA-positive cells were detected in the mucosa and lamina propria of the trachea in SF. Nevertheless, both MAA- and SNA-positive cells were observed in the trachea of WL, suggesting that WL may provide more opportunities than SF for infection by human influenza virus in these two breeds. Besides the respiratory tract, the gastrointestinal tract is another key route for influenza virus infection. Both MAA- and SNA-positive cells were observed in the mucosal epithelial cells, glandular cells, and cells in the lamina propria of the stomach and intestines of both breeds (Figures 1A and 2A). These results indicated that the gastrointestinal tracts of SF and WL could potentially support infection by both avian and human influenza viruses. Significantly fewer SA-α-2, 3-GAL– and SA-α-2, 6-GAL–positive cells were observed in the cecum of SF than were found in WL (P < 0.01, Figure 1B; P < 0.05, Figure 2B). So, we conclude that different from WL, there is lower
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Figure 1. MAA expression in the lung and cecum of Silky Fowl and White Leghorn by immunofluorescence (A). Bar = 100 μ m. MAA in the cecum of SF is significantly lower than in that of WL (∗∗ P < 0.01) (B).
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susceptibility in SF during influenza virus infection by gastrointestinal tract, especially the cloaca. As a peripheral immune organs in birds, the cecum contains a large number of immune cells. In order to investigate whether expression of the different influenza virus receptors was associated with the distribution of immune cells in the cecum, we stained and calculated the number of CD3-, Bu-1b– and F4/80-positive cells in the cecum of SF and WL. As shown in Figure 3A, significantly lower number of CD3+ cells were observed in the lamina propria of the cecum of SF than in WL (Figure 3B, P < 0.01). Bu-1b+ cells were mostly detected in the lymphoid nodule in the lamina propria of the cecum (Figure 3A) and their numbers were similar between SF and WL (Figure 3B). Significantly more F4/80-positive cells were observed in the cecum of WL than in that of SF (Figure 3, P < 0.01). Influenza virus receptors have been detected on macrophages, and direct infection of macrophages by influenza virus was reported (Ibricevic
et al., 2006; van Riel et al., 2007). So the significant differences of sialic acid receptors expressions between SF and WL might be related to the aberrant immune-cell development in SF (Han et al., 2015; Li et al., 2014). Compared with WL, fewer SNA-positive cells were detected in the cecum of SF, and the most SNA-positive cells were observed in the lamina propria, not in the mucosa and intestinal gland, which was likely a result in there being fewer numbers of macrophages. In addition, fewer numbers of MAA-positive cells were detected in SF, which also may be correlated with a lower number of immune cells. As reported in our previous work (Han et al., 2015), SF had widely distributed melanocytes in the trachea, lung, glandular stomach, muscular stomach and intestines. No melanocytes were observed in the corresponding tissues of WL. In this study, the differences of sialic acid receptor expression were only detected in the cecum between SF and WL, but there were no
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Figure 2. SNA expression in the lung and cecum of Silky Fowl and White Leghorn by immunofluorescence (A). Bar = 100 μ m. Obvious lower SNA expression in the cecum of SF than in that of WL (∗ P < 0.05) (B).
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Figure 3. Immune cells in the cecum of Silky Fowl and White Leghorn. Immunohistochemistry of Bu-1b, F4/80 and CD3 cells (A). Bar = 100 μ m. Numbers of Bu-1b, F4/80 and CD3 cells in the cecum of SF and WL (∗∗ P < 0.01) (B).
differences of melanocyte distribution in the duodenum, jejunum, ileum, cecum, and colon of SF. Moreover, the melanocytes embedded in organs of SF did not stain positively for either sialic acid receptor. So, melanocytes may not serve directly as host cells for the virus. In our work, the melanocytes in the air-
ways and gastrointestinal tracts of SF were mainly located around blood vessels, similar to mast cells (Han et al., 2015); thus, we predicted that melanocytes may not only act as constitutive cells, but also have some role in the immune response to certain diseases.
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Taken together, these results suggest that the lower quantity of sialic acid receptors in the cecum of SF is a reflection of a lower abundance of immune cells. Given that influenza virus infection can cause an exaggerated inflammatory response, an increase in the number of immune cells could increase the abundance of sialic acid receptors and cooperatively increase the likelihood of virus infection.
ACKNOWLEDGMENTS
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This work was supported by the Natural Science Foundation of China (31472082), Chinese University Scientific Fund (2014BH003), Program for Changjiang Scholar and Innovation Research Team in University (IRT1191), and National System for Layer Production Technology (CARS-41).
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