Distribution patterns of influenza virus receptors and viral attachment patterns in the respiratory and intestinal tracts of seven avian species

RESEARCH Open Access Distribution patterns of influenza virus receptors and viral attachment patterns in the respiratory and intestinal tracts of seven avian species Taiana Costa 1† , Aida J Chaves 1,2† , Rosa Valle 2 , Ayub Darji 2,3 , Debby van Riel 4 , Thijs Kuiken 4 , Natàlia Majó 1,2 and Antonio Ramis 1,2* Abstract This study assessed the presence of sialic acid a-2,3 and a-2,6 linked glycan receptors in seven avian species. The respiratory and intestinal tracts of the chicken, common quail, red-legged partridge, turkey, golden pheasant, ostrich, and mallard were tested by means of lectin histochemistry, using the lectins Maackia amurensis agglutinin II and Sambucus nigra agglutinin, which show affinity for a-2,3 and a-2,6 receptors, respectively. Additionally, the pattern of virus attachment (PVA) was evaluated with virus histochemistry, using an avian-origin H4N5 virus and a human-origin seasonal H1N1 virus. There was a great variation of receptor distribution among the tissues and avian species studied. Both a-2,3 and a-2,6 receptors were present in the respiratory and intestinal tracts of the chicken, common quail, red-legged partridge, turkey, and golden pheasant. In ostriches, the expression of the receptor was basically restricted to a-2,3 in both the respiratory and intestinal tracts and in mallards the a-2,6 receptors were absent from the intestinal tract. The results obtained with the lectin histochemistry were, in general, in agreement with the PVA. The differential expression and distribution of a-2,3 and a-2,6 receptors among various avian species might reflect a potentially decisive factor in the emergence of new viral strains. Introduction Wild aquatic birds are generally considered to be the source of all influenza viruses found in mammal and avian species, including humans, pigs,horses,minks,marine mammals, cats, and a great number of domestic avian species [1]. Phylogenetic studies indicate that all human influenza viruses, including the predominant strains associated with the seasonal flu (H1, H2, and H3 subtypes) originated from an avian ancestor [2]. More recently, a swine-origin influenza A H1N1 virus (pH1N1), which contains genes of human, avian and swine influenza viruses, caused the first pandemic of the 21 st century [3]. The host restriction of influenza A viruses is in part determined by specific sialic acid receptors on the surface of susceptible cells. These receptors are composed of nine carbon monosaccharides, usually found on the outermost terminal position of glycan chains, linked to cell-surface glycoproteins and glycolipids [4]. The N-acetylneuraminic acid (Neu5Ac), one of the most common sialic acids, is usually bound to galactose (Gal) in an a-2,3 (Neu5ACa2,3Gal) or a-2,6 configuration (Neu5ACa-2,6Gal), and their expression and distribution are cell specific [5]. The affinity of influenza viruses for these receptors varies according to the species from which they are isolated. Influenza viruses of avian origin preferentially bind to Neu5Aca-2,3Gal (a-2,3 receptors, avian-like receptors), the form that predominates in the duck enteric tract where these viruses replicate [6,7]; whereas human influenza strains recognize Neu5ACa-2,6Gal (a-2,6 receptors, human-like receptors) [8,9]. For many years, it was thought that the inter-species barrier could only be crossed after adaptation of an avian influenza virus in pigs, since pigs were shown to harbor both a-2,3 and a-2,6 receptors [6,10]. Later on, it was observed that the H5 and H7 avian influenza virus subtypes could be directly transmitted from poultry to humans, in spite of having a-2,3 receptor specificity [11-13]. This observation encouraged investigators to * Correspondence: [email protected] †Contributed equally 1 Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain Full list of author information is available at the end of the article Costa et al.Veterinary Research 2012, 43:28 http://www.veterinaryresearch.org/content/43/1/28 VETERINARY RESEARCH © 2012 Costa et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. study the role of a-2,3 and a-2,6 receptors in the species barrier, and led to the description of a-2,3 receptors in the human lower respiratory tract, which may partially explain the localization and severity of H5N1-associated pneumonia in humans [14,15]. The expression of influenza receptors in avian and mammal species have been studied by means of lectin histochemistry, based on the binding affinity of Maackia amurensis agglutinin II (MAAII) for a-2,3 receptors [16], and of the plant-derived lectin Sambucus nigra agglutinin (SNA), which preferentially detects a-2,6 receptors [17]. The avian species that have been studied include the following: the chicken (Gallus gallus domesticus), common quail (Coturnix coturnix), Japanese quail (Coturnix japonica), bobwhite quail (Colinus virginianus), Chinese ring-necked pheasants (Phasianus colchicus), turkey (Meleagris gallopavo), pearl guinea fowl, Pekin duck (Anas platythynchos domestica), mallard (Anas platyrhynchos), Tolousse goose (Anser anser domesticus), black-headed gull (Larus ridibundus), mew gull (Larus canus), herring gull (Larus argentatus), domestic pigeon (Columba livia), common wood pigeon (Columba palumbus), dunlin (Calidris alpina), and common murre (Uria aalge) [7,18-26]. The presence of both the a-2,3 and a-2,6 receptors has been reported in some domestic avian species such as the chicken, bobwhite quail, turkey, Chinese ring neck pheasant, white midget turkey, Pearl guinea fowl, and Pekin duck [18,19,23,25,27]. However, the information available in these studies is generally restricted to a few tissues in the respiratory or intestinal tracts, and accompanied by a concise description of the pattern expression of influenza receptors. The expression of influenza virus receptors can also be evaluated with virus histochemistry to determine the pattern of viral attachment (PVA) [9]. Virus histochemistry is a binding assay based on the attachment of concentrated fluorescein-labeled virus on host cells, and visualized by routine immunohistochemical techniques. The PVA is a valuable tool to determine the affinity of a specific virus to certain species and host cells, which are critical factors for an effective infection [15]. Several recent studies have evaluated the PVA of influenza viruses in avian species [28], as well as in humans [15,29-31] and other mammal species [29]. However, current knowledge of the PVA in avian species is still very limited [28]. The combined use of both techniques, lectin histochemistry and virus histochemistry, could be useful to elucidate the role of domestic species in the transmission of influenza viruses and help to understand the evolutionary pressures exerted by different poultry species over influenza viruses, discerning for instance, why these viruses evolve faster in chickens and turkeys than in wild birds [32]. Furthermore, understanding viral and host barriers that prevent transmission may be critical in establishing rational control measures as well as predicting and stratifying risk for individual strains of influenza [33]. The present study extensively assessed the expression of a-2,3 and a-2,6 receptors in the respiratory (nasal cavity, trachea, and lung) and intestinal (duodenum, jejunumileum, cecum, and colon) tracts of seven domestic avian species. Furthermore, the PVA of an avian-origin H4N5 virus and a human-origin H1N1 influenza virus on respiratory and intestinal tracts was also evaluated. The species studied include the chicken, common quail, redlegged partridge (Alectoris rufa), turkey, golden pheasant (Chrysolophus pictus), ostrich (Struthio camelus), and mallard. These domestic species were selected because they are commonly held commercially for egg, meat, feather or leather production, or for ornamental purposes. The influence of the a-2,3 and a-2,6 receptor-distribution and PVA on the emergence and perpetuation of influenza viruses is discussed herein. Materials and methods Animals and tissues The seven domestic avian species included in this study were the following: chicken, common quail, red-legged partridge, turkey, golden pheasant, ostrich, and mallard. Three individuals of each species were used. Samples of the nasal cavity (including both middle and posterior turbinates), trachea, lung, duodenum, jejunum, ileum, cecum and colon were obtained from archival formalin-fixed paraffin-embedded tissues of the Veterinary Pathology Service of the Universitat Autònoma de Barcelona (Barcelona, Spain). Tissues free from any histopathological lesions were selected for this study. Human, pig and mice tissue samples were used as positive controls for the detection of a-2,3 and a-2,6 receptors. Human lung samples, obtained from an adult patient that died without previous pulmonary disease, were kindly provided by Hospital Universitari Vall d’Hebrón (Barcelona, Spain) in accordance with protocols approved by the Ethics Committee on Clinic Investigations of the Hospital. Respiratory and intestinal tracts of pigs and mice were obtained from animals that died without previous respiratory or digestive diseases, submitted for necropsy at the Veterinary Pathology Service of the Universitat Autònoma de Barcelona. This study was carried out in strict accordance with the recommendations of the Ethics Committee of Animal and Human Experimentation of the Universitat Autònoma de Barcelona. Lectin histochemistry Lectin histochemistry was performed as previously described [34] with minor modifications. Briefly, 3 μmthick sections were deparaffinized and treated with 3% H 2 O 2 in methanol to eliminate endogenous peroxidase Costa et al.Veterinary Research 2012, 43:28 http://www.veterinaryresearch.org/content/43/1/28 Page 2 of 13 activity, washed with Tris-NaCl-Tween buffer (TNT) (0.1 M Tris HCl, 0.15 M NaCl, pH 7.5), and blocked with TNB (TNT plus blocking reagent) (Perkin Elmer, US) for 30 min at room temperature (RT). Tissue sections were then incubated with biotinylated SNA (10 μg/mL) and MAAII (15 μg/mL) (Vector Laboratories Inc, CA, USA) in TNB at 4 °C, overnight. After washing with TNT, sections were incubated with streptavidin-horse radish peroxidase (SA-HRP) 1:100 for 1 h, followed by incubation with Tyramide Signal Amplification (TSA™Biotin System, Perkin Helmer, USA) at 1:50 in dilution Buffer, and again incubated with SA-HRP for 30 min at RT. The reaction was developed with diaminobenzidine (SigmaAldrich, MO, USA) at RT for 30 s followed by counterstaining with Mayer’s haematoxylin. The expression of the receptors was visible by light microscopy as brown staining. To rule out the non-specific binding of lectins, two sequential slides were used as negative controls. One slide was pretreated with neuraminidase (NA), which cleaves both a-2,3 and a-2,6 residues, as previously described [34]; and the other was incubated with phosphate buffered saline instead of the lectins. After careful examination of each slide, and in order to compare receptor expression patterns among the tissues and species included in this study, the relative intensity of receptor expression was scored based on the percentage of cells in a section showing positivity, and was graded as: negative (-); low (+), when 1% or more, but less than 10% of the cells were positive; moderate (++), when 10% or more, but less than 50% of the cells were positive; strong (+++), when 50% or more of the cells were positive. Photomicrography of the lectin histochemistry was taken using a Leica DM6000B microscope, Leica DFC480 digital camera, and Leica Application Suite software program. Virus histochemistry The attachment of influenza virus to epithelial cells in respiratoryanddigestivetracts was visualized by virus histochemistry as previously described [29]. With the exception of the chicken and mallard, where three individuals were included, one individual of each species was evaluated using the virus histochemistry technique. The viruses used were A/Mallard/Netherlands/13/08 (H4N5) and the seasonal A/Netherlands/35/05 (H1N1). Both viruses were prepared as previously described [29]. Briefly, the viruses were inoculated in chicken embryo chorioallantoic membrane (H4N5) or in Madin-Darby canine kidney cells (H1N1), and harvested two days later. Viruses were concentrated and purified by centrifugation on sucrose gradient, inactivated by dialysis against 0.1% formalin, and labeled with fluorescein isothiocyanate (FITC). Tissue sections were incubated with 50-100 hemagglutinating units per 50 μL of FITC-labeled virus. Attachment of virus was detected with a peroxidaselabeled anti-FITC antibody, and amplified with a tyramide signal amplification system. Peroxidase was revealed with 3-amino-9-ethyl-carbazole, which resulted in a bright red precipitate. Attachment of influenza viruses to tissues was visible by light microscopy as granular to diffuse red staining on the apical surface and in the cytoplasm of epithelial cells. As for the lectin histochemistry, the relative intensity of the viral attachment to epithelial cells was scored based on the percentage of cells in a section showing virus attachment, as follows: negative (-); low (+), when 1% or more, but less than 10% of the cells were positive; moderate (++), when 10% or more, but less than 50% of the cells were positive; strong (+++), when 50% or more of the cells were positive. Photomicrography of the virus histochemistry was taken using a Leica DM6000B microscope, Leica DFC480 digital camera, and Leica Application Suite software program. Results Lectin histochemistry The pattern of receptor expression in the control tissues used in this study (human lung, pig and mice respiratory and digestive tracts) was in agreement with previously published literature. The NA pretreatment, used as a control, removed all the binding sites for the SNA; however, very low levels of staining for MAAII remained after the NA treatment in connective tissue of the lamina propriaintherespiratoryandintestinal tracts of all the species studied, and was considered as non-specific staining (data not shown). Respiratory tract There was a marked variation on the distribution and expression of influenza receptors among the tissues and avian species studied. The distribution of influenza receptors in the respiratory tract is given in Table 1 and the results from nasal cavity and trachea are illustrated in Figures 1 and 2, respectively. Chicken In the nasal cavity, strong a-2,3 receptors were observed on respiratory ciliated epithelial cells as well as on olfactory epithelial cells, but were low on respiratory non-ciliated epithelial cells. In contrast, positivity for a2,6 receptors was moderate on respiratory ciliated epithelial cells and low on olfactory epithelial cells. Chicken trachea showed low levels of staining for both receptors on ciliated epithelial cells. The chicken lung manifested strong staining for both receptors in the bronchial epithelial cells, whereas the parabronchial epithelial cells were strongly positive for a-2,3 receptors only. Common quail In common quails, low levels of a-2,3 receptors were observed in the nasal cavity (respiratory epithelium and olfactory epithelial cells), trachea (ciliated Costa et al.Veterinary Research 2012, 43:28 http://www.veterinaryresearch.org/content/43/1/28 Page 3 of 13 epithelial cells) and lung (bronchial and parabronchial epithelial cells), whereas strong expression of a-2,6 receptors was observed in nasal, tracheal and bronchial epithelial cells. Expression of a-2,6 receptors on respiratory non-ciliated epithelial cells and olfactory epithelium was relatively moderate. Red-legged partridge The partridge respiratory tract showed moderate staining for a-2,3 receptors on respiratory ciliated epithelial cells and the olfactory epithelium, low staining on respiratory non-ciliated epithelial cells and tracheal ciliated epithelial cells, and negative staining in the lung. Likewise, expression of a-2,6 receptors was moderate on olfactory epithelial cells, low on the respiratory epithelium, and negative on tracheal and pulmonary epithelial cells. Turkey The turkey nasal cavity expressed moderate staining for both receptors on respiratory ciliated epithelial cells and olfactory epithelial cells, while low levels of staining for a-2,6 receptors was observed on non-ciliated epithelial cells. Tracheal ciliated epithelial cells showed strong expression of a-2,3 receptors and negative expression for a-2,6 receptors. Moderate positivity for a-2,3 receptors was observed on bronchial epithelial cells, while strong positivity for a-2,6 receptors was observed in bronchial and parabronchial epithelial cells. Golden pheasant Moderate a-2,3 receptor expression was observed on respiratory ciliated epithelial cells while low levels of a-2,3 receptor expression were observed on respiratory non-ciliated epithelial cells, olfactory epithelium and lung (bronchial and parabronchial epithelial cells). Similarly, moderate positivity for a-2,6 receptors was observed on respiratory ciliated epithelial cells, olfactory epithelial cells, and tracheal ciliated epithelial cells; while low levels of staining were observed on respiratory non-ciliated epithelial cells and bronchial epithelial cells. Ostrich Low levels of a-2,3 receptor expression were observed in the trachea (ciliated epithelial cells), lung (bronchial and parabronchial epithelial cells), as well as respiratory non-ciliated epithelial cells. Moderate expression of a-2,3 receptors was noticed on respiratory ciliated epithelial cells and olfactory epithelial cells. Interestingly, a-2,6 receptors were not expressed in any segment of the ostrich respiratory tract. The receptor expression was not determined on the ostrich nasal gland epithelium due to the lack of this structure on the samples evaluated. Mallard Moderate expression of a-2,3 receptors was observed in respiratory non-ciliated epithelial cells and tracheal ciliated epithelial cells; while low levels of expression of a-2,3 receptors was noted on nasal respiratory ciliated epithelial cells, and bronchial and parabronchial epithelial Table 1 Distribution of Sia a-2,3 Gal and Sia a-2,6 Gal receptors a in the upper and lower respiratory tracts of seven avian species. Tissue, cell type Species, receptor type b Chicken Common Quail Red-legged Partridge Turkey Golden Pheasant Ostrich Mallard a-2,3 a-2,6 a-2,3 a-2,6 a-2,3 a-2,6 a-2,3 a-2,6 a-2,3 a-2,6 a-2,3 a-2,6 a-2,3 a-2,6 Nasal Cavity Respiratory Epithelium Ciliated epithelial cells +++ ++ + +++ ++ + ++ ++ ++ ++ ++ - + + Non-ciliated epithelial cells + - + ++ + + - + + + + - ++ - Olfactory Epithelium Olfactory epithelial cells +++ + + ++ ++ ++ ++ ++ + ++ ++ - - - Bowman gland epithelium +++ + ++ ++ ++ + - + + - - - ++ + Adjacent structures Nasal gland epithelium + ++ ++ + + + ++ ++ ++ + nd nd ++ + Salivary gland epithelium ++ + +++ ++ + + ++ ++ ++ + +++ - ++ + Trachea Ciliated epithelial cells + + + +++ + - +++ - - ++ + - ++ ++ Goblet cells - + + + + - - + + + + - - ++ Mucous gland epithelium ++ + + + + + + + + + + - - - Lung Bronchial epithelial cells +++ +++ + +++ - - ++ +++ + + + - + + Parabronchial epithelial cells +++ - + - - - - +++ + - + - + - Air capillary cells - - + - - + - - - + + - - - a The distribution of a-2,3 and a-2,6 influenza receptors was evaluated using lectin immunohistochemistry. b -: negative; +: low; ++: moderate; +++: strong; nd: not determined. Costa et al.Veterinary Research 2012, 43:28 http://www.veterinaryresearch.org/content/43/1/28 Page 4 of 13 Figure 1 Influenza receptor distribution and pattern of viral attachment in the nasal cavity. Composite bright field microscope images comparing the distribution of a-2,3 and a-2,6 receptors, demonstrated by means of MAAII and SNA lectin histochemistry, with the pattern of viral attachment of the avian influenza A/Mallard/Netherlands/13/08 (H4N5) virus and the human influenza A/Netherlands/35/05 (H1N1) virus, demonstrated by means of virus histochemistry, in the nasal cavity of the chicken (A1-A4), common quail (B1-B4), red-legged partridge (C1-C4), turkey (D1-D4), golden pheasant (E1-E4), ostrich (F1-F4), and mallard (G1-G4). Costa et al.Veterinary Research 2012, 43:28 http://www.veterinaryresearch.org/content/43/1/28 Page 5 of 13 Figure 2 Influenza receptor distribution and pattern of viral attachment in the trachea. Composite bright field microscope images comparing the distribution of a-2,3 and a-2,6 receptors, demonstrated by means of MAAII and SNA lectin histochemistry, with the pattern of viral attachment of the avian influenza A/Mallard/Netherlands/13/08 (H4N5) virus and the human influenza A/Netherlands/35/05 (H1N1) virus, demonstrated by means of virus histochemistry, in the trachea of chicken (A1-A4), common quail (B1-B4), red-legged partridge (C1-C4), turkey (D1-D4), golden pheasant (E1-E4), ostrich (F1-F4), and mallard (G1-G4). Costa et al.Veterinary Research 2012, 43:28 http://www.veterinaryresearch.org/content/43/1/28 Page 6 of 13 cells. Moderate expression of a-2,6 receptors was observed in tracheal ciliated epithelial cells and low levels of a-2,6 receptor expression were observed in respiratory ciliated epithelial cells and bronchial epithelial cells. Intestinal tract The expression and distribution of influenza receptors in the intestinal tract is described in detail in Table 2 and the results from the large intestine are illustrated in Figure 3. Chicken Moderate positivity for a-2,3 receptors was observed on columnar epithelial cells of the small intestine (duodenum and jejunum-ileum), and low levels of staining were recorded on columnar epithelial cells from the large intestine (cecum and colon). Expression of a2,6 receptors was low on columnar epithelial cells of the jejunum-ileum and cecum. Common quail Moderate expression of a-2,3 receptors was observed on columnar epithelial cells from the large intestine, while low levels of expression of a-2,3 receptors were observed in columnar epithelial cells from the small intestine. Regarding the expression of a-2,6 receptors, a moderate staining was visualized in cecal columnar epithelial cells, and low levels of staining were present in columnar epithelial cells from the small intestine and colon. Red-legged partridge Both receptors were expressed in low levels in red-legged partridge intestinal tract. The partridge intestinal tract showed expression of a-2,3 receptors along the columnar epithelial cell from the jejunum-ileum, cecum and colon, while a-2,6 receptors were only observed in cecal columnar epithelial cells. Turkey Both receptors were expressed in low levels in turkey intestinal tract. Expression of a-2,3 receptors was noted on columnar epithelial cells from the small intestine and colon, while expression of a-2,6 was restricted to columnar epithelial cells from the large intestine. Golden pheasant Moderate expression of a-2,3 receptors was observed in columnar epithelial cells from the small and large intestines, while expression of a-2,6 receptors was low on columnar epithelial cells from the small and large intestines. Ostrich The a-2,3 receptors were predominant on the intestinal tract. The expression of a-2,3 receptors was low on columnar epithelium cells from the small intestine and cecum, while a-2,6 receptors were absent from intestinal epithelial cells. Mallard Strong expression of a-2,3 receptors was observed throughout the columnar epithelial cells from the small and large intestines. The a-2,6 receptors were not expressed in the intestinal tract of mallards. Virus histochemistry The expression of a-2,3 and a-2,6 receptors, as determined by lectin histochemistry, was compared with the PVA of avian-origin H4N5 and human-origin H1N1 influenza viruses in the respiratory tract (Table 3) and intestinal tract (Table 4) of the seven avian species used in this study. In order to facilitate the comparison between the lectin histochemistry and virus histochemistry, the lectin histochemistry results in these tables were summarized; a media of the scoring of the epithelia lining Table 2 Distribution of Sia a-2,3 Gal and Sia a-2,6 Gal receptors a in the intestinal tract of seven avian species. Tissues, cell type Species, receptor type b Chicken Common Quail Red-legged Partridge Turkey Golden Pheasant Ostrich Mallard a-2,3 a-2,6 a-2,3 a-2,6 a-2,3 a-2,6 a-2,3 a-2,6 a-2,3 a-2,6 a-2,3 a-2,6 a-2,3 a-2,6 Duodenum Columnar epithelial cells ++ - + + - - + - ++ + + - +++ - Goblet cells +++ - ++ + - + - - + + ++ - - - GALT lymphocytes - + - + + + + + + + - + + - Jejunum-Ileum Columnar epithelial cells ++ + + + + - + - ++ + + - +++ - Goblet cells +++ - ++ - - - + - + + ++ - - - Cecum Columnar epithelial cells + + ++ ++ + + - + ++ + + - +++ - Goblet cells - - - + - - - - + ++ ++ - - - GALT lymphocytes - ++ - + - + - + - + - + - - Colon Columnar epithelial cells + - ++ + + - + + ++ + - - +++ - Goblet cells - - - + - + - - - ++ ++ - - - GALT lymphocytes - ++ - - - - + - + + - + - - a The distribution of a-2,3 and a-2,6 influenza receptors was done using lectin immunohistochemistry. b -: negative; +: low; ++: moderate; +++: strong. Costa et al.Veterinary Research 2012, 43:28 http://www.veterinaryresearch.org/content/43/1/28 Page 7 of 13 Figure 3 Influenza receptor distribution and pattern of viral attachment in the large intestine. Composite bright field microscope images comparing the distribution of a-2,3 and a-2,6 receptors, demonstrated by means of MAAII and SNA lectin histochemistry, with the pattern of viral attachment of the avian influenza A/Mallard/Netherlands/13/08 (H4N5) virus and the human influenza A/Netherlands/35/05 (H1N1) virus, demonstrated by means of virus histochemistry, in the large intestine of the chicken (A1-A4), common quail (B1-B4), red-legged partridge (C1C4), turkey (D1-D4), golden pheasant (E1-E4), ostrich (F1-F4), and mallard (G1-G4). Costa et al.Veterinary Research 2012, 43:28 http://www.veterinaryresearch.org/content/43/1/28 Page 8 of 13 (ciliated and non-ciliated epithelial cells) and goblet cells of the different tissues from the respiratory and intestinal tracts was calculated and expressed as follows: nasal cavity (nasal turbinates and infraorbital sinuses), trachea, lung, small intestine, and large intestine. It is remarkable that the H1N1 virus did not attach to the respiratory tract of the ostrich, nor did the H4N5 virus on the intestinal tract of turkeys. The results obtained with the receptor expression and the PVA were in general comparatively consistent among the different tissues and avian species studied (Tables 3 and 4, Figures 1, 2 and 3). With the exception of several cases, the avian-origin A/Mallard/ Netherlands/13/08 (H4N5) virus bound to tissues where a-2,3 receptors were observed, and the human-origin A/ Netherlands/35/05 (H1N1) virus bound to tissues where a-2,6 receptors were observed. In the respiratory tract of all the species evaluated, the attachment of A/Mallard/ Netherlands/13/08 (H4N5) was more evident than the attachment of A/Netherlands/35/05 (H1N1), particularly in the nasal cavity and trachea (Table 3 Figures 1 and 2). However, this tendency was not observed for the intestinal tract (Table 4 Figure 3). Minor inconsistencies between lectin histochemistry and PVA results were detected, and consisted in one grade of scoring. In some cases, there was viral attachment despite the absence of receptor expression, as observed in the partridge lung for a-2,3/H4N5 (Table 3) and in turkey, ostrich, and mallard small intestine for a-2,6/H1N1 (Table 4); in all these cases, the tissue was negative for the presence of the receptor but showed a PVA graded as low. Conversely, in other cases there was no viral attachment despite the presence of receptor expression, as that occurring for a-2,6/ H1N1 in the mallard nasal cavity, turkey trachea, and partridge, pheasant and mallard lungs (Table 3); as well as turkey small and large intestines for a-2,3/H4N5, and partridge large intestine for a-2,6/H1N1 (Table 4). In these cases, the tissue showed low expression of the receptor, but was negative for the PVA. Discussion In this study, a lectin histochemistry technique was used to extensively assess the expression of a-2,3 and a-2,6 receptors in the respiratory and intestinal tracts of seven domestic avian species. The staining for these two types Table 3 Influenza receptor distribution a and pattern of viral attachment b in the respiratory tract c of different avian species. Tissues, types of receptor/virus binding d Species Nasal Cavity e Trachea Lung a-2,3 H4N5 a-2,6 H1N1 a-2,3 H4N5 a-2,6 H1N1 a-2,3 H4N5 a-2,6 H1N1 Chicken ++ ++ f + ++ + ++ + + +++ + ++ + Quail + + ++ ++ ++ ++ ++ + + ++ g ++ Partridge ++ ++ + + + ++ - - - + + - Turkey + ++ ++ + ++ ++ + - + + +++ + Pheasant + +++ ++ + + +++ ++ + + + + - Ostrich ++ +++ - - + ++ - - + + - - Mallard + + + - + +++ ++ + + ++ g +- a The distribution of a-2,3 and a-2,6 influenza receptors was done using lectin immunohistochemistry. b The pattern of viral attachment was performed using virus histochemistry and the A/Mallard/Netherlands/13/08 (H4N5) and A/Netherlands/35/05 (H1N1) viruses. c Media of the results obtained in the epithelia lining (ciliated and non-ciliated epithelial cells and goblet cells). d -: negative; +: low; ++: moderate; +++: strong. e ’Nasal Cavity’includes the following: nasal turbinates and infraorbital sinuses. f Virus binding mainly in infraorbital sinuses. g Virus binding mainly in bronchi. Table 4 Influenza receptor distribution a and pattern of viral attachment b in the intestinal tract c of different avian species. Species Tissues, types of receptor/virus binding d Small Intestine Large Intestine a-2,3 H4N5 a-2,6 H1N1 a-2,3 H4N5 a-2,6 H1N1 Chicken +++ ++ + + + ++ + + Quail ++++++++++ Partridge +++++++ - Turkey +- -++-++ Pheasant ++ + + + ++ +++ ++ + Ostrich ++ + - + ++ + - - Mallard ++ + - + ++ + - - a The distribution of a-2,3 and a-2,6 influenza receptors was evaluated using lectin immunohistochemistry. b The pattern of viral attachment was performed using virus histochemistry and the A/Mallard/Netherlands/13/08 (H4N5) and A/Netherlands/35/05 (H1N1) viruses. c Media of the results obtained in the epithelia lining (columnar epithelial cells and goblet cells). d -: negative; +: low; ++: moderate; +++: strong. Costa et al.Veterinary Research 2012, 43:28 http://www.veterinaryresearch.org/content/43/1/28 Page 9 of 13