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Induction of mucosal immunity in poultry after oral immunization with a single dose of microencapsulated eds-76 virus Dandapat S.*, Banerjee D., Rajan, Kataria J.M., Ram G.C.1, Yadav M.P. Director's Laboratory Indian Veterinary Research Institute, Izatnagar-243 122 (U.P.) *Corresponding author
1Immunology Section
Abstract Formalin inactivated EDS-76 virus was encapsulated in the poly-lactide-co-glycolide (PLG) microspheres by solvent evaporation technique. White Leghorn layer chicks were orally immunized with a single dose of 75 µg of the microencapsulated EDS-76 virus and the immune responses induced at mucosal sites were evaluated. In the intestinal and tracheal washings of the immunized chicks, considerably higher levels of virus specific IgA were detected with the peak titres of 30 and 24, respectively at 3 wk PI as compared to the conventional inactivated EDS-76 vaccine which showed very negligible IgA level (titre <2.0). The cell-mediated immune responses induced at mucosal sites were evaluated by assessing the lymphoproliferative and cytotoxic activities of the intestinal intraepithelial lymphocytes (iIEL). It was found that the lymphoproliferative responses induced by microencapsulated EDS-76 virus were significantly higher (p<0.05) with a peak stimulation index value (1.314 ± 0.022) at 3rd wk PI as compared to the microsphere alone (control) and the conventional EDS-76 vaccine. The cytotoxic activities were also induced by the iIEL after oral delivery of microspheres containing the EDS-76 virus. Top | Most of the viruses including egg drop syndrome (EDS)-76 virus of poultry gain access to the host through the mucosal tissues. Administration of antigens by parenteral injection induces systemic immune responses, but does not efficiently induce immunity at mucosal sites. This fact has posed new challenges in the development of mucosal vaccines for induction of local immunity, which acts as the first line of defence. It is reported that there is significant contribution from the local T-cells in the induction of locally produced IgA antibodies, rather than serum antibodies and induction of such responses requires direct mucosal immunization (Mestecky, 1987). |
Oral vaccines have the attractions for no side effects and their ease of administration. There is also potential to incorporate the vaccines into feed and water, which would reduce the cost of immunization particularly in case of large organized poultry flocks. However, oral immunization with soluble antigens has largely been ineffective because of antigen degradation by acids and lytic enzymes in the gut (Mestecky et al., 1978). For effective mucosal immunization a protective vehicle for delivering the vaccine antigen is very essential. |
Among the various methods for delivering vaccines, microparticle based antigen delivery system has emerged as a potential tool for successful elicitation of mucosal immune responses. Microparticles with a diameter 10 µm or less are taken up by the M-cells in the Payer's patches of small intestine (Eldridge et al., 1990; Beier and Gebert, 1998). There are many studies on mucosal immunization using microspheres made from poly-lactide-co-glycolide (PLG), which resulted in induction of strong mucosal as well as systemic immunity (Challacombe et al., 1992 & 1997; ’Hagan et al., 1994; Greenway et al., 1998). Because of their biodegradability, biocompatibility and essential attributes of a good mucosal adjuvant, PLG microspheres have been considered to be superior to many conventional mucosal adjuvants (Eldridge et al., 1991). |
Poultry industry faces a stiff challenge from the threats posed by the infectious agents such as viruses whose portal of entry is almost invariably the exposed mucosal surfaces. EDS-76 virus is one of such infectious agents having faeco-oral route of entry and causing drops in the egg production to the extent ranging from 10-50% (McFerran et al., 1977; Mohanty et al., 1984; Kumar et al., 1992). Thus, EDS-76 is not only an economically important disease of poultry but also serves as a good model for studies on mucosal immunization. Hence, in the present study the polymer microspheres (poly-lactide-co-glycolide) has been used for mucosal (oral) delivery of EDS-76 virus in poultry with an objective to determine whether oral immunization can induce virus specific immune responses induced at local/mucosal sites. |
Top Materials and Methods Preparation of viral antigen The EDS-76 virus (a field isolate) of chicken origin at 3rd passage level, obtained from the Division of Avian Diseases, IVRI, Izatnagar, U. P., was propagated in the 13-day-old, susceptible embryonated duck eggs through intra-allantoic route. The HA titre of EDS-76 virus in the infected allantoic fluid was determined by performing haemagglutination test as per the method described by Higashihara et al. (1983). The infectivity titre was determined by inoculating serial 10-fold dilutions of virus material in duck embryos. The end point titre was expressed as 50 per cent embryo infective dose (EID50) per ml, as calculated by the method of Reed and Muench (1938). Inactivation of virus was carried out as described by Kumar et al. (1988) by treatment with 0.4% formalin at 37°C overnight. Then the virus was concentrated by direct pelleting at 1,00,000x g for 1.5 h using fixed angle rotor in the ultracentrifuge (Sorval Pro-80). Experimental chicks Day-old White Leghorn chicks (Strain-H) procured from Central Avian Research Institute (CARI), Izatnagar were used for immunization experiments after obtaining approval of the Animal Ethics Committee. The chicks were kept initially in deep litter system, then transferred to battery cages and reared under standard managemental practices with feed and water ad libitum. Preparation of PLG microspheres entrapped with EDS-76 virus Microspheres entrapped with EDS-76 virus were prepared by solvent evaporation technique as described by Greenway et al. (1998) with some modifications. Briefly, a 6% (w/v) solution of the polymer, i.e., poly-lactide-co-glycolide (PLG; molar ratio 50:50, MW 40,000-75000, Sigma chemicals, USA) in the organic solvent, dichloromethane (DCM; Sigma, USA) was emulsified with an aqueous solution of EDS-76 virus (10mg/ml) at 10000 rpm for 6 min using a homogenizer (PRO-260, HAVARD Apparatus, U.K) to produce a water-in-oil-in-water (w/o/w) emulsion. The emulsion was then added to an aqueous solution (2% w/v) of polyvinyl alcohol (PVA, 88% hydrolyzed, Aldrich Chemical Company, USA) and again homogenized in order to produce a stable water-in-oil-in-water (w/o/w) double emulsion. This emulsion was then stirred overnight after adding sufficient distilled water to allow solvent evaporation, with resultant microparticles formation. The microparticles were then collected by centrifugation at 10,000 rpm for 10 min, washed three times in distilled water and then freeze dried immediately and stored at 4°C for further use. The empty microspheres without the viral antigen were also prepared following the same protocol as described above. Immunization of chicks A total of 120 White Leghorn day-old chicks were randomly divided into 3 groups having 40 chicks in each group and immunization of chicks of different groups at 10th day of age was done as follows:
Group-I (O) : | = | Microencapsulated EDS-76 virus at the dose of 75 µg/chick, through oral route. |
Group-II (CV) : | = | Inactivated conventional EDS-76 vaccine through intramuscular route. |
Group-III (C) : | = | Unvaccinated control, sham-immunized with empty microspheres by oral route. |
Top Assessment of virus specific mucosal immune responses Humoral immune response The levels of virus specific IgA at the local mucosal site (i.e., in the intestinal secretion) and distant mucosal site (tracheal washings) of different groups of chicks were assessed at weekly intervals up to 4-wk post immunization (PI).
Collection of intestinal and tracheal secretions
Samples of intestinal and tracheal washings were separately collected from three randomly selected chicks of each group at weekly intervals post-immunization after sacrificing the chicks. The intestinal washings were collected as per the method described by Eldridge et al. (1991) and tracheal secretions were collected as per the method of Hawkes et al. (1983). Assessment of virus specific IgA levels by indirect ELISA
The virus specific IgA antibody levels in the pooled samples of intestinal and tracheal secretions were assessed by ELISA as per the method described by Mukiibi-Muka and Jones (1999) and Loa et al. (2002). The ELISA plates were coated with purified EDS-76 virus antigen (2 µg/well). After giving 3 washings, different 2-fold serial dilutions of intestinal and tracheal washings were added in triplicate wells to separate plates (50 µl/well) and then incubated at 37°C for 2 h. The IgA was detected by adding goat anti-chicken IgA-HRPO conjugate (Serotec, U.K.) at a dilution of 1:20,000 (50 µl/well). The O.D. values of the test samples, which were above 0.1 than the mean OD of the secretions taken from healthy control chicks, were considered for determining the titre.
Cell mediated immune responses The cell mediated immunity was determined by assessing the mean lymphoproliferative and cytotoxic activity of the intestinal intraepithelial lymphocytes (iIELs), collected from three randomly selected chicks of each group at weekly intervals.
Isolation of intestinal intraepithelial cells (iIELs)
Chicken iIELs were isolated as per the techniques described by Chai and Lillehoj (1988) and Hoggenmueller (1993) with slight modifications. Briefly, the intestinal tract from duodenum to illeal loop was cut open longitudinally and made into small pieces of 1cm long. After giving washings in chilled PBS, these intestinal pieces were treated with pre-warm DTT solution (5mM) in serum free RPMI medium at 41°C for 10 min. Then the tissue segments were washed twice with RPMI medium and the cleaned pieces were treated with pre-warmed (41°C) 1mM EDTA (in RPMI medium) by gently stirring for 20 min. The supernatant was collected and cells were pelleted by centrifugation at 1600 rpm for 10 min. Final cell pellet was suspended in 3ml of RPMI medium and passed through a Nylon wool column (soaked with medium). The filtrate was collected and centrifuged at 1200 rpm for 10 min. and lymphocytes were further purified by density gradient centrifugation using histopaque-1077 (Sigma). The final cell concentration was adjusted to 5x106 cells per ml. The intestinal segments before and after EDTA treatment were fixed and subjected to histological examination. Blastogenic response of iIELs
Blastogenic or proliferative response of iIELs to mitogen (Con-A) and virus antigen (EDS-76) was measured by MTT colorimetric assay as described by Lillehoj (1989) and Agrawal and Reynolds (1999). Briefly, in triplicate wells, 100 µl of iIEL suspension (5x106 cells per ml) was dispensed and then another 100 µl of growth medium containing either Con-A (25 µg/ml) for positive control or viral antigen (5x106.0 EID50/ml) was added. For negative control growth medium was added without any mitogen or antigen. After 96 h of incubation, MTT solution (25 µl/well) was added and further incubated for 4 h. Finally, the intensity of colour development was assessed by taking O.D. at 550 nm and the stimulation index was calculated. Cytotoxicity assay of iIELs
Cytotoxicity assay of iIELs was performed according to the method described by Mosmann (1983) and Espevik & Nissen-Meyer (1986) with slight modifications. Briefly, the vero cells (target cells) suspension in RPMI growth medium (2x105 cells/ml) was first dispensed into the wells (100 µl/well) of a flat bottom 96 wells tissue culture plate and incubated at 37°C in a CO2 incubator. The non-adherent iIELs were used as effectors cells (E) at the effector and target ratio (E:T) of 50:1. After discarding the supernatant medium from the wells with vero cell monolayer, 100 µl of effector cells from each donor chick was added to triplicate wells. Appropriate controls were also taken, i.e. for 0% lysis, no effector cells were added and for 100% lysis control, 100 µl of 10% Triton X-100 in growth medium was added. Similarly, effector cell control was taken with no target cell. While taking OD in the microplate reader, 100% lysis control wells were taken as blank. The percentage of cytotoxicity was calculated.
Top Results Viral antigen The EDS-76 virus, obtained at 3rd passage level initially showing the HA titre of 26 was given further 7 passages in duck embryos. At the end of the 7th passage the virus infected allantoic fluid showed HA titre was 216 and the infectivity titre of 1.5 x 107 EID50/ml. Top Assessment of mucosal immune responses Virus specific IgA response The levels of virus specific IgA in the intestinal and tracheal washings of the immunized chicks were assessed at different intervals and the kinetics of IgA titres are shown in Fig. 1 and 2, respectively. The IgA antibody response in the intestinal washings of group-I (oral) was considerably higher than that of group-II (conventional) and the unvaccinated control. In group-I, the IgA titres were 16 and 30 at 2nd and 3rd week PI, respectively, which was slightly declined to 24 at 4th wk PI (Fig. 1). The conventional group (II) did not show any considerable IgA titre. In the tracheal washings also, higher levels of IgA antibody were found in group-I with a peak titre of 24 at 3rd wk PI, as compared to the control group and group-II, which received the conventional EDS-76 vaccine (Fig. 2).
Cell mediated immune response The proliferative response as well as cytotoxic activity of the intestinal intraepithelial lymphocytes was investigated at different intervals post immunization. Intestinal IELs were isolated from the intestine of the chicks by DTT and EDTA treatment. Removal of iIELs from the mucosal epithelium was revealed by the appearance of broken epithelium on histological examination of the section of intestinal segments after DTT-EDTA treatments (Fig. 3B), whereas the intestinal section before treatment showed intact epithelium (Fig. 3A).
Blastogenic response of iIELs
The blastogenic responses of the iIELs of different groups of chicks were assessed by MTT colorimetric method. The mean stimulation indices (S.I.) with standard errors are shown in Fig. 4. It was found that the group-I (oral) showed significantly higher proliferative responses (p<0.05) as compared to the unvaccinated control (group-III), showing the peak value of mean S.I. as (1.314 ± 0.022) at 3rd wk PI. Cytotoxic activity of iIELs
The mean percentage of cytotoxic activity of iIELs of different groups at different intervals PI has been shown in Table-1. It was found that in group-I (oral) the cytotoxicity was 68.72 %, which was significantly higher (p< 0.05) as compared to the unvaccinated control, which showed cytotoxicity up to 47.38% at 2nd wk PI. The conventional vaccine showed a moderate cytotoxicity of 51.5% at 3rd wk PI.
Top Discussion The present study demonstrates that both IgA response as well as cell mediated immunity at mucosal sites were induced effectively by the virus loaded microspheres even without use of other adjuvants. On the other hand, intramuscular injection of the oil adjuvanted killed EDS-76 vaccine could not induce any marked level of IgA response at mucosal sites. This indicated that parenteral administration of antigen may be efficient in inducing systemic immune response, but it is not a suitable approach to address mucosal immunity. Hence, it can be assumed that after oral delivery there might be direct uptake of the microspheres by the antigen presenting cells (APC) and constant release of antigens in the gut associated lymphoid tissue (GALT) due to which a satisfactory level of IgA could be elicited at the inductive site (intestine) as well as at the distant sites (respiratory tract) indicating the existence of a common mucosal immune system. Enhanced IgA responses have also been reported by other workers (Challacombe et al., 1992; O'Hagan et al., 1994) after oral immunization with antigens incorporated in PLG microparticles, and the existence of common mucosal system has also been reported (Walker, 1994; Challacombe et al., 1997). |
It was also found that the IgA responses were comparatively stronger at nearby (inductive) sites and weaker at the distant mucosal effector sites. This finding correlated with that of Moldoveanu et al. (1992) and Greenway et al. (1998). The IgA response at mucosal sites is one of the essential features of protective immunity as the IgA plays major role in prevention of adherence of microorganisms to mucosal surfaces (Walker et al., 1972; Williams and Gibbons, 1972) and also causes neutralization of viruses (Waldmann et al., 1986). The intestinal intraepithelial lymphocytes of immunized chicks showed higher proliferative response as compared to the control. The iIELs also exhibited cytotoxicity against the vero cell target. This NK-cell like activity of iIEL might be due to the release of cytokines like IFN-γ and IL-2 after antigen stimulation. The finding was in agreement with other workers (Challacombe et al., 1997 and Mutwiri et al., 2002), who had found CTL response to the antigen delivered by microparticles. |
The present study shows that a single dose of killed EDS-76 virus loaded microspheres in comparison to the conventional EDS-76 vaccine elicited significantly higher IgA as well as cell mediated mucosal immune responses. Perusal of the available literature revealed no report on mucosal immunization against EDS-76 virus using PLG microspheres. Our findings indicate that oral immunization against egg drop syndrome would help in combating infection of mucosae, i.e., the invasion sites more effectively. Further studies are advocated to consider the efficacy of microencapsulated antigen as a novel mucosal immuno-prophylactic agent to replace the conventional vaccine for EDS in particular and also for other poultry diseases in general. |
Top Figures | Fig. 1: Kinetics of IgA responses in the intestinal washings of the immunized chicks at different period post immunization as measured by indirect ELISA.
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| | Fig. 2: Kinetics of IgA responses in the tracheal washings of the immunized chicks at different period post immunization as measured by indirect ELISA.
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| | Fig. 3.(A): Histological appearance of small intestine (duodenum) showing intact epithelium before DTT and EDTA treatment (H & E stain, 160 X)
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| | Fig. 3.(B): Histological appearance of small intestine (duodenum) showing broken epithelium after DTT and EDTA treatment (H & E stain, 160 X)
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| | Fig. 4: Kinetics of blastogenic response of the intestinal intraepithelial lymphocytes (iIEL) at different period post immunization.
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Table | Table 1: Cytotoxic activity of chicken intestinal intraepithelial lymphocytes (iIELs) at different period post immunization.
| | Group of chicks | Percent cytotoxicity (Mean ± SE)* at different period PI |
| | 1 wk | 2 wk | 3 wk | 4 wk |
| | Group-I (O) | 59.36 ± 0.724 | 68.72 ± 0.382 | 62 ± 0.661 | 58.1 ± 0.422 | | Group-II (CV) | 48.23 ± 0.559 | 47.38 ± 0.426 | 51.5 ± 0.662 | 46.6 ± 0.4 | | Group-III (C) | 41.72 ± 0.638 | 44.32 ± 0.437 | 48.28 ± 0.811 | 42.42 ± 0.522 |
| *:Cytotoxicity was assessed by MTT dye reduction at Effector (E) : Target (T) ratio of 50:1. | | -Each value represents mean percentage of cytotoxicity of iIELs of three chicks (n = 3). | | Group-I (O): chicks were immunized orally with microspheres containing EDS-76 virus; Group-II (CV): chicks received conventional inactivated EDS-76 vaccine and Group-III (C): unvaccinated control chicks. |
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| Acknowledgements The authors thank the Director, IVRI, Izatnagar for valuable suggestions and providing infrastructural facilities. This research work was financially supported by the ICAR under National Agricultural Technology Programme (NATP) with the project code number 28 (1)/2000-NATP/CGP-II/220 dated 25.5.2001. Top References | | AgrawalP.K., ReynoldsD.L.
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