Antibody isotype profiles in serum and circulating antibody-secreting cells following mucosal and peripheral immunisations of sheep

The isotype-specific antibody responses of sheep immunised with keyhole limpet hemocyanin by a peripheral route (intramuscular (i.m.) injection) were compared to those induced by immunisation via different mucosal routes: (1) intra-nasal spray; (2) rectal deposition with cholera toxin; (3) injection into the mucosa of the small intestine or rectum. Antigen-specific IgG1 antibodies were induced in the i.m., intra-intestinal and intra-rectal injection groups and in a proportion of the cholera toxin immunised sheep, but not in the intra-nasal immunisation group. IgA was the only antibody isotype detected in serum collected from the intra-nasal immunisation group. No significant differences in serum IgA levels were detected in any of the mucosal immunisation groups as compared to the i.m. injection group. In contrast, analysis of the in vitro antibody profiles secreted by circulating antibody-secreting cells (ASC) revealed significantly higher IgA responses in the supernatants from all mucosal immunisation groups. This suggests that the measurement of antibodies secreted by circulating ASCs may be a better correlate of local mucosal responses in ruminants, as has been previously demonstrated in human studies. In addition to IgG1 and IgA responses, immunisation by direct injection of antigen formulations into the intestinal and rectal mucosa were the only groups to induce consistently high IgG2 antibodies in serum and ASC cultures.     

Assessment of T-dependent and T-independent immune responses in cattle using a B cell ELISPOT assay

Understanding the mechanisms that maintain protective antibody levels after immunisation is important for vaccine design. In this study, we have determined the kinetics of plasma and memory B cells detectable in the blood of cattle immunised with model T-dependent or T-independent antigens. Immunisation with the T-D antigen resulted in an expansion of TNP-specific plasma cells post-TNP primary and booster immunisations, which was associated with increased titres of TNP-specific IgG antibodies. Although no TNP-specific memory B cells were detected in the T-D group following the primary immunisation, we detected an increase in the number of TNP-specific memory B cells post-TNP boost. In contrast, no TNP-specific plasma or memory B cells were detected after primary or secondary immunisation with the T-I antigen. We then investigated if immunisation with a third party antigen (tetanus toxin fragment C, TTC) would result in a bystander stimulation and increase the number of TNP-specific plasma and memory B cells in the T-D and/or T-I group. TTC immunisation in the T-D group resulted in a small increase in the number of TNP-specific plasma cells post-TTC primary immunisation and boost, and in an increase in the number of TNP-specific memory B cells post-TTC boost. This bystander effect was not observed in the animals previously immunised with the T-I antigen. In conclusion, the present study characterised for the first time the B cell response in cattle to immunisation with T-D and T-I antigens and showed that bystander stimulation of an established T-D B cell memory response may occur in cattle.     

Humoral immunity in the ewe. 1. The influence of adjuvancy and immunogenic substitution on immune reactivity following immunisation with protein antigens

The nature of antigen and presence of adjuvant are major factors which influence the level of immune reactivity following immunisation. This study examines the quantity and isotype of antibody produced in ewes immunised with different proteins in combination with different adjuvants. Results using indirect ELISA assays show that animals immunised with keyhole limpet haemocyanin (KLH) in adjuvant produced lower levels (P less than 0.05) of immunoglobulin M (IgM) and had increased levels and persistence of immunoglobulin G1 (IgG1) compared with ewes immunised with antigen in saline (P less than 0.005). The anti-KLH immunoglobulin G2 (IgG2) titre was significantly higher in animals given oil-emulsion adjuvant than all other groups (P less than 0.005). Ewes were also immunised with bovine serum albumin (BSA) or BSA haptenated with trinitrophenyl (TNP) for the primary injection and carrier BSA alone for the secondary inoculation. Chemical haptenation of BSA antigen reduced primary IgM (P less than 0.005), IgG1 (P less than 0.005) and IgG2 (P less than 0.005) levels compared with animals immunised with pure BSA. There was an increased secondary anti-BSA IgM response in all animals first immunised with TNP-BSA (P less than 0.05). The class of anti-hapten antibody produced to TNP determinants was influenced by the degree of TNP haptenation of the carrier BSA. Mid-range molar ratios of TNP produced the strongest IgM, IgG1 and IgG2 anti-TNP responses compared with all other groups (P less than 0.01).     

Protective immunity following immunisation of pigs with aerosol of Actinobacillus pleuropneumoniae serotype 2

To introduce antigen to the respiratory mucosa, killed Actinobacillus pleuropneumoniae with quil A as adjuvant was administered to pigs as an aerosol. Immunisation by this aerosol induced a marked IgA response in the bronchoalveolar and nasal fluids, and in the serum. Following challenge with live bacteria two weeks after the last exposure to the aerosol, the immunised pigs were protected from the severe pleuropneumonia which developed in non-immunised pigs. The immunised pigs had lower antibody titres in the mucosal fluids and serum after exposure to the challenge. The immune response after experimental infection of non-immunised animals was a weak IgA antibody response in the bronchoalveolar and nasal fluids, whereas the systemic immune response after challenge included both IgA and IgG antibodies.     

Effect of transcutaneous immunization with co-administered antigen and cholera toxin on systemic and mucosal antibody responses in sheep

Direct application of antigens to skin together with an adjuvant, a procedure called transcutaneous immunization (TCI), can induce systemic immune responses in mice, humans, cats and dogs. In previous studies we found that cholera toxin (CT) applied topically on unbroken skin induces systemic antibody and lymphocyte proliferative responses in sheep. The current study examined whether concurrent administration of CT and tetanus toxoid (TT) delivered transcutaneously could induce specific antibody responses to both antigens in sheep. Antibodies to both TT and CT were induced by TCI although antibody titres in serum to TT were higher in sheep receiving TT plus alum by intramuscular injection (n=5) than TT plus CT by TCI (n=5). The ratio of IgG1/IgG2 antibody to TT in serum was near unity, and the route of immunization, TCI versus injection, did not influence this ratio. In contrast, the ratio of IgG1/IgG2 antibody differed significantly between the two antigens, TT and CT, delivered by TCI, with a higher proportion of IgG1 antibody in serum to CT than TT. Antibody to TT was detected in lung washes from TCI and injection groups, with IgG1 predominating over IgG2 in both groups. IgA antibodies to CT and TT were detected in sera of CT and TT-immunized groups respectively but in lung washes IgA antibody to TT was detected only in the injection group. Results show that TCI induced systemic antibody responses to CT and the co-administered antigen TT, whereas no evidence was obtained for mucosal IgA responses following TCI.     

A peptide vaccine administered transcutaneously together with cholera toxin elicits potent neutralising anti-FMDV antibody responses

In this study a synthetic peptide representing residues 141-159 from the GH loop of VP1 protein of foot-and-mouth disease virus was tested for its capacity to elicit virus neutralising antibodies in mice after transcutaneous immunisation. Topical application of the peptide conjugated to bovine serum albumin together with cholera toxin as an adjuvant elicited anti-peptide antibody responses with strong virus neutralising activity. The combination of cholera toxin with an immunostimulatory CpG motif resulted in the induction of IgG1 and IgG2a anti-peptide antibodies with significantly enhanced virus neutralising activity. To shed more light on the mechanisms of cholera toxin adjuvanticity we demonstrated its binding to keratinocytes via GM(1)-gangliosides. This was followed by an increase of the intracellular cAMP and the rapid diffusion of cholera toxin throughout the epidermis. These findings demonstrate that peptide-based vaccines when combined with the appropriate adjuvant(s) can elicit potent virus neutralising antibody responses after transcutaneous immunisation. However, experiments in target species will be required to confirm the potential of this simple vaccination procedure in livestock.     

Adjuvant effect of ginseng extracts on the immune responses to immunisation against Staphylococcus aureus in dairy cattle

A crude ginseng extract (GS) and the purified ginsenoside R(b1) (R(b1)) were evaluated for their adjuvant effects in dairy cattle at immunisation with ovalbumin (OVA) and/or a Staphylococcus aureus bacterin used for prevention of bovine mastitis. To evaluate a suitable dose of GS as an adjuvant, 36 lactating cows were randomly divided into six groups. The cows were inoculated twice intramuscularly with a 2-week interval, with saline solution, OVA in saline, or OVA in combination with 4, 16 or 64 mg GS, or Al(OH)(3). The level of specific antibodies to OVA in serum and milk whey was measured before immunisations and 1-5 weeks after the second immunisation. The antibody response in serum was significantly higher in animals immunised with OVA and GS than in animals immunised with OVA alone. A significant increase in milk antibody titres compared with OVA only was only found 2 weeks after the second immunisation in the group immunised with OVA and 4 mg GS. In the second part of the study, 18 heifers were randomly divided into three groups and were immunised twice intramuscularly with a two week interval, with the S. aureus bacterin (control), or with the bacterin in combination with 4 mg GS or 1mg R(b1). The specific antibody response to S. aureus and the lymphocyte proliferation after stimulation with PWM, concanavalin A (Con A) or a specific S. aureus antigen was evaluated in blood samples taken before and after immunisations as specified above. Addition of R(b1) resulted both in significantly higher antibody production and lymphocyte proliferation in response to PWM, Con A and S. aureus antigens than in the control group. Addition of GS induced a significantly higher lymphocyte proliferation in response to PWM and Con A than the control, but had no additional effect on the antibody production. In conclusion, both GS and R(b1) were safe adjuvants, and R(b1) had the strongest adjuvant effects, when used for immunisation against S. aureus in dairy cattle. Field trials are warranted to test the ability of GS and R(b1) to enhance the efficacy of mastitis vaccines in protection against intramammary infections.     

IgA immune responses in the respiratory tract of pigs

Experiments were conducted in a group of pigs to determine the ontogeny of antigen specific IgA in the trachea. The results showed that following intraperitoneal immunisation with ovalbumin (OVA) there were antigen specific IgG and IgA responses in both serum and respiratory tract secretion (RTS). After intratracheal challenge the IgA response in RTS was greater than that in serum as judged by the IgA/IgG ELISA ratios. Furthermore, following intratracheal challenge the IgA/IgG RTS ratio remained elevated for about 30 days. At slaughter, the anti-OVA containing-cell (AOCC) response and the corresponding immunoglobulin class were assessed using double fluorochrome labelling techniques. Pigs challenged intratracheally on four occasions between 11 and 67 days after intraperitoneal immunisation had a population of AOCC in the trachea which was 49 +/- 15 per cent IgA. In contrast, groups of pigs intraperitoneally immunised but challenged intratracheally on fewer occasions had significantly lower proportions of AOCC that were IgA. In conjunction with previous findings these results suggest that following intraperitoneal immunisation regular antigenic challenge of the trachea over an extended period leads to an increase in the proportion of AOCC which are IgA. These results are discussed in terms of the mechanisms involved in protecting the respiratory tract.     

Specific antibody-containing cells in the mammary gland of non-lactating sheep after intraperitoneal and intramammary immunisation

Ewes were immunised intraperitoneally with ovalbumin and Brucella abortus in Freund's complete adjuvant, followed seven days later by intramammary immunisation in which ovalbumin was presented to one mammary gland and Brucella abortus to the other. Mammary tissue taken after a further seven days contained more antigen-specific plasma cells than ewes given intraperitoneal or intramammary immunisation alone. These cells were found predominantly in the specifically immunised gland and only a few were found in the contralateral gland. Most of these cells were of the IgG1 isotype. There was also an increase in the total number of IgG1- and IgG2-containing cells in mammary gland tissues of these ewes, indicating a non-specific response to immunisation. Following either intraperitoneal or intramammary immunisation there was also a significant increase in the number of antigen-specific IgA cells in the lamina propria of the jejunum. The gut response following intramammary immunisation alone was abrogated by chronic drainage of intestinal lymph but not mammary lymph. This suggests that antigen may relocate from the mammary gland to the intestine where an IgA response is generated from gut associated lymphoid tissue. These data provide evidence for interaction between the gut and mammary gland of sheep in response to antigen.     

Immunisation of goats against contagious caprine pleuropneumonia using sonicated antigens of F-38 strain of mycoplasma

Three groups of 15 goats each were immunised against contagious caprine pleuropneumonia (CCPP) using sonicated antigens of the F-38 strain of mycoplasma incorporated in incomplete Freund's adjuvant (IFA), emulsified in aluminium hydroxide and phosphate buffered saline respectively. Three months after immunisation, five goats from each group were challenged by the in-contact method. The goats immunised with the antigen incorporated in IFA were all solidly immune to the challenge whereas only two of five of the goats in the other two groups were protected. When the remaining 10 animals from each group were challenged six months after immunisation, those immunised with the antigen in IFA were still solidly immune while only two goats from each of the other two groups were protected. These results show that effective immunity against CCPP caused by the F-38 strain can be induced by vaccination with sonicated F-38 antigens emulsified in IFA.     

Modulation of IgG subclass expression during antibody responses in sheep

Experiments were undertaken to investigate IgG subclass expression during epitope-specific antibody responses in sheep. Animals were immunised with conjugates of bovine serum albumin-DNP (BSA-DNP), or killed Staphylococcus aureus-DNP (Sa-DNP) alone or with muramyl dipeptide (MDP), dextran sulphate (DXS), or staphylococcal exotoxins (toxin). Sheep received two injections of the same preparation intracutaneously at six weeks interval. Total and IgG subclass-specific anti-DNP, and anti-carrier (BSA or S aureus) antibody levels were measured by ELISA in blood taken at weekly intervals before and after immunisation. Anti-DNP antibody levels in animals given Sa-DNP alone were considerably greater than in those immunised with BSA-DNP alone. Toxin suppressed antibody responses to DNP and both carriers; MDP suppressed anti-hapten antibody responses below the levels obtained with antigen alone. Neither toxin nor MDP significantly altered the IgG subclass profile of antibody to DNP bound to either carrier. DXS did not significantly change total levels of anti-DNP antibody measured in sheep given BSA-DNP or S aureus-DNP. However, DXS promoted IgG1 and suppressed IgG2 anti-DNP antibody responses during the secondary response to Sa-DNP but not to BSA-DNP.     

Cholera toxin improves the F4(K88)-specific immune response following oral immunization of pigs with recombinant FaeG

Oral immunization of both humans and animals with non-replicating soluble antigens often results in the induction of oral tolerance. However, receptor-dependent uptake of orally administered soluble antigens can lead to the induction of an antigen-specific immune response. Indeed, oral immunization of pigs with recombinant FaeG (rFaeG), the adhesin of the F4(K88) fimbriae of enterotoxigenic Escherichia coli (ETEC), induces an F4-specific humoral and cellular immune response. This response is accompanied with a reduction in the excretion of F4(+)E. coli following challenge. To improve the immune response against F4, rFaeG was orally co-administered with the mucosal adjuvant cholera toxin (CT). Oral immunization of pigs with rFaeG and CT significantly improved the induction of an F4-specific humoral and cellular immune response and also significantly reduced the faecal F4(+)E. coli excretion following F4(+) ETEC challenge as compared to rFaeG-immunized pigs. Therefore, the present study demonstrates that CT can act in pigs as a mucosal adjuvant for antigens that bind to the intestinal epithelium by a CT-receptor-independent mechanism.     

Immunisation of chickens to reduce intestinal colonisation with Campylobacter jejuni

1. Systemic and intestinal antibody titres were measured in chickens following subcutaneous, intraperitoneal (IP), oral (po) and combined IP/po administration of antigen, in soluble, emulsified or microparticulate form. Antigens tested included keyhole limpet haemocyanin (KLH), killed Campylobacter jejuni whole cells and purified campylobacter flagellin protein.

2. The effect of immunisation with purified flagellin protein or with killed C. jejuni whole cells in reducing intestinal colonisation was assessed. The ability of newly‐hatched chicks to respond to immunisation was limited, possibly because of the immaturity of the immune system rather than maternal suppression of an immune response. Only 5 of 13 birds that were first immunised when 1‐d‐old with KLH showed a systemic response, even after 4 immunisations, whereas 10 of 11 birds that were first immunised at 24 d‐old responded systemically.

3. In an immunisation and challenge experiment, birds that were immunised twice intraperitoneally, at 16 and 29 d‐old, with killed C. jejuni whole cells, had fewer C. jejuni, in the caecal contents than unimmunised control birds. This reduction in intestinal colonisation, to less than 2% of bacterial numbers in control birds, was associated with an increase in specific IgG in intestinal secretions. There was no significant increase in specific IgA or IgM in intestinal secretions following immunisation and challenge.

4. These results indicate that immunisation can reduce the level of intestinal infection with C. jejuni. The protection may be enhanced by developing improved methods of immunisation that stimulate production of increased titres of specific antibody in intestinal secretions, particularly specific IgA antibody.     

The effect of adjuvants on active and passive immunity in pregnant deer and their offspring.

Experiments were carried out to determine the effects of a range of adjuvants on immunoglobulin M (IgM) and immunoglobulin G (IgG) serum concentrations to a protein antigen administered subcutaneously to farmed female deer following mating. The antibody responses of animals immunised with keyhole limpet hemocyanin (KLH) in Freund's Incomplete Adjuvant (FIA), diethylaminoethyl dextran (DEAE-dextran) and aluminium hydroxide (alum) were compared with the response to antigen administered in the absence of adjuvants. Animals were subsequently challenged with a subcutaneous immunisation of the antigen in saline. Following parturition, the concentration of passively transferred antigen-specific antibody was measured in the serum of the offspring. The polyionic adjuvant, DEAE-dextran, produced the greatest enhancement of both primary and secondary IgG responses to KLH. Offspring suckling from mothers immunised with antigen in DEAE-dextran consequently had higher concentrations of specific antibodies in their serum than other fawns in the experiment. The adjuvants FIA and alum were approximately 20-fold less effective in enhancing antigen-specific IgG than DEAE-dextran but induced greater amounts of antigen-specific IgM. From the results presented in this paper, there is evidence that immunisation of deer during pregnancy may be an effective way of reducing morbidity in both mothers and offspring.     

Specific antibody containing cells in the porcine respiratory tract following intraperitoneal and intratracheal immunisation

Pigs were immunised intraperitoneally with ovalbumin in Freund's complete adjuvant and subsequently challenged intratracheally with ovalbumin. The results show that the group receiving two intratracheal challenges had a significantly greater antiovalbumin-containing cell response in the lamina propria of the respiratory tract. With intraperitoneal immunisation or intratracheal challenge alone the response was negligible. The immunoglobulin isotype of the anti-ovalbumin containing cells was predominantly IgG. There was also a substantial antibody containing cell response in the lung which was also IgG. These findings are discussed in relation to immunity within the porcine respiratory tract.     

Systemic adjuvant effect of cholera toxin in the chicken.

Cholera toxin (CT) is a well-known mucosal adjuvant in mammals, but it does not give conclusive results in chickens. Cells from the chicken immune system may be insensitive to CT activity. Our results showed that intravenously administered CT had strong immunomodulatory effects on chicken antigen-specific T- and B-cell immune responses. Seven and eight days post-inoculation (p.i.), chickens immunized with KLH and CT exhibited a faster and higher specific proliferative response in the spleen after in vitro restimulation than chickens immunized with KLH alone. At the same time, the specific antibody response in serum was significantly higher, with a strong IgG enhancement and a peak of IgA in chickens immunized with KLH and CT. The anti-KLH splenic antibody response in vitro involved a significant increase in specific IgG and IgA isotypes when CT was used as adjuvant. In conclusion, as in mammals, systemic CT demonstrated strong adjuvant properties in chickens enhancing T-cell priming in vivo and, thus, leading to increased specific antibody production, including IgA.     

The intestinal and serum humoral immune response of mice to orally administered antigens in liposomes: II. The response to liposome-entrapped bacterial proteins.

Effective oral adjuvants are needed to improve the intestinal immune responses to oral vaccines that are based on relatively low molecular weight antigens refined from veterinary pathogens. Liposomes prepared by different methods and composed of phospholipids of varying transition temperature were used to entrap cholera toxin (CT) and fed to mice. No significant increase in the intestinal antibody nor the serum IgA antibody response was detected but levels of serum IgG anti-CT antibody were significantly elevated in the group fed CT in phosphatidylcholine-based liposomes. Levels of antibody were significantly reduced in the groups fed CT in dipalmitoylphosphatidylcholine liposomes. Escherichia coli wall extract (ECWE) entrapped in certain liposome types and fed to mice elicited significantly increased serum anti-ECWE antibody responses but intestinal antibody responses were insignificantly different from the controls. These results suggest that orally administered liposomes fail to act as potent intestinal adjuvants for the entrapped antigens of bacterial origin used in this study.     

Subunit vaccines based on intimin and Efa-1 polypeptides induce humoral immunity in cattle but do not protect against intestinal colonisation by enterohaemorrhagic Escherichia coli O157:H7 or O26:H-

Enterohaemorrhagic Escherichia coli (EHEC) infections in humans are an important public health concern and are commonly acquired via contact with ruminant faeces. Cattle are a key control point however cross-protective vaccines for the control of EHEC in the bovine reservoir do not yet exist. The EHEC serogroups that are predominantly associated with human infection in Europe and North America are O157 and O26. Intimin and EHEC factor for adherence (Efa-1) play important roles in intestinal colonisation of cattle by EHEC and are thus attractive candidates for the development of subunit vaccines. Immunisation of calves with the cell-binding domain of intimin subtypes beta or gamma via the intramuscular route induced antigen-specific serum IgG1 and, in some cases salivary IgA responses, but did not reduce the magnitude or duration of faecal excretion of EHEC O26:H- (Int(280)-beta) or EHEC O157:H7 (Int(280)-gamma) upon subsequent experimental challenge. Similarly, immunisation of calves via the intramuscular route with the truncated Efa-1 protein (Efa-1') from EHEC O157:H7 or a mixture of the amino-terminal and central thirds of the full-length protein (Efa-1-N and M) did not protect against intestinal colonisation by EHEC O157:H7 (Efa-1') or EHEC O26:H- (Efa-1-N and M) despite the induction of humoral immunity. A portion of the serum IgG1 elicited by the truncated recombinant antigens in calves was confirmed to recognise native protein exposed on the bacterial surface. Calves immunised with a mixture of Int(280)-gamma and Efa-1' or an EHEC O157:H7 bacterin via the intramuscular route then boosted via the intranasal route with the same antigens using cholera toxin B subunit as an adjuvant were also not protected against intestinal colonisation by EHEC O157:H7. These studies highlight the need for further studies to develop and test novel vaccines or treatments for control of this important foodborne pathogen.     

Salmonella flagellin enhances mucosal immunity of avian influenza vaccine in chickens

Flagellin, a bioactive Toll-like receptor (TLR) 5 ligand, may trigger the innate immunity that in turn is important for subsequent adaptive immune responses. In the present study, the adjuvant effects of the monomeric and polymeric forms of Salmonella flagellin (mFliC and pFliC, respectively) were examined in specific-pathogen free (SPF) chickens immunized intramuscularly (i.m.) or intranasally (i.n.) with formalin-inactivated avian influenza virus (AIV) H5N2 vaccines. Results showed that mFliC cooperating with the 64CpG adjuvant significantly induced influenza-specific antibody titers of plasma IgA in the i.m.-vaccinated animals. The nasal IgA levels in the i.n.-mFliC-coadministrated AIV vaccinated chickens were significantly elevated compared to levels observed in the control group (H5N2 vaccine alone). The pFliC cooperating with the 64CpG adjuvant significantly enhanced cell proliferation of splenocytes in the i.m.-vaccinated animals. TLR3 and TLR5 expressions were activated by flagellin stimulation in vitro and in vivo. These results suggest that flagellin can be used as an adjuvant in an AIV H5N2 vaccine, especially for mucosal immunity.     

B cell and macrophage response in chicks after oral administration of Salmonella typhimurium strains

Despite the fact that, in a number of countries, vaccination programmes are extensively used to control Salmonella infection in poultry, information on the immune mechanisms, especially the cellular response, is still needed. The aim of the study was to characterise the B cell and macrophage response in caecum (IgA+, IgM+, IgG+ cells, macrophages), bursa of Fabricius (IgM+ cells, macrophages), and spleen (IgM+ cells) of chicks after oral administration of a non-attenuated Salmonella (S.) typhimurium wild-type strain (infection) or an attenuated commercial live S. typhimurium vaccine strain (immunisation) to day-old chicks as compared to non-treated control birds using immunohistochemistry and image analysis. In caecum, higher counts of IgM-secreting cells were detected in infected animals compared with the controls from day 5 until day 12 of age. In contrast, in treated groups, IgA-secreting cells were found in higher numbers only between day 8 and 12 of age. Infected birds showed a higher number of IgA+ cells in spleen and bursa of Fabricius compared to the controls. In the bursa of Fabricius of immunised and infected birds, a depletion of strongly stained IgM+ cells and macrophages was established between day 5 and 9 indicating a possibly special and independent role of this organ during the immunological reaction against Salmonella organisms. The results suggest that IgM- and IgA-secreting cells are of importance in the caecal immune response of chickens against Salmonella strains. Immunised chickens always showed a weaker immune reaction compared to infected animals. Present findings regarding the B cell reaction within avian caeca prove a participation of both humoral and cellular immunity in defence against Salmonella strains. Immunohistochemical examination of the cellular response (B cells and macrophages) in relevant organs of chickens may be an important tool to evaluate the immunogenic characteristics of potential Salmonella live vaccine candidates.