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1. Effects of linoleic and linolenic acid provided via different oil sources on total antibody (Ab) titres, Ab isotypes after primary and secondary immunisation, and cutaneous hypersensitivity (CH) responses to bovine serum albumin (BSA) and maleyl-BSA, respectively, were studied in pullets fed on one of 4 diets. The diets were the basal control diet enriched with either sunflower oil or safflower oil as sources of linoleic acid, and linseed oil as a source of linolenic acid, tested against a control diet supplemented with animal fat. 2. Total Ab and immunoglobulin (Ig) isotype responses to BSA were affected by diet after primary, and diet x immunisation effects after secondary immunisation. Higher total Ab and IgG titres to BSA were found especially after primary immunisation in birds given the sunflower oil enriched diet, whereas birds given sunflower oil mounted significantly lower IgM titres to BSA after primary and secondary immunisation. The antibody responses to maleyl-BSA were affected by diet after primary, and immunisation x diet interactions after secondary immunisation. Sunflower oil enhanced total and IgG Ab titres to maleyl-BSA after primary immunisation, but decreased IgM titres to maleyl-BSA after primary and secondary immunisation. Cutaneous hypersensitivity responses to BSA and maleyl-BSA were not affected by the diet. 3. It is concluded that modulation of the magnitude and isotype of Ab responses of poultry to T cell-dependent antigens is affected not only by type of essential fatty acids, but also by their source. In the present study the n-6 source, sunflower oil, showed strong enhancement of primary Ab responses, directed to both Th2 and Th1 antigens. On the other hand, the different effects of safflower oil imply that constituents other than n-6 acids within dietary plant oils may affect immune responsiveness. 4. The relationship between magnitude and isotype of Ab responsiveness, type of antigen, and essential fatty acids is discussed.

1. Effects of dietary polyunsaturated fatty acids (PUFA) and vitamin E (VE) on an immune response may interact because VE may protect PUFA from in vivo oxidation. The present study was designed to study the presence of such an interaction in growing layer chickens. 2. Three dietary concentration of linoleic acid (LA, 3.3, 6.6 and 10%), in combination with 4 concentration of dietary VE (5, 20, 40 and 80 mg/kg) were used. Effects of LA and VE on circulating VE concentration, fatty acid composition of bursal and adipose fat, and antibody kinetics against keyhole limpet hemocyanin and Mycobacterim butyricum were established. 3. At high dietary LA concentration, bursal and adipose LA were higher but bursal arachidonic acid and long chain n-3 PUFA decreased. The dietary VE level did not consistently affect the deposition of PUFA in tissue. Plasma VE concentrations were affected by the dietary VE and LA content, but not by their interaction. Antibody responses before and 7 d after immunisation were affected by the dietary treatments. Antibody concentration were not affected by tissue fatty acid content. 4. In conclusion, the interaction effects of dietary PUFA and VE on fat deposition and immune responses are of minor importance compared to separate PUFA and VE effects. This implies that, within the studied range, adding extra VE to preserve or affect the effects of dietary PUFA on antibody responsiveness is unnecessary.

Effects of dietary polyunsaturated fatty acids (PUFA) on immune responses in poultry have been reported. However, effects on the underlying mechanisms, such as the role of cytokines, have not been documented because the necessary tools were lacking. Recently, primer sets for chicken interleukin (IL)-1beta, IL-2, interferon-gamma (IFN-gamma), myelomonocytic growth factor (MGF), and transforming growth factor (TGF)-beta2 have become available. Therefore, in the present study we first examined the in vivo effects of an inflammatory challenge with Salmonella typhimurium lipopolysaccharide (LPS) on cytokine profiles in growing laying-type chicks. Second, we examined whether dietary fat sources affected the observed cytokine profiles. Two hundred forty chicks were assigned in a 2 x 4 factorial design of treatments, with injection with LPS or saline and dietary fat source as factors. Factors were i.v. injection with S. typhimurium LPS or saline (control) and four dietary fat sources: corn oil, linseed oil, menhaden oil, and tallow. Two hours after injection, birds were killed, and their spleens were removed for RNA extraction. Reverse transcription polymerase chain reactions with primer sets for chicken IL-1beta, IL-2, IFN-gamma, MGF, TGF-beta2, and beta-actin were performed with RNA samples pooled by pen. The expression of cytokine mRNA was expressed relative to the level of beta-actin mRNA. Interleukin-1 (P < 0.001), MGF (P < 0.0001), IL-2 (P < 0.001), and IFN-gamma (P < 0.001) mRNA expressions were enhanced by challenge with LPS. Immunization treatment had no effect on TGF-beta2 or beta-actin expression. Dietary treatment did not affect mRNA expression of IL-1, MGF, IFN-gamma, TGF-beta2, or beta-actin. Interleukin-2 expression in LPS-injected birds that were fed the fish-oil-enriched diet was enhanced (P = 0.05). The present study indicates that in vivo effects of immune challenge on cytokine mRNA expression can be measured in poultry. The observation that mRNA level of IL-2, but not the mRNA levels of IFN-gamma or MGF, is enhanced by dietary fish oil at 2 h suggests that dietary PUFA at this moment initially affected naïve T lymphocytes.

Effects of four levels of dietary linoleic acid (LA), an n-6 fatty acid, and four levels of alpha-linolenic acid (LNA), an n-3 fatty acid, and their interactions on immune responses in growing layer hens were studied. Immune responses were induced by injection with keyhole limpet hemocyanin (KLH) or Mycobacterium butyricum particles at 35 d of age. Antibody (Ab) responses were measured until 21 d after immunization. In addition, delayed-type hypersensitivity, lymphocyte proliferation, weekly feed intake, and BW gain were studied. At Day 7 after immunization, anti-M. butyricum titers in the M. butyricum-immunized hens were decreased by the increase of dietary LA (P < 0.05). In the period from 10 to 14 d after immunization, anti-KLH Ab titers in KLH-immunized animals were affected by the interaction of dietary LA with LNA (P < 0.01). High dietary levels of LA or LNA increased the anti-KLH Ab response. However, at high levels of dietary LA and LNA, anti-KLH Ab titers were not increased. In the same period, anti-M. butyricum Ab titers of M. butyricum-immunized hens were affected by the interaction of dietary LA with LNA (P < 0.05). At low levels of LA and LNA, increased LA levels decreased the Ab response, whereas increased LNA levels at low LA levels hardly affected the anti-M. butyricum response. At a high level of LA, increased dietary LNA increased the anti-M. butyricum response. In vitro proliferation of peripheral blood leukocytes after stimulation with concanavalin A (ConA) was higher in chickens with a high level of dietary LNA. Feed intake decreased when the dietary levels of LA or LNA increased. However, BW gain was not affected by dietary treatments. Feed conversion was more efficient in birds fed high levels of LA and LNA. The present study indicates that various factors affect the Ab responses. First, the interaction of n-6 and n-3 polyunsaturated fatty acids (PUFA) is more important than the separate effects of n-3 or n-6. Second, the actions of dietary PUFA were different between antigens of a different nature. Third was the nature of the antigen affects when dietary PUFA exert their actions and the persistence of these effects. The presence of these multiple factors affecting immune responses should be considered when comparing effects of dietary PUFA on immune responses.

The effects of linoleic (LA)- and linolenic acid (LNA)-enriched diets on humoral and in vivo cellular immune responses to keyhole limpet hemocyanin (KLH)-dinitrophenyl (DNP) and Mycobacterium butyricum were studied in growing layer hens. Pullets were fed one of three diets: a control, LA enriched, or LNA enriched. Pullets were assigned to one of three immunization treatments: KLH-DNP, M. butyricum, or PBS. The LA-enriched diet enhanced the antibody response to KLH in pullets immunized with KLH-DNP. On the other hand, the antibody response to M. butyricum in M. butyricum-immunized birds was decreased by feeding an LA-enriched diet. In vitro lymphocyte proliferation in the presence of Concanavalin A was affected by the interaction between diet and immunization. Neither cutaneous hypersensitivity to KLH nor to M. butyricum was affected by the diet. The BW gain before immunization was not affected by the diet, but after immunization, the LA-enriched diet enhanced growth in birds immunized with M. butyricum. Diets had various effects on organ weights. We concluded that dietary linoleic acid enrichment of the diet has an antigen-dependent divergent effect on the antibody response. The dietary LNA effect on the antibody response is less pronounced and is opposite to that of the LA effect.

Effects of prostaglandin E2 (PGE2) and indomethacin, an inhibitor of PGE2 oxygenase, on primary and secondary antibody (Ab) responses to Mycobacterium butyricum protein or keyhole limpet hemocyanin (KLH) were studied in growing layer hens. Immunizations at 35 and 70 d of age were accompanied by immunomodulating treatments with PGE2, indomethacin, or PBS. In addition, we studied effects of various doses of indomethacin and PGE2 on mitogen-induced T-cell proliferation in vitro. Secondary Ab responses to KLH were enhanced by administration of indomethacin at secondary immunization and, to a lesser extent, by PGE2 administration at secondary immunization. Primary Ab responses to M. butyricum tended to decrease by administration of either PGE2 or indomethacin. Secondary Ab responses to M. butyricum were affected by administration of both PGE2 and indomethacin at primary immunization. Prostaglandin E2 increased phytohemagglutinin (PHA)-induced lymphocyte proliferation. Indomethacin decreased Concanavalin A (ConA)- and PHA-induced lymphocyte proliferation. The net effect of indomethacin on the Ab response could not be explained by inhibition of PGE2 oxygenase only. Our data rather suggest an inhibition by indomethacin of other immunosuppressing factors derived from arachidonic acid. We concluded that polyunsaturated fatty acid-derived products might especially affect secondary antibody responsiveness. This finding may depend on inhibition or enhancement of T-cell responsiveness. Consequently, immunomodulation by dietary polyunsaturated fatty acids may have profound effects at secondary rather than at primary exposure to pathogens.