Effect of induced molting on heterophil function in White Leghorn hens.

This study was undertaken to determine the effects of induced molt on basal functional activities of heterophils from aging hens. For this purpose, heterophils from both molted and unmolted hens were examined by in vitro bioassays for functional responsiveness and efficiency. We evaluated the ability of the heterophils to migrate to chemotactic stimuli, phagocytize opsonized and nonopsonized Salmonella-enteritidis (SE), and generate an oxidative burst in response to inflammatory agonists. A significant (P < 0.001) heterophilia was found in the molted hens within 2 days after feed withdrawal and remained throughout the length of the experimental feed withdrawal period. No significant differences were found in the random migration of heterophils from either group. The chemotactic movement of heterophils from molted hens was not affected until 8 days after feed withdrawal when compared with heterophil chemotaxis from unmolted hens. A significant decrease in chemotaxis by the heterophils from molted hens was observed days 8-12 after feed withdrawal (P < 0.05). Significantly (P < 0.05) fewer heterophils from molted hens were able to phagocytize opsonized (59% vs. 38%) and nonopsonized (26% vs. 15%) SE within 2 days after feed withdrawal. Likewise, significantly (P < 0.05) fewer bacteria were phagocytized per heterophil from the molted hens when compared with the number of bacteria per heterophil from the unmolted hens. The oxidative burst of heterophils stimulated by either opsonized zymosan A or phorbol myristate acetate of heterophils from molted hens was significantly (P < 0.05) reduced when compared with that generated by heterophils from the unmolted hens. These results indicate that feed withdrawal to induce molt alters the number and function of peripheral blood heterophils. This decreased efficiency of heterophil functional activity appears to play a role in the increased susceptibility of molting hens to SE infections.     

Enhancement of phagocytosis and bacterial killing by heterophils from neonatal chicks after administration of Salmonella enteritidis-immune lymphokines

During the first week post-hatch, chickens demonstrate an increased susceptibility to infection by bacteria such as Salmonella. The purpose of the present study was to evaluate the effects of immune lymphokines on phagocytosis and killing activities of heterophils in chicks during the first 1-7 days of life. Lymphokines isolated from chicken splenic T-cells harvested from Salmonella enteriditis (SE)-hyperimmunized hens (SE-ILK), have in past experiments, demonstrated augmentation of heterophil activity in day-of-hatch chicks resulting in protection from SE organ invasion. The present experiments reveal significant increases (p<0.05) in heterophil phagocytosis and killing when comparing chicks treated with SE-ILK to control groups in vitro. In SE-ILK-treated groups, a two-fold or greater increase is noted in heterophil phagocytosis within I h of incubation as compared to controls. Heterophils isolated from 1-day-old and 4-day-old chicks treated with SE-ILK killed significantly greater numbers (p<0.05) of SE than heterophils isolated from control groups. By Day 7 post-hatch, significance is not noted in the killing activity of heterophils from treated groups when compared to control groups. However, heterophils from SE-ILK groups continue to kill greater numbers of SE than control groups. These data support SE-ILK augmentation results in an enhanced heterophil function in chicks during the greatest period of susceptibility to Salmonella invasion.     

Heterophil function in healthy chickens and in chickens with experimentally induced staphylococcal tenosynovitis.

Heterophil function was evaluated in 16 healthy chickens and in 46 chickens with experimentally induced staphylococcal tenosynovitis. In paired blood samples, heterophils from chickens with tenosynovitis had a significant increase in adherence, chemotaxis, phagocytosis, and bacterial killing of Staphylococcus aureus compared to heterophils from healthy chickens. The percent adherence of heterophils to nylon fiber columns increased significantly from a 78.4% mean +/- 6.6% standard deviation to 87.6% +/- 3.2% after induction of staphylococcal tenosynovitis. Heterophil movement following in vitro exposure to saline or endotoxin was increased in chickens with tenosynovitis; 3 +/- 1 heterophils/0.25 mm2 to 10 +/- 6 heterophils/0.25 mm2 and 136 +/- 29 heterophils/0.25 mm2 to 340 +/- 74 heterophils/0.25 mm2, respectively. Endotoxin-activated serum was chemoattractive for heterophils from all chickens. Flow cytometry was used to define the heterophil population on light scatter histograms, evaluate individual cell phagocytosis of latex beads, and quantitate the number of beads phagocytosed per heterophil. When incubated with increased numbers of beads, only heterophils from chickens with tenosynovitis phagocytosed higher numbers of beads. At heterophil to bead ratios of 1:10, the percentage of heterophils that phagocytosed beads increased from baseline values of 37.8% +/- 9.0% to post-infection values of 67.3% +/- 7.5%. Using 1:20 heterophil to bead ratios, heterophil phagocytosis increased from 38.7% +/- 9.9% to post-infection values of 79.8% +/- 7.3%. Heterophils from all chickens were able to phagocytose and kill log phase staphylococcal bacteria. After phagocytosis, the heterophils from chickens with staphylococcal tenosynovitis rapidly decreased the number of viable bacterial colony forming-units per milliliter by approximately one log.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of oral, subcutaneous, and nasal administration of Salmonella enteritidis-immune lymphokines on the potentiation of a protective heterophilic inflammatory response to Salmonella enteritidis in day-old chickens.

We have previously reported that the prophylactic administration of factor(s) from T-cell supernatants derived from Salmonella enteritidis-immune chickens (ILK) have a favorable effect in controlling or eliminating salmonellosis in neonatal poultry. Experimentally, we have used the intraperitoneal injection as the standard method of administering ILK to neonatal poultry. However, this method is neither easy, practical, nor economical for the poultry industry. In the present study, we evaluated the effectiveness of oral (p.o.), intranasal (i.n.), and subcutaneous (s.c.) administration of ILK for ease of delivery, induction of protective resistance against Salmonella enteritidis (Se) organ invasion, and the ability to activate peripheral blood heterophils in day-old chickens. In the first experiments, delivery of ILK p.o., i.n., and s.c. significantly (P < 0.01) increased the resistance of day-old chickens to Se organ invasion. The level of protection was equivalent to that induced by the i.p. route. Administration of a comparable protein control (bovine serum albumin, BSA) by the 3 routes induced no protective effect against Se organ invasion. Likewise, a significant increase was found in the number of circulating heterophils within 4 h of administration of the ILK by all routes. In the 2nd experiment, the function of the heterophils from ILK-treated birds was compared with that of the control cells in adherence, chemotaxis, and phagocytosis assays. The heterophils from birds given ILK i.p., s.c., p.o., or i.n. had significantly (P < 0.01) increased functional activities when compared to the activities of the heterophils from the control birds. These studies indicate that the delivery of ILK either orally or parenterally, routes which can be used by the poultry industry, can confer protection to chickens against a localized enteric Se organ invasion by potentiating the systemic heterophilic innate response.     

Comparison of heterophil phagocytosis for heterophil-adapted Salmonella enteritidis (HASE) and wild-type Salmonella enteritidis (SE).

Serial passage of Salmonella enteritidis (SE) yields heterophil-adapted SE (HASE) strains that have resulted in decreased shedding of SE in feces and reduced egg contamination. Additionally, increasing the number of heterophil passages further reduced the number and frequency of fecal shedding. To evaluate SE and heterophil interaction, nine SE strains were fluorescein isothiocyanate-labeled when viable. There were six wild-types: SE TK 474, SE TK 584, SE TK 599, SE TK 600, SE TK 655, and SE TK 657; and three HASE strains: TK 499 heterophil adapted five times, TK 598 heterophil adapted six times, and TK 605 heterophil adapted 11 times. Trials were repeated seven times in duplicate with heterophils isolated from seven healthy chickens. Heterophils were incubated with the bacterial strains at 41 C for 15 min, and 10,000 heterophils were analyzed by flow cytometry. Percentage of phagocytosis and mean channel number of fluorescence were compared. Both parameters were significantly increased for all HASE-type strains compared with wild-type, nonadapted SE strains. Increased phagocytosis of HASE bacterial strains may be significant in processing and elimination of the HASE strains and may be related to the protective effect of HASE by decreased shedding of wild-type SE challenge strains.     

Proportion of circulating chicken heterophils and CXCLi2 expression in response to Salmonella enteritidis are affected by genetic line and immune modulating diet

Genetic line and diet affect chicken heterophil activity and gene expression, and the combination of these factors can enhance disease resistance. This study evaluated the effects of immune modulating diets on heterophil/lymphocyte (H/L) ratio and heterophil chemokine expression in distinct genetic lines. Fayoumi and Leghorn chickens were fed a basal diet or immune modulating diets enhanced with β-glucans, ascorbic acid, or corticosterone. H/L ratios and heterophil gene expression in response to in vitro stimulation with Salmonella enteritidis (SE) were evaluated on days 1, 3, 7, and 21 of diet treatment. The stress-mimicking corticosterone diet influenced H/L ratio in the Leghorn line, but not the Fayoumi line, suggesting resistance to stress-induced immunosuppression in the Fayoumi line. Leghorn line H/L ratios were increased on days 1 and 3 of corticosterone diet treatment, but not days 7 or 21. Expression of CXCLi2 by SE stimulated heterophils was higher in the Leghorn line, suggesting that Leghorns rely more heavily on inflammatory response than do Fayoumis. Corticosterone diet was associated with reduced CXCLi2 expression in heterophils from both lines. Dietary β-glucan or ascorbic acid did not affect H/L ratio or CXCLi2 expression, suggesting that benefits of these immunomodulators may not be evident in healthy birds.     

Turkey heterophil chemotaxis to Pasteurella multocida (serotype 3,4)-generated chemotactic factors

The purpose of this study was to determine the ability of three strains or isolates of Pasteurella multocida (serotype 3,4) to generate chemotactic factors for heterophils when exposed to pooled turkey serum. Results indicated that each bacterial strain or isolate (M-9, CU, and 86-1913) was associated with the production of chemotactic factors, but the more pathogenic bacterial isolate (86-1913) elicited greater heterophil migration in chemotaxis studies.     

Upregulation of NRAMP1 mRNA confirms its role in enhanced host immunity in post-artificial infections of Salmonella enteritidis in chicks

1. Salmonella enteritidis (SE) is reported as the most common food-borne pathogen transmitted through poultry products. The natural resistance-associated macrophage protein 1 (NRAMP1) is a candidate gene associated with SE-mediated immune response and is related to the phagocytosis of SE. In this study, the classical single-nucleotide polymorphism (SNP) G2357A in exon 8 of the NRAMP1 gene was detected. The expression of NRAMP1 mRNA was first investigated in heterophil granulocytes and spleen in chicks from two different Chinese native breeds at 1, 3 and 10 d post-infection. In addition, the association with the effect of SE challenge was identified.

2. The G2357A SNP showed no significant association with Salmonella natural infection in birds from two different Chinese native breeds.

3. The upregulation of NRAMP1 mRNA in heterophils and spleen was involved in the response to pathogenic SE colonisation during the acute infection period in chicks. The results suggest that genetics, age, gender and interactions among these factors play important roles in the modulation of NRAMP1 mRNA expression and copy number by SE-mediated immune response in different Chinese chickens.

4. In conclusion, the enhancement of host immunity mediated by the upregulation of NRAMP1 mRNA in heterophil granulocytes and spleen might be more obvious and earlier in the chicks resistant to infections with SE than in susceptible chicks.     

Acute airsacculitis in untreated and cyclophosphamide-pretreated broiler chickens inoculated with Escherichia coli or Escherichia coli cell-free culture filtrate.

Ninety commercial broiler chickens were divided into three equal groups; 30 were injected with brain-heart-infusion broth into the cranial thoracic air sacs (controls), 30 were similarly inoculated with a culture of Escherichia coli, and 30 were similarly inoculated with E. coli cell-free culture filtrate. Birds were examined from 0 to 6 hours post-inoculation. E. coli-inoculated and cell-free culture filtrate-inoculated chickens reacted similarly, with exudation of heterophils into the air sac. Microscopically, heterophils were present in low numbers perivascularly 0.5 hour after inoculation and became more numerous by 3 hours post-inoculation. By 6 hours post-inoculation, there was severe swelling of air sac epithelial cells and thickening of the air sac by proteinaceous fluid and heterophils. Ultrastructurally, air sac epithelial cells were swollen and vacuolated, and interdigitating processes were separated. Histologically and ultrastructurally, all features in control chickens were normal, with only rare heterophils in the air sac interstitium. In E. coli-inoculated and cell-free culture filtrate-inoculated chickens, cell counts (predominantly heterophils) in air sac lavage fluids increased markedly at 3 and 6 hours, with only slight increases in counts from lavages of controls. Heteropenia was observed in E. coli-inoculated chickens, whereas heterophilia was observed in cell-free filtrate chickens and controls. Ninety additional chickens were pretreated with cyclophosphamide, subdivided into three equal groups, and inoculated and examined similarly as above. Cyclophosphamide pretreatment reduced inflammatory changes in air sacs, lowered cell numbers in lavage fluids, and abolished hematologic changes; however, it did not prevent epithelial cell changes. These results indicate that cell-free culture filtrate of E. coli induces changes similar to those induced by cultures of E. coli.     

Heterophil function and resistance to staphylococcal challenge in broiler chickens naturally infected with avian leukosis virus subgroup J

Avian leukosis virus subgroup J has a high tropism for myeloid lineage cells and frequently induces neoplastic transformation of myelocytes. The impact of congenital avian leukosis virus subgroup J infection on the function of circulating heterophils and susceptibility to staphylococcal infection was investigated. Six-week-old broiler chickens negative for exogenous avian leukosis viruses or congenitally infected with avian leukosis virus subgroup J were inoculated intravenously with 10(6) colony-forming units of Staphylococcus aureus, and pre- and postinoculation heterophil function was assessed. All chickens developed a leukocytosis with heterophilia after inoculation, but total leukocyte and heterophil counts were significantly higher in leukosis-negative chickens than in virus-infected chickens. Tenosynovitis was more severe in leukosis-negative chickens, and 2/10 (20%) of the virus-infected chickens had no histologic evidence of tenosynovitis. Osteomyelitis in the tibiotarsus or tarsometatarsus developed in 5/10 (50%) of the chickens in each group. S. aureus was recovered from the hock joint of 6/10 (60%) of the chickens in each group. Heterophils from all chickens exhibited similar phagocytic ability pre- and postinoculation. Heterophils from virus-infected chickens exhibited less bactericidal ability preinoculation than did heterophils from leukosis-negative chickens. However, postinoculation bactericidal ability was similar in both groups. Avian leukosis virus subgroup J provirus was present in heterophils isolated from congenitally infected chickens. Heterophils isolated from broiler chickens congenitally infected with avian leukosis virus subgroup J exhibit no significant functional deficits, and infected and uninfected chickens exhibit similar susceptibility to staphylococcal infection.     

The importance of interleukin-8 as a neutrophil chemoattractant in the lungs of cattle with pneumonic pasteurellosis.

Interleukin-8 (IL-8), an in vitro and in vivo neutrophil chemoattractant, is expressed at high levels in the lesions observed in bovine pneumonic pasteurellosis. Because of the role of neutrophils in the pathogenesis of pneumonic pasteurellosis, we investigated the relative importance of IL-8 as a neutrophil chemoattractant in this disease. Bronchoalveolar lavage (BAL) fluid was harvested from calves experimentally infected with bovine herpesvirus-1 and challenged with Mannheimia haemolytica. Neutrophil chemotactic activity was measured in pneumonic BAL fluid samples treated with a neutralizing monoclonal antibody to ovine IL-8, and compared to the activity in samples treated with an isotype-matched control antibody. Bronchoalveolar lavage fluid was analyzed at a dilution which induced a half-maximal response, and the concentrations of antibody were optimized in a preliminary experiment. Following incubation of replicate samples of diluted pneumonic bovine BAL fluid with 70 microg/mL of IL-8-neutralizing antibody or control antibody, the neutrophil chemotactic activities of the samples were determined using an in vitro microchemotaxis assay. Overall, pretreatment of BAL fluid samples with neutralizing anti-IL-8 antibody reduced neutrophil chemotactic activity by 15% to 60%, compared to pretreatment with control antibody. This effect was highly significant (P < 0.001), and was present in 5 of 5 samples. These data indicate that IL-8 is an important neutrophil chemoattractant in calves with pneumonic pasteurellosis, but that mediators with actions redundant to those of IL-8 must also be present in the lesions.     

Loss of virulence by heterophil-adapted Salmonella pullorum.

We report that reduction of virulence for day-old chicks was achieved after eight-times-repeated heterophil passage of Salmonella pullorum (SP). The virulent source strain SP-V caused 64% mortality and 89% internal organ as well as 89% cecal colonization 10 days after administration of 10(7) colony-forming units (CFU) to day-old chicks. Eight-times-repeated passage of SP in heterophils resulted in attenuated strain SP-A that was nonlethal and reduced colonization of internal organs from 89% for SP-V strain to 4.3% for SP-A strain 10 days after administration of 10(7) or 10(8) CFU to day-old chicks. Cecal colonization was reduced from 89% for SP-V strain to 0 for SP-A strain 10 days after administration of 10(7) or 10(8) CFU to day-old chicks.     

The effects of ascorbic acid on in vitro heterophil function

As a feed additive, ascorbic acid has been shown to have a protective effect against bacterial and viral diseases and to reduce the impact of detrimental stress in chickens. This study examined the effect of ascorbic acid treatment on in vitro heterophil function by examining random migration and phagocytosis and bacterial killing of Staphylococcus aureus. Heterophils were evaluated in broiler chickens ranging from 5 to 16 wk of age, and age differences were seen. Significant increases in bacterial killing were found in heterophils treated with ascorbic acid, and this difference tended to be greater in chickens from 5 to 10.5 wk of age. No significant differences were found in phagocytosis or random migration, but ascorbic acid tended to decrease random migration. The most significant effect on in vitro heterophil function was an increase in bacterial killing.     

Characterisation of colony-stimulating activity in the avian T cell-derived factor, Salmonella enteritidis-immune lymphokine

This investigation was designed to characterise the specific cytokine activity from the conditioned medium of concanavalin A-stimulated avian T cells derived from Salmonella enteritidis-immune chickens, S enteritidis-immune lymphokine (ILK). Studies were designed to determine first, whether colony-stimulating activity was present in ILK, second, the type(s) of colonies from the bone marrow that were supported in vitro by the potential colony-stimulating factors in ILK and, third, whether colony-stimulating activity was present in serum from chicks treated with ILK and challenged with S enteritidis, and to use physicochemical treatment as a means of identifying the potential colony-stimulating factor(s) in ILK. Both ILK alone and serum from chicks treated with ILK and challenged with S enteritidis caused significant increases in the number of colony-forming units (CFU) from the bone marrow in vitro. After 10 days of incubation, ILK alone supported the in vitro growth of granulocytic bone marrow colonies. The colony-stimulating activity from serum derived from chicks treated with ILK and challenged with S enteritidis peaked two hours after the challenge. When ILK was either heated at 100°C or treated with trypsin or acid and then injected into chicks, all the chicks responded with significant increases in circulating polymorphonuclear leucocytes (PMNS). However, when assayed for in vitro colony-stimulating activity, only trypsinisation destroyed the activity in ILK. The results indicate that a colony-stimulating factor which preferentially supported the growth of granulocytic bone marrow colonies was present in ILK and that the factor was stable to heat and acid but sensitive to trypsin.     

Acute airsacculitis in turkeys inoculated with cell-free culture filtrate of Pasteurella multocida

Twenty-six female and 26 male turkeys, inoculated into the caudal thoracic air sacs with cell-free culture filtrate of Pasteurella multocida strain R44/6, were examined from 0 to 6 hours post-inoculation and compared with 26 female and 26 male sham-inoculated control turkeys given brain-heart-infusion broth. The air sac reacted rapidly with exudation of heterophils. Microscopically, low numbers of heterophils were present within air sac blood vessels and also perivascularly by 0.5 hour after inoculation. These became more numerous by 1.5 and 3 hours post-inoculation. By 6 hours post-inoculation, there was severe swelling of air sac epithelial and mesothelial cells and thickening of the air sac by proteinaceous fluid and heterophils. Ultrastructurally, mesothelial and air sac epithelial cells were vacuolated, and interdigitating processes of epithelial cells were separated. Microscopically, in control turkeys, rare heterophils were present perivascularly at 1.5, 3, and 6 hours after inoculation. Ultrastructurally, all features were normal. In turkeys given cell-free culture filtrate, total cell counts in air sac lavage fluids increased markedly by 3 hours post-inoculation in which heterophils predominated (greater than 97%). There were only slight increases in cell counts of air sac lavages from control turkeys. The circulating blood heterophil cell count dropped transiently at 1.5 hours post-inoculation, followed by a return to normal 3 hours after inoculation, and by heterophilia by 6 hours post-inoculation in turkeys given either cell-free culture filtrate or brain-heart-infusion broth. These results indicate cell-free culture filtrate of P. multocida induces hematologic, cytologic, and morphologic changes indistinguishable from those induced by cultures of P. multocida.     

Evaluation of the heterophil/lymphocyte ratio as a measure of stress in chickens

The number of lymphocytes in chicken blood samples decreased and the number of heterophils increased in response to stressors and to increasing levels of corticosterone in the chicken feed. The ratio of heterophils to lymphocytes was less variable than the number of heterophil or lymphocyte cells, and the range of values for this ratio was greater than the range of values for heterophils and lymphocytes among control and experimental groups. The heterophil/lymphocyte ratio appears to be a more reliable indicator of levels of corticosterone in the feed and to social stress than were the plasma corticosteroid levels.     

Production and functional characterization of recombinant bovine interleukin-8 as a specific neutrophil activator and chemoattractant

Interleukin-8, a member of the chemokine family of cytokines, is a potent neutrophil chemoattractant in many non-rodent species. In this study, recombinant bovine interleukin-8 (rbIL-8) was expressed in bacteria as a glutathione-S-transferase fusion protein. The fusion protein was purified by glutathione-Sepharose affinity chromatography and recombinant rbIL-8 was eluted by cleaving with thrombin. The purified rbIL-8 molecule was approximately 8 kDa and was confirmed as authentic IL-8 by Western analysis. Recombinant bovine IL-8 induced specific dose-dependent in vitro chemotaxis of neutrophils at doses as low as 1.0 ng/ml, and this activity was inhibited by pre-treatment of rbIL-8 with a monoclonal antibody to ovine IL-8. Neutrophils exposed to rbIL-8 developed pseudopodia and became elongated as determined by microscopic analysis and flow cytometry. Injection of 3.3 ng to 3.3 microg of rbIL-8 into the skin of a normal calf induced dose-dependent recruitment of neutrophils but not eosinophils. Intravascular margination of neutrophils was obvious at the injection sites from 15 to 60 min after administration of rbIL-8, and extravascular neutrophil numbers increased steadily from 1 to 18 h after injection. Neutrophils with morphologic features of apoptosis were detected in these lesions at 18 and 30 h after injection, and this correlated with reduction in the number of dermal neutrophils. These results confirm unequivocally that bovine IL-8 functions as a neutrophil, but not an eosinophil, chemoattractant in vitro and in vivo.     

The effects of environmental enrichment and transport stress on the weights of lymphoid organs,cell‐mediated immune response,heterophil functions and antibody production in laying hens

The effects of environmental enrichment and transport stress on the immune system were investigated in laying hens. A total of 48 1‐day‐old chickens were used, half of the chickens were reared in conventional cages (RCC) and the rest in enriched cages (REC). Transport stress was applied in the 17th week. Liver weight decreased, spleen and bursa of Fabricius weights, white blood cell count, CD4+ and CD8+ cell proportions increased due to the transport. Environmental enrichment significantly increased antibody production and tended to increase monocyte percentage and CD8+ cell proportion. The effect of transport on, heterophil (H) and lymphocyte (L) percentages was not significant in RCC chickens. While heterophil percentage and H:L ratio increased, lymphocyte percentage decreased in REC chickens subjected to transport. Transport stress increased heterophil functions both in REC and RCC chickens, but the increase was higher in REC hens than in RCC hens. In conclusion, although environmental enrichment did not neutralize the effect of transport on lymphoid organs, it activated the non‐specific immune system, cellular and the humoral branches of the specific immune system by increasing heterophil functions, CD8+ cells and antibody production, respectively. Therefore, environmental enrichment suggested for improving animal welfare may also be beneficial to improve the immune system of birds exposed to stress.     

Improved immune responses against avian influenza virus following oral vaccination of chickens with HA DNA vaccine using attenuated Salmonella typhimurium as carrier

This study evaluates the immune responses of single avian influenza virus (AIV) HA DNA vaccine immunization using attenuated Salmonella enterica sv. Typhimurium as an oral vaccine carrier and intramuscular (IM) DNA injection. One-day-old specific-pathogen-free (SPF) chicks immunized once by oral gavage with 10(9) Salmonella colony-forming units containing plasmid expression vector encoding the HA gene of A/Ck/Malaysia/5858/04 (H5N1) (pcDNA3.1.H5) did not show any clinical manifestations. Serum hemagglutination inhibition (HI) titer samples collected from the IM immunized chickens were low compared to those immunized with S. typhimurium.pcDNA3.1.H5. The highest average antibody titers were detected on day 35 post immunization for both IM and S. typhimurium.pcDNA3.1.H5 immunized groups, at 4.0±2.8 and 51.2±7.5, respectively. S. typhimurium.pcDNA3.1.H5 also elicited both CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells (PBMCs) of immunized chickens as early as day 14 after immunization, at 20.5±2.0 and 22.9±1.9%, respectively. Meanwhile, the CD4(+) and CD8(+) T cells in chickens vaccinated intramuscularly were low at 5.9±0.9 and 8.5±1.3%, respectively. Immunization of chickens with S. typhimurium.pcDNA3.1.H5 enhanced IL-1β, IL-12β, IL-15 and IL-18 expressions in spleen although no significant differences were recorded in chickens vaccinated via IM and orally with S. typhimurium and S. typhimurium.pcDNA3.1. Hence, single oral administrations of the attenuated S. typhimurium containing pcDNA3.1.H5 showed antibody, T cell and Th1-like cytokine responses against AIV in chickens. Whether the T cell response induced by vaccination is virus-specific and whether vaccination protects against AIV infection requires further study.