Establishment of a microplate assay for flow cytometric assessment and it is use for the evaluation of age-related phenotypic changes in canine whole blood leukocytes

The effectiveness of flow cytometric assays for canine use is still requiring standardization. Despite several studies using purified mononuclear cells, no methodology or reference ranges are available for immunophenotyping of whole blood leukocytes (WBL). Fresh and pre-fixed WBL were used to identify cell-subsets, (Thy-1(+)/CD5(+)/CD4(+)/CD8(+)/CD21(+) and CD14(+)) and measure MHC-II, CD45RA/CD45RB expression. We described here an efficient method for fast quantification of canine-WBL, using pre-fix in a microplate assay, which allows long-term sample storage prior to phenotyping. Decreased percentage of CD5(+)-T-cells within the lymphocyte-gate and increased percentage of CD21(+)-B-cells were observed in young animals, which led to higher T/B cell ratios in middle-aged dogs. Lower numerical counts of Thy-1(+), CD4(+), CD8(+) and CD21(+) lymphocyte were observed when compared to young animals. In addition, we identified an age-related decline of MHC-II/CD45RA expression by lymphocytes. We proposed an improved method for phenotyping of canine peripheral blood mononuclear cells (PBMC) that has significant use for researchers and veterinary clinicians. The hematological changes of senescence previously identified on PBMC could be adequately reproduced on features identified by whole blood. Furthermore, this study supplies normal range references as baseline standards for clinical purposes, besides specific immunological parameters to monitor canine aging process.     

Gamma/delta T cell response of chickens after oral administration of attenuated and non-attenuated Salmonella typhimurium strains

Poultry represents an important source of Salmonella infection in man. Despite intensive research on immunity, little is known about the involvement of T cell sub-populations in the immunological response of chickens against infection with non-host-adapted Salmonella (S.) serovars. In this study, the T cell composition of blood lymphocytes (CD4(+)CD8(+); CD4(+)CD8(-); CD4(-)CD8(+); CD8(+)TcR1(+); CD8(-)TcR1(+), CD8(+)TcR1(-)) after oral administration of the non-attenuated S. typhimurium wild-type strain 421 (infection) or the attenuated vaccine strain Salmonella vac((R)) T (immunization) to day-old chicks was investigated and compared with non-treated chickens by flow cytofluorometry. Additionally, the occurrence of T cell sub-populations (CD4(+); CD8(+); TcR1(+)(gammadelta); TcR2(+)(alphabeta(1))) in ceca, spleen and bursa of Fabricius of the birds was studied immunohistologically. Blood samples and tissues were examined between days 1 and 12 of age.Chicks inoculated with S. typhimurium 421 or Salmonella vac((R)) T showed significantly elevated percentages of CD8(+)TcR1(+) in blood on days 7, 8 and 9, or on day 8 in comparison to control animals. The CD4 to CD8 cell ratio was about 3:1 in infected animals on day 5 of age. In the organs of treated chicks the numbers of CD8(+)(gammadelta) and TcR1(+)(gammadelta) cells had markedly increased on days 4 and 5 in ceca, 8 and 9 in the bursa and 9 and 12 in the spleen. Moreover, infected or vaccinated birds revealed larger quantities of CD4(+) and TcR2(+) T cells in ceca on days 4 and 5. As shown by double staining, the TcR1(+) cells in the organs of infected animals additionally carried the CD8 antigen.In conclusion, immunization of day-old chicks with the attenuated Salmonella live vaccine strain resulted in the same changes in T cell composition as seen after infection with the non-attenuated Salmonella wild-type strain, but at a lower level. The remarkable increase of CD8(+)TcR1(+)(gammadelta) double positive cells in treated birds indicates an important role of this cell sub-population in the immunological defense of chickens against Salmonella exposure.     

Effect of in ovo administered reovirus vaccines on immune responses of specific-pathogen-free chickens

We investigated the effect of in ovo administered reovirus vaccines on the immune responses of specific-pathogen-free chickens. T-cell mitogenic responses to concanavalin A were numerically lower at 9 and 12 days of age and significantly lower at 6 days of age in birds vaccinated with a commercial reovirus vaccine compared with unvaccinated birds or birds vaccinated with an experimental reovirus-antibody complex vaccine. There were no significant differences in proportions of subpopulations of helper (CD4+CD8-) or cytotoxic (CD4-CD8+) T cells except at 12 days of age, when the percentages of CD4-CD8+ cells in the two vaccinated groups were statistically higher than in the nonvaccinated group. B-cell populations were not different among vaccine groups except at 9 days of age, when the vaccinated groups had the highest level of B cells. This commercial reovirus vaccine should not be given in ovo to embryos having little or no maternal antibody, otherwise immunosuppression may occur in the chicks. The addition of the antibody complex to the vaccine prevented this T-cell immunosuppression.     

Alterations of lymphocyte subpopulations in healthy dogs with aging and in dogs with cancer

Changes in an individual's immune status are considered major contributing factors towards the morbidity of cancer and mortality of aging. To evaluate age-related changes in the immune status of dogs, the immunophenotypes (CD3, CD4, CD8 and CD21) of peripheral blood lymphocytes were measured in 160 healthy dogs aged from 1 to 17 years, and in 365 dogs with various tumors and at various stages. In healthy dogs, the absolute numbers of white blood cells, lymphocytes, and CD3(+), CD4(+) and CD21(+) lymphocytes decreased significantly with age. The relative percentages of lymphocytes and CD4(+) cells decreased significantly, while CD8(+) cells increased significantly with age. The CD4:CD8 ratio showed a significant age-related decrease. In contrast, dogs with tumors possessed significantly lower absolute numbers and relative percentages of all lymphocyte phenotypes, while the CD4:CD8 ratio was significantly higher than in age-matched controls. The relative percentages of CD3(+) and CD8(+) lymphocytes were significantly lower in dogs with distant metastases compared with dogs without metastases, and the CD4:CD8 ratio increased with advanced stage. These observations illustrate the significant changes in immune status with age and the presence of marked immunological defects in a large-scale study of dogs with advanced tumors.     

Effect of ascorbic acid supplementation on the immune response of chickens vaccinated and challenged with infectious bursal disease virus

One-day-old chickens were divided into two groups and reared under similar conditions. One group was fed a diet supplemented with 1000ppm ascorbic acid and the other group was fed an identical diet, but not supplemented with ascorbic acid. Both groups were vaccinated against infectious bursal disease (IBD) at 7 days of age and challenged orally with 4x10(5) of 50% embryo-lethal-dose IBDV 14 days later. The number of anti-IBDV antibody secreting cells, production of interleukin-2 (IL-2) by splenocytes, number of CD4(+), CD8(+) and IgM(+) cells in spleen and IgM(+) cells in bursa of Fabricius were compared between the two groups at 7 days (prior to vaccination), 21 days (14 days post-vaccination and prior to challenge) and 31 days (10 days post-challenge) of age. The number of CD8(+) in spleen at 7 days of age and IgM(+) cells in bursa at 7, 21 and 31 days of age were significantly higher in ascorbic acid supplemented group (P<0.05). Production of IL-2 by splenocytes was higher as indicated by higher stimulation indices in ascorbic acid supplemented group. The number of anti-IBDV IgG antibody secreting cells in spleen at 21 and 31 days of age were significantly higher in ascorbic acid supplemented group (P<0.05). Dietary supplementation of ascorbic acid may ameliorate the immunosuppression caused by IBDV vaccination and improve humoral and cellular immune responses.     

Peripheral lymphocyte subsets as a prognostic indicator of mortality and morbidity in healthy dogs

To evaluate the relationship among immune status and increased morbidity and mortality, peripheral blood lymphocytes (CD3(+), CD4(+), CD8(+) and CD21(+) cells) from 32 healthy dogs over 8 years of age were analyzed. Twenty-five of the 32 dogs were followed-up for 3 years after the analysis; and 14 dogs were found to be diseased, and nine dogs died. There was no notable difference between the ages of the dogs that died compared with the ones that survived. The relative percentage of CD4(+) and the CD4(+):CD8(+) ratio decreased notably in dogs falling ill compared with healthy dogs. The relative percentage of CD3(+) lymphocytes showed a notable decrease in dogs that died within 3 years in comparison with dogs that survived. In a discriminant analysis of morbidity and mortality, most patients were correctly classified as diseased or not and surviving or dead, respectively. These results indicate that the immunophenotypes of peripheral blood lymphocytes in older dogs offer promise as parameters for evaluating mortality and morbidity.     

Cellular immune response of pigeons in the conditions of endotoxin fever and pyrogenic tolerance

The aim of this study was to investigate changes in selected parameters of cellular immune response in the conditions of endotoxin fever and pyrogenic tolerance in pigeons. On the first day of observation the experimental birds (n = 18) were intravenously injected with Escherichia coli LPS at a dose of 10 microg/kg b.w., while the control animals (n = 6) received apyrogenic physiological saline also in the form of injection. On the second and the third day of the experiment LPS was injected additionally at 24 h intervals. Four and a half hours after the saline and pyrogen administration blood samples were collected from the control and experimental pigeons. The following immunological assays were performed: WBC, leucogram and immunophenotyping of lymphocyte subsets in peripheral blood, i.e. CD 3+ (T lymphocytes), CD 4+ (T helper lymphocytes) and CD 8+ (T suppressor/cytotoxic lymphocytes) cells. In the conditions of endotoxin fever (i.e. after the first LPS injection) leucopenia, monocytopenia, heterophilia and eosinophilia were observed. Additionally, the immunophenotyping of peripheral blood lymphocytes indicated an increase in percentage of CD 3+, CD 4+ and CD 8+ cells in response to the single injection of LPS. In contrast, the consecutive injections of LPS, which created a pyrogenic tolerance effect, caused a decrease in WBC value, heteropenia, eosinopenia and lymphocytosis. Moreover, during this state an increase in percentage of CD 3+ and CD 8+ cells was demonstrated in contrast to the percentage of CD 4+ lymphocytes. The general tendencies in cellular immune response of the affected pigeons in the conditions of endotoxin fever and pyrogenic tolerance aim at activation of defence mechanisms against LPS for its prompt elimination from the animal's organism.     

Chicken peripheral blood CD3+CD4-CD8- cells are regulated by endocrine and nerve systems

The existence of CD3(+)CD4(-)CD8(-) T cells in thymus and spleen has already been known. However, because of the presence of large amounts of thrombocytes in peripheral blood (PB), the proportion of CD3(+)CD4(-)CD8(-) T cells in PB has yet to be investigated. Therefore, the proportion of peripheral T cell-subsets was investigated in 6-week-old chickens. The percentage of CD3(+) cells, CD4(+) cells, CD8 alpha(+) cells, CD8 beta(+), and CD3(+)CD4(-)CD8(-) cells was 76%, 41%, 14%, 5%, and 15%, respectively. The proportion of CD3(+)CD4(-)CD8(-) cells in PB increased during egg-laying periods and in chickens treated with an analog of estrogen, while it decreased with age and in response to restraint stress. All of the CD3(+)CD4(-)CD8(-) cells expressed TCR1, and did not have NK activity. CD3(+)CD4(-)CD8(-) cells represent about 60% of peripheral TCR1(+) cells. These findings indicate that the proportion of CD3(+)CD4(-)CD8(-) cells is regulated by the endocrine and nerve systems.     

Intestinal exposure to a parasite antigen in utero depresses cellular and cytokine responses of the mucosal immune system

The response of the mucosal immune system of 4-6-week old lambs to viable Trichostrongylus colubriformis larvae was compared in two groups of animals, one exposed to T. colubriformis antigen and the other to saline while in utero. Exposure to larval antigen two-thirds of the way through gestation resulted in significant reduction in the frequency of jejunal goblet cells and of ileal eosinophils, CD 1b(+) antigen-presenting cells and CD4(+), CD5(+) and CD8(+) cells. However, it resulted in a significant increase in the jejunal CD8(+) response to postnatal challenge. The expression of the cytokines TNF-alpha and IL-1 beta in the ileum, and of jejunal NSE, was significantly reduced by in utero exposure, whereas those of jejunal TNF-alpha and ileal TGF-beta were increased. The observed changes in cellular and cytokine responses to challenge with viable larvae, in those lambs previously exposed in utero, indicated that the intestinal mucosal immune system remains susceptible to down-regulation until considerably later in foetal development than is the case for other components of the immune system.     

Postnatal development of T-lymphocyte subpopulations in the intestinal intraepithelium and lamina propria in chickens.

Postnatal development of various T-lymphocyte subpopulations expressing CD3, CD8, CD4, and antigen-specific TCR heterodimers alpha beta (TCR2) or gamma delta (TCR1) was investigated in two different inbred chicken strains, SC and TK. The ratios of jejunum T-cells expressing TCR1 to TCR2 in the intraepithelium of SC and TK strains gradually increased after hatching and were 3.40 and 4.28 by 12 weeks in TK and SC chickens respectively. The ratios of TCR1+ to TCR2(+)-cells in intraepithelium and the lamina propria in SC chickens were 0.96 and 1.23 at 8 weeks and 4.29 and 2.15 at 12 weeks, respectively. Jejunum intraepithelial lymphocytes expressing the CD8 antigen increased gradually until 4-6 weeks of age and subsequently declined as chickens aged. CD4(+)-cells represented a minor subpopulation among the intestinal lymphocyte subpopulations. Therefore, the composition of various T-cell subpopulations in the intestine depended upon host age, the regions of the gut examined and host genetic background. These results suggest that changes in T-cell subpopulations in the intestine may reflect age-related maturation of the gut-associated lymphoid tissues.     

Montanide IMS 1313 N VG PR nanoparticle adjuvant enhances antigen-specific immune responses to profilin following mucosal vaccination against Eimeria acervulina

This study investigated protection against Eimeria acervulina (E. acervulina) following vaccination of chickens with an Eimeria recombinant profilin in conjunction with different adjuvants, or by changing the route of administration of the adjuvants. Day-old broilers were immunized twice with profilin emulsified in Montanide IMS 1313 N VG PR adjuvant (oral, nasal, or ocular routes), Montanide ISA 71 VG adjuvant (subcutaneous route), or Freund's adjuvant (subcutaneous route) and orally challenged with virulent E. acervulina parasites. Birds orally immunized with profilin plus IMS 1313 N VG PR, or subcutaneously immunized with profilin plus ISA 71 VG, had increased body weight gains compared with animals nasally or ocularly immunized with profilin plus IMS 1313 N VG PR, or subcutaneously immunized with profilin plus Freund's adjuvant. All adjuvant formulations, except for IMS 1313 N VG PR given by the nasal or ocular routes, decreased fecal parasite excretion and/or reduced intestinal lesions, compared with non-vaccinated and infected controls. Compared with animals vaccinated with profilin plus Freund's adjuvant, chickens immunized with profilin plus IMS 1313 N VG PR or ISA 71 VG showed higher post-infection intestinal levels of profilin-reactive IgY and secretary IgA antibodies. Finally, immunization with profilin in combination with ISA 71 VG was consistently better than profilin plus IMS 1313 N VG PR or Freund's adjuvant for increasing the percentages of CD4(+), CD8(+), BU1(+), TCR1(+), and TCR2(+) intestinal lymphocytes. These results indicate that experimental immunization of chickens with the recombinant profilin subunit vaccine in conjunction with IMS 1313 or ISA 71 VG adjuvants increases protective mucosal immunity against E. acervulina infection.     

Peripheral T lymphocyte changes in neonatal piglets: Relationship with growth hormone (GH), prolactin (PRL) and cortisol changes

Taking into account the role played by the neuroendocrine network in affecting the early development of the immune response, the present study aims to assess neonatal immunity in piglets by testing peripheral lymphocyte age-related changes in relationship to plasma levels of some relevant immunoregulatory hormones, such as growth hormone (GH), prolactin (PRL) and cortisol. For this purpose, we studied the peripheral lymphocyte age-related changes in relationship to plasma levels of GH, PRL and cortisol in conventional piglets from birth (day 0) to 41 days of age. A significant decrease was observed in the total number of lymphocytes at day 0, with a subsequent constant increment up to 41 days of age. Concomitantly, the number of T cell subsets (mainly CD8(+) cells and double positive CD4(+)CD8(+)) was low at birth, with strong increments between the 19th and 41st days of life. The CD4(+) T cell number subset was less diminished at birth than that of CD8(+), albeit with significant increments in the post-weaning period. Of interest, gammadelta T cells, which are more involved in innate immune efficiency, displayed the same trend as CD8(+) T cells from birth to the 41st day of life. From day 0 up to the 19th day, significant inverse correlations were found between T cell subsets and GH or PRL or cortisol, albeit with more significant inverse correlations with cortisol. The high levels of GH and PRL in the pre-weaning period may be due to the fact that they have to counteract the cortisol-mediated negative effect on lymphocyte production and development. These findings suggest that stress condition occurs at birth with decreases in the immune parameters, in the same way as in human newborns, with a subsequent gradual normalisation and immune development, as shown by decreased cortisol, GH and PRL normalisation and concomitant increments in T cell subsets.     

IBDV-induced bursal T lymphocytes inhibit mitogenic response of normal splenocytes

We examined the suppressive activity of bursal T cells induced by infectious bursal disease virus (IBDV) in inbred (15x7) and outbred commercial specific-pathogen-free (SPF) chickens. The suppressive activity was measured by the ability of bursal and splenic T cells from IBDV-infected chickens to inhibit mitogenic responses of normal splenocytes. The bursacytes but not the splenocytes of IBDV-infected chickens inhibited the mitogenic responses of normal splenocytes. The mitogenic inhibition by the bursacytes of IBDV-infected chickens was dose-dependent. The suppression was observed both in inbred and non-inbred chickens, and thus, was non MHC-restricted. Cell-sorting experiments revealed that both CD4(+) and CD8(+) cells from the bursa of IBDV-infected chickens, as well as cell-culture supernatants conditioned by these cells, mediated suppression. Suppressor T (Ts) cells may therefore be involved in the immunosuppression induced by IBDV.     

Fowl adenovirus (FAdV) serotype 4 causes depletion of B and T cells in lymphoid organs in specific pathogen-free chickens following experimental infection

In the present investigation flow cytometric analysis and immunohistochemistry were applied together for the first time to gain new insights into the interaction between virulent fowl adenoviruses (FAdV) and the immune system of chickens. As a model for virulent FAdV infections a FAdV-4 strain was used, known as the aetiological agent of Hepatitis-Hydropericardium syndrome (HHS) in broilers sometimes also named Angara Disease. Specified pathogen-free chickens (SPF) were divided into three different groups. Group I was infected at first day of life with an attenuated form of the virus obtained through continuous cell culture passage with the virulent virus and then re-infected 3 weeks later with the virulent progenitor virus. Group II was solely infected with the virulent virus at 3 weeks and group III served as a negative control. Following infection with the virulent virus a decrease of CD3+, CD4+ and CD8+ cells was noticed in the spleen. This was accompanied by a decrease of CD4+ and CD8+ T-lymphocytes in the thymus. Those birds infected with the attenuated virus in first instance and challenged with the virulent virus did not show these pathological effects in the thymus. In the bursa of Fabricius a severe depletion of lymphocytes was observed by immunohistochemistry in birds, infected with the virulent virus. Taken together it can be concluded that an infection with FAdV-4 has profound effects on cells, of the humoral and cell-mediated immune responses. The effects are much more severe in the birds infected with the virulent virus only indicating that the preceding infection with the attenuated virus reduces significantly the adverse effects induced by the virulent virus.     

Flow cytometric analysis of peripheral blood mononuclear cells induced by experimental endotoxemia in horse

Cellular activation and functional cell surface markers were evaluated during experimentally-induced endotoxemia in healthy horses. Eight healthy adult horses were infused a low dose of endotoxin (lipopolysaccharide from Escherichia coli O26: B6, 30 ng/kg of body weight, IV) and five control horses were given an equivalent volume of sterile saline solution. Venous blood samples were collected for flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and to measure plasma endotoxin concentrations. Clinical signs of endotoxemia were recorded at 10, 20, 30, 40, 50 min, 1, 2, 3, 4, 8, 16, 24 and 48 hr after endotoxin or saline solution administration. Clinical findings characteristic of endotoxemia (tachycardia, tachypnea, increased rectal temperature, and leukopenia) occurred transiently in all horses administered endotoxin; however, plasma endotoxin concentrations were detectable in only 50% (4/8) of the endotoxin-infused horses. The percentage of CD4(+), CD5(+), and CD8(+) cells decreased while the percentage of CD14(+), IgM(+), and MHC class II(+) cells increased significantly after endotoxin infusion. Alterations in the immunophenotype of PBMCs from horses with experimentally-induced endotoxemia were associated with changes in vital signs, indicating that endotoxin altered the immuno balance.     

Evaluation of the infectivity, length of infection, and immune response of a low-pathogenicity H7N2 avian influenza virus in specific-pathogen-free chickens

The H7N2 subtype of avian influenza virus (AIV) field isolate (H7N2/chicken/PA/3779-2/97), which caused the 1997-98 AIV outbreak in Pennsylvania, was evaluated for its infectivity, length of infection, and immune response in specific-pathogen-free (SPF) chickens. The composite findings of three clinical trials with various concentrations of virus indicated that this H7N2 subtype contained minimal pathogenicity for chickens. The concentration of the virus in the inoculum proved critical in the establishment of a productive infection in a chicken. Seven-day-old SPF chickens were not infected when inoculated with 10(0.7-2.0) mean embryo lethal dose (ELD50) of the H7N2 virus per bird. At this dose level, the immune response to this virus was not detected by the hemagglutination-inhibition (HI) test. Nonetheless, chickens at ages of 5 and 23 wk old tested were successfully infected when exposed to 10(4.7-5.7) ELD50 of H7N2 infectious doses per bird by various routes of administration and also by direct contact. Infected birds started shedding virus as early as 2 days postinoculation, and the period of virus shedding occurred mostly within 1 or 2 wk postinoculation (WPI). This H7N2 subtype of AIV induced a measurable immune response in all birds within 2 wk after virus exposure. Antibody titers were associated with AIV infectious doses and age of exposure of birds. Challenge of these infected birds with the same H7N2 virus at 5 and 10 WPI indicated the infective virus was recoverable from cloacal swabs at 3 days postchallenge and disappeared thereafter. In these challenged birds, the antibody levels as measured by the HI test spiked within 1-2 wk.     

Distribution and activation of T-lymphocyte subsets in tuberculous bovine lymph-node granulomas

The immune response against mycobacterial infections is dependant upon a complex interaction between T lymphocytes and macrophages in the context of the granuloma. For this study, we performed the analysis of 18 stage I or II, and 13 stage III or IV granulomas found in lymph nodes from 8 experimentally and 2 naturally infected cattle. T-cell subpopulations (CD3(+), CD4(+), CD8(+), WC1(+), CD25(+)) were investigated by immunohistochemistry. In the majority of stage I/II lesions, CD8(+) and CD25(+) cells were predominantly found in the lymphocytic outer region of the granuloma, suggesting a possible role for activated CD8(+) cells in the initial attempt to restrain the granuloma growth. CD4(+) T cells appeared equally distributed in the lymphocytic mantle and in the internal areas of the granulomas. WC1(+) cells appeared interspersed among the macrophages. We speculated that this could indicate a role for these 2 subsets in the maintenance and the maturation of the granuloma. In stage III/IV lesions, all of the T-cell subsets investigated appeared interspersed among the mononuclear component of the granulomas. In general terms, there was a higher density of CD8(+) cells compared with CD4(+) cells. However, there was no sense of rimming effect for any of the investigated cell populations.     

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.     

AA肉鸡免疫禽流感油乳剂灭活疫苗细胞免疫应答的变化

为了解肉鸡生长期内免疫前后的细胞免疫反应,本实验采用CD3/CD4/CD8三色流式细胞术、MTT法和Griess法依次检测了AA肉鸡免疫禽流感灭活疫苗和非免疫状态下外周血、胸腺和脾脏中T淋巴细胞及其亚群的相对含量、外周血中T淋巴细胞对PHA-P的增殖反应能力和诱导巨噬细胞NO分泌量的变化。结果表明,非免疫状态下,AA肉鸡外周血CD4+、CD8+、CD3+T淋巴细胞相对百分含量在7日龄时均为最低,脾脏中CD4+T淋巴细胞相对百分含量在整个试验期一直呈下降趋势,免疫后外周血、脾脏和胸腺中CD4+、CD8+、CD3+T淋巴细胞相对百分含量均明显高于非免疫组,且脾脏CD4+T淋巴细胞呈阶段性上升状态;各时期淋巴细胞刺激指数(SI)值和巨噬细胞NO分泌量检测结果表明,免疫组均高于同期对照组(p<0.05)。研究结果还表明:肉鸡在7日龄时细胞免疫水平最低,灭活疫苗可以激发肉鸡产生较强的细胞免疫应答。