Comparison of disease susceptibility and subclass-specific antibody response in SC and FP chickens experimentally inoculated with Eimeria tenella, E. acervulina, or E. maxima

Susceptibility to disease and the subclass-specific antibody response to Eimeria tenella, E. acervulina, and E. maxima were compared in two inbred strains of chickens, FP (B15B21) and SC (B2B2). FP strain was more susceptible to coccidiosis than SC chickens based on oocyst production, lesion score, and clinical signs. FP chickens infected with E. tenella had more severe cecal lesions and a significantly lower hematocrit level than SC chickens. FP chickens infected with E. acervulina excreted five times as many oocysts at 6 days postinfection as SC and showed a 71% reduction in plasma carotenoid level compared with controls (56% reduction in SC chickens). Body-weight change did not correlate with other signs of disease. Both SC and FP chickens produced high levels of serum IgM and IgG and biliary IgA. Although SC chickens had a slightly higher antibody response than FP chickens at 7 days postinoculation, both strains maintained high levels of IgM, IgG, and IgA for a prolonged period post primary inoculation. Although SC and FP chickens show different disease susceptibility to coccidiosis, they demonstrate similar antibody response.     

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.     

Serological and bacteriological observations on experimental infection with Salmonella hadar in chickens

Over the past 3 years the frequency of Salmonella hadar infections has increased in Belgium in both poultry and humans. Therefore, the course of infection with S. hadar in poultry was investigated. One day-old and 4 week-old specific pathogen-free chickens were orally infected with one of two S. hadar strains, SH1 or SH2. Mortality was 6% (SH1) and 17% (SH2) in birds infected at 1 day-old. Chickens infected at 1 day-old with SH2 showed a mild diarrhoea. The S. hadar faecal excretion in birds infected at 1 day-old remained high throughout the experiment until 12 weeks post-inoculation (pi). Faecal excretion was lower in older birds. Antibodies to S. hadar were observed from 2 weeks pi (SH2, infected at 1 day-old) or 4 weeks pi (SH1, both groups; SH2, chickens infected at 4 weeks of age). The percentage of chickens with antibodies was higher after infection at 1 day-old than after infection at 4 weeks of age. In a second experiment 1 day-old chicks were infected with SH1 and autopsied at regular intervals until 42 days pi. SH1 was isolated from the caeca from 3 h pi onwards and from the liver and spleen from 18 h until 14 days pi. Serous typhlitis and omphalitis were the main lesions. The number of macrophages in the lamina propria of the caecal tonsils was slightly increased from 18 h until 2 weeks pi. In the liver, inflammation was observed in the portal triads and in the sinusoids. This study indicates that infections with S. hadar lead to intense colonisation of the gut and extensive faecal shedding. It may also cause invasive infections in 1 day-old chickens.     

Interleukin-2 production in SC and TK chickens infected with Eimeria tenella

SC and TK inbred chicken strains display differential protective immunity to coccidiosis, SC being more resistant and TK susceptible to disease. In this study, the association between interleukin (IL)-2 and disease phenotype was assessed by cytokine quantification in serum, duodenum, cecum, and spleen cell cultures of SC and TK chickens experimentally infected with Eimeria tenella. In general, after primary infection, SC and TK strains produced equivalent amounts of IL-2 in all sources examined. However, after secondary infection, SC animals displayed significantly greater IL-2 levels in serum and the duodenum compared with strain TK. IL-2 production after reinfection with Eimeria may be an important factor contributing to the genetic differences in coccidiosis between SC and TK chickens and provides a rational foundation for cytokine-based immunotherapeutic approaches to disease control strategies.     

Dynamics of lymphocyte subpopulation changes in the cecal tonsils of chickens infected with Salmonella enteritidis

Salmonella enteritidis (SE)-induced changes in various T and B lymphocyte subpopulations in the cecal tonsils of chickens were analyzed using flow cytometry. At 1 day post-SE inoculation, the percentages of CD3(+) and CD8(+) T lymphocytes were significantly decreased in the group inoculated with 1x10(9) SE colony-forming units (CFU) (SE high) and in the group inoculated with 1x10(6) SE CFU (SE low) compared with the uninfected control group. The percentage of CD4(+) T lymphocytes was significantly increased in the SE high group compared to the uninfected and the SE low groups at 4 days after SE inoculation. The percentage of IgG(+) B lymphocytes was also significantly increased in both SE high and low groups compared to the uninfected control at 6 days post-SE inoculation. In contrast, the SE low group showed significantly fewer IgM(+) B lymphocytes compared to the uninfected and SE high groups. These results show that SE infection induces significant changes in the cecal tonsil lymphocytes subpopulations shortly following SE inoculation.     

Modulation of innate immunity in chickens induced by in vivo administration of baculovirus

Baculoviruses stimulate cytokine production in mammalian cells. They induce a strong innate immune response in animals and have adjuvant properties. The purpose of this work was to study the in vivo effect of baculovirus on chicken innate immune response. SPF chickens were inoculated intravenously with Autographa californica nuclear polyhedrosis virus (BV). Three hours later, chickens were bled, euthanized and their spleen, duodenum and cecal tonsils were excised in order to take samples for RNA extraction and real time PCR, and to isolate lymphocytes, which were stained and analyzed by flow cytometry. The results obtained showed that baculovirus inoculation up-regulates the expression of IFN-γ, IL-6 and LITAF in spleen cells. This result (IFN-γ) correlated with that obtained by ELISA which showed a very strong increase of IFN-γ in chicken plasma. Flow cytometry analysis revealed that BV inoculation induced in spleen an increase in the percentage of monocyte/macrophage population together with an increase in CD3(+)CD4(+) T lymphocytes. On the other hand, BV inoculation decreased the percentage of CD3(+)CD4(+) T lymphocytes and increased the percentage of NK cells in cecal tonsils. However, intraepithelial lymphocytes of the gut did not show differences between BV and control treated animals. Even though further studies in order to understand the mechanisms by which BVs affect the avian immune response are needed, results obtained in the present work demonstrate the ability of BVs to stimulate the innate immunity in chickens, modifying the expression pattern of related genes and the profile of the immune cells involved.     

Energy and nitrogen metabolism of chickens infected with either Eimeria acervulina or Eimeria tenella.

1. The effects of sublethal infections of E. acervulina and E. tenella on the energy and nitrogen metabolism of groups of five broilers aged 16 d were studied for 16 d in respiration chambers. 2. The metabolisable energy content of the diet for chickens infected with E. acervulina was 0.689 of its gross energy content and N retention was 42.5 g/100 g N intake compared with 0.738 and 47.1 g respectively, in uninfected pair-fed controls. Chickens infected with E. tenella were similarly affected. 3. Efficiency of utilisation of ME by chickens infected with E. acervulina was 0.43 during the first 8 d after infection, and 0.52 during the second 8 d compared with an overall efficiency by non-infected chickens fed ad libitum of 0.73. Maintenance energy requirement of infected chickens was higher during the first 8 d after infection than during the second 8-d period. 4. Body composition measurements showed that of the total gain in weight of chickens infected with E. acervulina, only 7.5 g/kg gain was fat and 213 g/kg was protein compared with 45 g and 210 g respectively for non infected chickens fed ad libitum. 5. E. acervulina and E. tenella infections reduced the apparent digestibility of total mineral, calcium and phosphorus.     

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.     

Influence of inoculation dose, inoculation schedule, chicken age, and host genetics on disease susceptibility and development of resistance to Eimeria tenella infection

Various parasite- and host-related factors influencing disease susceptibility and development of protective immunity against Eimeria tenella infection were investigated in two inbred strains of chickens. Chickens that received a primary inoculation of 10(3), 10(4), or 10(5) oocysts showed a significant reduction in packed cell volume and produced significantly more oocysts than chickens inoculated with fewer oocysts. Younger chickens were as susceptible as older chickens to identical parasite doses. However, upon a secondary inoculation 5 weeks following primary inoculation, FP chickens 1 to 21 days old at the time of primary inoculation developed resistance to reinfection, whereas SC chickens less than 3 weeks old at the time of primary inoculation were highly susceptible to secondary infection. Flow cytometric analysis of spleen lymphocytes showed a substantial reduction in T-cell number in 1-day-old SC but not FP chickens. Furthermore, 1-week-old SC chickens showed depressed mitogenic responses to concanavalin A compared with 1-week-old FP chickens. There was no significant difference between SC and FP chickens in speen B-cell number, regardless of age.     

Viral nucleoprotein localization and lesions of Newcastle disease in tissues of indigenous ducks

Localization of Newcastle disease viral nucleoprotein and pathological lesions was evaluated in tissues of 55 indigenous ducks (45 experimentally infected and 10 sentinel ones). In addition, ten Newcastle disease infected chickens were used to ensure that the virus inoculum administered to the ducks produced the disease in chickens, the susceptible hosts. Ducks were killed on day 1, 4, 8 and 14 post-infection. Post-mortem examination was done with six tissues (liver, spleen, lung, caecal tonsils, kidneys and brain) being collected from each bird. The tissues were preserved in 10% neutral formalin for 24 h. They were then transferred to 70% ethanol for histology and immunohistochemical staining. Airsacculitis, necrotic splenic foci, congested intestines, lymphoid depleted caecal tonsils and focal infiltrations by mononuclear cells were the main pathological lesions in infected ducks. Over 28.9% of the infected ducks had Newcastle disease viral nucleoprotein in macrophage-like large mononuclear cells in the caecal tonsils and kidney tubular epithelium. The viral antigens were located in the cytoplasm and nucleolus of the cells. The other organs had no detectable viral antigens. This study shows that the kidneys and caecal tonsils are the likely predilection sites for the virus in ducks. They thus need to be considered as diagnostic indicators for the viral carriage in ducks.     

Comparative susceptibility of chickens, turkeys and ducks to infectious bursal disease virus using immunohistochemistry

Twenty chicks, 12 turkey poults and 10 ducklings, all 5 weeks old were infected with 2 × 10^3.5 chick LD₅₀ IBD virus to determine the course of the virus in the 3 poultry species. Uninfected control birds were kept separately. Two infected and 2 control birds/species were euthanized at time intervals between 3 and 168 hours post infection (pi). Sections of thymus, bursa of Fabricius, spleen, liver, kidney, proventriculus and ceacal tonsil were stained for the detection of IBD virus antigen using immunoperoxidase technique. IBD virus antigen positive cells stained reddish-brown and the amount of such cells in tissue sections were noted and scored. Stained cells were present in all organs examined for up to 168 hours pi in the 3 poultry species except ceacal tonsils of ducks at 72 and 120 hours pi. Antigen score was highest in chickens and least in ducks as reflected by average of total scores/sampling time of 12, 10.8 and 8 in chickens, turkeys and ducks respectively. Total antigen score/sampling time in infected chickens peaked twice; 24/48 and 144 hours pi, whereas such bi-phasic peaks were absent in turkeys and ducks. Range of total antigen score at different sampling times was 7–17.5 in chickens, 10–13 in turkeys and 7–10 in ducks indicative of marked viral replication in chickens. Presence of IBD viral antigen in organs of all 3 poultry species is indicative of infections. The innate ability of turkeys and ducks to prevent appreciable replication of IBD virus after infection requires further investigation.     

葡萄球菌性关节炎肉鸡免疫器官的病理学变化

利用鸡源致病性金黄色葡萄球菌复制鸡葡萄球菌性关节炎的病理模型,研究免疫器官的主要病理学变化及其内CD4 和CD8 T淋巴细胞的动态变化。结果表明:鸡接种细菌后呈典型的关节炎症状。脾脏肿大,表面呈网格状,法氏囊黏膜增厚,盲肠扁桃体和胸腺上散在出血点。光镜下脾脏淋巴小结早期数目增多,后期呈灶状坏死,法氏囊水肿,淋巴小结坏死液化。CD4 和CD8 T淋巴细胞的变化为:脾脏在接种后7 d淋巴小结内的CD4 和CD8 T淋巴细胞已明显多于对照组,与对照组比差异极显著(P<0.01);接种后7 d,法氏囊的淋巴滤泡周围的间隙中检出较多的阳性细胞,在接种后14 d CD4 T淋巴细胞达到高峰,随后下降,与对照组相比在接种后7 d差异显著(P<0.05),在接种后14 d差异极显著(P<0.01);而CD8 T淋巴细胞在接种后7 d就达到高峰,与对照组相比差异极显著(P<0.01);盲肠扁桃体的CD8 T淋巴细胞在接种后7 d上升,21 d达到最高值;胸腺组织中阳性细胞数对照组和试验组都很少,试验组的阳性细胞数的变化没有明显的规律。鸡接种金黄色葡萄球菌后免疫器官内CD4 和CD8 T淋巴细胞数目增多,表明T淋巴细胞参与了金黄色葡萄球菌引起鸡的关节炎的发生发展过程。     

Differential expression of homing receptors and vascular addressins in tonsils and draining lymph nodes: Effect of Brucella infection in sheep

The differential expression of homing receptors (HR) and complementary vascular addressins was studied in T and B lymphocytes from ovine tonsils and draining lymph nodes (LN) in uninfected and Brucella melitensis-infected sheep. In uninfected sheep, CD4+CD25+ T cells expressed proportionally more L-selectin and beta1 integrin than beta7 integrin in pharyngeal and palatine tonsils and in parotid LN (PLN), retropharyngeal LN (RLN) and the peripheral prescapular LN (PSLN). In contrast, memory CD4+CD45RA- T cells expressed an equivalent proportion of the three HR in PLN and PSLN, whereas beta1 and beta7 integrins were proportionally more expressed than L-selectin in pharyngeal tonsil. beta7 integrin was proportionally more expressed than beta1 integrin or L-selectin in palatine tonsils, RLN and the mucosal mesenteric LN (MLN). beta1 integrin was proportionally more expressed in IgG+ and IgA+ cells than beta7 integrin and L-selectin in tonsils, PLN and RLN. The main endothelial addressin expressed on venules in both pharyngeal and palatine tonsils, the PLN and RLN, as well as in the PSLN, was the peripheral PNAd, while in the MLN it was MAdCAM-1. Conjunctival infection by Brucella resulted in an increase of CD4+CD25+ and CD4+CD45RA- T cell subsets, which was associated to modifications of HR expression. CD4+CD45RA- T cells expressed proportionally more beta1 and beta7 integrins than L-selectin in regional PLN and RLN, but also in PSLN. The infection induced an increase of IgG+ and IgA+ cell percentages expressing beta1 integrin in all LN, and also beta7 integrin in the RLN. PNAd continued to be expressed on venules of tonsils and draining LN after Brucella infection, and MAdCAM-1 was also weakly expressed on RLN venules. These results suggest that lymphocyte trafficking through tonsils and draining LN could involve L-selectin/PNAd interactions, as well as beta1 or beta7 integrin, possibly in interaction with VCAM-1 or MAdCAM-1. The homing of antigen-specific lymphocytes in these tissues could be modulated after conjunctival infection with Brucella, which induces the recruitment of lymphocytes that express both beta1 and/or beta7 integrin in regional and more distant LN.     

Lymphocyte subpopulations and apoptosis of immune cells in rabbits experimentally infected with a strain of the RHD virus having a variable haemagglutination capacity

The paper describes the immunological response in the matter of percentage of T cells (receptor CD5+) and subpopulations (Th with receptor CD4+, Tc/Ts with receptor CD8+, T with receptor CD25+) and B cells with receptor CD19+, as well as the percentage of apoptotic granulocytes and lymphocytes, in rabbits experimentally infected with the Hagenow strain of the RHD virus. The material chosen for the experiment is special, as among all strains of RHD virus, there are only two strains which carry the variable haemagglutination capacity of red cells. The results of the study show that the Hagenow strain gives an untypical picture of T and B lymphocytes, whereas the results in inducing apoptosis seems to corespond with previous data, confirming the inclusion of apoptosis from 4 h p.i. and the intensity of the phenomenon being higher in granulocytes.     

Nitric oxide production by macrophages stimulated with Coccidia sporozoites, lipopolysaccharide, or interferon-gamma, and its dynamic changes in SC and TK strains of chickens infected with Eimeria tenella

Nitric oxide (NO) is an important mediator of innate and acquired immunities. In the studies reported here, we quantified NO produced in vitro by chicken leukocytes and macrophages and in vivo during the course of experimental infection with Eimeria, the causative agent of avian coccidiosis, and identified macrophages as the primary source of inducible NO. Eimeria tenella-infected chickens produced higher levels of NO compared with noninfected controls. In Eimeria-infected animals, SC chickens produced greater amounts of NO compared with infected TK chickens, particularly in the intestinal cecum, the region of the intestine infected by E. tenella. Macrophages that were isolated from normal spleen were a major source of NO induced by interferon (IFN)-gamma, lipopolysaccharide (LPS), and E. tenella sporozoites. Macrophage cell line MQ-NCSU produced high levels of NO in response to Escherichia coli or Salmonella typhi LPS, whereas the HD-11 macrophage cell line was more responsive to IFN-gamma. These findings are discussed in the context of the genetic differences in SC and TK chickens that may contribute to their divergent disease phenotypes.     

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.     

Coccidiosis: a radiological study of sulphaquinoxaline distribution in infected and uninfected chickens

Using scintillation counting and autoradiographical techniques, the whole-body distribution in week-old uninfected chickens of the anticoccidial agent sulphaquinoxaline (SQ) labelled with ³⁵S was established at various time intervals after a single oral dose either alone or following continuous in-feed medication with unlabelled SQ, and after a single intravenous dose. The distribution was also established in chickens infected with the coccidia Eimeria acervulina or E. tenella , after a single oral dose of radiolabelled SQ administered either alone or following continuous in-feed medication with unlabelled SQ, as for uninfected chicks. In all uninfected chicks, SQ was rapidly absorbed from the gut and was distributed to all tissues. It appeared at high concentrations in the bile and kidneys 0.5 h after dosing. In chickens that had previously received unlabelled SQ in the diet, a radiolabelled dose maintained steadier tissue concentrations than the sharp rise and fall detected after a single oral dose. Intravenous dosing of uninfected chicks showed that SQ was secreted by the crop, gizzard and caecal epithelia into their lumina. Infection with E. acervulina or E. tenella coincided with an overall 3.5-fold sustained increase of SQ concentration in chick tissues. An updated hypothesis including these new observations for the anticoccidial mode of action of SQ in chickens is expounded.     

Increase of intestinal intraepithelial lymphocytes expressing CD8 antigen following challenge infection with Eimeria acervulina.

Eimerian infection-induced changes in the intestinal intraepithelial lymphocyte (IEL) subpopulations expressing CD8 antigen (cytotoxic/suppressor T cells) or antigen-specific T cell receptor (TCR) heterodimer alpha beta (TCR2) or gamma delta (TCR1) were investigated in F2 crosses of 15I5 B-congenic chickens differing for the major histocompatibility complex (MHC). Duodenum TCR2+ IEL were increased in B2 B2 and B5 B5 chickens 7 days following secondary infection. Two-color immunofluorescence revealed that the majority of CD8+ cells in the duodenum intraepithelium of immune chickens expressed TCR2. A significant increase in the duodenum TCR2+CD8+ and TCR1+CD8+ IEL occurred in B2 B2 chickens, which developed significantly less oocyst production than the B5 B5 chickens following challenge infection. The results suggest that a significant increase in the duodenum CD8+ IEL may reflect an enhanced acquired immunity of B2 B2 chickens.