Differences in highly pathogenic avian influenza viral pathogenesis and associated early inflammatory response in chickens and ducks.

We studied the immunological responses in the lung, brain and spleen of ducks and chickens within the first 7 days after infection with H7N1 highly pathogenic avian influenza (HPAI). Infection with HPAI caused significant morbidity and mortality in chickens, while in ducks the infection was asymptomatic. The HPAI viral mRNA load was higher in all investigated tissues of chickens compared with duck tissues. In the lung, brain and spleen of HPAI-infected chickens, a high, but delayed, pro-inflammatory response of IL-6 and IL-1ß mRNA was induced, including up-regulation of IFN-ß, IFN-γ, TLR3 and MDA-5 mRNA from 1 day post infection (p.i.). Whereas in ducks already at 8 h p.i., a quicker but lower response was found for IL-6, IL-1ß and iNOS mRNA followed by a delayed activation of TLR7, RIG-I, MDA5 and IFN-γ mRNA response. Virus-infected areas in the lung of chickens co-localized with KUL-01⁺ (macrophages, dendritic cells), CD4⁺, and CD8α⁺ cells, during the first day after infection. However, only KUL-01⁺ cells co-localized with the virus after 1 day p.i. In ducks, CVI-ChNL-68.1⁺ (macrophage-like cells), CD4⁺ and CD8α⁺ cells and apoptosis co-localized with the virus within 8 h p.i. Apoptosis was detected in the brain and lung of HPAI-infected chickens after 2 days p.i. and apoptotic cells co-localized with virus-infected areas. In conclusion, excessive delayed cytokine inflammatory responses but inadequate cellular immune responses may contribute to pathogenesis in chickens, while ducks initiate a fast lower cytokine response followed by the activation of major pattern recognition receptors (TLR7, RIG-I, MDA5) and a persistent cellular response.

Sensitivity of broiler performance, organ weights and plasma constituents to amino acid supplementation and reused litter exposure using ideal protein-formulated rations.

Effects of amino acid supplementation to ideal protein (IP) formulated rations were investigated on growth performance, plasma metabolites and organ weights of broilers placed on 100% recycled (reused) litter. Day-old Ross308 male broilers were raised on either clean or reused litter and fed for three weeks on one of five isoenergetic diets, where an IP-based control diet (C) was compared with diets containing threonine (T) or arginine (A) at 25% above requirements, or with 1% supplemented glutamine (G), or with each amino acid added (TAG). Litter and diet treatments did not strongly interact on outcomes. Reused litter placement resulted in greater weight gain, smaller feed conversion ratio and heavier bursal weights (P < 0.05) compared to clean litter placement. Relative to C and T birds, TAG birds reduced weight gain and feed intake (P < 0.05). Plasma uric acid levels in G birds were greater than in C, T and A birds (P < 0.001). Collectively, since the outcomes of placement on reused litter increased performance and the control diet was IP formulated, the absence of increased growth performance in response to amino acid supplementation would be consistent with amino acids tested being excess to requirements.

Differential innate responses of chickens and ducks to low-pathogenic avian influenza.

Ducks and chickens are hosts of avian influenza virus, each with distinctive responses to infection. To understand these differences, we characterized the innate immune response to low-pathogenicity avian influenza virus H7N1 infection in chickens and ducks. Viral RNA was detected in the lungs of chickens from day 0.8 to 7, in ducks mainly at day 4. In both species, viral RNA was detected in the bursa and gut. Infection in chickens resulted in up-regulation of interferon (IFN)-α and IFN-ß mRNA, while in the ducks IFN-γ mRNA was strongly up-regulated in the lung and bursa. In chickens and ducks, all investigated pathogen recognition receptor (PRR) mRNAs were up-regulated; however, in the chicken lung Toll-like receptor (TLR)7 and melanoma differentiation-associated protein (MDA)-5 mRNA were strongly induced. TLR3, TLR7 and MDA-5 responses correlated with IFN-α and IFN-ß responses in chickens, but in ducks a correlation between IFN-α and TLR7, retinoic acid-inducible gene-I and MDA-5 was absent. We studied the responses of duck and chicken splenocytes to poly(I:C) and R848 analogues to analyse the regulation of PRRs without the interfering mechanisms of the influenza virus. This revealed IFN-α and IFN-γ responses in both species. MDA-5 was only strongly up-regulated in chicken splenocytes, in which time-related PRR responses correlated with the IFN-α and IFN-ß response. This correlation was absent in duck splenocytes. In conclusion, chickens and ducks differ in induction of MDA-5, TLR7 and IFN-α mRNA after an influenza virus infection in vivo and after in vitro stimulation with TLR antagonists.

ELISPOT and intracellular cytokine staining: novel assays for quantifying T cell responses in the chicken.

The measurement of T cell responses in chickens, not only for quantitative aspects but also for the qualitative nature of the responses, becomes increasingly important. However, there are very few assays available to measure T cell function. Therefore, we have developed enzyme-linked immunosorbent spot assay (ELISPOT) and an intracellular cytokine staining (ICCS) assay. ELISPOT assay for the detection of chicken interferon-gamma (ChIFN-gamma) production was set up and shown to be reproducible for both polyclonal and antigen-specific stimuli such as Newcastle disease virus (NDV). However, the ELISPOT assay lacks the ability to identify individual cytokine-producing cells. Separation of CD4+ and CD8+ T cell populations gave additional information, but appeared to have the disadvantage of a loss of cell interactions during stimulation. In a further refinement, individual cells were identifiable by ICCS, which gives the possibility to characterize for multiple characteristics, such as cytokine production and phenotype of the cell. Using ICCS, ChIFN-gamma production was evaluated. Although cells were detected at only low frequencies, polyclonal stimulation of peripheral blood mononuclear cell (PBMC) or spleen cells resulted in a significant increase in ChIFN-gamma production by CD4+ and CD8+ cells.

Quantification and phenotypic characterisation of peripheral IFN-γ producing leucocytes in chickens vaccinated against Newcastle disease.

The aim of this study was to optimise and evaluate an intracellular cytokine staining (ICS) assay for assessment of T cell IFN-γ responses in chickens vaccinated against Newcastle disease (ND). We aimed to validate currently available antibodies to chicken IFN-γ using transfected CHO cells. Moreover, this ICS assay was evaluated for use to detect mitogen and antigen induced IFN-γ production in chicken peripheral blood leucocytes. Chickens from an inbred white leghorn line containing two MHC haplotypes, B19 and B21, were divided into three experimental groups; one group was kept as naive controls, one group was vaccinated intramuscularly twice with a commercial inactivated ND virus (NDV) vaccine, and the last group was vaccinated orally twice with a commercial live attenuated NDV vaccine. PBMC were ex vivo stimulated with ConA or with NDV antigen. The ICS assay was used to determine the phenotype and frequency of IFN-γ positive cells. ConA stimulation induced extensive IFN-γ production in both CD3+TCRγδ+ (γδ T cells) cells and CD3+TCRγδ- cells (αß T cells), but no significant differences were observed between the experimental groups. Furthermore, a large proportion of the IFN-γ producing cells were CD3- indicating that other cells than classic T cells, secreted this cytokine. NDV antigen stimulation induced IFN-γ production but to a lower extent than ConA and with a large variation between individuals. The CD3+TCR1γδ-CD8α+ (CTL) population produced the highest NDV specific IFN-γ responses, with significantly elevated levels of IFN-γ producing cells in the B19 chickens vaccinated orally with live attenuated NDV vaccine. This was not the case in the B21 animals, indicating a haplotype restricted variation. In contrast, the CD3+TCR1γδ-CD4+ (Th) population did not show a significant increase in IFN-γ production in NDV stimulated samples which was in part due to a high number of IFN-γ producing cells after incubation with medium alone. In conclusion, an ICS assay for phenotyping of IFN-γ producing chicken leukocytes was set up that proved useful in identifying cytokine producing cells upon either mitogen or antigen-specific stimulation.

Modelling the Innate Immune Response against Avian Influenza Virus in Chicken.

At present there is limited understanding of the host immune response to (low pathogenic) avian influenza virus infections in poultry. Here we develop a mathematical model for the innate immune response to avian influenza virus in chicken lung, describing the dynamics of viral load, interferon-α, -ß and -γ, lung (i.e. pulmonary) cells and Natural Killer cells. We use recent results from experimentally infected chickens to validate some of the model predictions. The model includes an initial exponential increase of the viral load, which we show to be consistent with experimental data. Using this exponential growth model we show that the duration until a given viral load is reached in experiments with different inoculation doses is consistent with a model assuming a linear relationship between initial viral load and inoculation dose. Subsequent to the exponential-growth phase, the model results show a decline in viral load caused by both target-cell limitation as well as the innate immune response. The model results suggest that the temporal viral load pattern in the lungs displayed in experimental data cannot be explained by target-cell limitation alone. For biologically plausible parameter values the model is able to qualitatively match to data on viral load in chicken lungs up until approximately 4 days post infection. Comparison of model predictions with data on CD107-mediated degranulation of Natural Killer cells yields some discrepancy also for earlier days post infection.

Core alpha1-->3-fucose is a common modification of N-glycans in parasitic helminths and constitutes an important epitope for IgE from Haemonchus contortus infected sheep.

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex-type N-glycans from Arabidopsis thaliana, which contain R-GlcNAcbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-Asn (core alpha1-->3-Fuc) and Xylbeta1-->2Manbeta1-->4GlcNAcbeta1-R (core beta1-->2-Xyl) modifications, and honeybee phospholipase A2, which carries N-glycans that contain the core alpha1-->3-Fuc epitope. Evidence is presented that core alpha1-->3-fucosylated N-glycans bind a substantial part of the parasite specific IgE in serum of H. contortus infected sheep. These results suggest that the core alpha1-->3-Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE. In addition we show here that N-glycans carrying core alpha1-->3-Fuc and beta1-->2-Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since N-glycans containing the core alpha1-->3-Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope.

In situ immunocytochemical detection of cells containing antibodies specific for Eimeria tenella antigens.

A three-step immunocytochemical method for the in situ detection of antibodies specific for Eimeria tenella has been developed. The method is based on the binding of E. tenella antigens to antibodies in cryostat sections of chicken tissues and the recognition of these antigens by rabbit antiserum specific for E. tenella or mouse monoclonal antibodies specific for E. tenella. The rabbit antiserum and mouse monoclonal antibodies were revealed by the immunoperoxidase technique. Suspensions of sonicated sporulated oocysts, incubated with or without various concentrations of the non-ionic detergents Triton X-100 (TX-100) or Nonidet P-40 (NP-40), were used as antigen. Cells containing antibodies specific for E. tenella were detected only when detergent extracts of sonicated sporulated oocysts were used. After chickens were intravenously immunized with a suspension of sonicated sporulated oocyst antigen, cells containing antibodies specific for E. tenella antigens were detected in the red pulp of the spleen. After simultaneous immunoenzyme staining for isotype and antigen specificity, the E. tenella-specific antibody-containing cells were of the IgM isotype after the primary immunization and of the IgM and IgG isotype after the booster immunization. Immune complexes specific for E. tenella on the surfaces of follicular dendritic cells in the germinal centers were also stained. Chickens were also orally infected with sporulated oocysts. In these experiments, cells containing antibodies specific for E. tenella were detected in the lamina propria of the ceca and in the red pulp of the spleen. Specific immune complexes were also detected in the germinal centers of the cecal tonsils. When detergent extracts of sonicated sporulated oocysts were characterized by immunoblotting, rabbit antiserum specific for E. tenella reacted with proteins ranging in size from 16 kDa to 200 kDa, with major bands of 20 kDa, 24 kDa, 45 kDa, and 100 kDa. Monoclonal antibodies specific for E. tenella recognized only proteins of low molecular weight (20 kDa and 24 kDa) or high molecular weight (80-100 kDa). Immune chicken serum reacted with proteins of low and high molecular weight but especially with proteins of 100 kDa and 113 kDa. This method is the first by which immune complexes and cells containing antibodies specific for parasitic antigens can be detected in situ and may be of value for studies of the local humoral immune response to E. tenella in the mucosa of chickens.

Immunocytochemical techniques to investigate the pathogenesis of infectious micro-organisms and the concurrent immune response of the host.

For poultry as well as for mammalian species used for scientific research, many immunocytochemical techniques have been developed to investigate in detail the interaction between infectious micro-organisms and the nonspecific and specific immune systems of the host. In this review three techniques have been described with all technical details necessary to perform them correctly: (1) single immunocytochemical staining to detect the infectious micro-organisms in situ at their site of infection, (2) double immunocytochemical staining to visualize simultaneously the infectious micro-organism and the host cellular response to investigate their interactions, and (3) detection of plasma cells producing antibodies specific to the micro-organism. Of the three techniques the results are described when applied on chicken tissues infected with various micro-organisms, such as Marek's disease virus, chicken anemia virus, infectious bursal disease virus and Eimeria tenella.

Immune expulsion of the trichostrongylid Cooperia oncophora is associated with increased eosinophilia and mucosal IgA.

Previous experiments have shown that a primary infection with 100000 infective larvae of the trichostrongylid Cooperia oncophora allows discrimination between different type of responder animals based on the speed by which the parasite is expelled from the host. In most of the animals (intermediate responders) the expulsion occurs 35-42 days after infection. This experiment was carried out to investigate which mechanisms contribute to the clearance of the parasite from the intestine. Sequential necropsy of the animals 14, 28 and 42 days after infection together with a segmental division of the small intestine, allowed us to characterise essential components associated with development of immunity and expulsion of the parasite from its niche. The results show that during the patent phase of the infection the parasite preferentially resides in the proximal gut. Forty-two days after infection ongoing expulsion is characterised by a migration of the worms to the more distal part of the intestine. Expulsion of the adult worm population appears to be mast-cell independent and is associated with a significant increase in parasite-specific mucous IgA and IgG1 as well as with an influx of eosinophils in the intestinal lamina propria. Although we did not observe a specific lymphocyte recruitment into the intestinal mucosa, the accumulation of eosinophils seems to be mediated by CD4+ cells. We measured significant negative correlations between the number of eosinophils and the expulsion rate of the parasite expressed by sex ratio and ratio eggs per gram faeces. Parasite-specific mucosal IgA levels were negatively correlated to the fecundity of the worms, expressed as number of eggs per female worm. Our results describe the involvement of both eosinophils and mucosal IgA in the regulation of C. oncophora expulsion and suggest the development of a Th2 effector immune response.

T-cell mediated immune responses in calves primary-infected or re-infected with Cooperia oncophora: similar effector cells but different timing.

Cooperia oncophora is the most prevalent intestinal nematode of cattle occurring in Western Europe. Primary infection with 100000 third stage infective larvae (L3) induces acquired immunity in a high proportion of the animals but there is little information on immunity against re-infection. In the current experiment, the contribution of the T-cell mediated immunity in protection against re-infection with C. oncophora was investigated in detail. Priming elicited long-lasting protective immunity that was evidenced by a significantly decreased worm burden and egg excretion in primed animals compared to challenge control animals. Lymphocyte proliferation tests with excretory/secretory products (ESP) of C. oncophora and with three distinct ESP fractions indicated an enhanced reactivity in primed animals and suggested that by fractionating of ESP we selected for proteins involved in protective immunity against re-infection with C. oncophora. Phenotypic analysis of T cell subsets at diverse anatomical locations revealed that the enhanced reactivity of lymphocytes from peripheral blood and lymph nodes of the infected animals coincided with a significantly increased frequency of CD4(+) cells at these locations but a deceased frequency of CD4(+) cells in the lamina propria. These findings were independent of the immune status of the animals but more pronounced in the primed animals than in the challenge control animals. In addition we demonstrated that primary and secondary infections with C. oncophora were associated with two waves of eosinophils and that the kinetics of this cell population differed as a result of priming. Based on the observed correlations we propose that the early increase of eosinophils is T cell independent and merely a consequence of inflammation in the parasitised gut. In contrast, the second wave of eosinophils depends upon CD4(+) cells and correlations with parasitological parameters at this time point support a role of eosinophils as effector cells against adult stages of C. oncophora.

B cells and antibody response in calves primary-infected or re-infected with Cooperia oncophora: influence of priming dose and host responder types.

We investigated whether the generation of protective memory humoral immunity in Cooperia oncophora infected calves occurs in a dose-dependent way and whether it depends on the animal responder types. To this end, serum and mucus antibody responses were measured in animals primary-infected with 30000 or 100000 L3, treated with anthelmintics and subsequently challenged with 100000 L3. A detailed phenotypic and functional analysis of B cells was done in animals infected once or twice with 100,000 L3. Based on the similarity in parasitological variables of animals primed with 30000 or 100000 L3, we concluded that with these doses priming conferred protection in a dose-independent way. Upon challenge significant increases in Cooperia-specific serum and mucus IgG1 and IgA and total serum IgE titres were induced in primed animals in a dose-independent way. In contrast, intermediate and low responders differed in the onset of the production of Cooperia-specific serum IgG1. Furthermore, not only the onset but also the level of total serum IgE significantly differed between intermediate and low responders. Phenotypic and functional analysis of B lymphocytes revealed that (i). priming induced the generation of memory B cells which upon challenge readily differentiated into antibody secreting cells; (ii). sensitised B cells were more efficiently recruited to the intestinal effector sites; (iii). based on the expression of CD62L and CD86 two distinct B cell subpopulation could be differentiated. CD62L(+)CD86(-) B cells that were likely lymphocytes not yet activated and with an enhanced recirculation capacity, and CD62L(-)CD86(+) B cells that were activated B cells with a reduced recirculation ability; and finally (iv). the increased expression of CD86 and subsequent correlations with parameters of the T helper 2 immune response induced by C. oncophora, suggested that CD86- interactions are involved in the generation of protective immunity against Cooperia.

Protection in lambs vaccinated with Haemonchus contortus antigens is age related, and correlates with IgE rather than IgG1 antibody.

Protection by vaccination with excretory-secretory products (ES) from Haemonchus contortus, containing predominantly proteins of 15 and 24 kDa, against an experimental challenge infection depends on the age of the sheep. Vaccinated sheep 9, 6 or 3 months of age were protected for 83%, 77% and -34%, respectively. There was a significant difference in ES-specific serum IgE response but not in IgG1 response, after the last vaccination between the different age groups. In the protected 9-month-old animals, there was an increase up to 18 times the prevaccination levels, while the increase in the unprotected 3-month-old animals was at most 1.4 times. The 6-month-old animals showed an intermediate increase of approximately six times the prevaccination level. There was no correlation within the 9-month-old sheep between ES-specific IgE levels and protection, measured as worm burden. However, when the different age groups were combined, there was a positive correlation (r = 0.38) between protection and ES-specific IgE levels 1 week after the vaccination. At the end of the experiment, peripheral blood eosinophils and mast cell counts in abomasal tissue were also significantly higher in the vaccinated and challenged 9-month-old sheep than in the vaccinated and challenged 3-month-old or than in the 9-month-old sheep with challenge, but without vaccination. Increased serum IgE levels, eosinophilia and mucosal mast cell hyperplasia are the hallmarks of a Th2 response and were all demonstrated in protected, older sheep, but not in unprotected, younger sheep.

Identification of chicken Bu-1 alloantigens using the monoclonal antibody AV20.

The genetically polymorphic chicken antigen Bu-1 (chB6) has been identified by alloantisera raised against RPL line 6(3) (Bu-1a) and line 7(2) (Bu-1b) birds and subsequently by monoclonal antibodies (mAbs) which identify individual alloantigens. We have produced a monoclonal antibody, AV20, which recognises a monomorphic determinant on the antigen Bu-1. AV20 identifies a marker on both bursal and peripheral B cells. Staining characteristics on bursa, spleen, thymus and peripheral blood lymphocytes are similar to those of the allotypic antibodies which identify Bu-1a and Bu-1b. However, AV20 identified B cells in partially inbred birds as well as inbred lines including line 6(1) and line 7(2), indicating that it recognises a monomorphic determinant, AV20 immunoprecipitated an antigen with a Mwr of 150 kDa under non-reducing conditions and 70-75 kDa under reducing conditions indicating it is a homodimer. Serial immunoprecipitations or bursal-cell lysates from line 6(1) or line 7(2) confirmed that AV20 recognised the same antigen as mAbs against Bu-1a and Bu-1b in the respective lines.

Eimeria tenella infections in chickens: aspects of host-parasite: interaction.

Intestinal coccidiosis, caused by various species of Eimeria, has become an economically important disease of poultry and livestock throughout the world. Infection of chickens starts after ingestion of oocysts when sporozoites penetrate the epithelium of the villi. After passage through the lamina propria, they enter crypt epithelial cells where they undergo several rounds of asexual and sexual proliferation, thus forming merozoites and later, gametocytes. When macrogametes are fertilized by microgametes, oocysts are formed that are shed in the faeces. Nowadays, coccidiosis is prevented by anticoccidial drugs that are added to food, but the prolonged use of these drugs leads inevitably to the emergence of resistant Eimeria strains. During infection, there are three stages when the chicken immune system can inhibit parasitic development. The first is when the sporozoite searches for a site of penetration and binds to the epithelium. The second is when the sporozoite is in the villus epithelium amongst intra-epithelial leucocytes. The third is during its passage through the lamina propria to the crypt epithelium. To investigate this, the decisive factors in the induction and effector phase of immunity against coccidiosis have been investigated in situ. Our studies have revealed that three phenomena are responsible for immunity against Eimeria infections. First, the actual passage and presence of parasites in the lamina propria to induce immunity. Second, the sporozoite seems to be the most important parasite stage for immunity, and third, cytotoxic T cells are necessary to inhibit parasites.

Induction of a local and systemic immune response using cholera toxin as vehicle to deliver antigen in the lamina propria of the chicken intestine.

In this study, the humoral mucosal immune response to a recombinant Eimeria antigen (Ea1A) was enhanced using cholera toxin (CT). Chickens were primed intra-intestinally with Ea1A either conjugated or not to CT. The local and systemic antibody responses to both Ea1A and CT were determined to find out whether the chickens could respond to CT and whether both antigens had reached the lamina propria. In addition the effects of CT on lamina propria leukocytes were examined. The results showed that chickens had receptors on the caecal epithelium that could bind CT. At day 7 after administration, the number of CD4+ and CD8+ T lymphocytes in the lamina propria of the caecum had increased, indicating that CT had a specific immunological effect. At this timepoint, anti-CT antibody containing cells were detected locally in the lamina propria of the caecum. In serum all antigen preparations containing CT induced IgM and IgG antibody titres specific for CT within 10 days after priming. In addition, the recombinant Ea1A antigen also induced serum responses when administered together with CT or conjugated to CT, thus both CT and the antigen had reached the lamina propria. Nevertheless, the Ea1A specific response was much higher in the primary response and after booster immunization when the antigen was conjugated to CT than when only mixed with CT. Therefore, we conclude that CT is a suitable adjuvant for intra-intestinal application in chickens, especially when the antigen is conjugated to it.

Estimating genetic differences in natural resistance in Rhön and Merinoland sheep following experimental Haemonchus contortus infection.

Genetic parameters of natural resistance were estimated in Rhön and Merinoland (German Merino) sheep following experimental infection with Haemonchus contortus. A total of 133 Rhön and 244 Merinoland lambs descending from 5 and 6 rams, respectively, were evaluated. Each helminth-naive lamb was orally infected with 5000 infective third-stage larvae (L(3)) of the nematode H. contortus at 12 weeks of age. Faecal egg counts (FEC) and haematocrit values were measured in all lambs at 16 and 20 weeks of age. Seventy-nine Merinoland and 29 Rhön male lambs were slaughtered immediately after the second sampling and worms were collected. Mean worm burden was calculated and the length of adults worms from an aliquot was measured.FEC of Rhön sheep was higher compared with Merinoland sheep (P<0.01). H. contortus L(3)-larvae specific antibody (IgL) level was higher in Rhön sheep (P<0.05). However, no differences in haematocrit, worm burden and IgG antibody values could be found between the breeds. Heritabilities for log FEC (+/-S.E.) were 0.0 and 0.07 (+/-0.07) for the first sample in Rhön and Merinoland sheep, respectively. Values for the second sample were higher in both breeds (Rhön 0.35+/-0.14, P<0.05; Merinoland 0.17+/-0.07, P<0.05). Corresponding heritabilities for haematocrit were higher in Merinoland (0.56+/-0.20 and 0.51+/-0.27) compared with Rhön (0.29+/-0.12 and 0.08+/-0.13). Heritabilities for worm burden were high in Rhön (0.54+/-0.2) and low in Merinoland (0.06+/-0.14 and 0.11+/-0.15). Estimated values for IgL were between 0.13 (+/-0.11) for the first sample in both breeds and 0.30 (+/-0.18) for the second sample in Rhön sheep. Corresponding heritabilities for IgG were not different from 0.0 in both breeds (P>0.05). Positive phenotypic correlations were estimated for IgG and IgL values in both breeds (P<0.01). IgG was significantly (P<0.05) and positively correlated with worm burden in male Merinoland and IgL with worm burden in male Rhön sheep.

In vitro inhibition of Eimeria tenella invasion of epithelial cells by phytochemicals.

Resistance to coccidiostats and possible future restrictions on their use raise the need for alternative methods of reducing coccidiosis in poultry. The aim of this study was to evaluate the effect of selected phytochemicals on Eimeria tenella sporozoite invasion in vitro. Four phytochemicals were selected on the basis that they reduce the virulence of Eimeria spp. and/or provide immune modulatory benefits to host cells: betaine, carvacrol, curcumin and Echinacea purpurea extract (EP). Madin-Darby bovine kidney (MDBK) cells were covered by medium containing phytochemicals at the highest concentration which was non-toxic to the cells. Salinomycin 50 µg/ml was positive control; negative control was medium only. E. tenella (Houghton strain) sporozoites were added to wells and after incubation for 2, 4 or 20 h at 37°C, cells were fixed and stained with hematoxylin-eosin. Ten evenly spaced fields per well were photographed and the percentage of cells invaded by sporozoites was calculated and normalized to the control. At 2h, carvacrol, curcumin and EP showed a significantly lower percentage of sporozoite invasion than the untreated control; in contrast, betaine treatment represented a significantly higher invasion percentage. Combining carvacrol with EP inhibited E. tenella invasion more effectively than applying the compounds individually, but the further addition of curcumin did not reduce invasion further. In conclusion, this study shows that invasion of MDBK epithelial cells by E. tenella sporozoites is inhibited in the presence of carvacrol, curcumin, or EP and enhanced by betaine. There may be potential for developing these phytochemicals as anti-coccidial feed or water additives for poultry.

Rapid NK-cell activation in chicken after infection with infectious bronchitis virus M41.

Natural killer (NK) cells are cytotoxic lymphocytes and play an important role in the early defence against viruses. In this study we focussed on NK cell and interferon (IFN) responses after infection with infectious bronchitis virus (IBV). Based on surface expression of CD107+, enhanced activation of lung NK cells was observed at 1 dpi, whereas in blood prolonged NK-cell activation was found. IFN-α and IFN-ß mRNA and proteins were not rapidly induced whereas IFN-γ production in lung, measured by Elispot assay, increased over time at 2 and 4 dpi. In contrast, IFN-γ production in blood was highest at 1 dpi and decreased over time down to levels comparable to uninfected birds at 4 dpi. Collectively, infection with IBV-M41 resulted in activation of NK cells in the lung and blood and rapid production of IFN-γ and not IFN-α and IFN-ß compared to uninfected birds.