Lonneke Vervelde

In situ characterization of leucocyte subpopulations after infection with Eimeria tenella in chickens

We characterized the leucocyte subpopulations after infection with Eimeria tenella in both naive and immune chickens. Immunocytochemical staining was used to characterize the cells in situ, so that the interaction between host and parasite could be studied. More leucocytes were detected in the lamina propria of immune chickens, and leucocytes infiltrated the ceca more rapidly than in naive chickens, but the infiltration was less pronounced than in naive chickens. In naive chickens, most infiltrated leucocytes were macrophages and T cells. Two days after inoculation the number of CD4+ cells had increased greatly. In immune chickens, mainly T cells (CD4+ and CD8+) infiltrated the lamina propria, and in contrast to naive chickens, the number of CD8+ cells exceeded the number of CD4+ cells. Furthermore, we characterized which cells contained a parasite and which cells were detected next to the parasites, because these cells are probably involved in the arrested development of the parasites. In naive chickens, sporozoites were significantly more often located within or next to macrophages than in immune chickens. In immune chickens, sporozoites were significantly more often located within or next to CD3+, CD8+, and TCR2+ cells. In conclusion, the marked increase of CD4+ cells after primary infection suggests that these cells are involved in the induction of the immune response, whereas the increase of CD8+ cells after challenge infection suggests that these cells act as effector cells.

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.

Structure of the Avian Lymphoid System

Our ability to understand the function, pathology and regeneration ability of an organ system is handicapped without knowledge of its normal structure. Electron microscopy and, later, immunocytochemistry made it possible to extend our knowledge of the structure of cells and the communication between them. These novel techniques helped to recognize the avian dendritic cells. Contemporary research on the structure of the lymphoid system contributed to recognition of the microenvironment and secretory functions of the primary lymphoid organs. Determining the histology of organs, using electron microscopy and immunocytochemistry of cells, will catalyze interactions between morphologists and immunologists to create novel ideas and hypotheses, and will result in a more comprehensive understanding of avian immunology. This chapter is a comprehensive immune-morphological report on the primary and secondary lymphoid organs of birds.

Postnatal development of intra-epithelial leukocytes in the chicken digestive tract: phenotypical characterization in situ

In the present study, we characterized intra-epithelial leukocytes in the digestive tract of chickens during postnatal development. Their phenotype was characterized by monoclonal antibodies in cryostat sections and the numbers of the different cell-types were counted in the epithelium of the esophagus, proventriculus, duodenum, jejunum, cecum, and colon. All intra-epithelial leukocytes bore the leukocyte-common antigen CD45; 35% were T lymphocytes, and 50% bore a B-cell marker. However, no immunoglobulin-bearing cells were detected in the epithelium. Monocytes and macrophages were found only in the epithelium of the esophagus. A remaining population of non-B, non-T, non-monocyte cells (15%) was present in all parts of the digestive tract. The number of intra-epithelial leukocytes was greatest in the duodenum and jejunum, and decreased in the proximal part of the cecum and in the colon. Intra-epithelial leukocytes were only sporadically detected in the proventriculus. The total number of intra-epithelial leukocytes increased until 8 weeks after hatching and then decreased at 18 months. In the esophagus, the total number of intra-epithelial leukocytes changed little during aging. We found that the intra-epithelial leukocytes of chickens and rodents are distinct in that chicken intra-epithelial leukocytes comprise a cell population that bears a B-cell antigen but that lacks surface immunoglobulins.

Functional CD1d and/or NKT cell invariant chain transcript in horse, pig, African elephant and guinea pig, but not in ruminants

CD1d-restricted invariant natural killer T cells (NKT cells) have been well characterized in humans and mice, but it is unknown whether they are present in other species. Here we describe the invariant TCR α chain and the full length CD1d transcript of pig and horse. Molecular modeling predicts that porcine (po) invariant TCR α chain/poCD1d/α-GalCer and equine (eq) invariant TCR α chain/eqCD1d/α-GalCer form complexes that are highly homologous to the human complex. Since a prerequisite for the presence of NKT cells is the expression of CD1d protein, we performed searches for CD1D genes and CD1d transcripts in multiple species. Previously, cattle and guinea pig have been suggested to lack CD1D genes. The CD1D genes of European taurine cattle (Bos taurus) are known to be pseudogenes because of disrupting mutations in the start codon and in the donor splice site of the first intron. Here we show that the same mutations are found in six other ruminants: African buffalo, sheep, bushbuck, bongo, N’Dama cattle, and roe deer. In contrast, intact CD1d transcripts were found in guinea pig, African elephant, horse, rabbit, and pig. Despite the discovery of a highly homologous NKT/CD1d system in pig and horse, our data suggest that functional CD1D and CD1d-restricted NKT cells are not universally present in mammals.

Highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses

Highly pathogenic avian influenza (HPAI) H5N1 viruses cause severe infection in chickens at near complete mortality, but corresponding infection in ducks is typically mild or asymptomatic. To understand the underlying molecular differences in host response, primary chicken and duck lung cells, infected with two HPAI H5N1 viruses and a low pathogenicity avian influenza (LPAI) H2N3 virus, were subjected to RNA expression profiling. Chicken cells but not duck cells showed highly elevated immune and pro-inflammatory responses following HPAI virus infection. HPAI H5N1 virus challenge studies in chickens and ducks corroborated the in vitro findings. To try to determine the underlying mechanisms, we investigated the role of signal transducer and activator of transcription-3 (STAT-3) in mediating pro-inflammatory response to HPAIV infection in chicken and duck cells. We found that STAT-3 expression was down-regulated in chickens but was up-regulated or unaffected in ducks in vitro and in vivo following H5N1 virus infection. Low basal STAT-3 expression in chicken cells was completely inhibited by H5N1 virus infection. By contrast, constitutively active STAT-3 detected in duck cells was unaffected by H5N1 virus infection. Transient constitutively-active STAT-3 transfection in chicken cells significantly reduced pro-inflammatory response to H5N1 virus infection; on the other hand, chemical inhibition of STAT-3 activation in duck cells increased pro-inflammatory gene expression following H5N1 virus infection. Collectively, we propose that elevated pro-inflammatory response in chickens is a major pathogenicity factor of HPAI H5N1 virus infection, mediated in part by the inhibition of STAT-3.

Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage

We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens.

Vaccination-induced IgG response to Galα1–3GalNAc glycan epitopes in lambs protected against Haemonchus contortus challenge infection

Lambs vaccinated with Haemonchus contortus excretory/secretory (ES) glycoproteins in combination with the adjuvant Alhydrogel are protected against H. contortus challenge infection. Using glycan micro-array analysis we showed that serum from such vaccinated lambs contains IgG antibodies that recognise the glycan antigen Galalpha1-3GalNAc-R and GalNAcbeta1-4(Fucalpha1-3)GlcNAc-R. Our studies revealed that H. contortus glycoproteins contain Galalpha1-3Gal-R as well as significant levels of Galalpha1-3GalNAc-R, which has not been previously reported. Extracts from H. contortus adult worms contain a galactosyltransferase acting on glycan substrates with a terminal GalNAc, indicating that the worms possess the enzymatic potential to synthesise terminal Gal-GalNAc moieties. These data illustrate that glycan micro-arrays constitute a promising technology for fast and specific analysis of serum anti-glycan antibodies in vaccination studies. In addition, this approach facilitates the discovery of novel, antigenic parasite glycan antigens that may have potential for developing glycoconjugate vaccines or utilization in diagnostics.

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.

Progression of lesions in the respiratory tract of broilers after single infection with Escherichia coli compared to superinfection with E. coli after infection with infectious bronchitis virus

The progression of Escherichia coli lesions was studied in the respiratory tract of 4-week-old commercial broilers. Lesions were induced after a single intratracheal E. coli infection, and after an infection with E. coli preceded 5 days earlier by an oculo-nasal and intratracheal infectious bronchitis virus (IBV) infection of either the virulent M41 strain or the H120 vaccine strain. Trachea, lung and thoracic airsac lesions were examined macroscopically and microscopically. Tissue samples were taken at 3h post-inoculation (hpi), and 1, 2, 4 and 7 days post-inoculation (dpi) with E. coli. The location of both pathogens was assessed by immunohistochemistry. Single E. coli inoculation induced pneumonia and airsacculitis; in case it was preceded by IBV infection, the same macroscopical lesions and also viral tracheitis were found. No clear difference existed between the single and dual infected birds with respect to inflammatory reactions in the lung, which had disappeared within 7 days, except for the presence of more follicles in dual infected birds. IBV antigen was detected in secondary bronchi and airsacs up to 2 dpi and in the trachea up to 4 dpi. E. coli bacteria were found in the tracheal lumen included in purulent material, the parabronchi and airsacs. In lung tissue E. coli antigen was found up to 4 dpi. No clear difference existed between single and dual inoculated birds regarding the presence of E. coli in the lung. In the airsacs, a few bacteria were found from 0.5 hpi up to 4 dpi in E. coli and IBV-E. coli inoculated birds. Although both pathogens were cleared beyond detection at 7 dpi, in IBV-E. coli inoculated birds lesions in the airsac persisted, in contrast to broilers inoculated with E. coli only. In the present study it is shown that 4-week-old broilers are not resistant to intratracheal E. coli inoculation, however, these birds can overcome the induced E. coli infection within a short time span. Moreover, a preceding infection with vaccine or virulent IBV does not seem to impair the clearance of E. coli in the respiratory tract of broilers, but rather induces an exaggerated inflammatory response in the airsacs only, which seems to be the mechanism behind the pattern of airsacculitis in commercial poultry in the field.