Visualisation and characterisation of mononuclear phagocytes in the chicken respiratory tract using CSF1R-transgenic chickens.

The respiratory tract is a key organ for many avian pathogens as well as a major route for vaccination in the poultry industry. To improve immune responses after vaccination of chickens through increased uptake of vaccines and targeting to antigen presenting cells, a better understanding of the avian respiratory immune system is required. Transgenic MacReporter birds were used expressing a reporter gene (eGFP or mApple) under the control of the CSF1R promoter and enhancer in cells of the mononuclear phagocyte (MNP) lineage to visualize the ontogeny of the lymphoid tissue, macrophages and dendritic cells, in the trachea, lung and air sac of birds from embryonic day 18-63 weeks of age. Small aggregates of CSF1R-transgene+ cells start to form at the openings of the secondary bronchi at 1 week of age, indicative of the early development of the organised bronchus-associated lymphoid tissue. Immunohistochemical staining revealed subpopulations of MNPs in the lung, based on expression of CSF1R-transgene, CD11, TIM4, LAMP1, and MHC II. Specialised epithelial cells or M cells covering the bronchus-associated lymphoid tissue expressed CSF1R-transgene and type II pneumocytes expressed LAMP1 suggesting that these epithelial cells are phagocytic and transcytose antigen. Highly organised lymphoid tissue was seen in trachea from 4 weeks onwards. Throughout the air sacs at all ages, CSF1R-transgene+ cells were scattered and at later stages, CSF1R-transgene+ cells lined capillaries. These results will serve as a base for further functional characterization of macrophages and dendritic cells and their role in respiratory diseases and vaccine responses.

A zebrafish larval model reveals early tissue-specific innate immune responses to Mucor circinelloides.

Mucormycosis is an emerging fungal infection that is clinically difficult to manage, with increasing incidence and extremely high mortality rates. Individuals with diabetes, suppressed immunity or traumatic injury are at increased risk of developing disease. These individuals often present with defects in phagocytic effector cell function. Research using mammalian models and phagocytic effector cell lines has attempted to decipher the importance of the innate immune system in host defence against mucormycosis. However, these model systems have not been satisfactory for direct analysis of the interaction between innate immune effector cells and infectious sporangiospores in vivo. Here, we report the first real-time in vivo analysis of the early innate immune response to mucormycete infection using a whole-animal zebrafish larval model system. We identified differential host susceptibility, dependent on the site of infection (hindbrain ventricle and swim bladder), as well as differential functions of the two major phagocyte effector cell types in response to viable and non-viable spores. Larval susceptibility to mucormycete spore infection was increased upon immunosuppressant treatment. We showed for the first time that macrophages and neutrophils were readily recruited in vivo to the site of infection in an intact host and that spore phagocytosis can be observed in real-time in vivo. While exploring innate immune effector recruitment dynamics, we discovered the formation of phagocyte clusters in response to fungal spores that potentially play a role in fungal spore dissemination. Spores failed to activate pro-inflammatory gene expression by 6 h post-infection in both infection models. After 24 h, induction of a pro-inflammatory response was observed only in hindbrain ventricle infections. Only a weak pro-inflammatory response was initiated after spore injection into the swim bladder during the same time frame. In the future, the zebrafish larva as a live whole-animal model system will contribute greatly to the study of molecular mechanisms involved in the interaction of the host innate immune system with fungal spores during mucormycosis.

Acute aerocystitis in Nile tilapia bred in net cages and supplemented with chromium carbochelate and Saccharomyces cerevisiae.

Oreochromis niloticus bred in net cages were supplemented with cell wall of Saccharomyces cerevisiae (Sc) (0.3%) or chromium carbochelate (Cr) (18 mg/kg of feed) or in association (Sc + Cr), for 90 days. After this period, acute inflammation was induced in the swim bladder by inoculation of 3 × 10(8) CFU of inactivated Streptococcus agalactiae, and another group received 0.65% saline solution (control). Twelve, 24, and 48 h after stimulation, the inflammation was evaluated through total and differential counting of accumulated cells, and through leukocyte respiratory burst in the blood, cortisolemia, glycemia and serum lysozyme concentration. The results showed that there were greater total numbers of cells in the exudate of fish inoculated with inactivated bacterium than in those injected with saline solution, with predominance of lymphocytes, thrombocytes, macrophages and granulocytes. Tilapia supplemented with Cr presented increased total numbers of cells with significant accumulation of lymphocytes and reductions in cortisolemia and glycemia, but the different treatments did not have any influence on leukocyte respiratory burst or serum lysozyme concentration. Tilapia supplemented with Sc and the Cr + Sc association did not present significant changes to the variables evaluated, despite higher accumulation of lymphocytes in the inflammatory exudate from fish treated with Sc. The results indicate that tilapia bred in net cages and supplemented with Cr presented higher total accumulation of cells at the inflammatory focus, thus indicating an increase in the inflammatory response induced by the bacterium, probably due to the reduction in cortisolemia and higher glucose consumption. Thus, supplementation with Cr had beneficial action, which facilitated development of acute inflammation induced by the bacterium, but did not affect neither leukocyte respiratory burst in the blood nor serum lysozyme concentration.

Recombinant Iss as a potential vaccine for avian colibacillosis.

Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a disease which is responsible for significant losses in poultry. Control of colibacillosis is problematic due to the restricted availability of relevant antimicrobial agents and to the frequent failure of vaccines to protect against the diverse range of APEC serogroups causing disease in birds. Previously, we reported that the increased serum survival gene (iss) is strongly associated with APEC strains, but not with fecal commensal E. coli in birds, making iss and the outer membrane protein it encodes (Iss) candidate targets for colibacillosis control procedures. Preliminary studies in birds showed that their immunization with Iss fusion proteins protected against challenge with two of the more-commonly occurring APEC serogroups (O2 and O78). Here, the potential of an Iss-based vaccine was further examined by assessing its effectiveness against an additional and widely occurring APEC serogroup (O1) and its ability to evoke both a serum and mucosal antibody response in immunized birds. In addition, tissues of selected birds were subjected to histopathologic examination in an effort to better characterize the protective response afforded by immunization with this vaccine. Iss fusion proteins were administered intramuscularly to four groups of 2-wk-old broiler chickens. At 2 wk postimmunization, chickens were challenged with APEC strains of the O1, O2, or O78 serogroups. One week after challenge, chickens were euthanatized, necropsied, any lesions consistent with colibacillosis were scored, and tissues from these birds were taken aseptically. Sera were collected pre-immunization, postimmunization, and post-challenge, and antibody titers to Iss were determined by enzyme-linked immunosorbent assay (ELISA). Also, air sac washings were collected to determine the mucosal antibody response to Iss by ELISA. During the observation period following challenge, 3/12 nonimmunized chickens, 1/12 chickens immunized with 10 microg of GST-Iss, and 1/12 chickens immunized with 50 microg of GST-Iss died when challenged with the O78 strain. No other deaths occurred. Immunized chickens produced a serum and mucosal antibody response to Iss and had significantly lower lesion scores than nonimmunized chickens following challenge, regardless of the challenge strain. This study expands on our previous report of the value of Iss as an immunoprotective antigen and demonstrates that immunization with Iss can provide significant protection of chickens against challenge with three different E. coli strains.

The European eel--the swim bladder-nematode system provides a new view of the invasion paradox.

It is widely assumed that the likelihood of invasion decreases with increased species richness in the recipient community. However, the invasion paradox supports a negative and a positive relationship between native biodiversity and the success of an invader. Here, we show that for a host-parasite system (Anguilla anguilla as host and Anguillicoloides crassus as parasitic invader), invasion increases with native micro- and macroparasitic species richness. In fact, about 30% of the A. crassus intensity in eels could be explained by the number of both micro- and macroparasite species. This pattern could be due to the fact that A. crassus exploits a niche (the swim bladder) that is unoccupied by native parasite species and by the Th1/Th2 trade-off between native microparasites and the invader. We conclude that the host-parasite system resistance to invasion may depend on both niche availability and the Th1/Th2 trade-off. As well, we encourage researchers to incorporate native parasite richness as a risk factor in epidemiological models of A. crassus.

Course of infection and immune responses in the respiratory tract of IBV infected broilers after superinfection with E. coli.

Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. The aim of this study was to investigate how IBV affects the course of events upon infection with E. coli. Broilers were inoculated with IBV H120 vaccine virus or virulent M41 and challenged 5 days later with E. coli 506. A PBS and E. coli group without previous virus inoculation were included. Sections of trachea, lung and airsacs were stained for CD4, CD8, gammadelta-TCR, alphabeta1-TCR, and for macrophages (KUL-01) and both pathogens. Changes in the mucociliary barrier of trachea, lung and airsacs did not predispose for bacterial superinfection. The disease in the lungs of the E. coli group and both IBV/E. coli groups was similar. Lesions in the airsacs were more pronounced and of longer duration in the IBV/E. coli groups. The immunocytological changes differed substantially between the E. coli group and both IBV/E. coli groups. In trachea, lungs and airsacs the CD4+ and CD8+ populations were significantly larger than in the E. coli and PBS groups. In the lungs and the airsacs the macrophages were more numerous in the IBV/E. coli and the E. coli groups than in the PBS group. The presence of high numbers of T cells and macrophages in IBV infected birds most likely induced an altered immune response, which is responsible for the enhanced clinical signs of colibacillosis.

Migratory response of European eel (Anguilla anguilla) phagocytes to the eel swimbladder nematode Anguillicola crassus.

The migratory response of peripheral blood granulocytes and monocytes from the European eel Anguilla anguilla (L.) to infective larvae of the swimbladder nematode Anguillicola crassus Kuwahara, Niimi and Hagaki, 1974 was examined by means of light microscopical histology and with an in vitro assay using a modified Boyden chamber. Histological examination of experimentally infected eels revealed that, already 8 days postinfection, an infiltration of inflammatory cells around L3 of A. crassus in the swimbladder tissue can occur. In the Boyden chamber, in presence of infective larvae of A. crassus (L3), neutrophil granulocytes and monocytes showed a higher migration activity than in the absence of L3. In conclusion, infection of European eels with A. crassus leads to an activation of the defence cells resulting in an increased migration activity compared to uninfected eels.

IFN-gamma enhances immune responses to E. coli infection in the chicken.

Escherichia coli infection of the respiratory system in chickens occurs as a sequel to a variety of environmental stressors or microbial infections, culminating as chronic respiratory disease (CRD) syndrome or colibacillosis. These diseases cause significant production losses in poultry. With the growing concerns about the use of antibiotics in animal production, for diseases such as CRD, alternative natural agents, like cytokines, may be considered for enhancing health by stimulating the immune system. The current study was aimed at understanding the in vivo effects of recombinant chicken interferon-gamma (ChIFN-gamma) treatment on a variety of immunologic parameters during E. coli infection in chickens. Administration of ChIFN-gamma to chickens increased the percentage of phagocytes in lung and blood of E. coli-infected birds. At the phenotypic level, there was an increase in the percentage of cells expressing MHC II in the air sac, with a concomitant reduction in the proportion of these cells in blood. Furthermore, the blood plasma from ChIFN-gamma-treated infected birds showed an increased level of interleukin-6 (IL-6) activity. Cumulatively, these findings are indicative of in vivo enhancement of immune responses due to ChIFN-gamma. However, administration of ChIFN-gamma protein did not mitigate the development of air sac lesions following E. coli infection.

The avian lung-associated immune system: a review.

The lung is a major target organ for numerous viral and bacterial diseases of poultry. To control this constant threat birds have developed a highly organized lung-associated immune system. In this review the basic features of this system are described and their functional properties discussed. Most prominent in the avian lung is the bronchus-associated lymphoid tissue (BALT) which is located at the junctions between the primary bronchus and the caudal secondary bronchi. BALT nodules are absent in newly hatched birds, but gradually developed into the mature structures found from 6-8 weeks onwards. They are organized into distinct B and T cell areas, frequently comprise germinal centres and are covered by a characteristic follicle-associated epithelium. The interstitial tissue of the parabronchial walls harbours large numbers of tissue macrophages and lymphocytes which are scattered throughout tissue. A striking feature of the avian lung is the low number of macrophages on the respiratory surface under non-inflammatory conditions. Stimulation of the lung by live bacteria but not by a variety of bacterial products elicits a significant efflux of activated macrophages and, depending on the pathogen, of heterophils. In addition to the cellular components humoral defence mechanisms are found on the lung surface including secretory IgA. The compartmentalisation of the immune system in the avian lung into BALT and non BALT-regions should be taken into account in studies on the host-pathogen interaction since these structures may have distinct functional properties during an immune response.

Composite cellular defence stratagem in the avian respiratory system: functional morphology of the free (surface) macrophages and specialized pulmonary epithelia.

Qualitative and quantitative attributes of the free respiratory macrophages (FRMs) of the lung--air sac systems of the domestic fowl (Gallus gallus variant domesticus) and the muscovy duck (Cairina moschata) were compared with those of the alveolar macrophages of the lung of the black rat (Rattus rattus). The birds had significantly fewer FRMs compared to the rat. In the birds, the FRMs were found both in the lungs and in the air sacs. Under similar experimental conditions, the most robust FRMs were those of the domestic fowl followed by those of the rat and the duck. Flux of macrophages onto the respiratory surface from the subepithelial compartment and probably also from the pulmonary vasculature was observed in the birds but not in the rat. In the duck and the domestic fowl, a phagocytic epithelium that constituted over 70% of the surface area of the blood-gas (tissue) barrier lines the atrial muscles, the atria and the infundibulae. The epithelial cells of the upper respiratory airways contain abundant lysosomes, suggesting a high lytic capacity. By inference, the various defence strategies in the avian lung may explain the dearth of FRMs on the respiratory surface. We counter-propose that rather than arising directly from paucity of FRMs, an aspect that has been over-stressed by most investigators, the purported high susceptibility of birds (particularly table birds) to respiratory ailments and afflictions may be explained by factors such as inadequate management and husbandry practices and severe genetic manipulation for fast growth and high productivity, manipulations that may have weakened cellular and immunological defences.

Safety, immunogenicity, and efficacy of two Escherichia coli cya crp mutants as vaccines for broilers.

Attenuated derivatives (delta cya delta crp mutants) of an O2 and an O78 avian septicemic Escherichia coli strain were used to immunize broiler chickens by spray to determine the safety, immunogenicity, and efficacy of the derivatives in single- and double-dose regimens. In the safety and immunogenicity studies, groups of 10 chickens were vaccinated by spray (droplet size approximately 20 microm) with the parent E. coli, the mutant organisms, or phosphate-buffered saline (PBS) at 14 days of age and euthanatised 21 days later. There was no deaths or gross pathologic finding in any of the chickens immunized with the vaccine strains. Compared with the levels in chickens exposed to PBS, there were significantly higher levels of immunoglobulin (Ig) G antibody in serum and air sac washings and of IgA antibody in air sac washings in response to the virulent parent strains than to the vaccine strains. In efficacy studies, chickens were immunized with the O2 or the O78 vaccine strain or PBS at day 14 and with the O2 vaccine strain or PBS at days 10 and 14 and challenged with the parent strain 10 days after the last vaccination. There was no significant difference in local IgA and IgG and serum IgG responses between vaccinated and control groups. Chickens vaccinated with the O2 strain, but not the O78 strain, had significantly lower air sac lesion scores compared with those of the unvaccinated groups in both single- and double-dose regimens. We conclude that the mutant O2 strain provided moderate protection against airsacculitis.

Evaluation of an ELISA and immunoblotting for studying the humoral immune response in Anguillicola crassus infected European eel Anguilla anguilla.

The applicability of an enzyme-linked immunosorbent assay (ELISA) for the detection of anguillicolosis in feral eels was examined using a crude antigen preparation from the body wall of adult Anguillicola crassus. The screening consisted of samples from 100 feral European eels Anguilla anguilla. As a reference the actual status of infection was determined by dissection of the eels' swim-bladders. The ELISA results were compared with a background value calculated from the results obtained from 43 non-infected farm eels. The screened samples had a high prevalence of A. crassus (83 %); however, the specificity and the negative predictive value of the ELISA were low compared to the high positive predictive value. Nonetheless, the reproducibility (precision) of the test was satisfactory, and for the non-infected reference group specificity was 97.7 %. Although the ELISA, as used in the present study, is not applicable for diagnostic purposes, it represents a useful tool for the investigation of the specific humoral immune response of eels against A. crassus under controlled experimental conditions. Immunoblots using crude antigen preparations from different parts of adult A. crassus as well as a crude somatic third-stage (L3) antigen preparation illustrated that only antigens associated with the body wall of adult A. crassus are potentially suitable for diagnostic purposes. Despite the fact that antibodies against Raphidascaris acus cross-reacted with 3 body wall antigens of A. crassus, the most encouraging results were obtained with the antigen preparation from the outer cuticle of adult A. crassus which yielded a conspicuous, broad band at about 100 kDa.

Degranulation of eosinophilic granular cells with possible involvement in neutrophil migration to site of inflammation in tilapia.

Observations were made on stretched preparations from the swim bladder and peritoneum obtained from tilapia (Oreochromis niloticus) after injection of formalin-killed Escherichia coli, proteose peptone, compound 48/80 or HBSS into the swim bladder. The eosinophilic granular cells (EGCs) in the swim bladder degranulated rapidly after inoculation. However, the peritoneal EGCs did not degranulate, indicating that degranulation occurs only at the injected site. There was also a correlation between the ratio of the degranulated EGCs and number of the exudate neutrophils. Killed E. coli (1 mg/fish) produced the greatest degranulation response of EGCs and migration of neutrophils into the inflammatory site. Additionally, the rate of the degranulation and number of the neutrophils were lowest when HBSS was injected. The results of this study verify that degranulation of EGCs is involved in the neutrophil migration and suggest that fish EGCs are analogous to mammalian mast cells.

Avian influenza A virus induced stunting syndrome-like disease in chicks.

Two-day-old specific-pathogen free chicks were inoculated with type A influenza virus (A/whistling swan/Shimane/499/83 (H5N3) through the air sac. Inoculated chicks showed mild to severe diarrhea and lesions of pancreatitis and atrophy of the pancreas, thymus and bursa of Fabricius. One chick died on each of days 4, 6 and 14 postinoculation (PI). Reduced weight gain was conspicuous from day 22 PI. Positive immunoreaction to the virus antigen was detected in the pancreas, kidneys, liver, lungs and air sacs, and cecal lamina propria. Virus recovery persisted longer in the pancreas. Some of these findings conformed to those of stunting syndrome.

Cellular defense of the avian respiratory system: protection against Escherichia coli airsacculitis by Pasteurella multocida-activated respiratory phagocytes.

The concept of nonspecific cellular defense of the respiratory system of poultry against respiratory pathogens by "preventive activation" of avian respiratory phagocytes (ARPs) was tested in an in vivo protection trial. Chickens were stimulated intratracheally by Pasteurella multocida Choloral vaccine strain. Seven hours later, these and mock-inoculated control chickens were challenged with pathogenic Escherichia coli via the air-sac route. Stimulated chickens had a 25-fold-elevated number of ARPs compared with mock-inoculated control chickens. The proportion of active phagocytes and the phagocytic capacity of these cells was higher in the ARP populations of stimulated chickens than in the ARP populations of control chickens. In vivo protection against E. coli air-sac infection was demonstrated by reduction of morbidity and mortality rates, diminished weight loss, and lower scores of gross and histopathological lesions of P. multocida-stimulated chickens compared with mock-inoculated controls.

Studies on immunological air pouch inflammation in the rat.

The development of a novel immune-based model of inflammation in the subcutaneous air pouch of the rat is described. Rats were sensitized by injection with methylated bovine serum albumin emulsified with Freund's complete adjuvant and challenged 14 days later by injection of heat-aggregated antigen in saline into a 6-day subcutaneous air pouch. Time course studies revealed a peak polymorph cell infiltration at 24 h with no exudate formation, followed by a separate mononuclear cell response, rapid exudate formation, and granuloma formation over the 2- to 7-day period. Separate peaks of increased vascular permeability were observed in the acute and chronic phases of the response. This reaction was considered to be an example of delayed-type hypersensitivity based on its extended time course, the characteristic time course of oedema formation in foot-pad-challenged rats, and the failure of serum from sensitized rats to elicit a reversed passive Arthus-type reaction. The sensitization schedule was also shown to enhance the acute inflammatory response to carrageenan. In addition, the 'flare-up' reaction to secondary antigenic challenge was investigated in 7-day postchallenge lesions.

Local immunity against Newcastle disease virus in the newly hatched chicken's respiratory tract.

Inoculation of 200 mean egg infectious doses (EID(50)) of lentogenic Newcastle disease virus strain B1 (NDV-B1) into the air sac of 4-day-old specific-pathogen-free chicks provided significant protection against challenge of the air sac with 100 chicken mean lethal doses (LD(50)) of velogenic NDV-H but no protection against reinfection when the challenge was by the eye. Conversely, inoculation of the eye with 200 EID(50) of NDV-B1 provided significant protection against challenge of the eye but not of the air sac with 100 chicken LD(50) of NDV-H. Birds that received both antiserum and intraocular immunization were subsequently protected against both eye and air-sac challenge. On the other hand, birds that received antiserum and air-sac immunization were protected only against air-sac challenge but not against ocular challenge. Low levels of passively administered antibody did not prevent infection of the eye or air sac but greatly reduced the mortality rate after inoculation of either the vaccine or the challenge viruses. Passively administered antibody also suppressed hemagglutination-inhibiting and virus-neutralizing antibody formation stimulated by air-sac infection but not antibody formation stimulated by ocular infection. These data are consistent with the hypothesis that local immunity is responsible for prevention of infection, since birds were immune to reinfection at one site and simultaneously susceptible at the other site of infection.