Analysis of antibody levels in egg yolk for detection of exposure to Ascaridia galli parasites in commercial laying hens.

Ascaridia galli is one of the most abundant nematode parasites in poultry. A. galli infections can significantly impact the profitability of egg farms and have negative implications for bird health and welfare. The main objectives of this study were to determine whether A. galli specific antibodies in egg yolks can be used to detect prior or current exposure to A. galli in laying hens, and to distinguish between eggs obtained from caged and free-range hens. Twenty-two laying hen flocks from different production systems (10 free-range, 2 barn-housed, and 9 caged flocks) were enrolled in the study. An in-house enzyme-linked immunosorbent assay was used to analyze levels of A. galli specific antibodies in yolk. The numbers of A. galli eggs in hen excreta were also determined in a subset of farms. Free-range flocks had higher and also more variable levels of anti-A. galli antibodies in the egg yolk compared to those of the cage flocks (0.50 ± 0.39 vs. 0.16 ± 0.13 OD units) (P < 0.001). Results also confirmed that excreta from free-range and barn-housed flocks contained higher numbers of A. galli eggs than did excreta from caged flocks in which no A. galli eggs were detected. In conclusion, analysis of anti-A. galli antibodies in the egg yolk can be used to detect worm exposure in commercial layer flocks. However, the method used in this study cannot be used in isolation to distinguish between eggs from cage and free-range production systems as anti-A galli antibodies were detected in egg yolk samples from all production systems, and the range of antibody levels overlapped between production systems.

Co-expulsion of Ascaridia galli and Heterakis gallinarum by chickens.

Worm expulsion is known to occur in mammalian hosts exposed to mono-species helminth infections, whilst this phenomenon is poorly described in avian hosts. Mono-species infections, however, are rather rare under natural circumstances. Therefore, we quantified the extent and duration of worm expulsion by chickens experimentally infected with both Ascaridia galli and Heterakis gallinarum, and investigated the accompanying humoral and cell-mediated host immune responses in association with population dynamics of the worms. Results demonstrated the strong co-expulsion of the two ascarid species in three phases. The expulsion patterns were characterized by non-linear alterations separated by species-specific time thresholds. Ascaridia galli burden decreased at a daily expulsion rate (e) of 4.3 worms up to a threshold of 30.5 days p.i., followed by a much lower second expulsion rate (e = 0.46), which resulted in almost, but not entirely, complete expulsion. Heterakis gallinarum was able to induce reinfection within the experimental period (9 weeks). First generation H. gallinarum worms were expelled at a daily rate of e = 0.8 worms until 36.4 days p.i., and thereafter almost no expulsion occurred. Data on both humoral and tissue-specific cellular immune responses collectively indicated that antibody production in chickens with multispecies ascarid infections is triggered by Th2 polarisation. Local Th2 immune responses and mucin-regulating genes are associated with the regulation of worm expulsion. In conclusion, the chicken host is able to eliminate the vast majority of both A. galli and H. gallinarum in three distinct phases. Worm expulsion was strongly associated with the developmental stages of the worms, where the elimination of juvenile stages was specifically targeted. A very small percentage of worms was nevertheless able to survive, reach maturity and induce reinfection if given sufficient time to complete their life cycle. Both humoral and local immune responses were associated with worm expulsion.

Immune responses following experimental infection with Ascaridia galli and necrotic enteritis in broiler chickens.

Broilers commonly suffer from necrotic enteritis (NE). Other gastrointestinal infectious diseases affect poultry, including nematode infections which are considered a re-emerging disease in barn and free-range systems. The aim of this study was to characterize the immune response of broilers after artificial infection with NE and contrast these with responses to the nematode Ascaridia galli and determine whether immune parameters measured during the course of infection can be used to distinguish infected from uninfected birds. A total of 96 one-day-old male Ross 308 broiler chickens were used in this study. At 10 days of age, broilers were randomly assigned to one of the following treatment groups: control birds (n = 32), A. galli infected birds (n = 32), or NE infected birds (n = 32) and inoculated with the appropriate infective agents. The immune response of birds was monitored through evaluation of haematology parameters, acute phase protein production, and intraepithelial intestinal lymphocyte population changes at 11, 16, 20, and 32 days of age. T-helper cells (CD4+CD8-) increased significantly over time, and were significantly higher in A. galli and NE compared to day 10 controls. In conclusion, α-1 glycoprotein levels can distinguish birds with NE from other birds, including those infected with A. galli; also T-helper cell numbers can distinguish both NE and A. galli from uninfected birds and thirdly, 10 days post infection is the best time point to evaluate the bird's immune response for A. galli infections.

A comprehensive evaluation of an ELISA for the diagnosis of the two most common ascarids in chickens using plasma or egg yolks.

BACKGROUND: Classical faecal egg counts (FEC) provide less reliable diagnostic information for nematode infections in chickens. We developed an ELISA based on Ascaridia galli antigens and tested two hypotheses, as follows: (i) IgY antibodies developed against A. galli will also be useful to identify Heterakis gallinarum infections, and (ii) circulating antibodies stored in egg yolks are as good as plasma samples, so a non-invasive diagnosis is possible. The aim of this study, therefore, was to compare the diagnostic accuracy of the ELISA system with FEC, using both plasma and egg yolks from experimentally infected hens. In addition, naturally infected animals were evaluated to validate the assay. RESULTS: The assay quantified large differences (P < 0.001) in plasma or in egg-yolk IgY concentrations between infected and uninfected animals in two experiments, each performed with either of the nematode species. The assay performed with high accuracy as quantified with the area under the ROC curve (AUC) values of > 0.90 for both nematodes using either plasma or egg yolks. Sensitivity of the assay was 94 and 93% with plasma and egg yolk samples, respectively, whereas FEC yielded in a sensitivity of 84% in A. galli experiment. Total test accuracy of the assay with plasma samples (AUC = 0.99) tended to be higher (P = 0.0630) than FEC (AUC = 0.92) for A. galli, while the assay with either sample matrix performed similar to FEC (AUC ≥ 0.91) for H. gallinarum. Among the three tests, the FECs correlated better with A. galli burden than the ELISA. Although 90% of naturally infected hens were correctly identified by the ELISA, 45% of the infected hens tested negative with FEC, indicating the validity of the higher test accuracy of the ELISA. CONCLUSIONS: Antigens of A. galli can be used successfully to identify H. gallinarum-infected animals, indicating that chickens develop cross-reactive antibodies against the two closely related species. Egg yolks are as informative as plasma samples, so that animal welfare-friendly sampling is possible. Although the assay with plasma samples reveals qualitative information of higher quality than FECs on the infection status of naturally infected birds, the latter is still a better tool to assess the intensity of A. galli but not of H. gallinarum infections.

Maternal protection against Ascaridia galli?

Maternally derived antibodies can provide partial protection against certain bacterial and viral infections. We investigated whether chicks descending from nematode-infected hens are more resistant against Ascaridia galli, a prevalent gastrointestinal nematode, than chicks from nematode-free mothers. One-day-old chicks (N=153) from infected (mab+; maternal antibody+) or uninfected control dams (mab-) were experimentally infected with A. galli at two different levels (100 or 1000 eggs/chick). The worm burdens of the chicks were determined at 6 weeks post infection. There was a high correlation (r=0.89) between A. galli-specific antibody concentrations in dam plasma and egg yolk. There was no difference between worm burdens of chicks descending from infected or uninfected dams (P=0.892), indicating no maternally derived protection against A. galli. Chicks receiving the higher infection dose had higher worm burdens (P<0.05). Although there was no difference (P>0.05) between worm counts of female and male chicks infected with 100 eggs, females chicks infected with 100 eggs harboured longer and heavier female worms. We conclude that there is no protective maternal immunity against A. galli infection.

Immune gene expression in the spleen of chickens experimentally infected with Ascaridia galli.

Ascaridia galli is a gastrointestinal nematode infecting chickens. Chickens kept in alternative rearing systems or at free-range experience increased risk for infection with resulting high prevalences. A. galli infection causes reduced weight gain, decreased egg production and in severe cases increased mortality. More importantly, the parasitised chickens are more susceptible to secondary infections and their ability to develop vaccine-induced protective immunity against other diseases may be compromised. Detailed information about the immune response to the natural infection may be exploited to enable future vaccine development. In the present study, expression of immune genes in the chicken spleen during an experimental infection with A. galli was investigated using the Fluidigm(®) BioMark™ microfluidic qPCR platform which combines automatic high-throughput with attractive low sample and reagent consumption. Spleenic transcription of immunological genes was compared between infected chickens and non-infected controls at week 2, 6, and 9 p.i. corresponding to different stages of parasite development/maturation. At week 2 p.i. increased expression of IL-13 was observed in infected chickens. Increased expression of MBL, CRP, IFN-α, IL-1ß, IL-8, IL-12ß and IL-18 followed at week 6 p.i. and at both week 6 and 9 p.i. expression of DEFß1 was highly increased in infected chickens. In summary, apart from also earlier reported increased expression of the Th2 signature cytokine IL-13 we observed only few differentially expressed genes at week 2 p.i. which corresponds to the larvae histotrophic phase. In contrast, we observed increased expression of pro-inflammatory cytokines and acute phase proteins in infected chickens, by week 6 p.i. where the larvae re-enter the intestinal lumen. Increased expression of DEFß1 was observed in infected chickens at week 6 p.i. but also at week 9 p.i. which corresponds to a matured stage where adult worms are present in the intestinal lumen.

Cytokine gene expression profiles in chicken spleen and intestinal tissues during Ascaridia galli infection.

In the poultry production industry, chickens with access to outdoor areas are exposed to a wide range of parasites e.g. the helminth Ascaridia galli. By real-time quantitative RT-PCR, the relative gene expression of the T helper 1 (Th1) cytokine IFN-γ, the T helper 2 (Th2) cytokine IL-13, the anti-inflammatory cytokines IL-10 and TGF-ß4 and the pro-inflammatory cytokine IL-17F were determined over a period of 3 weeks in A. galli and non-A. galli-infected chickens. A characteristic Th2 response was observed in the jejunum of A. galli-infected chickens with increased expression of IL-13 and decreased expression of IFN-γ from day 14 post infection. At the putative time of larvae invasion into the intestinal mucosa (day 7), an increased expression of IFN-γ, IL-10, and TGF-ß4 was observed in the spleen. At the putative onset of the innate immune response (day 10), a decreased expression of jejunal IFN-γ and IL-13 was observed. Finally, at the expected period of an adaptive immune response (days 14-21) a general decreased expression of IFN-γ and TGF-ß4 in spleen and IFN-γ in jejunum was followed by a decreased expression of IFN-γ and IL-10 at day 21 in caecal tonsils.

Ascaridia galli infection influences the development of both humoral and cell-mediated immunity after Newcastle Disease vaccination in chickens.

Potent vaccine efficiency is crucial for disease control in both human and livestock vaccination programmes. Free range chickens and chickens with access to outdoor areas have a high risk of infection with parasites including Ascaridia galli, a gastrointestinal nematode with a potential influence on the immunological response to vaccination against other infectious diseases. The purpose of this study was to investigate whether A. galli infection influences vaccine-induced immunity to Newcastle Disease (ND) in chickens from an MHC-characterized inbred line. Chickens were experimentally infected with A. galli at 4 weeks of age or left as non-parasitized controls. At 10 and 13 weeks of age half of the chickens were ND-vaccinated and at 16 weeks of age, all chickens were challenged with a lentogenic strain of Newcastle disease virus (NDV). A. galli infection influenced both humoral and cell-mediated immune responses after ND vaccination. Thus, significantly lower NDV serum titres were found in the A. galli-infected group as compared to the non-parasitized group early after vaccination. In addition, the A. galli-infected chickens showed significantly lower frequencies of NDV-specific T cells in peripheral blood three weeks after the first ND vaccination as compared to non-parasitized chickens. Finally, A. galli significantly increased local mRNA expression of IL-4 and IL-13 and significantly decreased TGF-ß4 expression in the jejunum two weeks after infection with A. galli. At the time of vaccination (six and nine weeks after A. galli infection) the local expression in the jejunum of both IFN-? and IL-10 was significantly decreased in A. galli-infected chickens. Upon challenge with the NDV LaSota strain, viral genomes persisted in the oral cavity for a slightly longer period of time in A. galli-infected vaccinees as compared to non-parasitized vaccinees. However, more work is needed in order to determine if vaccine-induced protective immunity is impaired in A. galli-infected chickens.

No protection in chickens immunized by the oral or intra-muscular immunization route with Ascaridia galli soluble antigen.

In chickens, the nematode Ascaridia galli is found with prevalences of up to 100% causing economic losses to farmers. No avian nematode vaccines have yet been developed and detailed knowledge about the chicken immune response towards A. galli is therefore of great importance. The objective of this study was to evaluate the induction of protective immune responses to A. galli soluble antigen by different immunization routes. Chickens were immunized with a crude extract of A. galli via an oral or intra-muscular route using cholera toxin B subunit as adjuvant and subsequently challenged with A. galli. Only chickens immunized via the intra-muscular route developed a specific A. galli antibody response. Frequencies of γδ T cells in spleen were higher 7 days after the first immunization in both groups but only significantly so in the intra-muscularly immunized group. In addition, systemic immunization had an effect on both Th1 and Th2 cytokines in caecal tonsils and Meckel's diverticulum. Thus both humoral and cellular immune responses are inducible by soluble A. galli antigen, but in this study no protection against the parasite was achieved.

Comparison of parasite-specific immunoglobulin levels in two chicken lines during sustained infection with Ascaridia galli.

Increasingly large numbers of poultry are held in production systems with access to outdoor areas. In these systems intestinal helminths are found with flock prevalences of up to 100%. Helminth infections influence chicken health negatively, which is why the following investigation has been performed. In the present experiment, 20 chickens of two inbred chicken lines containing the major histocompatibility complex (MHC) haplotypes, B14 and R5, were inoculated with 500 embryonated Ascaridia galli eggs. The A. galli-specific IgG titres of serum samples and the excretion of A. galli eggs in chicken faeces were measured for a period of 81 weeks. The level of excreted A. galli eggs measured as eggs per gram chicken faeces (EPG) varied greatly between chickens in each line. Significant differences were found between the two lines and with the R5 chickens reaching the highest levels. Likewise, the A. galli-specific IgG titres in serum differed significantly between the two lines, and an inverse relationship between infection level (EPG) and antibody titres was found. Although this inverse relationship suggests that humoral immunity may be involved in protection against A. galli infection, the high antibody titres did not prevent continued infection.

Immunopathogenesis of Ascaridia galli infection in layer chicken.

Gastro-intestinal nematode infections in mammals are associated with local T lymphocyte infiltrations, Th2 cytokine induction, and alterations in epithelial cell secretion and absorption. This study demonstrates that Ascaridia (A.) galli infection in chicken also elicits local gut-associated immune reactions and changes in the intestinal electrogenic nutrient transport. In A. galli-infected birds we observed infiltrations of different T cell populations in the intestinal lamina propria and accumulation of CD4+ lymphocytes in the epithelium. The Th2 cytokines IL-4 and IL-13 dominated the intestinal immune reactions following A. galli infection. A. galli-specific systemic IgY antibodies were detected after two weeks post infection, and did only poorly correlate with detected worm numbers. Electrogenic transport of alanin and glucose was impaired in A. galli-infected chicken. Our data provide circumstantial evidence that local immune responses and electro-physiological intestinal functions may be connected and contribute to the elimination of worm infection.

MHC haplotype and susceptibility to experimental infections (Salmonella Enteritidis, Pasteurella multocida or Ascaridia galli) in a commercial and an indigenous chicken breed.

In three independent experimental infection studies, the susceptibility and course of infection of three pathogens considered of importance in most poultry production systems, Ascaridia galli, Salmonella Enteritidis and Pasteurella multocida were compared in two chicken breeds, the indigenous Vietnamese Ri and the commercial Luong Phuong. Furthermore, the association of the Major Histocompatibility Complex (MHC) with disease-related parameters was evaluated, using alleles of the LEI0258 microsatellite as markers for MHC haplotypes. The Ri chickens were found to be more resistant to A. galli and S. Enteritidis than commercial Luong Phuong chickens. In contrast, the Ri chickens were more susceptible to P. multocida, although production parameters were more affected in the Luong Phuong chickens. Furthermore, it was shown that the individual variations observed in response to the infections were influenced by the MHC. Using marker alleles of the microsatellite LEI0258, which is located within the MHC region, several MHC haplotypes were identified as being associated with infection intensity of A. galli. An association of the MHC with the specific antibody response to S. Enteritidis was also found where four MHC haplotypes were shown to be associated with high specific antibody response. Finally, one MHC haplotype was identified as being associated with pathological lesions and mortality in the P. multocida experiment. Although not statistically significant, our analysis suggested that this haplotype might be associated with resistance. These results demonstrate the presence of local genetic resources in Vietnamese chickens, which could be utilized in breeding programmes aiming at improving disease resistance.

Antibody and inflammatory responses in laying hens with experimental primary infections of Ascaridia galli.

Ascaridia galli, an intestinal nematode that affects hens and other domestic and wild birds, causes economic losses in avian exploitations. The present work shows that A. galli stimulates a strong antibody response as well as an intense inflammatory reaction, in the intestinal mucous of experimentally infected Lohmann Brown laying hens. IgG antibodies against soluble extracts of A. galli embrionated eggs and adult worms, were detected in both blood and yolks eggs from infected hens during a period of 105 days after the infection. This indicates that hens transfer to their offspring a part of the IgG antibodies produced when they become infected. The antigens responsible for the stimulation of specific IgG were molecules of 30-34, 44-54 and 58-90 kDa, while in the yolk eggs of infected hens a reactivity directed against antigens of molecular weight (M(w)) lower than 50 kDa was detected. Histology revealed traumatic lesions with leukocyte infiltration, and inflammation of the intestinal wall of the infected hens after 105 days of initial infection. The possible influence of the immune and inflammatory response on the population dynamics of the parasite is discussed.

Case of retinochoroiditis associated with porcine ascarids diagnosed by antibodies in subretinal fluid.

PURPOSE: To determine the cause of retinochorioditis in a patient with a granulomatous retinal exudate and an exudative retinal detachment. CASE: A 45-year-old man presented at another hospital with increased visual disturbances of the left eye. He was diagnosed with uveitis, and treated with topical steroids for 1 month. However, the uveitis worsened, and he was referred to our hospital. Ophthalmoscopy showed a yellowish-white granulomatous exudate, and an exudative retinal detachment in the lower peripheral retina. The retinal detachment worsened and affected the macula. Pars plana vitrectomy was performed, and the retina was reattached. During the surgery, ocular samples were collected for further examinations. The titers of antibodies against 12 kinds of ascaridis were examined, and elevated titers of specific antibodies against porcine ascarids were detected in the subretinal fluid, but not in the aqueous humor, vitreous, or serum. CONCLUSIONS: Vitrectomy with the collection of ocular samples, especially subretinal fluid, was a key procedure in the diagnosis and treatment of retinochoroiditis associated with the porcine ascarids.

A coprological and serological survey for the prevalence of Ascaridia spp. in laying hens.

Ascaridia galli is a common nematode found in the intestine of domesticated chickens. The objectives of the study were to conduct a coprological and serological survey on the prevalence of ascaridiosis in laying hens of commercial farms. The farms recently adopted a breeding programme, where the hens have access to outdoor pens. Different amounts of Ascaridia eggs were detected in five of seven studied farms, while the other two farms were found to be free from the parasite. Serological tests revealed a seroprevalence of 21.8% (range 7.6-95%). No positive serum samples were detected in the same farms with previous negative coprological analysis. Western blot analyses confirmed the results obtained by the enzyme-linked immunosorbent assay (ELISA) test. In four experimentally infected hens, a progressive increase of the IgG antibody levels was observed, surpassing the cut-off point established for ELISA test 6 weeks post-infection. Serological tests are able to detect the infection before the eggs of the parasite appear in the faeces of infected hens, providing a useful tool to detect infections with Ascaridia spp. in avian farms.

Partial purification and characterization of Ascaridia galli diagnostic worm antigen.

Partial purification of Ascaridia galli whole worm extract was conducted by Cyanogen bromide Sepharose 4B immunoaffinity column chromatography. The resulted fraction was characterized by sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The fraction was found to be consisted of six bands of 207 KDa, 157 KDa. 110 KDa, 103 KDa, 76 KDa and 41 KDa. This profile was compared with that of whole worm and excretory-secretory antigens. Both antigens were resolved into multiple bands in both high and low molecular weight ranges. The isoelectric focusing of the fraction displayed 8 bands of isoelectric points 7.5, 7.0, 6.8, 6.5, 6.2, 5.8. 5.3 and 4.6. The potency of this fraction in the diagnosis of natural ascaridiosis in chickens was assessed by ELISA compared with that of whole worm and ES antigens. The affinity purified fraction showed higher potentials in the diagnosis of A. galli infection in chickens than whole worm antigen at any sera dilution and than ES antigen at high sera dilutions. While ES antigen of the worms revealed higher diagnostic capabilities than whole worm extract. The current research recommends utilization of the affinity isolated fraction in the diagnosis of natural ascaridiosis in chickens.

Th1/Th2 polarization by viral and helminth infection in birds.

Mammals developed an immune system able to functionally polarize into so-called type 1 or type 2 immune pathways, to resolve infections with intracellular and extracellular pathogens, respectively. In the well-studied avian immune system of the chicken, however, no evidence for polarized immunity could be found, as yet. To investigate whether these two major arms of mammalian immunity, regulated by a T helper (Th)1/Th2 cytokine balance, evolved similarly in birds, chickens were exposed to a prevalent intracellular (viral) or extracellular (helminth) infection. By using semi-quantitative RT-PCR analysis we provide evidence that polarization of Th1/Th2 type immunity extends beyond mammalian species, and, therefore, has been evolutionary conserved for more than 300 million years, when the lineages of mammalian and avian vertebrates are assumed to have segregated.

Host age only partially affects resistance to primary and secondary infections with Ascaridia galli (Schrank, 1788) in chickens.

Two experiments were conducted to compare the effect of chickens' age on resistance to primary and secondary infections with Ascaridia galli. In Experiment I, three groups, each of 80 female Lohman Brown chickens, aged one day, one month, or four months were compared. Within each group, 54 chickens were infected orally with 500 embryonated eggs and 26 were kept as non-infected controls. Weights were recorded weekly and five chickens in each group were slaughtered every 2 weeks for worm counts. At week 10 post-infection, 17 of the infected chickens and 18 of the controls were challenged with 500 eggs. In a replicate experiment (Experiment II), 35 one-day-old and 53 one-month-old female Lohman Brown chickens were infected orally with 500 A. galli eggs. Weights and fecal egg counts were recorded every week and infected chickens were necropsied every two weeks for determination of the worm burden. Chickens infected at one month of age excreted significantly fewer A. galli eggs when measured at 14 weeks of inoculation. The worms recovered from the one-month-old age group were significantly shorter than those from the chickens infected at one day of age in the first experiment. Worm burden and female fecundity values, however, were not significantly different between age groups in both Experiments I and II. Weight gains of infected chickens were not significantly different from the controls' and only a few chickens exhibited occasional slight diarrhea in both experiments. The results from these experiments demonstrate that the chickens' age only partially influences resistance to A. galli infection.

Immunization of chicks at various ages with irradiated infective eggs of Ascaridia galli.

The possibility of safe immunization of chicks at an appropriate age with a double-dose irradiated Ascaridia galli vaccine given orally at two weeks interval was explored. Chicks immunized at 7 or 10 days of age were not affected adversely since they did not develop any clinical signs and there was no worm establishment after challenge infection. Immunization also elicited detectable circulating antibody titres, with IHA and the conglutinating complement absorption test having a tendency to be enhanced after the booster dose.

Effect of irradiated Ascaridia galli eggs on growth and cell-mediated immune responses in chickens.

Growth and cell-mediated immune (CMI) responses were studied in 7-day-old chicks given orally 1000 irradiated (12.5 kR) or normal infective eggs of Ascaridia galli. Chicks immunised with irradiated eggs showed normal weight gains. CMI responses, as assessed by dinitrofluorobenzene (DNFB)-induced contact and delayed hypersensitivity reactions, were enhanced in the immunised group as compared with healthy controls, suggesting stimulation of CMI responses due to irradiation of A. galli eggs. CMI as well as growth responses were, however, found to be depressed in the birds administered normal infective eggs of A. galli. The present study highlights the role of the CMI response in protection against A. galli infection.