Ascaridia galli populations in chickens following single infections with different dose levels

In all, 3 groups of 20 Lohman Brown chickens aged 1 day were orally infected with doses of 100, 500, or 2,500 embryonated Ascaridia galli eggs, respectively. After 8 weeks, egg counts (eggs per gram of feces, EPG) were determined for all animals prior to slaughter. The gastrointestinal tracts were examined for the presence of adult and immature stages of A. galli. All groups had roughly similar worm burdens and, hence, significantly different establishment rates of 14.2%, 2.9%, and 0.5%, respectively. A significantly lower mean female worm burden was seen in the high-dose group (P = 0.02), which also showed a significantly lower level of egg excretion (P = 0.01). However, fecundity (EPG per female) did not significantly differ between the groups (P = 0.55). The mean lengths of adult worms as well as the weight of the mean worm burdens were significantly smaller in the high-dose group. This study demonstrated that single infections with varying doses of A. galli eggs influenced the establishment rate, sex ratio, egg excretion, and worm size and weight but not the worm fecundity. Received: 8 December 1996 / Accepted: 29 January 1997     

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.     

Molecular and parasitological tools for the study of Ascaridia galli population dynamics in chickens

Experiments were first conducted to compare and evaluate different methods of Ascaridia galli larval recovery from the chicken intestine. The number of larvae recovered from the intestinal wall of chickens infected with 1000 embryonated A. galli eggs and killed 15 days post infection (p.i.) by three methods (ethylenediamine tetraacetic acid [EDTA], pepsin digestion and scraping) were compared. The EDTA and pepsin digestion were found to be the most efficient methods with no significant difference (P > 0.05) in the number of recovered larvae between the two. Subsequently, three different A. galli cohorts were established using the polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) technique. A 533-bp long region of the cytochrome c oxidase subunit 1 gene of the mitochondrial DNA was targeted and 22 A. galli females were allocated to three different haplotypes. The four females with the highest embryonation rate from each haplotype group (total 12 females) were selected and used to inoculate each of 12 chickens with a dose of 1000 embryonated eggs. The chickens were killed 15 days p.i. and A. galli larvae were recovered from the small intestinal wall by the EDTA method and by sieving the lumen content on a 90 µm sieve. DNA of 40 larvae from each of the three different haplotypes was extracted using a worm lysis buffer, and PCR-RFLP analysis of these larvae revealed the same haplotype as that of their maternal parent. The identification of distinguishable cohorts may be a powerful tool in population studies of parasite turnover within the animal host.     

Adjuvant activity of monophosphoryl lipid A for nasal and oral immunization with soluble or liposome-associated antigen

The effectiveness of monophosphoryl lipid A (MPL) as a mucosal adjuvant was investigated following oral or intranasal (i.n.) administration of an aqueous adjuvant formulation of MPL (MPL-AF) added to soluble antigen or liposomal antigen or incorporated into liposomal antigen membranes. Groups of BALB/c female mice were immunized with 50 to 100 microg of free or liposomal Streptococcus mutans crude glucosyltransferase (C-GTF) with or without MPL-AF added to the vaccine or incorporated into the liposomal membrane. Plasma, saliva, vaginal wash, and fecal extract samples were collected biweekly following immunization and assessed for antigen-specific antibody activity by enzyme-linked immunosorbent assay (ELISA). Mice immunized by the i.n. route had higher levels of salivary, plasma, and vaginal immunoglobulin A (IgA) anti-C-GTF responses and higher levels of plasma IgG anti-C-GTF than the orally immunized groups. A second administration of the vaccine 14 weeks after the initial immunization resulted in an anamnestic response to C-GTF resulting in 10- and 100-fold increases in saliva and plasma IgA and plasma IgG, respectively (in the i.n. immunized groups). Mice receiving a second i.n. immunization with liposomal antigen and MPL-AF had higher salivary IgA anti-C-GTF responses than mice immunized with antigen plus MPL-AF or liposomal antigen (P < 0.05). Plasma IgG anti-C-GTF activity was highest in mice immunized by the i.n. route with antigen formulations containing MPL-AF (P < 0.05). These results demonstrate the effectiveness of MPL-AF as an adjuvant for potentiating mucosal and systemic immune responses to liposomal C-GTF following i.n. immunization.     

Effect of the pH of Ascaridia galli egg culture medium on the experimental infection in chickens]

1. A prolonged preservation of Ascaridia galli eggs in acid medium when cultivating them under laboratory conditions inhibits considerably the establishment of Ascaridia galli in the organism of chickens. The neutralization of medium of egg cultivation 3 days before their application for infesting facilitates the increase of larvae established in the organism. 2. In chickens invaded with Ascaridia galli eggs cultivated in medium with pH 8.0, larvae develop in the period of 7--21 days of invasion in the amount of 2.5--69 times more than in chickens receiving the same eggs but which have been cultivated in acid medium (pH 2.2) for the whole cultivation period. 3. With the increase in invasion period, the number of established larvae decreases in both test groups, but in chickens receiving eggs which have been cultivated in medium with pH 8.0, the number of larvae is considerably higher.     

Ascaridia galli in chickens: intestinal localization and comparison of methods to isolate the larvae within the first week of infection

This study was conducted to observe the localization and to compare methods for isolation of minute Ascaridia galli larvae in chicken intestine. Firstly, six 7-week-old layer pullets were orally infected with 2,000 embryonated A. galli eggs and necropsied either at 3, 5 or 7?days post infection (dpi). More than 95?% of the recovered larvae were obtained from the anterior half of the jejunoileum, suggesting this part as the initial predilection site for A. galli larvae. Secondly, the intestinal wall of one layer pullet infected with 20,000 A. galli eggs 3?days earlier was digested in pepsin?CHCl for 90?min. The initial 10?min of digestion released 51?% of the totally recovered larvae and the last 30?min of continuous digestion yielded only 5?%. This indicates that the majority of larvae were located superficially in the intestinal mucosa. Thirdly, 48 7-week-old layer pullets were infected with 500 A. galli eggs and necropsied at 3 dpi to compare three different larval isolation methods from the intestinal wall, viz., EDTA incubation, agar-gel incubation and pepsin?CHCl digestion, resulting in mean percentages of the recovered larvae: 14.4, 18.2 and 20.0?%, respectively (P?=?0.15). As conclusion, we recommended Pepsin?CHCl digestion as the method of choice for larval recovery from the intestinal wall in future population dynamics study due to high efficiency and quick and simple detection. The agar-gel method was considered to be a prerequisite for molecular and immunological investigations as the larvae were more active and fully intact.     

Passive immunization with milk produced from an immunized cow prevents oral recolonization by Streptococcus mutans.

Cell surface protein antigen (PAc) and water-insoluble glucan-synthesizing enzyme (GTF-I) produced by cariogenic Streptococcus mutans are two major factors implicated in the colonization of the human oral cavity by this bacterium. We examined the effect of bovine milk, produced after immunization with a fusion protein of functional domains of these proteins, on the recolonization of S. mutans. To prepare immune milk, a pregnant Holstein cow was immunized with the fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc and the glucan-binding (GB) domain of GTF-I. After eight adult subjects received cetylpyridinium chloride (CPC) treatment, one subgroup (n = 4) rinsed their mouths with immune milk and a control group (n = 4) rinsed with nonimmune milk. S. mutans levels in saliva and dental plaque decreased after CPC treatment in both groups. Mouth rinsing with immune milk significantly inhibited recolonization of S. mutans in saliva and plaque. On the other hand, the numbers of S. mutans cells in saliva and plaque in the control group increased immediately after the CPC treatment and surpassed the baseline level 42 and 28 days, respectively, after the CPC treatment. The ratios of S. mutans to total streptococci in saliva and plaque in the group that received immune milk were lower than those in the control group. These results suggest that milk produced from immunized cow may be useful for controlling S. mutans in the human oral cavity.     

Visualizing the T-cell response elicited by oral administration of soluble protein antigen

Oral administration of soluble protein antigen induces tolerance, while particulate antigens encountered in the intestine provoke active immunity. Although the events that lead to these distinct outcomes are not yet fully characterized, they may reflect differences at the antigen-presenting cell (APC) level. The role of dendritic cells (DC) in regulating responses at mucosal sites has remained largely undefined because of the low frequency of DC in mucosal-associated tissues. In this study we have used the growth factor Flt3-ligand (Flt3L) to expand DC populations in vivo, in combination with an adoptive transfer system, in order to track antigen-specific T cells during oral tolerance induction. We observed rapid T-cell activation, localized particularly in the mucosal tissues, within hours after feeding the soluble protein antigen, ovalbumin (OVA). The response was enhanced in Flt3L-treated mice, indicating an important role for DC during the inductive phase of tolerance.     

Humoral immune response in mice immunized by oral route with phaseolamine extracted from common bean (Phaseolus vulgaris)

The prevalence of obesity is increasing in Brazil, generating high costs for the health system. In the search for alternative therapies to control the obesity, several researches seek active compounds in natural products in order to find better pharmacological effects. Inhibitors of α-amylase such as phaseolamine extracted from common beans (Phaseolus vulgaris) have shown satisfactory results in weight loss. The present study aimed to verify the possibility of phaseolamine-causing allergic reactions through the humoral response in mice sensitized with phaseolamine. Swiss female mice (n?=?10) were sensitized orally with 100?µg of lyophilized phaseolamine diluted in water for 10 consecutive days and reinforced on the 21th and 35th days after the beginning of this experiment. The production of immunoglobulins IgG, IgG1 and IgE was measured by ELISA and western blotting. The immunological response was investigated and it was found that phaseolamine induced the synthesis of IgG and did not induce the synthesis of IgG1 and IgE. The present study indicates that the product did not cause allergic reactions.     

Crescentic glomerulonephritis induced in the goat by immunization with homologous or heterologous glomerular basement membrane antigen.

Crescentic glomerular changes developed in goats 16 to 46 weeks after immunization with homologous or heterologous glomerular basement membrane (GBM) antigens incorporated in Freund's complete adjuvant. Mesangial widening, accumulation of migrant cells, fibrin deposition, and GBM thickening became manifest with time. The GBM was then attenuated and ruptured at a local area. Through the ruptured GBM, the influx of fibrin and blood cells occurred in the Bowman's space (BS) accompanying proliferation of epithelial cells lining the BS. The crescent thus formed was mainly composed of the capsular epithelial cells. The visceral epithelial cells (podocytes) and migrated blood cells contributed to the crescent formation to a lesser extent. Basement menbrane-like material appeared later between the crescentic cells and the crescent underwent a fibroepithelial form. The results indicate that the crescent develops in relation to the characteristic GBM damage with a resultant severe exudative change in the BS.     

Intestinal mucosal immune response in chickens following intraocular immunization with liposome-associated Salmonella enterica serovar enteritidis antigen

Induction of intestinal mucosal immune responses against Salmonella enterica serovar enteritidis was studied by immunizing chickens with liposome-associated antigen. An ultrasonicated whole cell extract of the bacteria was used for immunizing antigen. Intraocular immunization induced serum IgA, IgG and IgM responses. Also, significant IgA and IgG antibodies were detected in the intestinal tract. Immunization with antigen alone induced only IgG response in the intestine. Salmonella enteritidis-specific antibody-secreting lymphocytes were detected in the spleen and lamina propria of the intestinal tract of immunized chickens. Immunoglobulin (Ig) fractions extracted from intestines of immunized chickens inhibited the adherence of S. enteritidis to cultured HeLa cells. These results indicate that intraocular immunization with liposome-associated S. enteritidis elicits specific antibody-producing lymphocytes in the intestinal tract, and that Ig secreted in the intestine inhibits adherence of the bacteria to intestinal epithelial cells, suppressing the spread of bacterial infection in the host.     

In immunization with Plasmodium falciparum apical membrane antigen 1, the specificity of antibodies depends on the species immunized

At least a million people, mainly African children under 5 years old, still die yearly from malaria, and the burden of disease and death has increased. Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is one of the most promising blood-stage malarial vaccine candidates. However, the allelic polymorphism observed in this protein is a potential stumbling block for vaccine development. To overcome the polymorphism- and strain-specific growth inhibition in vitro, we previously showed in a rabbit model that vaccination with a mixture of two allelic forms of PfAMA1 induced parasite growth-inhibitory antisera against both strains of P. falciparum parasites in vitro. In the present study, we have established that, in contrast to a single-allele protein, the antigen mixture elicits primarily antibodies recognizing antigenic determinants common to the two antigens, as judged by an antigen reversal growth inhibition assay (GIA). We also show that a similar reactivity pattern occurs after immunization of mice. By contrast, sera from rhesus monkeys do not distinguish the two alleles when tested by an enzyme-linked immunosorbent assay or by GIA, regardless of whether the immunogen is a single AMA1 protein or the mixture. This is the first report that a malarial vaccine candidate induced different specificities of functional antibodies depending on the animal species immunized. These observations, as well as data available on human immune responses in areas of endemicity, suggest that polymorphism in the AMA1 protein may not be as formidable a problem for vaccine development as anticipated from studies with rabbits and mice.     

Humoral immune response of carp (Cyprinus carpio) induced by oral immunization with liposome-entrapped antigen

To study the value of liposomes as carriers of antigens for oral vaccination in fish, humoral immune responses were analyzed after immunizing carp (Cyprinus carpio) with liposome-entrapped bovine serum albumin (BSA) as a model antigen. Oral immunization of BSA (100 microg)-containing liposomes that were stable in carp bile induced significant antibody responses against BSA in serum as well as in intestinal mucus and bile. By contrast, no serum antibody responses were observed when fish were orally immunized with the same dose of BSA-containing unstable liposomes or BSA alone. BSA-specific antibody-secreting lymphocytes were detected in the spleen and head kidney of immunized fish. Furthermore, when we applied Vibrio cholerae toxin B subunit (CT-B)-conjugated liposomes containing BSA for oral immunization we found significant increases of anti-BSA antibodies in serum. Our results suggest that delivery systems using liposomes or liposomes with CT-B to the intestinal tract are essential for inducing effective humoral immune responses following oral vaccination.     

Comparative study of the immune response in mice immunized with four live attenuated strains of mumps virus by intranasal or intramuscular route

Summary.  The observation of many cases of mumps and mumps-associated CNS complications in vaccinees prompted us to perform an evaluation of the efficacy of four attenuated mumps virus (Urabe, Jeryl Lynn, Rubini and S12) vaccines. Two doses of vaccine were necessary to induce a good immunity in animals. The humoral and cell-mediated response induced in mice immunized intramuscularly or intranasally with these vaccines has been evaluated. Although the Urabe and Jeryl Lynn strains appear more immunogenic than the other strains and induce higher levels of IgG when administered intramuscularly, the S-12 strain administered intranasally induces a good IgG response. A marked specific CTL activity against mumps virus was observed in mice immunized intranasally with all the strains and, particularly, with the S12 strain. Thus, the intranasal immunization could be considered a possible alternative and efficient route of vaccination against mumps. Received October 30, 2000 Accepted March 17, 2001     

Properties of neutrophils recruited into inflammatory foci with homologous or heterologous antigen in immunized ewes

Studies were undertaken to determine functional properties of neutrophils attracted into involuted mammary glands of Staphylococcus aureus--immunized ewes by soluble antigens prepared from S. aureus (homologous antigen) or from Bacillus cereus (heterologous antigen). The ewes were immunized with an S. aureus vaccine known to stimulate synthesis of cytophilic IgG2 antibody, and the inflammatory responses were elicited 4-8 weeks later by infusing homologous antigen into one gland and heterologous antigen into the other. Inflammatory cells were collected at 2, 4, and 8 h postinfusion. The magnitude of the cellular responses was similar in both glands with high proportions of viable neutrophils. There were no significant differences between neutrophil populations from each gland for proportions of cells bearing cytophilic immunoglobulin, although the proportions of cytophilic immunoglobulin-positive cells from both glands were lower in 2-h exudates than in 4-h or 8-h exudates. In invitro phagocytosis assays using 3H-labeled S. aureus and B. cereus no differences could be detected between the two populations of neutrophils in terms of phagocytic efficacy using a range of bacteria-neutrophil ratios and various opsonizing treatments.     

人源轮状病毒转基因马铃薯口服免疫小鼠的免疫应答研究

目的分析人源轮状病毒转基因植物抗原的口服免疫应答反应。方法在根癌脓杆菌介导获得系列转基因马铃薯植株的基础上，用ELISA分析转基因马铃薯中目的蛋白表达水平。马铃薯块茎直接口服免疫Balb／c小鼠，ELISA分析免疫小鼠血清、唾液、粪便提取物特异抗体水平。结果获得一株最高表达量的转化株；口服免疫可诱导较强的血清IgG反应和强烈的黏膜sIgA反应，粪便的sIgA最高，唾液次之，尿液中无sIgA；加霍乱毒素B亚单位（CTB）佐剂免疫组小鼠和霍乱毒素（CT）佐剂免疫组小鼠抗体水平无显著差异，无佐剂免疫组小鼠抗体水平略低。结论人源轮状病毒转基因植物疫苗联合黏膜佐剂免疫动物可诱导特异的系统与黏膜免疫应答，且黏膜免疫强度略高于系统免疫。     

Control of colonization by virulent Salmonella typhimurium by oral immunization of chickens with avirulent delta cya delta crp S. typhimurium

Oral immunization with a delta cya delta crp Salmonella typhimurium strain has been shown to preclude colonization by wild-type, virulent S. typhimurium and induces humoral and cellular immune response in chickens. Intestinal tract colonization by the virulent challenge strain was used to determine the level of protection conferred by immunization with the delta cya delta crp mutant. The associated humoral and cellular immune responses were measured by ELISA and delayed-type hypersensitivity (DTH) tests, respectively. The levels of colonization by both Salmonella strains were determined by enumeration of viable cells in the intestinal tract. A reduction in faecal excretion of the wild-type strain was observed with a single oral immunization with the delta cya delta crp mutant, but caecal colonization was not affected. However, double oral immunization with the delta cya delta crp mutant precludes caecal colonization by the virulent strain. IgM, IgA and IgG were detected against sonicated Salmonella whole-cell antigens. Outer membrane and flagella proteins induced DTH responses, whereas lipopolysaccharide failed to do so. The effectiveness of the delta cya delta crp strain in reducing caecal colonization by the highly virulent challenge strain in chickens demonstrates that oral vaccination with the delta cya delta crp S. typhimurium should aid in eliminating Salmonella carriers in chickens. The elimination of these carriers on the poultry farm should help to control Salmonella contamination of poultry products, therapy improving public health.     

Local active gingival immunization by a 3,800-molecular-weight streptococcal antigen in protection against dental caries.

Local gingival immunization was attempted in an effort to confine the immune response to the oral cavity and bypass the systemic immune response. A low-molecular-weight (3.8K) streptococcal antigen (SA) I/II was applied 10 times over a period of 1 year to the gingival crevices of rhesus monkeys. The antigen was maintained in situ by means of silicone rubber appliances. Serial examinations over a period of 1 year showed that topical gingival immunization with the 3.8K SA results in a significantly lower incidence of dental caries and colonization of Streptococcus mutans compared with that of the sham-immunized controls. This was associated with an increase in gingival crevicular immunoglobulin G and salivary immunoglobulin A anti-SA I/II antibodies, whereas no change occurred in serum antibodies to SA I/II. The immune mechanism which prevents the colonization of S. mutans and the development of caries may involve antibodies that prevent the adherence of S. mutans to the teeth and facilitate phagocytosis and killing by the local neutrophils. This novel route of local immunization is noninvasive, does not cause side effects, and bypasses systemic immunization.