Montanide IMS 1313 N VG PR nanoparticle adjuvant enhances antigen-specific immune responses to profilin following mucosal vaccination against Eimeria acervulina

This study investigated protection against Eimeria acervulina (E. acervulina) following vaccination of chickens with an Eimeria recombinant profilin in conjunction with different adjuvants, or by changing the route of administration of the adjuvants. Day-old broilers were immunized twice with profilin emulsified in Montanide IMS 1313 N VG PR adjuvant (oral, nasal, or ocular routes), Montanide ISA 71 VG adjuvant (subcutaneous route), or Freund's adjuvant (subcutaneous route) and orally challenged with virulent E. acervulina parasites. Birds orally immunized with profilin plus IMS 1313 N VG PR, or subcutaneously immunized with profilin plus ISA 71 VG, had increased body weight gains compared with animals nasally or ocularly immunized with profilin plus IMS 1313 N VG PR, or subcutaneously immunized with profilin plus Freund's adjuvant. All adjuvant formulations, except for IMS 1313 N VG PR given by the nasal or ocular routes, decreased fecal parasite excretion and/or reduced intestinal lesions, compared with non-vaccinated and infected controls. Compared with animals vaccinated with profilin plus Freund's adjuvant, chickens immunized with profilin plus IMS 1313 N VG PR or ISA 71 VG showed higher post-infection intestinal levels of profilin-reactive IgY and secretary IgA antibodies. Finally, immunization with profilin in combination with ISA 71 VG was consistently better than profilin plus IMS 1313 N VG PR or Freund's adjuvant for increasing the percentages of CD4(+), CD8(+), BU1(+), TCR1(+), and TCR2(+) intestinal lymphocytes. These results indicate that experimental immunization of chickens with the recombinant profilin subunit vaccine in conjunction with IMS 1313 or ISA 71 VG adjuvants increases protective mucosal immunity against E. acervulina infection.     

巨型艾美耳球虫感染鸡空肠粘膜肥大细胞数量和组织胺含量的变化

本文借助于改良甲苯胺蓝染色法和荧光测定法，分别检测了巨型艾美耳球虫感染鸡空肠粘膜肥大细胞（ＭＭＣ）数量和组织胺（ＨＡ）含量的变化。结果表明，初次感染巨型艾美耳球虫之后，空肠ＭＭＣ呈减少趋势，至感染后第１４天回复至不感染对照组水平。而初次感染组相应空肠ＨＡ水平呈现出不显著波动后，在感染后第４天达最大值。在再次攻毒试验中，免疫攻毒组ＭＭＣ呈现水升趋势，尔后回复至不感染对照组水平；免疫攻毒组织相应肠道Ｈ     

Development of protective immunity against Eimeria tenella and E. acervulina in White Leghorn chickens inoculated repeatedly with high doses of turkey coccidia

Repeated inoculation (immunization) of 2-week-old white leghorn chickens with 10(6) oocysts of the turkey coccidia Eimeria adenoeides or E. meleagrimitis partially protected chickens against moderate challenge with E. tenella or E. acervulina oocysts, but not with E. necatrix oocysts. After challenge, mean weight gains of the immunized chickens and the unchallenged controls did not differ significantly, but weight gains of unimmunized chickens were significantly lower. The mean feed-conversion ratio of the immunized challenged chickens was 3.14, as compared with 4.42 for unimmunized challenged control chickens. In general, immunization did not markedly reduce intestinal lesions. Repeated inoculation of chickens with the turkey coccidium E. gallopavonis failed to produce statistically significant protection against challenge with E. tenella, E. acervulina, or E. necatrix, as determined by weight gain, feed-conversion efficiency, and lesion scores. Antibody profiles of individual chickens did not correlate with protection.     

Avian eimeria: invasion in foreign host birds and generation of partial immunity against coccidiosis

Four species of avian Eimeria invaded the intestine of foreign host birds in the same areas in which they invaded the natural host. Repeated inoculation (immunization) of chickens with the turkey coccidian, Eimeria adenoeides, partially protected the chickens against a subsequent challenge with 5.8 x 10(4) E. tenella oocysts. At 6 days post-challenge, the weight gain and feed conversion efficiency of the immunized chickens was significantly better than those of the chickens that were not immunized with E. adenoeides. Lesion scores and cellular invasion by the sporozoites were significantly lower in the immunized birds than in the unimmunized group. Electrophoresis and Western blot analysis identified changes in the serum antibody profiles of the chickens that appeared to be associated with the immunization and challenge programs. An antibody or antibodies recognizing a 60,000-molecular-weight antigen of E. tenella sporozoites disappeared when chickens immunized with E. adenoeides were challenged with E. tenella; an antibody or antibodies recognizing a 23,000-molecular-weight sporozoite antigen appeared within 6 days of challenge. Reciprocal studies, in which turkeys were immunized with E. tenella and challenged with E. adenoeides, showed little evidence of protection.     

Effects of necrotic enteritis challenge on intestinal micro-architecture and mucin profile

1. This study investigated the effect of Eimeria spp./Clostridium perfringens induced necrotic enteritis and traditional antibiotic preventatives on intestinal micro-architecture and mucin profile. 2. A total of 600 Cobb 500 broiler chickens were randomly assigned to the following three groups: (i) unchallenged, (ii) challenged, and (iii) zinc bacitracin/monensin (ZnB/monensin) (n = 25 chickens/pen, 8 pens/group). The challenged and ZnB/monensin chickens were individually inoculated with Eimeria acervulina, E. maxima and E. tenella and C. perfringens type A (EHE-NE18) at 9 and 15 d post-hatch respectively, to induce necrotic enteritis. 3. The challenge procedure significantly decreased villus height, increased villus width and increased crypt depth in the challenged compared to the unchallenged chickens. Zinc bacitracin and monensin maintained villus-crypt structure similar to that of the unchallenged chickens. 4. Mucin profile was not affected by Eimeria spp./C. perfringens challenge as demonstrated by periodic acid-Schiff and high iron diamine-alcian blue pH 2 x 5 staining. Zinc bacitracin and monensin decreased the number of intestinal mucin-containing goblet cells. 5. Lectin histochemistry showed a trend towards greater Arachis hypogea (PNA) reactivity in unchallenged chickens. 6. In summary, Eimeria spp./C. perfringens challenge disrupted intestinal micro-architecture; however, challenge did not appear to affect intestinal mucin profile. Traditional antibiotics, zinc bacitracin and monensin maintained micro-architecture.     

Gel-Bead Delivery of Eimeria oocysts protects chickens against coccidiosis

Vaccines composed of either virulent or attenuated Eimeria spp. oocysts have been developed as an alternative to medication of feed with ionophore drugs or synthetic chemicals. The purpose of this study was to evaluate the use of gel-beads containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts as a vaccine against coccidiosis. Newly hatched chicks (Gallus gallus domesticus) were either sprayed with an aqueous suspension of Eimeria oocysts or were allowed to ingest feed containing Eimeria oocysts-incorporated gel-beads. Control day-old chicks were given an equivalent number of Eimeria oocysts (10(4) total) by oral gavage. After 3 days, chicks were randomly assigned to individual cages, and feces were collected between days 5 and 8 postinfection. All samples were processed for total Eimeria oocysts. At 4 wk of age, all chickens and a control nonimmunized group received a high-dose E acervulina, E maxima, and E. tenella challenge infection. Oocyst excretion by chicks fed gel-beads or inoculated by oral gavage was 10- to 100-fold greater than that of chicks spray-vaccinated with the Eimeria oocysts mixture (log 6.3-6.6 vs. log 4.8). Subsequent protection against challenge as measured by weight gain and feed conversion efficiency was significantly greater (P < 0.05) in gel-bead and oral gavage groups compared with spray-vaccinated or nonimmunized groups. Also, gel-bead and oral gavage groups showed no significant difference (P > 0.05) in weight gain and feed conversion efficiency compared with nonchallenged controls. These findings indicate that incorporation of Eimeria spp. oocysts in gel-beads may represent an effective way to deliver live oocyst vaccines to day-old chicks for preventing subsequent outbreaks of coccidiosis in the field.     

Protective immunity enhanced by chimeric DNA prime-protein booster strategy against Eimeria tenella challenge

In an attempt to investigate the immune efficacy ofa DNA prime-protein booster strategy against avian coccidiosis with a chimeric construct, the Eimeria tenella antigen gene (3-1E) and chicken interferon gamma gene (ChIFN-gamma) were subcloned into the mammalian expression vector proVAX forming the plasmids proE and prol, and then linked by splicing overlap extension by polymerase chain reaction to construct the chimeric plasmid prolE; the chimeric protein (rlE) was expressed in Escherichia coli harboring the constructed plasmid pGEX/IE. Broilers were administered two intramuscular injections with the constructed DNA vaccines (50 microg); in the protein booster groups 100 microg of the rlE were given following the proIE prime. After challenge the proIE-vaccinated chickens showed the protective immunity as demonstrated by significantly reduced oocyst shedding compared with chickens immunized with proE, but the prolE vaccine did not have an additive effect of increasing antibody titer and body weight gain. The chickens in the rlE booster groups had significantly higher specific antibody responses than those immunized with prolE, and displayed further decreased oocyst shedding and increased body weight gain. Taken together, these results indicate that ChIFN-gamma exerts an adjuvant effect coexpressed with 3-1E and provide the first evidence that the DNA prime-protein booster strategy is able to augment the protective efficacy of chimeric DNA vaccine against challenge with Eimeria tenella.     

CpG寡核苷酸增强鸡新城疫疫苗免疫效力的研究

为了探讨CpG寡核苷酸（CpG oligonucleotide,CpG ODN）对鸡新城疫疫苗免疫效力的影响,将CpG2007与鸡淋巴细胞共孵育,测定淋巴细胞增殖率,结果发现CpG2007对鸡淋巴细胞具有显著的刺激活性。将CpG2007与不同浓度的新城疫抗原混合,制备灭活疫苗,免疫健康雏鸡。分别于免疫后不同时间采血,测定抗体效价和细胞因子表达量,并进行攻毒保护试验。结果发现,添加CpG ODN佐剂的试验组均比对应相同抗原剂量的免疫对照组的抗体水平高,产生抗体速度快;抗原剂量降低10倍的佐剂试验组与高抗原剂量免疫对照组抗体水平和攻毒保护率均相当,表明CpG ODN能显著增强新城疫疫苗的免疫效力,能促进机体产生更强烈的免疫应答,是有效的疫苗佐剂候选物质。     

Evaluation of serum and secretory antibody responses to an immunodominant recombinant merozoite surface antigen, p150, using a sensitive enzyme-linked immunosorbent assay

An equation obtained from linear-regression analysis of positive/negative ratios and log of enzyme-linked immunosorbent assay (ELISA) titers of coccidia-immune serum samples was used to accurately predict the antibody titers of chickens immunized with a recombinant merozoite surface protein (p150). Chickens immunized twice intramuscularly with the recombinant p150 antigen emulsified in complete Freund's adjuvant developed a dose-dependent anti-p150 antibody response 14 days after primary immunization. Serum IgG and IgM and secretory biliary IgA antibodies were detected 2 months after primary immunization. Oral challenge with live Eimeria parasites significantly enhanced both the serum and secretory anti-p150 antibody titers. These results indicate that vaccination of chickens with the p150 recombinant merozoite antigen can induce a parasite-specific host immune response.     

Changes in local IFN-gamma and TGF-beta4 mRNA expression and intraepithelial lymphocytes following Eimeria acervulina infection

Inbred chickens SC (B2B2) and TK (B15B21) display different levels of susceptibility to Eimeria acervulina infection. Following primary and secondary infections, SC chickens showed significantly lower oocyst production compared to TK chickens. Both strains produce significantly fewer oocysts during secondary infection (si) indicating that a protective host immune response had developed subsequent to primary infection (pi). To elucidate the immunologic differences between SC and TK chickens that may account for their different levels of disease susceptibility, cellular and molecular parameters of intestinal immunity were compared. CD4 T-lymphocytes increased significantly and more rapidly post-pi and si in SC relative to TK chickens during the later stages of infections. However, later during the infections, CD4 cells were higher in TK compared to SC chickens. Although the percentage of CD8 lymphocytes increased in both strains after pi, following si the percentage of these cells continued to increase in SC chickens but showed a marked decrease in TK chickens. Contrary to the effects on CD4 cells, the percentage of TCR1 cells was higher in TK chickens early after pi while the same cell subset was higher in SC chickens later following infection. The percentages of TCR2 cells were significantly higher in both strains following pi. At the molecular level, IFN-gamma mRNA expression in caecal tonsils and splenic lymphocytes was generally higher in SC compared to TK chickens following E. acervulina infection, while intraepithelial lymphocytes from the duodenum demonstrated reduced levels of this cytokine in both the strains, particularly following pi. TGF-beta4 mRNA levels generally increased in lymphocytes from the caecal tonsils, spleen and duodenum from both the strains. These differences in lymphocyte subpopulations and cytokine mRNA expression between SC and TK chickens following E. acervulina infection indicate a complex genetic control of the native immune response to coccidiosis.     

Anticoccidial effect of green tea-based diets against Eimeria maxima

Anticoccidial effects of green tea (GT)-based diets were evaluated in chickens following oral infection with Eimeria maxima an ubiquitous intestinal parasite of poultry that impairs the growth and feed efficiency of infected birds. Five-week-old chickens were assigned to four groups (GT 0.5%, GT 2.0%, untreated/infected and non-infected control) and each group consisted of 15 chickens. Chickens were fed a standard diet supplemented with ground green tea for 2 weeks prior to infection with E. maxima (10,000 sporulated oocysts per bird). The effects of green tea on E. maxima infection were assessed by two parameters, fecal oocyst shedding and body weight gain. The green tea-fed chickens produced significantly reduced fecal oocysts (P<0.05) when compared to the E. maxima-infected group fed standard diet. The green tea-based diet, however, did not improve body weight loss caused by E. maxima infection. This study is the first to demonstrate anticoccidial effect of green tea on Eimeria parasites.     

Characterization of adaptive immune responses induced by a new genetically inactivated Salmonella Enteritidis vaccine

The superior conservation of antigenic determinants on the surface of genetically inactivated bacterial ghosts makes them attractive immunogenic inactivated vaccine candidates. The efficacy of Salmonella Enteritidis (SE) ghost vaccination was evaluated in chickens by characterizing the nature of the adaptive immune response. Chickens from the immunized group demonstrated significant increases in SE-specific plasma IgG, intestinal secretory IgA, and lymphocyte proliferative response. The populations of CD4, CD8, and TCR γδ T-cells in immunized chickens were significantly greater than in the controls. Increased levels of IFN-γ, IL-2, IL-6 and IL-10 were observed in peripheral blood mononuclear cells stimulated with SE specific antigen. After virulent SE challenge, the immune system of immunized chickens was rapidly stimulated, as indicated by significantly increased population of CD4 and CD8 T-cells. Furthermore, the immunized group exhibited decreased challenge strain recovery of the internal organs compared to the non-immunized group. Together, these data indicate that the immunization induced humoral and cell-mediated immunity might be responsible for significant reduction of the virulent challenge strain load in the internal organs of immunized chickens.     

Role of oregano and Citrus species-based essential oil preparation for the control of coccidiosis in broiler chickens

Background: Due to presence of drug-resistant Eimeria strains and raised public health safety concerns about drug residues in the meat, there is renewed interest in the search for natural alternatives to the coccidiosis control agents. This study was conducted to test the anticoccidial efficacy of oregano and Citrus spp.-based essential oils for broilers.Methods: A total of 280 7-day-old broiler chicks were fed a control diet or diets with salinomycin or essential oils for up to 35 d of age. On d 14, half of the control groups and the treated groups were orally challenged with a coccidiosis vaccine at 25 times higher than the recommended vaccine dose. Control diet-fed chickens that were gavaged with phosphate-buffered saline were considered non-challenged control group.Results: Eimeria challenge or dietary additives failed to affect growth performance during the 7 to 20 d growth period although essential oil-fed chickens exhibited the lowest body wight gain(P = 0.332) and the highest feed conversion ratio(P = 0.062). Oocysts in the litter were detected in the challenged control diet group and the challenged/essential oil-fed groups at 21 and 35 d, respectively. Superoxide dismutase activity in the serum was elevated(P = 0.059) in the salinomycin-fed chickens compared to the challenged controls. Alpha-1-acid glycoprotein was decreased by 28.7% in the salinomycin-fed chickens but increased by 38.1% in the essential oil group compared with the challenged control group. Challenged control group exhibited a significantly higher cooking loss of the thigh meat, compared to the non-challenged control diet group, which was marginally mitigated by dietary supplementation with essential oils. Chickens fed essential oil-added diet had the highest branched-chain fatty acids contents in the cecum.Conclusions: In conclusion, this study shows that oregano and Citrus-based essential oil preparation did not affect growth performance in broiler chickens challenged with the coccidiosis vaccine nor did Eimeria-specific duodenal lesion. However, dietary essential oil preparation lowered oocysts present in litter materials and altered branchedchain fatty acids in cecal digesta. Beneficial findings of the essential oil preparation on volatile fatty acids and oocysts output may warrant further research into assessing its effectiveness and its efficacy in pathogenic fieldisolate Eimeria spp.-induced coccidiosis disease model.     

细菌CpG DNA对鸡球虫弱毒苗Immucox的免疫增强效应

采用细菌CpG DNA作鸡球虫弱毒苗Immucox佐剂以探索其应用的可行性。将制备的细菌CpG DNA和球虫疫苗Immuncox单独或联合免疫接种肉鸡,第2次免疫后7d进行攻虫,口服接种柔嫩艾美儿球虫孢子化卵囊105个/只。感染后第7天,对试验动物进行称重并屠杀,分别观察盲肠病变积分、每克粪便卵囊排出数和体增重情况。结果:在相对增重方面,细菌CpG DNA+疫苗组高于感染非免疫组、疫苗组和CpG组,且与它们均差异显著(P<0.05);在卵囊排出总数和盲肠病变平均记分方面,细菌CpG DNA+疫苗组低于感染非免疫组、疫苗组和CpG组,且与之比较差异显著(P<0.05);在抗球虫综合指数方面,疫苗+细菌CpGDNA组达到172。结果表明用细菌CpG作鸡球虫弱毒苗Immucox的佐剂可提高该疫苗免疫保护效果。     

The role of an early Salmonella Typhimurium infection as a predisposing factor for necrotic enteritis in a laboratory challenge model

Necrotic enteritis (NE) caused by Clostridium perfringens (CP) in poultry is an important bacterial disease in terms of economic implications. The disease is multifactorial and is invariably associated with predisposing factors. In the present experiments, we investigated the potential predisposing role of neonatal Salmonella Typhimurium (ST) infection for NE-associated mortality in a laboratory challenge model. In two experiments, day-of-hatch chicks were randomly assigned to four groups: Group 1, nonchallenged control; Group 2, chickens received Eimeria maxima (EM) and CP; Group 3, chickens received EM and CP and were also challenged with ST at day 1 of age; Group 4, chickens received EM and CP and were also challenged with ST at day 17 of age. Challenged groups received an oral dose of EM at 18 days of age and CP (10(8) colony-forming units/chick) at 22-23 days of age. When compared to EM and CP, chicks challenged with ST (day 1) had increased NE-associated mortality and CP-associated lesion scores (P < 0.05) in both experiments. Furthermore, body weight and body weight gain were lower (P < 0.05) in chicks infected with ST (day 1) in the first experiment, even though no differences (P > 0.05) were observed in weight gain in the second experiment. Chicks challenged with ST (day 17) were similar to the EM and CP group in all of the above-mentioned parameters, indicating that a paratyphoid infection in younger chicks remarkably alters the susceptibility to secondary bacterial infections. Based on this work, the authors suggest that an ST infection early in the age of a chick may be important for altering susceptibility to NE, an observation that may be useful from the perspective of experimental reproduction of this disease and, perhaps, as an economically important reason to address the problem of paratyphoid Salmonella infections in young chicks.     

Protective effects of sugar cane extracts (SCE) on Eimeria tenella infection in chickens

The effects of oral administration of sugar cane extracts (SCE) on Eimeria tenella oocysts infection in chickens were studied with 2 different experiments. In Experiment 1, 3-week-old inbred chickens (MHC; H.B15) were inoculated into the crop with SCE (500 mg/kg/day) for 1 day or 3 consecutive days, and then challenged with E. tenella sporulated oocysts (2 x 10(4) cells/chicken). In Experiment 2, 1-week-old chickens were orally administered SCE at the same dose for 3 consecutive days, and then initially infected with E. tenella sporulated oocysts (2 x 10(3) cells/chicken). At 2 and 3 weeks of age, these chickens were immunized intravenously with the mixed antigens of sheep red blood cells (SRBC) and Brucella abortus (BA). At 4 weeks of age, chickens were challenged with E. tenella sporulated oocysts (1 x 10(5)/chicken). Challenged chickens with E. tenella oocysts showed markedly decreased body weight gain/day, severe hemorrhage and great number of shedding oocysts in feces and high lesion scores. Oral administration of SCE and initial infection with oocysts (2 x 10 (3)/chicken) resulted in a remarkable improvement in body weight gain/day, hemorrhage, the number of shedding oocysts and lesion score, compare to other infected groups. In addition, SCE-inoculated chickens with the initial infection showed a significant increase in antibody responses against SRBC and BA and also improvement in decreased relative proportions of Bu-1a(+) and CD4( )cells in cecal tonsil lymphocytes of E. tenella-challenged chickens. Cecal tissues of chickens administered SCE and initially infected with E. tenella oocysts showed lower numbers of schizonts, gametocytes and oocysts than those of infected control chickens. These results suggest that SCE have immunostimulating and protective effects against E. tenella infection in chickens.     

Immunization of young chicks using graded dose of wild strain of Eimeria tenella

A wild strain of Eimeria tenella was isolated and utilized for immunization studies. Its optimal sporulation was attained at room temperature 24-25 degrees C after 24-48 h. Two groups of chicks were immunized by dosing a graded dose of five oocysts/chick/day for 6 days followed by 50 oocysts/chick/day for 7 days. A third group was not immunized and served as a negative control. Immunized chicks gained mass at the same rate as unimmunized ones, but when challenged with 200,000 oocysts/chick, mass gains declined in the unimmunized group. The growth rate of immunized chicks was not affected by challenge (P > 0.05). Upon challenge, unimmunized chicks produced significantly more oocysts than immunized chicks (P < 0.005). Immunized chicks withstood a challenged with 200,000 oocysts/chick without developing any clinical signs whereas the unimmunized chicks developed typical clinical signs of coccidiosis. Unimmunized chicks had significantly more severe lesions in the caecum than any other group (P > 0.005) and also produced significantly more oocysts than any other group (P > 0.005).     

Adjuvant effect of chicken interferon-gamma for inactivated Salmonella Enteritidis antigen

The adjuvant effect of chicken interferon-gamma (ChIFN-gamma) was examined for protecting chickens against intestinal colonization of Salmonella Enteritidis (SE) following oral exposure. Ten 7-week-old chickens per group were immunized with inactivated SE twice with or without co-administration of ChIFN-gamma intramuscularly, and all chickens were challenged with SE. Sera collected from immunized groups with or without ChIFN-gamma, and from unimmunized group were measured for SE antibody by agglutination test. The levels of antibodies were raised by 1 week post-immunization and did not show any difference between groups with and without ChIFN-gamma. No antibodies were detected in unimmunized group before challenge. Fecal samples from each group were cultured at 1, 4, 7, and 13 days post-challenge to determine the incidence of intestinal colonization and the numbers of SE shed into the environment. Co-administration of ChIFN-gamma, significantly reduced the incidence of intestinal colonization (P<0.05). At 13 days post-challenge, the bacterial counts of SE in organs were also reduced in ChIFN-gamma administered group. These data suggest co-administration of ChIFN-gamma with SE antigen enhances protection against SE challenge without acceleration of antibody production.