Co-expression of EtMic2 protein and chicken interleukin-18 for DNA vaccine against chicken coccidiosis

In the present study, a naked EtMIC2 DNA vaccine, a ChIL-18 expression vector and a EtMIC2 and ChIL-18 co-expression DNA vaccine were constructed and their protective efficacies against homologous challenge were compared and evaluated by examining the body weight gain, oocyst shedding, cecal lesion, ACI as well as specific anti-EtMic2 antibody level, the proliferation ability and percentages of CD4+ and CD8+ of splenocytes. The results showed the naked EtMIC2 DNA vaccine could increase the weight gain and decrease the oocyst shedding, but could not alleviate the cecal lesion of immunized chickens compared to unimmunized chickens. Chickens immunized with the co-expression vector pVAX1-MIC2-IL-18 exhibited much improved immune protection against challenge compared to chickens immunized with naked EtMIC2 DNA vaccine, or with naked EtMIC2 DNA vaccine and ChIL-18 expression vector applied separately. These results suggest that the co-expression of ChIL-18 with EtMic2 together could significantly improve the immune protection of the EtMic2 protein.     

Vaccination of chickens with DNA vaccine expressing Eimeria tenella MZ5-7 against coccidiosis

A chimeric DNA vaccine co-expressing Eimeria tenella MZ5-7 and chicken IL-17 gene was constructed and its efficacy against E. tenella challenge was evaluated. The ORF of MZ5-7 from E. tenella's second generation merozoite and the mature interleukin 17 gene of chicken were cloned into the expression vector of pcDNA4.0 to construct DNA vaccine pcDNA4.0-MZ and pcDNA4.0-MZ-IL17. The expression of aim gene products in vivo was detected by western blotting. The expression of IL-2 and IFN-γ in chicken splenocytes was detected by RT-PCR 7 days post-immunization. The expression levels of the two cytokines in the pcDNA4.0-MZ-IL17 DNA vaccine immunized group were significantly higher than that in the pcDNA4.0-MZ immunized group (p<0.05). Either pcDNA4.0-MZ or pcDNA4.0-MZ-IL17 DNA vaccine could obviously alleviate cecal lesions, body weight loss and increase oocyst decrease ratio. The ACI of pcDNA4.0-MZ-IL17 group was 190, which is higher than that of pcDNA4.0-MZ group and all the three control groups. In short, MZ5-7 was an effective candidate antigen for vaccine and co-expression of cytokine with antigen was an alternative method to enhance the immunity DNA vaccine.     

Potential of a recombinant antigen as a prophylactic vaccine for day-old broiler chickens against Eimeria acervulina and Eimeria tenella infections.

A genetically engineered Eimeria tenella antigen (GX3262), produced as a fusion protein with beta-galactosidase and identified with a monoclonal antibody, induced partial but significant protection in young broiler chickens against experimental E. tenella and Eimeria acervulina infections. The antigen appears to share a T-helper cell epitope with the parasite as evidenced by (a) booster inoculation with either the recombinant antigen or with a small number of live oocysts enhanced the protective immunity in GX3262 primed chickens, and (b) ability of the antigen to induce in vitro stimulation of T-cells from chickens immunized with antigen or parasite. These observations suggest the feasibility of a single vaccination of 1 or 2-day-old broilers with GX3262 to induce an acceptable degree of protective immunity. The implications of the observations reported here are far reaching in terms of a practical coccidiosis vaccine for poultry, and show for the first time that 1-day-old broiler chickens can be efficiently vaccinated with a recombinant antigen against one or more species of Eimeria.     

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.     

Resistance to intestinal coccidiosis following DNA immunization with the cloned 3-1E Eimeria gene plus IL-2, IL-15, and IFN-gamma

A cloned Eimeria acervulina gene (3-1E) was used to vaccinate chickens in ovo against coccidiosis, both alone and in combination with genes encoding interleukin (IL)-1, IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or interferon (IFN)-gamma. Vaccination efficacy was assessed by increased serum anti-3-1E antibody titers, reduced fecal oocyst shedding, and enhanced body weight gain following experimental infection with E. acervulina. When used alone, anti-3-1E antibody titers were transiently, but reproducibly, increased at 2 wk and 3 wk posthatching in a dose-dependent manner. Similarly, significantly reduced oocyst shedding and increased weight gain were observed at relatively high-dose 3-1E vaccinations (> or =25 microg/egg). Combined immunization with the 3-1E and IL-1, IL-2, IL-15, or IFN-gamma genes induced higher serum antibody responses compared with immunization with 3-1E alone. Following parasite infection, chickens hatched from embryos given the 3-1E gene plus the IL-2 or IL-15 genes displayed significantly reduced oocyst shedding compared with those given 3-1E alone, while 3-1E plus IL-15 or IFN-gamma significantly increased weight gain compared with administration of 3-1E alone. Taken together, these results indicate that in ovo immunization with a recombinant Eimeria gene in conjunction with cytokine adjuvants stimulates protective intestinal immunity against coccidiosis.     

A recombinant DNA vaccine encoding C. andersoni oocyst wall protein induces immunity against experimental C. parvum infection

Cryptosporidium andersoni parasited in the abomasum has been demonstrated as a cause of reduction of milk production in dairy cow. In this study, a novel chimeric DNA vaccine pVAX1-AB was constructed and the efficacy against Cryptosporidium parvum was determined. BALB/c mice were divided into 3 groups and immunized with DNA vaccine expressing the oocyst wall protein, AB protein of C. andersoni, the recombinant plasmid containing the AB gene, respectively. After inoculation of 1 × 10(6) oocysts of C. parvum, the humoral and cellular immune responses were detected. Experimental results showed that the recombinant plasmid can induce corresponding specific antibody response, simultaneously influenced cellular immune responses, and provided greater protection rate (48.6%) than the other groups. These results indicated that chimeric DNA vaccine has a potential in Cryptosporidium vaccine development.     

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.     

巨型艾美球虫重组蛋白Gam82-Y和重组质粒pcDNA-gam82的免疫保护效果研究

为研究巨型艾美球虫NT株重组蛋白Gam82-Y和重组质粒pcDNA-gam82对鸡的免疫保护效果,本研究以平均增重、相对增重率及卵囊减少率等作为评价指标,检测结果显示:中剂量重组蛋白佐剂组(0.5 mg-FCA)和中剂量重组蛋白组(0.5 mg)的免疫保护力均低于卵囊免疫组(p＜0.05),但比其他各免疫组高(p＜0.05);重组质粒(pcDNA-gam82)免疫组的平均增重、相对增重率、卵囊减少率等方面与重组蛋白免疫组相同,两者均优于未免疫攻虫组(p＜0.05),但仍未能够达到卵囊免疫组的免疫保护水平(p＜0.05).     

Coccidiosis immunization: effects of mushroom and herb polysaccharides on immune responses of chickens infected with Eimeria tenella

An experiment was conducted to investigate the effects of polysaccharide extracts (E) of two mushrooms, Lentinus edodes (LenE) and Tremella fuciformis (TreE), and an herb, Astragalus membranaceus (AstE), on the immune responses of chickens infected with Eimeria tenella. A total of 180 broiler chickens were assigned to nine groups: three groups were fed with each of the extracts (LenE, TreE, and AstE), three groups were fed with the extracts and immunized with live oocyst vaccine (LenE+V, TreE+V, and AstE+V), a group was immunized with the vaccine only, and there were two controls (E. tenella-infected and noninfected groups). The oocyst vaccine was given at 4 days of age, and the extracts (1 g/kg of the diet) were supplemented from 8 to 14 days of age. At 18 days of age, all birds except those in the noninfected group were infected with 9 x 10(4) sporulated oocysts. The results showed that at 7 days postinfection (p.i.), birds fed the extracts without vaccination had lower body weight (BW) gain than those given the vaccine only. However, the extracts in conjunction with the vaccine significantly enhanced BW gain of the infected chickens compared with the vaccine group. Of the three extracts, LenE and TreE showed a better growth-promoting effect. The extracts largely increased oocyst excretion of droppings during the primary response postvaccination. The cecal peak oocyst output and lesion scores measured at 7 days p.i. were higher in the groups fed the extracts than in the group immunized with the vaccine only, whereas those of the groups fed with the extracts and immunized with the vaccine were not significantly different from the vaccine group. Of the three extracts, both LenE- and AstE-fed groups showed lower cecal oocyst output. Thus, as compared with the extracts, the live, attenuated vaccine showed better results with significantly increased immune response in coccidial infected birds. The polysaccharide extracts may prove useful against avian coccidiosis, and, particularly when they are used in conjunction with vaccine, they have shown preliminary promise against the experimental coccidial infection.     

鸡胚接种柔嫩艾美耳球虫和堆形艾美耳球虫活卵囊的免疫保护试验

给18日龄鸡胚接种一定剂量的柔嫩艾美耳球虫(Eim eria tenella)和/或堆形艾美耳球虫(E.acervulina)孢子化卵囊,出雏后在无球虫环境中笼养,1~10日龄每天收集各组粪便样本,计数克粪便卵囊数(OPG),并于14日龄时以大剂量同源孢子化卵囊攻虫,以相对增重率(RWG)、饲料转化率(FCR)、相对卵囊产量(ROP)评价免疫保护效果。结果显示,以E.tenella或E.acervulina卵囊免疫18日龄鸡胚,其卵囊排出的潜隐期及达到峰值的时间与1日龄雏鸡接种组相一致,有相似的排卵囊曲线,提示其诱导免疫的建立是在出雏后开始建立的。攻虫后各免疫组的RWG由攻虫对照组的31.9%~51.7%提高到了76.5%~83.6%,RCR由攻虫对照组的4.11~4.89改善为2.72~2.96,ROP降至4.7%~23.5%。结果表明以一定剂量E.tenella和E.acervulina卵囊单独或混合经羊膜腔免疫18日龄鸡胚都可以建立起针对出雏后14日龄同源攻虫的良好免疫保护力。比较混合免疫E.tenella和E.acervulina卵囊组与单一接种E.tenella或E.acervulina卵囊组的免疫效果发现,混合免疫组的各项指标均稍优于后者。     

Limited heterogeneity between strains of Eimeria tenella isolated from Britain and Bangladesh

Single-oocyst-derived field strains of Eimeria tenella isolated from Rugby in the United Kingdom (E tenella R) and from Mymensingh and Dhaka in Bangladesh (E tenella M and D, respectively) and a laboratory strain (E tenella, Houghton, H) were compared by isoenzyme electrophoresis, reactivity with antisporozoite monoclonal antibodies and, for some pairs of strains, cross-protection in vivo. The three field strains conformed to one zymodeme with respect to six isoenzymes. For glucose phosphate isomerase (GPI) all field strains were characterised by GPI-9. A panel of six different monoclonal antibodies raised against sporozoites of E tenella H did not discriminate between strains by titration in an immunofluorescence assay against air-dried, acetone fixed sporozoites. In cross-protection experiments involving immunisation and challenge of young chickens, two immunisation schedules were used which, after homologous challenge, provided complete immunity either by the criterion of oocyst output, or by the criterion of weight gain (and more than 94 per cent protection by the criterion of oocyst output). While strain heterogeneity was minimal in the former situation, there was poor cross protection between some strains in the latter case. Under those conditions, heterologous challenge with E tenella M resulted in dysentery and in significantly (P less than 0.05) increased oocyst output and decreased weight gain. The results suggested that E tenella M was immunologically superior to E tenella R and H strains. The results show that a limited degree of immunogenic variability exists between these strains of E tenella and that, unless homologous strain immunity is complete by the criterion of oocyst output, challenge with heterologous strains may result in depressed weight gain.     

Effects of Pediococcus- and Saccharomyces-based probiotic (MitoMax) on coccidiosis in broiler chickens

Coccidiosis is the major parasitic disease of poultry. In this study, the role of the commercial probiotic MitoMax which contains Pediococcus acidilactici and Saccharomyces boulardii was evaluated by measuring body weight gain, fecal oocyst shedding, and serum antibody responses as an alternative control method of prophylactic drug against coccidiosis. Day-old broiler chicks were fed regular or probiotic diets supplemented with MitoMax at 0.01%, 0.1%, or 1.0% of diet, and challenged 2 weeks later with 5000 oocysts of either Eimeria acervulina (EA) or Eimeria tenella (ET). Birds fed 1.0% or 0.1% MitoMax-supplemented diets in EA- or ET-infected groups shed less (P<0.05) oocysts than control-infected chickens. Also, chickens fed 0.1% MitoMax-supplemented diet and infected with EA exhibited higher (P<0.001) serum Eimeria-specific antibodies than other groups. These results demonstrate that MitoMax may enhance the resistance of birds against coccidiosis by enhancing humoral immunity when included at > or = 0.1% of the broiler diet.     

鸡柔嫩艾美耳球虫SO7基因的原核表达及动物免疫试验

为了研究鸡柔嫩艾美耳球虫SO7基因原核表达重组蛋白的免疫效力,分析其在鸡抗柔嫩艾美耳球虫感染中对鸡平均增重、抗球虫指数(ACI)、减少盲肠病变和卵囊数量中的作用,从柔嫩艾美耳球虫孢子化卵囊中提取总RNA作为模板,用RT-PCR扩增出SO7基因片段,再应用DNA重组技术将SO7基因克隆到原核表达载体pET32a(+)中并用IPTG诱导表达。SDS-PAGE结果显示,pET32a(+)-SO7表达的目的蛋白约为40ku,主要以包涵体形式存在。用纯化的重组蛋白进行动物免疫试验显示,SO7重组蛋白在鸡抗柔嫩艾美耳球虫感染中有较好的免疫保护作用,能够显著提高鸡柔嫩艾美耳球虫感染鸡的平均增重和抗球虫指数(ACI),对减少盲肠病变和卵囊数量也有部分作用。     

An enzyme-linked immunosorbent assay for coccidiosis in chickens: correlation of antibody levels with prior exposure to coccidia in the laboratory and in the field

An enzyme-linked immunosorbent assay was developed for detecting antibody to coccidia to facilitate the survey of laboratory and field infections. Serum antibody levels in chickens were measured against soluble Eimeria tenella oocyst antigen. Sera from breeders aged 10, 23, 37, and 43 weeks were positive with uniformly high antibody titers. Broiler chick sera showed high maternal antibody titer at hatch, decreasing to an almost negligible response at 3 weeks of age. Two-week-old broiler chicks had variable responses to a single infection of E. tenella: titers were elevated at 8 to 10 days postinfection and generally increased through day 24. Weekly reinfection of 2-week-old broiler chickens produced an antibody titer in proportion to the number of oocysts per dose and stimulated protection against challenge with 2 x 10(5) E. tenella. Inbred birds raised in a pathogen-free environment for 6 weeks had no detectable antibody titers.     

Vaccination of chickens with a chimeric DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2 induces protective immunity against coccidiosis

A fusion DNA vaccine co-expressed Eimeria tenella TA4 and chicken IL-2 (chIL-2) was constructed and its efficacy against E. tenella challenge was observed. TA4 gene of E. tenella and chIL-2 gene were cloned into expression vector pcDNA3.1 and pcDNA4.0c in different forms, producing vaccines pcDNA3.1-TA4-IL-2, pcDNA3.1-TA4 and pcDNA4.0c-IL-2. The expression of aim genes in vivo was detected by RT-PCR and western blot. Animal experiment was carried out to evaluate the immune efficacy of the vaccines. Results indicated these DNA vaccines were successfully constructed and the antigen genes could be expressed effectively in vivo. The animal experimental results showed that DNA vaccines could obviously alleviate cecal lesions, body weight loss and increase oocyst decrease ratio. The ACI of pcDNA3.0-TA4-IL-2 group was 192, higher than that of pcDNA3.1-TA4 group. The results suggested that TA4 was an effective candidate antigen for vaccine and co-expression of cytokine with antigen was an alternative method to enhance DNA vaccine immunity.     

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.     

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.     

Genetics of resistance to coccidiosis: response of inbred chicken lines to infection by Eimeria tenella and Eimeria maxima

1. Experiments have been carried out to compare weight gain, mortality and oocyst production in 7 inbred and partially inbred lines of chickens after challenge with the coccidial parasites Eimeria tenella and E. maxima. 2. There were large differences between lines in the effects of challenge on weight gain and mortality for both species of parasite. However, the lines suffering the greatest mortality were not those showing the greatest effects on weight gain, indicating that mortality alone is not an adequate criterion in selection for resistance. 3. Although oocyst production differed between lines there was no correlation with mortality or with weight loss, implying that the variation observed in these traits was not due to a restriction of the parasite but to an accommodation of its effects. 4. Mortality and weight loss resulting from challenge with E. tenella in the different lines did not correlate with that caused by E. maxima. 5. There was evidence of an association of genes of the major histocompatibility complex genes with mortality, but not with weight loss or oocyst production: there was no indication of association of resistance to coccidiosis with resistance to Marek's disease.