The optimal immunization procedure of DNA vaccine pcDNA–TA4–IL-2 of Eimeria tenella and its cross-immunity to Eimeria necatrix and Eimeria acervulina

The immunization procedure of DNA vaccine pcDNA–TA4–IL-2 of Eimeria tenella, including route, dose, time of immunization and age of primary immunization of chicken, was optimized. The stability and the cross-species protection of the vaccine were also analyzed. Efficacy of immunization was evaluated on the basis of oocyst decrease ratio, lesion score, body-weight gain and the anti-coccidial index (ACI). Chinese Yellow chickens were randomly distributed into corresponding groups (30/group). The challenged, unchallenged and vector control groups were designed. The results illustrated that 25 μg was the optimal dose and intramuscular injection was the most effective route to induce protective immunity. There were no significant differences of ACIs between boosting and non-boosting groups. Storage time and temperature had little effect on the immunizing efficacy of the vaccine. The vaccine could provide partial cross-protection against the challenge with E. necatrix and E. acervulina, but not with E. maxima.     

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.     

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.     

Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-��

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-γ were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-γ (ChIFN-γ) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-γ did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.     

Development of a DNA vaccine against chicken anemia virus by using a bicistronic vector expressing VP1 and VP2 proteins of CAV

In the present study, we describe the development of a DNA vaccine against chicken anemia virus. The VP1 and VP2 genes of CAV were amplified and cloned into pBudCE4.1 to construct two DNA vaccines, namely, pBudVP1 and pBudVP2-VP1. In vitro and in vivo studies showed that co-expression of VP1 with VP2 are required to induce significant levels of antibody against CAV. Subsequently, the vaccines were tested in 2-week-old SPF chickens. Chickens immunized with the DNA-plasmid pBudVP2-VP1 showed positive neutralizing antibody titer against CAV. Furthermore, VP1-specific proliferation induction of splenocytes and also high serum levels of Th1 cytokines, IL-2 and IFN-γ were detected in the pBudVP2-VP1-vaccinated chickens. These results suggest that the recombinant DNA plasmid co-expressing VP1 and VP2 can be used as a potential DNA vaccine against CAV.     

Construction of DNA vaccines encoding Eimeria acervulina cSZ-2 with chicken IL-2 and IFN-γ and their efficacy against poultry coccidiosis

The study describes vaccination experiments with highly immunogenic sporozoite E. acervulina cSZ-2 co-administered with chicken IL-2 (chIL-2) and interferon-γ (chIFN-γ) to determine their efficacies against homologue challenge. The entire coding sequence of cSZ2, chIL-2 and chIFN-γ were cloned into eukaryotic expression vector pVAX1, constructing DNA vaccines pVAX1-cSZ2, pVAX1-chIL-2, pVAX1-chIFN-γ, pVAX1-cSZ2-chIL-2 and pVAX1-cSZ2-chIFN-γ. The expression of target genes in vivo was detected by RT-PCR and Western blot. Chicken experiments were carried out by vaccinating chickens two times at dose rate of 100 μg intramuscularly. At 28 days of age, all chickens were inoculated orally with 1 × 10⁵ sporulated oocysts of E. acervulina except the unchallenged control group. Seven days after challenge, all chickens were weighted and slaughtered for duodenum collection. The results indicated that these DNA vaccines were successfully constructed and the antigen genes could be expressed effectively in vivo. The findings also demonstrated best synergistic effect of IL-2 with this protein which suggested that co-administration of cytokines with this antigen was a powerful method to enhance immunity by alleviating intestinal lesions, body weight loss and oocyst count imparting partial protection against homologous challenge.     

DNA immunization against very virulent infectious bursal disease virus with VP2-4-3 gene and chicken IL-6 gene

The present study was to investigate the feasibility and efficiency of the DNA vaccine to protect chickens against very virulent infectious bursal disease virus (vvIBDV) infection. A plasmid DNA carrying VP2-4-3 genes of vvIBDV SH95 and a plasmid DNA carrying chicken interleukin-6 (ChIL-6) genes were constructed and designated as pALTER-MAX-VP2-4-3 and pALTER-MAX-ChIL-6 respectively. Several DNA vaccination experiments were performed: 1-week-old chickens were intramuscularly injected with only plasmid pcDNA3-VP2, pALTER-MAX-VP2-4-3 or mixture with pALTER-MAX-ChIL-6. The chickens at 4 weeks old were orally inoculated with vvIBDV SH95. The results showed that immunization with the mixture of pALTER-MAX-VP2-4-3 and pALTER-MAX-ChIL-6 three times conferred protection for 90% of chickens. Enzyme-linked immunosorbent assay (ELISA) antibody titres in chickens immunized together with pALTER-MAX-ChIL-6 were higher than those immunized simply with plasmid pcDNA3-VP2 or pALTER-MAX-VP2-4-3. IBDV was not detected in the bursa of the protected chickens at 8 days after challenge by RT-PCR. The results indicate that protection against vvIBDV can be achieved by using the VP2-4-3 gene of vvIBDV as a DNA vaccine. Furthermore, the simultaneous injection of ChIL-6 plasmid significantly increased the protection after challenge with the very virulent strain.     

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.     

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.     

Effects of Artemisia annua and Foeniculum vulgare on chickens highly infected with Eimeria tenella (Phylum Apicomplexa)

Background

Intensive poultry production systems depend on chemoprophylaxis with anticoccidial drugs to combat infection. A floor-pen study was conducted to evaluate the anticoccidial effect of Artemisia annua and Foeniculum vulgare on Eimeria tenella infection. Five experimental groups were established: negative control (untreated, unchallenged); positive control (untreated, challenged); a group medicated with 125 ppm lasalocid and challenged; a group medicated with A. annua leaf powder at 1.5% in feed and challenged; and a group treated with the mixed oils of A. annua and Foeniculum vulgare in equal parts, 7.5% in water and challenged. The effects of A. annua and oil extract of A. annua + F. vulgare on E. tenella infection were assessed by clinical signs, mortality, fecal oocyst output, faeces, lesion score, weight gain, and feed conversion.

Results

Clinical signs were noticed only in three chickens from the lasalocid group, six from the A. annua group, and nine from the A. annua + F. vulgare group, but were present in 19 infected chickens from the positive control group. Bloody diarrhea was registered in only two chickens from A. annua group, but in 17 chickens from the positive control group. Mortality also occurred in the positive control group (7/20). Chickens treated with A. annua had a significant reduction in faecal oocysts (95.6%; P = 0.027) and in lesion score (56.3%; P = 0.005) when compared to the positive control. At the end of experiment, chickens treated with A. annua leaf powder had the highest body weight gain (68.2 g/day), after the negative control group, and the best feed conversion (1.85) among all experimental groups.

Conclusions

Our results suggest that A. annua leaf powder (Aa-p), at 1.5% of the daily diet post-infection, can be a valuable alternative for synthetic coccidiostats, such as lasalocid.     

DNA vaccine encoding avian influenza virus H5 and Esat-6 of Mycobacterium tuberculosis improved antibody responses against AIV in chickens

The H5 gene of avian influenza virus (AIV) strain A/chicken/Malaysia/5744/2004(H5N1) was cloned into pcDNA3.1 vector, and Esat-6 gene of Mycobacterium tuberculosis was fused into downstream of the H5 gene as a genetic adjuvant for DNA vaccine candidates. The antibody level against AIV was measured using enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition (HI) test. Sera obtained from specific-pathogen-free chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5/Esat-6 demonstrated antibody responses as early as 2 weeks after the first immunization. Furthermore, the overall HI antibody titer in chickens immunized with pcDNA3.1/H5/Esat-6 was higher compared to the chickens immunized with pcDNA3.1/H5 (p < 0.05). The results suggested that Esat-6 gene of M. tuberculosis is a potential genetic adjuvant for the development of effective H5 DNA vaccine in chickens.     

Protective immunity of recombinant Mycobacterium bovis BCG expressing rhomboid gene against Eimeria tenella challenge

Two recombinant Mycobacterium bovis BCG (rBCG) strains carrying the Eimeria tenella rhomboid gene (Rho) delivered by extrachromosomal vector pMV261 and integrative vector pMV361 were evaluated for their ability to protect chickens against E. tenella challenge. The chickens were immunized intranasal with BCG, rBCG pMV261-Rho, or rBCG pMV361-Rho twice at a 2-week interval. All the recombinant BCG immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4⁺ and CD8⁺ cells compared to the control (P < 0.05). Challenge experiments demonstrated that the two rBCG strains could provide significant protection against E. tenella challenge. But vaccination with rBCG pMV261-Rho induced higher specific antibody titers and produced greater protection rate (56.04%) than rBCG pMV361-Rho group (P < 0.05). These results indicated that M. bovis BCG is a novel vaccine vector to express and present antigens of E. tenella, and rBCG has a potential as vaccine in chickens.     

Concurrent response to challenge infection with Cryptosporidium parvum in immunosuppressed C57BL/6N mice

We investigated the response to challenge infection with Cryptosporidium parvum oocysts in immunosuppressed C57BL/6N mice. In the primary infection, fecal oocyst shedding and parasite colonization were greater in immunosuppressed mice than in nonimmunosuppressed mice. Compared with primary infection, challenge infection with C. parvum didn''t show any oocyst shedding and parasite colonization. Especially, oocyst shedding and parasite colonization from the mice infected with heat-killed oocysts were not detected. After challenge infection with C. parvum oocysts, however, these mice were shedding small numbers of oocysts and parasite colonization. Except normal control and uninfected groups, the antibody titers of other groups appear similar. Based on the fecal oocyst shedding, parasite colonization of ilea, and antibody titers in the mice, these results suggest that the resistance to challenge infection with C. parvum in immunosuppressed C57BL/6N mice has increased.     

Evaluation of protection conferred by a Salmonella Typhimurium inactivated vaccine in Salmonella-infected finishing pig farms

The efficacy of an inactivated S. Typhimurium vaccine administered to pigs at the beginning of the fattening period was evaluated in four clinical trials (trials A, B, C and D). Faecal shedding and the systemic antibody response during fattening, as well as, the cecal contents and mesenteric lymph nodes collected after slaughtering were used to assess the outcome. Salmonella shedders prevalence in the control groups was six times higher than in the treated groups in trials A and D, both herds infected by S. Typhimurium. The risk of positive pens was also four or five times higher for the pens housing control pigs in trials A and C. Lower prevalence of Salmonella was observed in the slaughter samples from the vaccinated pigs in trial D and in the cecal content samples in trial A, when just the S. Typhimurium results were compared. The results suggest the effective homologous protection of the vaccinated pigs; however, the high humoral response elicited in the vaccinated pigs complicates their use in farms under serological surveillance programmes.     

Orally administered attenuated Salmonella enteritidis reduces chicken cecal carriage of virulent Salmonella challenge organisms

Chickens were immunized orally with 10(9)cfu of the temperature-sensitive (T(s)) mutant E/1/3 of Salmonella enteritidis at 1, 2, 3 and 7 days of age. The animals were challenged with wild-type strains of Salmonella of different serotypes 7 or 14 days following immunization. Chickens receiving multiple oral doses of the vaccine strain showed no signs of disease. Immunized animals shed the vaccine strain for at least 2 weeks after the last inoculation; on the other hand, colonization by the attenuated mutant of internal organs such as spleen and liver was limited. Early exposure of the immunized animals to the virulent bacteria resulted in a reduced cecal colonization by the pathogen. Visceral invasion by the wild-type strain of S. enteritidis or S. gallinarum was drastically diminished in birds challenged 14 days after immunization. Significant differences in the number of these Salmonella were found in the cecal contents, spleen and liver of immunized birds compared with the control animals. In addition, cecal colonization by the virulent strain was reduced in birds challenged with S. typhimurium. These results demonstrate that immunization of newly hatched chickens with live attenuated T(s) mutant E/1/3 of S. enteritidis is safe and reduces Salmonella shedding.     

Immunogenicity of a DNA vaccine of Avian Reovirus orally delivered by attenuated Salmonella typhimurium

This study aimed to assess the ability of a Salmonella typhimurium-mediated Avain Reovirus DNA vaccine in eliciting antibody production. Six-day-old SPF chickens were orally immunized with SL7207 (pVAX-σC) twice at 2-week interval, detectable antibody was generated 2 weeks after immunization and was significantly higher than the control groups (P < 0.01) and ten chickens (66.7%) were considered safe in the subsequent challenge. These results show that SL7207 (pVAX-σC) can induce protective antibody in chickens and the newly-constructed vaccine is also effective in protection chickens against ARV infection.     

Protectivity conferred by immunization with intranasal recombinant outer membrane protein H from Pasteurella multocida serovar A:1 in chickens

Recombinant outer membrane protein H (rOmpH) is a potential fowl cholera vaccine candidate. The present study was aimed at developing rOmpH formulations for intranasal administration. The rOmpH was purified and formulated with either Escherichia coli enterotoxin B (LTB) or CpG oligodeoxynucleotides (ODN) as an adjuvant. Antibody responses in chickens intranasally immunized with rOmpH in combination with 2 different adjuvants were significantly increased (P<0.05) post immunization. Chicken survival rates showed that rOmpH formulated with ODN and LTB elicited 90% and 70% protection, respectively. Our findings indicated that rOmpH formulated with ODN elicited protection better than that formulated with LTB. Therefore, the vaccines formulations in the present study can be considered new intranasal vaccine formulations for fowl cholera in chickens.     

Enhanced protective immune responses against Salmonella Enteritidis infection by Salmonella secreting an Escherichia coli heat-labile enterotoxin B subunit protein

Escherichia coli heat-labile enterotoxin B subunit (LTB) protein is a potent mucosal adjuvant. In this study, the effect of an attenuated Salmonella secreting LTB protein as an adjuvant strain (JOL1228) for a live Salmonella Enteritidis (SE) vaccine candidate (JOL919) was evaluated. In a single immunization experiment, chickens immunized with a mixture of JOL919 (5 parts) and JOL1228 (1 part) showed enhanced mucosal and cellular immune responses and efficient protection against salmonellosis as compared to those unimmunized control chickens. In further analysis, chickens were primed at one day of age and were boosted at the fifth week of age to prolong immune responses and to maximize the protection efficacy against salmonellosis. The immunized groups B (prime and booster with JOL919), C (prime with JOL919-JOL1228 mixture and booster with JOL919), and D (prime and booster with JOL919-JOL1228 mixture) showed significantly higher humoral and cellular immune responses as compared to those in the unimmunized control group A. In addition, immunized groups C and D showed fewer gross lesions in the liver and spleen and a lower number of SE-positive organs, with the lowest bacterial counts in the SE challenge strain as compared to the control group. These results indicate that SE vaccination with the LTB strain can have an adjuvant effect on the vaccine candidate by enhancing immune responses, and that a prime-boost strategy with the addition of the adjuvant strain can efficiently protect birds against salmonellosis.     

巨型艾美球虫gam56基因的截短原核表达及其产物的免疫保护效果

为评价gain56基因重组表达产物作为亚单位疫苗预防鸡巨型艾美球虫(E.maxima),感染的效果,以E.maxima NT株配子体总RNA为模板,RT.PCR扩增和克隆gain56基因,选择该基因两段丰富抗原表位的编码区片段.利用原核表达载体pGEX-6P-1在大肠杆菌中进行截短表达.以可溶性的GST-gain56-2融合蛋白为免疫原,设立高、中、低(1.0 mg、0.5 mg、0.25 mg)3个剂量,单独或使用弗氏完全佐剂,于5 d、12 d和19 d对雏鸡进行3次免疫,26 d用5 ×10~4个E.maxima孢子化卵囊进行攻虫,8 d后迫杀,对各组的存活率、相对增重率、卵囊减少率、ACI等指标进行统计分析.结果显示:就相对增重率而言各免疫组比未免疫攻毒组的高27.6%以上,而各免疫组之间差异不显著(p>0.05);就卵囊减少率指标而言,各免疫组较未免疫攻毒组均有不同程度减少,但是均小于75%;就ACI指标而言,各免疫组较未免疫攻毒组均有不同程度增加,以中剂量蛋白佐剂组为最高.GST-gam56-2融合蛋白对于E.maxima感染具有一定的免疫保护效力.