Anticoccidial Index Research Articles

Prevalence and drug resistance of avian Eimeria species in broiler chicken farms of Zhejiang province, China

In this study, coccidia were isolated and identified from 5 main poultry farms located in Zhejiang province, eastern China. The overall prevalence of Eimeria spp. was 30.7% (95 of 310). Five common species were observed: E. tenella, E. acervulina, E. maxima, E. necatrix, and E. mitis. Two isolates (HZ and QZ) were tested for sensitivity to 8 anticoccidial drugs using 4 indexes including anticoccidial index (ACI), percent of optimum anticoccidial activity (POAA), reduction of lesion scores (RLS), and relative oocyst production (ROP): sulfachloropyrazine, toltrazuril, diclazuril, sulfamonomethoxine/trimethoprim, and amprolium; sulfaquinoxaline/sulfadimethoxine, nicarbazin, and halofuginone. The results showed that the 2 isolates have developed various degrees of resistance to most of the tested drugs. The multi-resistance coccidia are a potential threat to local poultry farming. Rotation of anticoccidial drugs and shuttle programs are recommended to prevent further economic losses caused by coccidiosis.

Immunization of Chicken with Live Eimeria tenella Sporulated Oocysts for Control of Caecal Coccidiosis

Eimeria tenella is the most pathogenic and one of the commonest species of Eimeria infecting broiler chickens raised under deep litter system throughout the world, causing caecal coccidiosis and incurring huge economic losses to the poultry industry. In the present study, the immunization potential of live Eimeria tenella oocysts was evaluated following homologous oocyst challenge in broilers. The birds were immunized at seven and 21 days of age orally with 1000 live sporulated oocyst of Eimeria tenella and challenged with homologous strain of parasite on 28 days of age. The immunization potential was evaluated in terms of relative weight gain, caecal lesion scoring, oocyst output and the anti-coccidial index (ACI). The results revealed that immunization with live oocysts of Eimeria tenella resulted in signifcant relative weight gain (82.47%), reduction in oocyst output (93.74%) and ACI of 161.47, indicating that oral immunization of chickens against E. tenella was effective in preventing the clinical disease and decreasing the oocyst burden in poultry farms.

Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella.

The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE) based on the expressed sequence tag (EST). The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF) that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites). The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.

Evaluation of ionophore sensitivity of Eimeria acervulina and Eimeria maxima isolated from the Algerian to Jijel province poultry farms

The present study represents the first description of ionophore resistance in recovered from commercial Algerian (Jijel-Algeria) broiler farms. Microscopy and intervening transcribed sequence 1 PCR (ITS1 PCR) revealed only 2 Eimeria species present in litter from these farms- namely Eimeria acervulina and Eimeria maxima. A pool of these isolates were evaluated in broiler chickens (Cobb 500) for sensitivity to 5 anticoccidial compounds-diclazuril (1ppm), lasalocid (125ppm), monensin (125ppm), narasin (70ppm) and salinomycin (60ppm). As indicated by anticoccidial sensitivity profiles based on lesion scores and anticoccidial index (ACI), complete resistance to monensin and narasin, partial resistance to salinomycin and lasalocid, and complete sensitivity to diclazuril was observed. While lack of sensitivity to monensin is not surprising given its use for years as the sole anticoccidial compound, the resistance to monoether (narasin) and polyether (lasalocid) ionophores suggests that cross-resistance has developed in a segment of the Eimeria population. The fairly uniform Eimeria species composition among all poultry farms suggests that E. acervulina and E. maxima more rapidly develop resistance to ionophore drugs.

The Molecular Characterization and Immunity Identification of Microneme 3 of Eimeria acervulina.

The gene of Eimeria acervulina microneme protein 3 (EaMIC3) was cloned and characterized. According to the conserved sequence, the 3'- and 5'-ends of EaMIC3 were amplified by the rapid amplification of cDNA ends (RACE). The full length cDNA of this gene was obtained by overlapping the sequences of 3'- and 5'-extremities and amplification by reverse transcription PCR. The sequence analysis revealed that the opening reading frame (ORF) of EaMIC3 was 2,607bp and encoded a protein of 868 amino acids with 93.04kDa. Western blotting assay showed that the recombinant protein was successfully recognized by the sera of chickens experimentally infected with E.acervulina, whereas the native protein in the somatic extract of sporozoites was as well detected by sera from rats immunized with the recombinant protein of EaMIC3. Immunofluorescence analysis indicated that EaMIC3 was expressed in the sporozoites and merozoites stages of E.acervulina. Animal challenge experiments demonstrated that the recombinant protein of EaMIC3 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented anticoccidial index (ACI) more than 165. Moreover, EaMIC3 protein produced significantly high level of IgG antibody, IFN-γ, IL-10, IL-17 TGF-β, CD4+ , and CD8+ .

Field trial of medicinal plant, Bidens pilosa, against eimeriosis in broilers.

Eimeriosis is a severe protozoan disease in poultry. Because of increasing concern about drug residue and drug resistance with the use of anticoccidial drugs, natural products are emerging as an alternative and complementary approach to control avian eimeriosis. Our previous publication showed that feed supplemented with B. pilosa (BP) was effective at combating chicken eimeriosis in experimental settings. However, its efficacy against chicken eimeriosis under field conditions is not known. Here, we investigated the efficacy of BP against eimeriosis on an organic chicken farm. We found that feed supplemented with BP, at the dose of 0.025% of feed or more, significantly reduced Eimeria infection. This treatment increased body weight gain and reduced feed conversion ratio, leading to superior growth performance. It lowered morbidity/mortality rate, decreased oocysts per gram of feces and gut pathology and augmented the anticoccidial index. Collectively, these data demonstrated the potential of BP to control chicken eimeriosis on chicken farms. BP can, therefore, be used as an effective means to control eimeriosis.

Identification and immunogenicity of microneme protein 2 (EbMIC2) of Eimeria brunetti

There have been only a few antigen genes of Eimeria brunetti reported up to now. In this study, the gene encoding the microneme protein 2 (EbMIC2) was isolated from oocysts of E. brunetti by RT-PCR and the immunogenicity of recombinant EbMIC2 was observed. The EbMIC2 was cloned into vector pMD19-T for sequencing. The sequence was compared with the published EbMIC2 gene from GenBank revealed homology of the nucleotide sequence and amino acids sequence were 99.43 and 98.63%, respectively. The correct recombinant pMD-EbMIC2 plasmid was inserted into the pET-28a (+) expressing vector and transformed into competent Escherichia coli BL21 cells for expression. The expressed product was analyzed using SDS-PAGE and Western-blot. The results indicated that the recombinant EbMIC2 protein was recognized strongly by serum from naturally infected chicken with E. brunetti. Rat rcEbMIC2 antisera bound to bands of about 36 kDa in the somatic extract of E. brunetti sporozoites. The recombinant plasmid pVAX1-EbMIC2 was constructed and then the efficacies of recombinant plasmid and recombinant protein were evaluated. The results of IgG antibody level and cytokines concentration suggested that recombinant EbMIC2 could increase the IgG antibody level and induce the expressions of cytokines. Animal challenge experiments demonstrated that the recombinant EbMIC2 protein and recombinant plasmid pVAX1-EbMIC2 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented high anti-coccidial index. All results suggested that EbMIC2 could become an effective candidate for the development of new vaccine against E. brunetti infection.

Immune protection of microneme 7 (EmMIC7) against Eimeria maxima challenge in chickens

In the present study, the immune protective effects of recombinant microneme protein 7 of Eimeria maxima (rEmMIC7) and a DNA vaccine encoding this antigen (pVAX1-EmMIC7) on experimental challenge were evaluated. Two-week-old chickens were randomly divided into five groups. Experimental groups of chickens were immunized with 100 μg DNA vaccine pVAX1-MIC7 or 200 μg rEmMIC7, while control groups of chickens were injected with pVAX1 plasmid or sterile phosphate buffered saline (PBS). The results showed that the anti-EmMIC7 antibody titres in chickens of both rEmMIC7 and pVAX1-MIC7 groups were significantly higher as compared to PBS and pVAX1 control (P < .05). The splenocytes from both vaccinated groups of chickens displayed significantly greater proliferation response compared with the controls (P < .05). Serum from chickens immunized with pVAX1-MIC7 and rEmMIC7 displayed significantly high levels of interleukin-2, interferon-γ, IL-10, IL-17, tumour growth factor-β and IL-4 (P < .05) compared to those of negative controls. The challenge experiment results showed that both the recombinant antigen and the DNA vaccine could obviously alleviate jejunum lesions, body weight loss and enhance oocyst decrease ratio. The anti-coccidial index (ACI) of the pVAX1-MIC7 group was 167.84, higher than that of the recombinant MIC7 protein group, 167.10. Our data suggested that immunization with EmMIC7 was effective in imparting partial protection against E. maxima challenge in chickens and it could be an effective antigen candidate for the development of new vaccines against E. maxima.

Construction of Eimeria tenella multi-epitope DNA vaccines and their protective efficacies against experimental infection

The search for effective vaccines against chicken coccidiosis remains a challenge because of the complex organisms with multiple life cycle stages of Eimeria. Combination of T-cell epitopes from different stages of Eimeria life cycle could be an optimal strategy to overcome the antigen complexity of the parasite. In this study, 4 fragments with concentrated T-cell epitopes from the sporozoite antigen SO7 and the merozoite antigen MZ5-7 of Eimeria tenella were cloned into eukaryotic expression vector pVAX1 in different forms, with or without chicken cytokines IL-2 or IFN-γ genes as genetic adjuvants, to construct multistage, multi-epitope DNA vaccines against Eimeria tenella. Transcription and expression of the multi-epitope DNA vaccines in vivo were detected by reverse transcription-PCR (RT-PCR) and Western blot. On the basis of survival rate, lesion score, body weight gain, oocyst decrease ratio and the anti-coccidial index (ACI), Animal experiments were carried out to evaluate the protective efficacy against Eimeria tenella. Results showed the constructed DNA vaccines were transcribed and translated successfully in vivo. Animal experiment showed that the multi-epitopes DNA vaccines were more effective to stimulate immune response than single fragment. Compared with the DNA vaccines composed with less T-cell epitopes, DNA vaccine pVAX1-m1-m2-s1-s2 containing 4 fragments with concentrated T-epitopes provided the highest ACI of 180.39. DNA vaccines composed of antigens from two developmental stages were more effective than the single-stage ones. Especially DNA vaccine pVAX1-m1-m2-s1-s2 provided the most effective protection with the ACI of 180.39. Furthermore, cytokines IL-2 or IFN-γ could improve the efficacy of the multi-epitope DNA vaccines significantly. Overall, pVAX1-m1-m2-s1-s2-IFN-γ provided the most effective protection with the ACI of 189.92. The multi-epitope DNA vaccines revealed in this study provide new candidates for Eimeria vaccine development.

Efficacy of chimeric DNA vaccines encoding Eimeria tenella 5401 and chicken IFN-γ or IL-2 against coccidiosis in chickens

Chimeric DNA vaccines encoding Eimeria tenella (E. tenella) surface antigen 5401 were constructed and their efficacies against E. tenella challenge were studied. The open reading frame (ORF) of 5401 was cloned into the prokaryotic expression vector pGEX-4T2 to express the recombinant protein and the expressed recombinant protein was identified by Western blot. The ORF of 5401 and chicken cytokine gene IFN-γ or IL-2 were cloned into the eukaryotic expression vector pVAX1 consecutively to construct DNA vaccines pVAX-5401-IFN-γ, pVAX-5401-IL-2 and pVAX-5401. The expression of aim genes in vivo was detected by reverse transcription-polymerase chain reaction and Western blot. Fourteen-day-old chickens were inoculated twice at an interval of 7 days with 100 µg of plasmids pVAX-5401, pVAX-5401-IFN-γ and pVAX-5401-IL-2 or 200 µg of recombinant 5401 protein by leg intramuscular injection, respectively. Seven days after the second inoculation, all chickens except the unchallenged control group were challenged orally with 5 × 104 sporulated oocysts of E. tenella. Seven days after challenge, all chickens were weighted and slaughtered to determine the effects of immunization. The results showed the recombinant protein was about 90 kDa and reacted with antiserum against soluble sporozoites. The animal experiment showed that all the DNA vaccines pVAX-5401, pVAX-5401-IFN-γ or pVAX-5401-IL-2 and the recombinant 5401 protein could obviously alleviate body weight loss and cecal lesions as compared with non-vaccinated challenged control and empty vector pVAX1control. Furthermore, pVAX-5401-IFN-γ or pVAX-5401-IL-2 induced anti-coccidial index (ACI) of 180.01 or 177.24 which were significantly higher than that of pVAX-5401. The results suggested that 5401 was an effective candidate antigen for vaccine. This finding also suggested that chicken IFN-γ or IL-2 could effectively improve the efficacies of DNA vaccines against avian coccidiosis.

Protective immunity against Eimeria tenella infection in chickens following oral immunization with Bacillus subtilis expressing Eimeria tenella 3-1E protein.

The current experiment was conducted to construct recombinant Bacillus subtilis WB600 expressing Eimeria tenella 3-1E protein to investigate the oral immunization protective effects against E. tenella. The merozoite surface antigen 3-1E gene of E. tenella was introduced into the pBS-H1 expression vector with a novel signal peptide sequence. After the electro-transformation, the expression of objective protein in B. subtilis WB600 was detected by Western blot. The results showed that the recombinant B. subtilis strain with the ability of high-level secretion of 3-1E was constructed successfully. Seven-day-old broiler chickens were orally vaccinated with B. subtilis WB600 harboring 3-1E (B.S-pBS-H1-3-1E) or B. subtilis WB600 with empty plasmid (B.S-pBS-H1) 10 days prior to challenge with sporulated E. tenella oocysts. The results showed the recombinant B. subtilis strain with the ability of high-level secretion of 3-1E was constructed successfully. Vaccination with B.S-pBS-H1-3-1E strain significantly increased the anti-coccidial index and reduced cecal lesion scores compared with the positive control group (chickens were challenged with sporulated E. tenella oocysts without oral administration of B.S-pBS-H1-3-1E strain) and B.S-pBS-H1 group. Ceca mucosal sIgA, secretion, and IL-2, IL-12, IFN-γ, and IL-10 level after challenge were greater in the B.S-pBS-H1-3-1E group than in the positive control group. Taken together, these results indicated that B. subtilis WB600 harboring 3-1E protein induces protective immunity against E. tenella.

Evaluation of immune protective efficacies of Eimeria tenella EtMic1 polypeptides with different domain recombination displayed on yeast surface

In the present study, three different live oral vaccines using the EBY100/pCTCON-2 yeast surface display system with different Eimeria tenella microneme-1 (EtMic1) protein domain recombination were constructed and their protective efficacies against homologous challenge were compared by evaluating the body weight gains, relative growth rate, cecal lesion scores, oocyst output, oocyst decrease ratio, anti-coccidial index, the serum antibody levels and the proliferation ability of blood lymphocytes. The results indicated that all the three constructed live oral vaccines expressing different EtMic1 polypeptides provided excellent protection against homologous challenge by significantly increasing weight gains, reducing cecal lesions, achieving a high ACI, elevating specific antibody response and splenocyte proliferation ability compared with controls. The yeasts displaying the EtMic1 polypeptide-III (expressed TSP-2, TSP-3 and TSP-4 domains) provided better protection against challenge than the yeasts displaying either the EtMic1 polypeptide-I (expressed I-domain, TSP-1 and TSP-2) or polypeptide-II (expressed I-domain and all the five TSP domains) did. Considering the exclusion of antibiotic resistant gene in the system, the strain EBY100 of Saccaromyces cerevisiae may be a better choice for coccidian antigen delivery.

Induction of protective immunity against Eimeria tenella, Eimeria necatrix, Eimeria maxima and Eimeria acervulina infections using multivalent epitope DNA vaccines

Avian coccidiosis is mostly caused by mixed infection of several Eimeria species under natural conditions and immunity to avian coccidiosis is largely dependent on T-cell immune response. In this study, 14 T-cell epitope fragments from eight antigens of Eimeria tenella (E. tenella), Eimeria necatrix (E. necatrix), Eimeria maxima (E. maxima) and Eimeria acervulina (E. acervulina) were ligated with pVAX1 producing 14 monovalent DNA vaccines, respectively. Protective immunity of the monovalent DNA vaccines was assessed by in vivo challenge experiments and then four most protective fragments of each species were chosen to construct multivalent epitope DNA vaccines with or without chicken IL-2 as genetic adjuvant. Protective efficacies of the epitope DNA vaccines on chickens against E. tenella, E. necatrix, E. maxima and E. acervulina were evaluated. The results showed that the constructed multivalent epitope DNA vaccines significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds. Especially, the multivalent epitope DNA vaccines of pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1 and pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2 not only significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds, but also resulted in anti-coccidial index (ACI) more than 170 against E. tenella, E. necatrix, E. maxima and E. acervulina, which indicated they could induce protective immunity against E. tenella, E. necatrix, E. maxima and E. acervulina. Our findings suggest the constructed multivalent epitope DNA vaccines are the potential candidate multivalent vaccines against mixed infection of Eimeria.

Comparsion Between Anti Coccidial Effectes of Artemisia Siberi Extract and Apacox Copmplex and Lasalosid in Experimental Infected Broilers with Coccidiosis

Increasing resistance against conventional anticoccidial drugs and consequence of their residues has paid the attention toward more effective and safe compounds. Artemisia plant and the apacox complex are potential candidates that their anticoccidial effects has been previously discusse: This study aimed to determin an appropriate dosage from the extract of Artemisia siberi and evaluate its anticcocidial effects compared to apacox complex and lasalosid drug. Artemisinin extract was extracted from Artemisia siberi by petroleum ether the apacox complex and lasalosid bought from the pharmacy by our order.105 chicks were divided in to 7 group tree replicates each (n=15).artemisia extract in two dosages 0/5 and 1/5 mg/kg for group A &B and the apacox complex in two dosages 0/5 and 1/5 gr/kg for group C&D and lasalosid in 75 mg/kg were used for the E group of broilers from the first day as feed additive . infected group(F) and non infected group(G) didn’t recive any medication . Experimental coccidiosis was induced in chicks by oral administration of 100000 oocysts/chick on 14 th day . fecal samples of each group were collected for 5 days from 15th day until 19th day and the OPG (oocyst per gram) was determined as anticoccidial index. Artemisia and apacox complex significantly decreased OPG value in treated group although it was lower than lasalosid group .artemisia extract in 1/5 mg /kg had stronger power than apacox in its two dosage and Artemisia in 0/5 mg/kg .this means Artemisia have dependent dosage effect in dosage lower than 1/5 mg/kg. This study showed that the Artemisia siberi extract can be considered as a new effective and safe anticoccidial drug although it is weaker than lasalosid

Identification and molecular characterization of microneme 5 of Eimeria acervulina.

In the present study, the microneme 5 gene of Eimeria acervulina (E. acervulina) (EaMIC5) was cloned and characterized. Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expressed sequence tag (EST, GenBank Accession No. EH386430.1) to amplify the 3′- and 5′-ends of EaMIC5. The full length cDNA of this gene was obtained by overlapping the sequences of 3′- and 5′-extremities and amplification by reverse transcription PCR. Sequence analysis revealed that the open reading frame (ORF) of EaMIC5 was 336 bp and encoded a protein of 111 amino acids with 12.18 kDa. The ORF was inserted into pET-32a (+) to produce recombinant EaMIC5. Using western blotting assay, the recombinant protein was successfully recognized by the sera of chicks experimentally infected with E. acervulina, while the native protein in the somatic extract of sporozoites was as well detected by sera from rats immunized with the recombinant protein of EaMIC5. Immunofluorescence analysis using antibody against recombinant protein EaMIC5 indicated that this protein was expressed in the sporozoites and merozoites stages of E. acervulina. Animal challenge experiments demonstrated that the recombinant protein of EaMIC5 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens, and presented anti-coccidial index (ACI) more than 160. All the above results suggested that the EaMIC5 was a novel E. acervulina antigen and could be an effective candidate for the development of a new vaccine against this parasite.

Effect of Bidens pilosa on infection and drug resistance of Eimeria in chickens

Extensive use of current anti-coccidial drugs together with drug resistance and residue has raised concerns about public health and poultry development. Here, we studied the anti-coccidial properties of Bidens pilosa. A phytochemical approach was developed for analysis of B. pilosa utilized as a feed additive. The protective effects of B. pilosa supplemented chicken diet were evaluated chickens infected with Eimeria tenella. B. pilosa, at doses of 0.5%, 1% and 5% of the chicken diet, significantly protected against E. tenella as measured by reduction in mortality, weight loss, fecal oocyst excretion and gut pathology in chickens. Finally, drug resistance of E. tenella to B. pilosa was assessed in chickens using the anti-coccidial index. This index showed that B. pilosa induced little, if any, drug resistance to Eimeria in chickens. Collectively, this work suggests that B. pilosa may serve as a novel, natural remedy for coccidiosis with low drug resistance in chickens.

Protective efficacy in chickens of recombinant plasmid pET32a(+)-ADF-3-1E of Eimeria acervulina.

This experiment was conducted to study the protective efficacy of recombinant plasmid pET32a(+)-ADF-3-1E in coccidian-infected breeding chickens. The 7-day-old chickens were randomly divided into five groups: a recombinant plasmid pET32a(+)-ADF-3-1E group, a pET32a(+)-ADF group, a pET32a(+)-3-1E group, a control group, and an infection control group. The chickens were immunized intramuscularly with recombinant plasmid DNA in a dose of 200 μg, respectively, and a booster vaccination was given at the same dosage 1 week later. The peripheral blood T lymphocyte proliferation, serum IgG antibody response, and levels of interleukin 2 (IL-2) and interferon gamma (IFN-γ) were detected, respectively. The chickens were inoculated with 4 × 10(6) Eimeria acervulina-sporulated oocysts (Baoding strain) on the seventh day after the last immunization to evaluate the protective efficiency of the recombinant plasmid DNA. The results showed that the lymphocyte proliferation, serum IgG antibody, and IL-2 and IFN-γ levels in recombinant plasmid DNA group were significantly higher than those in control group (P < 0.01). The lymphocyte proliferation, serum IgG antibody, and IL-2 and IFN-γ levels in pET32a(+)-ADF-3-1E group were significantly higher than those in pET32a(+)-3-1E group and pET32a(+)-ADF group, respectively (P < 0.05). It indicated that the pET32a(+)-ADF-3-1E could produce stronger immune responses. The relative body weight gain rate in pET32a(+)-ADF-3-1E group was 88.36 %, which was significantly higher than that in control group (P < 0.05) and infection control group (P < 0.01). The reductions of oocyst production and lesion scores in pET32a(+)-ADF-3-1E group were 67.88 and 67.13 %, respectively. The oocyst excretion and the lesion score of chickens in pET32a(+)-ADF-3-1E group were lower than those in infection control group, respectively. Anticoccidial index (ACI) value in group immunized with pET32a(+)-ADF-3-1E was 169.82. ACI value of 160-179 was considered as effective. These results demonstrated that the pET32a(+)-ADF-3-1E recombinant plasmid DNA could effectively improve the cellular responses and humoral immune responses of the chickens, and it might provide protection against coccidiosis in chickens.

Anticoccidial effects of a novel triazine nitromezuril in broiler chickens

The anticoccidial efficacy of 2-(3-methyl-4-(4-nitrophenoxy)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (nitromezuril, NZL), a novel triazine compound, was evaluated in three different studies under experimental conditions. The anticoccidial efficacy was chiefly evaluated using the anticoccidial index (ACI). The resistance level was determined by calculating ACI, percentage optimum anticoccidial activity (POAA), reduction in lesion scores (RLS) and relative oocyst production (ROP). In the dose determination study (study A), NZL was added to the diet at doses of 1, 2, 3, 4, 5 and 6mg/kg to test its efficacy against coccidiosis caused by Eimeria tenella. Groups treated with NZL 1mg/kg feed could observe the faecal dropping scores and caecal lesions. ACIs of NZL-treated groups reached 179–199. In the study on the anticoccidial efficacy of 3mg/kg NZL in the diet (study B), only a few faecal oocysts and slight lesions were observed. NZL significantly promoted weight gain (WG) and reduced lesion scores (LS) compared to controls receiving diclazuril (DZL) (P<0.05). ACIs of NZL-treated groups were 193, 192, 191 and 163 for E. tenella, Eimeria necatrix, Eimeria acervulina and Eimeria maxima, respectively, whereas those of DZL-treated groups were 185, 176, 176 and 148. In the cross-drug resistance study (study C), ACIs of NZL and toltrazuril (TZL)-treated groups ranged from 188 to 204, which were significantly higher than those of DZL-treated groups (P<0.05). NZL- and TZL-treated groups were sensitive to experimentally induced DZL-resistant E. tenella, whereas DZL-treated groups showed complete resistance. No cross-resistance was observed between DZL and NZL or TZL. Based on the abovementioned studies, it was concluded that diets containing 3mg/kg NZL had an excellent efficacy in preventing coccidiosis in broiler chickens. The activity of 3mg/kg NZL in the diet was equal or superior to that of 1mg/kg DZL. These results are of great significance for the future applications of NZL; however, its actual mechanism of action remains unknown. NZL is a potential novel anticoccidial agent suitable for further development.

Immunization of Broiler with Dead Sporozoites as Vaccine Against Eimeria tenella Parasite

2 Abstract: Chickens were protected against coccidiosis induced by Eimeria tenella by using 125, 000 dead sporozoites that were injected in neck subcutaneously with two doses at 3rd and 16th days of age. Efficacy of this vaccine was estimated after challenge by determination some parameters like protection rate which was around 96.1-96%. Number of oocysts and cecal lesion score from the chickens in the immunized groups were decreased significantly (p<0.05<0.01). This vaccine was not effected change on the body weight gain. Also, the immunized groups were higher body weight gain as compare with control groups after challenge. The Anti coccidial index measurement revealed the effectiveness of vaccine considerably. The immunogenicity of vaccine was studied by using SDS-PAGE and Western blot. 7 polypeptides had been estimated more immunogenic after probed with chickens serum at 39 days of age, their molecular weight are (12.3, 13.68, 18.7, 39, 59.5 and 77.3) KD. The results were obtained the feasibility of immunizing Broiler against E. tenella infection by using dead sporozoites as a vaccine.

Partial protective of chickens against Eimeria tenella challenge with recombinant EtMIC-1 antigen

Eimeria tenella microneme protein 1 (EtMIC-1) is highly conserved with TgMIC-2, which is involved in parasite binding specifically to host cells. Little is known about the immune responses and protective efficacy against E. tenella infection with EtMIC-1 antigen. In the present study, the recombinant proteins of E. tenella mature MIC-1 and adhesive domain (von Willebrand factor type A domain, EtMIC-1-VD) were obtained, protective efficacy against E.tenella infection and the mucosal immune response, which is induced in broilers was evaluated. The antibody levels and the transcription profiles of cytokine of chickens, such as interleukin-12 (IL-12) and interferon-γ (IFN-γ), were detected after being immunized three times with the recombinant EtMIC-1 and EtMIC-1-VD by ELISA assay and quantitative real-time PCR, respectively. The results showed that both groups of chickens, after being immunized with 100μg EtMIC-1 or EtMIC-1-VD antigen, induced about tenfold higher IgG levels compared to the nonimmune groups. The transcription profiles of IL-12 and IFN-γ of the immunized groups were significantly higher than the control groups as well. The anticoccidial index of the group immunized with 100μg EtMIC-1 and the group immunized with 100μg EtMIC-1-VD were 167.2 and 165.5, respectively, which are significantly higher than low-dose immunized groups and challenged control groups. Our data suggests that VD domain is the key functional structure of EtMIC-1 that could trigger a significant humoral and cellular response against E. tenella infection, and EtMIC-1 had the potential in imparting partial protection in chickens against homologous challenge.