Fecal Oocyst Shedding Research Articles

Eimeria maxima: efficacy of recombinant Mycobacterium bovis BCG expressing apical membrane antigen1 against homologous infection

Coccidiosis is one of the most important protozoan diseases and inflicts severe economic losses on the poultry industry. The aim of this study was to evaluate the capacity of Bacillus Calmette-Guerin (BCG) to deliver apical membrane antigen1 (AMA1) of Eimeria maxima to stimulate specific cellular and humoral immune responses in chickens. Day-old birds were immunized twice with rBCG/pMV261-AMA1, rBCG/pMV361-AMA1, or BCG via oral, intranasal, and subcutaneous routes and then orally challenged with homologous E. maxima sporulated oocysts. Gain of body weight, fecal oocyst output, lesion scores, serum antibody responses, numbers of splenocyte CD4(+) and CD8(+) T cells, and gut cytokine transcript levels were assessed as measures of protective immunity. Challenge experiments demonstrated that rBCG vaccination via intranasal or subcutaneous routes could increase weight gain, decrease intestinal lesions, and reduce fecal oocyst shedding, and the subcutaneous and intranasal routes were superior to the oral route based on the immune effects. Furthermore, intranasal rBCG immunization could also lead to a significant increase in serum antibody, the percentage of CD4+ and CD8+ T lymphocyte cells, and the levels of IL-1β, IFN-γ, IL-15, and IL-10 mRNAs compared with the control group. These results suggested that intranasal rBCG immunization could induce a strong humoral and cellular response directed against homologous E. maxima infection. This study provides data for the use of rBCG to develop a prophylactic vaccine against coccidiosis.

Cryptosporidium parvum: Determination of ID50 and the dose–response relationship in experimentally challenged dairy calves

The objectives were to determine the median infective dose (ID50) of Cryptosporidium parvum and to describe the dose–response relationship including associated clinical illness in experimentally challenged dairy calves. Within the first 24h of life, 27 test calves were experimentally challenged with C. parvum oocysts and 3 control calves were sham dosed. Test calves received 1 of 8 possible doses (25, 50, 100, 500, 1×103, 1×104, 1×105, and 1×106 oocysts). All 27 test calves developed diarrhea. Fecal oocyst shedding occurred in 25 (92.6%) test calves and in 0 control calves. The 2 non-shedding test calves both received 25 oocysts. There was an inverse relationship between dose and time to onset of fecal oocyst shedding (P=0.005). There was no relationship found between dose and duration (P=0.2) or cessation (P=0.3) of fecal oocyst shedding. In addition, there was not a significant relationship between log-dose and the log-peak oocysts (P=0.2) or log-total oocysts (P=0.5) counted/g of feces across the dose groups. There was a positive dose–response relationship between log-dose and diarrhea (P=0.01). However, when controlling for other factors, such as onset and cessation of fecal oocyst shedding, dose was not a significant predictor of diarrhea (P=0.5). Onset and cessation of fecal oocyst shedding were found to be the best predictors of diarrhea (P=0.0006 and P=0.04, respectively). The ID50 for fecal oocyst shedding was 5.8 oocysts, for diarrhea was 9.7 oocysts, and for fecal oocyst shedding with diarrhea was 16.6 oocysts. Given that the ID50 of C. parvum is far less than would be excreted into the environment by a naturally infected calf, prevention and control of cryptosporidiosis is a formidable challenge.

Parasiticidal activity of a novel synthetic peptide from the core α-helical region of NK-lysin

NK-lysin is an anti-microbial peptide that plays a critical role in innate immunity against infectious pathogens through its selective membrane disruptive property. We previously expressed and purified a full-length chicken NK-lysin (cNKL) recombinant protein, and demonstrated its in vitro anti-parasitic activity against the apicomplexan protozoan, Eimeria, the etiologic agent of avian coccidiosis. This study evaluated the in vitro and in vivo anti-parasitic properties of a synthetic peptide (cNK-2) incorporating a predicted membrane-permeating, amphipathic α-helix of the full-length cNKL protein. The cNK-2 peptide exhibited dose- and time-dependent in vitro cytotoxic activity against E. acervulina and E. tenella sporozoites. The cytotoxic activity of 1.5μM of cNK-2 peptide against E. acervulina following 6h incubation was equal to that of 2.5μM of melittin, the principal active component of apitoxin (bee venom) that also exhibits anti-microbial activity. Even greater activity was detected against E. tenella, where 0.3μM of cNK-2 peptide was equivalent to 2.5μM of melittin. Against Neospora caninum tacyzoites, however, the cytotoxic activity of cNK-2 peptide was inferior to that of melittin. Transmission electron microscopy of peptide-treated E. tenella sporozoites revealed disruption of the outer plasma membrane and loss of intracellular contents. In vivo administration of 1.5μM of cNK-2 peptide increased protection against experimental E. acervulina infection, as measured by greater body weight gain and reduced fecal oocyst shedding, compared with saline controls. These results suggest that the cNK-2 synthetic peptide is a novel anti-infective peptide that can be used for protection against avian coccidiosis during commercial poultry production.

Description of fecal shedding of Cryptosporidium parvum oocysts in experimentally challenged dairy calves

The objective was to describe the probability of Cryptosporidium parvum fecal oocyst shedding at different magnitudes of exposure, the pattern of fecal shedding over time, and factors affecting fecal shedding in dairy calves. Within the first 24 h of life, 36 calves were experimentally challenged with C. parvum oocysts at one of four possible magnitudes of oral exposure (1 × 103, 1 × 104, 1 × 105, and 1 × 106 oocysts), and 7 control calves were sham dosed. Fecal shedding occurred in 33 (91.7 %) experimentally challenged calves and in none of the control calves. There was a difference in the log-total number of oocysts counted per gram of feces dry weight among the four exposure groups; calves with the lowest magnitude of exposure (1 × 103 oocysts) shed less than the other three groups. At higher magnitudes of exposure, there was more variability in the range of fecal oocyst shedding. There was an inverse relationship between the log-total amount of oocysts counted per gram of feces dry weight and the number of days to the onset of fecal shedding per calf, i.e., the more time that elapsed to the onset of fecal shedding, the fewer oocysts that were shed. The pattern of fecal shedding over time for all calves shedding oocysts was curvilinear; the number of oocysts increased with time, reached a peak, and declined. Therefore, the dynamics of oocyst shedding can be influenced in part by limiting exposure among calves and delaying the onset of fecal oocyst shedding.

Anticoccidial Activity of Allium sativum and Aloe Vera in Broiler Chickens

Allium sativum (garlic) and Aloe Vera have been widely used for their broad range of pharmacological activities, including antiparasitic activities. Avian coccidiosis is the most costly and wide-spread parasitic disease in the poultry industry, and has been mainly controlled by the use of different chemotherapeutic agents. Due to the emergence of drug-resistant strains, alternative control strategies are needed. In this study, the protective effects of Allium sativum or Aloe vera aqueous extracts-based diets were investigated in broiler chickens following infection with mixed Eimeria species. One hundred and twenty five Cobb chicks were allocated into 5 equal groups each of 25 birds named A, B, C, D, and E. The group A served as control non-infected nontreated, group B was infected non-treated control, group C was on fed on diet supplemented with 1% aqueous extract or Allium sativum, group D was fed on diet supplemented with1% aqueous extract Aloe vera and group E was fed on diet supplemented with 0.5 % of a combination of Allium sativum and Aloe vera aqueous extract. Birds in groups B, C, D, and E, were experimentally infected orally with 50000 mixed sporulated oocysts at day 14 of age. The obtained results showed that, fecal oocyst shedding decreased significantly (p

Eimeria tenella: Interleukin 17 contributes to host immunopathology in the gut during experimental infection

Although IL-17 is a key factor in Th17 lineage host responses and plays critical roles in immunological control of a variety of infectious diseases, the contribution of IL-17 to immune function during Eimeria tenella infection is unknown. In the present study, we used an experimental E. tenella infection model to clarify the role of Th17-associated response in the resulting immune response by quantitative real-time PCR assays. We observed robust production of STAT-3 (the transcription factors), IL-1β, IL-6 and IL-17 in cecal intraepithelial lymphocytes during the early infection, peaking at 6h p.i. and declining thereafter. The expression of TGF-β was moderately upregulated and had 2 peaks at 6 and 72h p.i. during the early infection. To further investigate the role of chIL-17 during the infection, we treated the infected chickens with IL-17 and its neutralized antibody. As a result, the reduced fecal oocyst shedding and cecal lesion scores, but enhanced body weight gains were observed in IL-17 neutralized chickens. The results of histopathology showed that the neutrophils recruitment diminished and the parasite burden in IL-17 neutralized chickens decreased. These results may be due to the significant decrease in the production of IL-17, IL-6 and TGF-β, but enhanced IL-12 and IFN-γ expression in IL-17 neutralized chickens. The converse results were shown in IL-17 treated infected-chickens in which chickens showed increased fecal oocyst shedding, exacerbated lesion scores, and reduced body weight gains. These results suggested that chicken IL-17 might mediate E. tenella - induced immunopathology during the infection.

Effects of in ovo interleukin-4-plasmid injection on anticoccidia immune response in a coccidia infection model of chickens

Two experiments were conducted to study the effects of an in ovo interleukin (IL)-4 plasmid injection in a coccidia infection model. In experiment I, chicks were hatched from eggs that had been injected in ovo with an empty vector or with 10 or 15 μg of IL-4 plasmid, and then challenged posthatch with coccidia. In experiment II, chicks were hatched from eggs that had been vaccinated with coccidia and injected in ovo with an empty vector or with 10 or 15 μg of IL-4 plasmid, and then challenged posthatch with coccidia. In experiment II, the BW gain of birds hatched from eggs vaccinated with live oocysts plus 15 μg of IL-4 plasmid was 25% higher than the BW gain of birds hatched from eggs vaccinated with live oocysts plus empty plasmid. In both experiments I and II, a 15-μg IL-4-plasmid injection decreased fecal oocyst shedding, decreased the number of CD8(+) cells in the cecal tonsils, and decreased cecal tonsil lymphocyte cell proliferation postcoccidia challenge. In experiment I, splenic macrophages of chicks hatched from eggs injected with 15 μg of IL-4 plasmid had higher nitric oxide production than those of chicks hatched from eggs injected with the empty plasmid. In experiment II, a 15-μg IL-4-plasmid injection increased serum anticoccidia IgG postcoccidia challenge. It could be concluded that 15 μg of IL-4 plasmid improved anticoccidia immune responses synergistically with in ovo coccidia vaccination in chickens.

Eimeria tenella heat shock protein 70 enhances protection of recombinant microneme protein MIC2 subunit antigen vaccination against E. tenella challenge

Heat shock proteins have been reported to stimulate the immune system via innate receptors. Our study found that the novel immunopotentiator, Eimeria tenella (E. tenella) heat shock protein 70 (HSP70), enhanced protective immunity elicited by E. tenella antigen microneme protein 2 (EtMIC2) against avian coccidiosis. It demonstrated that the expression of TLR2 and TLR4 were strongly upregulated in EtHSP70 and EtMIC2 plus EtHSP70 stimulated chicken embryo fibroblasts (CEF) compared with untreated controls and EtMIC2 alone. In addition, the same treatment induced high levels of interleukin (IL)-12 and interferon (IFN)-γ that are critical cytokines of innate immunity. In vivo experiments involved using broiler chickens subcutaneously immunized with EtMIC2 alone or EtMIC2 plus EtHSP70 at 7 and 14 days post-hatch, which were then orally challenged with live E. tenella at 7 days following secondary immunization. Body weight gains, cecal lesion scores, fecal oocyst shedding, serum antibody responses against MIC2, and intestinal cytokine transcript levels were assessed as measures of protective immunity. Chickens immunized with EtMIC2 plus EtHSP70 showed increased body weight gains, decreased oocyst shedding, increased serum antibody responses, and high levels of IL-12, IFN-γ, and IL-17 compared with the EtMIC2 only or control groups. Moreover, chickens immunized with EtHSP70 alone showed significantly protective effect against E. tenella infection. In summary, this study provides the first evidence of the immunoenhancing activities of EtHSP70 in poultry.

Effects of Novel Vaccine/Adjuvant Complexes on the Protective Immunity Against Eimeria acervulina and Transcriptome Profiles

SUMMARY. This study investigated the ability of two novel adjuvant formulations, QCDC (Quil A/cholesterol/DDA/ Carbopol) and QCDCR (QCDC/Bay R1005), in combination with a recombinant profilin vaccine, to modulate host protective immunity and to alter gene expression during experimental avian coccidiosis. Vaccination with profilin plus QCDCR significantly reduced the severity of intestinal lesions and increased mitogen-induced lymphocyte proliferation in infected chickens compared with immunization with profilin alone or profilin plus QCDC. Immunization with profilin plus QCDC or profilin plus QCDCR increased body weight gain but had no effect on fecal oocyst shedding of chickens infected with Eimeria acervulina compared with birds vaccinated with profilin alone. The results of global gene expression analysis revealed that, compared with PBS controls, (a) chickens vaccinated with profilin alone had 71 up-regulated and 56 down-regulated mRNA transcripts, (b) chickens immunized with profilin plus QCDC had 198 up-regulated and 247 down-regulated mRNAs, and (c) birds immunized with profilin plus QCDCR had 210 up-regulated and 267 down-regulated mRNAs. Compared with birds vaccinated with profilin alone, (a) chickens given profilin plus QCDC had 60 up-regulated and 104 down-regulated transcripts and (b) chickens immunized with profilin plus QCDCR had 103 up-regulated and 130 down-regulated mRNAs. Finally, chickens vaccinated with profilin plus QCDCR had 193 up-regulated and 204 down-regulated transcripts compared with birds given profilin plus QCDC. Biological function and network analysis revealed that the majority of altered transcripts were encoded by immune-related genes.

Induction of Protective Immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina Infections Using Dendritic Cell-Derived Exosomes

This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting T(h)1 cytokines, T(h)2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the T(h)1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the T(h)2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible.

Control strategies using diclazuril against coccidiosis in goat kids

Coccidiosis is probably the main parasitic disease affecting goat kids around the weaning period, leading to high economic losses in goat production due to deaths and delayed growth rates of infected animals. A total of 101 kids of 2-4 weeks of age, naturally infected with Eimeria spp., were divided into five groups and studies were conducted to analyse the effects of metaphylactic administration of diclazuril (Vecoxan®) on parasitological and productive parameters. Two different doses of diclazuril (1 and 2 mg/kg BW, p.o.) were given either at 3 weeks (single treatment) or at 3 and 5 weeks of life (double treatment). The faecal oocyst shedding and the body weights of the animals were monitored at 2-weeks intervals for 6 consecutive weeks. Treatments of goat kids with diclazuril were effective against the three most predominant Eimeria species recorded in this study (E. arloingi, E. ninakohlyakimovae and E. christenseni) and also against other minor species found in faecal examinations, including E. alijevi, E. caprina, E. jolchijevi, E. caprovina, E. hirci and E. aspheronica). In consequence, OPG values lower than 1 × 10(3) were detected in 90 to 100% of the animals up to 15-20 days post-treatment depending on the treatment regimen. Even a single dose of 1 mg/kg BW p.o. resulted in an increase of growth rates in treated animals and therefore should be considered as a control strategy in farms precluding coccidian infections, whilst double and multiple dose treatments could be the recommendation for environments heavily contaminated with Eimeria oocysts. In relation to the OPG reduction and increased growth rates, the severity of the clinical signs (i.e., diarrhoea) was ameliorated in treated animals during the course of infection compared to that of non-treated or control kids. The precise timing of treatment appears crucial in order to prevent severe clinical coccidiosis and thereby enabling the adequate development of protective immune response against Eimeria challenge infections.

Effects of dietary supplementation with phytonutrients on vaccine-stimulated immunity against infection with Eimeria tenella

Two phytonutrient mixtures, VAC (carvacrol, cinnamaldehyde, and Capsicum oleoresin), and MC (Capsicum oleoresin and turmeric oleoresin), were evaluated for their effects on chicken immune responses following immunization with an Eimeria profilin protein. Chickens were fed with a non-supplemented diet, or with VAC- or MC-supplemented diets, immunized with profilin, and orally challenged with virulent oocysts of Eimeria tenella. Immunity against infection was evaluated by body weight, fecal oocyst shedding, profilin antibody levels, lymphocyte recall responses, cytokine expression, and lymphocyte subpopulations. Following immunization and infection, chickens fed the VAC- or MC-supplemented diets showed increased body weights, greater profilin antibody levels, and/or greater lymphocyte proliferation compared with non-supplemented controls. Prior to Eimeria infection, immunized chickens on the MC-supplemented diet showed reduced IFN-γ and IL-6 levels, but increased expression of TNFSF15, compared with non-supplemented controls. Post-infection levels of IFN-γ and IL-6 were increased, while IL-17F transcripts were decreased, with MC-supplementation. For VAC-supplemented diets, decreased IL-17F and TNFSF15 levels were observed only in infected chickens. Finally, immunized chickens fed the MC-supplemented diet exhibited increased MHC class II+, CD4+, CD8+, TCR1+, or TCR2+ T cells compared with nonsupplemented controls. Animals on the VAC-containing diet only displayed an increase in K1+ macrophages. In conclusion, dietary supplementation with VAC or MC alters immune parameters following recombinant protein vaccination against avian coccidiosis.

Induction of protective immunity against Eimeria tenella infection using antigen-loaded dendritic cells (DC) and DC-derived exosomes

Current methods for sustainable control of avian coccidiosis, whether by prophylactic medication or parasite vaccination, are suboptimal. In this study, we describe an alternative immunization strategy against Eimeria tenella infection using parasite antigen (Ag)-loaded dendritic cells (DCs), or their derived exosomes, in the absence of free Ag. CD45 + intestinal DCs were isolated from E. tenella-infected chickens and loaded ex vivo with an extract of sporozoites as parasite Ag. Extracellular vesicles purified from the Ag-pulsed DCs expressed surface proteins associated with DC-derived exosomes, including major histocompatibility complex proteins (MHC I and MHC II), CD80, flotillin, and heat shock protein (HSP70). Following intramuscular immunization of chickens with Ag-pulsed DCs or Ag-pulsed DC-derived exosomes, Ag-containing cells were observed diffusely localized in the lymphoid tissue and concentrated in germinal centers of caecal tonsils, and restricted to germinal centers (GC) in the spleen. Chickens immunized with pulsed DCs or exosomes exhibited (a) higher numbers of caecal tonsil and spleen cells expressing IgG and/or IgA antibodies that were reactive with E. tenella Ag, (b) greater numbers of IL-2-, IL-16-, and IFN-γ-producing cells, and (c) higher E. tenella Ag-driven cell proliferation, compared with chickens immunized with Ag in the absence of DCs or exosomes. Chickens immunized with Ag-pulsed DCs or Ag-pulsed DC-derived exosomes and subsequently given a live E. tenella challenge infection at 10 d post-immunization displayed (a) increased body weight gains, (b) decreased feed conversion ratios, (c) reduced fecal oocyst shedding, (d) diminished intestinal lesions, and (e) lower mortality, compared with animals given Ag alone. This is the first demonstration of Ag-specific protective immunity against avian coccidiosis using parasite Ag-loaded DCs or DC-derived exosomes.

Embryo vaccination of chickens using a novel adjuvant formulation stimulates protective immunity against Eimeria maxima infection

Our previous study demonstrated that chickens immunized subcutaneously with an Eimeria recombinant profilin protein vaccine emulsified in a Quil A/cholesterol/DDA/Carbopol (QCDC) adjuvant developed partial protection against experimental avian coccidiosis compared with animals immunized with profilin alone. Because in ovo vaccination is presently used in commercial applications worldwide throughout the poultry industry, the current study was undertaken to investigate chicken embryo vaccination with profilin plus QCDC adjuvant. Eighteen day-old embryos were immunized with isotonic saline (control), profilin alone, QCDC alone, or profilin plus QCDC, and orally challenged with live Eimeria maxima at 7 days post-hatch. Body weight gain, fecal oocyst output, and intestinal cytokine transcript levels were assessed as measures of protective immunity. While immunization with profilin alone or QCDC alone did not alter body weight gain of infected chickens compared with the saline control group, vaccination with profilin plus QCDC increased body weight gain such that it was equal to the uninfected controls. Immunization with profilin plus QCDC also reduced fecal oocyst shedding compared with unimmunized controls, although in this case QCDC failed to provide an adjuvant effect since no difference was observed between the profilin-only and profilin/QCDC groups. Finally, increased levels of transcripts encoding IL-1β, IL-15, and IFN-γ were seen in the intestinal tissues of animals given profilin plus QCDC compared with the profilin-only or QCDC-only groups. In summary, this study demonstrates an adjuvant effect of QCDC on body weight gain and intestinal cytokine responses following in ovo vaccination of chickens with an Eimeria profilin vaccine.

Montanide™ ISA 71 VG adjuvant enhances antibody and cell-mediated immune responses to profilin subunit antigen vaccination and promotes protection against Eimeria acervulina and Eimeria tenella

The present study was conducted to investigate the immunoenhancing effects of Montanide™ ISA 71 VG adjuvant on profilin subunit antigen vaccination. Broiler chickens were immunized subcutaneously with a purified Eimeria acervulina recombinant profilin protein, either alone or mixed with ISA 71 VG, and host immune responses were evaluated. After secondary immunization, antigen-specific antibody and T-cell responses were higher in the group which received profilin plus ISA 71 VG compared with the other groups. Furthermore, body weight gains and fecal oocyst shedding were evaluated following oral challenge infection with live E. acervulina or Eimeria tenella oocysts. Vaccination with profilin plus ISA 71 VG reduced oocyst shedding compared with animals immunized with profilin alone. These results demonstrate that the recombinant profilin subunit vaccine, when given in combination with Montanide™ ISA 71 VG, augments protective immunity against E. acervulina and E. tenella.

Protective effects of Aloe vera-based diets in Eimeria maxima-infected broiler chickens

Aloes have been widely used for a broad range of pharmacological activities, including parasitic problems. Avian coccidiosis is the most costly and wide-spread parasitic disease in the poultry industry, and has been mainly controlled by the use of chemotherapeutic agents. Due to the emergence of drug-resistant strains, alternative control strategies are needed. In this study, the protective effects of Aloe vera-based diets were assessed in broiler chickens following oral infection with Eimeria maxima. Chickens were fed a regular diet supplemented with ground Aloe vera throughout the duration of the experiment beginning 2 days prior to infection with 1 × 10 4 sporulated oocysts of E. maxima. No significant differences were found in body weight gain or loss between the Aloe vera-supplemented and unsupplemented groups with or without E. maxima infections. Fecal oocyst shedding decreased significantly ( p < 0.05) in all of the treatment groups that were supplemented with Aloe vera as compared to the unsupplemented group. Furthermore, the Aloe vera-supplemented group showed significantly fewer intestinal lesions ( p < 0.05) than the unsupplemented group following infection. The findings of this study suggest that Aloe vera could be used an alternative treatment for controlling avian coccidiosis.

The effects of a novel adjuvant complex/ Eimeria profilin vaccine on the intestinal host immune response against live E. acervulina challenge infection

The effects of a novel adjuvant composed of Quil A, cholesterol, dimethyl dioctadecyl ammonium bromide, and Carbopol (QCDC) on protective immunity against avian coccidiosis following immunization with an Eimeria recombinant protein were determined. Broiler chickens were subcutaneously immunized with isotonic saline (control), Eimeria recombinant profilin alone, or profilin emulsified with QCDC at 1 and 7 days post-hatch, and orally challenged with live Eimeria acervulina at 7 days following the last immunization. Body weight gains, gut lesion scores, fecal oocyst outputs, profilin serum antibody titers, lymphocyte proliferation, and intestinal cytokine transcript levels were assessed as measures of protective immunity. Chickens immunized with profilin plus QCDC showed increased body weight gains and decreased intestinal lesion scores compared with the profilin only or control groups. However, no differences were found in fecal oocyst shedding among the three groups. Profilin serum antibody titers and antigen-induced peripheral blood lymphocyte proliferation in the profilin/QCDC group were higher compared with the profilin only and control groups. Finally, while immunization with profilin alone or profilin plus QCDC uniformly increased the levels of intestinal transcripts encoding all cytokines examined (IL-1β, IL-10, IL-12, IL-15, IL-17F, and IFN-γ) compared with the control group, transcripts for IL-10 and IL-17F were further increased in the profilin/QCDC group compared with the profilin only group. In summary, this study provides the first evidence of the immunoenhancing activities of QCDC adjuvant in poultry.

Immunoenhancing effects of Montanide™ ISA oil-based adjuvants on recombinant coccidia antigen vaccination against Eimeria acervulina infection

The current study was conducted to investigate the immunoenhancing effects of Montanide™ adjuvants on protein subunit vaccination against avian coccidiosis. Broiler chickens were immunized subcutaneously with a purified Eimeria acervulina recombinant profilin protein, either alone or mixed with one of four adjuvants (ISA 70 VG, ISA 71 VG, ISA 201 VG or ISA 206 VG), and body weight gains, fecal oocyst shedding, and humoral and innate immune responses were evaluated following oral challenge infection with live E. acervulina oocysts. Immunization with profilin plus ISA 70 VG or ISA 71 VG increased body weight gains compared with vaccination with profilin alone. Profilin plus ISA 71 VG also reduced fecal oocyst shedding compared with vaccination in the absence of adjuvant. All adjuvants enhanced profilin serum antibody titers. Increased levels of gene transcripts encoding IL-2, IL-10, IL-17A, and IFN-γ, but decreased levels of IL-15 mRNAs, were seen in intestinal intraepithelial lymphocytes of chickens immunized with profilin plus adjuvants compared with immunization with profilin alone. Finally, increased infiltration of lymphocytes, especially CD8 + lymphocytes at the site of immunization was observed in birds given profilin plus ISA 71 VG compared with profilin alone. These results demonstrate that vaccination with the E. acervulina profilin subunit vaccine in combination with Montanide™ adjuvants enhances protective immunity against avian coccidiosis.

Genetic effects analysis of myeloid leukemia factor 2 and T cell receptor-β on resistance to coccidiosis in chickens

Associations between the parameters of resistance to coccidiosis and SNP in 3 candidate genes located on chromosome 1 [T cell receptor-beta (TCR-beta), myeloid leukemia factor 2 (MLF2), and lymphotactin] were determined. Single nucleotide polymorphisms were genotyped in 24 F1 generation and 290 F2 generation birds. Four SNP were identified in the lymphotactin gene, 12 were located in the TCR-beta gene, and 4 in the MLF2 gene. At various times after experimental infection of the F2 generation with Eimeria maxima, BW, fecal oocyst shedding, and biochemical parameters were measured as parameters of coccidiosis resistance. Single marker association test was applied to determine the associations between the 20 SNP and the parameters of coccidiosis resistance. The maximum additive genetic effect on disease resistance of an SNP in MLF2 was explained by BW (P = 0.0002). The SNP in MLF2 significantly associated with BW was also associated with fecal oocyst shedding (P = 0.001). Four SNP associated with oocyst shedding were found within the coding region of TCR-beta (P < 0.05). Although none of the lymphotactin SNP were associated with oocyst shedding, interferon-gamma level was associated with 2 SNP in lymphotactin. These results suggest that the SNP in the MLF2 gene can be one of the markers to select coccidiosis resistance in chickens.

Efficacy and Characteristics of different Methods of Coccidiosis Infection in Broiler Chickens

Problem statement: Different methods of experimental infection of broiler chickens with Eimeria species have been described in the literature. These methods had not been compared and contrasted so as to contribute to the selection of the most appropriate model of coccidiosis in broiler chickens. Identifying such a model was important to speed up the screening of potential coccidiostatics. Approach: In five different experiments with broiler chickens, we used different methods of infection with Eimeria species. In this paper the different methods and the results have been evaluated. Results: Administration through gavage into the crop of relatively low doses of either Eimeria tenella alone, or in combination with Eimeria acervulina and Eimeria maxima, did not influence body-weight gain and feed intake, but did induce intestinal lesions and faecal shedding of oocysts. The administration of an identically high number of sporulated oocysts in the form of a mixture of the three Eimeria species, either through a single dose by gavage or through the litter, produced similar lowering effects on body-weight gain or feed intake, similar degrees of severity of intestinal lesions and similar rates of faecal oocyst shedding. Conclusion: Depending on the variables considered of interest, the present data may indicate the most appropriate model. The model using infection with oocysts through the litter may optimally mimic the field situation in combination with controlled conditions and allowing experimental flexibility and a high number of experimental units within the research facility.