Efficacy of an attenuated vaccine against avian coccidiosis in combination with feed additives based on organic acids and essential oils on production performance and intestinal lesions in broilers experimentally challenged with necrotic enteritis.

Several factors predisposing to necrotic enteritis (NE) have been identified, including diet and Eimeria spp. infestations. Coccidiosis vaccines are indicated to decrease the intestinal lesions caused by specific Eimeria species that are a known predisposing factor to NE and, consequently, these vaccines could be a holistic approach to the control of NE disease and an alternative solution to coccidiostats. Besides, feed additives have also gained special attention from the poultry industry as an alternative solution to antibiotics to prevent NE as well as other bacterial enteritis. Then, the combination of vaccination against coccidiosis and the supplementation of the diet with feed additives could be a composite approach to the control of NE problems triggered by Eimeria spp. infestation. The objective of this study was to test the efficacy of an attenuated coccidiosis vaccine (EVANT) in combination with different feed additives to prevent the loss of production performance and intestinal lesions in broilers challenged with NE. Healthy day-old broilers (n = 960) were randomly allocated to 6 groups (8 cages/group). Groups 1-2 were left unvaccinated. Groups 3-6 were vaccinated following the manufacturer's instructions. Chickens were grown using a diet favoring the intestinal proliferation of Clostridium perfringens. Moreover, the diets of groups 4-6 were supplemented with medium chain fatty acids (MCFA), butyric acid or phytogenic feed additives (PFA), respectively. A NE infection model was used to challenge groups 2-6; chickens were orally infected with Eimeria maxima (4,500 oocysts) and then C. perfringens (108 CFU) at 15 and 20 d, respectively. Birds were monitored and productive parameters recorded until 42 d; intestinal lesions were scored. Results showed that coccidiosis vaccination, with or without the addition of feed additives, decreased intestinal lesions associated with NE and improved the performance of the birds. Besides, the addition of MCFA to the diet decreased intestinal lesions associated to NE in vaccinated animals compared to all treatment groups. Moreover, the same additive improved the feed conversion rate. Therefore, vaccination with a live attenuated coccidiosis vaccine together with in-feed inclusion of MCFA might be a solution to reduce NE in broilers raised antimicrobial- and coccidiostat-free.

Antibody and cytokine response to Cystoisospora suis infections in immune-competent young pigs.

BACKGROUND: To date, investigations on the immune response to Cystoisospora suis infections focused on suckling piglets, the age group clinically most affected. Actively immunizing piglets is unfeasible due to their immature immune system and the typically early infection in the first days after birth. Therefore, understanding and possibly enhancing the immune response of immune-competent animals is the prerequisite to develop a passive immunization strategy for piglets which currently rely on very limited treatment options. METHODS: To investigate antibody and cytokine responses of immune-competent animals and the impact of the oral immunization protocol on their immune response, growers with unknown previous exposure to C. suis (10-11 weeks-old) were infected one or three times with different doses (600 and 6000 or 200 and 2000, respectively) of C. suis oocysts, and compared to uninfected controls. Oocyst excretion was evaluated, and blood and intestinal mucus antibody titers were determined by IFAT. Systemic production of Th1, Th2, inflammatory and regulatory cytokines was determined in different immune compartments at mRNA and (after stimulation with a recombinant merozoite-protein) at protein level by PCR and multiplex fluorescent immunoassay, respectively. RESULTS: Infection generated significantly increased serum IgA and IgG levels against C. suis sporozoites and merozoites, irrespective of infection mode, with IgG against merozoites showing the strongest increase. No clinical signs and only occasional excretion were observed. The systemic cytokine response to C. suis was only weak. Nonetheless, in white blood cells, IL-4, IL-6 and IL-10 mRNA-levels significantly increased after infection, whereas IFN-É£, IL-2 and TGF-ß expression tended to decrease. In mesenteric lymph nodes (MLN), IL-10 and TNF-α levels were elevated while splenic cytokine expression was unaltered upon infection. Stimulated MLN-derived lymphocytes from infected pigs produced slightly more IL-12 and less IFN-α than controls. CONCLUSIONS: An infection and a subsequent systemic immune response can be induced in immune-competent animals by all evaluated infection models and growers can be used as models to mimic sow immunizations. The immune response to C. suis, although mild and with considerable variation in cytokine expression, was characterized by a Th2-associated and regulatory cytokine profile and antibody production. However, none of the parameters clearly stood out as a potential marker associated with protection. Antibody titers were significantly positively related with oocyst excretion and might thus serve as correlates for parasite replication or severity of infection.

Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein.

BACKGROUND: The genome of the apicomplexan parasite Cystoisospora suis (syn. Isospora suis) has recently been sequenced and annotated, opening the possibility for the identification of novel therapeutic targets against cystoisosporosis. It was previously proposed that a 42 kDa uncharacterized merozoite protein, encoded by gene CSUI_005805, might be a relevant vaccine candidate due to its high immunogenic score, high expression level and species-specificity as determined in silico. METHODS: The 1170 bp coding sequence of the CSUI_005805 gene was PCR amplified and cloned into the bacterial expression vector pQE-31. The specificity of the expressed recombinant protein was evaluated in an immunoblot, and relative levels of expression in different developmental stages and subcellular localization were determined by quantitative real-time PCR and indirect immunofluorescence assay, respectively. RESULTS: The CSUI_005805 gene encoded for a 389 amino acid protein containing a histidine-rich region. Quantitative RT-PCR showed that CSUI_005805 was differentially expressed during the early development of C. suis in vitro, with higher transcript levels in merozoites compared to sporozoites. The recombinant protein was specifically recognized by sera from chicken immunized with recombinant CSUI_005805 protein and sera from piglets experimentally infected with C. suis, all of which suggested that despite prokaryotic expression, the recombinant CSUI_005805 protein maintained antigenic determinants and could elicit an immune response in the host. Immunofluorescence labelling and confocal microscopy revealed localization primarily at the surface of the parasite. CONCLUSIONS: The results suggest that CSUI_005805 is highly expressed in merozoites and might thus be critical for their survival and establishment inside host cells. Owing to its specificity, localization and expression pattern, CSUI_005805 could be exploited as an attractive candidate for alternative control strategies against C. suis such as vaccines.

Pathogenicity of Eimeria praecox alone or associated with Eimeria acervulina in experimentally infected broiler chickens.

Eimeria praecox and Eimeria acervulina are two species of coccidia parasites infecting chickens, which develop in the duodenum. The purpose of this study was to evaluate the pathogenicity of E. praecox and to study interactions of this coccidium with E. acervulina. The results showed that the pathogenicity of E. praecox was related to the infective dose, and that its impact on individual weight and weight gain was significant from the lowest administered dose: 5000 oocysts per bird. No morbidity was observed, even with the highest infective dose, but faecal consistency alteration was higher with increasing infective doses. No consistent lesion was observed. When E. praecox was associated with E. acervulina with a low infective dose, performance deterioration seemed to be an additional effect of the two species. However, in the case of heavy infections, signs worsened along with duration of negative impact on growth, compared to a mono-infection.

Effects of in ovo vaccination and anticoccidials on the distribution of Eimeria spp. in poultry litter and serum antibody titers against coccidia in broiler chickens raised on the used litters.

The present study reports the effects of various field anticoccidial programs on the distribution of Eimeria spp. in poultry litter and serum antibody titers against coccidia in broiler chickens raised on the used litters. The programs included in ovo vaccination and various medications with either chemicals, ionophores, or both. In general, serum samples from these chickens showed anticoccidial antibody titers when tested at days 7 and 14 post hatch with the peak response at day 43. Serum anticoccidial titers were highest in birds fed a non-medicated diet compared with those vaccinated or fed medicated diets. Total number of Eimeria oocysts and the composition of Eimeria spp. present in the litter samples from different treatment groups varied depending on the type of anticoccidial program. Oocyst counts in general ranged from 3.7×10(3) to 7.0×10(4) per g of litter. Importantly, both morphological and molecular typing studies revealed four major predominant Eimeria spp., E. acervulina, E. maxima, E. praecox, and E. tenella in the litter samples. Collectively, these results indicate that the field anticoccidial programs influenced the type and abundance of Eimeria spp. present in the litter samples and also modulated host immune response to Eimeria.

Distinct immunoregulatory properties of macrophage migration inhibitory factors encoded by Eimeria parasites and their chicken host.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays an important role in host defense against a variety of microorganisms including protozoan parasites. Interestingly, some microbial pathogens also express a MIF-like protein, although its role in disease pathogenesis is not well understood. The aim of this study was to compare an Eimeria-encoded MIF (E.MIF) protein with chicken MIF (C.MIF) on the basis of their structural, immunological, and biological properties. E.MIF and C.MIF proteins, each with a glutathione S-transferase epitope tag, were expressed in Escherichia coli or COS-7 cells and purified by glutathione affinity chromatography. Rabbit antisera against the purified proteins demonstrated their mutual immunological cross-reactivity on Western blots, and immunolocalized intracellular native E.MIF to the Eimeria schizont, merozoite, and oocyst life cycle stages. HD11 chicken macrophages treated in vitro with C.MIF recombinant protein expressed increased levels of transcripts encoding interleukin-6 (IL-6), IL-17, and tumor necrosis factor superfamily member 15 (TNFSF15), but decreased levels of IL-8 transcripts, compared with cells treated with the PBS control; similar treatment with E.MIF only down-regulated IL-8 transcripts. Unlike recombinant E.MIF, C.MIF exhibited in vitro chemotactic activity for HD11 cells. Conversely, E.MIF, but not C.MIF, enhanced protection against experimental Eimeria infection, compared with the PBS control. These studies provide evidence for overlapping structural and antigenic properties, but distinct immunoregulatory roles, of E.MIF and C.MIF.

Location of RAD51-like protein during meiotic prophase in Eimeria tenella.

This study focuses on reporting events in Eimeria tenella oocysts from early to late prophase I in terms of RAD51 protein in association with the synaptonemal complex formed between homologous chromosomes. The aim of the study was the sequential localization of RAD51 protein, which is involved in the repair of double-strand breaks (DSBs) on the eimerian chromosomes as they synapse and desynapse. Structural Maintenance of Chromosome protein SMC3, which plays a role in synaptonemal complex formation, was labeled to identify initiation and progress of chromosome synapsis and desynapsis in parallel with the appearance and disappearance of RAD51 foci. Antibodies directed against RAD51 and cohesin subunit SMC3 proteins were labeled with either fluorescence or colloidal gold to visualize RAD51 protein foci and synaptonemal complexes. RAD51 protein localization during prophase I was studied on meiotic chromosomes spreads obtained from oocysts at different points in time after the start of sporulation. The present findings showed that foci detected with the antibody directed against RAD51 protein first appeared at the pre-leptotene stage before homologous chromosomes began pairing. Subsequently, the foci were detected in association with the lateral elements at the precise sites where synapsis were in progress. These findings lead us to suggest that in E. tenella, homologous chromosome pairing was a DSB-dependent mechanism and reinforced the participation of RAD51 protein in meiotic homology search, alignment and pairing of chromosomes.

Meiotic chromosome pairing and bouquet formation during Eimeria tenella sporulation.

In Eimeria tenella, meiotic division occurs exclusively in oocysts within the first 8h of sporulation. Difficulties with the wall-oocyst breakage in gaining access to chromosomes during meiosis have resulted in a scarcity of morphological data on Eimeria chromosomes. This study tracks the general behaviour of telomeres, attachment plaques and synaptonemal complexes in the nucleus of the meiotic oocyst of E. tenella. Fluorescence microscopy methods, in combination with immunoelectron microscopy techniques, were applied to obtain a series of time-lapse images during oocyst sporulation. Antibodies to Structural Maintenance of Chromosome proteins SMC1 and SMC3, and lamin were labelled with either fluorescence or colloidal gold to visualise the telomeres, central elements of the synaptonemal complex (SC) and nuclear periphery, respectively, at both the structural and ultrastructural levels. Using oocyst spreads and ultrathin sections of fixed oocysts it was possible to study telomere dynamics at stages during meiosis. The stages of the meiotic prophase I are delineated on the basis of the telomere position and the SC synapsis and desynapsis. During the leptotene stage, at 4h following the start of sporulation, meiotic chromosomes attached to the nuclear envelope. At that stage, chromosome synapsis was initiated in the telomeric regions but no interstitial synapsis pairing was observed. In the zygotene stage, telomere signals were clustered in a limited area of the nuclear envelope. Bouquet formation occurred at 5h after the start of sporulation, whereas chromosomes did not appear completely synapsed until the pachytene stage at 6h of sporulation. Desynapsis was observed at 8h of sporulation during the diplotene stage. This study provides the first morphological description of both the behaviour of the chromosomes and the timing of the prophase I stages in the meiotic nucleus of E. tenella.

HSP70 is part of the synaptonemal complex in Eimeria tenella.

In the current study the expression and ultrastructural localization of heat shock protein 70 (HSP70) was analyzed by immunogold labelling of surface spreads of meiotic chromosomes from Eimeria tenella oocysts. The authors used a previously reported method that overcomes the difficulties of the resistance of Eimeria oocysts to disruption and permits the release of intact meiotic chromosomes. HSP70 was localized at the ultrastructural level using an anti-HSP70 monoclonal antibody in combination with a secondary antibody coupled to colloidal gold. Synaptonemal complexes (SCs) were visualized by means of the surface spreading technique to study both HSP70 expression and the consequences of the lack of HSP70 in the behaviour of the eimerian chromosomes during meiosis. For that purpose E. tenella oocysts were treated with quercetin, a flavonoid that is known to inhibit the synthesis of HSP70. The results showed a close association of HSP70 with the lateral elements (LEs) of the SCs. That association began at the time that SCs were formed and persisted until disassemble. Comparison between distribution of immunogold label over the SCs from non-treated and treated oocysts revealed a decreasing number of gold particles as the concentration of quercetin increased. The current results demonstrated three dose-dependent effects of the quercetin treatment of Eimeria oocysts: a reduction in the HSP70 synthesis; defects in SC formation or desynapsis, and inhibition of sporulation. HSP70, as a structural component of the SCs, may be involved in SC functions such as chromosomal pairing, recombination, or disjunction.

Synaptonemal complex karyotype of Eimeria tenella.

In most organisms, biological variability rests on the behaviour of the chromosomes in the meiotic context. Despite the importance of meiosis, very little is known about the meiotic behaviour of the Eimeria chromosomes. The aim of the present study is to describe the standard synaptonemal complex karyotype from Eimeria tenella oocyst spreads by electron microscopy. For that purpose, complete sets of pachytene synaptonemal complexes were obtained and the morphological pachytene karyotype was determined. The authors used a previously reported method that overcomes the difficulty of the extreme resistance of protozoan oocysts to disruption and permits the release of intact meiotic chromosomes. The chromosomes were selected under a light microscope and those selected were stained with phosphotungtic acid and studied by transmission electron microscopy. The authors confirmed 14 chromosomes, which were observed as synaptonemal complexes, and the karyotype was constructed by arranging synaptonemal complexes according to their relative lengths and kinetochore position. Components of the synaptonemal complex, lateral elements, central element, recombination nodules and kinetochore were observed. Measures of the kynetochore, width of the synaptonemal complex, diameter of the recombination nodule and length of the telomeres are given. Minimal and no significant differences were found between measures of chromosomes isolated from different Eimeria tenella strains. To the best of our knowledge, the present investigation for the first time identifies and describes the morphological characteristics of the synaptonemal complex of Eimeria tenella during the meiosis that occurs within the oocysts. In addition, the authors provide evidence of the presence of recombination nodules, suggesting that the recombination process may play an important role in the molecular evolution of this parasite.