Transcriptomic responses of rabbits to infections by precocious line and wild-type Eimeria media: revealing molecular signatures and pathway differences in liver and duodenum during the peak and terminal phases of oocyst production.

Eimeria media is a principal pathogen responsible for rabbit coccidiosis, targeting the rabbit's intestinal epithelial cells. This parasitism damages the intestinal mucosal barrier, initiating a systemic immune and inflammatory response that jeopardizes the sustainable growth of rabbit farming. To understand the implications of infection on the host's immune and metabolic responses, we employed RNA-Seq to analyze RNA from the liver and duodenum tissues of post-infected rabbits infected with both the precocious line and wild-type strain of E.media. Comprehensive transcriptomic analysis revealed that the two parasites exhibit divergent transcriptomic imprints on host tissues. While the precocious line predominantly modulates immune-centric pathways with significant differential gene enrichment, wild-type strain favors pathways that affect metabolism. In addition, our study pinpointed a set of genes that undergo significant modifications in response to these effects. These revelations grant a fresh avenue to probe deeper into the symbiotic intricacies of the E.media and its rabbit host.

Comparative molecular analyses of Eimeria Schneider (Apicomplexa: Eimeriidae) species from rock ptarmigan in Iceland, Svalbard-Norway, and Japan.

The rock ptarmigan (Lagopus muta) has a Holarctic breeding distribution and is found in arctic and sub-arctic regions. Isolated populations and glacial relicts occur in alpine areas south of the main range, like the Pyrenees in Europe, the Pamir mountains in Central Asia, and the Japanese Alps. In recent decades considerable effort has been made to clarify parasite infections in the rock ptarmigan. Seven Eimeria spp. have been reported parasitizing rock ptarmigan. Two of those species, E. uekii and E. raichoi parasitizing rock ptarmigan (L. m. japonica) in Japan, have been identified genetically. Here we compare partial sequences of nuclear (18S rRNA) and mitochondrial (COI) genes and we detail the morphology of sporulated oocysts of E. uekii and E. raichoi from Japan, E. muta and E. rjupa, from the rock ptarmigan (L. m. islandorum) in Iceland, and two undescribed eimerian morphotypes, Eimeria sp. A, and Eimeria sp. B, from rock ptarmigan (L. m. hyperborea) in Norway (Svalbard in the Norwegian Archipelago). Two morphotypes, ellipsoidal and spheroidal, are recognized for each of the three host subspecies. Our phylogenetic analysis suggests that the ellipsoidal oocyst types, E. uekii, E. muta, and Eimeria sp. A (Svalbard-Norway) are identical and infects rock ptarmigan in Japan, Iceland, and Svalbard-Norway, respectively. Eimeria uekii was first described in Japan in 1981 so that E. muta, described in Iceland in 2007, and Eimeria sp. A in Svalbard-Norway are junior synonyms of E. uekii. Also, phylogenetic analysis shows that the spheroidal oocyst types, E. rjupa and Eimeria sp. B (Svalbard-Norway), are identical, indicating that rock ptarmigan in Iceland and Svalbard-Norway are infected by the same Eimeria species and differ from E. raichoi in Japan.

Eimeria atricillae n. sp. (Apicomplexa: Eimeriidae) from the laughing gull Leucophaeus atricilla (Linnaeus) (Aves: Charadriiformes: Laridae) in Port Isabel, Texas.

A new coccidian species, Eimeria atricillae n. sp. (Apicomplexa: Eimeriidae) collected from the laughing gull Leucophaeus atricilla, is reported from Port Isabel, Texas, USA. Sporulated oöcysts of the new species are spherical to subspherical, 16.0-18.1 × 14.4-16.6 (17.1 × 15.4) µm, with a length/width (L/W) ratio of 1.0-1.1; polar granules are present, but micropyle and oöcyst residuum are absent. Sporocysts are ovoid, 9.1-9.7 × 6.1-7.1 (9.4 × 6.6) µm, with a L/W ratio of 1.3-1.5; Stieda body present, but sub-Stieda and para-Stieda bodies are absent; sporocyst residuum diffuse.

The impact of within-host coinfection interactions on between-host parasite transmission dynamics varies with spatial scale.

Within-host interactions among coinfecting parasites can have major consequences for individual infection risk and disease severity. However, the impact of these within-host interactions on between-host parasite transmission, and the spatial scales over which they occur, remain unknown. We developed and apply a novel spatially explicit analysis to parasite infection data from a wild wood mouse (Apodemus sylvaticus) population. We previously demonstrated a strong within-host negative interaction between two wood mouse gastrointestinal parasites, the nematode Heligmosomoides polygyrus and the coccidian Eimeria hungaryensis, using drug-treatment experiments. Here, we show this negative within-host interaction can significantly alter the between-host transmission dynamics of E. hungaryensis, but only within spatially restricted neighbourhoods around each host. However, for the closely related species E. apionodes, which experiments show does not interact strongly with H. polygyrus, we did not find any effect on transmission over any spatial scale. Our results demonstrate that the effects of within-host coinfection interactions can ripple out beyond each host to alter the transmission dynamics of the parasites, but only over local scales that likely reflect the spatial dimension of transmission. Hence there may be knock-on consequences of drug treatments impacting the transmission of non-target parasites, altering infection risks even for non-treated individuals in the wider neighbourhood.

Intestinal parasites of the endangered European ground squirrel (Spermophilus citellus) populations in Slovakia.

The present pilot research was focused on the detection of intestinal parasites in the ground squirrel populations in various regions of Slovakia. Only a very little information is currently available on the parasitic species composition of the European ground squirrel in Slovakia and across Europe. In the Slovak Republic, there are 70 locations where the ground squirrel populations are present. A total of 600 faecal samples of the European ground squirrels, collected from 36 locations all over Slovakia, were examined by applying the coprological method. The presence of the protozoan coccidian parasite of the Eimeria genus was confirmed in all of the analysed locations. The presence of eggs of four helminths were confirmed: Capillaria spp. (66.6% of locations); the Trichostrongylidae family (42.8% of locations); Hymenolepis spp. (11.9% of locations); and Citellina spp. (7.14% of locations). Dead individuals that were found in the analysed localities were subjected to necropsy and the tissues scraped off their small intestines were stained in order to confirm the presence of parasites. The post-mortem examination of the intestines and the sedimentation of the intestinal contents in a saline solution did not result in the confirmation of the presence of the eggs, adults or the larval stages of parasites. Spermophilus citellus is one of the strictly protected animal species in Slovakia. In recent years, numerous projects aimed at supporting and protecting ground squirrels have been implemented. The present pilot study on intestinal parasites and the subsequent cooperation with environmental activists will contribute to the support and stabilisation of the presence of these animals in our country.

Natural intraepithelial lymphocyte populations rise during necrotic enteritis in chickens.

Intraepithelial lymphocytes (IEL) reside in the epithelium at the interface between the contents of the intestinal lumen and the sterile environment of the lamina propria. Because of this strategic location, IEL play a crucial role in various immunological processes, ranging from pathogen control to tissue stability. In mice and humans, IEL exhibit high diversity, categorized into induced IEL (conventional CD4 and CD8αß T cells) and natural IEL (TCRαßCD8αα, TCRγδ, and TCRneg IEL). In chickens, however, the subpopulations of IEL and their functions in enteric diseases remain unclear. Thus, we conducted this study to investigate the role of IEL populations during necrotic enteritis (NE) in chickens. At 14 days of age, sixty-three Specific-pathogen-free (SPF) birds were randomly assigned to three treatments: Control (sham challenge), Eimeria maxima challenge (EM), and Eimeria maxima + Clostridium Perfringens (C. Perfringens) co-challenge (EM/CP). The EM and EM/CP birds were infected with Eimeria maxima at day 14 of age, and EM/CP birds were additionally orally inoculated with C. perfringens at days 18 and 19 of age. Birds were weighed at days 18, 20, and 26 of age to assess body weight gain (BWG). At 20 days of age (1 day-post C. perfringens infection; dpi), and 26 days of age (7 dpi), 7 birds per treatment were euthanized, and jejunum was harvested for gross lesion scores, IEL isolation, and gene expression. The EM/CP birds exhibited subclinical NE disease, lower BWG and shorter colon length. The Most changes in the IEL populations were observed at 1 dpi. The EM/CP group showed substantial increases in the total number of natural IEL subsets, including TCRαß+CD4-CD8-, TCRαß+CD8αα+, TCRγδ+, TCRneg and innate CD8α (iCD8α) cells by at least two-fold. However, by 7 dpi, only the number of TCRαß+CD4-CD8- and TCRαß+CD8αα+ IEL maintained their increase in the EM/CP group. The EM/CP group had significantly higher expression of proinflammatory cytokines (IL-1ß and IFN-γ) and Osteopontin (OPN) in the jejunum at 1 dpi. These findings suggest that natural IEL with innate and innate-like functions might play a critical role in the host response during subclinical NE, potentially conferring protection against C. perfringens infection.

Host specific Eimeria genus diagnosis and qPCR development in Ovis aries and Capra hircus.

The objective of the present study was to develop a real-time PCR (qPCR) technique for the diagnosis of Eimeria spp. in Ovis aries and Capra hircus. The qPCR technique was developed using SYBR Green, resulting in a PCR with high sensitivity, specificity, and reproducibility.

CHARACTERIZATION OF EIMERIA LANCASTERENSIS AND EIMERIA ONTARIOENSIS (APICOMPLEXA: EIMERIIDAE) FOUND IN EASTERN GRAY SQUIRRELS, SCIURUS CAROLINENSIS (RODENTIA: SCIURIDAE), FROM ARKANSAS AND OKLAHOMA.

We report the morphological characteristics of oocysts of Eimeria lancasterensisJoseph, 1969, collected from 6 of 6 (100%) eastern gray squirrels, Sciurus carolinensis, collected in Arkansas (n = 3) and Oklahoma (n = 3), and Eimeria ontarioensisLee and Dorney, 1971, recovered from an individual of S. carolinensis from Arkansas. Oocysts of E. lancasterensis were ovoidal to ellipsoidal, measuring (L × W) 24.0 × 14.6 (18-29 × 12-16) µm; shape index (L/W) was 1.6 (1.3-1.8). A micropyle and an oocyst residuum were absent, but up to 2 polar granules were present. Oocysts of E. ontarioensis were piriform and measured 40.6 × 26.0 (37-44 × 23-28); L/W was 1.6 (1.5-1.7). These oocysts possessed a distinct micropyle and rarely a polar granule but lacked an oocyst residuum. The DNA was isolated from both eimerians, and the 18S rDNA genetic markers were PCR-amplified, cloned, sequenced, and analyzed. To our knowledge, this study represents the first time 18S DNA sequence data have been generated from E. lancasterensis and E. ontarioensis found in North American sciurid hosts, as well as new geographic distribution records for these coccidians. In addition, we also include a tabular summary of these 2 species of Eimeria from Sciurus spp. worldwide, with information on their hosts, distribution, and taxonomically important morphological characteristics, including key measurements of oocysts and sporocysts.

Host transcriptome response to heat stress and Eimeria maxima infection in meat-type chickens.

Eimeria (E.) maxima parasite infects chickens' midgut disrupting the jejunal and ileal mucosa causing high morbidity and mortality. Heat stress (HS) is a seasonal stressor that impacts biological functions leading to poor performance. This study elucidates how HS, E. maxima infection, and their combination affect the ileum transcriptome. Two-hundred and forty 2-week-old males Ross708 chickens were randomly allocated into four treatment groups: thermoneutral-control (TNc), thermoneutral-infected (TNi), heat-stress control (HSc), and heat stress-infected (HSi), with 6 replicates each of 10 birds. Infected groups received 200x103 sporulated E. maxima oocysts/bird, and heat-treated groups were raised at 35°C. At 6-day post-treatment, ileums of five randomly selected chickens per group were sampled, RNA was extracted and sequenced. A total of 413, 3377, 1908, and 2304 DEGs were identified when applying the comparisons: TNc vs HSc, TNc vs TNi, HSi vs HSc, and TNi vs HSi, respectively, at cutoff ≥1.2-fold change (FDR: q<0.05). HSc vs TNc showed upregulation of lipid metabolic pathways and degradation/metabolism of multiple amino acids; and downregulation of most immune-related and protein synthesis pathways. TNc vs TNi displayed upregulation of most of immune-associated pathways and eukaryotic mRNA maturation pathways; and downregulation of fatty acid metabolism and multiple amino acid metabolism pathways including tryptophan. Comparing HSi versus HSc and TNi revealed that combining the two stressors restored the expression of some cellular functions, e.g., oxidative phosphorylation and protein synthesis; and downregulate immune response pathways associated with E. maxima infection. During E. maxima infection under HS the calcium signaling pathway was downregulated, including genes responsible for increasing the cytoplasmic calcium concentration; and tryptophan metabolism was upregulated, including genes that contribute to catabolizing tryptophan through serotonin and indole pathways; which might result in reducing the cytoplasmic pool of nutrients and calcium available for the parasite to scavenge and consequently might affect the parasite's reproductive ability.

Effects of phytase supplementation on broilers fed with calcium and phosphorus-reduced diets, challenged with Eimeria maxima and Eimeria acervulina: influence on growth performance, body composition, bone health, and intestinal integrity.

An experiment was conducted to evaluate the effects of phytase in calcium (Ca) and available phosphorous (avP)-reduced diet on growth performance, body composition, bone health, and intestinal integrity of broilers challenged with Eimeria maxima and Eimeria acervulina. A total of 672 14-day-old male broilers were allocated to a 2 × 4 factorial arrangement with 6 replicates per treatment and 14 birds per replicate. Two factors were Eimeria challenge and 4 dietary treatments: 1) a positive control (PC; 0.84% Ca and 0.42% avP); 2) a negative control (NC; 0.74% Ca and 0.27% avP); 3) NC + 500 FTU/Kg of phytase (NC + 500PHY); and 4) NC + 1,500 FTU/Kg of phytase (NC + 1500PHY). On d 14, birds in the Eimeria-challenged groups received a solution containing 15,000 sporulated oocysts of E. maxima and 75,000 sporulated oocysts of E. acervulina via oral gavage. At 5 d postinoculation (DPI), the challenged birds showed a higher (P < 0.01) FITC-d level than the unchallenged birds. While the permeability of the NC group did not differ from the PC group, the phytase supplementation groups (NC + 500PHY and NC + 1500PHY) showed lower (P < 0.05) serum FITC-d levels compared to the NC group. Interaction effects (P < 0.05) of Eimeria challenge and dietary treatments on feed intake (FI), mucin-2 (MUC2) gene expression, bone ash concentration, and mineral apposition rate (MAR) were observed. On 0 to 6 and 0 to 9 DPI, Eimeria challenge decreased (P < 0.01) body weight (BW), body weight gain (BWG), FI, bone mineral density (BMD), bone mineral content (BMC), bone area, fat free bone weight (FFBW), bone ash weight, bone ash percentage and bone ash concentration; and it showed a higher FCR (P < 0.01) compared to the unchallenged group. The reduction Ca and avP in the diet (NC) did not exert adverse effects on all parameters in birds, and supplementing phytase at levels of 500 or 1,500 FTU/Kg improved body composition, bone mineralization, and intestinal permeability, with the higher dose of 1,500 FTU/Kg showing more pronounced enhancements. There was an observed increase in FI (P < 0.01) when phytase was supplemented at 1,500 FTU/Kg during 0 to 6 DPI. In conclusion, results from the current study suggest that dietary nutrients, such as Ca and avP, can be moderately reduced with the supplementation of phytase, particularly in birds infected with Eimeria spp., which has the potential to save feed cost without compromising growth performance, bone health, and intestinal integrity of broilers.

Identification and characterization of the receptors of a microneme adhesive repeat domain of Eimeria maxima microneme protein 3 in chicken intestine epithelial cells.

Eimeria maxima microneme protein 3 (EmMIC3) is pivotal in the initial recognition and attachment of E. maxima sporozoites to host cells. EmMIC3 comprises 5 tandem Type I microneme adhesive repeat (MAR) domains, among which MAR2 of EmMIC3 (EmMAR2) has been identified as the primary determinant of EmMIC3-mediated tissue tropism. Nonetheless, the mechanisms through which EmMAR2 guides the parasite to its invasion site through interactions with host receptors remained largely uncharted. In this study, we employed yeast two-hybrid (YTH) screening assays and shotgun LC-MS/MS analysis to identify EmMAR2 receptors in chicken intestine epithelial cells. ATPase H+ transporting V1 subunit G1 (ATP6V1G1), receptor accessory protein 5 (REEP5), transmembrane p24 trafficking protein (TMED2), and delta 4-desaturase sphingolipid 1 (DEGS1) were characterized as the 4 receptors of EmMAR2 by both assays. By blocking the interaction of EmMAR2 with each receptor using specific antibodies, we observed varying levels of inhibition on the invasion of E. maxima sporozoites, and the combined usage of all 4 antibodies resulted in the most pronounced inhibitory effect. Additionally, the spatio-temporal expression profiles of ATP6V1G1, REEP5, TMED2, and DEGS1 were assessed. The tissue-specific expression patterns of EmMAR2 receptors throughout E. maxima infection suggested that ATP6V1G1 and DEGS1 might play a role in early-stage invasion, whereas TMED2 could be involved in middle and late-stage invasion and REEP5 and DEGS1 may participate primarily in late-stage invasion. Consequently, E. maxima may employ a multitude of ligand-receptor interactions to drive invasion during different stages of infection. This study marks the first report of EmMAR2 receptors at the interface between E. maxima and the host, providing insights into the invasion mechanisms of E. maxima and the pathogenesis of coccidiosis.

Growth performance, organs weight, intestinal histomorphology, and oocyst shedding in broiler chickens offered novel single strain Bacillus subtilis isolated from camel dung and challenged with Eimeria.

We evaluated a single strain Bacillus subtilis BS-9 direct-fed microbial (BSDFM) isolated from camel dung in Eimeria challenged broiler chickens. Seven-hundred d-old Ross 708 male chicks were placed in pens (25 birds/pen) and allocated to 2 treatments (n = 14). From d 0 to 13, control pens received untreated water (-BSDFM), and 2 treated pens received water and 2 mL x 108 colony forming unit/bird/d (+BSDFM); daily water intake (WI) was recorded. On d 9, birds in half (+Eimeria) of pens per treatment received of 1 mL of Eimeria maxima and Eimeria acervulina oocysts orally, and the other half (-Eimeria) sterile saline solution. Birds had ad libitum access to feed and a water line from d 14. Feed intake (FI), body weight (BW) and mortality were recorded for calculating BW gain (BWG) and feed conversion ratio (FCR). On d 14 and 35, samples of birds were necropsied for organ weight and intestinal measurements. Excreta samples were collected from d 14 to 19 for oocyst count. There was no treatment effect (P > 0.05) on growth performance or WI on d 0 to 9. There were interactions between BSDFM and Eimeria on d 19 (P = 0.014) and 29 (P = 0.036) BW with unchallenged +BSDFM birds being heavier than birds in the other treatments. The main effects (P < 0.05) on d 10 to 35 FI, BW, and BWG were such that +BSDFM increased and Eimeria decreased (P < 0.01) these parameters. There was interaction (P = 0.022) between BSDFM and Eimeria on d 10 to 35 FCR such that the FCR of challenged -BSDFM birds was poor than that of unchallenged counterparts, but none differed with +BSDFM birds. There was an interaction (P = 0.039) between BSDFM and Eimeria on d 14 bursa weight with challenged birds exhibiting heavier bursa than unchallenged +BSDFM birds. Eimeria reduced (P = 0.01) and BSDFM (P = 0.002) increased the villi height to crypt depth ratio. Results showed that BSDFM supplementation via water can support the growth performance of broiler chickens challenged with Eimeria and may be a strategy to reduce adverse effects of coccidiosis.

Evaluation of the immunoprotective effect of the recombinant Eimeria intestinalis rhoptry protein 25 and rhoptry protein 30 on New Zealand rabbits.

BACKGROUND: Rabbit coccidiosis is a parasitism caused by either one or multiple co-infections of Eimeria species. Among them, Eimeria intestinalis is the primary pathogen responsible for diarrhea, growth retardation, and potential mortality in rabbits. Concerns regarding drug resistance and drug residues have led to the development of recombinant subunit vaccines targeting Eimeria species as a promising preventive measure. The aim of this study was to assess the immunoprotective efficacy of recombinant subunit vaccines comprising EiROP25 and EiROP30 (rhoptry proteins (ROPs)) against E. intestinalis infection in rabbits. METHODS: Cloning, prokaryotic expression, and protein purification were performed to obtain EiROP25 and EiROP30. Five groups of fifty 35-day-old Eimeria-free rabbits were created (unchallenged control group, challenged control group, vector protein control group, rEiROP25 group, and rEiROP30 group), with 10 rabbits in each group. Rabbits in the rEiROP25 and rEiROP30 groups were immunized with the recombinant proteins (100 µg per rabbit) for primary and booster immunization (100 µg per rabbit) at a two-week intervals, and challenged with 7 × 104 oocysts per rabbit after an additional two-week interval. Two weeks after the challenge, the rabbits were euthanized for analysis. Weekly collections of rabbit sera were made to measure changes in specific IgG and cytokine level. Clinical symptoms and pathological changes after challenge were observed and recorded. At the conclusion of the animal experiment, lesion scores, the relative weight increase ratio, the oocyst reduction rate, and the anticoccidial index were computed. RESULTS: Rabbits immunized with rEiROP25 and rEiROP30 exhibited relative weight gain ratios of 56.57% and 72.36%, respectively. Oocysts decreased by 78.14% and 84.06% for the rEiROP25 and rEiROP30 groups, respectively. The anticoccidial indexes were 140 and 155. Furthermore, there was a noticeable drop in intestinal lesions. After the primary immunization with rEiROP25 and rEiROP30, a week later, there was a notable rise in specific IgG levels, which remained elevated for two weeks following challenge (P < 0.05). Interleukin (IL)-2 levels increased markedly in the rEiROP25 group, whereas IL-2, interferon gamma (IFN-γ), and IL-4 levels increased substantially in the rEiROP30 group (P < 0.05). CONCLUSION: Immunization of rabbits indicated that both rEiROP25 and rEiROP30 are capable of inducing an increase in specific antibody levels. rEiROP25 triggered a Th1-type immune protection response, while rEiROP30 elicited a Th1/Th2 mixed response. EiROP25 and EiROP30 can generate a moderate level of immune protection, with better efficacy observed for EiROP30. This study provides valuable insights for the promotion of recombinant subunit vaccines targeting rabbit E. intestinalis infection.

Developing efficient strategies for localizing the enhanced yellow fluorescent protein subcellularly in transgenic Eimeria parasites.

Eimeria species serve as promising eukaryotic vaccine vectors. And that the location of heterologous antigens in the subcellular components of genetically modified Eimeria may determine the magnitude and type of immune responses. Therefore, our study aimed to target a heterologous fluorescent protein to the cell surface or microneme, two locations where are more effective in inducing protective immunity, of Eimeria tenella and E. acervulina sporozoites. We used an enhanced yellow fluorescent protein (EYFP) as a tagging biomarker, fusing variously with some localization or whole sequences of compartmental proteins for targeting. After acquiring stable transgenic Eimeria populations, we observed EYFP expressing in expected locations with certain strategies. That is, EYFP successfully localized to the surface when it was fused between signal peptides and mature products of surface antigen 1 (SAG1). Furthermore, EYFP was efficiently targeted to the apical end, an optimal location for secretory organelle known as the microneme, when fused to the C terminus of microneme protein 2. Unexpectedly, EYFP exhibited dominantly in the apical end with only weak expression on the surface of the transgenic sporozoites when the parasites were transfected with plasmid with EYFP fused between signal peptides and mature products of E. tenella SAG 13. These strategies worked in both E. tenella and E. acervulina, laying a solid foundation for studying E. tenella and E. acervulina-based live vaccines that can be further tailored to the inclusion of cargo immunogens from other pathogens.

Prediction of coccidiosis prevalence in extensive backyard chickens in countries and regions of the Horn of Africa.

Coccidiosis is one of the leading morbidity causes in chickens, causing a reduction of body weight and egg production. Backyard chickens are at risk of developing clinical and subclinical coccidiosis due to outdoor housing and scavenging behaviour, jeopardizing food security in households. The objectives of this study were to estimate clinical prevalence of coccidiosis at country and regional levels in the Horn of Africa in extensive backyard chickens. A binomial random effects model was developed to impute prevalence of coccidiosis. Previously gathered prevalence data (n = 40) in backyard chickens was used to define the model. Precipitation (OR: 1.09 (95% CI: 1.05-1.13) and the presence of seasonal rainfall (OR: 1.85, 95% CI: 1.27-2.70) significantly increase prevalence. Results showed an overall prevalence of coccidiosis in the Horn of Africa of 0.21 (95% CI: 0.15-0.29). Ethiopia, the Republic of South Sudan and Kenya showed the highest prevalence and Djibouti the lowest. Significant differences between Djibouti and the countries with highest prevalence were found. However, no evidence of a significant difference between the rest of the countries. Kenya and Ethiopia showed larger prevalence differences between regions. Results could assist with the targeting of testing for coccidiosis, the observation for clinical disease of chickens living in specific regions and as a baseline for the evaluation of future control measures.

Association of Bacillus subtilis and Bacillus amyloliquefaciens: minimizes the adverse effects of necrotic enteritis in the gastrointestinal tract and improves zootechnical performance in broiler chickens.

This study aimed to evaluate the efficiency and capacity of the probiotic composed of Bacillus subtilis and Bacillus amyloliquefaciens, in improving the zootechnical performance of broiler chickens challenged with Eimeria spp. and Clostridium perfringens. The broilers were distributed in a completely randomized design in poultry isolators (12 birds each), resulting in 3 treatments: T1 (control, no challenge and no Bacillus in diet), T2 (challenged with Eimeria spp., followed by Clostridium perfringens infection and no Bacillus in the diet), and T3 (challenged with Eimeria spp., Clostridium perfringens and treated with Bacillus subtilis and Bacillus amyloliquefaciens). They were evaluated for a period of 29 d, divided into preinitial (1-7 d of age), initial (8-21 d), and growth (22-29 d) phases. Assessments of body weight, weight gain, feed consumption, and feed conversion were conducted, along with the classification of the scores and optical microscopy of the tract gastrointestinal. The animals challenged and treated with the probiotic containing Bacillus spp. showed improved indicators of zootechnical performance. Additionally, the animals challenged and treated (T3) had a better score for intestinal lesions compared to the other treatment groups. Therefore, the probiotic consisting of Bacillus subtilis and Bacillus amyloliquefaciens could be considered an effective option for disease prevention and improving the zootechnical performance of broiler chickens.

Bioefficacy of dietary inclusion of Nannochloropsis oculata on Eimeria spp. challenged chicks: clinical approaches, meat quality, and molecular docking.

Although anticoccidial drugs have been used to treat avian coccidiosis for nearly a century, resistance, bird harm, and food residues have caused health concerns. Thus, Nannochloropsis oculata was investigated as a possible coccidiosis treatment for broilers. A total of 150 1-day-old male Cobb broiler chicks were treated as follows: G1-Ng: fed a basal diet; G2-Ps: challenged with Eimeria spp. oocysts and fed basal diet; G3-Clo: challenged and fed basal diet with clopidol; G4-NOa: challenged and fed 0.1% N. oculata in diet, and G5-NOb: challenged and fed 0.2% N. oculata. Compared to G2-Ps, N. oculata in the diet significantly (P < 0.05) decreased dropping scores, lesion scores, and oocyst shedding. Without affecting breast meat colour metrics, N. oculata improved meat quality characters. At 28 days of age, birds received 0.2% N. oculata had significantly (P < 0.05) higher serum levels of MDA, T-SOD, HDL, and LDL cholesterol compared to G2-Ps. Serum AST, ALT, and urea levels were all decreased when N. oculata (0.2%) was used as opposed to G2-Ps. Histopathological alterations and the number of developmental and degenerative stages of Eimeria spp. in the intestinal epithelium were dramatically reduced by 0.2% N. oculata compared to G2-Ps. Molecular docking revealed a higher binding affinity of N. oculata for E. tenella aldolase, EtAMA1, and EtMIC3, which hindered glucose metabolism, host cell adhesion, and invasion of Eimeria. Finally, N. oculata (0.2%) can be used in broiler diets to mitigate the deleterious effects of coccidiosis.

Microneme-located VP2 in Eimeria acervulina elicits effective protective immunity against infectious bursal disease virus.

Using transgenic Eimeria spp. to deliver exogenous antigens is a viable option for developing multivalent live vaccines. Previous research revealed that the location of antigen expression in recombinant Eimeria dictates the magnitude and type of immune responses. In this study, we constructed genetically modified Eimeria acervulina that expressed VP2 protein, a protective antigen from infectious bursal disease virus (IBDV), on the surface or in the microneme of sporozoites. After vaccination, VP2-specific antibody was readily detected in specific pathogen-free chickens receiving transgenic E. acervulina parasites expressing VP2 in microneme, but animals vaccinated with which expressing VP2 on surface failed to produce detectable antibody after two times immunizations. Moreover, the bursal lesion of microneme-located VP2 transgenic E. acervulina immunized chickens was less severe compared with un-immunized animals after IBDV challenge infection. Therefore, genetically modified E. acervulina that express IBDV-derived VP2 in micronemes are effective in inducing specific antibody responses against VP2, while parasites that have VP2 expression on cell surface are not suitable. Thus, the use of Eimeria parasites as vaccine vectors needs to consider the proper targeting of exogenous immunogens. Our results have implications for the design of other vector vaccines.

Toltrazuril + fenbendazole for cattle: Pharmacokinetics and efficacy against Eimeria spp. and gastrointestinal nematodes.

The present work evaluated the pharmacokinetics and efficacy of the association of 15cmg/kg toltrazuril +5cmg/kg fenbendazole against Eimeria spp. and gastrointestinal nematodes (GINs) in calves of different regions of Brazil (Center-West, Southeast, and South). A total of seven experiments were carried out, five of which determined formulation efficacy against Eimeria spp., considering the following aspects: therapeutic, preventive, metaphylactic, and residual efficacy. Therapeutic efficacy experiments for GINs were carried out by parasitological necropsy. The toltrazuril + fenbendazole association demonstrated ≥95% efficacy against Eimeria spp. for 21 days post-treatment (DPT). When used preventively and metaphylatically, the same association demonstrated ≥97% efficacy against E. zuernii, E. ellipsoidalis, E. cylindrica, E. bovis, E. wyomingensis and E. auburnensis. Toltrazuril + fenbendazole administered seven days before challenge was 100% effective against all these Eimeria species. Results of therapeutic, preventive, metaphylactic and residual efficacies can be related to the pharmacokinetic results, especially considering toltrazuril sulfone, which was detected in animal plasma for a longer period than the parent compound. Toltrazuril + fenbendazole achieved 100% anthelminthic efficacy against the GINs Haemonchus placei (L4), Cooperia pectinata and Oesophagostomum radiatum; 99.94% against adult H. placei; and 99.98% against C. puntacta. The association of toltrazuril + fenbendazole, associated with other measures, is an important and suitable tool for the control and treatment of Eimeria spp. and GINs in young cattle.

A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species.

BACKGROUND: The gastrointestinal epithelium plays an important role in directing recognition by the immune system, and epithelial cells provide the host's front line of defense against microorganisms. However, it is difficult to cultivate avian intestinal epithelial cells in vitro for lengthy periods, and the lack of available cell lines limits the research on avian intestinal diseases and nutritional regulation. Chicken coccidiosis is a serious intestinal disease that causes significant economic losses in the poultry industry. In vitro, some cell line models are beneficial for the development of Eimeria species; however, only partial reproduction can be achieved. Therefore, we sought to develop a new model with both the natural host and epithelial cell phenotypes. METHODS: In this study, we use the SV40 large T antigen (SV40T) gene to generate an immortalized cell line. Single-cell screening technology was used to sort positive cell clusters with epithelial characteristics for passage. Polymerase chain reaction (PCR) identification, immunofluorescence detection, and bulk RNA sequencing analysis and validation were used to check the expression of epithelial cell markers and characterize the avian intestinal epithelial cell line (AIEC). AIECs were infected with sporozoites, and their ability to support the in vitro endogenous development of Eimeria tenella was assessed. RESULTS: This novel AIEC consistently expressed intestinal epithelial markers. Transcriptome assays revealed the upregulation of genes associated with proliferation and downregulation of genes associated with apoptosis. We sought to compare E. tenella infection between an existing fibroblast cell line (DF-1) and several passages of AIEC and found that the invasion efficiency was significantly increased relative to that of chicken fibroblast cell lines. CONCLUSIONS: An AIEC will serve as a better in vitro research model, especially in the study of Eimeria species development and the mechanisms of parasite-host interactions. Using AIEC helps us understand the involvement of intestinal epithelial cells in the digestive tract and the immune defense of the chickens, which will contribute to the epithelial innate defense against microbial infection in the gastrointestinal tract.