Immunological studies on the effects of toltrazuril and neem extract in broiler chickens suffering from coccidiosis.

Background: The prevalence of avian coccidiosis in the poultry industry has grown, resulting in substantial financial losses from high mortality, stunted growth, reduced productivity, and expensive medical expenses. Aim: The purpose of the current study was to assess the immunological effects of neem leaf extract and toltrazuril on broilers that had contracted coccidiosis. Methods: In this investigation, 100 one-day-old Cobb broiler chicks without sexes were employed. The chicks were divided into five equal groups, with 20 birds in each. On the 14th day of life, the birds in groups 2, 3, 4, and 5 received an oral inoculation with 1 × 105 sporulated oocysts of Eimeria tenella (E. tenella) (field isolate). The first group (Gp), which consists of 20 healthy broilers, served as a negative control. Gp (2) contains experimentally infected broilers and nontreated (served as a positive control). Gp (3) contains experimentally infected broilers treated with toltrazuril (1 ml/l drinking water) for two consecutive days. Gp (4) contains experimentally infected broilers treated with neem leaf extract 4% (50 ml/l drinking water) for 5 successive days, and Gp (5) contains experimentally infected broilers treated with toltrazuril (1 ml/l drinking water) and a half dose of neem leaves extract 4% (25 ml/l drinking water) for 5 successive days. For the purpose of estimating body weight growth and feed conversion ratio, each broiler was weighed separately at the start of the trial and again on the 1st and 10th day after treatment. In addition to obtaining intestinal samples for immunohistochemistry, blood samples were also obtained for immunological examination. Results: As compared to the negative control group, the experimentally infested broilers with E. tenella showed significant decreases in serum nitric oxide, lysosome, phagocytic percent, and phagocytic index, along with significant increases in white blood cells (WBCs), lymphocyte, heterophilis, eosinophilis, basophilis, monocyte, serum total protein, γ globulin, fibrinogen, and haptoglobin. When compared to the control positive group, experimentally infested broilers treated with either neem or toltrazuril alone or in combination demonstrated significant increases in serum total protein, nitric oxide, lysozyme, phagocytic percent, and phagocytic index, but significant decreases in WBCs, lymphocytes, heterophile, eosinophile, basophile, and monocyte. The intestinal peroxidase stain of broilers infected with E. tenella exhibited a significant positive expression for CD4, but the infected broilers treated with toltrazuril and half a dosage of neem displayed a negative expression for CD4, identical to the negative control. Conclusion: The broiler chickens infested with E. tenella may have a variety of negative impacts on their immune systems and immunohistopathological findings. Nonetheless, toltrazuril and neem extract, either separately or in combination, function as anticoccidial medications that may enhance the broiler chicks' immune state.

Efficacy of dietary supplements of Glycyrrhiza glabra (Licorice) and maduramicin alone or in combination with Eimeria tenella infected chicks: A clinical study and molecular docking.

Background: Coccidiosis is one of the most economically significant poultry diseases worldwide, caused by the pathogenic Eimeria species, and is characterized by decreased weight gain (WG) and failure to grow due to malabsorption, low feed conversion rate, bloody diarrhea, and dehydration. Aim: This study investigated the effectiveness of licorice root extract (LRE) in controlling cecal coccidiosis to determine whether its combination with maduramicin could help alleviate the pathological, biochemical, and histopathological effects of cecal coccidiosis in Sasso broiler chicks. Methods: A total of 125 one-day-old Sasso broiler chicks were categorized into five equal groups (n = 25), each consisting of five replicates (n = 5 per replicate). G1-LE received a basal diet supplemented with LRE (3 g/kg); G2-ME received a basal diet containing maduramycin (0.5 g/kg); and G3-LME received a basal diet containing LRE and maduramicin together with the same rates. G4-E (positive control) and G5-N (negative control) received no additives in their feed. Birds in groups (G1-4) were challenged on day 14 of the experiment by orally intercropping a 1 ml suspension of Eimeria tenella sporulated oocysts. Results: Groups of birds fed on LRE and maduramicin separately or together appeared to be in good condition where no deaths or clinical abnormalities were observed, based on the analysis of clinicopathological examination. Compared with the G4-E positive control, the dropping scoring and oocyst shedding of groups G1-LE, G2-ME, and G3-LME along the 10th-day post-challenge (dpc), as well as macroscopic and microscopic lesions scoring at the 7th dpc, was considerably lower. The dual supplementation use of LRE and maduramicin in G3-LME's reduced the harmful effects of coccidian, which appeared only as a mononuclear cellular infiltration and a small number of oocysts invading the intestinal glands. Molecular docking revealed that LRE and maduramicin interacted with E. tenella DNA polymerase, E. tenella apical membrane antigen 1, and microneme protein binding sites resulting in reduced E. tenella replication and invasion. Conclusion: The inclusion of LRE and maduramicin, individually or in combination, in the diet might effectively mitigate the detrimental effects of coccidiosis.

Molecular detection of Toxoplasma gondii and Neospora caninum in seabirds collected along the coast of Santa Catarina, Brazil.

Toxoplasma gondii and Neospora caninum are two closely related protozoans that infect a wide range of animals, including birds. However, the occurrence of N. caninum and T. gondii in seabirds is unknown. Therefore, this study aimed to determine the presence of T. gondii and N. caninum DNA in tissue samples of seabirds. Tissue samples of the pectoral muscles, heart, and brain were collected from 47 birds along the coastline of Santa Catarina State, SC, Brazil. The DNA was extracted from the tissues and screened using nested-PCR (nPCR) targeting internal transcribed spacer 1 (ITS1). T. gondii DNA was detected in tissues from seven seabirds (7/47, 14.8%), kelp gull (Larus dominicanus) (5/21), and Manx shearwater (Puffinus puffinus) (2/8). N. caninum DNA was detected in tissues of nine seabirds (9/47, 19.1%), the kelp gull (L. dominicanus) (4/21), Manx shearwater (P. puffinus) (2/8), neotropic cormorant (Phalacrocorax brasilianus) (1/4), brown booby (Sula leucogaster) (1/5), and white-chinned petrel (Procellaria aequinoctialis) (1/1); however, no co-infection was observed. In conclusion, this study showed the circulation of N. caninum and T. gondii in seabirds along the coastline of Santa Catarina State. Further studies are required to clarify the role of these birds in the epidemiology of neosporosis and toxoplasmosis.

The seroprevalence and risk factors for exposure to Neospora caninum and Neospora hughesi in Ontario broodmares.

The seroprevalence and risk factors for exposure to Neospora caninum and Neospora hughesi in broodmares in Ontario were investigated. Sixty of the 219 (27.4%) study broodmares were seropositive for N. caninum and 65/219 (29.7%) for N. hughesi with cut-offs of ≥1:40 and ≥1:160, respectively. Thirty-one of 63 participating farms (49.2%) had at least 1 broodmare seropositive for N. caninum. Thirty-three of the 63 (52.4%) participating farms had at least 1 broodmare positive for N. hughesi. Risk factors for N. caninum included presence of farm dogs (OR = 6.70; 95% CI = 2.14-20.97; p = 0.001), and high stocking density (OR = 2.83; 95% CI = 1.27-6.30; p = 0.011). Presence of livestock, excluding cattle, was associated with reduced risk of exposure (OR = 0.17; 95% CI = 0.06-0.53; p = 0.002). The only risk factor for exposure to N. hughesi was feeding hay on the ground in the paddock (OR = 4.31; 95% CI = 1.65-11.22; p = 0.003). This study demonstrated widespread exposure to Neospora spp. in broodmares in Ontario.

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.

Recombinant production of SAG1 fused with xylanase in Pichia pastoris induced higher protective immunity against Eimeria tenella infection in chicken.

Chicken coccidiosis is an intestinal disease caused by the parasite Eimeria, which severely damages the growth of chickens and causes significant economic losses in the poultry industry. Improvement of the immune protective effect of antigens to develop high efficiency subunit vaccines is one of the hotspots in coccidiosis research. Sporozoite-specific surface antigen 1 (SAG1) of Eimeria tenella (E. tenella) is a well-known protective antigen and is one of the main target antigens for the development of subunit, DNA and vector vaccines. However, the production and immunoprotective effects of SAG1 need to be further improved. Here, we report that both SAG1 from E. tenella and its fusion protein with the xylanase XynCDBFV-SAG1 are recombinant expressed and produced in Pichia pastoris (P. pastoris). The substantial expression quantity of fusion protein XynCDBFV-SAG1 is achieved through fermentation in a 15-L bioreactor, reaching up to about 2 g/L. Moreover, chickens immunized with the fusion protein induced higher protective immunity as evidenced by a significant reduction in the shedding of oocysts after E. tenella challenge infection compared with immunized with recombinant SAG1. Our results indicate that the xylanase enhances the immunogenicity of subunit antigens and has the potential for developing novel molecular adjuvants. The high expression level of fusion protein XynCDBFV-SAG1 in P. pastoris holds promise for the development of effective recombinant anti-coccidial subunit vaccine.

Construction of luciferase-expressing Neospora caninum and drug screening.

BACKGROUND: Neospora caninum is an apicomplexan parasite that is particularly responsible for abortions in cattle and neuromuscular disease in dogs. Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing N. caninum and evaluate anti-N. caninum drugs. METHODS: Luciferase-expressing N. caninum (Nc1-Luc) was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9). After testing the luciferase expression and phenotype of the Nc1-Luc strains, the drug sensitivity of Nc1-Luc strains was determined by treating them with known positive or negative drugs and calculating the half-maximal inhibitory concentration (IC50). The selective pan-rapidly accelerated fibrosarcoma (pan-RAF) inhibitor TAK-632 was then evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo studies. RESULTS: The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parental strains Nc1, and Nc1-Luc strains can be used to determine the IC50 for anti-N. caninum drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against N. caninum, with an IC50 of 0.6131 µM and a selectivity index (SI) of 62.53. In vitro studies demonstrated that TAK-632 inhibited the invasion, proliferation, and division of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burden in the brain. CONCLUSIONS: In conclusion, a luciferase-expressing N. caninum strain was successfully constructed, which provides an effective tool for drug screening and related research on N. caninum. In addition, TAK-632 was found to inhibit the growth of N. caninum, which could be considered as a candidate lead compound for new therapeutics for neosporosis.

New insights on the phylogeography of Hepatozoon canis in Brazil.

This study aimed to molecularly characterize the Hepatozoon spp. infecting domestic and wild dogs in Brazil. A total of 22 whole blood samples tested positive for Hepatozoon spp., and five samples were sequenced for the 18S rDNA gene from H. canis after PCR amplification with four primer sets. Phylogenetic analysis using Bayesian inference showed that the three H. canis isolates from domestic dogs were not monophyletic; however, they were more closely related to each other than to other H. canis sequences. The isolate from the hoary fox (Lycalopex vetulus) was phylogenetically more distant. Two haplotype networks were constructed, identifying 10 haplotypes of H. canis in Brazil, with H10 constituting the largest group. It contains nine isolates, including three from domestic dogs. The H5 haplotype grouped the sequence of L. vetulus with two additional sequences from hosts Tapirus terrestris and L. vetulus, representing the sole haplotype with wild hosts. Bayesian analysis suggested the possible existence of two genetic groups of H. canis in Brazil, indicating gene flow of this agent within the country. These findings contribute valuable insights for a more comprehensive understanding of the molecular diversity of Hepatozoon spp. in Brazil and may help in the development of effective control measures.

Integrated application of transcriptomics and metabolomics provides insight into the mechanism of Eimeria tenella resistance to maduramycin.

Avian coccidiosis, caused by Eimeria parasites, continues to devastate the poultry industry and results in significant economic losses. Ionophore coccidiostats, such as maduramycin and monensin, are widely used for prophylaxis of coccidiosis in poultry. Nevertheless, their efficacy has been challenged by widespread drug resistance. However, the underlying mechanisms have not been revealed. Understanding the targets and resistance mechanisms to anticoccidials is critical to combat this major parasitic disease. In the present study, maduramycin-resistant (MRR) and drug-sensitive (DS) sporozoites of Eimeria tenella were purified for transcriptomic and metabolomic analysis. The transcriptome analysis revealed 5016 differentially expressed genes (DEGs) in MRR compared to DS, and KEGG pathway enrichment analysis indicated that DEGs were involved in spliceosome, carbon metabolism, glycolysis, and biosynthesis of amino acids. In the untargeted metabolomics assay, 297 differentially expressed metabolites (DEMs) were identified in MRR compared to DS, and KEGG pathway enrichment analysis indicated that these DEMs were involved in 10 pathways, including fructose and mannose metabolism, cysteine and methionine metabolism, arginine and proline metabolism, and glutathione metabolism. Targeted metabolomic analysis revealed 14 DEMs in MRR compared to DS, and KEGG pathway analysis indicated that these DEMs were involved in 20 pathways, including fructose and mannose metabolism, glycolysis/gluconeogenesis, and carbon metabolism. Compared to DS, energy homeostasis and amino acid metabolism were differentially regulated in MRR. Our results provide gene and metabolite expression landscapes of E. tenella following maduramycin induction. This study is the first work involving integrated transcriptomic and metabolomic analyses to identify the key pathways to understand the molecular and metabolic mechanisms underlying drug resistance to polyether ionophores in coccidia.

Effects of a coccidiosis challenge on dietary methionine recommendations in broilers.

Broilers are commonly exposed to coccidiosis infections, and the use of dietary strategies to reduce losses in growth performance has practical implications for the poultry industry. Methionine (Met) is typically the first limiting amino acid for broilers and is involved in metabolic and immunological pathways; however, literature is conflicting on how dietary Met requirements are affected by environmental stressors. Our objective was to assess how the Met requirement changes during coccidiosis based on results of growth performance, carcass traits, and health outcomes. Two trials were conducted using 780 male Ross 308 broiler chicks in floor pens randomly assigned to 1 of 12 experimental treatments. All birds received common starter (d 0-10) and finisher (d 24-35, Trial 2 only) diets, and only differed based on their assigned experimental grower diet (d 10-24). Trial 1 experimental grower diets ranged from 2.61 to 6.21 g/kg digestible Met. Trial 2 experimental grower diets were formulated to contain 15% below, at, or 15% above the Met requirement determined in Trial 1. Birds were exposed to a coccidiosis challenge on d 11, with blood and tissue collection (1 bird/pen) on d 18 and carcass processing on d 35 (2 birds/pen) in Trial 2. Data were analyzed using a 1- or 2-way ANOVA. A non-linear regression analysis was conducted in Trial 1 to determine the Met requirement of 4.32 g of digestible Met/kg of diet using BW gain. Coccidiosis infection reduced (P < 0.05) growth performance during the experimental grower and overall study periods in Trial 2. Increasing dietary Met from below requirement to meeting requirement during the grower period improved (P < 0.001) BW gain and feed conversion ratio (FCR), but this effect was only significant between treatments below and above the requirement for the overall study period. There was an interactive effect (P = 0.038) on FCR for the overall study period. These findings provide evidence that the Met requirement is likely increased during coccidiosis based on growth performance outcomes.

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.

Bromelain can reduce the negative effects of a subclinical necrotic enteritis in broiler chickens.

This study was conducted to examine the efficacy of a bromelain-based supplementation coded ANR-pf on growth performance and intestinal lesion of broiler chickens under necrotic enteritis (NE) challenge. A total of 540 Ross 308 day-old male chicks were randomly allocated into 6 treatments of 6 replicates. The bromelain formulation was delivered to chickens through gavaging or in drinking water method twice, on d 8 and 13. Nonchallenged groups included 1) without or 2) with the specific bromelain formulation gavaged at 0.8 mL/kg. NE-challenged groups included 3) without the specific bromelain formulation; 4) gavaged with 0.4 mL/kg; 5) gavaged with 0.8 mL/kg and 6) supplemented with 0.8 mL/kg via drinking water. Birds were challenged with Eimeria spp. on d 9 and Clostridium perfringens (NE-18 strain) on d 14 and 15. On d 14 and 19, fresh faecal contents were collected for the determination of oocyst counts. Intestinal lesion scores were determined on d16. Performance and mortality were recorded throughout the entire experiment. Among challenged groups, birds received additive via drinking water had higher weight gain (WG) compared to the remaining groups (P < 0.001) in the grower phase and had lower FCR compared to 0.4 mL/kg inoculated group in the grower and finisher phases (P < 0.001). Bromelain supplementation via drinking water improved the WG of challenged birds, similar to that of the nonchallenged birds (P < 0.001), and lowered FCR compared to other challenged groups (P < 0.001). Nonchallenged birds and birds that received bromelain formulation in drinking water did not have lesions throughout the small intestine whereas challenged birds, either un-supplemented or supplemented with bromelain via inoculation route recorded similar lesion score levels in the jejunum. At d 19, birds received bromelain in drinking water had lower fecal oocyst numbers compared to challenged birds without additive (P < 0.001). In conclusion, bromelain administration via drinking water could ameliorate the negative impacts of NE-infection in broilers by improving performance, lowering the oocyst numbers and lesion scores.

Coccidiosis and necrotic enteritis model may have a greater impact than dietary anti-interleukin-10 on broiler chicken systemic immunometabolic responses.

Dietary egg yolk-derived anti-interleukin (IL)-10 may preserve broiler chicken performance during coccidiosis due to Eimeria spp. infection while effects on secondary Clostridium perfringens (necrotic enteritis) are unknown. Some necrotic enteritis models implement Salmonella Typhimurium to improve repeatability; however, Salmonella upregulation of IL-10 may be a confounder when evaluating anti-IL-10. The study objective was to investigate anti-IL-10 effects on systemic cytokine concentrations and immunometabolism during E. maxima ± C. perfringens challenge in models ± S. Typhimurium. Three 25 d replicate studies using Ross 308 chicks were conducted in wire-floor cages (32 cages/ replicate) with chicks assigned to diets ± 0.03% anti-IL-10. 640 chicks (20/ cage; replicates 1 and 2) were inoculated with sterile saline ± 1×108 colony forming units (CFU) S. Typhimurium while 480 chicks (15/ cage) were placed in replicate 3. In all replicates, blood samples were collected on d 14 (6 chicks/treatment) before administering 15,000 sporulated E. maxima M6 oocysts to S. Typhimurium-inoculated (replicates 1 and 2) or challenge-designated chicks (replicate 3). Half the E. maxima-challenged chicks received 1×108 CFU C. perfringens on d 18 and 19. Blood samples were collected at 1, 3, 7, and 11 d post-inoculation (dpi) with E. maxima and 1, 3, and 7 dpi with secondary C. perfringens. Plasma cytokines were determined by ELISA while immunometabolic assays evaluated peripheral blood mononuclear cell ATP production and glycolytic rate responses. Data were analyzed with diet and challenge fixed effects plus associated interactions (SAS 9.4; P ≤ 0.05). Replicates 1 and 2 showed few immunometabolic responses within 3 dpi with E. maxima, but 25 to 31% increased ATP production and 32% increased compensatory glycolysis at 1 dpi with C. perfringens in challenged vs. unchallenged chicks (P ≤ 0.04). In replicate 3, total ATP production and compensatory glycolysis were increased 25 and 40%, respectively, by the E. maxima main effect at 1dpi (P ≤ 0.05) with unobserved responsiveness to C. perfringens. These outcomes indicate that model type had greater impacts on systemic immunity than anti-IL-10.

Macrophage migration inhibitory factor favors Neospora caninum infection in mice.

Neospora caninum is a protozoan parasite with worldwide incidence, acting as a major cause of reproductive failures in ruminants and neuromuscular symptoms in dogs. Macrophage Migration Inhibitory Factor (MIF) is produced by several cell types and exhibits a central role in immune responses against intracellular pathogens. The present study aimed to comprehend the role of MIF in the relationship between N. caninum and its host. We used in vivo, in vitro and ex vivo experiments in a model of infection based on genetically deficient mice to analyze the infection kinetics and inflammatory markers. MIF production was measured in response to N. caninum during the acute and chronic phases of the infection. While Mif-/- mice survived lethal doses of NcLiv tachyzoites, sublethal infections in these mice showed that parasite burden was controlled in target tissues, alongside with reduced inflammatory infiltrates detected in lung and brain sections. TNF was increased at the initial site of the infection in genetically deficient mice and the MIF-dependent reduction was confirmed in vitro with macrophages and ex vivo with primed spleen cells. In sum, MIF negatively regulated host immunity against N. caninum, favoring disease progression.

CRISPR-Cas9-based method for isolating microgametes of Eimeria tenella.

Eimeria tenella infections are known to cause severe caecal damage and death of the infected chicken. Gamogony is an essential stage in E. tenella life cycle and in the establishment of coccidiosis. Prior research had extensively explored isolation and separation of the parasite gametes - microgamete (male) and macrogamete (female). However, there is little information on the efficient, highly purified and distinctly separated male and female gametes. In this study, we generated a genome editing line expressing mCherry fluorescent protein fused with GCS1 protein in E. tenella by using Toxoplasma gondii CRISPR-Cas9 system, flow cytometry and fluorescence microscopy. This allowed precise separation of E. tenella male and female gametes in the transgenic parasite population. The separation of male and female gametes would not only build on our understanding of E. tenella transmission, but it would also facilitate development of gametocidal compounds as drug targets for E. tenella infection.

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.

Cystoisospora spp. infection at a dog breeding facility in the Madrid region: Infection rate and clinical management based on toltrazuril metaphylaxis.

Canine coccidiosis caused by Cystoisospora canis and Cystoisospora ohioensis-complex is common in kennels. While often underestimated, coccidiosis may cause severe clinical signs in puppies and sometimes even lead to death, so preventative measures are important. This study examines Cystoisospora spp. infection at a Labrador retriever breeding facility in Madrid, Spain. To identify environmental factors associated with infection, dams were examined throughout a reproductive cycle (from oestrus to 60 days postpartum) and their puppies during their first 60 days of life. Also assessed was the efficacy of combined treatment with emodepside (0.9 mg/ml) and toltrazuril (18 mg/ml) at a dose of 0.5 ml/kg of weight, equivalent to 0.45 mg/kg and 9 mg/kg, respectively, in puppies on day 35 of life. Oocyst shedding was detected in 4.6-18.6% of 45 dams examined and in 2.2-9.1% of their litters (315 puppies). In both cases, peak opg elimination was recorded on day 30 postpartum/of life. The species of Cystoisospora detected were C. canis (91.3%) and C. ohioensis-complex (8.7%). While in both dams and puppies opg counts were higher in autumn when rainfall was at its highest, correlation between opg and rainfall emerged as significant only in puppies (p = 0.031). The treatment of 35 day-old puppies with toltrazuril was 100% effective in controlling this infection in the kennel. Our findings therefore suggest the need for a strict hygiene regime and the use of toltrazuril as blanket treatment to reduce Cystoisospora transmission in dog breeding facilities.

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.

Immunoprotective effects of DNA vaccine against Eimeria tenella based on EtAMA3 and EtRON2L2.

Eimeria tenella is the most pathogenic and harmful intestinal parasitic protozoan. Recombinant DNA vaccines open options for promising strategies for preventing avian coccidiosis, replacing chemical drugs and live oocyst vaccines. Two important antigenic proteins, EtAMA3 (also known as SporoAMA1) and EtRON2L2, act together to promote the invasion of E. tenella sporozoites. In this study, a recombinant DNA vaccine, designated pcDNA3.1(+)-AR, was constructed based on EtAMA3DII, EtRON2L2D3, and EtRON2L2D4. Chickens were intramuscularly immunized with different doses (25, 50, or 100 µg) of pcDNA3.1(+)-AR to evaluate its immunoprotective effects in vivo. The chickens in the 50 µg and 100 µg groups had higher cytokine concentrations (interleukin 2, interferon-gamma, and interleukin 10), and lesion scores (81.9% and 67.57%, respectively) and relative oocyst production (47% and 19%, respectively) reduced compared with the unchallenged group, indicating partial protection against E. tenella. These results suggest that pcDNA3.1(+)-AR is a promising vaccine candidate against avian coccidiosis.