Krameria lappacea root extract's anticoccidial properties and coordinated control of CD4 T cells for IL-10 production and antioxidant monitoring.

Introduction: Recently, the use of botanicals as an alternative to coccidiostats has been an appealing approach for controlling coccidiosis. Therefore, this study was conducted to evaluate the potential role of aqueous methanolic extract (200 mg/kg) of Krameria lappacea (roots) (KLRE) against infection induced by Eimeria papillata. Methods: A total of 25 male C57BL/6 mice were divided into five groups (I, II, III, IV, and V). On 1st day of the experiment, all groups except groups I (control) and II (non-infected-treated group with KLRE), were inoculated orally with 103 sporulated E. papillata oocysts. On the day of infection, group IV was treated with KLRE. Group V served as an infected-treated group and was treated with amprolium (coccidiostat). Results: Treatment with extract and coccidiostat was continued for five consecutive days. While not reaching the efficacy level of the reference drug (amprolium), KLRE exhibited notable anticoccidial activity as assessed by key criteria, including oocyst suppression rate, total parasitic stages, and maintenance of nutrient homeostasis. The presence of phenolic and flavonoid compounds in KLRE is thought to be responsible for its positive effects. The Eimeria infection increased the oxidative damage in the jejunum. KLRE treatment significantly increased the activity of catalase and superoxide dismutase. On the contrary, KLRE decreased the level of malondialdehyde and nitric oxide. Moreover, KLRE treatment decreased macrophage infiltration in the mice jejunal tissue, as well as the extent of CD4 T cells and NFkB. E. papillata caused a state of systemic inflammatory response as revealed by the upregulation of inducible nitric oxide synthase (iNOs)-mRNA. Upon treatment with KLRE, the activity of iNOs was reduced from 3.63 to 1.46 fold. Moreover, KLRE was able to downregulate the expression of pro-inflammatory cytokines interferon-γ, nuclear factor kappa B, and interleukin-10 -mRNA by 1.63, 1.64, and 1.38 fold, respectively. Moreover, KLRE showed a significant reduction in the expression of IL-10 protein level from 104.27 ± 8.41 pg/ml to 62.18 ± 3.63 pg/ml. Conclusion: Collectively, K. lappacea is a promising herbal medicine that could ameliorate the oxidative stress and inflammation of jejunum, induced by E. papillata infection in mice.

Working towards the development of vaccines and chemotherapeutics against neosporosis-With all of its ups and downs-Looking ahead.

Neospora caninum is an apicomplexan and obligatory intracellular parasite, which is the leading cause of reproductive failure in cattle and affects other farm and domestic animals, but also induces neuromuscular disease in dogs of all ages. In cattle, neosporosis is an important health problem, and has a considerable economic impact. To date there is no protective vaccine or chemotherapeutic treatment on the market. Immuno-prophylaxis has long been considered as the best control measure. Proteins involved in host cell interaction and invasion, as well as antigens mediating inflammatory responses have been the most frequently assessed vaccine targets. However, despite considerable efforts no effective vaccine has been introduced to the market to date. The development of effective compounds to limit the effects of vertical transmission of N. caninum tachyzoites has emerged as an alternative or addition to vaccination, provided suitable targets and safe and efficacious drugs can be identified. Additionally, the combination of both treatment strategies might be interesting to further increase protectivity against N. caninum infections and to decrease the duration of treatment and the risk of potential drug resistance. Well-established and standardized animal infection models are key factors for the evaluation of promising vaccine and compound candidates. The vast majority of experimental animal experiments concerning neosporosis have been performed in mice, although in recent years the numbers of experimental studies in cattle and sheep have increased. In this review, we discuss the recent findings concerning the progress in drug and vaccine development against N. caninum infections in mice and ruminants.

Effects of dietary water-soluble extract of rosemary supplementation on growth performance and intestinal health of broilers infected with Eimeria tenella.

This study was conducted to explore the effect of dietary supplementation of water-soluble extract of rosemary (WER) on growth performance and intestinal health of broilers infected with Eimeria tenella (E. tenella), and evaluate the anticoccidial activity of WER. 360 1-d-old Chinese indigenous male yellow-feathered broiler chickens were randomly allocated to six groups: blank control (BC) group and infected control (IC) group received a basal diet; positive control (PC) group, received a basal diet supplemented with 200 mg/kg diclazuril; WER100, WER200, and WER300 groups received a basal diet containing 100, 200, and 300 mg/kg WER, respectively. On day 21, all birds in the infected groups (IC, PC, WER100, WER200, and WER300) were orally gavaged with 1 mL phosphate-buffered saline (PBS) of 8 × 104 sporulated oocysts of E. tenella, and birds in the BC group were administrated an aliquot of PBS dilution. The results showed that dietary supplementation of 200 mg/kg WER increased the average daily gain of broilers compared to the IC group from days 22 to 29 (P < 0.001). The anticoccidial index values of 100, 200, and 300 mg/kg WER were 137.49, 157.41, and 144.22, respectively, which indicated that WER exhibited moderate anticoccidial activity. Compared to the IC group, the groups supplemented with WER (100, 200, and 300 mg/kg) significantly lowered fecal oocyst output (P < 0.001) and cecal coccidia oocysts, alleviated intestinal damage and maintained the integrity of intestinal epithelium. Dietary supplementation with WER significantly improved antioxidant capacity, elevated the levels of secretory immunoglobulin A, and diminished inflammation within the cecum, particularly at a dosage of 200 mg/kg. The results of this study indicated that dietary supplementation with 200 mg/kg WER could improve broiler growth performance and alleviate intestinal damage caused by coccidiosis.

Avian coccidiosis, a prevalent parasitic disease caused by Eimeria protozoa, leads to significant economic losses in the global poultry industry. Currently, the control of coccidiosis in chickens primarily relies on chemical and ionophore anticoccidials. However, the long-term use of these compounds has resulted in the development of drug-resistant strains, presenting a critical challenge. Additionally, the toxic and side effects of ionophore anticoccidials have become increasingly apparent. Thus, there is an urgent need to find economical and environmentally friendly measures to control coccidiosis in chickens. In this study, we established a model of Eimeria tenella infection in broilers to explore whether the water-soluble extract of rosemary (WER) could serve as an alternative method for controlling avian coccidiosis. Our results showed that dietary supplementation with WER (100, 200, and 300 mg/kg) had a beneficial anticoccidial effect, alleviating intestinal damage caused by coccidiosis by enhancing the intestinal antioxidant defense and activating the immune function of the infected broilers. Specifically, dietary supplementation with 200 mg/kg WER emerged as a promising strategy for controlling avian coccidiosis in the poultry industry.

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.

The impact of different levels of functional oil supplementation in combination with salinomycin on growth performance and intestinal microbiota of broilers undergoing Eimeria challenge: An analysis of dynamics.

The effect of salinomycin sodium alone and in combination with functional oils on performance and microbiota of broiler infected Eimeria were evaluated. 512 broilers were randomly assigned to 4 treatments (8 replicates, 16 birds/pen): a Control group (any additives); Ionophore group: salinomycin supplementation at 66 ppm (SS66); Ionophore +0.075% Functional oil (FO) group (SS66 + FO supplementation at 750 ppm); and Ionophore +0.10% FO group (SS66 + FO supplementation at 1000 ppm). At 14 days of age, birds were gavaged with 1 mL of a saline solution containing sporulated oocysts of E. tenella, E. acervulina and E. maxima. Performance indices were measured weekly. At 28 days, intestinal content was collected for microbiota analysis. Broilers of Control group presented the worst performance indices. Broilers of Ionophore + FO (0.075% and 0.10%) groups exhibited a higher BW at 28 days of age. The supplementation of Ionophore +0.075% FO resulted in a higher relative proportion of Firmicutes and a lower proportion of Actinobacteria in the ileum-jejunum. Lactobacillaceae was the dominant family in the jejunal, and ileal microbiotas of broilers fed diets supplemented with Ionophore, Ionophore +0.075% FO and Ionophore +0.10% FO. The supplementation of ionophore yielded higher numbers of Lactobacillaceae, Enterobactereaceae and Cloritridiaceae in the cecal. Ionophore associated with FO controlled the Lactobacillaceae, Enterobactereaceae and Cloritridiaceae families present in the cecum. Therefore, the combination of salinomycin with functional oil showed synergistic effect on performance and modulation of intestinal microbiota of broilers challenged with Eimeria.

The effect of an injectable toltrazuril - gleptoferron (Forceris®) on Cystoisospora suis oocyst excretion and growth of neonatal piglets pre- and post-weaning.

In this study the efficacy of an intramuscular formulation of toltrazuril combined with gleptoferron for the control of porcine cystoisosporosis caused by Cystoisospora suis was investigated. The study was carried out on three Belgian farms with a confirmed history of C. suis infections. As none of the farms implemented a standardized toltrazuril treatment regimen for their piglets, the presence of resistant C. suis strains seems improbable. In total 90 litters, representing 1249 piglets, were included in the study and randomly allocated to either the treatment or control group. Piglets in the treatment group received a single intramuscular injection, containing 45 mg toltrazuril and 200 mg gleptoferron, between 1 and 3 days of age. Piglets in the control group received a single injection with only 200 mg gleptoferron. The effect of treatment on oocyst excretion, expressed in oocysts per gram of feces (OPG), average daily weight gain (ADG) and mortality was determined both pre- and post-weaning. A significant decrease in OPG as well as a decrease in the number of litters (pre-weaning) and pens (post-weaning) that tested positive for cystoisosporosis, was observed in the treated animals compared to the controls. Furthermore, treatment resulted in an increased ADG during the period from day 1 to day 21 (p-value: 0.03881). There was no significant difference in mortality observed between the treatment group to the control group (p-value: 0.2167). To our knowledge, this is the first report on the effect of toltrazuril on oocyst excretion after weaning. This finding highlights the potential long-term benefits of the treatment beyond the initial administration.

Prophylactic and Therapeutic Efficacy of Ultrasonicated Rosmarinus officinalis Ethanolic Extract and its Chitosan-Loaded Nanoparticles Against Eimeria tenella Infected Broiler Chickens.

PURPOSE: The in vivo efficacy of ultrasonicated Rosmarinus officinalis ethanolic extract (UROEE) and its chitosan-loaded nanoparticles (UROEE-CsNPs) was investigated as a dietary prophylactic agent and as a therapeutic treatment against Eimeria tenella infected broiler chickens. METHODS: Chickens were infected with 4 × 104 E. tenella oocysts at 21 days old for primary infection and with 8 × 104 oocysts at 35 days old for secondary infection. Eleven experimental groups were conducted. Dietary addition of 100 mg/kg UROEE and 20 mg/kg for CsNPs as well as UROEE-CsNPs were included for prophylactic groups from day 1 to 42. The same doses were used for therapeutic treatment groups for 5 constitutive days. Oocyst output in feces was counted. Histopathological and immunohistochemical studies were conducted. Gene expression of pro-inflammatory cytokines as IFN-γ, IL-1ß and IL-6 as well as anti-inflammatory cytokines as IL-10 and TGF-ß4 was analyzed using semi-quantitative reverse transcriptase-PCR. RESULTS: The results showed an efficacy of UROEE, CsNPs and UROEE-CsNPs in reduction of oocyst excretion and improving the cecal tissue architecture. CD4+ and CD8+ T lymphocytes protein expression were reduced. E. tenella infection lead to upregulation of pro-inflammatory cytokines as IFN-γ, IL-1ß, IL-6 and anti-inflammatory cytokines as TGF-ß4 following primary infection, while their expression was downregulated following secondary infection. CONCLUSION: The dietary prophylactic additives and therapeutic treatments with UROEE, CsNPs and UROEE-CsNPs could decrease the inflammatory response to E. tenella as indicated by oocyst output reduction, histopathological improvements, CD4+ and CD8+ T cells protein expression reduction as well as reducing mRNA expression levels of the tested cytokines following primary and secondary infections. Consequently, these results will help to develop better-combating strategies for the control and prevention of coccidiosis on poultry farms as a dietary prophylactic agent or as a therapeutic treatment.

Matrine reduced intestinal stem cell damage in eimeria necatrix-infected chicks via blocking hyperactivation of Wnt signaling.

BACKGROUND: Coccidiosis is a rapidly spreading and acute parasitic disease that seriously threatening the intestinal health of poultry. Matrine from leguminous plants has anthelmintic and anti-inflammatory properties. PURPOSE: This assay was conducted to explore the protective effects of Matrine and the AntiC (a Matrine compound) on Eimeria necatrix (EN)-infected chick small intestines and to provide a nutritional intervention strategy for EN injury. STUDY DESIGN: The in vivo (chick) experiment: A total of 392 one-day-old yellow-feathered broilers were randomly assigned to six groups in a 21-day study: control group, 350 mg/kg Matrine group, 500 mg/kg AntiC group, EN group, and EN + 350 mg/kg Matrine group, EN + 500 mg/kg AntiC group. The in vitro (chick intestinal organoids, IOs): The IOs were treated with PBS, Matrine, AntiC, 3 µM CHIR99021, EN (15,000 EN sporozoites), EN + Matrine, EN + AntiC, EN + Matrine + CHIR99021, EN + AntiC + CHIR99021. METHODS: The structural integrity of chicks jejunal crypt-villus axis was evaluated by hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM). And the activity of intestinal stem cells (ISCs) located in crypts was assessed by in vitro expansion advantages of a primary in IOs model. Then, the changes of Wnt/ß-catenin signaling in jejunal tissues and IOs were detected by Real-Time qPCR,Western blotting and immunohistochemistry. RESULTS: The results showed that dietary supplementation with Matrine or AntiC rescued the jejunal injury caused by EN, as indicated by increased villus height, reduced crypt hyperplasia, and enhanced expression of tight junction proteins. Moreover, there was less budding efficiency of the IOs expanded from jejunal crypts of chicks in the EN group than that in the Matrine and AntiC group, respectively. Further investigation showed that AntiC and Matrine inhibited EN-stimulated Wnt/ß-catenin signaling. The fact that Wnt/ß-catenin activation via CHIR99021 led to the failure of Matrine and AntiC to rescue damaged ISCs confirmed the dominance of this signaling. CONCLUSION: Our results suggest that Matrine and AntiC inhibit ISC proliferation and promote ISC differentiation into absorptive cells by preventing the hyperactivation of Wnt/ß-catenin signaling, thereby standardizing the function of ISC proliferation and differentiation, which provides new insights into mitigating EN injury by Matrine and AntiC.

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.

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.

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.

Protective effect of Inonotus obliquus polysaccharide on mice infected with Neospora caninum.

The study aimed to explore the protective effects of Inonotus obliquus polysaccharide (IOP) on Neospora caninum (N. caninum) infection. Our data showed that the survival rate of the mice was the highest and the survival time was the longest when the IOP was 2 mg/10 g. In agreement with these observations, IOP alleviated the pathological damage in the various organs and tissues of the mice. Compared with that in the Neosporidium infection model group, the content of N. caninum in the heart, liver, spleen, lung, kidney and brain, determined through HE staining, was significantly lower. In addition, IOP inhibited the levels of immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2a) from the 21st to 42nd day of the administration group, whereas the levels of interleukin-12 (IL-12) and serum tumor necrosis factor alpha (TNF-α) were down-regulated at 7 d - 42 d. The production of CD4+ T lymphocytes was promoted, the number of CD8+ T lymphocytes were significantly lower and the CD4+/CD8+ ratio was significantly elevated. Furthermore, IOP effectively balanced the levels of hormones including gonadotropin-releasing hormone (GnRH), luteotropic hormone (LH) and testosterone (T) in male mice, and progesterone (PROG), estradiol (E2) and prolactin (PRL) in female mice. These findings demonstrate that IOP exerts protective effects against pathological damage caused by N. caninum infection in mice, and improve the immune function of the organism and regulate the secretion balance of sex hormones.

Effects of natural extract from medicinal herbs on broilers experimentally infected with Eimeria tenella.

This study aimed to evaluate the effects of natural extracts from nine medicinal herbs (SMA) on the growth performance, immunity, and intestinal integrity of broilers experimentally infected with Eimeria tenella. A total of 252 one-day-old broiler chicks were divided into 7 groups with 3 replicates per group and 12 broilers per cage. The groups were uninfected-untreated blank control group (BC), infected-untreated negative control group (NC), SMA treatment groups, Chinese medicine positive control group (CM), and chemical drug positive control group (CD). The SMA groups were infected and fed a basal diet supplemented with 0.6 (SMA-L), 0.8 (SMA-M), and 1.0 (SMA-H) g/kg SMA. The CM and CD groups were infected and fed a basal diet supplemented with 15 g/kg Jiqiuchong San and 0.2 g/kg Diclazuril, respectively. Results showed that feeding SMA could significantly reduce the number of oocysts in infected chickens, especially 1.0 g/kg SMA, which exhibited moderate anticoccidial efficacy. When infected with E. tenella, the supplementation of 1.0 g/kg SMA increased the renal index; restored the hepatic, splenic, and bursal indexes to BC levels; increased the levels of immunoglobulin A (IgA), IgM, and IgY; and reduced the contents of tumor necrosis factor (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6), and IL-10 of the infected chickens. Moreover, treatment with 1.0 g/kg SMA alleviated the pathological changes in cecal tissue and increased the contents of zonula occludens-1 (ZO-1), occludin, claudin-1, and mucoprotein 2 (mucin-2) in cecal tissues of E. tenella-infected chickens. We found that 1.0 g/kg SMA reduced the number of oocysts, improved immunity, and alleviated intestinal barrier damage, which could improve the growth performance of infected chickens. Thus, SMA proved to be an effective natural extract against E. tenella and has the potential to be used as an efficient anticoccidial drug or additive.

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.

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.

Ponazuril: Clinical efficacy, ultrastructure, and histopathology studies of in vivo anticoccidial action against Eimeria tenella.

Ponazuril, a novel antiprotozoal drug in the class of triazine, has shown a promising application on apicomplexan infections in poultry and livestock. However, the effect and mechanism of action of ponazuril against Eimeria tenella (E. tenella) are unclear. The efficacy against E. tenella was initially studied by administering different doses of ponazuril in drinking water. The treated stage and site of ponazuril on E. tenella were observed through ultrastructural and histopathological analyses. Chicks were orally treated with a dose of 15 mg/kg body weight of ponazuril at different endogenous stages of E. tenella post-infection. According to the clinical study, the values of anticoccidial indices (ACI) were 157.0, 162.3, 196.9, 194.5, and 190.9, respectively, when the ponazuril was administered in drinking water at doses of 5, 10, 20, 40, and 50 mg/L for two consecutive days after infection. Among them, the 20 mg/L ponazuril group showed the best anticoccidial effect, which was superior to that of the toltrazuril treatment group, with an ACI value of 191.7. Histological analysis indicated that ponazuril effectively relieved cecal lesions, and decreased the number of merozoites. Transmission electron micrographs (TEM) observed that merozoites became irregular in shape, and some apparent protrusions of the outer membrane were presented especially the second-generation merozoites. Additionally, abnormalities in the development of WFBI and WFBII in the macrogametocyte were observed, which may affect the formation of the ovule wall. Moreover, merozoites in the treated group showed uneven and marginalized chromatin and mitochondrial swelling. These results suggested ponazuril is a potential anticoccidial drug, providing information on the mechanism of anticoccidial effects.

Metaphylactic strategies using toltrazuril against coccidiosis in goat kids.

Goat coccidiosis compromises animal welfare, reduces productivity and may cause mortality and delayed growth rates in goat kids around the weaning period worldwide. This field study was conducted to evaluate the efficacy of metaphylactic treatments with two doses of toltrazuril (20 or 40 mg/kg body weight - BW, p. o.), at different timing, in kids naturally infected with Eimeria spp. A total of 97 healthy goat kids (Majorera milk aptitude breed) were divided into five groups, depending on the age of treatment (2 or 7 weeks). One group remained untreated as a negative control until the end of the study. Faecal oocyst shedding, faecal consistency, and body weight of the animals were monitored at day 0 and at weekly intervals. Counts of oocysts per gram of faeces (OPG) were determined by a modified McMaster technique. Morphometric identification of Eimeria species was carried out on individual faecal samples from each experimental group after oocyst sporulation. Goat kids treated at two weeks of age maintained OPG values close to zero during the 5 weeks post-treatment and, overall, had lower faecal oocyst counts than untreated control animals. No significant differences were observed between the two doses of toltrazuril used in two-week-old treated animals. By contrast, when treatment was carried out at seven weeks of age, the dose of 40 mg/kg BW of toltrazuril reduced oocyst levels for longer and to a greater extent than the 20 mg/kg dose. Irrespectively of the treatment and dose, toltrazuril delayed the appearance of pathogenic Eimeria species, i. e. Eimeria ninakohlyakimovae and Eimeria arloingi. As a whole, Eimeria christenseni, with a rather moderate pathogenicity, was highly predominant throughout the study period, including the untreated control group, which was probably the reason why clinical signs of coccidiosis were barely observed throughout the experiment. Under these circumstances, the positive effect of toltrazuril on body weight condition observed in some treated groups was difficult to correlate to the timing and doses. Metaphylactic treatments with 20 mg/kg BW toltrazuril given at two weeks of age are sufficient to control oocyst excretion in goat kids; whereas if administered later in 7-week-old animals, thereby coinciding with the frequently observed peak of oocyst elimination in goat kids under field conditions, a higher dose might be advisable to prevent environmental contamination with infectious oocysts.

Synthesis and evaluation of febrifugine derivatives as anticoccidial agents.

A series of new febrifugine derivatives with a 4(3H)-quinazolinone scaffold were synthesized and evaluated for their anticoccidial activity both in vitro and in vivo. The targets' in vitro activity against Eimeria tenella was studied using quantitative real-time reverse transcription polymerase chain reaction and Madin-Darby bovine kidney cells. Most of these compounds demonstrated anticoccidial efficacy, with inhibition ratios ranging from 3.3% to 85.7%. Specifically, compounds 33 and 34 showed significant inhibitory effects on the proliferation of E. tenella and exhibited lower cytotoxicity compared to febrifugine. The IC50 values of compounds 33 and 34 were 3.48 and 1.79 µM, respectively, while the CC50 values were >100 µM for both compounds. Furthermore, in a study involving 14-day-old chickens infected with 5 × 104 sporulated oocysts, treatment with five selected compounds (22, 24, 28, 33, and 34), which exhibited in vitro inhibition rate of over 50% at 100 µM, at a dose of 40 mg/kg in daily feed for 8 consecutive days showed that compound 34 possessed moderate in vivo activity against coccidiosis, with an anticoccidial index of 164. Structure-activity relationship studies suggested that spirocyclic piperidine may be a preferable substructure to maintain high effectiveness in inhibiting Eimeria spp., when the side chain 1-(3-hydroxypiperidin-2-yl)propan-2-one was replaced.

Anticoccidial activity of Aloe Vera Leafs' aqueous extract and vaccination against Eimeria tenella: pathological study in broilers.

This study aimed to assess the efficacy of an anticoccidial vaccine and the anticoccidial activity of Aloe vera in broiler chickens infected with Eimeria tenella (E. tenella). A total of 225 healthy, sexless, one-day-old broiler chicks (avian48) from a commercial broiler company were randomized into nine experimental groups of 25 chicks. The groups were as follows: Group 1 (control, vaccinated, non-infected), Group 2 (vaccinated and infected with 5 × 104 sporulated oocysts), Group 3 (vaccinated, infected with 5 × 104 sporulated oocysts, and treated with Aloe vera), Group 4 (infected with 5 × 104 sporulated oocysts and treated with Aloe vera), Group 5 (positive control, infected with 5 × 104 sporulated oocysts), Group 6 (challenged with 5 × 104 sporulated oocysts and then treated with amprolium), Group 7 (treated with amprolium), Group 8 (blank control negative group), and Group 9 (treated with Aloe vera gel).Various parameters were evaluated, including clinical signs, growth performance, oocyst shedding, hematological and immunological parameters, and pathological lesion scoring. The results demonstrated that Aloe vera improved growth performance, reduced oocyst shedding, and decreased caecal lesion scores in E. Tenella-infected broiler chicks. The use of Aloe vera in combination with either amprolium or anticoccidial vaccines provided a potential solution to the issues of drug resistance and drug residues.In conclusion, this study provides valuable insights regarding the control of coccidiosis in broilers. Supplementing the chicken diet with Aloe vera had beneficial effects on the pathogenicity and infectivity of E. tenella, making it a cost-effective alternative as an herbal extract with no adverse side effects for coccidiosis control. These findings suggest that Aloe vera can be considered a potential candidate for inclusion in broiler diets for effective coccidiosis control.

Effects of Radix dichroae extract supplementation on growth performance, oocysts output and gut microbiota in growing lambs with coccidiosis.

Coccidiosis is an intestinal protozoan disease of sheep, that causes substantial economic losses in the industry due to its intestinal protozoan origins. Many anti-protozoan drugs including ionophores, triazines, and sulfonamides have been widely used to treat sheep coccidiosis. Still, anticoccidial resistance and drug residues in edible tissues have prompted an urgent search for alternatives. In this study, the anti-coccidial effectiveness of the Radix dichroae extract was compared to that of the conventional anti-coccidial drug diclazuril. Here, eighteen 45-day-old lambs naturally-infected with Eimeria spp. were randomly allocated in three groups: control group, Radix dichroae extract group and diclazuril group. The results showed that the body weight gain (BWG) during the treatment and withdrawal periods was considerably improved in the coccidiosis-infected sheep treated with Radix dichroae extract and diclazuril compared to the control group, respectively. Additionally, the Radix dichroae extract and diclazuril had fewer oocysts per gram (OPG) than the control group, showing similar anti-coccidial effects on days 14, 21, 28, 35 and 78, respectively. Furthermore, Radix dichroae extract and diclazuril treatment altered the structure and composition of gut microbiota, promoting the relative abundance of Actinobacteriota, Firmicutes, Alistipes, and Bifidobacterium, while decreasing the abundance of Bacteroidota, Marinilaceae, Helicobacteraceae, and Prevotella. Moreover, Spearman's correlation analysis further revealed a correlation between the OPG and BWG and gut microorganisms. Collectively, the results indicated that Radix dichroae extract had similar anti-coccidial effects as diclazuril, and could regulate gut microbiota balance in growing lambs.