Assessment of the feed additive consisting of diclazuril (Clinacox® 0.5%) for chickens for fattening and chickens reared for laying for the renewal of its authorisation (Elanco GmbH).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of diclazuril (Clinacox® 0.5%) as a coccidiostat for chickens for fattening and chickens reared for laying. The additive currently on the market complies with the existing conditions of authorisation. The additive remains safe for the target species and the consumer under the authorised conditions of use. The additive is irritant to skin, eyes and respiratory tract but is not a skin sensitiser. Exposure by inhalation cannot be excluded. The FEEDAP Panel cannot conclude on the safety for the environment of diclazuril from Clinacox® 0.5% due to lack of data. Diclazuril from Clinacox® 0.5% at a concentration of 1 mg diclazuril/kg complete feed has the potential to control coccidiosis in chickens for fattening. This conclusion is extended to chickens reared for laying. Development of resistance to diclazuril of field Eimeria spp. strains isolated from chickens should be monitored.

Effects of prebiotic (lactoferrin) and diclazuril on broiler chickens experimentally infected with Eimeria tenella.

Introduction: Avian coccidiosis presents a significant challenge to the poultry industry in Egypt, highlighting the urgent need for validating new drug targets offering promising prospects for the development of advanced anticoccidials. Although numerous reports highlight the activity of lactoferrin (LF) against various microorganisms, its potential against Eimeria has not been explored. The present study evaluated the potential anticoccidial effect of LF and diclazuril in broiler chickens experimentally infected with Eimeria tenella. Methods: A total of 100 one-day-old broiler chicks were divided into five equal groups (20 each) as follows: Group 1 (G1) served as the normal healthy control group, Group 2 (G2) consisted of chickens infected with 1 × 105 sporulated E. tenella oocysts at 14 days of age, Group 3 (G3) comprised infected chickens treated with diclazuril (0.5 mL/L in drinking water) for 3 days successively, Group 4 (G4) included infected chickens treated with LF (at a dose of 250 mg/kg of diet) from one day of age until the end of the study, and Group 5 (G5) comprised infected chickens treated with both LF and diclazuril. Results: The positive control group (G2) experienced significant reductions in body weight (BW), BW gain, serum glucose, lipase, amylase, total antioxidant capacity, several hematological indices, and total proteins, along with alterations in various antioxidant enzymes. Conversely, serum levels of aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatases (ALP), urea, creatinine, nitric oxide, mean corpuscular volume (MCV), White blood cells (WBCs), heterophils, alpha 2, beta 1, and liver contents of malondialdehyde were elevated in this group. Moreover, higher oocyst counts and lesion scores, along with histopathological alterations, were observed in G2. Remarkably, treatment with diclazuril and/or LF demonstrated potent antioxidant and anticoccidial effects, resulting in reduced shedding of oocysts, lesion scores, and lymphocytic infiltrates in the cecum. Additionally, these treatments improved the antioxidant and immune systems in chickens and restored all histopathological changes reported in the infected non-treated group (G2). Conclusion: This study offers novel perspectives on the potential anticoccidial effects of the combination of LF and diclazuril in broiler chickens infected with E. tenella, highlighting the potential synergistic actions of LF in treating poultry coccidiosis.

A facile and intelligent detection method for diclazuril based on a stable dual emissive Eu3+-dopped metal-organic framework.

Diclazuril (DIC) is a broad-spectrum anti-coccidiosis drug of the triazine class, widely used in poultry farming. The overuse of DIC may lead to its accumulation in animal bodies, which may enter the food chain and threaten human health. In this work, we fabricated a stable Eu3+-doped UiO-66 fluorescence sensor (EuUHIPA-30) for the sensitive detection of DIC. Among 20 veterinary drugs, the fluorescence of EuUHIPA-30 selectively responds to DIC, with a low detection limit (0.19 µM) and fast response (10 s). EuUHIPA-30 is recyclable and can detect DIC in chicken and eggs with good recoveries. Moreover, a smartphone-integrated paper-based sensor enables the instrument-free, rapid, visual, and intelligent detection of DIC in chickens and eggs. This work provides a promising candidate for practical fluorescent DIC sensing in animal-derived food to promote food safety.

Diclazuril-induced expression of CDK-related kinase 2 in the second-generation merozoites of Eimeria tenella.

Diclazuril is a classic anticoccidial drug. The key molecules of diclazuril in anticoccidial action allows target screening for the development of anticoccidial drugs. Cyclin-dependent kinases (CDK) are prominent target proteins in apicomplexan parasites. In this study, a diclazuril anticoccidiosis animal model was established, and the transcription and translation levels of the CDK-related kinase 2 of Eimeria tenella (EtCRK2) were detected. mRNA and protein expression levels of EtCRK2 decreased in the infected/diclazuril group compared with those in the infected/control group. In addition, immunofluorescence analysis showed that EtCRK2 was localised in the cytoplasm of the merozoites. The fluorescence intensity of EtCRK2 in the infected/diclazuril group was significantly weaker than that in the infected/control group. The anticoccidial drug diclazuril against E.tenella affects the expression pattern of EtCRK2 molecule, and EtCRK2 is a potential target for new drug development.

Safety for the environment of a feed additive consisting of diclazuril (Coxiril®) for chickens reared for laying and pheasants (Huvepharma NV).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety for the environment of diclazuril (Coxiril®) as a coccidiostat feed additive for chickens reared for laying and pheasants. In its previous assessments, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) could not reach a final conclusion on the risk resulting from the use of diclazuril in acid soil from Coxiril®. On the basis of the new data provided, the FEEDAP Panel updates the previous conclusions as follows: no risk is expected for the terrestrial compartment and for sediment when diclazuril is used in chickens reared for laying and to pheasants at the proposed condition of use (in both acidic and non-acidic soils). No concern for groundwater is expected for both acidic and non-acidic soils. Due to the lack of data, no conclusions can be drawn for the aquatic compartment. Diclazuril does not have the potential for bioaccumulation; therefore, a risk of secondary poisoning is unlikely.

Forward genetic analysis of monensin and diclazuril resistance in Eimeria tenella.

Worldwide distributed coccidiosis is caused by infection of both Eimeria species and Cystoisospora in the host intestine and causes huge economic losses to the livestock industry, especially the poultry industry. The control of such diseases relies mainly on chemoprophylaxis with anticoccidials, which has led to a very common drug resistance in this field. However, the genetic mechanisms underlying resistance to many anticoccidial drugs remain unknown. In this study, strains of E. tenella resistant to 250 mg/kg monensin were generated and characterized. Forward genetic approaches based on pooled genome sequencing, including experimental evolution and linkage group selection, were used to locate candidate targets responsible for resistance to monensin and diclazuril in E. tenella. A total of 16 nonsynonymous mutants in protein-coding genes were identified in monensin-resistant strains, and two genomic regions with strong selection signals were also detected in diclazuril-resistant strains. Our study reveals the genetic characterization of the experimental evolution and linkage group selection in Eimeria species, and also provides important information that contributes to the understanding of the molecular mechanism of drug resistance in coccidia.

Development and Application of a Physiologically Based Pharmacokinetic Model for Diclazuril in Broiler Chickens.

Withdrawal periods for diclazuril in broilers have traditionally been determined through regression analysis. However, over the last two decades, the physiologically based pharmacokinetic (PBPK) model has gained prominence as a predictive tool for veterinary drug residues, which offers an alternative method for establishing appropriate withdrawal periods for veterinary drugs. In this current study, a flow-limited PBPK model was developed to predict diclazuril concentrations in broilers following long-duration administration via medicated feed and water. This model consists of nine compartments, including arterial and venous plasma, lung, muscle, skin + fat, kidney, liver, intestine contents, and the rest of the body compartment. Physiological parameters such as tissue weights (Vcxx) and blood flow (Qcxx) were gathered from published studies, and tissue/plasma partition coefficients (Pxx) were calculated through the area method or parameter optimization. Published diclazuril concentrations were compared to the predicted values, indicating the accuracy and validity of the model. The sensitivity analysis showed that parameters associated with cardiac output, drug absorption, and elimination significantly affected diclazuril concentrations in the muscle. Finally, a Monte Carlo analysis, consisting of 1000 iterations, was conducted to calculate the withdrawal period. Based on the Chinese MRL values, we calculated a withdrawal period of 0 days for both recommended dosing regimens (through mediated water and feed at concentrations of 0.5-1 mg/L and 1 mg/kg, respectively). However, based on the European MRLs, longer periods were determined for the mediated feed dosing route. Our model provides a foundation for scaling other coccidiostats and poultry species.

Tulathromycin and Diclazuril Lack Efficacy against Theileria haneyi, but Tulathromycin Is Not Associated with Adverse Clinical Effects in Six Treated Adult Horses.

Equine theileriosis, caused by Theileria haneyi and Theileria equi, leads to anemia, exercise intolerance, and occasionally, death. Theileriosis-free countries prohibit the importation of infected horses, resulting in significant costs for the equine industry. Imidocarb dipropionate is the only treatment for T. equi in the United States, but lacks efficacy against T. haneyi. The goal of this study was to assess the in vivo efficacy of tulathromycin and diclazuril against T. haneyi. Fourteen T. haneyi-infected horses were utilized. Six were treated with eight weekly 2.5 mg/kg doses of tulathromycin. Three were treated daily for eight weeks with 2.5 mg/kg diclazuril. Three were pre-treated with 0.5 mg/kg diclazuril daily for one month to determine whether low-dose diclazuril prevents infection. Following infection, the dose was increased to 2.5 mg/kg for eight weeks. Two infected horses remained untreated as controls. The horses were assessed via nested PCR, physical exams, complete blood counts, serum chemistry panels, and cytology. Tulathromycin and diclazuril failed to clear T. haneyi and the treated and control groups exhibited similar parasitemia and packed cell volume declines. To obtain additional safety data on tulathromycin use in adult horses, necropsy and histopathology were performed on tulathromycin-treated horses. No significant lesions were detected.

Molecular characterization and analysis of drug resistance-associated protein enolase 2 of Eimeria tenella.

Eimeria tenella, an intestinal parasite, has brought huge economic losses to the poultry industry. The prevalence and severity of the development of drug resistance has increased the challenge of coccidiosis control. We previously identified the enolase 2 of E. tenella (EtENO2) was differentially expressed in drug-sensitive (DS) and drug-resistant strains using RNA-seq. In this study, the expression of EtENO2 in diclazuril-resistant (DZR), maduramicin-resistant (MRR), and salinomycin-resistant (SMR) strains was analyzed by quantitative real-time PCR (qRT-PCR) and western blots. EtENO2 was highly expressed in several drug-resistant strains compared with the DS strain. The qRT-PCR showed that the transcription level of EtENO2 in the field-isolated resistant strains was upregulated compared with the DS strain. The enzyme activity results indicated that the catalytic activity of EtENO2 in the drug-resistant strains was higher than in the DS strain. In addition, qRT-PCR and western blots showed that the expression level of EtENO2 was higher in second generation merozoites (SM) and unsporulated oocysts (UO) than that in sporozoites (SZ) and sporulated oocysts (SO). Immunofluorescence localization revealed that EtENO2 was distributed throughout SZ and SM and on the surface of the parasites. After the SZ invasion DF-1 cells, it was also observed on the parasitophorous vacuole membrane. Our secretion experiments found that EtENO2 could be secreted outside the SZ. This study indicated that EtENO2 might be related to the interaction between E. tenella and host cells and be involved in the development of E. tenella resistance to some anticoccidial drugs.

Pharmacokinetics of a FDA-labeled dose of diclazuril administered orally once weekly to adult horses.

Equine protozoal myeloencephalitis (EPM) has remained a devastating neurological disease of the Americas, especially in young performance horses. Prophylactic treatment strategies with diclazuril have shown to reduce seroprevalence and titer levels to Sarcocystis neurona in healthy horses continuously exposed to the apicomplexan parasite. The goal of this study was to determine if the FDA-labeled dose of 1 mg/kg of 1.56% diclazuril (ProtazilTM) given once weekly to healthy adult horses would achieve steady-state concentrations in plasma known to be inhibitory to S. neurona in cell culture. Five individual diclazuril doses were administered at weekly intervals to 8 adult horses. Blood was collected via venipuncture immediately before (trough concentration) and 10 hours after (peak concentration) each diclazuril administration. Following the fifth dose, additional blood samples were collected every 24 hours after the peak blood collection for 7 days. All plasma samples were analyzed by high-pressure liquid chromatography. The pharmacokinetic analysis was performed using a nonlinear mixed effects model. The mean population-derived peak concentration was 264 ng/mL and the mean terminal half-life was 3.6 days. Thus, the oral administration of an FDA-labeled dose of diclazuril to healthy horses once a week was able to produce steady-state plasma drug concentrations known to inhibit S. neurona in vitro.

Tetrabromobisphenol A and Diclazuril Evoke Tissue-Specific Changes of Thyroid Hormone Signaling in Male Thyroid Hormone Action Indicator Mice.

Thyroid hormone (TH) signaling is a prerequisite of normal tissue function. Environmental pollutants with the potential to disrupt endocrine functions represent an emerging threat to human health and agricultural production. We used our Thyroid Hormone Action Indicator (THAI) mouse model to study the effects of tetrabromobisphenol A (TBBPA; 150 mg/bwkg/day orally for 6 days) and diclazuril (10.0 mg/bwkg/day orally for 5 days), a known and a potential hormone disruptor, respectively, on local TH economy. Tissue-specific changes of TH action were assessed in 90-day-old THAI mice by measuring the expression of a TH-responsive luciferase reporter in tissue samples and by in vivo imaging (14-day-long treatment accompanied with imaging on day 7, 14 and 21 from the first day of treatment) in live THAI mice. This was followed by promoter assays to elucidate the mechanism of the observed effects. TBBPA and diclazuril impacted TH action differently and tissue-specifically. TBBPA disrupted TH signaling in the bone and small intestine and impaired the global TH economy by decreasing the circulating free T4 levels. In the promoter assays, TBBPA showed a direct stimulatory effect on the hdio3 promoter, indicating a potential mechanism for silencing TH action. In contrast, diclazuril acted as a stimulator of TH action in the liver, skeletal muscle and brown adipose tissue without affecting the Hypothalamo-Pituitary-Thyroid axis. Our data demonstrate distinct and tissue-specific effects of TBBPA and diclazuril on local TH action and prove that the THAI mouse is a novel mammalian model to identify TH disruptors and their tissue-specific effects.

Toltrazuril and diclazuril: comparative evaluation of anti-coccidial drugs using a murine model.

Intestinal coccidiosis caused by Eimeria protozoan species is an economically important disease, especially in poultry and cattle. Anti-coccidial drugs commonly used for controlling coccidiosis are toltrazuril (TTZ) and diclazuril (DCZ). In this study, the efficacies of TTZ and DCZ were compared using a murine model, and the effect of these treatments on the induction of acquired resistance was evaluated. Male C57BL/6J mice were inoculated with 1,000 sporulated E. vermiformis oocytes and treated with TTZ or DCZ. The recommended TTZ dose for cattle (15 mg/kg) completely prevented oocyte excretion. But, mice required 5 mg/kg of DCZ, which is five times the recommended dose for cattle, to reduce oocyte excretion. In E. vermiformis re-infection, TTZ (15 mg/kg) and DCZ (5 mg/kg) treatments did not interfere with the development of acquired resistance. Bodyweight gain was significantly higher in the TTZ-treated group than in the control (untreated/infected) group and the DCZ-treated group, and no significant difference in bodyweight gain was observed between the TTZ-treated group and the healthy (uninfected/untreated) group. Analysis of T lymphocyte subsets in the spleen and mesenteric lymph nodes indicated that the relative populations of CD4+ and CD8+ T cells were reduced in the DCZ-treated and control (untreated/infected) groups, suggesting there was immunosuppression during the infection. However, no reductions in T cell populations were observed in the TTZ-treated group. The results indicated that an optimal anti-coccidial drug is one that can completely break the parasite life cycle in the host animal.

Plasma concentrations of diclazuril following oral administration of diclazuril and diclazuril sodium salt to cattle.

Diclazuril is a triazine-based antiprotozoal agent widely used in veterinary practice that may have clinical application in the treatment of bovine protozoal diseases. The present study reports on the bioavailability, pharmacokinetics, and metabolism of diclazuril and diclazuril sodium salt in cattle following administration of diclazuril suspended in water and by direct application of diclazuril sodium salt to the oral mucosa. Compared with diclazuril itself, the sodium salt formulation of diclazuril applied to the oral mucosa was rapidly and reliably absorbed. Plasma concentrations of diclazuril peaked at around 8 h after oral-mucosal administration of diclazuril sodium salt. On the contrary, application of diclazuril itself orally resulted in delayed and variable absorption. The mean bioavailability of diclazuril as pure powder was 42.5% relative to diclazuril sodium salt indicating approximately 2.5-fold increase in bioavailability of diclazuril as a sodium salt relative to diclazuril as a pure compound in cattle. The present study also reports finding of a previously unreported diclazuril metabolite at high concentrations in plasma especially after oral administration of diclazuril. Further studies, including synthesis and characterization of the novel described metabolite, are required to accurately determine aspects of the metabolism of diclazuril in cattle.

Ameliorative effect of Psidium guajava essential oil supplemented feed on chicken experimental coccidiosis.

Present study investigated the therapeutic efficacy of Psidium guajava essential oil (EO) on chicken experimental coccidiosis in comparison to the diclazuril. Seventy-five 1-day-old Ross 308 broiler chickens were allocated into 5 groups: CEO1: received EO at 1 mg kg-1 of feed; CEO5: received EO at 5 mg kg-1 of feed; CT: received diclazuril as standard treatment; CNT: received only basal diet; NC: control chickens; all of the groups except NC were challenged with mixed Eimeria spp. on d14 and received supplemented diet from d1 to d42. Zootechnical records and oocyst per gram (OPG) of feces samples were analyzed on weekly basis. On the last day of the study, blood samples were taken to measure serum concentrations of biochemical parameters and also activities of antioxidant enzymes. ß-caryophyllene and α-pinene were determined as major constituents of the EO. On the 3rd, 4th, and 5th weeks, a significant difference was noted in feed conversion ratio (FCR) between CEO1, CEO5, and CT in comparison to NC and CNT chickens (p < 0.05). No significant difference was observed in OPG between CEO1 and CEO5 (p > 0.05); however, CT showed a lower number of OPG relative to EO supplemented groups (p < 0.05). The highest serum activity of glutathione peroxidase was observed in CEO5 which was higher than other groups (p < 0.05). Results of the present study showed that supplementation of P. guajava EO especially at 5 mg kg-1 of feed may have beneficial effects on prevention of coccidiosis and improvement of health in broilers.

Parasitological and histopathological examination of Cocktail lovebirds infected with Eimeria aratinga (Apicomplexa: Eimeriidae).

Coccidiosis is a devastating worldwide disease and is considered a dreadful disease in lovebirds. Indeed, a problem has been appeared cocktail lovebirds kept in a private pet birdhouse in Sheikh Zayed City, Giza, Egypt, in the shape of blood-tinged diarrhea, birds huddled together and showing signs of inappetence, ruffled feathers, unable to fly, general weakness and emaciation associated with high mortalities. Therefore, this study aimed to diagnose and find a suitable treatment to overcome such problems. To achieve this aim, blood and droppings samples were collected from infected and healthy birds for parasitological and hematological examinations, and tissue samples were collected from freshly dead birds for postmortem and histopathological examinations. A treatment trial was adopted on 50 infected birds and 25 healthy and parasitological negative birds and groups were classified as follows: group 1) 25 infected birds treated with Diclazuril, group 2) infected birds treated with Coccicure, and group 3) 25 birds kept as control negative reference birds. The parasitological identification revealed the presence of Eimeria aratinga (E. aratinga) oocysts in the infected bird intestine. Finally, we concluded that E. aratinga is a serious protozoon parasite infesting lovebirds revealing severe clinical signs, high mortalities, histopathological changes in the intestine and alteration in blood parameters. Diclazuril is an effective drug in treating E. aratinga in cocktail lovebirds.

Use of Zinc Oxide Nanoparticles as Anticoccidial Agents in Broiler Chickens along with Its Impact on Growth Performance, Antioxidant Status and Hematobiochemical Profile.

The impact of zinc oxide nanoparticles (ZnO-NPs) on the pathogenesis of coccidiosis in broiler chickens was tested. A total of 160 1-day-old broiler chicks (Ross 308) were randomly allocated into 4 groups (n = 40). Group 1: unchallenged, unmedicated; Group 2: challenged, unmedicated; Group 3: challenged, supplemented with diclazuril (1 ppm); Group 4: challenged, supplemented with ZnO-NPs (20 ppm). Mixed Eimeria species (E. maxima, E. acervulina, E. mivati, and E. tenella) of a commercial coccidial vaccine (FORTEGRA®) were used to perform the coccidial challenge by 15× of its vaccinal dose on the 14th day of age. Diclazuril and ZnO-NPs supplementation in Group 3 and 4, respectively, reduced the mortality rate due to coccidial challenge to 5.8% compared to 11.9% in Group 2. The growth performance was improved by ZnO-NPs in coccidiosis-infected group (p ≤ 0.05) compared to Group 2 and was comparable to that of Group 3 (p ≥ 0.05). The average oocyst count was lower in Groups 3 and 4 (7.8 × 103 and 14.3 × 103, respectively) than in Group 2 (67 × 103 oocysts). Group 3 had a decreased gross lesion score in duodenum and caecum (p ≤ 0.05) as well as jujenum and ileum (p ≥ 0.05) compared to Group 2; while the average lesion scores of all intestinal parts in Group 4 were significantly decreased (p ≤ 0.05). However, diclazuril was superior to ZnO-NPs in reducing caecal lesion score (p ≤ 0.05). Plasma carotenoids levels were increased by diclazuril (p ≥ 0.05) and ZnO-NPs (p ≤ 0.05) supplementation compared to Group 2. Oxidative stress appeared on the fourth week post-challenge (pc) in Group 2 (p ≤ 0.05) compared to Group 1, while the dietary supplementation with either diclazuril or ZnO-NPs numerically decreased Malondialdhyde (p ≥ 0.05) and statistically increased antioxidant activity (p ≤ 0.05). Both medications significantly improved the PCV%, Hb% and RBCs count on the 6th-day and 4th-week pc (p ≤ 0.05) compared to Group 2, though this improvement was higher significantly in Group 4 than Group 3 on the 6th day pc (p ≤ 0.05). Neither coccidial challenge nor medications had an impact on the total WBCs count as well as organ index, except Bursa of fabricious index that significantly improved by ZnO-NPs on the 4th-week pc compared to Group 2. Coccidial challenge reduced total protein and globulin levels and increased the serum alanine aminotransferase, serum cholesterol, and low-density lipoprotein levels (p ≤ 0.05) compared to Group 1, while those of both medicated groups (Group 3 and 4) were comparable to Group 1 (p ≥ 0.05). In conclusion, ZnO-NPs were found to be as effective as diclazuril against coccidiosis. However, further research is needed to fully comprehend its anticoccidial mechanisms.

Interactions of diclazuril enantiomers with serum albumins: Multi-spectroscopic and molecular docking approaches.

In this work, multi-spectroscopic and molecular docking methods have been conducted in the investigation of enantioselective interactions between diclazuril enantiomers and human/bovine serum albumins (HSA/BSA). The binding constants between serum albumins (SAs) and diclazuril enantiomers revealed that SAs exhibited stronger binding affinity for (R)-diclazuril than (S)-enantiomer. In addition, the fluorescence quenching of SAs induced by diclazuril enantiomers was ascribed to static quenching mechanism, in which hydrogen bonds and Van der Waals forces were the main interactions. According to the thermodynamic study, binding of diclazuril enantiomers and SAs was an exothermic process driven by enthalpy change. Then, circular dichroism spectroscopy of SAs with diclazuril enantiomers revealed that the SAs conformation had changed in the presence of diclazuril. Moreover, molecular docking technology was applied in exploration of interactions between SAs and diclazuril enantiomers. The docking energy between SAs and (R)-diclazuril was larger than (S)-diclazuril, which indicated that the affinity of SAs with (R)-diclazuril was stronger than (S)-enantiomer. This work may provide valuable information for explaining differences in pharmacokinetics and residue elimination of diclazuril enantiomers in living organisms.

Immunomodulatory and ameliorative effects of probiotic in combination with diclazuril on broilers under coccidia infection.

AIM: This study aimed to determine the potential prophylactic efficacy of probiotic individually and/or in combination with anti-coccidial drug on the performance and immunity of broilers under an induced coccidial infection over a 28-day of experimental trial. METHODS: One hundred and eighty 1-day-old Cobb broiler chicks were randomly divided into five groups, included control group (CG), control positive group (CPG), probiotic-treated group (Prob), diclazuril-treated group (Dic), and probiotic + diclazuril-treated group (Prob + Dic). On day 21 of age, all birds, except group CG, were orally inoculated with 1 ml of tap water containing 25,000 Eimeria tenella sporulated oocysts. RESULTS: Our results showed that the probiotic treatment did not influence pre-challenge body weight, feed intake and feed conversion ratio (FCR). During the post-challenge period, chickens in groups probiotic and diclazuril individually and in combination exhibited higher body weight and lower (better) FCR, reduced oocyst shedding (throughout the day four, five, six and seven post-infection), cecal lesions and mortality compared with control positive chickens. Moreover, Compared to CPG group, Prob + Dic group showed increased (p < 0.05) serum levels of interleukin-10 (IL-10) and immunoglobulin M (IgM) and decreased the concentrations of interferon gamma (IFN-γ). On the other hand, individual treatment with probiotic exhibited highest serum levels of IL-10 and IgM, while diclazuril alone increased the blood concentrations of IL-10 and decreased the levels of IFN-γ compared to control positive group; however, there was no significant effect of Prob on IFN-γ, Dic on IgM and all groups on interleukin-17. CONCLUSION: In conclusion, supplementation of probiotic, with and/or without anti-coccidial drug, enhances immunity and inhibits the negative effects of Eimeria infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals the anti-coccidial mechanisms of probiotic in the presence and absence of anti-coccidial drug in preventing the coccidia infection.

Investigation of the Bi-Weekly Administration of Diclazuril on the Antibody Kinetics to Sarcocystis Neurona in Healthy Horses.

The aim of this study was to determine if bi-weekly administration of diclazuril at half the label dose would reduce seroprevalence and magnitude of titers to S. neurona in healthy horses naturally exposed to the apicomplexan protozoal parasite. 12 healthy adult horses were moved from a low-risk exposure to a farm with high exposure rate to S. neurona in their horse population. The horses were randomly assigned to either a treatment or a control group. Treatment consisted in the administration of half the label dose (0.5 mg/kg) of diclazuril (Protazil) pelleted top dress twice weekly (every 3-4 days) for 12 months. Prior to initiation of treatment and monthly thereafter, blood was collected for the detection of antibodies to S. neurona using a quantitative immunoassay. Further, trough plasma diclazuril levels were determined every 60 days. All 20 horses remained healthy during the entire study period. Seroprevalence to S. neurona decreased initially in the treatment group to 50% at 30 days post-treatment commencement. This was followed by a slow increase in seroprevalence in the treatment group before reaching 100% in both groups by 90 days post-treatment commencement. The seroprevalence remained 100% in both groups from 90 to 360 study days. While titer distribution between the two groups was similar at study commencement, treated horses had significantly lower titers throughout the treatment period (P < 0.05). All treated study horses had detectable plasma trough diclazuril levels at the 6 time points and the levels were above the concentration known to inhibit S. neurona in vitro (1.0 ng/mL). The administration of diclazuril pelleted top dress at half the label dose twice weekly was able to maintain low titers to S. neurona in healthy adult horses naturally exposed to the protozoal parasite. Further, trough diclazuril levels were in excess of the minimal concentration known to inhibit S. neurona.

Diclazuril Inhibits Biofilm Formation and Hemolysis of Staphylococcus aureus.

Biofilm formation and hemolysis induced by Staphylococcus aureus are closely related to pathogenicity. However, no drugs exist to inhibit biofilm formation or hemolysis induced by S. aureus in clinical practice. This study found diclazuril had antibacterial action against S. aureus with minimum inhibitory concentrations (MICs) at 50 µM for both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Diclazuril (at 1/4× or 1/8× MICs) significantly inhibited biofilm formation of S. aureus under static or flow-based conditions and also inhibited hemolysis induced by S. aureus. The RNA levels of transcriptional regulatory genes (agrA, agrC, luxS, sarA, sigB, saeR, saeS), biofilm formation-related genes (aur, bap, ccpA, cidA, clfA, clfB, fnbA, fnbB, icaA, icaB, sasG), and virulence-related genes (hla, hlb, hld, hlg, lukDE, lukpvl-S, spa, sbi, alpha-3 PSM, beta PSM, coa) of S. aureus were decreased when treated by diclazuril (at 1/4× MIC) for 4 h. The diclazuril nonsensitive clones of S. aureus were selected in vitro by induction of wildtype strains for about 90 days under the pressure of diclazuril. Mutations in the possible target genes of diclazuril against S. aureus were detected by whole-genome sequencing. This study indicated that there were three amino acid mutations in the diclazuril nonsensitive clone of S. aureus, two of which were located in genes with known function (SMC-Scp complex subunit ScpB and glyceraldehyde-3-phosphate dehydrogenase 1, respectively) and one in a gene with unknown function (hypothetical protein). Diclazuril showed a strong inhibition effect on planktonic cells and biofilm formation of S. aureus with the overexpression of the scpB gene.