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.

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.

Comparing the effect of phytobiotic, coccidiostat, toltrazuril, and vaccine on the prevention and treatment of coccidiosis in broilers.

This study compared 2 herbal anticoccidiosis drugs (water-soluble and feed-additive drugs) with monensin coccidiostat, toltrazuril (TTZ, anticoccidiosis drug), and Livacox Q (anticoccidiosis vaccine) in terms of their effects on the prevention and treatment of coccidiosis in broilers. In this study, 280 Ross 308 broiler chickens (a mix of both genders) were used in a completely randomized design with 7 treatments and 5 replications each including 8 chickens per replicate. On d 21 of rearing, all experimental groups, except for the negative control group (NC), were challenged with a mixed suspension of common strains of Eimeria, and the intended indices were assessed, including performance indices, number of oocysts per gram (OPG) of feces, intestinal injuries, and the total number of intestinal bacteria. In addition, the NC and the group receiving the monensin had greater body weight gain (BWG) (P < 0.05). At the end of week 6, the monensin group had the highest feed intake (FI), while the water soluble medicine treatment resulted in the lowest feed intake (P < 0.05). Regarding the lesion scores on day 28, the highest and lowest rates of jejunal injuries were observed in the positive control group (PC), the monensin and vaccine group respectively. The rate of oocysts excretion (oocysts per gram of feces = OPG) on different days was higher in the PC group, and the use of monensin could further reduce excretion compared to the other groups (P > 0.05). Based on a comparison of the population of lactic acid bacteria between the NC and both medicinal plant treated groups, the use of these products could increase the population of these types of bacteria. Moreover, the population of Escherichia coli was less considerable in the NC and herbal powder groups (P < 0.05). Overall, similar to commercial medicines, the herbal medicines used in this project can be effective in the prevention and treatment of coccidiosis and can improve profitability in broiler rearing centers by improving intestinal health.

A solid-liquid microextraction approach from a spot in an adsorbent layer of a chromatographic plate.

A new approach to extracting substances from a spot on a chromatographic plate for subsequent liquid chromatography-mass spectrometry analysis is described. This method involves extraction in a solid phase (an adsorbent layer of a chromatographic plate) - a liquid system using a simple device. For a single extraction of six selected coccidiostats from the adsorbent layer on the chromatographic plate with silica gel, 50 µL of methanol was used for 5 min. The data from the extraction experiments and liquid chromatography-mass spectrometry measurements demonstrated a good correlation between the ratio of the peak areas of the coccidiostats to the internal standard and the concentration of the substances in the range of two orders of magnitude. The coefficients of determination for the mentioned correlations range from 0.962 to 0.999. Moreover, the repeatability and reproducibility, expressed as the percentage values of relative standard deviation, do not exceed 7.5 % for any of the coccidiostats.

Simultaneous determination of selected ionophoric coccidiostats and amino acids in feed premixes using high-performance liquid chromatography-ultraviolet detection method.

The combination of ionophoric coccidiostats and amino acids (AAs) is important in poultry feeding to enhance immunity and improve the growth and feed efficiency of birds suffering from coccidiosis. A simple, rapid, and economical high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method for the simultaneous determination of three ionophoric coccidiostats, namely salinomycin (SAL), maduramicin (MAD), and monensin (MON) in addition to three AAs; L-tryptophan (L-TRP), alpha-ketoleucin (KLEU), and L-valine (L-VAL) in feed premixes was developed and validated. Chromatographic separation was achieved in less than 12 min using a phenyl hexyl column with a mobile phase consisting of acetonitrile/methanol/water (25:20:55, v/v/v) adjusted to pH 3 using phosphoric acid. Isocratic elution was performed at a flow rate of 1 mL/min with UV detection at 210 nm. The method showed good linearity in the ranges 0.50-5.0 mg/mL for MON, 0.20-2.0 mg/mL for MAD and SAL, 10.0-100.0 µg/mL for L-TRP and KLEU, and 50.0-500.0 µg/mL for VAL. The developed method was successfully applied to determine the studied analytes in feed premixes with good recoveries and precision. The good validation criteria of the proposed method allow its utilization in quality control laboratories.

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.

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.

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.

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.

Pharmacokinetics of toltrazuril and its metabolite, toltrazuril sulfone, in suckling piglets following oral and intramuscular administrations.

Toltrazuril (TZR) is currently the only registered chemotherapeutic drug in the European Union for the treatment of Cystoisospora suis. This study investigated the comparative pharmacokinetics and tissue concentration-time profiles of TZR and its active metabolite, toltrazuril sulfone (TZR-SO2 ), after oral (per os, p.o.) and intramuscular (i.m.) administration to suckling piglets. Following a single administration of TZR orally at 50 mg/piglet or intramuscularly at 45 mg/piglet, higher concentrations of TZR and TZR-SO2 were observed in all three investigated tissues after p.o. administration. The mean TZR concentration in serum peaked at 14 µg/mL (34.03 h) and 5.36 µg/mL (120 h), while TZR-SO2 peaked at 14.12 µg/mL (246 h) and 9.92 µg/mL (330 h) after p.o. and i.m. administration, respectively. TZR was undetectable in the liver after p.o. administration (18 days) and in the jejunum (24 days) after i.m. injection, while TZR-SO2 was still detectable in all three tissues after 36 days regardless of administration routes. This study showed that p.o. formulation exhibited faster absorption and higher serum/tissue TZR/TZR-SO2 concentrations than i.m. formulation. Both formulations generated sufficient therapeutic concentrations in the serum and jejunum, and sustained enough time to protect against Cystoisospora suis infection in the piglets.

LC-MS/MS analysis of coccidiostats in meat supply chain safety.

The presence of coccidiostats in meat products represents an important topic because of the animal administration of these substances, authorized as feed additives for targeted species, in order to prevent and inhibit coccidiosis. Coccidiostats include both ionophores and synthetic molecules characterized by different chemical-physical properties such as polarity. Meat is a matrix characterized by many interfering compound groups, such as proteins, phospholipids, and fats. High-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) analysis allows the required selectivity and sensitivity for discriminating analytes and matrix interferences. For these reasons, an LC-MS/MS method for the analysis of coccidiostats in meat products was developed without SPE purification steps. The correct analyte quantification is allowed by matrix-matched calibration. The method validation was performed by the replicated analysis of spiked meat samples at two different concentration levels (limit of quantification-LOQ-and a 10 times LOQ) in order to evaluate method recovery and repeatability, plus spiked samples at higher concentrations up to 10,000 µg/kg. Moreover, the metrological approach was used for the calculation of method uncertainty. The application of the developed method to real samples evidenced the presence of some non-ionophores coccidiostats in the meat and liver of chicken and rabbit species. Although, the determined concentration was below the established MRLs, the monitoring of coccidiostats in the meat supply chain is confirmed as a good strategy in order to safeguard consumer health.

Therapeutic effect of a self-made herbal formula on a multi-drug resistant Eimeria tenella isolate infection in broiler chickens.

In-feed prophylactic chemotherapy is widely considered the mainstay of avian coccidiosis control, while serious drug resistance strictly restricts its application. Confronted with the urgent need for an alternative strategy, a traditional Chinese medicine formula (TCMF) was developed. Meanwhile, its potential to iron out complicated clinical coccidiosis was scrutinized in vivo with a field-isolated multi-drug resistant Eimeria tenella (E. tenella) isolate. Birds were inoculated with 5 × 104 sporulated oocysts and administrated with TCMF supplementation in water from 72 h post-infection to the end of the experiment, diclazuril (DIC) was set as a positive control. As a result, TCMF intervention reduced oocyst shedding, cecal lesion and mortality, and enhanced body weight gain. According to the above, anticoccidial index (ACI) was calculated and TCMF exerted a moderate anticoccidial activity. Besides, macroscopic, histopathological, and ultrastructural observations revealed the safeguarding effects of TCMF on E. tenella-induced cecal injury. Following, TCMF treatment presented an obvious inhibition effect on E. tenella caused oxidative stress and inflammatory response. Moreover, TCMF supplementation restored the cecal flora abundance and diversity, reduced the colonization of harmful bacteria, and increased the probiotics abundance. In conclusion, TCMF exhibited a moderate anticoccidial effect along with alleviating E. tenella-induced cecal injury, redox imbalance, and inflammatory response which may be associated with the microflora modulatory effect.

Analysis of authorized coccidiostats in chicken feces and environmental water by ultra-performance liquid chromatography-tandem mass spectrometry based on dispersive solid-phase extraction and lyophilization.

A simple, sensitive, and efficient method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of 8 coccidiostats in chicken feces and environmental water (including sewage, pond water, and lake water) surrounding the farm. Target analytes in chicken feces were extracted with 2% acetic acid in acetonitrile solution, followed by a dispersive solid-phase extraction (DSPE) cleanup step using the mixture of PSA and C18 adsorbents. Environmental water samples were pretreated using a lyophilization approach. Analysis was carried out on a UPLC-MS/MS with the combination of methanol and 0.1% formic acid aqueous solution as the mobile phase under multiple reaction monitoring in positive and negative ionization modes. Results showed that 8 coccidiostats were linear with correlation coefficients higher than 0.99. Method validation was performed using fortified samples, reaching satisfactory recoveries of 75.9%-97.8% in chicken feces and 71.9%-108.2% in environmental water. Limits of detection for 8 analytes in chicken feces and environmental water were 0.03∼2 µg/kg and 0.005∼1 µg/L, respectively. Matrix effects were calculated and strong signal suppression (>50%) for some coccidiostats was observed. The developed method was successfully applied to analyze coccidiostats in chicken feces and environmental water collected from local chicken farms.

Analysis of feed additives by DART mass spectrometry: method optimisation and applications for product traceability in the European Union focusing on coccidiostats and carotenoids.

In this study, direct analysis in real time high resolution mass spectrometry (DART-HRMS) was used to investigate the accurate characterisation of feed additive formulations containing coccidiostats or carotenoids. The study demonstrates the efficacy of DART-HRMS in identifying the active substances in these formulations and distinguishing between feed additives with the same active substance. The protocol for this method involves two simple steps that are extracting samples with organic solvents and measuring the extracts with DART-HRMS. The study also employs various statistical tools, including a factorial design approach, to optimise the DART-HRMS settings, and multivariate statistics, to establish classification models for feed additive formulations using nominal mass spectra. Our study demonstrates the potential of DART-HRMS in ensuring the correct identification of feed additives containing various coccidiostats or carotenoids and proposes this tool as an additional means for compliance checks with EU regulations.

A moderate anticoccidial effect of cedrol on Eimeria tenella in broiler chickens.

As drug-resistant strains of Eimeria have emerged and concerns about drug residues in poultry have grown, there is renewed interest in identifying natural alternatives to control coccidiosis. Cedrol, a natural sesquiterpene alcohol, was used in this study to test anticoccidial efficacy in chicks. Both the control and treatment groups were orally challenged with 2 × 104 oocysts per chicken. Chicks administered with cedrol had reduced oocyst count, an increase in the relative weight gain rate of chicks, and a decrease in severe swelling of the cecum. Based on the above, ACI was calculated and the cedrol group reached moderate anti-coccidial activity (169.34). In chickens treated with cedrol, there were no changes in serum biochemical parameters, but oxidative stress biomarkers and cytokine levels associated with anticoccidial response were altered. These changes suggest that the administered concentration of cedrol did not have any adverse effects on the chickens while enhancing their antioxidant capacity and immunity, leading to an improved anticoccidial ability. In conclusion, this study shows that the addition of cedrol in poultry production has an anticoccidial effect and successfully improves growth performance during the growth period.

Research Note: Isolation, speciation, and anticoccidial sensitivity of Eimeria spp. recovered from wild turkey feces in the United States.

Between 2018 and 2020, over 100 wild turkey fecal samples were collected from the Eastern and Central thirds of the United States, where commercial turkey production is uncommon. We hypothesized that anticoccidial-sensitive Eimeria spp. would be present in wild turkey fecal samples. Samples containing Eimeria spp. oocysts were amplified in vivo. If propagation was successful, the samples were PCR-speciated and subjected to anticoccidial sensitivity testing (AST) for key members of both ionophore and chemical categories of anticoccidial drugs. The purpose of this study was to isolate Eimeria spp. relevant to commercial turkey production that possessed sensitivity to monensin, zoalene, and amprolium. Future research would evaluate the efficacy of wild turkey Eimeria spp. as vaccine candidates for reducing coccidiosis in commercial turkey flocks utilizing single oocyst-derived stocks obtained in the present study.

The effect of early administration of antibiotics or feeding a diet containing a coccidiostat on inflammatory responses and the morphological structure of selected organs of the immune system in young meat-type turkeys.

It was assumed that early administration of enrofloxacin or doxycycline may impair immune function and alter the morphology of organs of the immune system in turkeys, and that diets containing the coccidiostat monensin, an ionophore antibiotic, can exert similar effects. The aim of this study was to determine whether early antibiotic administration or feeding a diet containing a coccidiostat affect immune function in young turkeys. The experiment had a completely randomized design, with 8 groups (a total of 3,080 one-day-old turkeys), 7 replicate pens per group and 55 birds per pen. The experiment had a 2-factorial design, with 4 treatments (C-control, M-monensin, E-enrofloxacin, and D-doxycycline) and 2 groups of birds (vaccinated and unvaccinated) per treatment. Control group birds did not receive the coccidiostat or antibiotics. Group M was administered monensin at 90 mg/kg feed for the first 5 d of life, group E received enrofloxacin at 10 mg/kg BW, added to drinking water, for the first 5 d of life, and group D received doxycycline at 50 mg/kg BW, added to drinking water, for the first 5 d of life. One-day old turkeys from groups C+, M+, E+, and D+ were administered live-attenuated vaccines against turkey rhinotracheitis (TRT) (Poulvac TRT; Zoetis, Parsippany, NJ) and Newcastle disease (ND) (Nobilis ND clone 30; Merck, Rahway, NJ) by coarse spray; 28-day-old birds were administered a subcutaneously injected inactivated vaccine against Ornithobacterium rhinotracheale (ORT) (Ornitin, Phibro, Poland). Turkeys from groups C-, M-, E-, and D- were not vaccinated. It was found that early administration of enrofloxacin or doxycycline, or feeding a diet containing monensin, did not weaken the immune system of turkeys. The administration of monensin, in particular when combined with vaccination, was least effective in inhibiting inflammatory responses. Histological changes in immunocompetent organs (fatty degeneration) were also most severe in birds receiving monensin, followed by those administered doxycycline and enrofloxacin. The observed changes were exacerbated by vaccination.