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Background Chicken coccidiosis is an intracellular parasitic disease that presents major challenges to the development of the commercial poultry industry. Perennial drug selective pressure has led to the multi-drug resistance of chicken coccidia, which makes the prevention and control of chicken coccidiosis extremely difficult. In recent years, natural plant products have attracted the attention of researchers due to their inherent advantages, such as the absence of veterinary drug residues. The development of these natural products provides a new direction for the prevention and treatment of chicken coccidiosis. Methods The anticoccidial effect of a natural plant product combination formulation (eucalyptus oil + apigenin + eugenol essential oil) was tested against Eimeria tenella in broilers. To search for the optimal concentration of the combination formulation, we screened 120 broilers in a chicken cage trial in which 100 broilers were infected with 5 × 10 4 sporulated Eimeria tenella oocysts; broilers receiving a decoquinate solution was set up as a chemical control. The optimal anticoccidial concentration was determined by calculating the anticoccidial index (ACI), and the suitable concentration was used as the recommended dose for a series of safety dose assessment tests, such as feed conversion ratio (FCR), hematological indices and serum biochemical indices, as well as liver and kidney sections, at onefold (low dose), threefold (medium dose) and sixfold (high dose) the recommended dose (RD). Results The results showed that this combination formulation of three plant natural products had a better anticoccidial effect than formulations containing two plant natural products or a single one, with an ACI of 169.3. The dose gradient anticoccidial test revealed that the high-dose formulation group had a better anticoccidial effect (ACI = 169.2) than the medium- and low-dose groups. The safety evaluation test showed that concentrations of the formulation at one-, three- and sixfold the RD were non-toxic to Arbor Acres broilers, indicating the high safety of the combination formulation. Conclusions The combination formulation showed not only a moderate anticoccidial effect but also had a high safety profile for broilers. The results of this study indicate a new alternative for the prevention and control of coccidiosis in broilers. Graphical Abstract

Inorganic arsenic (iAs) causes cancer and possibly other adverse health outcomes. Arsenic-based drugs are permitted in poultry production; however, the contribution of chicken consumption to iAs intake is unknown. We sought to characterize the arsenic species profile in chicken meat and estimate bladder and lung cancer risk associated with consuming chicken produced with arsenic-based drugs. Conventional, antibiotic-free, and organic chicken samples were collected from grocery stores in 10 U.S. metropolitan areas from December 2010 through June 2011. We tested 116 raw and 142 cooked chicken samples for total arsenic, and we determined arsenic species in 65 raw and 78 cooked samples that contained total arsenic at ≥ 10 µg/kg dry weight. The geometric mean (GM) of total arsenic in cooked chicken meat samples was 3.0 µg/kg (95% CI: 2.5, 3.6). Among the 78 cooked samples that were speciated, iAs concentrations were higher in conventional samples (GM = 1.8 µg/kg; 95% CI: 1.4, 2.3) than in antibiotic-free (GM = 0.7 µg/kg; 95% CI: 0.5, 1.0) or organic (GM = 0.6 µg/kg; 95% CI: 0.5, 0.8) samples. Roxarsone was detected in 20 of 40 conventional samples, 1 of 13 antibiotic-free samples, and none of the 25 organic samples. iAs concentrations in roxarsone-positive samples (GM = 2.3 µg/kg; 95% CI: 1.7, 3.1) were significantly higher than those in roxarsone-negative samples (GM = 0.8 µg/kg; 95% CI: 0.7, 1.0). Cooking increased iAs and decreased roxarsone concentrations. We estimated that consumers of conventional chicken would ingest an additional 0.11 µg/day iAs (in an 82-g serving) compared with consumers of organic chicken. Assuming lifetime exposure and a proposed cancer slope factor of 25.7 per milligram per kilogram of body weight per day, this increase in arsenic exposure could result in 3.7 additional lifetime bladder and lung cancer cases per 100,000 exposed persons. Conventional chicken meat had higher iAs concentrations than did conventional antibiotic-free and organic chicken meat samples. Cessation of arsenical drug use could reduce exposure and the burden of arsenic-related disease in chicken consumers.

Background Coccidiosis represents a serious threat to the poultry industry, affecting production and causing high morbidity, mortality and significant costs resulting from treatment and prophylaxis. In-feed anticoccidials have been used for decades for managing avian coccidiosis and were very effective until drug resistance emerged. The use of natural remedies has become a promising alternative in combating coccidiosis in chickens. Therefore, the purpose of the present study was to assess the efficiency of a commercial herbal formula (H), as oral liquid preparations, in experimental chicken coccidiosis. Methods Two independent controlled battery experiments (BE1 and BE2) were designed and the product was tested in 3 different formulas (H1, H2 and H3): H1 contained a propylene glycol extract of Allium sativum and Thymus serpyllum ; H2 contained Origanum vulgare , Satureja hortensis and Chelidonium majus ; and H3 contained Allium sativum , Urtica dioica , Inula helenium , Glycyrrhiza glabra , Rosmarinus officinalis , Chelidonium majus , Thymus serpyllum , Tanacetum vulgare and Coriandrum sativum . Chickens were divided into five groups for each BE as follows: (i) uninfected untreated control (UU1, UU2); (ii) infected untreated control (IU1, IU2); (iii) infected treated with amprolium (ITA1, ITA2); and (iv, v) two experimental groups infected treated with H1 (ITH1) and H2 (ITH2) formulas in the BE1 and with H3 (ITH3-5 and ITH3-10) formula in the BE2. The chickens from infected groups were challenged with 5000 (BE1) and 50,000 (BE2) sporulated oocysts of Eimeria spp. ( E. acervulina , E. tenella and E. maxima ), respectively. The anticoccidial efficacy was assessed by recording the following: oocysts output (OPG), lesion score (LS), weight gain (WG), feed conversion ratio (FCR) and anticoccidial index (ACI). Additionally, polyphenolics and flavonoids (caffeic-chlorogenic acid, apigenin, kaempferol, luteolin, quercitin, quercitrin) from herb extracts found in H3 formula were determined by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Results H1 and H2 reduced the WG, and increased the FCR and OPG compared with controls. H1 reduced the duodenal lesions, whilst H2 reduced the caecal lesions, compared with control. H3 decreased the OPG of Eimeria spp., reduced the total lesion score and improved the zootechnical performance (weight gain and feed conversion ratio). According to ACI value, H1 and H2 had no efficacy on Eimeria spp. infection, but H3 had good to marked anticoccidial effect, the ACI being slightly greater in the group ITH3-5. According to the results of LC-MS/MS, the concentration of polyphenols in H3 formula was the highest, the sum of chlorogenic acid and caffeic acid being 914.9 µg/ml. Conclusions H3 formula is a promising natural anticoccidial and field trials are recommended in order to validate the obtained data.

Background Coccidia are among the primary pathogens causing diarrhea and even fatalities in lambs. With the increasing use of chemical drugs to treat coccidiosis, the problem of drug resistance is becoming more and more threatening. Therefore, there is an urgent need to identify novel alternative drugs for the treatment of the lamb coccidia. In this study, the effect of different doses and extraction methods of Artemisia annua ( A. annua ) on anticoccidial activity and growth performance was assessed by oocysts output (OPG), fecal index, average daily gain (ADG) and the new production value of experimental lambs. High-throughput sequencing technology was employed to investigate the effect of A. annua on the intestinal microbiota and metabolites of lambs afflicted with coccidiosis. Results The results revealed that all A. annua treatment groups exhibited good anticoccidial effects. According to the soft stool index and ADG analysis, the Low-dose A. annua (AL) and A. annua alcohol extract (AA) groups demonstrated a better overall effect. The microbiota and metabolites of lambs changed after A. annua was administered. Unclassified_Muribaculaceae exhibited a significant positive correlation with ADG ( P  < 0.05) and a negative correlation with OPG, although the latter was not statistically significant ( P  > 0.05). Alistipes displayed a significant negative correlation with ADG ( P  < 0.05), and a positive correlation with OPG ( P  > 0.05). Additionally, UCG 005 exhibited a highly significant negative correlation with OPG ( P  < 0.01). Conclusion The above results demonstrated that AL and AA groups had more effective anticoccidial action. Unclassified_Muribaculaceae could be employed as a suitable probiotic to enhance weight gain in lambs, while UCG-005 could inhibit intestinal Eimeria colonization in lambs. Alistipes may serve as a biomarker for predicting the risk of intestinal coccidia outbreaks in lambs. A. annua induced significant changes in gut microbiota, accompanied by corresponding changes in metabolites. These differences in gut microbiota and metabolites provide valuable insights for subsequent research on the mechanisms underlying anticoccidial action.

The effects of a compound including the secondary metabolites of garlic, propyl thiosulphinate (PTS) and propyl thiosulphinate oxide (PTSO), on the in vitro and in vivo parameters of chicken gut immunity during experimental Eimeria acervulina infection were evaluated. In in vitro assays, the compound comprised of PTSO (67 %) and PTS (33 %) dose-dependently killed invasive E. acervulina sporozoites and stimulated higher spleen cell proliferation. Broiler chickens continuously fed from hatch with PTSO/PTS compound-supplemented diet and orally challenged with live E. acervulina oocysts had increased body weight gain, decreased faecal oocyst excretion and greater E. acervulina profilin antibody responses, compared with chickens fed a non-supplemented diet. Differential gene expression by microarray hybridisation identified 1227 transcripts whose levels were significantly altered in the intestinal lymphocytes of PTSO/PTS-fed birds compared with non-supplemented controls (552 up-regulated, 675 down-regulated). Biological pathway analysis identified the altered transcripts as belonging to the categories ‘Disease and Disorder’ and ‘Physiological System Development and Function’. In the former category, the most significant function identified was ‘Inflammatory Response’, while the most significant function in the latter category was ‘Cardiovascular System Development and Function’. This new information documents the immunologic and genomic changes that occur in chickens following PTSO/PTS dietary supplementation, which are relevant to protective immunity during avian coccidiosis.

Intestinal coccidiosis is a common parasitic disease in livestock, caused by the infection of Eimeria and Cystoisospora parasites, which results in great economic losses to animal husbandry. Triazine compounds, such as toltrazuril and diclazuril, are widely used in the treatment and chemoprophylaxis of coccidiosis. Unfortunately, widespread drug resistance has compromised their effectiveness. Most studies have focused on prophylaxis and therapeutics with toltrazuril in flocks, while a comprehensive understanding of how toltrazuril treatment alters the transcriptome of E. tenella remains unknown. In this study, merozoites of E. tenella were treated in vitro with 0.5 μg/mL toltrazuril for 0, 1, 2 and 4 h, respectively. The gene transcription profiles were then compared by high-throughput sequencing. Our results showed that protein hydrolysis genes were significantly upregulated after drug treatment, while cell cycle-related genes were significantly downregulated, suggesting that toltrazuril may affect parasite division. The expression of redox-related genes was upregulated and elevated levels of ROS and autophagosomes were detected in the parasite after toltrazuril treatment, suggesting that toltrazuril may cause oxidative stress to parasite cells and lead to its autophagy. Our results provide basic knowledge of the response of Eimeria genes to toltrazuril and further analysis of the identified transcriptional changes can provide useful information for a better understanding of the mechanism of action of toltrazuril against Eimeria.

A sensitive electrochemical (voltammetric; DPV) sensor has been developed for the determination of coccidiostat drug (roxarsone) based on the use of an SPCE (screen-printed carbon electrode) modified with tungsten disulfide nanosheets (WS 2 NSs). The electrochemical detection of roxarsone on the WS 2 -modified SPCE was examined by electrochemical strategies. XPS, XRD, Raman, SEM, TEM, EDS and EIS were used to characterize the nanosheets. The effects of scan rate, pH values (phosphate buffer) and buffer concentration were optimized. A selective roxarsone sensor was developed that works best at -0.64 V (vs. Ag/AgCl) and performs much better than the bare SPCE. Features include (a) a wider linear range (0.05 to 490 μM), (b) a nanomolar detection limit (0.03 μM) and (c) high sensitivity (29 μA·μM -1 ·cm -2 ). The modified SPCEs have been successfully applied to the determination of roxarsone in spiked meat samples where they gave high accuracy and good recoveries. Graphical abstract Synthesis of WS 2 nanosheets and electrochemical detection of roxarsone.

Background Coccidiosis remains a major challenge in poultry farming due to the drug-resistance phenomena in Eimeria strains and the possible risk of accumulation of anticoccidial residues in poultry-derived products. Essential oils (EOs) and their bioactive constituents are being considered for their potential role as alternative control strategies. The present study evaluated the in vitro efficacy of thymol, carvacrol, cinnamic aldehyde, eugenol, and a commercial EO blend (namely Energy Poultry, EP) against Eimeria spp., as well as the in vivo efficacy of thymol and EP (supplemented at 5 mg/kg and 50 mg/kg of feed, respectively) in growing chickens. Results In vitro assays demonstrated that thymol and carvacrol significantly disrupted oocyst integrity and inhibited sporulation at concentrations ≥ 2%, with maximal degeneration rates of 96% and 90%, respectively, at 10%. Cinnamic aldehyde reduced sporulation by 79%, whereas eugenol showed minimal efficacy. The potential synergistic effect between carvacrol and cinnamic aldehyde of EP resulted in a marked reduction in oocyst viability (~ 90%). Based on these findings, thymol and EP were selected for in vivo evaluation in a native Italian egg-type chicken breed, the Black Livorno. Despite not significant, dietary supplementation (thymol: 5 mg/kg; EP: 50 mg/kg) led to a reduction in oocyst per gram (OPG) values from day 78 onward compared to the control group in which the highest oocyst excretion persisting for an extended period. Growth performance (average daily gain and feed conversion ratio) was not affected, indicating no adverse effects of thymol and EP supplementation. Despite the presence of highly pathogenic Eimeria species ( E. tenella , E. necatrix ), no clinical signs were observed, probably due to the possible low parasitic burden and breed’s inherent resistance. Conclusions The obtained findings suggest that while evaluated EO constituents exhibited promising anticoccidial effects in vitro, their in vivo efficacy may be dose-dependent, influenced by infection pressure and host factors. Further research is warranted to optimise tested EOs inclusion levels and assess their long-term impact on coccidiosis control in poultry production systems.

There are concerns about residues of drugs in meat that are used to prevent coccidiosis in chickens. Natural compounds are an alternative to drugs. Two studies investigated the effect of an extract of Enterolobium cyclocarpum fruits (EEC) in the feed of male Leghorn chickens exposed to Eimeria spp. In the first experiment, the administration of EEC after infection with Eimeria spp. was investigated over 16 days. One thousand chickens were randomly housed in 20 pens of 1 m2 each. The pens were randomly assigned to each treatment. Five treatments were administered, containing 150, 300 and 450 mg/kg of EEC in the feed, the fourth treatment (C) contained 0.5 g/kg of a commercial anticoccidial, and the fifth treatment provided no treatment (WA). The second experiment lasted 18 days. Administration of the EEC began five days before the chickens were infected with Eimeria spp. Four hundred and eighty chickens were randomly allocated to 24 pens of 1 m2. The pens were randomly assigned to each treatment. In the second experiment, the same five treatments were tested and one additional treatment containing 300 mg EEC plus 1 g of polyethylene glycol (PEG)/kg of feed (E300PEG). In the experiment one chickens in the EEC treatments had lower faecal oocyst excretion (OE) on day 14 post infection with Eimeria spp., than chickens in the WA treatment (P < 0.05). A reduction in live weight gain (LWG) was observed in the EEC treatments (P < 0.05). In the second experiment, the excretion of oocysts in chickens from the EEC and E300PEG treatments on day 13 post-infection with Eimeria spp. was the same as in the C treatment and lower than in the WA treatment (P < 0.05). LWG was lower in the EEC treatments than in the C treatment (P < 0.05). However, the Chickens in the E300PEG and C treatments had similar LWG (P > 0.05) suggesting that PEG inhibits the negative effect of EEC tannins on LWG. In conclusion, the addition of EEC to chicken feed reduced both OE and LWG. Treatment with EEC and PEG (E300PEG) reduced the excretion of oocysts without negative effects on LWG.