Beagle dog 90-day oral toxicity study of a novel coccidiostat - ethanamizuril.

BACKGROUND: Triazine coccidiostats are widely used in chickens and turkeys for coccidiosis control. Ethanamizuril is a novel triazine compound that exhibits anticoccidial activity in poultry. This study was designed to evaluate the subchronic toxicity of ethanamizuril in beagle dogs at doses of 12, 60 or 300 mg/kg/day in diet for 90 days. RESULTS: Ethanamizuril was well tolerated at low and middle dosages in beagle dogs, and no drug-related toxical effects were observaed in terms of survival, clinical observations, organs weight and damage in these dose groups. However, in high dose administration group, food consumption and histologic changes in kidneys were noticed in both sexes of beagle dog, although the renal lesions were finally resolved at the end of 4 weeks exposure of ethanamizuril. CONCLUSIONS: No-observed-adverse-effect level (NOAEL) was considered for ethanamizuril at dose of 60 mg/kg/day in Beagle dog. This result added toxicity effects of ethanamizuril to the safety database, which might guide safely using of ethanamizuril as a novel coccidiostat.

Silver nanoparticles as a therapeutic agent in experimental cyclosporiasis.

Cyclosporiasis is an emerging worldwide infection caused by an obligate intracellular protozoan parasite, Cyclospora cayetanensis. In immunocompetent patients, it is mainly manifested by self-limited diarrhea, which is persistent and may be fatal in immunocompromised patients. The standard treatment for cyclosporiasis is a combination of two antibiotics, trimethoprim and sulfamethoxazole. Gastrointestinal, haematologic and renal side effects were reported with this combination. Moreover, sulfa allergy, foetal anomalies and recurrence were recorded with no alternative drug treatment option. In this study, silver nanoparticles were chemically synthesized to be evaluated for the first time for their anti-cyclospora effects in both immunocompetent and immunosuppressed experimental mice in comparison to the standard treatment. The effect of silver nanoparticles was assessed through studying stool oocyst load, oocyst viability, ultrastructural changes in oocysts, and estimation of serum gamma interferon. Toxic effect of the therapeutic agents was evaluated by measuring liver enzymes, urea and creatinine in mouse sera. Results showed that silver nanoparticles had promising anti-cyclospora potentials. The animals that received these nanoparticles showed a statistically significant decrease in the oocyst burden and number of viable oocysts in stool and a statistically significant increase in serum gamma interferon in comparison to the corresponding group receiving the standard treatment and to the infected non-treated control group. Scanning electron microscopic examination revealed mutilated oocysts with irregularities, poring and perforations. Biochemical results showed no evidence of toxicity of silver nanoparticles, as the sera of the mice showed a statistically non-significant decrease in liver enzymes in immunocompetent subgroups, and a statistically significant decrease in immunosuppressed subgroups. Furthermore, a statistically non-significant decrease in urea and creatinine was recorded in all subgroups. Thus, silver nanoparticles proved their effectiveness against Cyclospora infection, and this will draw the attention to its use as an alternative to the standard therapy.

Maduramicin induces apoptosis through ROS-PP5-JNK pathway in skeletal myoblast cells and muscle tissue.

Our previous work has shown that maduramicin, an effective coccidiostat used in the poultry production, executed its toxicity by inducing apoptosis of skeletal myoblasts. However, the underlying mechanism is not well understood. Here we show that maduramicin induced apoptosis of skeletal muscle cells by activating c-Jun N-terminal kinase (JNK) pathway in murine C2C12 and L6 myoblasts as well as skeletal muscle tissue. This is supported by the findings that inhibition of JNK with SP600125 or ectopic expression of dominant negative c-Jun attenuated maduramicin-induced apoptosis in C2C12 cells. Furthermore, we found that treatment with maduramicin reduced the cellular protein level of protein phosphatase 5 (PP5). Overexpression of PP5 substantially mitigated maduramicin-activated JNK and apoptosis. Moreover, we noticed that treatment with maduramicin elevated intracellular reactive oxygen species (ROS) level. Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, suppressed maduramicin-induced inhibition of PP5 and activation of JNK as well as apoptosis. The results indicate that maduramicin induction of ROS inhibits PP5, which results in activation of JNK cascade, leading to apoptosis of skeletal muscle cells. Our finding suggests that manipulation of ROS-PP5-JNK pathway may be a potential approach to prevent maduramicin-induced apoptotic cell death in skeletal muscle.

Development and optimization of confirmatory liquid chromatography-Orbitrap mass spectrometry method for the determination of 17 anticoccidials in poultry and eggs.

A new sensitive and selective multi-residue method based on liquid chromatography - Orbitrap high resolution mass spectrometry (LC-Orbitrap-HRMS) was developed and validated for the determination of 17 anticoccidials in poultry and eggs. Instrumental parameters were optimized by the means of statistical experimental designs to improve the sensitivity, precision, and repeatability of the method. Further optimization of auto-tuned MS parameters led to an increase of signal intensity by 10% to 99% for 16 out of 17 analytes. The sample preparation procedure included extraction from muscle tissue and egg samples with acetonitrile, followed by preconcentration, reconstitution, and filtration. Validation was performed according to the Commission Decision 2002/657/EC. The occurrence of anticoccidials in eggs and poultry was assessed by using the developed analytical procedure within the Latvian national monitoring program, revealing quantifiable residues for 6 analytes (marker residue of nicarbazin - 4,4'-dinitrocarbanilide (DNC), salinomycin, narasin, toltrazuril, and its two metabolites).

Safety evaluation of a triazine compound nitromezuril by assessing bacterial reverse mutation, sperm abnormalities, micronucleus and chromosomal aberration.

Nitromezuril (NZL) is a novel triazine compound that exhibits remarkable anticoccidial activity. However, mutagenicity and genotoxicity of NZL have not been evaluated to date. This study evaluated the potential risks of NZL by testing for bacterial reverse mutation (Ames), mouse sperm abnormality (SA), bone marrow micronucleus (MN) and chromosomal aberration (CA). Mice were orally administered with NZL at 385, 192 and 96 mg/kg, corresponding to 0.5 ×, 0.25 × and 0.125 × the LD50 of NZL, respectively. No significant increases in SA and CA were found in mice treated with NZL for 5d and 3d, respectively (P>0.05). NZL at 96-385 mg/kg did not have significant influence on micronucleated polychromatic erythrocyte counts (P>0.05). These results suggest that NZL is not genotoxic. However, Ames test results were positive both with and without the S9 system for Salmonella typhimurium TA98 and TA100, suggesting that NZL may be mutagenic. The mutagenic effects of NZL were different in in vitro and in vivo assays. Further studies should be conducted to confirm the safety of using and developing NZL as a novel anticoccidial drug.

Degradation of roxarsone in a silt loam soil and its toxicity assessment.

The land application of poultry or swine litter, containing large amounts of roxarsone, causes serious arsenic pollution in soil. Understanding biotransformation process of roxarsone and its potential risks favors proper disposal of roxarsone-contaminated animal litter, yet remains not achieved. We report an experimental study of biotransformation process of roxarsone in a silt loam soil under various soil moisture and temperature conditions, and the toxicity of roxarsone and its products from degradation. Results showed that soil moisture and higher temperature promoted roxarsone degradation, associating with emergent pentavalent arsenic. Analysis of fluorescein diacetate (FDA) hydrolysis activity revealed that roxarsone does not exert acute toxic on soil microbes. With the release of inorganic arsenic, FDA hydrolysis activity was inhibited gradually, as evidenced by ecotoxicological assessment using Photobacterium leiognathi. The results shade new lights on the dynamic roxarsone biotransformation processes in soil, which is important for guiding appropriate disposal of poultry or swine litter in the environment.

Anticoccidial effects of Morinda lucida acetone extracts on broiler chickens naturally infected with Eimeria species.

CONTEXT: The development of drug resistance to Eimeria species in poultry is a reality on farms. This necessitates chemotherapeutic control alternatives, and plant secondary metabolities with activity is one of those potential solutions. OBJECTIVE: This study was designed to evaluate the efficacy of acetone leaf extract of Morinda lucida Benth. (Rubiaceae) against coccidiosis in broiler chickens. MATERIALS AND METHODS: Acute toxicity of the plant was investigated using 30 one-day-old broiler chicks. Anticoccidial activity investigations were carried out in birds drenched with 2.0, 3.0, 4.0, 5.0 and 6.0 g/kg body weight (BW) of acetone extract of M. lucida leaves once a day for five days, with toltrazuril® and untreated controls. The activity was evaluated by means of oocyst inhibition in feces, fecal score, weight gain, mortality and hematological parameters. RESULTS: No sign of toxicity was observed during the acute toxicity test. Fecal oocyst counts decreased steadily in all the treatment groups with time. The anticoocidial efficacy of birds treated with 2.0, 3.0, 4.0, 5.0 and 6.0 g/kg BW of the extract was 91.2, 95.2, 98.7, 99 and 99.5%, respectively. The packed cell volumes, red blood cell counts and white blood cell of the treated birds were not significantly different (p > 0.05) from the untreated control. However, haemoglobin concentration and weight gain of the treated birds were significantly different (p < 0.05) from the untreated control. CONCLUSION: This study is the first to demonstrate the anticoccidial effect of M. lucida on Eimeria parasites. Morinda lucida leaves could therefore find application in anticoccidial therapy.

Pyrimethamine-loaded lipid-core nanocapsules to improve drug efficacy for the treatment of toxoplasmosis.

We propose an innovative product based on the nanoencapsulation of pyrimethamine (PYR), aiming an improvement of drug efficacy for the treatment of toxoplasmosis. The in vitro cytotoxicity effect of encapsulated PYR and PYR-colloidal suspension was concomitantly evaluated against LLC-MK2 lineage and mouse peritoneal macrophage showing that the cells had similar tolerance for both PYR encapsulated or in the aqueous suspension. CF1 mice acutely infected with tachyzoites of Toxoplasma gondii RH strain treated with different doses (5.0-10 mg/kg/day) of PYR-nanocapsules had survival rate higher than the animals treated with the same doses of non-encapsulated PYR. Thus, encapsulation of PYR improved the efficacy of this drug against an acute model of toxoplasmosis in mice and can be considered an alternative for reducing the dose of PYR, which, in turn, could also reduce the side effects associated to the treatment.

Evaluation of glutathione s-transferase as toxicity indicator for roxarsone and arsanilic acid in Eisenia fetida.

Different compounds can induce stress response by targeting specific genes. Studies related to elucidating the detoxification and adaptive responses of proteins like glutathione-s-transferase (GST) can be helpful in better understanding toxicity. Roxarsone and arsanilic acid, which have been exhaustively used as animal and poultry feed additives, pose a threat to the environment and human health. GST enzyme bioassay revealed fluctuations in response to different concentrations of roxarsone and arsanilic acid at different time intervals. The highest GST enzyme activity (40.51%) was observed on day 15 of treatment with roxarsone. On the other hand, arsanilic acid caused the maximum enzyme activity (52.11%) on day 10 of treatment. During this study, the full-length gene sequence of GST, having the size 984 bp (Genbankno. HQ693699), was achieved from Eisenia fetida and established as a biomarker to assess the toxicity of roxarsone and arsanilic acid. The deduced protein has a computed molecular mass of 23.56 kDa and a predicted isoelectric point of 9.92. Quantitative real-time PCR revealed significant differential gene expression in response to roxarsone and arsanilic acid treatment as compared with control treatment. Roxarsone caused the highest gene expression of 7.0-fold increase over control on day 15 of treatment, whereas arsanilic acid resulted in the highest gene expression reaching to 14.56-fold as compared with control. This study is helpful in understanding the role of GST as a potential biomarker for chemicals like roxarsone and arsanilic acid, which can pollute the food chain.

Assessing the genotoxic potentials of roxarsone in V79 cells using the alkaline Comet assay and micronucleus test.

Until recently, knowledge about the genotoxicity of roxarsone in vitro or in vivo was limited. This study assessed the genotoxicity of roxarsone in an in vitro system. Roxarsone was tested for potential genotoxicity on V79 cells by a Comet assay and a micronucleus (MN) test, exposing the cells to roxarsone (1-500 µM) and to sodium arsenite (NaAsO2, 20 µM) solutions for 3-48 h. Roxarsone was found to be cytotoxic when assessed with a commercial cell counting kit (CCK-8) used to evaluate cell viability, and moderately genotoxic in the Comet assay and micronucleus test used to assess DNA damage. The Comet metrics (percentages TDNA, TL, TM) increased significantly in a time- and concentration-dependent manner in roxarsone-treated samples compared with PBS controls (P<0.05), while the data from samples treated with 20 µM NaAsO2 were comparable to those from 500 µM roxarsone-treated samples. The MN frequency of V79 cells treated with roxarsone was higher than that in the negative control but lower than the frequency in cells treated with 20 µM NaAsO2. A dose- and time-dependent response in MN induction was observed at 10, 50, 100 and 500 µM doses of roxarsone after 12-48 h exposure time. The DNA damage in V79 cells treated with 500 µM roxarsone was similar to cells exposed to 20 µM NaAsO2. The uptake of cells was correlated with the DNA damage caused by roxarsone. This investigation depicts the genotoxic potentials of roxarsone to V79 cells, which could lead to further advanced studies on the genotoxicity of roxarsone.

Ionophore toxicity and tolerance.

The rapid growth of the biofuels industry in the Midwest in the past 10 years has created an increased supply of corn coproduct feed for animals. This article discusses the tolerance and toxicology of biofuels coproducts in ruminants, including polioencephalomalacia, sulfur toxicosis, sulfur metabolism, mycotoxins, antibiotic residue, and biodiesel by-product toxicosis.

Does monensin in chicken manure from poultry farms pose a threat to soil invertebrates?

Monensin is a carboxylic polyether ionophore used in the poultry industry as a coccidiostat. It enters the environment via manure from broiler farms. In spite of its potential presence in the environment, information concerning monensin residues in manure and soil and its toxicity to soil organisms are insufficient. In the present study, two beneficial soil invertebrate species, earthworms (Eisenia andrei) and woodlice (Porcellio scaber), were used to assess the toxicity of monensin. Animals were exposed to a range of monensin concentrations via soil or food. Earthworm reproduction was found to be the most susceptible endpoint (NOEC=3.5 mg kg(-1) dry soil; EC(50)=12.7 mg kg(-1) dry soil), while no adverse effects were recorded in isopods (NOEC⩾849mgkg(-1) dry soil, NOEC⩾357mgkg(-1) dry food). The obtained toxicity data were compared with potential concentrations of monensin in soil. In view of this, manure from broiler chickens treated with monensin at a poultry farm was sampled. According to monensin and nitrogen concentrations in the chicken manure and the degradation time of monensin, the predicted environmental concentration (PEC) was calculated. PEC of monensin is around 0.013 mg kg(-1) soil if manure is used after 3 months of composting and 0.05 mg kg(-1) soil if used without storage. Data for earthworm reproduction was used to estimate the predicted no-effect concentration (PNEC). If fresh chicken manure is applied to terrestrial ecosystems, the risk quotient (PEC/PNEC ratio) is above 1, which indicates that monensin might pose an environmental risk under certain conditions. To prevent this, it is strongly recommended to compost chicken manure for several months before using it as fertiliser.

Lasalocid awareness and sampling in Scotland.

Lasalocid is an ionophore antibiotic extensively used as a coccidiostat in poultry production. Lasalocid should not be fed to egg-laying hens as it accumulates in the eggs, and residues have often been found in eggs. Other ionophores are toxic to humans, but the exact level of lasalocid toxicity to humans has not been established. Approximately 250 egg samples were analysed for lasalocid each year from the 10 billion eggs consumed annually in the UK. A census of the 32 Scottish Local Authority Environmental Health Departments assessed awareness of lasalocid residues in eggs, and the results indicated that awareness of lasalocid was very low and no local authorities tested for lasalocid. The example of lasalocid revealed weaknesses in the current sampling regime surrounding foods of animal origin. Conclusions are drawn that central government should raise awareness within local authorities and provide financial support on local authority sampling to achieve proper representation.

Intoxicação por antibióticos ionóforos em animais:[revisão] / Ionophore poisoning in animals: [review]

O uso terapêutico de antibióticos ionóforos em medicina veterinária difundiu-se muito nos últimos anos, com conseqüente aumento no risco de intoxicação em animais. Antibióticos ionóforos são usados como coccidiostáticos e como aditivo em alimentos para animais, com o propósito de estimular o desenvolvimento e o ganho de peso. Os ionóforos mais utilizados na alimentação de animais são a monensina, lasalocida, nasarina e salinomicina. Há uma grande variação na susceptibilidade dos efeitos tóxicos dos ionóforos de acordo com a espécie animal. A intoxicação pode ocorrer quando dosagens elevadas de ionóforos são adicionadas aos alimentos, ou quando ionóforos são incluídos inadvertidamente ou acidentalmente em dosagens não corretas para determinada espécie animal. Casos de intoxicação têm sido descritos em bovinos, ovinos, suínos, eqüinos, cães e aves. Para os eqüinos os ionóforos são extremamente tóxicos. São considerados seguros quando usados nas espécies-alvo, dentro das dosagens recomendadas pelo fabricante.

The therapeutic use of ionophores in veterinary medicine has grown in the last years, with resultant increase in the risk of poisoning in animals. Ionophores are used as food additives as coccidiostacts in several animal species and growth promoter and bloat prevention in ruminants. The most often used ionophores are monensin, lasalocid, narasin and salinomycin. There is a great variation in the susceptibility to the toxic effect of ionophores in different animal species. Poisoning can occur when the dosage is too high or when not correct doses for a certain animal species are given. Cases of poisoning have been described in sheep, swine, horses, dogs and poultry. For horses ionophores are extremely toxic. The use of ionophores is only safe when used accordingly to the instructions of the manufacturer and especially for each animal species. In this paper the most important data regarding clinical-pathological and pathogenic aspects, and also the conditions in which the poisoning may occur are critically reviewed.

A case of salinomycin intoxication in turkeys.

A flock of 4287 heavy hybrid turkey hens were accidentally fed broiler premix containing salinomycin sodium and suffered a 34.5% death loss. Measures taken to ensure food safety for the remaining flock and consumer food safety included feed record studies, on-farm veterinary consultation, diagnostic laboratory studies, and CgFARAD and CFIA consultation. The remaining turkeys were processed 3 weeks after the initial toxicosis with no evidence of lesions that would render the product unfit for human consumption.

Assessment of feed additives and contaminants: an essential component of food safety.

Feed additives make the bulk of chemicals used in animal production, thus representing a major issue for safety of foods of animal origin. This paper summarizes the approaches adopted by the European Food Safety Authority to perform risk analysis of feed additives as regards the whole food production chain, including target species, consumers, occupational exposure and the environment. Feed safety must consider also environmental contaminants; in particular feeds can be a major vehicle for human dietary intake of persistent pollutants such as polychlorinated biphenyls. Critical issues include toxicological characterization, pathways of feed contamination as well as transfer to animal products. The possible effects of feed additives and contaminants on the overall safety and nutritional quality of human diet are discussed.

Inhibition of lipid peroxidation by IRFI 042, a vitamin E analogue, decreases monensin cardiotoxicity in chicks.

Monensin, a well-known ionophore antibiotic, may cause severe damage in myocardial cells. We investigated whether IRFI 042, a new analogue of vitamin E, may block lipid peroxidation in myocardial cells and in turn protect against monensin toxicity. Monensin toxicity was induced by repeated daily administration of the ionophore antibiotic (150 mg/kg/day for 7 days). Sham animals received by oral gavages only a saline solution and were used as controls. All animals were randomized to receive concomitantly by oral gavages IRFI 042 (20 mg/kg) or its vehicle. The experiment lasted 8 days. Survival rate, heart lipid peroxidation, studied by means of thiobarbituric acid-reactive substances (TBARs) levels, cardiac expression of endothelial nitric oxide (e-NOS) and histological analysis of the heart were performed. Monensin administration caused a decrease in survival rate. Mortality appeared following the second monensin injection and at day 7 caused a survival rate of 20%. Thereafter, no further mortality was observed. IRFI 042 administration improved survival rate. Injection of the ionophore antibiotic resulted in a marked cardiac lipid peroxidation and in a significant reduction in cardiac e-NOS message and protein expression. IRFI 042 decreased heart TBARs levels (Monensin + vehicle = 6.5 +/- 0.8 nmol/mg; Monensin + IRFI 042 = 3.2 +/- 1.1 nmol/mg; P < 0.001) and increased e-NOS message and protein expression. Histological analysis showed that IRFI 042 improved myocardial cells damage and enhanced the depressed e-NOS expression in chick heart samples following monensin administration. Our data suggest that IRFI 042 is a promising drug to reduce monensin cardio-toxicity in chicks.

Evaluation of zinc against salinomycin toxicity in broilers.

Salinomycin was studied for its toxicity and zinc (80 mg/kg) was assessed for prophylactic and therapeutic management in broiler chicks. Male broiler chicks were randomly divided into 7 groups consisting of 6 chicks in each. Group 1, 2 and 3 were maintained as control, therapeutic dose control (60 mg/kg feed) and toxic dose control (120 mg/kg feed), respectively. Group 4 was fed on feed containing salinomycin therapeutic dose and zinc. Group 5 received feed containing toxic dose of salinomycin. Group 6 and 7 were fed on feed containing toxic dose of salinomycin for the first 4 weeks for induction of ionophore toxicity and for the subsequent 2 weeks, group 6 received zinc and group 7 was fed on feed containing toxic dose of salinomycin along with zinc. Weekly body weights revealed a significant (P<0.01) decrease in toxic controls as compared to group 1, 2, 4 and 5. The activity of glutathione peroxidase, glutathione reductase and catalase, and the values of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total proteins, total cholesterol, triglycerides, low density lipoproteins (LDL), urea, creatinine and blood urea nitrogen (BUN) were significantly (P<0.01) elevated in toxic controls, whereas glutathione (GSH) and high density lipoproteins (HDL) were significantly (P<0.01) lowered as compared to group 1, 2, 4 and 5. Following toxicity, zinc supplementation in group 6 and 7, all serobiochemical parameters were revived to normal. Thus, it is enunciated that salinomycin toxicity is due to oxidative damage and use of zinc in feed tends to cure and avoid any accidental toxicity.

Effects of monensin feeding during development on female rats and their offspring.

Monensin, a growth-promoting agent and coccidiostat used in veterinary medicine, was studied in terms of its effects on the development of female rats and their offspring when added to the diet. Young female rats received 100 or 300 ppm monensin mixed with powdered chow diet until adulthood, when they were mated and their offspring were evaluated for physical and neurobehavioural development. The data showed that 1) exposure to the higher monensin concentration reduced female body weight; no deaths occurred after exposure to the two monensin concentrations; 2) the offspring of the experimental group receiving 300 ppm monensin presented a weight reduction from 10 to 21 days of lactation; 3) incisor eruption was delayed only in females after exposure to the 100 ppm concentration. We conclude that exposure to monensin during development induces toxicity in female rats and has a notable adverse effect on growth with some limited effects on selective milestones of physical and functional development of the offspring during the postnatal period.

Amprolium-induced cerebrocortical necrosis (CCN) in dromedary racing camels.

Amprolium was successfully used to induce cerebrocortical necrosis (CCN) in dromedary racing camels, only when they were fed on a barley diet. Camels which were fed on hay ad libitum did not suffer form CCN, although their thiamine pyrophosphate (TPP) reached similar levels as in camels fed on barley. The reason for this phenomenon is discussed. Five camels which suffered from CCN had TPP values of 80-115% and were euthanized on humane grounds when they were in lateral recumbency. Pathohistological investigations revealed a polioencephalomalacia of the dorsal cerebral cortex with oedema and status spongiosus. Cerebral autofluorescence was observed under ultraviolet light. The major clinicopathological changes were a slight anemia and a decreased potassium value whereas glucose, muscle enzymes, leucocyte counts and differential counts were elevated. A TPP effect of 12% was found during this study in healthy dromedary racing camels and symptoms were observed when TPP values reached 80-115%. The test is now being widely used during the camel racing season.