US20140161919A1 - Plant Parts and Extracts Having Anticoccidial Activity - Google Patents

Plant Parts and Extracts Having Anticoccidial Activity Download PDF

Info

Publication number
US20140161919A1
US20140161919A1 US13/928,504 US201313928504A US2014161919A1 US 20140161919 A1 US20140161919 A1 US 20140161919A1 US 201313928504 A US201313928504 A US 201313928504A US 2014161919 A1 US2014161919 A1 US 2014161919A1
Authority
US
United States
Prior art keywords
infectoria
coccidiosis
extracts
gallic acid
eimeria
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/928,504
Other languages
English (en)
Inventor
Gokila Thangavel
Rajalekshmi Mukkalil
Haridasan Chirakkal
Hannah Kurian
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kemin Industries Inc
Original Assignee
Kemin Industries Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kemin Industries Inc filed Critical Kemin Industries Inc
Priority to US13/928,504 priority Critical patent/US20140161919A1/en
Assigned to BANK OF AMERICA, N.A., AS COLLATERAL AGENT reassignment BANK OF AMERICA, N.A., AS COLLATERAL AGENT IP SUPPLEMENT (PATENTS) Assignors: KEMIN FOODS, L.C., KEMIN HOLDINGS, L.C., KEMIN INDUSTRIES, INC.
Assigned to KEMIN INDUSTRIES, INC. reassignment KEMIN INDUSTRIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MAST, ILSE, SCHOETERS, ELKE, WOUTERS, HILDE, CHIRAKKAL, HARIDASAN, KURIAN, Hannah, MUKKALIL, Rajalekshmi, THANGAVEL, Gokila
Publication of US20140161919A1 publication Critical patent/US20140161919A1/en
Priority to US14/675,180 priority patent/US10568923B2/en
Priority to US14/802,545 priority patent/US10426808B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/49—Fagaceae (Beech family), e.g. oak or chestnut
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/22—Anacardiaceae (Sumac family), e.g. smoketree, sumac or poison oak
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy

Definitions

  • the present invention relates generally to the control of coccidiosis and, more specifically, to the application of plant parts, plant extracts and compounds to control coccidiosis in poultry and other animals.
  • Coccidiosis is a major disease in the poultry industry and according to a recent survey, it is estimated that the global impact is greater than $3 billion USD annually (worldpoultry.net/Broilers/Health/2009/9/In-ovo-vaccination-against-coccidiosis-WP006949W/—accessed Jun. 18, 2013).
  • Coccidiosis is caused by a protozoan parasite, namely Eimeria, belonging to the phylum Apicomplexa, and the family Eimeriidae (Clare, R. A and Danforth, H. D (1989).
  • tenella are known to cause coccidiosis in poultry (Williams, 2005) and the species are highly host and site specific.
  • E. tenella is one of the major species causing coccidiosis in poultry, and their site of infection is the caecum (Khazandi, M and Tivey, D (2010). Developing an in vitro method for Eimeria tenella attachment to its preferred and non-preferred intestinal sites. Experimental Parasitology, 125 (2), 137-140). Coccidiosis is currently controlled by medication, but the increasing emergence of drug-resistant strains of Eimeria requires the development of an alternative control strategy. Since plants are known to possess antiparasitic and anticoccidial activity due to the presence of phenolic compounds (Tipu, M.
  • McCann et al. tested the effect of Sweet Chestnut Wood tannins on the performance of broiler chicks vaccinated with a live coccidia vaccine (M. E. E. McCann, E. Newell, C. Preston and K. Forbes. The Use of Mannan-Oligosaccharides and/or Tannin in Broiler Diets. Intl. J. of Poultry Sci. 5 (9): 873-879, 2006). They reported that supplementation with mannan-oligosaccharides or tannins, either individually or in combination, did not reduce the impact of the coccidiosis.
  • Wang et al. teach the use of a grape seed proanthocyanidin extract on coccidiosis (Wang, et al. Influence of Grape Seed Proanthocyanidin Extract in Broiler Chickens: Effect on Chicken Coccidiosis and Antioxidant Status. Poultry Science. 87:2273-2280, 2008). They attributed activity to the anti-inflammatory and antioxidant properties of the proanthocyanidins, a condensed tannin rather than a hydrolysable tannin.
  • Naidoo et al. teach an in vivo study using four plants selected based on their antioxidant activity (Naidoo et al. The value of plant extracts with antioxidant activity in attenuating coccidiosis in broiler chickens. Veterinary Parasitology. 153:214-219; 2008). They observed that one of the plants ( Tulbaghia violacea ) reduced the Eimeria oocyst counts in the chicken excreta and they speculate that this effect could be due to the antioxidant compound S (methylthiomethyl) cysteine sulfoxide.
  • McDougald et al. describe the use of a muscadine pomace to enhance resistance to coccidiosis in broiler chickens (McDougald et al. Enhancement of Resistance to Coccidiosis and Necrotic Enteritis in Broiler Chickens by Dietary Muscadine Pomace. Avian Diseases. 52: 646-651; 2008).
  • Muscadine pomace is a by-product of grapes used in wine production. They make no mention of efficacy of any specific compounds in the pomace.
  • the proposed anti-coccidial activity differs significantly from the activities proposed by Wang et al. and Naidoo et al.
  • the present invention consists of the identification and use of plant parts and plant extracts effective in the control of coccidiosis in animals, particularly in poultry.
  • plant parts and natural extracts of Quercus infectoria, Rhus chinensis gall nut, Terminalia chebula fruit have been found to control coccidiosis in poultry and, more specifically, coccidiosis caused by Eimeria spp.
  • the plant parts and natural extracts of gall nuts of Quercus infectoria, Rhus chinensis and fruits of Terminalia chebula result in a reduction of lesion score, oocysts per gram of fecal matter and mortality.
  • the plant parts/extract was also found to have a direct inhibitory effect on the sporozoites of Eimeria, as observed in the in vitro MTT assay.
  • Compounds selected gallic acid, gallotannins and hydrolys able tannins were also found to reduce lesion score, oocysts per gram of fecal matter and mortality.
  • the compounds were also found to have a direct inhibitory effect on the sporozoites of Eimeria, as observed in the in vitro MTT assay.
  • the present invention also consists of a method of controlling coccidiosis in poultry and other animals by administering a composition comprising plant parts or extracts of plants containing an efficacious amount of gall nuts of Quercus infectoria, Rhus chinensis, Terminalia chebula fruit and/or compounds such as gallic acid, gallotannins and hydrolysable tannins.
  • FIG. 1 are microphotographic images of caecal lesions of control birds, birds treated with the negative control, birds treated with the positive control, and birds treated with Quercus infectoria.
  • FIG. 2 is a chart of the oocysts per gram (OPG) of excreta of birds treated with Quercus infectoria on day 7 post infection; columns with different superscripts are statistically significant (p ⁇ 0.05).
  • OPG oocysts per gram
  • FIG. 3 are microphotographic images of the H and E stained sections of caecum of infected control birds and birds treated with Quercus infectoria.
  • FIG. 4 is a chart of the lesion score for E. acervulina, E. maxima and E. tenella for the birds treated with Q. infectoria water extract on day 5 post infection; columns with different superscripts are statistically significant (p ⁇ 0.05).
  • FIG. 5 is a chart of the oocysts per gram (OPG) of excreta of birds treated with Q. infectoria water extract on day 7 post infection; columns with different superscripts are statistically significant (p ⁇ 0.05).
  • OPG oocysts per gram
  • FIG. 6 is a chart of the MTT assay carried out for the evaluation of Q. infectoria at various dosage levels along with a coccidiostat (Salinomycin) as positive control; columns with different superscripts are statistically significant (p ⁇ 0.05).
  • FIG. 7 is a chart of PCR results after invasion of MDBK host cells with sporozoites and different concentrations of Q. infectoria.
  • FIG. 8 is a chart of fold changes in Eimeria DNA for different time points versus T4 within one treatment.
  • FIG. 9 is a chart of PCR results after invasion of MDBK host cells with sporozoites, pre-treated with different concentrations of Q. infectoria.
  • FIG. 10 is a chart of fold changes in Eimeria DNA for different time points versus T20 within one treatment.
  • FIG. 11 is a chart of the anti-sporozoite activity of the different fractions of Q. infectoria; columns with different superscripts are statistically significant (p ⁇ 0.05).
  • FIG. 12 is a High Performance Liquid Chromatogram (HPLC) chromatogram of water fraction of Q. infectoria.
  • FIG. 13 is a chart of the anti-sporozoite activity of the four major peaks of Q. infectoria; columns with different superscripts are statistically significant (p ⁇ 0.05).
  • FIG. 14 is the LC/MS/MS chromatogram of peak 1 of Q. infectoria.
  • FIG. 15 is a chart depicting the correlation between the concentration of gallic acid and the anti-sporozoite activity of Q. infectoria; columns with different superscripts are statistically significant (p ⁇ 0.05).
  • FIG. 16 is a chart of PCR results of MDBK host cells, pre-treated with 10 ppm gallic acid, invaded with sporozoites.
  • FIG. 17 is a chart of fold changes in Eimeria DNA for different time points versus T20 within one treatment.
  • FIG. 18 is a chart of PCR results after invasion of MDBK host cells with sporozoites, pre-treated with different concentrations of gallic acid.
  • FIG. 19 is a chart of fold changes in Eimeria DNA for different time points versus T20 within one treatment.
  • FIG. 20 is a chart of the lesion score on day 5 post infection of birds treated with gallic acid at different concentrations; columns with different superscripts are statistically significant (p ⁇ 0.05).
  • FIG. 21 is a chart of the OPG on day 7 post infection of birds treated with gallic acid at different concentrations; columns with different superscripts are statistically significant (p ⁇ 0.05).
  • FIG. 22 is a chart of the anti-sporozoite activity of Rhus chinensis and a chart of the anti-sporozite activity of Terminalia chebula; columns with different superscripts are statistically significant (p ⁇ 0.05).
  • FIG. 23 is a chart of PCR results after invasion of MDBK host cells with sporozoites, pre-treated with different concentrations of T. chebula.
  • FIG. 24 is a chart of fold changes in Eimeria DNA for different time points versus T20 within one treatment.
  • FIG. 25 is a chart of the lesion score on day 5 post infection of birds treated with R. chinensis and T. chebula; columns with different superscripts are statistically significant (p ⁇ 0.05).
  • FIG. 26 is a chart of the OPG on day 7 post infection of birds treated with R. chinensis and T. chebula; columns with different superscripts are statistically significant (p ⁇ 0.05).
  • a plant part, extract or compounds is termed to be efficacious if it can result in statistically significant reduction in the lesion score, the oocysts shed in the excreta, (Oocyst Per Gram (OPG)) or the mortality of the birds as compared to the infected control which is untreated.
  • OPG Olecyst Per Gram
  • administration of gallic acid and gallic acid containing formulations are described with formulations providing a dosage from 0.1 to 50 ppm, preferably from 2 to 20 ppm, and most preferably from 3 to 10 ppm through feed or water or an equivalent supplementation through other routes.
  • the plants, plant parts and/or extracts described contain around a mininimum of 0.1% of gallic acid.
  • infectoria in controlling coccidiosis caused by other species of Eimeria also.
  • an in vitro method based on 3-(4, 5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay was developed to evaluate the anti-sporozoite activity of plant extracts as a measure of the viability of the sporozoites.
  • MTT 3-(4, 5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
  • an in vitro assay was developed based on a co-culture of host cells and Eimeria parasites. Cells and parasite are combined in an assay with a positive control and different test products. The invasion and proliferation of the Eimeria parasites is measured by detecting Eimeria DNA using real-time PCR. For this, specific primers were selected and PCR conditions were optimised. The positive control and potential anticoccidial compounds are added to the in vitro assay in three different ways:
  • the products are combined with Eimeria sporozoites and added to the host cells.
  • the products are added to the sporozoites for a specific time, then removed and afterwards the sporozoites are added to the host cells.
  • the products are added to the host cells for a specific time, then removed and afterwards the sporozoites are added to the host cells.
  • Farm management Good farm managing practices were followed during the trial. The entire farm and the equipment used for the study were cleaned and disinfected before the arrival of the chicks. The birds were housed in cages organized on concrete flooring and a tray was provided at the bottom of the cages to facilitate collection of fecal samples. The temperature and humidity of the farm was monitored continuously.
  • NDV Newcastle Disease Virus
  • IBD Infectious Bursal Disease
  • Feed formulation A corn soya based mash diet was formulated. The feed ingredients were procured from Ponni feeds, Tamil Nadu, India. The mash feed was fed ad libitum to the birds throughout the study period. Three feed formulations were prepared according to the phases of the life of the bird; Prestarter (Day 1-10), Starter (Day 11-20), and Finisher feed (Day 21-42). No antimicrobials and supplements were used in the feed formulation.
  • Control 1 uninfected Normal feed control
  • Control 2 Negative infected Coccidiosis induction + Normal feed Control
  • Control 3 Pursitive Control
  • Coccidiosis induction + Feed with Q. infectoria at 100 g/ton *Coxistac is a product from Pfizer containing Salinomycin at 12% concentration.
  • addition of Coxistac at the mentioned dose of 500 g/ton of feed will enable delivery of Salinomycin at 60 ppm levels in the feed which is the recommended preventive dose for broilers.
  • the dose in this experiment was double the recommended concentration.
  • Parameters analyzed were the indices of pathogenesis namely excreta appearance, mortality, lesion scoring of the caecum for coccidiosis, and oocysts per gram (OPG) of excreta. The methods are detailed below.
  • excreta Examination of excreta. The excreta of the birds were monitored daily from the day 1 post infection to day 10 for their consistency, presence of blood, mucus, undigested feed, and orange color. Scoring of the excreta was carried out based on the severity of blood shedding.
  • OPG of excreta Triplicate samples of the excreta of the birds were collected randomly from the tray kept below the cages and the oocyst per gram was evaluated.
  • Lesion scoring of the caecum Lesion scoring of the caeca on day 5 and 7 post infection indicated that the lesions were severe on day 5, and the birds started recovering on day 7 post infection which was indicated by the formation of a caecal plug. This followed the normal pattern of infection enabling the removal of oocysts from the caeca.
  • the results of the lesion score showed that the positive control (Salinomycin control did not show any difference in the score as compared to the negative control due to inexplicable reasons.
  • the treatment with Q. infectoria reduced the lesion score as compared to the negative control (Table 4). The reduced lesion score correlated with reduced excreta score and absence of mortality.
  • OPG of excreta of the birds on day 7 post infection The counts of OPG of excreta of the birds on day 7 post infection are shown in Table 5.
  • the anticoccidial Salinomycin treated birds C3, Table 5
  • the values presented are an average of three replicates.
  • a 35 day in vivo challenge trial was conducted in broiler birds challenged with Eimeria tenella
  • the treatment groups included, 1) control, uninfected normal birds; 2) negative control, birds infected with E. tenella and fed normal diet without any anticoccidial compounds; 3) positive control, birds infected and fed diet containing Coxistac (anticoccidial agent, Salinomycin) at the recommended dose of 500 g/ton and 4) treatment group including infected birds administered diet containing Q. infectoria gall nut at 500 g/ton dose. No mortality was observed in the positive control group and treatment group supplemented with crude powder of gall nuts of Quercus infectoria.
  • the second in vivo experiment involved the following treatment groups.
  • Oocyst counts in excreta The OPG of excreta was estimated on day 7 post infection to evaluate the shedding of oocysts. The results of the study showed that the positive control, Q. infectoria had significantly lower counts of oocysts in the excreta as compared to the infected negative control (p ⁇ 0.05). Q. infectoria treatment was equally effective as the positive control ( FIG. 2 ). This correlates with the results of the lesion score.
  • Quercus infectoria is a potent candidate in controlling caecal coccidiosis in broiler birds caused by E. tenella.
  • the efficacy of the extract was found to be on par with that of the positive control in terms of reducing lesion score, OPG and rate of mortality.
  • Efficacy of Q. infectoria crude powder in controlling mixed infection of coccidiosis in broiler birds was evaluated.
  • a 35 day in vivo trial was conducted wherein the birds were challenged with field strains of mixed culture of oocysts of the species E. tenella, E. acervulina and E. maxima.
  • the mixed culture of oocysts was provided by Department of parasitology, Tamil Nadu Veterinary Research Institute, Namakkal, India.
  • the oocysts culture was a mixture of E. tenella, E. acervulina and E. maxima isolated from feces of birds with clinical coccidiosis infection.
  • Virulence of the oocysts obtained was evaluated in broiler birds and the dosage of the oocysts was finalized to be 5 ⁇ 10 5 based on the concentration that yields a lesion score of 3 and above for all the tested oocysts, E. tenella, E. maxima and E. acervulina.
  • NDV Newcastle Disease Virus
  • IBD Infectious Bursal Disease
  • Extracts of gall nut of Q. infectoria were prepared by mixing the crude powder (100-800 micron particle size) in distilled water at the ratio of 1:2, then extracting at 80 to 90° C. for one and half hour with agitation. The extract was filtered and again the residue was extracted in water in a similar manner. This was repeated for about 2 more times and the total liquid extract was freeze dried.
  • an in vitro method based on 3-(4, 5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay was developed to evaluate the anti-sporozoite activity of plant extracts as a measure of the viability of the sporozoites.
  • the optimized method included the preparation, sterilization and purification of sporozoites, followed by incubation of sporozoite suspension (minimum of 10 5 cells/ml) with required concentration of plant extract.
  • the plant samples were prepared by mixing crude powder into a known volume of distilled water to achieve the specific ppm, vortexed for 2 min and filtered through a 0.2 ⁇ syringe filter.
  • MTT-PMS solution (0.2 millimolar each) is incubated with the sporozoite suspension (at 1:10 ratio) for 2 h at 41° C. After incubation, the contents are centrifuged at 800 g for 5 min and the supernatant is carefully removed. The purple dye formazan is dissolved in 200 ul DMSO and the absorbance is measured at 530 nm against a reference wavelength of 630 nm.
  • MTT assay was carried out for the evaluation of Q. infectoria at various dosage levels along with Coccidiostac (Salinomycin) as positive control ( FIG. 6 ). There was a dose dependent reduction in the viability of sporozoites in the Q. infectoria treated samples as compared to the control.
  • Sporozoites were obtained from sporulated oocysts after glass bead grinding and enzymatic excystation.
  • As host cells Madin-Darby Bovine Kidney (MDBK) cells, were selected. Sporozoites and Quercus infectoria at 50 and 100 ppm were added to MDBK host cells for four hours. Afterwards, the medium was removed, cells were washed and fresh medium was added. After 4 (T4), 24 (T24), 48 (T48) and 72 (T72) hours the medium and MDBK cells were collected and stored at ⁇ 20° C.
  • MDBK Madin-Darby Bovine Kidney
  • the negative control was MDBK cells infected with Eimeria sporozoites, incubated in cell culture medium.
  • the positive control was MDBK cells infected with Eimeria sporozoites, incubated with a 5 ⁇ g/ml solution of Salinomycin.
  • DNA was extracted from the infected MDBK cells.
  • Real-time PCR to detect Eimeria DNA was performed on the samples for the different time points and different treatments.
  • the PCR results are presented in FIG. 7 .
  • the negative control shows a clear Eimeria proliferation since there is a 15 fold increase in Eimeria DNA at 72 hours versus the start at 4 hours.
  • the positive control was able to inhibit the proliferation completely.
  • Q. infectoria treatments a clear inhibition of the proliferation was observed versus the start of 4 hours, in a dose dependent manner.
  • Sporozoites were obtained from sporulated oocysts after glass bead grinding and enzymatic excystation.
  • As host cells Madin-Darby Bovine Kidney (MDBK) cells, were selected. Sporozoites were pre-treated with 50, 100 and 250 ppm of Quercus infectoria for three hours. Thereafter, the sporozoite suspension was washed and put onto a culture of MDBK cells for 20 hours. After incubation, the medium was removed, cells were washed and fresh medium was added. After 20, 72 and 96 hours the medium and MDBK cells were collected and stored at ⁇ 20° C.
  • MDBK Madin-Darby Bovine Kidney
  • the negative control was MDBK cells infected with Eimeria sporozoites, incubated in cell culture medium.
  • the positive control was MDBK cells infected with Eimeria sporozoites, incubated with a 5 ⁇ g/ml solution of Salinomycin.
  • DNA was extracted from the MDBK cells.
  • Real-time PCR to detect Eimeria tenella DNA was performed on the samples for the different time points and different treatments.
  • the PCR results are presented in FIG. 9 .
  • the negative control shows a clear Eimeria proliferation since there is a 20 fold increase in Eimeria DNA at 96 hours versus the start at 20 hours.
  • the positive control was able to inhibit the proliferation completely.
  • the different dosages of Q. infectoria all inhibited the Eimeria proliferation.
  • Bioassay Guided Fractionation assay (BGFA) of Q. infectoria gall nuts was carried out using the modified MTT reduction assay as the bioassay as we had identified that the crude extract possess anti-sporozoite activity and this could be one of the mode of action by which it is able to control coccidiosis.
  • Q. infectoria gall nut crude powder was fractionated using different solvent by column chromatography. The sample from each of the fractions was evaluated for their anti-sporozoite activity. Methanol and water fractions of Q. infectoria showed better reduction in the viability of sporozoites as compared to the other fractions and were comparable to the Salinomycin control ( FIG. 11 ).
  • Sporozoites were obtained from sporulated oocysts after glass bead grinding and enzymatic excystation.
  • As host cells Madin-Darby Bovine Kidney (MDBK) cells, were selected. MDBK cells were incubated with 10 ppm gallic acid for seven hours. Afterwards the medium was removed and a sporozoite suspension was added to the MDBK cells for 20 hours. After incubation, the medium was removed, cells were washed and fresh medium was added. After 20, 72 and 96 hours the medium and the MDBK cells were collected and stored at ⁇ 20° C.
  • MDBK Madin-Darby Bovine Kidney
  • the negative control was MDBK cells infected with Eimeria sporozoites, incubated in cell culture medium.
  • the positive control was MDBK cells infected with Eimeria sporozoites, incubated with a 5 ⁇ g/ml solution of Salinomycin.
  • DNA was extracted from the MDBK cells.
  • Real-time PCR to detect Eimeria DNA was performed on the samples for the different time points and different treatments.
  • the PCR results are presented in FIG. 16 .
  • the negative control shows a clear Eimeria proliferation since there is a 60 fold increase in Eimeria DNA at 96 hours versus the start at 20 hours.
  • the positive control was able to inhibit the proliferation almost completely.
  • gallic acid treatment a clear inhibition of the proliferation was observed in a dose dependent manner. This indicates that gallic acid at a low dose of 10 ppm is able to protect the host cells to some extend against Eimeria proliferation.
  • Sporozoites were obtained from sporulated oocysts after glass bead grinding and enzymatic excystation.
  • As host cells Madin-Darby Bovine Kidney (MDBK) cells, were selected. Sporozoites were pre-treated with 10, 25 and 50 ppm gallic acid monohydrate for three hours. Thereafter, the sporozoite suspension was washed and put onto a culture of MDBK cells for 20 hours. After incubation, the medium was removed, cells were washed and fresh medium was added. After 20, 72 and 96 hours the medium and MDBK cells were collected and stored at ⁇ 20° C.
  • MDBK Madin-Darby Bovine Kidney
  • the negative control was MDBK cells infected with Eimeria sporozoites, incubated in cell culture medium.
  • the positive control was MDBK cells infected with Eimeria sporozoites, incubated with a 5 ⁇ g/ml solution of Salinomycin.
  • DNA was extracted from the MDBK cells.
  • Real-time PCR to detect Eimeria DNA was performed on the samples for the different time points and different treatments.
  • the PCR results are presented in FIG. 18 .
  • the negative control shows a clear Eimeria proliferation since there is a 20 fold increase in Eimeria DNA at 96 hours versus the start at 20 hours.
  • the positive control as well as the different dosages of gallic acid inhibited the Eimeria proliferation. There was a slightly lower effect visible for 10 ppm gallic acid. But this is negligible in comparison to the increase in the negative control.
  • the efficacy of gallic acid at three different dosages of 11, 22 and 33 ppm in controlling coccidiosis in broiler birds was evaluated by an in vivo challenge trial.
  • the birds were induced with mixed infection of Eimeria using oocysts of E. tenella, E. maxima and E. acervulina. These oocysts were isolated from birds confirmed with clinical coccidiosis.
  • the trial design, oocysts dosage, vaccination schedule, farm maintenance were similar to that of example 3.
  • the lesion scoring showed that there was significant reduction in the score for all the three tested species of Eimeria as compared to the infected control and even the positive control, Salinomycin ( FIG. 20 ).
  • Rhus chinensis Choinese gall nut
  • Terminalia chebula Indian gall nut
  • Rhus chinensis contains about 70% hydrolysable tannins
  • Terminalia chebula contains around 0.28% free gallic acid.
  • T. chebula contains 25 to 40% hydrolysable tannins which can degrade to release gallic acid.
  • Sporozoites were obtained from sporulated oocysts after glass bead grinding and enzymatic excystation.
  • As host cells Madin-Darby Bovine Kidney (MDBK) cells, were selected. Sporozoites were pre-treated with 50, 100 and 250 ppm of Terminalia chebula for three hours. Thereafter, the sporozoite suspension was washed and put onto a culture of MDBK cells for 20 hours. After incubation, the medium was removed, cells were washed and fresh medium was added. After 20, 72 and 96 hours the medium and MDBK cells were collected and stored at ⁇ 20° C.
  • MDBK Madin-Darby Bovine Kidney
  • the negative control was MDBK cells infected with Eimeria sporozoites, incubated in cell culture medium.
  • the positive control was MDBK cells infected with Eimeria sporozoites, incubated with a 5 ⁇ g/ml solution of Salinomycin.
  • DNA was extracted from the MDBK cells.
  • Real-time PCR to detect Eimeria tenella DNA was performed on the samples for the different time points and different treatments.
  • the PCR results are presented in FIG. 23 .
  • the negative control shows a clear Eimeria proliferation since there is a 20 fold increase in Eimeria DNA at 96 hours versus the start at 20 hours.
  • the positive control as well as 250 ppm T. chebula completely inhibited the Eimeria proliferation. There was a dose-response effect visible although the lower effect for 100 ppm T. chebula is negligible in comparison to the increase in the negative control
  • the efficacy of plants containing gallic acid namely, Terminalia chebula and Rhus chinensis in controlling coccidiosis in broiler birds was evaluated by an in vivo challenge trial.
  • the birds were induced with mixed infection of Eimeria using oocysts of E. tenella, E. maxima and E. acervulina isolated from birds confirmed with clinical coccidiosis.
  • the trial design, oocysts dosage, vaccination schedule, farm maintenance were similar to that of example 3.
  • Rhus chinensis at 200 and 500 ppm and Terminalia chebula at 1000 ppm were able to reduce the score for all the three tested species of Eimeria as compared to the infected control and even the positive control, Salinomycin ( FIG. 25 ).
  • the oocysts per gram showed a similar trend ( FIG. 26 ), however, mortality was not observed in any of the treatment groups including the infected control. Dose dependent response was observed with Rhus chinensis.

Landscapes

  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Engineering & Computer Science (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
US13/928,504 2012-06-27 2013-06-27 Plant Parts and Extracts Having Anticoccidial Activity Abandoned US20140161919A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US13/928,504 US20140161919A1 (en) 2012-06-27 2013-06-27 Plant Parts and Extracts Having Anticoccidial Activity
US14/675,180 US10568923B2 (en) 2012-06-27 2015-03-31 Plant parts and extracts having anticoccidial activity
US14/802,545 US10426808B2 (en) 2012-06-27 2015-07-17 Plant extracts having anticoccidial activity

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201261664795P 2012-06-27 2012-06-27
IN177DE2013 2013-01-23
IN177/DEL/2013 2013-01-23
US13/928,504 US20140161919A1 (en) 2012-06-27 2013-06-27 Plant Parts and Extracts Having Anticoccidial Activity

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US14/675,180 Continuation-In-Part US10568923B2 (en) 2012-06-27 2015-03-31 Plant parts and extracts having anticoccidial activity
US14/802,545 Division US10426808B2 (en) 2012-06-27 2015-07-17 Plant extracts having anticoccidial activity

Publications (1)

Publication Number Publication Date
US20140161919A1 true US20140161919A1 (en) 2014-06-12

Family

ID=49784015

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/928,504 Abandoned US20140161919A1 (en) 2012-06-27 2013-06-27 Plant Parts and Extracts Having Anticoccidial Activity
US14/802,545 Active 2035-07-08 US10426808B2 (en) 2012-06-27 2015-07-17 Plant extracts having anticoccidial activity

Family Applications After (1)

Application Number Title Priority Date Filing Date
US14/802,545 Active 2035-07-08 US10426808B2 (en) 2012-06-27 2015-07-17 Plant extracts having anticoccidial activity

Country Status (9)

Country Link
US (2) US20140161919A1 (ja)
EP (1) EP2866794A4 (ja)
JP (1) JP6441794B2 (ja)
KR (1) KR102104921B1 (ja)
CN (1) CN104936583A (ja)
AU (1) AU2013280331C1 (ja)
BR (1) BR112014032632A2 (ja)
CA (1) CA2877884C (ja)
WO (1) WO2014004761A2 (ja)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017066777A1 (en) * 2015-10-15 2017-04-20 Kemin Industries, Inc. Compositions and methods of improving performance and intestinal health of poultry

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106107194A (zh) * 2016-06-28 2016-11-16 北京中农华正兽药有限责任公司 净化肉鸡小肠球虫病的组合物及使用方法
FR3078629B1 (fr) 2018-03-12 2020-07-31 Adisseo France Sas Additif alimentaire a base de saponines pour le traitement de la coccidiose
KR102408566B1 (ko) * 2020-04-09 2022-06-16 대한민국 터미날리아 비아라타 추출물을 이용한 피부 주름 개선용 조성물
KR102660905B1 (ko) * 2020-09-25 2024-04-24 씨제이제일제당 (주) 비올라세인을 포함하는 항콕시듐용 조성물 및 이의 용도

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101744839A (zh) * 2008-12-08 2010-06-23 杨建华 维药没食子及其提取物的抑制皮肤色素沉着和防晒美白作用

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0395294A3 (en) * 1989-04-25 1991-05-22 Takeda Chemical Industries, Ltd. Control of protozoal disease
CH688172A5 (fr) * 1994-10-26 1997-06-13 Crina Composition d'additif pour aliments destinés à la volaille.
SK14882002A3 (en) * 2000-04-27 2004-11-03 Pfizer Prod Inc The use of azalide antibiotic compositions for treating or preventing a bacterial or protozoal infection in mammals
WO2004052393A1 (en) * 2002-12-09 2004-06-24 University Of Georgia Research Foundation, Inc. Coccidial vaccine and methods of making and using same
DE102006038292A1 (de) 2006-08-16 2008-02-21 Bayer Healthcare Ag Transdermale Anwendung von Triazinen zur Bekämpfung von Coccidien-Infektionen
DE102007045724B4 (de) * 2007-09-24 2012-01-26 Evonik Stockhausen Gmbh Superabsorbierende Zusammensetzung mit Tanninen zur Geruchskontrolle, Verfahren zu deren Herstellung und Verwendung
CN102988764B (zh) * 2012-12-19 2014-07-23 西安市昌盛动物保健品有限公司 一种抗鸡球虫的中药制剂及其制备方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101744839A (zh) * 2008-12-08 2010-06-23 杨建华 维药没食子及其提取物的抑制皮肤色素沉着和防晒美白作用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
McDougald et al., Enhancement of Resistance to Coccidiosis and Necrotic Enteritis in BroilerChickens by Dietary Muscadine Pomace, 2008, Avian Diseases, 52: 646-651 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017066777A1 (en) * 2015-10-15 2017-04-20 Kemin Industries, Inc. Compositions and methods of improving performance and intestinal health of poultry

Also Published As

Publication number Publication date
US10426808B2 (en) 2019-10-01
EP2866794A4 (en) 2015-12-09
KR102104921B1 (ko) 2020-04-28
BR112014032632A2 (pt) 2017-06-27
AU2013280331A1 (en) 2015-01-22
JP2015522023A (ja) 2015-08-03
AU2013280331C1 (en) 2018-06-21
KR20150023899A (ko) 2015-03-05
CA2877884A1 (en) 2014-01-03
WO2014004761A2 (en) 2014-01-03
US20160015765A1 (en) 2016-01-21
CA2877884C (en) 2022-09-06
WO2014004761A3 (en) 2014-02-20
EP2866794A2 (en) 2015-05-06
CN104936583A (zh) 2015-09-23
AU2013280331B2 (en) 2017-12-14
JP6441794B2 (ja) 2018-12-19

Similar Documents

Publication Publication Date Title
US10426808B2 (en) Plant extracts having anticoccidial activity
Cejas et al. Evaluation of quebracho red wood (Schinopsis lorentzii) polyphenols vegetable extract for the reduction of coccidiosis in broiler chicks
Dragan et al. Effects of Artemisia annua and Pimpinella anisum on Eimeria tenella (Phylum Apicomplexa) low infection in chickens
Imran et al. Anticoccidial Efficacy of Citrus sinensis Essential Oil in Broiler Chicken.
Wang et al. Anticoccidial effects of areca nut (Areca catechu L.) extract on broiler chicks experimentally infected with Eimeria tenella
Acharya et al. Alternatives to fight against coccidiosis: A review
Adamu et al. Effect of Lepidium sativum L.(garden cress) seed and its extract on experimental Eimeria tenella infection in broiler chickens
Hayajneh et al. Anticoccidial effect of apple cider vinegar on broiler chicken: an organic treatment to measure anti-oxidant effect
Ahmad et al. Management and control of coccidiosis in poultry—A review
Meskerem et al. Protective effects of Moringa stenopetala leaf supplemented diets on Eimeria tenella infected broiler chickens in debre zeit, central, Ethiopia
KR100519720B1 (ko) 유해활성산소 및 방사선에 의한 생체손상 억제효과와면역활성 증진효과를 갖는 프로폴리스 함유 복합조성물 및그의 제조방법
US10568923B2 (en) Plant parts and extracts having anticoccidial activity
US20240024396A1 (en) Anticoccidial composition comprising ginkgo leaves and use thereof
Zyan et al. Molecular identification of Eimeria tenella in broiler chickens in Kalyoubia governorate and evaluation of different strategies for control cecal coccidiosis.
KR100836944B1 (ko) 돌외를 포함하는 콕시듐증의 예방 또는 치료용 조성물
Abbasi et al. In vitro and in vivo activity of cinnamaldehyde against Eimeria kofoidi in chukar partridge (Alectoris chukar)
KR20220035944A (ko) 항-콕시듐성 식물성 제제
Ahmadov et al. Effects of herbal plants on ducks and quail infected with Eimeria species
EP4302608A1 (en) Anticoccidial composition comprising coumaric acid and use thereof
Ferdji et al. Anticoccidial resistance in poultry: determination of ionophore sensitivity for Eimeria acervulina and Eimeria maxima isolated from broiler chicken farms in Tizi Ouzou province (Algeria)
EP3024470A1 (de) Zusatzstoff für futtermittel, nahrungsmittel, trinkwasser oder pharmazeutische präparate
Guven et al. Anticoccidal efficacy of usnic acid in broilers
Alqhtani et al. Efficacy and Optimal Feeding Level of Rumex Nervosus Leaves on Blood Biochemistry, Carcass Characteristics, Productivity Indices, and Anticoccidial Indicators of Broiler Chickens Infected or Not Infected with Eimeria Tenella
Tuvshintulga et al. Evaluation of inhibitory effect of methylene blue against Babesia and Theileria parasites
Jatau et al. COMBINED EFFECTS OF FEED SUPPLEMENTATION WITH PROBIOTIC AND ORAL ADMINISTRATION OF METHANOL LEAF EXTRACT OF Anacardium occidentale AGAINST EXPERIMENTAL Eimeria tenella INFECTION IN BROILERS

Legal Events

Date Code Title Description
AS Assignment

Owner name: BANK OF AMERICA, N.A., AS COLLATERAL AGENT, IOWA

Free format text: IP SUPPLEMENT (PATENTS);ASSIGNORS:KEMIN INDUSTRIES, INC.;KEMIN FOODS, L.C.;KEMIN HOLDINGS, L.C.;REEL/FRAME:031615/0812

Effective date: 20131104

AS Assignment

Owner name: KEMIN INDUSTRIES, INC., IOWA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SCHOETERS, ELKE;WOUTERS, HILDE;MAST, ILSE;AND OTHERS;SIGNING DATES FROM 20130710 TO 20140130;REEL/FRAME:032353/0801

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION