US 2014O161919A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0161919 A1 Thangavel et al. (43) Pub. Date: Jun. 12, 2014

(54) PARTS AND EXTRACTS HAVING Publication Classification ANTICOCCDAL ACTIVITY (51) Int. C. (71) Applicant: Kemin Industries, Inc., Des Moines, IA A61E36/49 (2006.01) (US) A61E36/85 (2006.01) A61E36/22 (2006.01) (72) Inventors: Gokila Thangavel, Hosur (IN); (52) U.S. C. Rajalekshmi Mukkalil, Cochin (IN); CPC ...... A61K 36/49 (2013.01); A61K 36/22 Haridasan Chirakkal, Nolambur (IN); (2013.01); A61K 36/185 (2013.01) Hannah Kurian, Benson Town (IN) USPC ...... 424/769; 424/725 (21) Appl. No.: 13/928,504 (57) ABSTRACT (22) Filed: Jun. 27, 2013 Natural plant parts and extracts of selected from the Related U.S. Application Data group consisting of , Rhus chinensis and (60) Provisional application No. 61/664,795, filed on Jun. containing compounds such as gallic 27, 2012. acid, derivative of gallic acid, gallotannins and hydrolysable have been found to control coccidiosis in poultry and, (30) Foreign Application Priority Data more specifically, coccidiosis caused by Eimeria spp. The plant parts and natural extracts result in a reduction of lesion Jan. 23, 2013 (IN) ...... 177/DELA2013 score, oocysts per gram of fecal matter and mortality. Patent Application Publication Jun. 12, 2014 Sheet 1 of 14 US 2014/O161919 A1

Positive control S

Negative control Positive Control Quercus infectoria Patent Application Publication Jun. 12, 2014 Sheet 2 of 14 US 2014/O161919 A1

as a 3. Q3 is niecifia

FIG. 3

3

9.

-

E. acervilia E. axifra E. ferrella

& Corto & fece: Otro : Satiromycin 60 ppm N Quercus infectoria water extract 100 ppm n =6, p<0.05 FIG. 4 Patent Application Publication Jun. 12, 2014 Sheet 3 of 14 US 2014/O161919 A1

n = 3, p<0.05

in Sal my Quercus 60ppm 100 ppm infectori 50ppm 100ppm 200ppm FG. 6 n = 3, p<0.05 Patent Application Publication Jun. 12, 2014 Sheet 4 of 14 US 2014/O161919 A1

36 wneg ctrl m\npos ctrl *Q. infectoria 100 ppm 34 assG. infectoria 50ppm SS - 32 w YM saway assass

Time (hours) FIG. 7

30 : & Neg Ctrl & POS Ctrl s Q. Infectoria 100 ppm N 25 : &Q. Infectoria 50 ppm ta 25 20 s E s

15

o s 5 10 s d : 5 : sess ŠsN O ------,s------,------WNSls------Sr. O 10 20 30 40 50 60 70 80 90 100 Incubation time (hours)

FIG. 8 Patent Application Publication Jun. 12, 2014 Sheet 5 of 14 US 2014/O161919 A1

36 Nneg ctrl Napos ctrl *::::: Q. infectoria 250 ppm 34 &Q. infectoria 100 ppm sasa Q. infectoria 50ppm 32

30 S ŠSS Sws N s sans.

28 ass ŠNaaS-i. 26

24 r

22

Time (hours) FIG. 9

40 N^neg ctrl &pos ctrl 35 . :x Q. infectoria 250 ppm NQ. infectoria 100 ppm 30 s&Q. infectoria 50 ppm

25

20

15

10 -

5 sssssssssss S

s&w\ &Sissssssssssss &SS O 20 40 60 80 Incubation time (hours)

FIG 10 Patent Application Publication Jun. 12, 2014 Sheet 6 of 14 US 2014/O161919 A1

8 .

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FIG 11

FIG. 12 Patent Application Publication Jun. 12, 2014 Sheet 7 of 14 US 2014/O161919 A1 ----|

;-->* Patent Application Publication Jun. 12, 2014 Sheet 8 of 14 US 2014/O161919 A1

i

n=3, p<0.05, Correlation Coefficient= -0.982226

32 Nneg ctrl w$mpos ctrl

& Gallic acid 10 ppm 30

28

& 2 6 ^ma& my s

24 T S. NSN & 22

20 2O 72 9 6 Time (hours) FG 16 Patent Application Publication Jun. 12, 2014 Sheet 9 of 14 US 2014/O161919 A1

wneg ctrl s 100 & NSnpos ctrl s 90 s as Gallic acid 10 ppm es R i 80 s N se R a. & 2 70 R ? &s s R 60 s s sS. s s s Ss S s S.s s XS 50 s S s s s S s S s 40 - R s s sR Ss Ss s s s s - Rs s s s s R s s C S.s SS s SS s O 20 s s s s s Rw s & SS s S s SS s S s SS S s S Sa& xxs SSS S s s's S s SsssSS sawn\Š samaš s SSSSSSSSSSS s Šy - - - -:------x------x------Sys N.------. S. O 20 40 60 80 100 Incubation time (hours)

FIG. 17

36 x^neg ctrl &pos ctrl x:x Gallic acid 50 ppm 34 & Gallic acid 25 ppm w\Gallic acid 10 ppm

32 SS Ssss i 30 SSS Sm. Ssss SX 28 Ša N N 26 NN N N 24 YomSSX \xass.

22

Time (hours) FIG. 18 Patent Application Publication Jun. 12, 2014 Sheet 10 of 14 US 2014/O161919 A1

40 - as a neg ctrl Napos ctrl 35 gallic acid 50 ppm $Šs gallic acid 25ppm 30 *&^gallic acid 10 ppm

25

20

15 -

10 -

5

O rurururururururururururururuvurr-uur-rurr-uur-rurur------O 20 40 Incubation time (hours)

FIG. 19 Patent Application Publication Jun. 12, 2014 Sheet 11 of 14 US 2014/O161919 A1

b &S S s O.5. . C 8 8

O c ------E. ferreia E. acervuilia E. iiiaxina

Š COtto Sece Cool & Sanomycin 60 ppi. & Galic acid 11 pm & Gallic acid 22 ppin & Galic acic 55 ppm

1.00E+07 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 . Patent Application Publication Jun. 12, 2014 Sheet 12 of 14 US 2014/O161919 A1

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36 &neg ctrl R Nanpos Ctrl T. chebula 250 ppm 34 NT. chebula 100 ppm wT.chebula 50ppm 32 9 30 5 S. N SS si Saxas 28 - sn \ s s s N\s \s s s N s ymna W 26 s N \ -- w Y s NN N& T 24 . to

20 72 96 Time (hours) FIG. 23

40 n^neg ctrl 2G wpos ctrl 35 T. chebula 250 ppm S NxT. chebula 100 ppm aw 30 &T. chebula 50 ppm Z s o

S. 25 &

in 20 s 3o 15 As 10 U 9. O 5 ss 0 O 2 O 40 60 8O 100 Incubation time (hours)

FIG. 24 Patent Application Publication Jun. 12, 2014 Sheet 14 of 14 US 2014/O161919 A1

3 2.5

s 4.

C 15i s

-

E. acervifa E. praxifra E. ferrella

8 COO 8 feted COO & Salinomycin 60 ppm & Rhus to ppm & Rhus 200 ppm & Rhus 500 ppm 6, p<0.05 : Terminalia chebula 1000 ppm

FIG. 25

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iE-g4 e US 2014/01 6 1919 A1 Jun. 12, 2014

PLANT PARTS AND EXTRACTS HAVING the anti-inflammatory and antioxidant properties of the ANTICOCCDAL ACTIVITY proanthocyanidins, a condensed rather than a . 0001. This application claims priority to U.S. Patent Application Ser. No. 61/664,795, filed Jun. 27, 2012 and 0006 Naidoo et al. teach an in vivo study using four plants selected based on their antioxidant activity (Naidoo et al. The Indian Application No. 177/DEL/2013, filed Jan. 23, 2013, value of plant extracts with antioxidant activity in attenuating both of which are incorporated herein by this reference. coccidiosis in broiler chickens. Veterinary Parasitology. 153: BACKGROUND OF THE INVENTION 214-219; 2008). They observed that one of the plants (Tul baghia violacea) reduced the Eimeria oocyst counts in the 0002 The present invention relates generally to the con chicken excreta and they speculate that this effect could be trol of coccidiosis and, more specifically, to the application of due to the antioxidant compound S (methylthiomethyl) cys plant parts, plant extracts and compounds to control coccidi teine sulfoxide. osis in poultry and other animals. 0007 McDougald et al. describe the use of a muscadine 0003 Coccidiosis is a major disease in the poultry indus pomace to enhance resistance to coccidiosis in broiler chick try and according to a recent Survey, it is estimated that the ens (McDougald et al. Enhancement of Resistance to Coc global impact is greater than $3 billion USD annually (world cidiosis and Necrotic Enteritis in Broiler Chickens by Dietary poultry.net/Broilers/Health/2009/9/In-Ovo-vaccination Muscadine Pomace. Avian Diseases. 52: 646-651; 2008). against-coccidiosis-WP006949W/ accessed Jun. 18, Muscadine pomace is a by-product of used in wine 2013). Coccidiosis is caused by a protozoan parasite, namely production. They make no mention of efficacy of any specific Eimeria, belonging to the phylum Apicomplexa, and the fam compounds in the pomace. The proposed anti-coccidial activ ily Eimeriidae (Clare, R.A and Danforth, H. D (1989). Major ity differs significantly from the activities proposed by Wang histocompatibility complex control of immunity elicited by et al. and Naidoo et al. genetically engineered Eimeria tenella (Apicomplexa) anti gen in chickens. Infection and immunity, 57 (3): 701-705). SUMMARY OF THE INVENTION The parasite invades the gut cells and causes necrosis in the intestine which leads to malabsorption, diarrhea, morbidity, 0008. The present invention consists of the identification reduction of weight gain, poor feed conversion, and, in severe and use of plant parts and plant extracts effective in the cases, mortality (Williams, R. B (2005). Intercurrent coccidi control of coccidiosis in animals, particularly in poultry. Spe osis and necrotic enteritis of chickens: Rational, integrated cifically, plant parts and natural extracts of Quercus infecto disease management by maintenance of gut integrity. Avian ria, Rhus chinensis nut, Terminalia chebula have Pathology, 34(3), 159-180). Seven different species of Eime been found to control coccidiosis in poultry and, more spe ria, E. acervulina, E. brunetti, E. maxima, E. mitis, E. neca cifically, coccidiosis caused by Eimeria spp. More specifi trix, E. praecox, and E. tenella are known to cause coccidiosis cally, plant parts or extracts containing efficacious amounts of in poultry (Williams, 2005) and the species are highly host compounds selected from the group consisting of gallic acid, and site specific. E. tenella is one of the major species causing gallotannins and hydrolysable tannins. coccidiosis in poultry, and their site of infection is the caecum 0009. The plant parts and natural extracts of gall nuts of (Khazandi, M and Tivey, D (2010). Developing an in vitro Quercus infectoria, Rhus chinensis and of Terminalia method for Eimeria tenella attachment to its preferred and chebula result in a reduction of lesion score, oocysts per gram non-preferred intestinal sites. Experimental Parasitology, of fecal matter and mortality. The plant parts/extract was also 125 (2), 137-140). Coccidiosis is currently controlled by found to have a direct inhibitory effect on the sporozoites of medication, but the increasing emergence of drug-resistant Eimeria, as observed in the in vitro MTT assay. Compounds strains of Eimeria requires the development of an alternative selected gallic acid, gallotannins and hydrolys able tannins control strategy. Since plants are known to possess antipara were also found to reduce lesion score, oocysts per gram of sitic and anticoccidial activity due to the presence of phenolic fecal matter and mortality. The compounds were also found to compounds (Tipu, M. A., Akhtar, M. S., Anjum, M. I and have a direct inhibitory effect on the sporozoites of Eimeria, Raja, M. L (2006). New dimension of medicinal plants as as observed in the in vitro MTT assay. animal feed. Pakistan vet.J., 26(3): 144-148), they could be 0010. The present invention also consists of a method of potential sources of bioactive molecules against coccidiosis controlling coccidiosis in poultry and other animals by in poultry. administering a composition comprising plant parts or 0004. Others have attempted to use plant parts or plant extracts of plants containing an efficacious amount of gall extracts in treating coccidiosis. For example, McCann et al. nuts of Quercus infectoria, Rhus chinensis, Terminalia che tested the effect of Sweet tannins on the bula fruit and/or compounds such as gallic acid, gallotannins performance of broiler chicks vaccinated with a live coccidia and hydrolysable tannins. vaccine (M. E. E. McCann, E. Newell, C. Preston and K. Forbes. The Use of Mannan-Oligosaccharides and/or Tannin BRIEF DESCRIPTION OF THE DRAWINGS in Broiler Diets. Intl.J. of Poultry Sci. 5 (9): 873-879, 2006). They reported that Supplementation with mannan-oligosac 0011 FIG. 1 are microphotographic images of caecal charides or tannins, either individually or in combination, did lesions of control birds, birds treated with the negative con not reduce the impact of the coccidiosis. trol, birds treated with the positive control, and birds treated 0005 Wang et al. teach the use of a seed proantho with Quercus infectoria. cyanidin extract on coccidiosis (Wang, et al. Influence of 0012 FIG. 2 is a chart of the oocysts per gram (OPG) of Grape Seed Proanthocyanidin Extract in Broiler Chickens: excreta of birds treated with Quercus infectoria on day 7 post Effect on Chicken Coccidiosis and Antioxidant Status. Poul infection; columns with different SuperScripts are statistically try Science.87:2273-2280, 2008). They attributed activity to significant (p<0.05). US 2014/01 6 1919 A1 Jun. 12, 2014

0013 FIG.3 are microphotographic images of the Hand E 0033 FIG. 23 is a chart of PCR results after invasion of stained sections of caecum of infected control birds and birds MDBK host cells with sporozoites, pre-treated with different treated with Quercus infectoria. concentrations of T. chebula. 0014 FIG. 4 is a chart of the lesion score for E. acervulina, 0034 FIG. 24 is a chart of fold changes in Eimeria DNA E. maxima and E. tenella for the birds treated with O. infec for different time points versus T20 within one treatment. toria water extract on day 5 post infection; columns with 0035 FIG. 25 is a chart of the lesion score on day 5 post different superscripts are statistically significant (p<0.05). infection of birds treated with R. Chinensis and T. chebula, 0015 FIG. 5 is a chart of the oocysts per gram (OPG) of columns with different SuperScripts are statistically signifi excreta of birds treated with O. infectoria water extract on day cant (p<0.05). 7 post infection; columns with different SuperScripts are sta 0036 FIG. 26 is a chart of the OPG on day 7 post infection tistically significant (p<0.05). of birds treated with R. Chinensis and T. chebula, columns 0016 FIG. 6 is a chart of the MTT assay carried out for the with different superscripts are statistically significant (p<0. evaluation of O. infectoria at various dosage levels along with 05). a coccidiostat (Salinomycin) as positive control; columns with different superscripts are statistically significant (p<0. DESCRIPTION OF THE INVENTION 05). 0037. A preliminary in vivo evaluation of crude powder of 0017 FIG. 7 is a chart of PCR results after invasion of O. infectoria gall nuts (100-800 micron particle size, pro MDBK host cells with sporozoites and different concentra cured from Pooja Herbs, Mumbai, India) in controlling coc tions of O. infectoria. cidiosis at 100 g/ton of feed dose gave some indication of 0018 FIG.8 is a chart offold changes in Eimeria DNA for promising results which urged further evaluation at higher different time points versus T4 within one treatment. dosage. A 35 day in vivo trial conducted in broiler birds 0019 FIG. 9 is a chart of PCR results after invasion of challenged with oocysts of Eimeria tenella showed that O. MDBK host cells with sporozoites, pre-treated with different infectoria gall nuts reduced the lesion score to 0 and mortality concentrations of O. infectoria. to 0%, comparable to the positive control (0%), whereas the 0020 FIG. 10 is a chart of fold changes in Eimeria DNA negative control showed a score of 4 and a mortality of 17%. for different time points versus T20 within one treatment. The histopathological analysis of the caecum Samples 0021 FIG. 11 is a chart of the anti-sporozoite activity of showed that the birds treated with O. infectoria showed lesser the different fractions of O. infectoria, columns with different area infected by the parasite, lower mononuclear infiltrations Superscripts are statistically significant (p<0.05). and hemorrhages of the caecum. 0022 FIG. 12 is a High Performance Liquid Chromato 0038. In general in this description, a plant part, extract or gram (HPLC) chromatogram of water fraction of O. infecto compounds is termed to be efficacious if it can result in C. statistically significant reduction in the lesion score, the 0023 FIG. 13 is a chart of the anti-sporozoite activity of oocysts shed in the excreta, (Oocyst Per Gram (OPG)) or the the four major peaks of O. infectoria, columns with different mortality of the birds as compared to the infected control SuperScripts are statistically significant (p<0.05). which is untreated. Generally, administration of gallic acid 0024 FIG. 14 is the LC/MS/MS chromatogram of peak 1 and gallic acid containing formulations are described with of O. infectoria. formulations providing a dosage from 0.1 to 50 ppm, prefer 0025 FIG. 15 is a chart depicting the correlation between ably from 2 to 20 ppm, and most preferably from 3 to 10 ppm the concentration of gallic acid and the anti-sporozoite activ through feed or water or an equivalent Supplementation ity of O. infectoria, columns with different superscripts are through other routes. The plants, plant parts and/or extracts statistically significant (p<0.05). described contain around a mininimum of 0.1% of gallic acid. 0026 FIG. 16 is a chart of PCR results of MDBK host 0039. The efficacy of O. infectoria crude powder in con cells, pre-treated with 10 ppm gallic acid, invaded with sporo trolling mixed infection of Eimeria inbroiler birds was evalu Zoites. ated. The results showed that there was significant reduction in the lesion score for E. tenella and E. acervulina as com 0027 FIG. 17 is a chart of fold changes in Eimeria DNA pared to the infected control and even the positive control, for different time points versus T20 within one treatment. Salinomycin. Whereas in case of E. maxima, a numerical 0028 FIG. 18 is a chart of PCR results after invasion of reduction in the lesion score was observed as compared to the MDBK host cells with sporozoites, pre-treated with different infected control and Salinomycin. The oocysts per gram of concentrations of gallic acid. treated groups were significantly lower than the infected con 0029 FIG. 19 is a chart of fold changes in Eimeria DNA trol and Salinomycin group, however, mortality was not for different time points versus T20 within one treatment. observed in any of the treatment groups including the infected 0030 FIG. 20 is a chart of the lesion score on day 5 post control. This proves the efficacy of O. infectoria in control infection of birds treated with gallic acid at different concen ling coccidiosis caused by other species of Eimeria also. trations; columns with different SuperScripts are statistically Further, to determine the mode of action of O. infectoria, an significant (p<0.05). in vitro method based on 3-(4,5-Dimethyl-2-thiazolyl)-2,5- 0031 FIG. 21 is a chart of the OPG on day 7 post infection diphenyl-2H-tetrazolium bromide (MTT) reduction assay of birds treated with gallic acid at different concentrations: was developed to evaluate the anti-sporozoite activity of plant columns with different SuperScripts are statistically signifi extracts as a measure of the viability of the sporozoites. Stud cant (p<0.05). ies with O. infectoria gall nut on the sporozoites of Eimeria 0032 FIG. 22 is a chart of the anti-sporozoite activity of tenella showed significant reduction in the viability of sporo Rhus chinensis and a chart of the anti-sporozite activity of Zoites compared to the sporozoite control. A dose dependent Terminalia chebula, columns with different superscripts are efficacy was observed in studies conducted with different statistically significant (p<0.05). dosages of O. infectoria and the results were validated by US 2014/01 6 1919 A1 Jun. 12, 2014 conducting experiments several times independently. Hence, tions were prepared according to the phases of the life of the directanti-sporozoite activity of O. infectoria could be one of bird; Prestarter (Day 1-10), Starter (Day 11-20), and Finisher the modes of actions which attributes to the efficacy of the feed (Day 21-42). No antimicrobials and supplements were extract in controlling in coccidiosis in vivo. used in the feed formulation. 0040 Similarly, to determine the mode of action of O. 0049. Details of treatment groups. Groups and the treat infectoria in controlling Eimeria in a host cell line, an in vitro ments are shown in Table 2. The treated birds were fed with assay was developed based on a co-culture of host cells and plant extract incorporated in the feed from day 1 (Table 2). Eimeria parasites. Cells and parasite are combinedinanassay The crude powders of O. infectoria gall nut were procured with a positive control and different test products. The inva from Pooja herbs, Mumbai, India. sion and proliferation of the Eimeria parasites is measured by detecting Eimeria DNA using real-time PCR. For this, spe TABLE 2 cific primers were selected and PCR conditions were opti mised. The positive control and potential anticoccidial com Details of treatment groups and feed. pounds are added to the in vitro assay in three different ways: Groups Treatments 0041. The products are combined with Eimeria sporozoi tes and added to the host cells. Control 1 (uninfected Normal feed control) 0042. The products are added to the sporozoites for a Control 2 (Negative infected Coccidiosis induction + Normal feed specific time, then removed and afterwards the sporozoites Control) are added to the host cells. Control 3 (Positive Control) Coccidiosis induction + Feed with Coxistac' at 1000 g/ton 0043. The products are added to the host cells for a specific Treatment Coccidiosis induction + Feed with time, then removed and afterwards the sporozoites are added P. infectoria at 100gton to the host cells. *Coxistac is a product from Pfizer containing Salinomycin at 12% concentration. Hence, 0044) The effect of Quercus infectoria was evaluated in addition of Coxistiac at the mentioned dose of 500gton of feed will enable delivery of the in vitro assay. 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. EXAMPLE1 0050 Induction of coccidiosis. Sporulated oocysts of E. tenella (Houghton strain Chapman, H.D. and Shirley, M. W. Efficacy of Quercus infectoria in Controlling Caecal 2003. The Houghton strain of Eimeria tenella. A review of Coccidiosis the type strain selected from genome sequencing. Avian 0045 Experimental facility and study design. The screen Pathol., 32: 115-127-propagated) were orally administered ing trial was conducted at a poultry farm facility located in to each bird on day 14, 15 and 16 of age through oral gavage Gummidipundi, India. Straight run commercial hybrid at a dose of 1x10 oocysts/bird/day. Feeding was stopped on broiler chickens, Gallus domesticus (Var. Vencobb 400) were the day of inoculation, for 2 h before and 2 hafter inoculation. used for the study. Day old male chicks were procured, 0051 Parameters analyzed. The parameters that were cho weighed individually, wing banded, and randomly segregated Sen for analyses were the indices of pathogenesis namely into groups. The experimental design is detailed in Table 1. excreta appearance, mortality, lesion scoring of the caecum for coccidiosis, and oocysts per gram (OPG) of excreta. The TABLE 1. methods are detailed below. 0.052 Examination of excreta. The excreta of the birds Study design. were monitored daily from the day 1 post infection to day 10 for their consistency, presence of blood, mucus, undigested Category Trial Parameter feed, and orange color. Scoring of the excreta was carried out Rearing type Cages based on the severity of blood shedding. Age of birds at the start of 1 day old 0053 Mortality. The mortality of the birds was recorded the trial Total no of Birds 56 on a daily basis and post mortem was carried out to confirm Number of groups 8 the cause of death. No of birds groups 7 0054 Lesion scoring of the caecum. On day 5 and 7 post Duration of the trial 35 days infection, 2 birds from each of the groups were sacrificed by cervical dislocation and the intestine was cut open. The caeca 0046 Farm management. Good farm managing practices of the birds were scored for coccidiosis lesions. The scoring were followed during the trial. The entire farm and the equip was done based on the severity of the lesions in the caecum ment used for the study were cleaned and disinfected before and the presence of blood (Johnson, J. K., and W. M. Reid. the arrival of the chicks. The birds were housed in cages (1970). Anticoccidial drugs: lesion scoring techniques in bat organized on concrete flooring and a tray was provided at the tery and floor-pen experiments with chickens. Experimental bottom of the cages to facilitate collection of fecal samples. Parasitology 28:30-36). The score for caecal coccidiosis was The temperature and humidity of the farm was monitored a scale of 0-4. continuously. 0055 OPG of excreta. Triplicate samples of the excreta of 0047. Vaccination schedule. The birds were vaccinated for the birds were collected randomly from the tray kept below Newcastle Disease Virus (NDV) and Infectious Bursal Dis the cages and the oocyst per gram was evaluated. ease (IBD). 0048 Feed formulation. A corn soya based mash diet was Results formulated. The feed ingredients were procured from Ponni 0056 Indices of pathogenesis. The observations on the feeds, Tamil Nadu, India. The mash feed was fedadlibitum to excreta of the birds showed that the blood shedding in the the birds throughout the study period. Three feed formula infected groups started by day 4 post infection and the sever US 2014/01 6 1919 A1 Jun. 12, 2014

ity peaked on the day 5. The results of the scoring of the groups. The lesion score and OPG data of the positive control excreta are given in Table 3. Day 7 post infection the excreta did not show any difference from that of the negative control. were found to be normal with no blood. The positive control 0060 Although the positive control did not perform well (C3, Table 3) on day 5 had a score of 3 as compared to the in this trial, the lesion scores of birds treated with plant negative control (C2, Table 3) of 4. Birds treated with O. extracts of O. infectoria were lower than the negative infected infectoria had a lower score of 2 and were better than the control which indicates that they could be candidates for positive control. further investigation. However, they showed no reduction in the OPG. TABLE 3 Scoring of excreta on day 5 post infection. EXAMPLE 2 Treatments Score Description Efficacy of Quercus infectoria in Controlling Caecal C1 - uninfected control Excreta normal consistency Coccidiosis C2 - Negative infected control Presence of heavy raw blood C3 - Positive Control Excreta with blood +++ T - Q. infectoria at 100 g/ton Excreta with blood ++ 0061. A 35 day in vivo challenge trial was conducted in broiler birds challenged with Eimeria tenella The treatment + - denotes the severity of blood loss and amount of blood in the excreta. groups included, 1) control, uninfected normal birds; 2) nega tive control, birds infected with E. tenella and fed normal diet 0057 Lesion scoring of the caecum. Lesion scoring of the without any anticoccidial compounds; 3) positive control, caeca on day 5 and 7 post infection indicated that the lesions birds infected and fed diet containing Coxistac (anticoccidial were severe on day 5, and the birds started recovering on day agent, Salinomycin) at the recommended dose of 500 g/ton 7 post infection which was indicated by the formation of a and 4) treatment group including infected birds administered caecal plug. This followed the normal pattern of infection diet containing O. infectoria gall nut at 500 g/ton dose. No enabling the removal of oocysts from the caeca. The results of mortality was observed in the positive control group and the lesion score showed that the positive control (Salinomy treatment group Supplemented with crude powder of gall nuts cin control did not show any difference in the score as com of Quercus infectoria. The caecal lesions indicated that the pared to the negative control due to inexplicable reasons. The negative control birds were highly infected with an average treatment with O. infectoria reduced the lesion score as com score of 4 whereas the positive control had score of 0. Birds pared to the negative control (Table 4). The reduced lesion treated with Quercus infectoria showed results similar to the score correlated with reduced excreta score and absence of positive control (O). O. infectoria showed reduction in the mortality. OPG counts comparable to the positive control. The histo pathological analysis of the caecum samples showed that the TABLE 4 birds treated with O. infectoria had lesser area affected by Eimeria, no hemorrhages and minimal mononuclear infiltra Lesion score of the caeca on day 5 post infection tions up to the mucosa. Treatments Lesion Score 0062. The second in vivo experiment involved the follow C1 - uninfected control O ing treatment groups. C2 - Negative infected control 3 C3 - Positive Control 3 T - Q. infectoria at 100 g/ton 2.5 TABLE 6 Description of treatment groups 0058. OPG of excreta of the birds on day 7 post infection. Groups Treatments The counts of OPG of excreta of the birds on day 7 post Control 1 (C1) Uninfected control - Normal feed without anticoccidial infection are shown in Table 5. Unexpectedly, the anticoc Control 2 (C2) Negative control - Coccidiosis induction + Normal feed cidial Salinomycin treated birds (C3, Table 5) did not show without anticoccidial any indication of reduction of oocysts as compared to the C2 Control 3 (C3) Positive Control - Coccidiosis induction + Normal feed (Table 5). The values presented are an average of three repli with Coxistac 12% (a) 500 g/ton* Cates. Treatment (T) Coccidiosis induction + Normal feed w Q. infectoria *Coxistac is a product from Pfizer containing Salinomycin at 12% concentration. Hence, addition of Coxistiac at the mentioned dose of 500gton of feed will enable delivery of TABLE 5 Salinomycin at 60 ppm levels in the feed which is the recommended preventive dose for broilers. Oocysts per gram of excreta on day 7 post infection Average Oocysts Treatments Per Gram Excreta CV Results C2 - Negative infected 2.SE-OS 1.23 0063 Caecal lesions on day 5 post infection. The lesion control C3 - Positive Control 4.OE--OS O.68 scoring for caecal coccidiosis was carried out on day 5 post T - Q. infectoria at 3.SE-OS 1.36 infection based on the criteria of scoring as before. The results 100 g/ton of the scoring showed that the positive control completely alleviated the effects of caecal coccidiosis as compared to the negative infected control. O. infectoria treated birds showed 0059 Mortality. The rate of mortality was 17% in control no lesions in the caecum and was comparable to the positive 2 (negative infected control). There was no mortality in other control and uninfected control C1 (Table 7, FIG. 1). US 2014/01 6 1919 A1 Jun. 12, 2014

TABLE 7 TABLE 10 Lesion scoring on day 5 post infection. Histopathological findings of the tissues of caecum of birds

Treatments Lesion Score Groups Histopathological findings

C1 - Uninfected control Oe Uninfected Cecum within normal histological limits. control C1 C2 - Negative infected control 4a Negative Cecum showed moderate load of different Eimerial C3 - Positive Control Oe control C2 stages (Oocyst, Schizont and merozoite) along with T - Q. infectoria Oe mild mucosal hemorrhages and mild to moderate mononuclear cell infiltration in mucosal, Submucosal Columns with different superscripts are statistically significant (p<0.05). and muscular layers. Positive Cecum showed minimal load of different Eimerial control C3 stages with Schizonts and merozoites contributing 0064 Oocyst counts in excreta. The OPG of excreta was the major load. Minimal mucosal hemorrhages and estimated on day 7 post infection to evaluate the shedding of necrosis was evident microscopically. Mild to oocysts. The results of the study showed that the positive moderate mononuclear cell infiltration in mucosal and Submucosal layers was seen. control, O. infectoria had significantly lower counts of P. infectoria Cecum showed minimal load of different Eimerial oocysts in the excreta as compared to the infected negative T stages with oocyst contributing the major load. control (p<0.05). O. infectoria treatment was equally effec Minimal mucosal necrosis and mononuclear cell tive as the positive control (FIG. 2). This correlates with the infiltration in mucosal layers was evident. results of the lesion score. 0067. Histopathological results showed that the birds 0065. Mortality. The rate of mortality was recorded during treated with O. infectoria had fewer regions of the caecum the experiment, and the data are given in Table 8. AS expected, infected with E. tenella, and the mononuclear infiltration was there was no mortality in the uninfected control group (C1) restricted only to the mucosa with a score of 1 indicating mild and the positive control group (C3). O. infectoria Supple infiltration (FIG. 3). The submucosa and muscular layers mented group showed no mortality. were free from infiltration (Table 10). In the negative control, mononuclear infiltration was observed in the mucosa, Sub TABLE 8 mucosa and even the muscular layer. There were no hemor rhages in the caecum of birds treated with Q. infectoria as Rate of mortality during the trial period compared to that of the negative control (2). This indicates Treatment Groups Rate of mortality (%) that the caecum of birds treated with O. infectoria was less C1 - Uninfected control O infected than the positive control. C2 - Negative infected control 33.33 0068. The in vivo screening of plant extracts revealed that C3 - Positive Control O Quercus infectoria is a potent candidate in controlling caecal T - Q. infectoria O 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 0066 Histopathological analysis of caecum samples. O. mortality. infectoria showed positive reductions in all parameters tested such as lesion score, OPG and rate of mortality and the data EXAMPLE 3 were comparable to the positive control, Salinomycin. Hence, histopathological analysis of the caecum samples of birds Efficacy of Water Extracts of Quercus infectoria in from this group was carried out in comparison to the unin Controlling mixed Infection of Coccidiosis fected control (C1), negative control (C2) and positive control 0069 Efficacy of O. infectoria crude powder in control (C3). The severity and distribution of the lesions in the cae ling mixed infection of coccidiosis in broiler birds was evalu cum were based on the grading provided in Table 9. ated. A 35 day in vivo trial was conducted wherein the birds were challenged with field strains of mixed culture of oocysts TABLE 9 of the species E. tenella, E. acervulina and E. maxima. The mixed culture of oocysts was provided by Department of Severity and distribution of lesions parasitology, Tamil Nadu Veterinary Research Institute, in the caecum of different groups Namakkal, India. The oocysts culture was a mixture of E. Uninfected Negative Positive Quercus tenella, E. acervulina and E. maxima isolated from feces of Histopathology control control control infectoria birds with clinical coccidiosis infection. Virulence of the Mononuclear cell O 3 2 1 oocysts obtained was evaluated inbroiler birds and the dosage infiltration-mucosa of the oocysts was finalized to be 5x10 based on the concen Mononuclear cell O 2 3 O tration that yields a lesion score of 3 and above for all the infiltration-Submucosa Mononuclear cell O 2 O O tested oocysts, E. tenella, E. maxima and E. acervulina. infiltration-muscular a. The screening trial was conducted at Kemin's in-house layer R&D poultry farm facility located in Gummidipundi, India. Hemorrhages O 2 1 O Necrosis-Villi O 1 1 1 Straight run commercial hybrid broiler chickens, Gallus Distribution of O 3 1 1 domesticus (Var. Vencobb 400) were used for the study. Day stages of Eimeria old male chicks were procured, weighed individually, wing banded, and randomly segregated into groups. The experi mental design is detailed in Table 11. Good farm managing US 2014/01 6 1919 A1 Jun. 12, 2014

practices and vaccination schedule were followed during the lowed by incubation of sporozoite Suspension (minimum of 3" in vivo trial as mentioned in example 1. 10 cells/ml) with required concentration of plant extract. The plant samples were prepared by mixing crude powder into a TABLE 11 known volume of distilled water to achieve the specific ppm, vortexed for 2 min and filtered through a 0.2L syringe filter. Study design Following 24 h of incubation with the plant extracts, the Category Trial Parameter sporozoites were thoroughly washed and then MTT assay was performed. MTTPMS solution (0.2 millimolar each) is Duration of the trial 35 days incubated with the sporozoite suspension (at 1:10 ratio) for 2 Breed Cobb 400 Rearing type Cages h at 41° C. After incubation, the contents are centrifuged at Age of birds at the start of 1 day old 800 g for 5 min and the supernatant is carefully removed. The the trial purple dye formazan is dissolved in 200 ul DMSO and the Total no of Birds 315 Number of groups 21 absorbance is measured at 530 nm against a reference wave No of birds groups 15 (male) length of 630 nm. 0074 MTT assay was carried out for the evaluation of O. infectoria at various dosage levels along with Coccidiostac 0070. The birds were vaccinated for Newcastle Disease (Salinomycin) as positive control (FIG. 6). There was a dose Virus (NDV) and Infectious Bursal Disease (IBD). A corn dependent reduction in the viability of sporozoites in the O. soya based mash diet was used for the study. The birds were infectoria treated samples as compared to the control. fed with the extract of O. infectoria gall nut incorporated in the feed from day 1. The treatment groups are given in Table EXAMPLE 5 12. In Vitro Effect of O. infectoria on Eimeria tenella TABLE 12 Sporozoite Invasion and Proliferation in Host Cells Details of treatment groups for the trial 0075 An experiment was conducted to evaluate the in Groups Diet vitro effect of O. infectoria on Eimeria tenella sporozoite invasion and proliferation of host cells. Control 1 No infection + normal feed Control 2 Coccidiosis induction + normal feed without anticoccidial 0076 Sporozoites were obtained from sporulated oocysts Control 3 Coccidiosis induction + Normal feed with Coxistiac 12% after glass bead grinding and enzymatic excystation. As host premix (500 g/ton) cells, Madin-Darby Bovine Kidney (MDBK) cells, were Treatment Coccidiosis induction + Normal feed with O. infectoria water selected. Sporozoites and Quercus infectoria at 50 and 100 extract at 100 g/ton ppm were added to MDBK host cells for four hours. After wards, the medium was removed, cells were washed and fresh 0071 Extracts of gall nut of O. infectoria were prepared medium was added. After 4 (T4), 24 (T24), 48 (T48) and 72 by mixing the crude powder (100-800 micron particle size) in (T72) hours the medium and MDBK cells were collected and distilled water at the ratio of 1:2, then extracting at 80 to 90° stored at -20°C. C. for one and half hour with agitation. The extract was (0077. The negative control (neg ctrl) was MDBK cells filtered and again the residue was extracted in water in a infected with Eimeria sporozoites, incubated in cell culture similar manner. This was repeated for about 2 more times and medium. The positive control (pos ctrl) was MDBK cells the total liquid extract was freeze dried. infected with Eimeria sporozoites, incubated with a 5ug/ml 0072 The results showed that there was significant reduc Solution of Salinomycin. tion in the lesion score for E. tenella and E. acervulina as compared to the infected control and even the positive con (0078. At the different collection time points, DNA was trol, Salinomycin. Whereas, in case of E. maxima, a numeri extracted from the infected MDBK cells. Real-time PCR to cal reduction in the lesion score was observed as compared to detect Eimeria DNA was performed on the samples for the the infected control and Salinomycin (FIG. 4). The oocysts different time points and different treatments. The PCR per gram of treated groups were significantly lower than the results are presented in FIG. 7. infected control and Salinomycin group (FIG. 5), however, mortality was not observed in any of the treatment groups Real-Time PCR Analyses including the infected control. This proves the efficacy of O. 0079. Differences in Ct values were calculated for each infectoria in controlling coccidiosis caused by other species time point versus T4 within one treatment (ACt). Fold of Eimeria also. changes were calculated for each time point versus T4 using the following equation: EXAMPLE 4 Fold change=2^ In Vitro Anti-Sporozoite Activity of O. infectoria by 0080 These results are presented in FIG. 8. MTT Assay I0081. The negative control shows a clear Eimeria prolif 0073. Further, an in vitro method based on 3-(4, 5-Dim eration since there is a 15 fold increase in Eimeria DNA at 72 ethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide hours versus the start at 4 hours. The positive control was able (MTT) reduction assay was developed to evaluate the anti to inhibit the proliferation completely. Also for the O. infec sporozoite activity of plant extracts as a measure of the viabil toria treatments, a clear inhibition of the proliferation was ity of the sporozoites. The optimized method included the observed versus the start of 4 hours, in a dose dependent preparation, sterilization and purification of sporozoites, fol a. US 2014/01 6 1919 A1 Jun. 12, 2014

EXAMPLE 6 known to possess 60 to 70% hydrolysable tannins which can hydrolyse to release gallic acid in addition to about 7% free In Vitro effect of O. infectoria on Eimeria tenella gallic acid present in it. Sporozoite Invasion and Proliferation in Host Cells 0091. These four compounds were separated by semi-pre parative HPLC and anti-sporozoite activity was evaluated in 0082 An experiment was conducted to evaluate the in comparison to the crude powder in equivalent concentrations. vitro effect of O. infectoria on Eimeria tenella sporozoite The anti-sporozoite activity of the compounds showed that invasion and proliferation in host cells. compound of peak 1 had the best anti-sporozoite activity. The 0083 Sporozoites were obtained from sporulated oocysts other compounds showed minimal activity against the sporo after glass bead grinding and enzymatic excystation. As host Zoites. However, the crude powder showed better activity cells, Madin-Darby Bovine Kidney (MDBK) cells, were than the peak 1 indicating synergistic activity of the com selected. Sporozoites were pre-treated with 50, 100 and 250 pounds from the extract (FIG. 13). ppm of Quercus infectoria for three hours. Thereafter, the 0092 LC/MS/MS analysis of the different peaks of the sporozoite Suspension was washed and put onto a culture of HPLC chromatogram confirmed that peak 1 was gallic acid MDBK cells for 20 hours. After incubation, the medium was (FIG. 14) and the other peaks were high molecular weight removed, cells were washed and fresh medium was added. compounds which could be degraded products of hydrolys After 20, 72 and 96 hours the medium and MDBK cells were able tannind. It was hypothesized that these compounds can collected and stored at -20°C. further breakdown to release gallic acid. 0084. The negative control (neg ctrl) was MDBK cells 0093. Further, to arrive at the correlation between gallic infected with Eimeria sporozoites, incubated in cell culture acid '% and anti-sporozoite activity, O. infectoria was medium. The positive control (pos ctrl) was MDBK cells extracted in water for 5 min, 2 and 12 h and their anti infected with Eimeria sporozoites, incubated with a 5ug/ml sporozoite activity was evaluated. The study showed that Solution of Salinomycin. there was a clear correlation (correlation coefficient=-0. I0085. At the different collection time points, DNA was 982226) between the concentration of gallic acid and anti extracted from the MDBK cells. Real-time PCR to detect sporozoite activity (FIG. 15). These results indicate that gallic Eimeria tenella DNA was performed on the samples for the acid is the active ingredient responsible for the anti-sporozo different time points and different treatments. The PCR results are presented in FIG. 9. ite activity of O. infectoria. Real-Time PCR Analyses EXAMPLE 8 0.086 Differences in Ct values were calculated for each In Vitro Protective Effect of Gallic Acid time point versus T20 within one treatment (ACt). Fold changes were calculated for each time point versus T20 using 0094. An experiment was conducted to evaluate the in the following equation: vitro protective effect of gallic acid monohydrate on host cells challenged with Eimeria tenella sporozoites. Fold change=2A 0.095 Sporozoites were obtained from sporulated oocysts 0087. These results for O. infectoria are presented in FIG. after glass bead grinding and enzymatic excystation. As host 10. cells, Madin-Darby Bovine Kidney (MDBK) cells, were 0088. The negative control shows a clear Eimeria prolif selected. MDBK cells were incubated with 10 ppm gallic acid eration since there is a 20 fold increase in Eimeria DNA at 96 for seven hours. Afterwards the medium was removed and a hours versus the start at 20 hours. The positive control was sporozoite suspension was added to the MDBK cells for 20 able to inhibit the proliferation completely. Also the different hours. After incubation, the medium was removed, cells were dosages of O. infectoria all inhibited the Eimeria prolifera washed and fresh medium was added. After 20, 72 and 96 tion. There was a slightly lower effect visible for 50 ppm O. hours the medium and the MDBK cells were collected and infectoria. But this is negligible in comparison to the increase stored at -20°C. in the negative control. 0096. The negative control (neg ctrl) was MDBK cells infected with Eimeria sporozoites, incubated in cell culture EXAMPLE 7 medium. The positive control (pos ctrl) was MDBK cells infected with Eimeria sporozoites, incubated with a 5ug/ml Identification of Active Ingredients of O. infectoria Solution of Salinomycin. 0089. Further to this, Bioassay Guided Fractionation (0097. At the different collection time points, DNA was assay (BGFA) of O. infectoria gall nuts was carried out using extracted from the MDBK cells. Real-time PCR to detect the modified MTT reduction assay as the bioassay as we had Eimeria DNA was performed on the samples for the different identified that the crude extract possess anti-sporozoite activ time points and different treatments. The PCR results are ity and this could be one of the mode of action by which it is presented in FIG. 16. able to control coccidiosis. O. infectoria gall nut crude pow der was fractionated using different solvent by column chro Real-Time PCR Analyses matography. The sample from each of the fractions was evalu ated for their anti-sporozoite activity. Methanol and water 0098. Differences in Ct values were calculated for each fractions of O. infectoria showed better reduction in the time point versus T20 within one treatment (ACt). Fold viability of sporozoites as compared to the other fractions and changes were calculated for each time point versus T20 using were comparable to the Salinomycin control (FIG. 11). the following equation: 0090 Phytochemical analyses of the active fractions were Fold change=2A carried out by High Performance Liquid Chromatography (HPLC) to identify the active ingredient/s responsible for the (0099. These results are presented in FIG. 17. anti-sporozoite activity. Four major peaks were observed in 0100. The negative control shows a clear Eimeria prolif the HPLC chromatogram of both methanol and water frac eration since there is a 60 fold increase in Eimeria DNA at 96 tions, with one peak corresponding to the retention time of a hours versus the start at 20 hours. The positive control was gallic acid standard (FIG. 12). O. infectoria gall nut are able to inhibit the proliferation almost completely. Also for 10 US 2014/01 6 1919 A1 Jun. 12, 2014 ppm gallic acid treatment, a clear inhibition of the prolifera mycin (FIG.20). The oocysts per gram showed a similar trend tion was observed in a dose dependent manner. This indicates (FIG. 21), however, mortality was not observed in any of the that gallic acid at a low dose of 10 ppm is able to protect the treatment groups including the infected control. Dose depen host cells to some extend against Eimeria proliferation. dent response was observed with no significant difference between gallic acid at 22 and 55 ppm. This shows that gallic EXAMPLE 9 acidisable to control mixed infection of coccidiosis inbroiler birds. It is also evident that gallic acid is the active ingredient In Vitro Effect of Gallic Acid on Eimeria tenella responsible for the anticoccidial activity of O. infectoria. Sporozoite Invasion and Proliferation in Host Cells 0101. An experiment was conducted to evaluate the in EXAMPLE 11 vitro effect of gallic acid monohydrate on Eimeria tenella sporozoite invasion and proliferation in host cells. Anti-Sporozoite Activity of Plants Containing Gallic 0102 Sporozoites were obtained from sporulated oocysts Acid after glass bead grinding and enzymatic excystation. As host cells, Madin-Darby Bovine Kidney (MDBK) cells, were 0109 Further, other plants that contain gallic acid were selected. Sporozoites were pre-treated with 10, 25 and 50 also evaluated for their anti-sporozoite activity and anticoc ppm gallic acid monohydrate for three hours. Thereafter, the cidial activity in broiler birds. The plants chosen were Rhus sporozoite Suspension was washed and put onto a culture of chinensis (Chinese gall nut) and Terminalia chebula (Indian MDBK cells for 20 hours. After incubation, the medium was gall nut). Rhus chinensis contains about 70% hydrolysable removed, cells were washed and fresh medium was added. tannins and Terminalia chebula contains around 0.28% free After 20, 72 and 96 hours the medium and MDBK cells were gallic acid. However, T. chebula contains 25 to 40% hydrolys collected and stored at -20°C. able tannins which can degrade to release gallic acid. These 0103) The negative control (neg ctrl) was MDBK cells plants have been reported for their antioxidant, anti-inflam infected with Eimeria sporozoites, incubated in cell culture matory, antibacterial, antifungal, antimutagenic and antican medium. The positive control (pos ctrl) was MDBK cells cer activities. infected with Eimeria sporozoites, incubated with a 5ug/ml 0110. Crude powder of fruit of Terminalia chebula and Solution of Salinomycin. gall nut of Rhus chinensis were obtained from Natural Rem 0104. At the different collection time points, DNA was edies, Bangalore, India and Xinjiang, respectively. The extracted from the MDBK cells. Real-time PCR to detect anti-sporozoite assay by MTT assay showed that both the Eimeria DNA was performed on the samples for the different tested plants were able to reduce the viability of sporozoites time points and different treatments. The PCR results are as compared to the control and better than the positive control, presented in FIG. 18. Salinomycin (FIG. 22). Real-Time PCR Analyses EXAMPLE 12 0105 Differences in Ct values were calculated for each time point versus T20 within one treatment (ACt). Fold In Vitro Effect of Plants Containing Gallic Acid on changes were calculated for each time point versus T20 using Eimeria tenella Sporozoite Invasion and the following equation: Proliferation in Host Cells Fold change=2^ 0106. These results are presented in FIG. 19. 0111. An experiment was conducted to evaluate the in 0107 The negative control shows a clear Eimeria prolif vitro effect of other sources of gallic acid on Eimeria tenella eration since there is a 20 fold increase in Eimeria DNA at 96 sporozoite invasion and proliferation in host cells. hours versus the start at 20 hours. The positive control as well 0112 Sporozoites were obtained from sporulated oocysts as the different dosages of gallic acid inhibited the Eimeria after glass bead grinding and enzymatic excystation. As host proliferation. There was a slightly lower effect visible for 10 cells, Madin-Darby Bovine Kidney (MDBK) cells, were ppm gallic acid. But this is negligible in comparison to the selected. Sporozoites were pre-treated with 50, 100 and 250 increase in the negative control. ppm of Terminalia chebula for three hours. Thereafter, the sporozoite Suspension was washed and put onto a culture of EXAMPLE 10 MDBK cells for 20 hours. After incubation, the medium was removed, cells were washed and fresh medium was added. Efficacy of Gallic Acid in Controlling Coccidiosis After 20, 72 and 96 hours the medium and MDBK cells were collected and stored at -20°C. 0108. The efficacy of gallic acid at three different dosages of 11, 22 and 33 ppm in controlling coccidiosis in broiler 0113. The negative control (neg ctrl) was MDBK cells birds was evaluated by an in vivo challenge trial. The birds infected with Eimeria sporozoites, incubated in cell culture were induced with mixed infection of Eimeria using oocysts medium. The positive control (pos ctrl) was MDBK cells of E. tenella, E. maxima and E. acervulina. These oocysts infected with Eimeria sporozoites, incubated with a 5ug/ml were isolated from birds confirmed with clinical coccidiosis. Solution of Salinomycin. The trial design, oocysts dosage, vaccination schedule, farm 0114. At the different collection time points, DNA was maintenance were similar to that of example 3. The lesion extracted from the MDBK cells. Real-time PCR to detect scoring showed that there was significant reduction in the Eimeria tenella DNA was performed on the samples for the score for all the three tested species of Eimeria as compared different time points and different treatments. The PCR to the infected control and even the positive control, Salino results are presented in FIG. 23. US 2014/01 6 1919 A1 Jun. 12, 2014

Real-Time PCR Analyses cious amounts of compounds selected from the group con sisting of gallic acid, derivatives of gallic acid, gallotannins 0115 Differences in Ct values were calculated for each and hydrolysable tannins time point versus T20 within one treatment (ACt). Fold 2. The compositions of claim 1, wherein the plants are changes were calculated for each time point versus T20 using selected from the group consisting of Quercus infectoria, the following equation: Rhus chinensis and Terminalia chebula. Fold change=2^ 3. The compositions of claim 1, wherein the extracts are 0116. These results for T. chebula are presented in FIG. 24. natural extracts. 0117 The negative control shows a clear Eimeria prolif 4. The compositions of claim 1, wherein the coccidiosis is eration since there is a 20 fold increase in Eimeria DNA at 96 caused by Eimeria spp. hours versus the start at 20 hours. The positive control as well 5. The compositions of claim 4, wherein the Eimeria spp. as 250 ppm T. chebula completely inhibited the Eimeria are selected from the group consisting of E. tenella, E. proliferation. There was a dose-response effect visible maxima and E. acervulina. although the lower effect for 100 ppm T. chebula is negligible 6. The compositions of claim 1, wherein controlling coc in comparison to the increase in the negative control cidiosis includes a reduction in caecal lesion score, oocysts per gram of fecal matter and mortality in poultry. EXAMPLE 13 7. A method of controlling coccidiosis in animals, com prising the step of administering an efficacious amount of Efficacy of Plants Containing Gallic Acid in either plant parts or extracts of plants selected from the group Controlling Coccidiosis consisting of Quercus infectoria, Rhus chinensis and Termi malia chebula. 0118. The efficacy of plants containing gallic acid namely, 8. The method of claim 7, wherein the plant parts or Terminalia chebula and Rhus chinensis in controlling coc extracts of plants contain efficacious amounts of compounds cidiosis in broiler birds was evaluated by an in vivo challenge selected from the group consisting of gallic acid, derivatives trial. The birds were induced with mixed infection of Eimeria of gallic acid, gallotannins and hydrolysable tannins. using oocysts of E. tenella, E. maxima and E. acervulina 9. The method of claim 7, wherein the extracts are natural isolated from birds confirmed with clinical coccidiosis. The eXtractS. trial design, oocysts dosage, vaccination schedule, farm 10. The method of claim 7, wherein the coccidiosis is maintenance were similar to that of example 3. The lesion caused by Eimeria spp. scoring showed that Rhus chinensis at 200 and 500 ppm and 11. The method of claim 10, wherein the Eimeria spp. are Terminalia chebula at 1000 ppm were able to reduce the score selected from the group consisting of E. tenella, E. maxima for all the three tested species of Eimeria as compared to the and E. acervulina. infected control and even the positive control, Salinomycin 12. The method of claim 7, wherein controlling coccidiosis (FIG.25). The oocysts per gram showed a similar trend (FIG. includes a reduction in lesion score, oocysts per gram of fecal 26), however, mortality was not observed in any of the treat matter and mortality. ment groups including the infected control. Dose dependent 13. A method of reducing the activity of sporozoites in an response was observed with Rhus chinensis. animal infected with Eimeria spp., comprising the step of 0119 The foregoing description and drawings comprise administering to the animal, plant parts or extracts of plants illustrative embodiments of the present inventions. The fore going embodiments and the methods described herein may containing an efficacious amount of at least one compound vary based on the ability, experience, and preference of those selected from the group consisting of gallic acid, derivatives skilled in the art. Merely listing the steps of the method in a of gallic acid, gallotannins and hydrolysable tannins certain order does not constitute any limitation on the order of 14. The method of claim 13, wherein the extracts are natu the steps of the method. The foregoing description and draw ral extracts. ings merely explain and illustrate the invention, and the 15. The method of claim 13, wherein the Eimeria spp. are invention is not limited thereto, except insofar as the claims selected from the group consisting of E. tenella, E. maxima are so limited. Those skilled in the art who have the disclosure and E. acervulina. before them will be able to make modifications and variations 16. The method of claim 13, wherein the reduction in the therein without departing from the scope of the invention. activity of sporozoites results in a reduction in caecal lesion We claim: score, oocysts per gram of fecal matter and mortality in poul 1. Compositions for controlling coccidiosis in animals, try. comprising plant parts or extracts of plants containing effica