WO2014004761A2 - Plant parts and extracts having anticoccidial activity - Google Patents

Abstract

Natural plant parts and extracts of plants selected from the group consisting of Quercus infectoria, Rhus chinensis and Terminalia chebula containing compounds such as gallic acid, derivative of gallic acid, gallotannins and hydrolysable tannins have been found to control coccidiosis in poultry and, more specifically, coccidiosis caused by Eimeria spp. The plant parts and natural extracts result in a reduction of lesion score, oocysts per gram of fecal matter and mortality.

Description

PLANT PARTS AND EXTRACTS HAVING ANTICOCCIDIAL ACTIVITY

[0001] This application claims priority to United States Patent Application Serial No. 61/664,795, filed June 27, 2012 and Indian Application No. 177/DEL/2013, filed January 23, 2013, both of which are incorporated herein by this reference.

Background of the Invention

[0002] 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.

[0003] 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- WP006949WV - accessed June 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). Major histocompatibility complex control of immunity elicited by genetically engineered Eimeria tenella (Apicomplexa) antigen in chickens. Infection and immunity, 57 (3): 701 - 705). The parasite invades the gut cells and causes necrosis in the intestine which leads to malabsorption, diarrhea, morbidity, reduction of weight gain, poor feed conversion, and, in severe cases, mortality (Williams, R.B (2005). Intercurrent coccidiosis and necrotic enteritis of chickens: Rational, integrated disease management by maintenance of gut integrity. Avian Pathology, 34(3), 159- 180). Seven different species of Eimeria, E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox, and E. 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.A., Akhtar, M.S., Anjum, M.I and Raja, M. L (2006). New dimension of medicinal plants as animal feed. Pakistan vet. J., 26(3): 144-148), they could be potential sources of bioactive molecules against coccidiosis in poultry. [0004] Others have attempted to use plant parts or plant extracts in treating coccidiosis. For example, 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.

[0005] 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.

[0006] 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.

[0007] 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.

Summary of the Invention

[0008] 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. Specifically, plant parts and natural extracts of Quercus injectoria, Rhus chinensis gall nut, Terminalia chebula fruit have been found to control coccidiosis in poultry and, more specifically, coccidiosis caused by Eimeria spp. More specifically, plant parts or extracts containing efficacious amounts of compounds selected from the group consisting of gallic acid, gallotannins and hydrolysable tannins. [0009] 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, gallo tannins and hydroly sable 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.

[00010] 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.

Brief Description of the Drawings

[00011] Fig. 1 is an image of caecal lesions of birds treated with Quercus infectoria

[00012] 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).

[00013] Fig. 3 is microphotographic images of the H and E stained sections of caecum of birds treated with Quercus infectoria.

[00014] 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).

[00015] 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).

[00016] 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).

[00017] Fig. 7 is a chart of PCR results after invasion of MDBK host cells with sporozoites and different concentrations of Q. infectoria.

[00018] Fig. 8 is a chart of fold changes in Eimeria DNA for different time points versus T4 within one treatment. [00019] Fig. 9 is a chart of PCR results after invasion of MDBK host cells with sporozoites, pre-treated with different concentrations of Q. infectoria.

[00020] Fig. 10 is a chart of fold changes in Eimeria DNA for different time points versus T20 within one treatment.

[00021] 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).

[00022] Fig.12 is a High Performance Liquid Chromatogram (HPLC) chromatogram of water fraction of Q. infectoria.

[00023] 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).

[00024] Fig. 14 is the LC/MS/MS chromatogram of peak 1 of Q. infectoria.

[00025] 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).

[00026] Fig. 16 is a chart of PCR results of MDBK host cells, pre-treated with 10 ppm gallic acid, invaded with sporozoites.

[00027] Fig. 17 is a chart of fold changes in Eimeria DNA for different time points versus T20 within one treatment.

[00028] Fig. 18 is a chart of PCR results after invasion of MDBK host cells with sporozoites, pre-treated with different concentrations of gallic acid.

[00029] Fig. 19 is a chart of fold changes in Eimeria DNA for different time points versus T20 within one treatment.

[00030] 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).

[00031] 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).

[00032] Fig. 22 is a chart of the anti -sporozoite activity of Rhus chinensis and Terminalia chebula; columns with different superscripts are statistically significant (p<0.05).

[00033] Fig. 23 is a chart of PCR results after invasion of MDBK host cells with sporozoites, pre-treated with different concentrations of T. chebula.

[00034] Fig. 24 is a chart of fold changes in Eimeria DNA for different time points versus T20 within one treatment. [00035] 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).

[00036] Fig. 21 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).

Description of the Invention

[00037] A preliminary in vivo evaluation of crude powder of Q. infectoria gall nuts (100- 800 micron particle size, procured from Pooja Herbs, Mumbai, India) in controlling coccidiosis at lOOg/ton of feed dose gave some indication of promising results which urged further evaluation at higher dosage. A 35 day in vivo trial conducted in broiler birds challenged with oocysts of Eimeria tenella showed that Q. infectoria gall nuts reduced the lesion score to 0 and mortality to 0%, comparable to the positive control (0%), whereas the negative control showed a score of 4 and a mortality of 17%. The histopathological analysis of the caecum samples showed that the birds treated with Q. infectoria showed lesser area infected by the parasite, lower mononuclear infiltrations and hemorrhages of the caecum.

[00038] In general in this description, 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. Generally, administration of gallic acid and gallic acid containing formulations are described with formulations providing a dosage from 0.1 to 50ppm, preferably from 2 to 20ppm, and most preferably from 3 to lOppm 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.

[00039] The efficacy of Q. infectoria crude powder in controlling mixed infection of Eimeria in broiler birds was evaluated. The results showed that there was significant reduction in the lesion score for E. tenella and E. acervulina as compared to the infected control and even the positive control, Salinomycin. Whereas in case of E. maxima, a numerical reduction in the lesion score was observed as compared to the infected control and Salinomycin. The oocysts per gram of treated groups were significantly lower than the infected control and Salinomycin group, however, mortality was not observed in any of the treatment groups including the infected control. This proves the efficacy of Q. infectoria in controlling coccidiosis caused by other species of Eimeria alsoFurther, to determine the mode of action of Q. infectoria, 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. Studies with Q. infectoria gall nut on the sporozoites of Eimeria tenella showed significant reduction in the viability of sporozoites compared to the sporozoite control. A dose dependent efficacy was observed in studies conducted with different dosages of Q. infectoria and the results were validated by conducting experiments several times independently. Hence, direct anti- sporozoite activity of Q. infectoria could be one of the modes of actions which attributes to the efficacy of the extract in controlling in coccidiosis in vivo.

[00040] Similarly, to determine the mode of action of Q. infectoria in controlling Eimeria in a host cell line, 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:

[00041] The products are combined with Eimeria sporozoites and added to the host cells.

[00042] The products are added to the sporozoites for a specific time, then removed and afterwards the sporozoites are added to the host cells.

[00043] The products are added to the host cells for a specific time, then removed and afterwards the sporozoites are added to the host cells.

[00044] The effect of Quercus infectoria was evaluated in the in vitro assay.

Example 1 - Efficacy of Quercus infectoria in controlling caecal coccidiosis

[00045] Experimental facility and study design. The screening trial was conducted at a poultry farm facility located in Gummidipundi, India. Straight run commercial hybrid broiler chickens, Gallus domesticus (Var. Vencobb 400) were used for the study. Day old male chicks were procured, weighed individually, wing banded, and randomly segregated into groups. The experimental design is detailed in Table 1. Table 1. Study design.

Category Trial Parameter

Rearing type Cages

Age of birds at the start of 1 day old

the trial

Total no of Birds 56 Number of groups 8

No of birds / groups 7

Duration of the trial 35 days

[00046] 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.

[00047] Vaccination schedule. The birds were vaccinated for Newcastle Disease Virus (NDV) and Infectious Bursal Disease (IBD).

[00048] 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.

[00049] Details of treatment groups. Groups and the treatments are shown in Table 2. The treated birds were fed with plant extract incorporated in the feed from day 1 (Table 2). The crude powders of Q. infectoria gall nut were procured from Pooja herbs, Mumbai, India. Table 2. Details of treatment groups and feed.

Groups Treatments

Control 1 (uninfected _{- T} ,„ .

Normal teed

control)

Control 2 (Negative infected „ _^

„ ° Coccidiosis induction + Normal teed

Control)

Coccidiosis induction + Feed with Coxistac* at

Control 3 (Positive Control) .„„„ ,

1000 g/ton

„ Coccidiosis induction + Feed with Q. infectoria at Treatment ,„„ ,

100 g/ton

* Coxistac is a product from Pfizer containing Salinomycin at 12 % concentration.

Hence, addition of Coxistac at the mentioned dose of 500g/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.

[00050] Induction of coccidiosis. Sporulated oocysts of E. tenella (Houghton strain

[Chapman, H.D. and Shirley, M.W. 2003. The Houghton strain of Eimeria tenella: A

1 of the type strain selected from genome sequencing. Avian Pathol., 32: 115-127] - propagated) were orally administered to each bird on day 14, 15 and 16 of age through oral gavage at a dose of 1 x 10⁵ oocysts/bird/day. Feeding was stopped on the day of inoculation, for 2 h before and 2 h after inoculation.

[00051] Parameters analyzed. The parameters that were chosen for analyses 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.

[00052] 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.

[00053] Mortality. The mortality of the birds was recorded on a daily basis and post mortem was carried out to confirm the cause of death.

[00054] Lesion scoring of the caecum. On day 5 and 7 post infection, 2 birds from each of the groups were sacrificed by cervical dislocation and the intestine was cut open. The caeca of the birds were scored for coccidiosis lesions. The scoring was done based on the severity of the lesions in the caecum and the presence of blood (Johnson, J. K., and W. M. Reid. (1970). Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Experimental Parasitology 28:30-36). The score for caecal coccidiosis was a scale of 0-4.

[00055] 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.

Results

[00056] Indices of pathogenesis. The observations on the excreta of the birds showed that the blood shedding in the infected groups started by day 4 post infection and the severity peaked on the day 5. The results of the scoring of the excreta are given in Table 3. Day 7 post infection the excreta were found to be normal with no blood. The positive control (C3, Table 3) on day 5 had a score of 3 as compared to the negative control (C2, Table 3) of 4. Birds treated with Q. infectoria had a lower score of 2 and were better than the positive control. Table 3. Scoring of excreta on day 5 post infection.

Treatments Score Description

CI - uninfected control 0 Excreta normal consistency

C2 - Negative infected control 4 Presence of heavy raw blood

C3 - Positive Control 3 Excreta with blood +++

T - Q.infectoria at lOOg/ton 2 Excreta with blood ++

+ - denotes the severity of blood loss and amount of blood in the excreta.

[00057] 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.

Table 4. Lesion score of the caeca on day 5 post infection

Treatments Lesion Score

CI - uninfected control 0

C2 - Negative infected control 3

C3 - Positive Control 3

T - Q. infectoria at lOOg/ton 2.5

[00058] 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. Unexpectedly, the anticoccidial Salinomycin treated birds (C3, Table 5) did not show any indication of reduction of oocysts as compared to the C2 (Table 5). The values presented are an average of three replicates.

Table 5. Oocysts per gram of excreta on day 7 post infection

Treatments Average Oocysts CV

Per Gram Excreta

C2 - Negative infected 2.5E + 05 1.23 control

C3 - Positive Control 4.0E + 05 0.68 T - Q.infectoria at 3.5E + 05 1.36 lOOg/ton [00059] Mortality. The rate of mortality was 17 % in control 2 (negative infected control). There was no mortality in other groups. The lesion score and OPG data of the positive control did not show any difference from that of the negative control.

[00060] Although the positive control did not perform well in this trial, the lesion scores of birds treated with plant extracts of Q. infectoria were lower than the negative infected control which indicates that they could be candidates for further investigation. However, they showed no reduction in the OPG.

Example 2 - Efficacy of Quercus infectoria in controlling caecal coccidiosis

[00061] 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 500g/ton and 4) treatment group including infected birds administered diet containing Q. infectoria gall nut at 500g/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 caecal lesions indicated that the negative control birds were highly infected with an average score of 4 whereas the positive control had score of 0. Birds treated with Quercus infectoria showed results similar to the positive control (0). Q. infectoria showed reduction in the OPG counts comparable to the positive control. The histopathological analysis of the caecum samples showed that the birds treated with Q. infectoria had lesser area affected by Eimeria, no hemorrhages and minimal mononuclear infiltrations up to the mucosa.

[00062] The second in vivo experiment involved the following treatment groups.

Table 6. Description of treatment groups

Groups Treatments

Control 1 (CI) Uninfected control - Normal feed without anticoccidial

Negative control - Coccidiosis induction + Normal feed without

Control 2 (C2)

anticoccidial

Positive Control - Coccidiosis induction + Normal feed with Coxistac 12

Control 3 (C3)

% @ 500 g/ton*

Treatment (T) Coccidiosis induction + Normal feed w Q. infectoria

* Coxistac is a product from Pfizer containing Salinomycin at 12 % concentration. Hence, addition of Coxistac at the mentioned dose of 500g/ton of feed will enable delivery of Salinomycin at 60 ppm levels in the feed which is the recommended preventive dose for broilers.

Results

[00063] Caecal lesions on day 5 post infection. The lesion scoring for caecal coccidiosis was carried out on day 5 post infection based on the criteria of scoring as before. The results of the scoring showed that the positive control completely alleviated the effects of caecal coccidiosis as compared to the negative infected control. Q. infectoria treated birds showed no lesions in the caecum and was comparable to the positive control and uninfected control CI (Table 7, Figure 1).

Table 7. Lesion scoring on day 5 post infection.

Treatments Lesion Score

CI - Uninfected control 0^e

C2 - Negative infected control 4^a

C3 - Positive Control 0^e

T - Q. infectoria f

Columns with different superscripts are statistically significant

(p<0.05).

[00064] 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.

[00065] Mortality. The rate of mortality was recorded during the experiment, and the data are given in Table 8. As expected, there was no mortality in the uninfected control group (CI) and the positive control group (C3). Q. infectoria supplemented group showed no mortality. Table 8. Rate of mortality during the trial period

[00066] Histopathological analysis of caecum samples. Q. infectoria showed positive reductions in all parameters tested such as lesion score, OPG and rate of mortality and the data were comparable to the positive control, Salinomycin. Hence, histopathological analysis of the caecum samples of birds from this group was carried out in comparison to the uninfected control (CI), negative control (C2) and positive control (C3). The severity and distribution of the lesions in the caecum were based on the grading provided in Table 9.

Table 9. Severity and distribution of lesions in the caecum of different groups

Uninfected Negative Positive Quercus

Histopathology

control control control infectoria

Mononuclear cell infiltration-

0 z 1

mucosa

Mononuclear cell infiltration-

0 z 0

submucosa

Mononuclear cell infiltration-

0 z 0 0

muscular layer

Hemorrhages 0 2 1 0

Necrosis- Villi 0 1 1 1

Distribution of stages of

0 1 1

Eimeria

Table 10. Histopathological findings of the tissues of caecum of birds

Groups Histopathological findings

Uninfected Cecum within normal histological limits,

control C 1

Negative Cecum showed moderate load of different Eimerial stages (oocyst, schizont control C2 and merozoite) along with mild mucosal hemorrhages and mild to moderate mononuclear cell infiltration in mucosal, submucosal and muscular layers.

Positive Cecum showed minimal load of different Eimerial stages with schizonts and control C3 merozoites contributing the major load. Minimal mucosal hemorrhages and necrosis was evident microscopically. Mild to moderate mononuclear cell infiltration in mucosal and submucosal layers was seen.

Q. infectoria Cecum showed minimal load of different Eimerial stages with oocyst T contributing the major load. Minimal mucosal necrosis and mononuclear cell infiltration in mucosal layers was evident.

[00067] Histopathological results showed that the birds treated with Q. infectoria had fewer regions of the caecum infected with E. tenella, and the mononuclear infiltration was restricted only to the mucosa with a score of 1 indicating mild infiltration (Fig. 3). The submucosa and muscular layers were free from infiltration (Table 10). In the negative control, mononuclear infiltration was observed in the mucosa, submucosa and even the muscular layer. There were no hemorrhages in the caecum of birds treated with Q. infectoria as compared to that of the negative control (2). This indicates that the caecum of birds treated with Q. infectoria was less infected than the positive control.

[00068] The in vivo screening of plant extracts revealed that 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.

Example 3 - Efficacy of water extracts of Quercus infectoria in controlling mixed infection of coccidiosis

[00069] 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 X 10⁵ 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.

a. The screening trial was conducted at Kemin's in-house R&D poultry farm facility located in Gummidipundi, India. Straight run commercial hybrid broiler chickens, Gallus domesticus (Var. Vencobb 400) were used for the study. Day old male chicks were procured, weighed individually, wing banded, and randomly segregated into groups. The experimental design is detailed in Table 11. Good farm managing practices and vaccination schedule were followed during the 3^rd in vivo trial as mentioned in example 1.

Table 11. Study design

Category Trial Parameter

Duration of the trial 35 days

Breed Cobb 400

Rearing type Cages

Age of birds at the start of 1 day old

the trial

Total no of Birds 315

Number of groups 21

No of birds / groups 15 (male)

[00070] The birds were vaccinated for Newcastle Disease Virus (NDV) and Infectious Bursal Disease (IBD). A corn soya based mash diet was used for the study. The birds were fed with the extract of Q. infectoria gall nut incorporated in the feed from day 1. The treatment groups are given in Table 12.

Table 12. Details of treatment groups for the trial

Coccidiosis induction + Normal feed with Q. infectoria water

Treatment

extract at 100 g/ton

[00071] 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.

[00072] The results showed that there was significant reduction in the lesion score for E. tenella and E. acervulina as compared to the infected control and even the positive control, Salinomycin. Whereas, in case of E. maxima, a numerical reduction in the lesion score was observed as compared to the infected control and Salinomycin (Fig. 4). The oocysts per gram of treated groups were significantly lower than the infected control and Salinomycin group (Fig. 5), however, mortality was not observed in any of the treatment groups including the infected control. This proves the efficacy of Q. infectoria in controlling coccidiosis caused by other species of Eimeria also.

Example 4 - In vitro anti-sporozoite activity of 0. infectoria by MTT Assay

[00073] Further, 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 10⁵ 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 μ syringe filter. Following 24 h of incubation with the plant extracts, the sporozoites were thoroughly washed and then MTT assay was performed. 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.

[00074] 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.

Example 5 - In vitro effect of Q. infectoria on Eimeria tenella sporozoite invasion and proliferation in host cells

[00075] An experiment was conducted to evaluate the in vitro effect of Q. infectoria on Eimeria tenella sporozoite invasion and proliferation of host cells.

[00076] 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.

[00077] The negative control (neg Ctrl) was MDBK cells infected with Eimeria sporozoites, incubated in cell culture medium. The positive control (pos Ctrl) was MDBK cells infected with Eimeria sporozoites, incubated with a 5 μg/ml solution of Salinomycin.

[00078] At the different collection time points, 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 Figure 7.

Real-time PCR analyses

[00079] Differences in Ct values were calculated for each time point versus T4 within one treatment (ACt). Fold changes were calculated for each time point versus T4 using the following equation:

Fold change = 2 ^{~ ct}

[00080] These results are presented in Figure 8.

[00081] 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. Also for the Q. infectoria treatments, a clear inhibition of the proliferation was observed versus the start of 4 hours, in a dose dependent manner.

Example 6 - In vitro effect of Q. infectoria on Eimeria tenella sporozoite invasion and proliferation in host cells. [00082] An experiment was conducted to evaluate the in vitro effect of Q. infectoria on Eimeria tenella sporozoite invasion and proliferation in host cells.

[00083] 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.

[00084] The negative control (neg Ctrl) was MDBK cells infected with Eimeria sporozoites, incubated in cell culture medium. The positive control (pos Ctrl) was MDBK cells infected with Eimeria sporozoites, incubated with a 5 μg/ml solution of Salinomycin.

[00085] At the different collection time points, 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 Figure 9.

Real-time PCR analyses

[00086] Differences in Ct values were calculated for each time point versus T20 within one treatment (ACt). Fold changes were calculated for each time point versus T20 using the following equation:

Fold change = 2 ^{"A t}

[00087] These results for Q. infectoria are presented in Figure 10.

[00088] 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. Also the different dosages of Q. infectoria all inhibited the Eimeria proliferation. There was a slightly lower effect visible for 50 ppm Q. infectoria. But this is negligible in comparison to the increase in the negative control.

Example 7 - Identification of active ingredient/s of Q. infectoria

[00089] Further to this, 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).

[00090] Phytochemical analyses of the active fractions were carried out by High Performance Liquid Chromatography (HPLC) to identify the active ingredient/s responsible for the anti-sporozoite activity. Four major peaks were observed in the HPLC chromatogram of both methanol and water fractions, with one peak corresponding to the retention time of a gallic acid standard (Fig. 12). Q. infectoria gall nut are known to possess 60 to 70 % hydrolysable tannins which can hydrolyse to release gallic acid in addition to about 7 % free gallic acid present in it.

[00091] These four compounds were separated by semi - preparative HPLC and anti- sporozoite activity was evaluated in comparison to the crude powder in equivalent concentrations. The anti-sporozoite activity of the compounds showed that compound of peak 1 had the best anti-sporozoite activity. The other compounds showed minimal activity against the sporozoites. However, the crude powder showed better activity than the peak 1 indicating synergistic activity of the compounds from the extract (Fig. 13).

[00092] LC/MS/MS analysis of the different peaks of the HPLC chromatogram confirmed that peak 1 was gallic acid (Fig. 14) and the other peaks were high molecular weight compounds which could be degraded products of hydrolysable tannins. It was hypothesized that these compounds can further breakdown to release gallic acid.

[00093] Further, to arrive at the correlation between gallic acid % and anti-sporozoite activity, Q. infectoria was extracted in water for 5 min, 2 and 12 h and their anti-sporozoite activity was evaluated. The study showed that there was a clear correlation (correlation coefficient= -0.982226) between the concentration of gallic acid and anti-sporozoite activity (Fig. 15). These results indicate that gallic acid is the active ingredient responsible for the anti-sporozoite activity of Q. infectoria.

Example 8 - In vitro protective effect of Gallic acid

[00094] An experiment was conducted to evaluate the in vitro protective effect of gallic acid monohydrate on host cells challenged with Eimeria tenella sporozoites.

[00095] 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.

[00096] The negative control (neg Ctrl) was MDBK cells infected with Eimeria sporozoites, incubated in cell culture medium. The positive control (pos Ctrl) was MDBK cells infected with Eimeria sporozoites, incubated with a 5 μg/ml solution of Salinomycin.

[00097] At the different collection time points, 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 Figure 16.

Real-time PCR analyses

[00098] Differences in Ct values were calculated for each time point versus T20 within one treatment (ACt). Fold changes were calculated for each time point versus T20 using the following equation:

Fold change = 2^{"A t}

[00099] These results are presented in Figure 17.

[000100] 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. Also for 10 ppm 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.

Example 9 - In vitro effect of gallic acid on Eimeria tenella sporozoite invasion and proliferation in host cells

[000101] An experiment was conducted to evaluate the in vitro effect of gallic acid monohydrate on Eimeria tenella sporozoite invasion and proliferation in host cells.

[000102] 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. [000103] The negative control (neg Ctrl) was MDBK cells infected with Eimeria sporozoites, incubated in cell culture medium. The positive control (pos Ctrl) was MDBK cells infected with Eimeria sporozoites, incubated with a 5 μg/ml solution of Salinomycin.

[000104] At the different collection time points, 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 Figure 18.

Real-time PCR analyses

[000105] Differences in Ct values were calculated for each time point versus T20 within one treatment (ACt). Fold changes were calculated for each time point versus T20 using the following equation:

Fold change = 2 ^{~ ct}

[000106] These results are presented in Figure 19.

[000107] 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.

Example 10 - Efficacy of gallic acid in controlling coccidiosis

[000108] 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). The oocysts per gram showed a similar trend (Fig 21), however, mortality was not observed in any of the treatment groups including the infected control. Dose dependent response was observed with no signifcant difference between gallic acid at 22 and 55 ppm. This shows that gallic acid is able to control mixed infection of coccidiosis in broiler birds. It is also evident that gallic acid is the active ingredient responsible for the anticoccidial activity of Q. infectoria. Example 11 - Anti-sporozoite activity of plants containing gallic acid

[000109] Further, other plants that contain gallic acid were also evaluated for their anti- sporozoite activity and anticoccidial activity in broiler birds. The plants chosen were Rhus chinensis (Chinese gall nut) and Terminalia chebula (Indian gall nut). Rhus chinensis contains about 70 % hydrolysable tannins and Terminalia chebula contains around 0. 28% free gallic acid. However, T. chebula contains 25 to 40% hydrolysable tannins which can degrade to release gallic acid. These plants have been reported for their antioxidant, antiinflammatory, antibacterial, antifungal, antimutagenic and anticancer activities.

[000110] Crude powder of fruit of Terminalia chebula and gall nut of Rhus chinensis were obtained from Natural Remedies, Bangalore, India and Xinjiang, China respectively. The anti-sporozoite assay by MTT assay showed that both the tested plants were able to reduce the viability of sporozoites as compared to the control and better than the positive control, Salinomycin (Fig 22).

Example 12 - In vitro effect of plants containing gallic acid on Eimeria tenella sporozoite invasion and proliferation in host cells

[000111] An experiment was conducted to evaluate the in vitro effect of other sources of gallic acid on Eimeria tenella sporozoite invasion and proliferation in host cells.

[000112] 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.

[000113] The negative control (neg Ctrl) was MDBK cells infected with Eimeria sporozoites, incubated in cell culture medium. The positive control (pos Ctrl) was MDBK cells infected with Eimeria sporozoites, incubated with a 5 μg/ml solution of Salinomycin.

[000114] At the different collection time points, 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 Figure 23.

Real-time PCR analyses [000115] Differences in Ct values were calculated for each time point versus T20 within one treatment (ACt). Fold changes were calculated for each time point versus T20 using the following equation:

Fold change = 2 ^{"A t}

[000116] These results for T. chebula are presented in Figure 24.

[000117] 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

Example 13 - Efficacy of plants containing gallic acid in controlling coccidiosis

[000118] 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. The lesion scoring showed that 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.

[000119] The foregoing description and drawings comprise illustrative embodiments of the present inventions. The foregoing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The foregoing description and drawings merely explain and illustrate the invention, and the invention is not limited thereto, except insofar as the claims are so limited. Those skilled in the art who have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention.

Claims

We claim:

1. Compositions for controlling coccidiosis in animals, comprising plant parts or extracts of plants containing efficacious amounts of compounds selected from the group consisting of gallic acid, derivatives of gallic acid, gallotannins and hydrolysable tannins.

2. The compositions of claim 1, wherein the plants are selected from the group consisting of Quercus infectoria, Rhus chinensis and Terminalia chebula.

3. The compositions of claim 1, wherein the extracts are natural extracts.

4. The compositions of claim 1, wherein the coccidiosis is caused by Eimeria spp.

5. The compositions of claim 4, wherein the Eimeria spp. are selected from the group consisting of E. tenella, E. maxima and E. acervulina.

6. The compositions of claim 1, wherein controlling coccidiosis includes a reduction in caecal lesion score, oocysts per gram of fecal matter and mortality in poultry.

7. A method of controlling coccidiosis in animals, comprising the step of administering an efficacious amount of either plant parts or extracts of plants selected from the group consisting of Quercus infectoria, Rhus chinensis and Terminalia chebula.

8. The method of claim 7, wherein the plant parts or extracts of plants contain efficacious amounts of compounds selected from the group consisting of gallic acid, derivatives of gallic acid, gallotannins and hydrolysable tannins.

9. The method of claim 7, wherein the extracts are natural extracts.

10. The method of claim 7, wherein the coccidiosis is caused by Eimeria spp.

11. The method of claim 10, wherein the Eimeria spp. are selected from the group consisting of E. tenella, E. maxima and E. acervulina.

12. The method of claim 7, wherein controlling coccidiosis includes a reduction in lesion score, oocysts per gram of fecal matter and mortality.

13. A method of reducing the activity of sporozoites in an animal infected with Eimeria spp., comprising the step of administering to the animal, plant parts or extracts of plants containing an efficacious amount of at least one compound selected from the group consisting of gallic acid, derivatives of gallic acid, gallotannins and hydrolysable tannins.

14. The method of claim 13, wherein the extracts are natural extracts.

15. The method of claim 13, wherein the Eimeria spp. are selected from the group consisting of E. tenella, E. maxima and E. acervulina.

16. The method of claim 13, wherein the reduction in the activity of sporozoites results in a reduction in caecal lesion score, oocysts per gram of fecal matter and mortality in poultry.