Abstract
Avian coccidiosis has a major economic impact on the poultry industry, it is caused by 7 species of Eimeria, and has been primarily controlled using chemotherapeutic agents. Due to the emergence of drug-resistant strains, alternative control strategies are needed. We assessed anticoccidial effects of berberine-based diets in broiler chickens following oral infection with 5 Eimeria species (E. acervulina, E. maxima, E. tenella, E. mitis, and E. praecox). When 0.2% berberine, a concentration that does not affect weight gain, was added to the diet, the 4 groups infected with E. acervulina, E. tenella, E. mitis, or E. praecox showed significant reductions in fecal oocyst shedding (P<0.05) compared to their respective infected and untreated controls. In chickens treated 0.5% berberine instead of 0.2% and infected with E. maxima, fecal oocyst production was significantly reduced, but body weight deceased, indicating that berberine treatment was not useful for E. maxima infection. Taken together, these results illustrate the applicability of berberine for prophylactic use to control most Eimeria infections except E. maxima. Further studies on the mechanisms underlying the differences in anticoccidial susceptibility to berberine, particularly E. maxima, are remained.
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Key words: Eimeria species, berberine, chicken, anticoccidial effect, different susceptibility
Coccidiosis is an enteric disease caused by infection with one or multiple species of
Eimeria and is the most costly and prevalent disease in the poultry industry worldwide [
1–
3]. Several studies have shown that the worldwide prevalence of
Eimeria infection varies from 10% to 90% in chicken farms [
1,
4]. The etiologic agents of avian coccidiosis are intracellular protozoan parasites of the genus
Eimeria that infect different locations of chicken intestinal tracts. The prevalent poultry
Eimeria species are
E. acervulina,
E. tenella,
E. maxima,
E. necatrix,
E. brunetti,
E. mitis, and
E. praecox [
3,
5,
6]. The infectious parasites invade intestinal epithelial cells, causing a variety of clinical signs, such as necrotic gut lesions, inefficient feed conversion rates, impaired growth rates, and, in severe cases, mortality [
2,
5,
7]. To date, the poultry industry relies mainly on prophylactic in-feed anticoccidial agents to suppress the infection cycle and prevent coccidiosis outbreaks [
8]. Although prophylactic drugs have been relatively successful in controlling outbreaks of avian coccidiosis, due to the development of drug-resistant parasites and increasing public health concerns about anticoccidial medication use, new approaches to fight this disease are needed [
8–
10].
Several natural products have been examined for potential therapeutic and prophylactic effects against
Eimeria, one of which is berberine [
9,
11–
14]. Berberine is a yellow isoquinoline alkaloid extracted from the stems and roots of various plants, such as
Berberis,
Hydrastis canadensis, and
Coptidis rhizoma, and is used in Chinese medicine to treat gastrointestinal diseases [
15,
16]. Additionally, berberine has several different bioactivities including antiviral, antibacterial, anticancer, antidiabetic, analgesic, anti-hyperlipidemic, cardio protective, and anti-inflammatory effects [
14,
15,
17–
19].
Recently, berberine has received more attention due to its potential antiparasitic effects [
20–
23]. A limited number of studies have examined the effect of berberine in mice infected with
E. papillata [
12] and chickens infected with only 1 species
E. tenella [
14,
24]. In chickens, coccidiosis can be caused by 7 species of
Eimeria and each
Eimeria strain invades the intestinal epithelium in a region-specific manner [
2,
3]. A commercial herbal formula containing a propylene glycol extract of
Allium sativum and
Thymus serpyllum was effective at reducing duodenal lesions caused by
E. acervulina but is not effective in reducing cecal lesions caused by
E. tenella [
25]. Therefore, natural products may have different effects on the clinical symptoms caused by different species of
Eimeria. Therefore, this study was conducted to further investigate anticoccidial activity of berberine-based diets in broiler chickens following oral infection with 5
Eimeria species, including
E. acervulina,
E. maxima,
E. tenella,
E. mitis, and
E. praecox.
All animal maintenance and experimental procedures were performed according to Gyeongsang National University Guidelines for the Care and Use of Experimental Animals and approved by the Institutional Animal Care and Use Committee (IACUC) of Gyeongsang National University (GNU-191111-C0058). Humane endpoint criteria were set for all animals such that severe moribund animals exhibiting severe weight loss and tremors or unresponsive and unaware of stimuli were euthanized immediately by atlanto-occipital dislocation. All remaining animals were euthanized at specific timepoints post-inoculation.
ROSS308 broiler chicks (Samhwa, Hongseong-gun, Korea) were raised in wire cages in a temperature-controlled environment with unlimited access to anticoccidial/antibiotic-free feed and water. Constant light was provided for the duration of the experiments, and infected and non-infected birds were housed separately in different rooms. The wild-type strains of E. acervulina, E. maxima, E. tenella, E. mitis, and E. praecox developed and maintained at the Gyeongsang National University (Jinju, Korea) were used in this study. Feces were collected from Eimeria-infected chickens and diluted with phosphate buffered saline (PBS). Fecal samples were then passed through gauze to remove debris and washed 3 times with PBS by centrifugation. Precipitates were suspended in 2.5% potassium dichromate (Daejung Chemicals and Metals Co. Ltd, Siheung, Korea) and incubated at 28°C for 2 days for sporulation. Sporulated oocysts for experimental infections were enumerated using a McMaster counting chamber.
Berberine hydrochloride was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) at the highest available purity (≥97%). Male chickens were inoculated orally with 1×104 sporulated Eimeria oocysts and fed a standard diet supplemented with powdered berberine beginning 2 days prior to infection and throughout the experimental period. Oocysts for infection were cleaned by flotation on 5.25% sodium hypochlorite and washed 3 times with PBS.
Fecal samples were collected from 6 to 9 days post-infection and homogenized in a blade grinder. Two 30-ml samples were collected from each suspension. Samples were diluted in saturated NaCl, and oocysts were counted microscopically in a McMaster counting chamber. Total oocyst numbers were calculated as oocyst count×dilution factor×(fecal sample volume/counting chamber volume).
Data were analyzed using Student’s t-test or one-way ANOVA, and Dunnett’s multiple comparison tests using InStat statistical software (GraphPad, San Diego, California, USA). Differences were considered significant at P<0.05. Data are expressed as the mean±standard error (SE).
Chickens were treated with 4 different concentrations of berberine (0.05, 0.1, 0.2, or 0.3%) and infected with
E. tenella. Fecal oocysts were collected from 6 to 9 days post-infection and oocysts were counted. The 0.2% and 0.3% treated groups showed a significant reduction in oocyst production compared to the untreated/infected group. However, the 0.05% and 0.1% groups had no suppressive effects on oocyst production (
Fig. 1A). To determine if berberine treatment affects sporulation rates, the collected fecal oocysts were sporulated at 28°C for 2 days. Sporulation rates of all treatment groups were similar to that of the untreated/infected group (
P>0.05) (
Fig. 1B).
Furthermore, fecal oocysts were collected from
E. maxima- or
E. tenella-infected chickens and then treated with 3 different concentrations of berberine.
E. tenella samples were treated with berberine at concentrations of 0.1%, 0.2%, and 0.3% (
Table S1).
E. maxima samples were treated with berberine at concentrations of 0.1%, 0.2%, and 0.5% (
Supplementary Fig. S1B). Sporulation rates of all treatment groups were similar to the untreated groups (
P>0.05) (
Supplementary Fig. S1).
Because berberine treatment has the potential to cause reductions in weight gain [
16,
26], we monitored weight gain in chickens after feeding 3 different concentrations of berberine (0.1, 0.2, and 0.5%) for 2 or 6 days (
Table 1). Body weight gain was not significantly affected by 0.1% and 0.2% berberine supplementation for 6 days. However, the 0.5% berberine-treated groups showed significant reductions (
P<0.001) in weight gain compared to the untreated chickens. The groups fed 0.5% berberine for 2 or 6 days showed weight loss of approximately 31.5% or 45.5%, respectively (
Table 1). It is speculated that the weight loss of berberine may be partly mediated by the reduced rate of glucose absorption through delayed carbohydrate digestion and extended digestion time and by alleviating proliferation and differentiation of adipose tissue [
16,
26]. Therefore, 0.2% berberine treatment was used for subsequent experiments.
Seven species of
Eimeria infect the intestinal epithelium in a region-specific manner. Thus, to evaluate the effect of berberine in chickens infected with different species of
Eimeria, chickens were treated with 0.2% berberine and infected with 5
Eimeria species (
E. acervulina,
E. maxima,
E. tenella,
E. mitis, or
E. praecox). Fecal oocyst shedding was significantly reduced (
P<0.001) in berberine-treated chickens infected with
E. acervulina (
Fig. 2A),
E. tenella (
Fig. 2B),
E. mitis (
Fig. 2C), or
E. praecox (
Fig. 2D) compared to the respective untreated/infected controls. However, in berberine-treated chickens infected with
E. maxima, fecal oocyst shedding was similar to that of the untreated and infected control; however, treatment with 0.5% berberine significantly reduced fecal oocyst shedding (
Fig. 2E).
The etiologic agents of chicken coccidiosis consist of more than 7 species of the genus
Eimeria, an intracellular protozoan parasite. The various
Eimeria species infect different areas of the chicken intestinal tract [
5,
6], thus
Eimeria species may exhibit differences in susceptibility to alternative products, such as herbal products, essential oils, organic minerals, and probiotics [
25]. One of the alternative products is berberine that has potential anticoccidial properties as demonstrated within a limited range, namely in
E. tenella infected-chickens [
14,
24]. Therefore, this study investigated whether berberine differentially affects chickens infected with various species of
Eimeria,
E. acervulina,
E. maxima,
E. tenella,
E. mitis, and
E. praecox. Furthermore, we monitored whether berberine affects sporulation with berberine-treated chicken stool samples or berberine-supplemented stool samples.
Berberine intercalates into viral DNA and inhibits DNA synthesis, protein biosynthesis, and enzyme activity, resulting in reduced virus replication [
10,
18]. Berberine also exhibits direct antimicrobial activity on Gram-negative bacteria, Gram-positive bacteria, filamentous fungi, and yeast [
27], and dose-dependent inhibition of promastigote cell growth in the parasite
Leishmania [
28]. Furthermore, the sporulation rate of fecal oocysts is a critical factor affecting the epidemiology of
Eimeria infection in a chicken flock because chickens can only be infected via ingestion of sporulated oocysts [
6].
In our study, berberine did not inhibit oocyst sporulation rates in experiments with fecal samples obtained after berberine treatment and
Eimeria infection (
Fig. 1B) or in fecal samples supplemented with berberine (
Supplementary Fig. S1). The oocyst sporulation rate was not inhibited in
E. maxima samples treated with 0.5% berberine (
Supplementary Fig. S1B). Therefore, oocysts excreted by berberine-treated birds did not have impaired sporulation and berberine at the concentrations used in the in vitro study did not affect the sporulation process of unsporulated oocysts. However, several natural extracts inhibit the formation of oocyst sporulation under in vitro conditions [
29,
30]. Artemisinin from
Artemisia annua extracts inhibit the sporulation rate of mixed oocysts of
E. acervulina,
E. necatrix, and
E. tenella; many of the sporulated oocysts were wrinkled or included abnormal sporocysts [
29]. Similarly,
Aloe debrana and
Aloe pulcherrima leaf gels inhibited the sporulation rate of mixed oocysts of
E. acervulina,
E. maxima,
E. necatrix, and
E. tenella [
30].
Although berberine did not affect sporulation rates under in vitro conditions, berberine treatment significantly reduced fecal oocyst shedding in chickens infected with different
Eimeria species (
Fig. 2), indicating that berberine had anticoccidial activity only in vivo. Interestingly, previous studies of
E. tenella showed that berberine-treated,
E. tenella-infected chickens significantly reduced fecal oocyst shedding [
14,
24]. It is speculated that anticoccidial activity of berberine may be partly mediated by generation of a redox imbalance and depolarization of mitochondrial membrane [
28], by impairment of intracellular development and multiplication of
Eimeria [
12] or by inhibition of telomerase activity [
21]. Further analysis of the mechanisms or metabolic pathways would be necessary to know the precise anticoccidial effects of berberine.
Chickens can be infected with 7 species of
Eimeria parasites that invade the intestinal epithelium in a region-specific manner [
2,
3]. In our study, berberine had differing anticoccidial activities depending on the
Eimeria specie. Fecal oocyst shedding was significantly reduced in chickens treated with 0.2% berberine that were infected with
E. acervulina,
E. tenella,
E. mitis, or
E. praecox; whereas
E. maxima oocyst shedding was only significantly reduced in chickens treated with 0.5% berberine. Interestingly, a commercially available herbal formula was effective in reducing duodenal lesions caused by
E. acervulian, but not cecal lesions caused by
E. tenella [
25]. Therefore, it is possible that natural products have different efficacies for the various
Eimeria species.
In conclusion, Berberine-based diets significantly inhibited fecal oocyst shedding in chickens infected with various Eimeria species. E. maxima unlike the other 4 species was more resistant to berberine treatment, suggesting that natural products may have different anticoccidial activities on the various Eimeria species. Taken together, our data illustrate the applicability of berberine for prophylactic use to control most eimeriosis in conventional and organic chicken industries. Further research remains on the mechanisms leading to differences in anticoccidial susceptibility to berberine, especially E. maxima.
Notes
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We declare that we have no conflict of interest related to this work.
Supplementary Information
Supplementary Fig. S1.
Effect of berberine on in vitro oocyst sporulation. One-week-old male chickens (ROSS308) (n=10/group) were orally infected with 1×104 sporulated E. tenella or E. maxima oocysts and fed a standard diet throughout the experimental period. Fecal oocysts were collected from 6 to 9 days post-infection. (A) E. tenella samples were supplemented with berberine at concentrations of 0.1%, 0.2%, and 0.3%. (B) E. maxima samples were supplemented with berberine at concentrations of 0.1%, 0.2%, and 0.5%. Fecal samples for sporulation were incubated at 28°C for 2 days following berberine treatment. Sporulated oocysts were enumerated using a McMaster counting chamber. Data represent the mean±SE from 3 replicates and 1 representative of 2 independent experiments with similar results.
ACKNOWLEDGMENT
This research was supported by the Basic Science Research Program through the NRF of Korea funded by the Ministry of Education (2018R1D1A1B07045179) and IPET in Food, Agriculture, Forestry and Fisheries through the Agriculture, Food and Rural Affairs Research Center Support Program, funded by MAFRA (716002-7).
Fig. 1Effect of berberine on oocyst shedding and sporulation. Twelve-day-old male chickens were inoculated orally with 1×104 sporulated oocysts of E. tenella and fed a standard diet supplemented with powdered berberine (0.05, 0.1, 0.2, or 0.3%) beginning 2 days prior to infection. (A) Fecal oocyst shedding to berberine treatment (n=48/group). Fecal materials were collected on days 6 to 9 post-infection and oocyst numbers were assessed. Data represent the mean±SE from 4 replicates, with 12 chickens in each replicate and one representative of 2 independent experiments. Control indicates uninfected, untreated healthy controls. ***P<0.001 compared to untreated and infected group. BBR, berberine. (B) Fecal materials were incubated at 28°C for 2 days for sporulation. Sporulated oocysts were enumerated using a McMaster counting chamber. Data represent the mean±SE from 3 replicates and one representative of 2 independent experiments.
Fig. 2Fecal oocyst shedding following berberine-based diets in chickens infected with Eimeria species. One week-old male chickens (n=28/group) were inoculated orally with 1×104 sporulated E. acervulina, E. tenella, E. mitis, E. praecox, or E. maxima oocysts and fed a standard diet supplemented with powdered berberine (0.2 or 0.5%) beginning 2 days prior to infection. Fecal oocysts were collected from 6 to 9 days post-infection and oocyst numbers were assessed. Data represent the mean±SE from 2 replicates, with 14 chickens in each replicate and one representative of 2 independent experiments. ***P<0.001 compared to untreated and infected group. BBR, berberine.
Table 1Effect of berberine treatment on body weight gain compared to untreated chickens
Table 1
|
Period of berberine treatment |
Concentrations of berberine |
|
Control |
0.1% |
0.2% |
0.5% |
|
2 days |
100±9.7 |
ND |
108.4±12.1**
|
68.5±10.4***
|
|
6 days |
100±22.6 |
103±21.8 |
98.3±22.2 |
55±12.2***
|
References
- 1. Lee BH, Kim WH, Jeong J, Yoo J, Kwon YK, Jung BY, Kwon JH, Lillehoj HS, Min W. Prevalence and cross-immunity of Eimeria species on Korean chicken farms. J Vet Med Sci 2010;72:985-989. https://doi.org/10.1292/jvms.09-0517
- 2. Min W, Kim WH, Lillehoj EP, Lillehoj HS. Recent progress in host immunity to avian coccidiosis: IL-17 family cytokines as sentinels of the intestinal mucosa. Deve Comp Immunol 2013;41:418-428. https://doi.org/10.1016/j.dci.2013.04.003
- 3. Soutter F, Werling D, Tomley FM, Blake DP. Poultry coccidiosis: Design and interpretation of vaccine studies. Front Vet Sci 2020;7:101. https://doi.org/10.3389/fvets.2020.00101
- 4. Venkatas J, Adeleke MA. A review of Eimeria antigen identification for the development of novel anticoccidial vaccines. Parasitol Res 2019;118:1701-1710. https://doi.org/10.1007/s00436-019-06338-2
- 5. Kim WH, Chaudhari AA, Lillehoj HS. Involvement of T cell immunity in avian coccidiosis. Front Immunol 2019;10:2732. https://doi.org/10.3389/fimmu.2019.02732
- 6. López-Osorio S, Chaparro-Gutiérrez JJ, Gómez-Osorio LM. Overview of poultry Eimeria life cycle and host-parasite interactions. Front Vet Sci 2020;7:384. https://doi.org/10.3389/fvets.2020.00384
- 7. Dalloul RA, Lillehoj HS. Poultry coccidiosis: recent advancements in control measures and vaccine development. Expert Rev Vaccines 2006;5:143-163. https://doi.org/10.1586/14760584.5.1.143
- 8. Blake DP, Knox J, Dehaeck B, Huntington B, Rathinam T, Ravipati V, Ayoade S, Gilbert W, Adebambo AO, Jatau ID, Raman M, Parker D, Rushton J, Tomley FM. Re-calculating the cost of coccidiosis in chickens. Vet Res 2020;51:115. https://doi.org/10.1186/s13567-020-00837-2
- 9. Kadykalo S, Roberts T, Thompson M, Wilson J, Lang M, Espeisse O. The value of anticoccidials for sustainable global poultry production. Int J Antimicrob Agents 2018;5:304-310. https://doi.org/10.1016/j.ijantimicag.2017.09.004
- 10. Li XL, Hu YJ, Wang H, Yu BQ, Yue HL. Molecular spectroscopy evidence of berberine binding to DNA: comparative binding and thermodynamic profile of intercalation. Biomacromolecules 2012;13:873-880. https://doi.org/10.1021/bm2017959
- 11. Chang CL, Yang CY, Muthamilselvan T, Yang WC. Field trial of medicinal plant, Bidens pilosa, against eimeriosis in broilers. Sci Rep 2016;6:24692. https://doi.org/10.1038/srep24692
- 12. Dkhil MA, Metwaly MS, Al-Quraishy S, Sherif NE, Delic D, Al Omar SY, Wunderlich F. Anti-Eimeria activity of berberine and identification of associated gene expression changes in the mouse jejunum infected with Eimeria papillata. Parasitol Res 2015;114:1581-1593. https://doi.org/10.1007/s00436-015-4344-z
- 13. Jang SI, Jun MH, Lillehoj HS, Dalloul RA, Kong IK, Kim S, Min W. Anticoccidial effect of green tea-based diets against Eimeria maxima. Vet Parasitol 2007;144:172-175. https://doi.org/10.1016/j.vetpar.2006.09.005
- 14. Malik TA, Kamili AN, Chishti MZ, Tanveer S, Ahad S, Johri RK. Synergistic approach for treatment of chicken coccidiosis using berberine--a plant natural product. Microb Pathog 2016;93:56-62. https://doi.org/10.1016/j.micpath.2016.01.012
- 15. Ye M, Fu S, Pi R, He F. Neuropharmacological and pharmacokinetic properties of berberine: a review of recent research. J Pharm Pharmacol 2009;61:831-837. https://doi.org/10.1211/jpp.61.07.0001
- 16. Ilyas Z, Perna S, Al-Thawadi S, Alalwan TA, Riva A, Petrangolini G, Gasparri C, Infantino V, Peroni G, Rondanelli M. The effect of Berberine on weight loss in order to prevent obesity: a systematic review. Biomed Pharmacother 2020;127:110137. https://doi.org/10.1016/j.biopha.2020.110137
- 17. Fernandez CP, Afrin F, Flores RA, Kim WH, Jeong J, Kim S, Chang HH, Lillehoj HS, Min W. Downregulation of inflammatory cytokines by berberine attenuates Riemerella anatipestifer infection in ducks. Dev Comp Immunol 2017;77:121-127. https://doi.org/10.1016/j.dci.2017.07.027
- 18. Warowicka A, Nawrot R, Goździcka-Józefiak A. Antiviral activity of berberine. Arch Virol 2020;165:1935-1945. https://doi.org/10.1007/s00705-020-04706-3
- 19. Habtemariam S. Berberine and inflammatory bowel disease: a concise review. Pharmacol Res 2016;113:592-599. https://doi.org/10.1016/j.phrs.2016.09.041
- 20. Calvo A, Moreno E, Larrea E, Sanmartín C, Irache JM, Espuelas S. Berberine-loaded liposomes for the treatment of Leishmania infantum-infected BALB/c mice. Pharmaceutics 2020;12:858. https://doi.org/10.3390/pharmaceutics12090858
- 21. Sriwilaijareon N, Petmitr S, Mutirangura A, Ponglikitmongkol M, Wilairat P. Stage specificity of Plasmodium falciparum telomerase and its inhibition by berberine. Parasitol Int 2002;51:99-103. https://doi.org/10.1016/s1383-5769(01)00092-7
- 22. Elizondo-Luévano JH, Castro-Ríos R, López-Abán J, Gorgojo-Galindo O, Fernández-Soto P, Vicente B, Muro A, Chávez-Montes A. Berberine: a nematocidal alkaloid from Argemone mexicana against Strongyloides venezuelensis. Exp Parasitol 2021;220:108043. https://doi.org/10.1016/j.exppara.2020.108043
- 23. Rahimi M, Seyyed Tabaei SJ, Ziai SA, Sadri M. Anti-leishmanial effects of chitosan-polyethylene oxide nanofibers containing berberine: An applied model for leishmania wound dressing. Iran J Med Sci 2020;45:286-297. https://doi.org/10.30476/ijms.2019.45784
- 24. Malik TA, Kamili AN, Chishti MZ, Tanveer S, Ahad S, Johri RK. In vivo anticoccidial activity of berberine [18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium]--an isoquinoline alkaloid present in the root bark of Berberis lycium. Phytomedicine 2014;21:663-669. https://doi.org/10.1016/j.phymed.2013.11.005
- 25. Pop LM, Varga E, Coroian M, Nedişan ME, Mircean V, Dumitrache MO, Farczádi L, Fülöp L, Croitoru MD, Fazakas M, Györke A. Efficacy of a commercial herbal formula in chicken experimental coccidiosis. Parasites Vectors 2019;12:343. https://doi.org/10.1186/s13071-019-3595-4
- 26. Zhao L, Cang Z, Sun H, Nie X, Wang N, Lu Y. Berberine improves glucogenesis and lipid metabolism in nonalcoholic fatty liver disease. BMC Endocr Disord 2017;17:13. https://doi.org/10.1186/s12902-017-0165-7
- 27. Cernáková M, Kostálová D. Antimicrobial activity of berberine--a constituent of Mahonia aquifolium. Folia Microbiol 2002;47:375-378. https://doi.org/10.1007/BF02818693
- 28. De Sarkar S, Sarkar D, Sarkar A, Dighal A, Staniek K, Gille L, Chatterjee M. Berberine chloride mediates its antileishmanial activity by inhibiting Leishmania mitochondria. Parasitol Res 2019;118:335-345. https://doi.org/10.1007/s00436-018-6157-3
- 29. Fatemi A, Razavi SM, Asasi K, Goudarzi MT. Effects of Artemisia annua extracts on sporulation of Eimeria oocysts. Parasitol Res 2015;114:1207-1211. https://doi.org/10.1007/s00436-014-4304-z
- 30. Desalegn AY, Ahmed MR. Anticoccidial activity of Aloe debrana and Aloe pulcherrima leaf gel against Eimeria oocysts. J Parasitol Res 2020;2020:8524973. https://doi.org/10.1155/2020/8524973
, Rochelle Alipio Flores, Paula Leona Taymen Cammayo, Suk Kim, Woo Hyun Kim, Wongi Min,*
