Anticoccidial Activity of Berberine against Eimeria-Infected Chickens

Article information

Korean J Parasito. 2021;59(4):403-408

Publication date (electronic) : 2021 August 18

doi : https://doi.org/10.3347/kjp.2021.59.4.403

Binh Thanh Nguyen, Rochelle Alipio Flores, Paula Leona Taymen Cammayo, Suk Kim, Woo Hyun Kim, Wongi Min

College of Veterinary Medicine & Institute of Animal Medicine, Gyeongsang National University, Jinju 52828, Korea

^*Corresponding author (wongimin@gnu.ac.kr)

Received 2021 May 29; Revised 2021 July 28; Accepted 2021 July 30.

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).

Fig. 1

Effect 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.

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.

Table 1

Effect of berberine treatment on body weight gain compared to untreated chickens

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).

Fig. 2

Fecal 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.

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.