To develop a natural herbal antimicrobial compound, over 1,000 kinds of herbal materials were tested by Center for Animal Resources Development, Wonkwang University. The herbal specimens were purchased at the University Oriental Drugstore, Iksan, Korea. Voucher specimens were deposited at the Herbarium of the College of Pharmacy, Wonkwang University. Plant material was extracted with ethanol under ultrasonic conditions for 3 h followed by paper filtration. The filtrates were evaporated in vacuum to yield water-soluble extracts. We employed in vitro antibacterial and in vitro antiviral screening tests to evaluate the effect of the medicinal herbal extracts on the pathogenic bacteria and viruses. We found that GR extract had very strong antibacterial and antiviral effects in vitro and in vivo [8-10]. In this study, we administered GR extract to the experimental animals.

The study was performed using 3-day-old chickens (n=30) in the animal facility of Center for Animal Resources Development, Wonkwang University, Korea. Animals were acclimatized and maintained in an animal facility room with regulated temperature (28±2℃), humidity (50±5%) and light/dark cycle (12/12 h). The animals were fed a commercial post-broiler diet without antibiotics and coccidiostat (Hanil Feed Co., Yongin, Korea) and tap water ad libitum. The chickens were maintained in wire-floored grower cages during the study period. All studies were performed in accordance with the Guide for Animal Experimentation by Wonkwang University, and the protocol was approved by the Institutional Animal Care and Use Committee of Wonkwang University (WKU11-007). All efforts were made to minimize pain or discomfort to the animals used.

Anticoccidial effects of GR extract were evaluated in chickens after infection with E. tenella via the oral route. This study was performed using 3-day-old chickens (n=30). The animals were divided into 3 groups: GR 0.5%/infected (n=10), untreated/infected (n=10), and non-infected control (n=10). Chickens were fed a standard diet supplemented with or without GR for 1 week before infection with E. tenella (10,000 sporulated oocysts per chicken). We examined the effects of GR on E. tenella infection using 2 parameters, number of fecal oocysts and body weight gain, and the findings of polymerase chain reaction (PCR).

Oocysts of E. tenella were cleaned by flotation on 5.25% sodium hypochlorite and washed 3 times using phosphate buffered saline. E. tenella was provided kindly by Professor Wongi Min at Gyeongsang National University in Korea. Chickens were treated by oral gavage using a 24-gauge stainless steel animal feeding tube (Popper & Sons, Inc., New York, USA) attached to a 3-mL syringe. The oral infectious dose of E. tenella has been approximated to be 10⁴ oocysts of E. tenella in 1 mL of saline. The control chickens (n=10) received saline through the same route.

During the study period, the animals were examined twice daily for morbidity and mortality. Further, we compared clinical signs and changes in body weight of experimental animals. Body weights were individually measured for 2 weeks before infection and for 10 days post-infection.

Fecal materials were collected from 6 to 10 days post-infection. The fecal samples were analyzed for the presence of coccidial oocysts using a standard fecal flotation technique [13]. Briefly, 1 g from each sample was suspended with 5 mL saline and pelleted by centrifugation at 1500×g for 5 min. The resulting pellet was resuspended in saturated sodium chloride (aqueous) and passed through a sieve with a mesh size of 1 mm to remove coarse fecal debris. The resulting filtrate was used in a standard gravity vial fecal flotation using 22 mm×22 mm cover slips. After flotation, the cover slip was mounted on a slide and examined in its entirety for the presence of coccidial oocysts. Total number of oocysts was calculated using the following formula: total number of oocysts=oocyst count×dilution factor×(fecal sample volume/counting chamber volume)/number of birds per cage.

For DNA extraction, fecal samples were collected at 10 days after inoculation and cleaned with sodium hypoclorite solution (5%-6% active chlorine) for 10 min at 4℃, washed 3 times with deionized water, and resuspended in extraction buffer (10 mM Tris-Cl, pH 8.0; 50 mM EDTA, pH 8.0). The oocysts and sporocysts were completely broken down by vortexing with half the volume of 425-600 µm acid-washed glass beads (Sigma-Aldrich, St. Louis, Mo., USA). The lysate was centrifuged at 14,000×g for 10 min to eliminate debris and then digested using DNAse-free RNAse A (20 µg/mL) at 37℃ for 1 h. Further digestion was performed using proteinase K (100 µg/mL) and sodium dodecyl sulphate (SDS, 0.5%) at 50℃ for 2 h. The DNA was then extracted once with one volume of phenol, phenol/chloroform, and chloroform and precipitated using ethanol and ammonium acetate. The pellet was washed using 70% ethanol. The DNA was eluted in Tris-EDTA buffer (pH 8.0), and an aliquot was used for PCR amplification. The template DNA (50 ng) and 20 pmol of each primer were added to a PCR mixture tube (AccuPower PCR PreMix; Bioneer Corp., Korea) containing 2.5 U of Taq DNA polymerase, 250 µM each deoxynucleoside triphosphate, 10 mM Tris-HCl (pH 8.3), 40 mM KCl, 1.5 mM MgCl₂, and the gel loading dye. The volume was adjusted using distilled water to 20 µL. The genomic DNA was amplified using species-specific forward primer (5'-AATTTAGTCCATCGCAACCCTTG-3') and reverse primer (5'-CGAGCGCTCTGCATACGACA-3') for the amplification of the 278-bp segment of E. tenella [13]. The reaction mixture was subjected to denaturation at 96℃ for 5 min followed by 35 cycles of denaturation at 95℃ for 30 s, annealing at 58℃ or 65℃ for 30 s, and extension at 72℃ for 1 min, and a final extension step of 72℃ for 3 min; the samples were maintained at 4℃ until analysis [13]. Reactions were performed using My Genie 32 Thermal Block PCR (Bioneer, Korea). Each sample (8 µL) was mixed with 2 µL loading buffer and was analyzed by electrophoresis in 2% agarose gels stained with 0.5 µg/mL ethidium bromide.

Differences in the mean oocyst production and mean weight gain between the 4 groups were tested using oneway analysis of variance (ANOVA; GraphPad InStat; GraphPad Software Inc., San Diego, CA) and considered significant at P<0.05.