Coccidiosis is caused by infection with Eimeria species and an significant parasitic disease in poultry [1]. Losses include mortality, morbidity and cost of prophylactic or therapeutic drugs and/or vaccination. Also, many medications such as in-feed drugs commonly added to feed to prevent infection with Eimeria spp. have become less effective because some parasite strains have reduced susceptibility to anticoccidial agents [2]. This suggests that coccidiosis may have a greater impact on the profitability of future broiler production [2].
Traditionally, H. cordata Thunb has been used as an herbal medicine for the treatment of inflammatory diseases such as ulcerative colitis in humans [3]. Previous studies have shown that H. cordata extracts exhibit antiviral and antibacterial [4, 5], antiallergic [6], antioxidant and antimutagenic activity [7]. The main components of H. cordata extract include methyl nonyl ketone, β-myrcene, β-pinene, α-pinene, α-terpineol and n-decanoic acid. And the anti-inflammatory effects of H. cordata have also been proven [8]. Thogh various types of natural products have been investigated to find alternative controls for coccidiosis in chickens [1], the effects of H. cordata on Eimeria infection has not been investigated.
The aim of this study is to study the anticoccidial effect of H. cordata extract (HCE) in chickens after oral infection by E. tenella.
The dried lump of H. cordata was purchased from an Oriental medicine pharmacy (Iksan, Korea), and it meets the official standards of Korean Pharmacopoeia and Korean Herbal Pharmacopoeia. The procedure for preparing HCE is as follows. Naturally dried H. cordata masses (100 g) were cut into pieces and extracted twice for 3 h using 50% (v/v) ethanol (6 times the weight of the dried plants at 80°C. After filtration through a 400-mesh filter cloth, it was filtered again with filter paper (No. 5, Whatman, Maidstone, UK) and concentrated with a rotary evaporator (EYELA, Tokyo, Japan) and the concentrated filtrate was vacuum-dried to dryness under vacuum with freezing dryer (Labconco, Kansas City, MO, USA). Finally, the solid residue was collected, bottled, sealed and stored at –20°C.
This study was performed on three-day-old chickens (n = 30) in the animal facility of Center for Animal Resources Development, Wonkwang University, Korea. Animals were acclimatized and housed in an animal facility with controlled temperature (28 ± 2°C), humidity (50 ± 5%), and light-dark cycle (12/12 h). These chicks were provided with a commercial post-broiler feed (Hanil Feed, Yongin, Korea) and tap water that did not include antibiotics and coccidiostat and tab water ad libitum. The chicks stayed in wire-floored cages during the study period. All studies were conducted in accordance with the guidelines for animal experiments and were approved by the Institutional Animal Care and Use Committee of Wonkwang University. Every effort was made to minimize the pain or discomfort of the animals participating in the experiment.
The research team evaluated the anticoccidial effect of HCE after oral infection in chicks by E. tenella. This study was performed on three-day-old chicks (n = 30). Three-day-old chicks were classified into three groups; HCE 0.2% treated/infected (n = 10), HCE untreated/infected (n = 10) and non-infected control (n = 10). We have determined the following subsequent doses of HCE as a concentrated additive to the recommended feed. Chicks were inoculated with or without HCE for a week prior to infection with E. tenella (10,000 sporulated oocysts per a chick). The effectiveness of HCE on E. tenella infection were assessed by two parameters, fecal oocysts numbers and weights gain.
Oocysts of E. tenella were cleaned by flotation on 5.25% sodium hypochlorite and washed three times with phosphate buffered saline. Chicks were treated orally by gavages using a 24 gauge, mouse stainless steel feeding tube (Popper & Sons, New York, NY, USA) attached to a 3 mL syringe. The oral infectious dose of has been approximated 104 oocysts of E. tenella in 1 mL of saline. The control chicken (n = 10) was provided with saline solution through the same route.
The disease rate and mortality rate of animals were confirmed twice a day during the study period. In addition, the clinical symptoms and weight gain changes in experimental animals were compared. Body weights were separately measured for 10 days after infection.
Feces were collected 6 to 10 days after infection. The fecal samples were analyzed for the presence of coccidial oocysts using standard fecal flotation techniques [9]. Briefly, 5 mL from each sample was centrifuged at 1,500 × g for 5 min. The produced pellets were redeposited in saturated sodium chloride (aqueous), passed through a 1 mm mesh size sieve to remove coarse fecal debris. The producesg filtrates were used for standard gravity vial stool suspension using a 22 mm × 22 mm cover slip. After floating, the coverslip was mounted on the slide and the entire presence of the coccidium follicle was examined. The 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].
Differences in average oocyst production and mean weight gain between the three groups were tested using one-way analysis of variance (ANOVA, GraphPad InStat, GraphPad Software, San Diego, CA, USA) and were considered significant at p<0.05.
The extract yield of H. cordata by 50% ethanol was 21.50%. The HCE composition was analyzed by LC. The concentration of decanoyl acetaldehyde in HCE was 121.6 ug/g.
The chicks treated with HCE showed a significant reduction in fecal oocyst excretion and strong anticoccidial activity compared to the untreated control group (p<0.01).
As shown in Table 1, oocyst shedding was significantly higher in inoculated chickens than in controls (p<0.05). The number of excreted fecal oocysts was highest at 7 days after inoculation (Table 1). Moreover, the weight gain was less in the animals of the inoculated group than the animals of the control group (Table 2).
Coccidiosis in poultry is a global disease caused by absolute intracellular protozoa of the genus Eimeria. This disease causes considerable economic loss to poultry production. E. tenella is an important pathogen that causes avian coccidiosis in laboratory avian animals and is known to affect experimental results from contaminated animals [1, 10]. The disease is characterized by intestinal lesions of varying extent and severity, which reduce the absorptive function of the intestinal mucosa, resulting in weight loss, diarrhea, low feed conversion and high mortality in affected flocks [11].
Results of the study show that HCE has a strong anticoccidial effect on E. tenella. HCE contains a various components (terpenoids, hydrocarbons, esters, alcohols, ketones, aldehydes, acids, phenols, ethers and mixed compounds) and flavonoids (quercitrin, isoquercitrin, aphzeline, hyperin, rutin) [12]. Among compounds contained in the extracts, aldehydes such as lauryl aldehyde and decanoyl acetaldehyde (houttuynin) have antimicrobial effects against Gram positive bacteria and antifungal effects [5, 13].
This study estimated the anticoccidial effect of HCE in chickens after oral infection by E. tenella. Chicks fed HCE significantly reduced fecal oocysts when compared to E. tenella-infected group fed standard feed (p<0.05). In addition, the HCE-based diet improved weight loss due to E. tenella infection. Our experimental results showed that HCE has significant antiprotozoal activity against E. tenella. This finding may have implications for the development of anticoccidial drugs.