Abstract: Background and Objective: Ascaridia galli (A. galli) infection can depress the body weight and egg production of domestic chickens. Synthetic anthelmintics are usually used as a treatment but A. galli resistance to synthetic anthelmintics is rising. The aim of this research was to understand the effects of combinations of Areca catechu seed and Anredera cordifolia leaf on the number of eggs per gram (EPG) of feces, body weight, number of Ascaridia galli worms, histopathology and immunoexpression of CD4+lymphocytes in the intestine. Materials and Methods: Three groups of fifteen chickens infected with A. galli were used in this research. For seven days, group 1 was orally administered a combination of 2 mg kg–1 b.wt., of Areca catechu and 1 mg kg–1 b.wt., of Anredera cordifolia, group 2 was given a combination of 5 mg kg–1 b.wt., of Areca catechu and 2.5 mg kg–1 b.wt., of Anredera cordifolia and group 3 was not treated. This research had ethical clearance No. 00040/04/LPPT/IV/2017. On the 10th day, the chickens were euthanized and examined for the number of EPG of feces, body weight, number of A. galli, histopathology and immunoexpression of intestinal CD4+lymphocytes. The data were analyzed statistically and the histopathological changes were analyzed descriptively. Results: The results showed that the combination of Areca catechu and Anredera cordifolia significantly reduced EPG of feces and eliminated A. galli in 60% of the chickens in group 1 but there was no significant difference in body weight among groups. In group 2, the alkaloids and saponins contained in Areca catechu and Anredera cordifolia display antagonistic activity. Thus, high doses do not reduce EPG levels in group 2. Immunohistochemistry showed that wound healing is improved in the treated groups. Conclusion: Ascaridia galli infection might be reduced by a combination of Areca catechu and Anredera cordifolia.
INTRODUCTION
The domestic chicken is widely reared in traditional farms. These chickens are the most important source of protein and economic income for rural people1-3. Intestinal parasitic nematodes cause severe diseases in poultry and decrease the productivity of poultry farms in developing countries, including Indonesia4. Ascaridia galli is a nematode commonly found in chickens that causes economic losses. Infection by Ascaridia galli can occur in chickens of all ages, but the greatest degree of damage is often found in young birds under 12 weeks of age5,6. Heavy infection by Ascaridia galli is characterized by retarded growth, emaciation, anorexia, anemia, diarrhea, dehydration and decreases in body weight and egg production7-9.
Ascaridia galli infection is most commonly treated using synthetic anthelmintics, but these anthelmintics have many problems. Such problems include the high cost of the anthelmintics, the negative impacts of carcinogenic drug residues in poultry meat and the development of anthelmintic resistance in helminths. Salam10 revealed that the main treatment for Ascaridia galli infection in native chicken was using synthetic anthelmintic. If the anthelmintic used for the worm treatment is of the same type and used for a long time, it may cause resistance to the worms11. Drug resistance causes the effectiveness of therapeutic drugs to be suboptimal3,12. The development of drug resistance in helminths to chemotherapeutical products has drawn attention to the need for alternative treatments. Anthelmintics from natural sources could play a key role in the treatment of parasitic infections. One of the plants that can be used as an anthelmintic is Areca catechu. Areca catechu, or betel nut, belongs to the family Arecaceae. Areca catechu contains active substances such as flavonoids, tannins, saponins, monoterpenes, sesquiterpenes, phenols, quinones and alkaloids (arecoline and arecaidine)13,14. Anredera cordifolia is a plant that can be used to improve the wound-healing process15. Infection by Ascaridia galli causes histopathological change. Immature Ascaridia galli penetrate intestinal mucosa and cause severe hemorrhagic enteritis16. The active substances in Anredera cordifolia such as saponins, alkaloids and flavonoids were capable of promoting wound-healing processes15. Considering the above facts, the present study was performed to evaluate the effect of Areca catechu seed and Anredera cordifolia leaf on the number of A. galli eggs per gram (EPG) of feces, body weight, number of A. galli in the intestine, histopathology and immunoexpression of intestinal CD4+lymphocytes. Research on the potential of Areca catechu powders to work as anthelmintics for local chickens in vivo has also been reported by Tangalin17 and Ozaraga et al.3. Areca catechu powder is able to decrease the number of EPG of feces by 66.51%3. Anredera cordifolia also shows potential as an anthelmintic that causes paralysis in the worms18.
MATERIALS AND METHODS
The study was carried out at the Department of Veterinary Pathology, Universitas Gadjah Mada, Yogyakarta, Indonesia. Powdered Areca catechu seed and Anredera cordifolia leaf seeds of Areca catechu and leaves of Anredera cordifolia were collected from the field. The collected seeds and leaves were dried in the shade at the ambient air temperature of the surrounding area, minced to powder using an electrical blender and stored17.
Sample size and experimental animals: Young domestic chickens were used in this research. Fifteen infected chickens were divided into 3 groups of 5 chickens each: Group 1, group 2 and group 3. Group 1 was orally administered a combination of 2 mg kg–1 b.wt., of Areca catechu seed and 1 mg kg–1 b.wt., of Anredera cordifolia leaf extract, group 2 was given a combination of 5 mg kg–1 b.wt., of Areca catechu seed and 2.5 mg kg–1 b.wt., of Anredera cordifolia leaf extract and group 3 was not given any treatment. Treatments were given for seven days19 and all chickens were then euthanized on the 10th day. This research had ethical clearance No. 00040/04/LPPT/IV/2017.
Fecal egg count (FEC): Fecal samples were collected from the initially selected birds on day 0 (for pretreatment FECs) and again on the 10th day of treatment. Fecal egg count was calculated as the number of eggs per gram (EPG) of feces using the McMaster technique20.
Number of A. galli in the intestine: Chickens were euthanized on the 10th day. During necropsies, all visible A. galli were collected. The number of worms in each part of the intestine (duodenum, jejunum and ileum) was counted. The efficacy of the treatments was determined by comparing the number of worms in the control and treated groups.
Body weight: The body weight of chickens was measured. Every chicken was examined on day 0 (pretreatment) and 10. The effect of Areca catechu and Anredera cordifolia was determined by comparing body weight in the control and treated groups.
Histological evaluation: The histological change of the intestine was evaluated using immunohistochemistry (IHC).
RESULTS AND DISCUSSION
Most medicinal plants show antiparasitic characteristics and have usually been applied as a remedy against parasites21. Many researchers demonstrated the effectiveness of numerous plants, including Areca catechu, against A. galli infection in poultry and some other animals3,13,17,22. The results of the present study revealed that, on day 0 (pretreatment), the EPG of groups 1, 2 and 3 were 570, 230 and 240, respectively. On the 10th day, the EPG in group 1 was significantly lower (Table 1). In group 2, these results illustrate that the combination of Areca catechu and Anredera cordifolia produces a negative effect. Areca catechu seeds contain active ingredients such as flavonoids, saponins, monoterpenes, sesquiterpenes, phenols, quinones and alkaloids such as arecoline, tannin and arecaine13,14, while Anredera cordifolia contains active ingredients on leaves such as saponins, flavonoids, triterpenes, steroids, quinones, monoterpenoids and sesquiterpenes23-26. According to Milugo et al.27, alkaloids and saponins can display antagonistic activity, as evidenced by the inhibition activity testing at each fraction of 80%, which reduced to 53% after merging the two fractions. Thus, high doses do not result in decreased EPG levels compared to low doses. Further studies on the combined antagonistic effects of both active ingredients that do not produce toxic results are needed28.
This result showed that powdered Areca catechu and Anredera cordifolia were effective against A. galli in chickens. Tangalin17 used Areca catechu extract and reported a decrease in A. galli EPG of feces in chickens. Present observations revealed the strong effectiveness of Areca catechu and Anredera cordifolia against A. galli infection and agree with the study of Ozaraga et al.3, who demonstrated the successful control of helminths, including A. galli, through the use of Areca catechu. The successful control of A. galli using Areca catechu extract was also reported by Febriani et al.13. The seeds of Areca catechu are used as an anthelmintic because they contain alkaloids, arecoline and tannins. Arecoline is toxic to some worms and can paralyze them. Proanthocyanidins (a group of condensed tannins) can inhibit enzymes and degrade membranes. The inhibition of enzymes causes metabolic failure, which decreases energy production and leads to worm death. Tannins also act as anthelmintics through their ovicidal properties29.
| Table 1: | Effect of treatment on EPG result |
| Same row with different superscripts are significantly different, p<0.05 | |
| Table 2: | Number of A. galli in intestine |
| Table 3: | Effect of Areca catechu seed and Anredera cordifolia leaf on body weight of chicken |
Post-mortem parasite counts indicated the anthelmintic activity of Areca catechu and Anredera cordifolia. The number of A. galli did not differ significantly between the untreated group and groups treated with Areca catechu and Anredera cordifolia, although A. galli was absent in 60% of the chickens in group 1, 40% of the chickens in group 2 and 100% of the chickens in group 3. This anthelmintic property represents a promising alternative to chemical treatments. This would be of great importance for poultry farms and will contribute to sustainable agriculture. Furthermore, the active compounds in plants are diverse and stable natural compounds with low molecular weights, which can prevent the development of anthelmintic resistance30. The numbers of A. galli in the intestine are shown in Table 2.
Body weight increased in all groups, but these increases were not significantly different among treated and untreated groups (Table 3). Body weight increased in all groups because the movement activity of chickens was lower, which was probably a direct effect of the increased amount of time used for food intake. The total time used for resting behavior in treated groups was similar to that in the control group.
| Fig. 1: | Expression of CD4+lymphocytes in jejunum (arrows, A = group 1), (B = group 2) and (C = group 3). Wound healing in process, normal tissues (arrow heads). Scale bar: 20 μm |
Because basic activities were similarly reduced among groups, these changes were probably caused in part by reduced energy availability or modulation of the neuroendocrine system that allowed body weight to increase31. This increase in body weight in the treated groups may have compensated for negative effects of A. galli on body weight throughout development. Some of the many sensory, neural and hormonal factors that modulate food intake in healthy animals are known to be affected by certain host-parasite relationships32.
The results of immunohistochemistry in the intestine indicated that powdered Areca catechu and Anredera cordifolia improve wound-healing processes (Fig. 1). Lymphocytes in the jejunum are dark brown in color. The number of lymphocytes increased with treatment in group 1 but decreased in group 2, which was treated with a higher dose of Anredera cordifolia and had normal intestinal villi. These findings proved that Anredera cordifolia can improve healing processes in the intestine. CD4+lymphocytes stimulate the differentiation of B cells that then secrete antibodies and these lymphocytes can also secrete cytokines that activate macrophages to phagocytize antigens33. CD4+lymphocytes are activated when an antigen is processed by an antigen presenting cell (APC). When an antigen is transported through lymphoid organs by APC cells with MHC class II molecules, the number of activated CD4+lymphocytes increases until the antigen is eliminated and the number of lymphocytes then decreases33-35. Anredera cordifolia contains substances that can improve the wound-healing process and it has also antimicrobial and anti-inflammatory effects. Saponins have an antimicrobial effect that disturbs bacterial membranes. Saponins can increase TGF-β1, which increases inflammatory cells in the wound area and can accelerate the inflammation process. Saponins are also used as agents of angiogenesis36. Miladiyah and Prabowo15 revealed that flavonoids and polyphenols can also be used to improve wound healing by inhibiting peroxidase lipids and antioxidants. The inhibition of peroxidase lipids increases the production of collagen, fibrin and blood vessels.
Previous studies have shown the effectiveness of Areca catechu in controlling A. galli infection in poultry17,22 and of Anredera cordifolia in accelerating wound-healing processes15,36. In the present study, powdered Areca catechu seed and Anredera cordifolia leaf were proven to be an effective treatment for A. galli infection. These findings agree with Ozaraga et al.3, who tested various medicinal plants against A. galli and demonstrated that the best control occurred with Areca catechu. Febriani et al.13 also reported that Areca catechu is effective against A. galli eggs.
CONCLUSION
It is concluded that, treating chickens infected by A. galli using a combination of Areca catechu and Anredera cordifolia significantly reduced the EPG of feces and eliminated A. galli in 60% of the chickens in group 1, but there was no significant difference in body weight among groups. CD4+lymphocytes were expressed in the intestine, which showed that wound healing improved in the treated groups.