Vet. Med. Austria / Wien. Tierärztl. Mschr. 94 (2007), 269 - 273

From the Veterinary Medical Aquatic Animal Research Center (Department of Veterinary Medicine), Faculty of Veterinary Science, Chulalongkorn University, Bangkok

Effects of dried Indian almond leaf ( catappa L.) extract on monogenean parasites in goldfish (Caras- sius auratus)

N. CHANSUE received May 5, 2006 accepted for publication January 24, 2007

Keywords: goldfish, Indian almond leaf, Terminalia catap- Schlüsselwörter: Goldfisch, indischer Mandelbaum, Ter- pa, Gyrodactylus sp., Dactylogyrus sp. minalia catappa, Gyrodactylus sp., Dactylogyrus sp.

Summary Wirkung eines Extrakts aus getrockneten Blättern des Goldfish (Carassius auratus), a popular ornamental indischen Mandelbaums (Terminalia catappa L.) auf fish, is often affected by external parasites such as Gyro- Parasitenbefall beim Goldfisch (Carassius auratus) dactylus sp. and Dactylogyrus sp. The effects of Indian almond leaf (IAL) extract on the elimination of these para- Zusammenfassung sites were investigated. The highest applied concentration Der als Zierfisch beliebte Goldfisch (Carassius auratus) of IAL (5.1 g/l of water) was the most effective, completely wird oftmals von Ektoparasiten wie Gyrodactylus sp. und eliminating all parasites after 2 weeks of treatment without Dactylogyrus sp. befallen. Die Auswirkungen eines Extraktes adverse side effects. A concentration of 3.4 g/l eliminated aus den Blättern des indischen Mandelbaumes (IAL) auf all Dactylogyrus sp. and virtually all Gyrodactylus sp. after einen Befall mit diesen Parasiten wurden untersucht. Die 3 weeks of treatment, whereas some parasites still per- höchste angewandte Konzentration des IAL (5,1 g/l Wasser) sisted after 4 weeks of treatment with 1.7 g/l. No significant war am effektivsten, wobei nach zweiwöchiger Behandlung reduction of parasites occurred in the control group without keine Parasiten mehr nachweisbar waren und nachteilige treatment. Nebenwirkungen nicht zu beobachten waren. Bei einer Konzentration von 3,4 g/l waren nach 3 Wochen keine Dacty- Abbreviations: ALT = alanine aminotransferase; ANOVA = one-way logyrus sp. und nahezu keine Gyrodactylus sp. mehr nach- analysis of variance; AST = aspartate aminotransferase; BUN = weisbar. Nach vierwöchiger Behandlung mit 1,7 g/l waren blood urea nitrogen; CDHP = conjugated diene hydroperoxide; jedoch noch einige Parasiten nachweisbar. In der Kontroll- Crea = creatinine; DPPH = 1,1-diphenyl-2-picryl-hydrazyl; IAL = gruppe ohne Zusatz des Blattextraktes war gleichzeitig kein Indian almond leaf; LSD = least significant difference; NRCT = National Research Council of Thailand; OD = optical density; RBC signifikanter Rückgang der Parasitenzahlen zu beobachten. = red blood cell count; WBC = white blood cell count

Gyrodactylus sp. is viviparous and has a distinct charac- Introduction teristic (many generations of offspring dormant inside the mother fluke). On the other hand, Dactylogyrus sp. is Goldfish (Carassius auratus) is a popular ornamental oviparous, and its eggs hatch 2 to 3 days after being fish in many countries, originating from China and Japan, deposited. After attaching to a fish, the young flukes will and later being brought to Europe and America in the sev- mature in about 4 to 5 days at a water temperature of 28 enteenth and nineteenth century, respectively. Goldfish are to 29 °C (SIRIKARNJANA, 1995). In warm water and ideal easy to keep, have high endurance and can reproduce conditions, they can reproduce very rapidly and quickly year-round, making them popular amongst fish breeders. overwhelm any ill or stressed fish (FISHDOC, 2004). Since The easiest and cheapest way to breed goldfish is to facil- the monogenic parasites cannot swim in water, they need itate the natural reproductive process, which is to allow to attach to fish to move and spread. This is why, in an male and female goldfish to copulate naturally in a tank overcrowded farming environment, it is easier for them to with aquatic . A female goldfish will lay around 500 to spread and infest fish (BELLONA FOUNDATION, 2004). A 1,000 eggs at a time. During this period, the fish are phys- fish infested with flukes will turn on its side (flashing), rub ically weakened, making them susceptible to diseases and against objects, float on the water surface, become emaci- parasitic infestation. ated and will rapidly close and open the gills and cheeks. The 2 most relevant external parasites that cause dis- Occasionally, there are lesions the size of the tip of a pin eases in goldfish originate from the families Gyrodactyli- on the body surface, and in severe infestation the body of dae and Dactylogyridae, which cause lesions and tissue the fish will appear as if covered with a whitish substance damage such as hyperplasia of the skin and gill epithelium (RITTHAISONG, 2001). Diagnosis includes scrape sam- and creation of sites susceptible to secondary infection in pling of skin and gill mucus for further examination. Indi- the form of ulcers. Although Gyrodactylus sp. and Dactylo- viduals affected with monogenic parasites are treated with gyrus sp. are classified as skin and gill flukes respectively, consecutive salt baths over 2 to 3 days. Chemical treat- they are not exclusive to these areas (FISHDOC, 2004). ment may be applied as well, such as bathing for 24 hours

269 Vet. Med. Austria / Wien. Tierärztl. Mschr. 94 (2007) in formaldehyde solution diluted to 25 to 40 ppm, or calagin and tercatein). Trichlorfon diluted to 0.25 to 0.50 ppm (NOGA, 2000). Various other parts of the Indian almond tree are used Chloramines-T has also been used, but it kills only mature traditionally for medicinal purposes in Thailand (SUCH- flukes while the eggs and embryos prevail (UNTER- ABUN, 1992; WUTITHAMVEJ, 1999), Suriname (TROPI- GASSER, 1989). There are numerous chemicals available LAB, 2002) and Sri Lanka (RATNASOORIYA and DHAR- for treating parasitic infestation, but these are costly and MASIRI, 2002). Moreover, the leaves (especially old mate- have pronounced side effects affecting both breeders and rial) and oil extracts have antioxidant properties and fish. Some herbal substances contain active compounds also increase scavenging activities of 1,1-diphenyl-2- which have been traditionally observed to be effective in picryl-hydrazyl (DPPH). In addition, the seed extracts inhib- treating, amongst others, fish diseases. it the production of conjugated diene hydroperoxide Dried Indian almond leaves are applied in the treatment (CDHP) and mildly activate DPPH (KO et al., 2002). In a of a dermal condition in fish (’’velvet disease’’), which is study to quantify the aphrodisiac effects of Indian almond caused by contaminated water or flagellates that are in mice, it was found that the fruit of Indian almond in imported with mosquito larvae (SUTTANANT, 2001). 1 % methyl cellulose at a dosage of 1,500 mg/kg has an Moreover, dried Indian almond leaf extract has a quantifi- aphrodisiac effect in that it increases ejaculation time, but able positive effect on the development of scales in has no effects on libido, sexual vigor (mounting and intro- (CHANSUE et al., 2004). mission frequency), or sexual performance (intercopulato- The chemicals contained in the Indian almond leaves ry interval) (RATNASOORIYA and DHARMASIRI, 2000). (Terminalia catappa) are violaxanthin, lutein, zeaxanthin, Ethanol extract of leaves can inhibit osmotically induced (such as quercetin and kamferol), calcium hemolysis in human red blood cells, displaying a potential oxalate, glycosides and (such as punicalin, puni- for treating sickle cell disorders (MGBEMENE and OHIRI,

Tab. 1: OD of each week for different concentrations of IAL

OD Control Tc1 Tc2 Tc3 week 1 0.000 0.502 0.951 1.152 week 2 0.027 0.647 0.979 1.183 week 3 0.031 0.732 0.986 1.198 week 4 0.039 0.873 0.994 1.202

Tab. 2: Average number of monogenic flukes (Gyro+Dac) for each week

Treatment Control Tc1 Tc2 Tc3 pre-treatment Gyro 1.8±0.94 3.67±2.41 2.4±1.24 2.8±1.32 Dac 0.8±0.94 1.07±0.96 0.93±1.53 0.6±1.05 all 2.6±1.18 4.73±2.73 3.3±2.17 3.4±1.68 week 1 Gyro 2.8±2.17 1.4±1.18 1.0±0.75 0.2±0.41 Dac 0.73±1.03 0.27±0.59 0.27±0.70 0.13±0.35 all 3.53±2.82 1.67±1.39 1.27±1.16 0.33±0.48 week 2 Gyro 1.454±2.6 0.93±1.38 0.4±0.91 0.00 Dac 0.73±1.03 0.53±0.83 0.13±0.51 0.00 all 3.33±1.83 1.4±1.63 0.53±1.12 0.00 week 3 Gyro 1.4±1.40 0.53±0.74 0.27±0.59 0.00 Dac 0.87±1.06 0.07±0.25 0 0.00 all 2.27±1.66 0.6±0.82 0.27±0.59 0.00 week 4 Gyro 1.4±1.35 0.33±0.61 0.07±0.25 0.00 Dac 0.53±0.74 0.13±0.51 0 0.00 all 1.93±1.94 0.47±0.91 0.07±0.25 0.00

Gyro = Gyrodactylus; Dac = Dactylogyrus; all = monogenic flukes

270 Vet. Med. Austria / Wien. Tierärztl. Mschr. 94 (2007)

Tab. 2a: p-values observed for Gyrodactylus count at Tc3 Table 2b: p-values observed for Dactylogyrus count at Tc3

week 0 week 1 week 2 week 3 week 4 week 0 week 1 week 2 week 3 week 4 week 0 - 0.03 0.002 0.002 0.001 week 0 - 0.035 0.012 0.035 0.006 week 1 - 0.189 0.221 0.121 week 1 - 0.595 1.000 0.405 week 2 - 0.923 0.793 week 2 - 0.595 0.759 week 3 - 0.720 week 3 - 0.405 week 4 - week 4 -

Tab. 3: Hematological findings after 4 weeks

control Tc1 Tc2 Tc3 p

ALT (IU/L) 109 ± 4 104 ± 8 101 ± 5 98 ± 4 0.083 AST (IU/L) 912 ± 35 894 ± 42 889 ± 65 884 ± 56 0.066 BUN (mg/dl) 18 ± 2 15 ± 4 14 ± 8 12 ± 3 0.080 Crea (mg/dl) 0.09 ± 0.05 0.08 ± 0.01 0.07 ± 0.05 0.07 ± 0.06 0.080 RBC (x106/μl) 1.5 ± 0.82 1.5 ± 0.54 1.5 ± 0.63 1.6 ± 0.06 0.391 WBC (x103/μl) 62.6 ± 5.23 58.9 ± 6.12 60.2 ± 4.38 59.45 ± 5.14 0.066

Tab. 4: p-values concerning the differencies in the overall mean fluke numbers in the evaluated groups (Gyrodactyla hori- zontal, Dactylogyra vertical)

control Tc1 Tc2 Tc3 control - 0.564 0.183 0.103 Tc1 0.131 - 0.434 0.271 Tc2 0.033 0.470 - 0.741 Tc3 0.010 0.199 0.557 -

1999). Oil extracted from young Indian almond leaves is ried out with 5 fish per group (total of 20 fish) and repeat- used by veterinarians to treat mange with high efficacy and ed 3 times. a low rate of side effects. It also decreases the need for All experiments were carried out in accordance with a chemicals that may be ineffective and produce pesticide draft for a law concerning animal welfare which is current- contamination in treated animals (FLORES, 2005). Indian ly being considered by the parliament (Thailand), as well almond is also used by fish breeders to generally boost as with the recommendations of the National Research fish health (SUPACHAI, 2001; HARIKRISHNAN et al., Council of Thailand (NRCT) under the ’’Code of conduct for 2003). the use of laboratory animals in biomedical research“. If effective in treating parasitic infestation in goldfish, Indi- an almond leaf extract would reduce the need for chemicals Water preparation that are dangerous to both breeders and aquatic animals, Three 100 litre water tanks were used for the immersion and subsequently the ecological systems involved. Chemi- of dried Indian almond leaves. The first tank contained cals employed in treating fish in Thailand are mainly import- 1.7.g of dried Indian almond leaves per 1 litre of water, the ed. IAL extract is economically attractive to breeders second tank contained twice this amount (3.4 g/l) and the because of its low price and ready availability. Moreover, it third tank contained 3 times this amount (5.1 g/l). The represents an integration of local knowledge and science, leaves were immersed for 7 days. Then the optical density leading to a new medication alternative in fish. (OD) of water in each tank was measured by means of a spectrophotometer (Spectronic® Genesys™, Thermo Elec- Material and methods tron Corporation, Massachusetts, USA) at a wavelength of 340 nm (CHANSUE, 2003). Then the water from each tank Specimen preparation was filtered and transferred to the experimental tanks of 30 60 goldfish of either sex (approximately 3 months of litres each. The optical density measurement was repeat- age) were purchased from the Chatujak fish Market, ed each week for a total of 4 weeks. Bangkok. The average height was 3 cm and the average length was 8.7 cm. All were kept in the same tank as fish Experiment conduction that were found to be infected with the 2 flukes for 14 days Goldfish that had been infected with flukes (verified in order to infect these previously healthy fish. The fish microscopically) were introduced to tanks with dimensions were subsequently separated into lots of 5 fish. Each of 40x60x45 cm. Each tank was filled with 30 litres of the experiment (3 water concentrations and control) was car- following:

271 Vet. Med. Austria / Wien. Tierärztl. Mschr. 94 (2007)

Statistical analysis In this experiment, the average fluke counts were sta- tistically analyzed by ANOVA (One-way analysis of vari- ance) to test for the differences of the variables by the Least Significant Difference (LSD) method (PONG- WICHAI, 2004). All computer analyses were done by SPSS® (Statistical Package for the Social Sciences) v. 10 (SPSS Inc., Chicago, Illinois, USA) and Microsoft Excel 2000®. In statistical evaluation, the outcome criterion test- ed for significant differences was the overall mean of the number flukes in the 4 groups (MATTHEWS et al., 1990). Results

The optical density of the water in each tank (group) is summarized in Tab. 1. It is evident that the OD of the control group and groups Tc3 and Tc2 remained virtually constant and group Tc1 showed only a marginal increase, indicating that there is no relevant environmental influence on the OD. Fig. 1: Average monogenic flukes (Gyro+Dac) of each Thus, the OD may be interpreted to indirectly represent the week quantity of the active ingredient of the IAL in the water. The average Gyrodactylus, Dactylogyrus, and com- Group 1 (control): filtered tap water bined species counts are shown in Tab. 2 and Fig. 1. Tab. Group 2 (Tc1): 1.7g/l dried IAL water 2 represents the statistical analysis of the total number of Group 3 (Tc2): 3.4g/l dried IAL water flukes over time (Dactylogyrus and Gyrodactylus, respec- Group 4 (Tc3): 5.1g/l dried IAL water tively) versus the different concentrations of IAL in order to Each of the 4 experiments was conducted 3 times with determine concentrations potentially suitable for treat- 5 goldfish (total of 60 fish) for 4 weeks at room tempera- ment. Statistical evaluation (comparative testing of fluke ture. Before the experiment was conducted, the goldfish count between weeks 0 to 4) was performed by means of were examined for flukes and the data recorded for later ANOVA. Tab. 2a shows p-values observed for Gyrodacty- comparison with post-experimental data. lus count at Tc3, Tab. 2b for Dactylogyrus count at Tc3. No negative side effects were seen even at the highest Diagnosis of fluke infection concentration of 5.1 g/l.  A glass cover slip (as used in preparing microscopy The hematological findings after 4 weeks are shown in slides) was slanted at 45 degrees to the fish body and Tab. 3. Histology of the liver revealed normal findings in the mucus of the skin, gills, and fins were scraped gen- both the control and the experimental groups. No tly. necroses, fatty degeneration or other pathological signs  The cover slip was then gently placed on a slide with were in evidence. one drop of water on the slide. The process was per- The scale and epidermal thickness of the individual formed slowly to allow the water to disperse evenly. groups revealed no relevant changes or variations in  The slide was examined microscopically to determine microscopy and were all within normal bounds. Specifical- the species of the flukes and to count the number of ly, cross sections of scales of all individuals were per- each species per square centimeter. formed and all skin layers were considered to be of normal  The data were recorded. configuration. During the experiment, every fish was examined for Tab. 4 indicates that only the differences in the overall monogenic parasites once every week. mean number of Dactylogyra between the control group and Tc2 and Tc3 showed p-values below 0.05. The other Feeding of fish differences could not be proved significant. Packaged commercial fish food was used to feed the fish in order to prevent external parasites that might be Discussion introduced with live food from influencing the experiment. The fish were fed once per day. In order to study the effectiveness of dried Indian Almond leaf extract water on the elimination of monogenic Clinical and pathological parameters parasites (Gyrodactylus sp. and Dactylogyrus sp.) in gold- Hematology was done for ALT, AST, BUN, Crea, RBC fish, infected fish were exposed to 3 different IAL concen- and WBC. trations. Corresponding to a previous study (CHANSUE et Histology of the liver and scales using hematoxylin / al., 2004), group Tc1 was exposed to 1.7 g of dried IAL per eosin staining was performed. litre of water. Group Tc2 was exposed to 3.4 g/l, and group The scale thickness and epidermal thickness were Tc3 to 5.1 g/l. The control group did not receive any treat- examined microscopically in all cases and compared to ment. Mucus samples were scraped from the skin and gills check for relevant changes over the course of the treat- of fish and subsequently examined microscopically for the ment. number of parasites per square centimetre. Data were recorded on a weekly basis.

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It was evident that the most effective concentration was study of the effects of Indian almond (Terminalia catappa) leaf Tc3 throughout. Concentrations Tc2 and Tc1 were less extract on Siamese fighting fish (Betta splendens) scale by effective but still displayed significant anti-parasitic effects scanning electron microscope. In: Proc. Thai : (p≤0.05). opportunity and alternative choice of animal production indus- try. Thai Veterinary Association. January 15-16, 2004, Siam Both species of parasites were completely eliminated City Hotel, Bangkok, p.140-144 (Thai). after 2 weeks of treatment at Tc3. Whilst Tc2 led to a total FISH DOC (2004): The home of fish health. Skin and gill flukes, elimination of Dactylogyrus sp. after 3 weeks of treatment, monogenean parasites. http://www.fishdoc.co.uk/disease/ some Gyrodactylus sp. were still in evidence after 3 weeks. flukes.htm; accessed 19.4.2006. Very small numbers of Gyrodactylus sp. could still be found FLORES, L. (2005): Integumentary system. Philippine herbs used after 4 weeks of treatment at Tc2. After 4 weeks of treat- in small animal practice. http://www.stuartxchange.org/Herbs ment at Tc1, relevant numbers of both Dactylogyrus sp. AnimalsSkin.html; accessed 19.4.2006. and Gyrodactylus sp. could still be found in fish. The con- HARIKRISHNAN, R., NISHA RANI, M., BALASUNDARAM, C. (2003): Hematological and biochemical parameters in common trol group, which was kept without any treatment, present- carp, Cyprinus carpio, following herbal treatment for ed with no significant reduction of number of parasites over Aeromonas hydrophila infection. Aquaculture 221, 41-50. the 4 week period (p>0.05). KO, T.F., WENG, Y.M., CHION, R.Y. (2002): Squalene content and It can be concluded that the Tc3 concentration is suit- antioxidant activity of Terminalia cattappa and . J. Agric. able for the treatment of infestation of goldfish with mono- Food Chem. 50, 5343-5348. genean parasites. Treatment for more than 3 weeks at Tc2 MATTHEWS, J.N., ALTMAN, D.G., CAMPBELL, M.J., ROYSTON, might lead to a complete elimination of parasites, as is P. (1990): Analysis of serial measurements in medical apparent from the data. Concentration Tc1 does not seem research. BMJ 300, 230-235. MGBEMENE, C.N., OHIRI, F.C. (1999): Anti-sickling potential of sufficient to completely eliminate parasites, although it Terminalia catappa leaf extract. Pharmac. Biol. 37, 152-154. does result in a continuous relevant reduction of the num- NOGA, E.J. (2000): Fish disease: diagnosis and treatment. Mos- ber of parasites. by-Year Book, Iowa State University Press Inc., St. Louis, Mis- The traditional knowledge of the antiparasitic effects of souri. IAL (enteral and dermatological) is substantiated by the PONGWICHAI, S. (2004): Statistic data analysis by computer.13th results of this study. It has been stipulated that among the ed., Chulalongkorn University Publishing, Bangkok, p. 191-228 many substances contained in the leaves, the active com- (Thai). pounds responsible for the effects observed by us are tan- RATNASOORIYA, W.D., DHARMASIRI, M.G. (2000): Effect of Ter- minalia catappa seeds on sexual behavior and fertility of male nins, flavonoids, , calcium oxalate and glycosides rats. Asian J. Androl. 2, 213-219. (FLORES, 2005). IAL extract is indeed applicable to the RATNASOORIYA, W.D., DHARMASIRI, M.G. (2002): Tender leaf treatment of infestation with monogenean parasites in extract of Terminalia catappa antinociceptive activity in rats. goldfish. Moreover, no negative side effects were seen Pharmac. Biol. 40, 60-66. even at the highest concentration of 5.1 g/l. Specifically, no RITTHAISONG, T. (2001): Complete book of Goldfish genetics negative effects were seen in blood chemistry (ALT, AST, and culture. Phet Karat Sudios Press, Bangkok, p. 143-146 BUN, creatinine and total blood count), histology as well as (Thai). scale and epidermal thickness. No clinical evidence of neg- SIRIKARNJANA, P. (1995): Aquatic animal parasites. Sahamitr Printing, Nonthaburi, p. 53-59 (Thai). ative side effects was observed. Further research on the SUCHABUN, P.(1992): Herbal medicinal . Aksarapiphat Pub- potential side-effects and dosage of IAL seems necessary. lisher, Bangkok, p. 63 (Thai). It is intended to carry out further experiments and to deter- SUPACHAI, N. (2001): Professional manual of how to raise perfect mine the LD50 of IAL in other species along with the rele- fighting fish. 1st ed., Matichon Publishing, Bangkok, p.87-105. vant histological examination. SUTTANANT, T. (2001): Wild type Siamese fighting fish. Fancy Chemical analysis of the pharmacologically active com- Fish J. 1, 15-29 (Thai). pounds in IAL is desirable. TROPILAB INC. (2002): Terminalia catappa. http://www.tropilab. com; accessed 19.4.2006. UNTERGASSER, D. (1989): Handbook of fish diseases. T.F.H Acknowledgements Publications Inc., New York, USA, p. 160. Many thanks to Prof. Dr. Harald J. Meyer for his advice WUTITHAMVEY, W. (1999): Principle of Thai pharmacology. 2nd and assistance in the preparation of this manuscript. ed., N.P. Screen Printing, Bangkok, p. 657 (Thai). I am also sincerely thankful to Assoc. Prof. Dr. J. Tangtrongpiros, Mr. Y. Wongsawan and Ms. K. Anek- Author’s address: thanakul and the Veterinary Medicine Aquatic Animal Assoc. Prof. Dr. Nantarika Chansue (DVM, MBA, Ph.D., LL.B.), Research Center, Department of Veterinary Medicine, Henri Dunant Rd., Patumwan, Bangkok 10330, Thailand. Chulalongkorn University, Bangkok, for the laboratory and e-mail: [email protected] the equipment. References

BELLONA FOUNDATION (2004): Gyrodactylus salaris, salmon and fish farm. http://www.Bellona.no/en/b3/biodiversity/wild_ salmon/12780.html; accessed 19.4.2006. CHANSUE, N. (2003): Effects of dried Indian almond (Terminalia catappa) leaf on hematology and blood chemistry of Siamese fighting fish (Betta splendens). Proc. Asian-Pacific Aquaculture 2003, World Aquaculture Society, Sept. 22-25, Bangkok, p.77. CHANSUE, N., MATADERM, T., SAILASUTR, A. (2004): Basic

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