Abstract

Flagellated protozoa of the genus Hexamita are the most commonly encountered enteric parasite in angelfish (Pterophyllum scalare). Many Hexamita infections are subclinical until the fish are subjected to stress such as poor environment, altered nutrition, or temperature fluctuation. Under these conditions, the parasite numbers increase dramatically resulting in anorexia, loss of condition, weakness and in extreme cases, catarrhal enteritis. This study compared the effectiveness of metronidazole treatments administered by two different routes. Oral medication was administered at 50mg/kg body weight for five consecutive days. Bath treatments were administered at concentrations of 5mg/L. Necropsies were performed before, during, and after administration to determine the parasite load of control fish, fish treated by bath, and fish treated with medicated feed. Medicated feed appeared to be the more effective method of treatment. The parasite number in the distal 1 cm of the gut dropped below 30 per 100x field following one treatment of medicated feed. Three bath treatments were necessary to achieve similar decreases in parasite load. At the end of the trial (5 days), the parasite number dropped to zero in all fish necropsied from the medicated feed group, but those from the bath treatment group remained mildly infected.

Introduction

The tropical freshwater angelfish Pterophyllum scalare is one of the most popular ornamentals in the pet fish industry today. Indigenous to the Amazon River Basin, the angelfish has been successfully cultivated here in the United States, especially in Florida. In 1986, reports of unusual and excessive mortality rates of angelfish were cited by Specht et al, 1989. Further investigation in our laboratory revealed that suboptimal water quality and severe parasitism were important causes of some of the angelfish mortalities.

The flagellated protozoan Hexamita was the most commonly encountered intestinal parasite (19.3%) cited by Specht et al, 1989. Three species of Hexamita have been associated with disease in fish; Hexamita salmonis in trout and salmon, Hexamita truttae in angelfish, discus fish, goldfish, and trout, and Hexamita intestinalis in trout and frogs (Post 1987). It is unknown whether these species or a new unidentified species are responsible for the disease in angelfish.

Transmission of the organism is probably accomplished by ingestion of fecal contents containing cysts and possibly trophozoites (Kreier 1977). Under normal circumstances, the parasite number is low and the disease process is subclinical. However, under stressful conditions such as poor water quality, overcrowding, shipping, malnutrition, or temperature fluctuations, the organism may rapidly reproduce resulting in massive infection and clinical disease.

Upon clinical presentation, anorexia, weight loss, listlessness, and erratic swimming may be observed. Severely infected individuals may lie horizontally on the water's surface with the abdomen visibly distended. In addition, infection in adult breeding angelfish may be associated with decreased hatchability of eggs or death of young fry. Internally, Hexamita infections can produce inflammation and pathological changes in the epithelial lining of the intestine and the gallbladder (Kreier 1977). Involvement of other organs such as the liver, spleen, and heart have been reported in discus (Symphysodon discus) (Amblacher 1970). This organism has also been implicated as the cause of "hole in the head disease" in Oscars (Astronotus ocellatus) and discus (Andrews et al, 1988).

Diagnosis can be made by making a squash preparation of the intestine and searching for the organism under low (l00x) magnification (Post 1988). Hexamita can be identified by its pyriform shape, its two anterior nuclei and its four pair of flagellae (3 anterior pair and 1 posterior pair). Also, its dimensions measure from 6 to 8 um wide by 10 to 12 um long (Post 1988).

Several treatment regimes have been cited for the use in both food and ornamental fish including simple water changes, medicated bath treatments, and medicated feed treatments. Several chemicals and dosages have been used such as dimetronidazole, magnesium sulfate, and carbarsone, but the most current and most widely used drug for pet fish is metronidazole. The recommended dosages are as follows: Bath treatment 5 mg/L every other day until the parasite count has decreased to zero (Gratzek 1988). Medicated feed treatment 50 mg/kg per body weight for five consecutive days (Stoskopf 1988).

To date, no comparison of the effectiveness of either route has been documented. The objective of this research was to compare the effectiveness of both routes and to determine the best method of treatment in angelfish using metronidazole.

Materials and Methods

Adult angelfish (average weight of 3.5 grams) infected with Hexamita were purchased from -a local Florida fish farm. None showed any clinical signs, but the severity and location of heaviest infection in the intestine was determined by necropsy of several fish prior to beginning the trial.

Individual fish were weighed and transferred into ten 34 liter aquariums and stocked at a rate of 18 fish per tank. Each tank was aerated and the water temperature was maintained at 25°C. Water quality parameters including pH, toxic ammonia, nitrite, water hardness, and alkalinity were measured 24 hours prior to treatment to establish baseline data. Again, the pH, toxic ammonia, and nitrite were measured following the end of the five day trial.

A special diet was created for maintenance and that could also be used to administer oral medication (Table 1). All fish were fed this diet once daily at 3% of their body weight.

The experiment involved a random block design consisting of four treatment groups: 1. untreated control with 25% water change every other day (2 groups), 2. untreated control without water change(2 groups), 3. bath treatment with 5 mg/L metronidazole for 3 hours followed by 25% water change(Gratzek 1988) (3 groups), 4. feed treatment with 50 mg/kg body weight metronidazole daily for 5 consecutive days(Stoskopf 1988) with a 25% water change every other day(3 groups).

The physical status, intestinal Hexamita count, and location of heaviest gut parasitism were determined prior to the trial by necropsy of three individuals from each tank. The subjects were euthanized with a lethal dose of MS 222. Also, liver and intestinal tissue was preserved in 10% neutral buffered formalin from the same individuals for histopathology.

The response to treatment was measured by estimating the Hexamita count as described by Post 1987(analysis of 1 cm of gut minced with O.10ml of water) on days 2, 4, and 6 (24 hours following the last treatment). The severity of infection in all groups and the decision to retreat the bath group was extrapolated from Post (1987) recommendation for salmon; retreatment was indicated when 15 or more organisms were seen per l00x field. Following the end of the trial, three fish per tank were necropsied and their liver and intestinal tissue was preserved for histopathology.

Results

Treatment Results: Three fish per group were sacrificed and necropsies were performed on days 2, 4, and 6 of the trial. The Hexamita count was estimated by averaging the number of organisms in four 100x fields (±5). The results are summarized in Figure 1.

Histopathology: Histopathology revealed mild intestinal inflammation in several of the individuals necropsied characterized by an increase in inflammatory cells within the lamina propria. Also, occasional nematodes were seen in the intestinal lumen of two fish. The liver tissue appeared normal in all specimens necropsied prior to treatment.

At the end of the trial, the intestinal lamina propria looked normal in all specimens necropsied. The liver tissue from three of the medicated feed group appeared to have an increase in vacuolization.

Subjects: No mortalities were encountered during the trial. The fish readily accepted the diet and their appetite remained normal throughout the trial.

Three fish were sacrificed and necropsied from each group to establish baseline data. The necropsy protocol included weight, standard and total body length, and total intestinal length. The averages were as follows: weight=3.5 gm, standard body length=4 cm, total body length=5 cm, total gut length=5.5 cm. The physical exam revealed healthy fish with occasional costia organisms on the skin and occasional encysted metacercariae on the gill epithelium. Internally, all organs grossly looked normal. Digesta and a yellow mucus film were observed in the intestine. The distal 1 cm of the intestine appeared to be the location of heaviest parasitism ranging from 50 to 100 organisms per l00x field.

Water Quality: Water quality averages before the trial included the following: water temperature=24°C (23-26°C), pH=8, toxic ammonia=0.096ppm (0.08534-0.1148ppm), nitrite=0.069ppm (0.033-0.36ppm), water hardness=253.08ppm (239.4-273.6ppm), water alkalinity=249.81ppm (239.4-273.6ppm). Water quality averages at the end of the 5 day trial included the following: water temperature=21°C, pH=7.7 (7.7-7.9), toxic ammonia=0.043ppm (0.0253-0.1226ppm), nitrite=0.074ppm (0.01-0.26ppm).

Discussion

The Hexamita count decreased dramatically following just one day with the medicated feed treatment, whereas it took three bath treatments to reach similar results. A decrease in parasite load was also evident in the water change group, values comparable to the bath treatment. As long as the fish continues to eat, the oral route is the best method of treatment for Hexamitiasis. If the fish are anorexic, at least three applications of the bath treatments are required. Partial water changes are helpful but are not recommended by this author because it is considered an unreliable sole method of treatment by aquarists.

The intestinal inflammation prior to treatment was minimal in most individuals with only a few individuals illustrating a response to the Hexamita infection. The nematodes encountered were probably an incidental finding. The liver vacuolization was minimal and considered by pathologists to be normal, possibly a response to stress (L. Roth 1991, personal communication).

The high toxic ammonia readings (normal<0.02ppm) on days 0 and 6 represents a normal finding with newly established tanks, "New Tank Syndrome" (Beleau 1988). Two tanks experienced high nitrite values (normal<0.lppm) which can also be explained by the same phenomena (Beleau 1988). All other water quality parameters were within normal limits.

Results of this study strongly suggest that medicated food is the preferred route for delivery of metronidazole to angel fish. Findings also confirm that metronidazole, administered in food or in bath, is effective for removal of Hexamita from the intestinal tract of angelfish.

Table 1. Recipe for Gelatinized Food used to Deliver Metronidazole to Angel Fish

Ingredients

Optional Ingredients: 0.5 tsp Liquid Vitamins

Procedure

1.  Blend 5 oz of water with shrimp, spinach, carrots, brewer's yeast, and vitamins.

2.  Boil remaining 10 oz of water.

3.  Add boiling water to gelatin in a bowl.

4.  Cool until gelatin mixture is hot but doesn't burn.

5.  Add contents of blender to partially cooled gelatin mixture, mix thoroughly.

6.  Add medication to cooled mixture.

7.  Pour into flat pan and refrigerate.

8.  When mixture has gelatinized, cut into cubes for feeding and store in freezer.

Figure 1. Comparison Between Bath and Feed Treatments for Hexamitiasis

Legend to Figure 1

a. Intensity of Infection represents the average number of Hexamita in four 100x fields.
b. Control 1 represents the average Hexamita count in the water change group (not measured on day 4).
c. Control 2 represents the average Hexamita count in the no water change group (not measured on day 4).

References

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2.  Andrews, C., Exell, A., and Carrington, N. The Manual of Fish Health. Tetra Press. Morris Plains, New Jersey. 1988; 122-123.

3.  Becker, D.C. Flagellate Parasites of Fish. In: Parasitic Protozoa, (J.P. Kreier, Ed.). Academic Press. New York, New York. 1977; Vol.II: 403-416.

4.  Beleau, M.H. Evaluating Water Problems. In: Tropical Fish Medicine, (M.K. Stoskopf, Ed.). Vet Clin No Am: Sm An Practice. 1988; 18(2): 291-304.

5.  Gratzek, J.B. Parasites Associated With Ornamental Fish. In: Tropical Fish Medicine, (M.K. Stoskopf, Ed.). Vet Clin No Am: Sm An Practice. 1988; 18(2): 393.

6.  Post, G. Textbook of Fish Health. T.F.H. Publications, Inc.. Neptune City, New Jersey. 1987; 165-169.

7.  Specht, D., and Floyd, R. Significance of Intestinal Parasitism in the Mortality of Domestically-Bred and Imported Angelfish, Pterophyllum scalare. In: Proceedings of the 20th Annual IAAAM Conference. San Antonio, Texas. 1989.

8.  Stoskopf, M.K. Fish Chemotherapeutics. In: Tropical Fish Medicine, (M.K. Stoskopf, Ed.). Vet Clin No Am: Sm An Practice. 1988; 18(2): 343.