Abstract

Larval nematodes belonging to the family are commonly found encysted in the musculature of wild South American tropical fish. In the fish examined, cysts were usually located in the musculature of the back and flank, were 3 to 5 millimeters in diameter and dark brown to red in color. In some of the smaller tetras, the nematode may he 2 to 3 times longer than the fish itself once the worm is extracted from the cyst.

The incidence of infestation appears to vary between species. During certain months of the year between 1% and 3 % of the imported rummy nose tetras may be affected. The number of infected fish in the wild populations may be much higher since affected fish are usually not marketable at the retail level and some effort is made to cull out infected fish in South America. Imported South American fish which have a noticeable incidence of infection include: the rummynose tetra (Hemigrammus bleheri), red phantom tetrad (Megalamphodus sweglesi), red pristella tetra (Pristella maxillaris), gold tetra (Hemigrammus rodwayi), and the transparent knife (Eigenmannia virescens). Two experimental methods of surgical nematode removal were attempted. These surgical techniques and the method of anesthesia used are discussed.

Introduction

The tropical pet fish industry in the United States is currently importing millions of freshwater teleost fish per year. Many of these animals are obtained from the will and shipped within a few days to wholesalers for sale to retail pet shops. Fish come from the Amazon Basin of South America, Africa and Asia. They are frequently accompanied by a tremendous range of viruses, bacteria, protozoa, and multicellular parasites. Many of these organisms are opportunistic pathogens which through the stresses of transport, overcrowding and reduced water quality, cause disease. Any diseased fish which does not die may be compromised in such a way as to reduce its retail value or make it unmarketable. Most of these individuals are treated prior to reaching the pet stores and some are culled.

We have noted that a significant number of tropical fish imported from South America have nematode larvae Encysted in their musculature. The larvae have been identified as Eustrongylides sp. Dioctophymatidae (Karmanova 1986). The life cycle of Eustrongylides sp. is complex and can involve several hosts. Fish-eating birds function as the definitive host, with the adult worm living in the proventriculus and esophagus. Adult Eustrongylides produce eggs which contain L₁ larvae. The ova are ingested from the bird's feces by aquatic oligochaetes, an intermediate host. While in the oligochaete, the L₁ hatches and matures to the L₃ larva. When the oligochaetes are eaten by fish, Eustrongylides encysts in the fish musculature or coelomic cavity and matures to the L₄ stage. L₃ and L₄ larvae can also be found in piscivorous fish, amphibians and reptiles, which function as reservoir hosts. Freshwater and estuarine fish are involved with this cycle. Adult worms have been reported in, cormorants, auks, grebes, mergansers, mallards and other bird species. (Karmanova 1986, Paperna 1974)

The nodules caused by the encysted larvae are grossly visible and render the fish unmarketable. Direct per cutaneous surgical extraction is possible. To date, we have performed several experimental surgeries to examine the efficacy of surgical larval extraction.

Materials and Methods

Animals and Holding Conditions

Affected fish were divided into two groups; the first group served as an anesthetic control group and were allowed to recover from anesthesia without surgery (15 fish total) and the second group was anesthetized and subsequently underwent surgical excision of the encysted nematode (15 fish total).

Four species of tropical freshwater fish were used. Adult rummynose tetras, red phantom tetras, red pristella tetras and gold tetras with encysted larvae were randomly assigned to either the anesthesia control or the experimental surgery group. Each group was maintained separately in 30 gallon glass aquaria with a flow-through water system. Water temperature was maintained at 23±1°C and the fish were fed a commercial flake food twice daily. All fish were allowed to acclimate to the holding aquaria for at least two days prior to the beginning of the experiment.

Anesthesia

The fish were anesthetized with tricaine methanesulfonate (MS-222) at a concentration of 100 ppm (Brown 1988). Fish were placed in the anesthesia bath until a surgical plane was reached (loss of equilibrium, slow ventilatory rate and a failure to respond to external stimuli). Induction took approximately thirty seconds and 29 of the 30 fish completely recovered within two minutes after being returned to their holding aquaria.

Surgical Techniques

Two methods of parasite excision were examined. In Method 1, a three millimeter long, full-thickness skin and cyst incision was made with a sterile #10 scalpel blade (7 fish received this treatment). The larva was located and extracted with watch-maker's forceps. The incision was not closed and the fish were returned to the holding aquarium and recovery monitored (Figure 1). Method 2 employed the use of a 23 gauge hypodermic needle to puncture the cyst wall (Figure 2). The larva was then extracted with watch-maker's forceps (8 fish in this group). The recovery procedures were the same as in Method 1.

All fish undergoing surgery were treated post-operatively with nitrofurazone at a concentration of approximately 15 ppm for 12 hours. This treatment was intended to reduce the incidence of secondary bacterial disease.

Click on the image to see a larger view

	Figure 1. Method 1, removal of encysted larvae through an incision created by scalpel blade.
	Figure 2. Method 2, removal of encysted larvae via hypodermic needle.

Gross and Histological Evaluation

Affected fish were examined grossly noting the size, number, color, location and progression of the lesions. A transparent knife, rummy nose tetra, and gold tetra were chosen at random for histological evaluation of the cyst lesions. Each fish was preserved in 10% buffered formalin. After fixation, tissues were embedded in paraffin, cut into 2-3pm thick sections, affixed to microscopic slides and stained with hematoxylin and eosin. All organ systems including the tissues surrounding the cyst lesions were examined.

Results

All but one fish receiving an experimental surgery recovered without incident from anesthesia (one fatality occurred). However, each fish which received a full-thickness incision made via a scalpel blade died within 24 hours. Two of the eight fish receiving the hypodermic needle extraction treatment survived for up to 40 hours post-operatively. The other six fish in this group died within 24 hours.

Gross examination of each affected fish revealed 1 dark brown to red, raised nodular lesion, ranging from 3 to 5 millimeters in diameter. These cysts were usually located subcutaneously or intramuscularly in the back or flank region. Each cyst contained one Eustrongylides larva which, upon extraction, was up to two to three times the length of the fish (15-35mm). A group of 10 affected tetras (mixed species), which were not in the anesthesia control or surgical excision groups, were observed for 100 days and remained otherwise healthy and showed no progression of the lesions created by the cysts.

Histopathologic changes were noted in tissues from a parasitized transparent knife fish. Several transverse sections of nematode larvae were seen in the coelomic cavity between the liver and the pyloric stomach. A thin (approximately 15-20µm) connective tissue capsule surrounded these nematodes. A moderate, predominantly mononuclear cell, inflammatory response was seen around the parasites. A much thicker (50-60µm) connective tissue capsule disrupted epaxial muscle architecture in the trunk and Caudal peduncle region, just dorsal to the spinal cord. Although this connective tissue layer was extremely well vascularized with numerous arborizing capillaries, there was no associated inflammation in this area.

An additional pathologic change was noted in this fish. The gills were extensively parasitized by a piriform-shaped, flagellated protozoan, Ichthyabodo necatrix. Approximately 75 of these organisms were noted per high-power field. They were attached to the gill epithelium with a destruction and necrosis of the gill filaments, sloughing of epithelial cells and scattered areas of hemorrhage. Ichthyabodo were not seen in the other two fish examined.

A parasitized rummy nose tetra was also examined histologically. A thin walled (20-25µm) connective tissue cyst surrounded the larval nematode and extended the width of the caudal peduncle. Although the lesion was extensive, there was little associated inflammation. Multifocal areas of melanin accumulation were present around the cyst.

Histopathologic findings differed on examination of a parasitized gold terra. Extensive multifocal to coalescing necrosis of the body wall musculature was evident secondary to parasite migration. Moderate mononuclear cell inflammation and cell debris was also evident. The parasite was not encapsulated in connective tissue in this fish.

Discussion

In our experience, several South American fish species have a notable incidence of Eustroylides sp. infection: the rummy nose tetra, red phantom tetra, red pristella tetra, gold terra, and the transparent knife. The incidence of infection appears to vary between species. Between 1% and 3% of the imported rummy nose tetras may be affected. The number of infected fish in the wild populations may be much higher since these fish are usually not marketable at the retail level and are culled prior to leaving South America.

We are unaware of any attempts at medical treatment for this condition. Killing the encysted larvae using an appropriate anthelmintic has potential, but a severe foreign body reaction to the decomposing nematode is likely. Surgical removal would avoid such a complication, however, the results of this study suggest that surgical excision methods are of little value. It is likely that the fish treated with a full-thickness incision via scalpel blade succumbed due to secondary protozoal/bacterial/viral infection(s) or to disrupted osmoregulation from the large breach of their integument. The increased survival time of two of the fish receiving the hypodermic needle extraction method is presumed to be due to the relatively small integument defect created by the needle This method may be useful for the removal of other encysted parasites, such as metacercaria, found in other fish species.

The infectious potential created by these infested fish entering the tropical fish trade, i.e., being purchased by a hobbyist, is insignificant. The affected species are not routinely used as "feeder fish" for other fish, reptiles, birds or mammals so the possibility of these larvae reaching a definitive host is unlikely. Surgical excision may be more appropriate for the correction of similar conditions in larger fish where the survival rate may be greater and the remaining defect is not noticeable.

Acknowledgements

The authors would like to thank Dr. Alan Kelly of the University of Pennsylvania School of Veterinary Medicine, for his support and Mr. David O'Beirne of Greenway Farms for supplying the fish.

References

1.  Brown, L.A. 1988. Anesthesia in fish in The Veterinary Clinics of North America, Tropical Fish Medicine. ed. M. Stoskopf, Volume 18 (2): 315-328.

2.  Karmanova, E. M. 1986. Dioctophymidea of animals and man and diseases caused by them. (Fundamentals of Nematology, Vol. 20). New Delhi, India; Amerind Publ. Co. Pvt. Ltd. 383 pp. (Translated from /968 Russian publication)

3.  Paperna, I. 1974. Hosts, distribution and pathology of infections with larvae of Eustrongylides (Dioctophymidea Nematoda) in fishes from East African lakes. J. Fish Biol. 6: 67-76.