Abstract

Aeromonas salmonicida, generally considered to be a salmonid pathogen, was isolated from paddlefish (Polyodon spathula). Bacterial isolates were cultured from gill and kidney tissue of mortalities collected in 1992 during a disease epizootic at the Spring River State Hatchery, Arkansas.

Introduction

Life History and Culture of Paddlefish

Paddlefish, Polyodon spathula, are endemic to river systems of the Mississippi River and Gulf Slope basins. Paddlefish were abundant in these large rivers but within the last century both the range and the population of the species diminished (Gengerke, 1986; Russell, 1986). Six states currently list paddlefish in protective status. Overexploitation and pollution have contributed to the decline in paddlefish populations. Also, paddlefish populations have been adversely affected by man-made alterations that destroy or prevent access to spawning sites (Russell, 1986; Unkenholz, 1986).

Paddlefish prefer open water environment of large rivers. Today, many of these large rivers have been dammed or channelized to increase water flow which has altered natural temperature and flow patterns of the river, as well as destroyed optimum spawning habitat for paddlefish (Unkenholz, 1986). Paddlefish now concentrate in pools formed below sand bars, islands, bridge supports and other structures that impede water currents of rivers. Large populations of paddlefish are also found in reservoirs but they move into streams to spawn (Russell 1986).

Paddlefish reach sexual maturity when they are between 8-12 years-old and weigh between 15-30 lbs. Paddlefish do not exhibit external sexual characteristics and are difficult to sex, except by examination of the gonads. Male paddlefish can spawn yearly, but females cannot and are thought to spawn only once every 4 to 5 years. Egg production for females varies between locations but averages near 10,000 eggs per pound of body weight. Natural spawning of paddlefish occurs over gravel substrate during late March through June when water temperatures are near 55° F and water flows increase (Russell, 1986). Eggs attach to gravel substrate and hatch within 7 days. Young paddlefish swim up and take approximately 5 days to absorb there yolk. At this stage, young paddle fish do not resemble adults, and they do not have a rostrum or paired fins.

The rostrum begins to develop within ten days and within a month the juveniles begin to resemble adult paddlefish (Purkett, 1961).

The first attempts to culture paddlefish were first reported in the early 1900's (Alexander, 1914); however, the first successful collection and hatching of eggs did not occur until 1961 (Purkett, 1963). Recently, culture of paddlefish important not only to enhance existing populations in the wild but to meet commercial demand for roe (caviar) and flesh from paddlefish (Semmens and Shelton, 1986; Ottinger et al., 1992). Because of spawning requirements and extended time between ovulations for females, broodfish are generally obtained yearly from wild populations. Broodfish are held in small ponds or tanks with a continuous supply of water for approximately 2 months. Males and females have traditionally been given hormone injections to insure optimal release of sex products. Fertilized eggs are then maintained using standard procedures (Graham et al., 19846). Kurten et al. (1992) and Ottinger et al., (1992) reported culture methods for rearing and maintenance of juvenile paddlefish. Fingerlings raised by these methods have been used for research and to supplement recovery of wild populations. As with other fish, rearing conditions for paddlefish are often overcrowded which contributes to poor water quality and disease epizootics.

Diseases of Paddlefish

Most of the published literature on diseases of paddlefish concerns parasitic infestations. A parasitic coelenterate, Polypodium hydriforrne, and a microsporidian, Pleistophora sp., have been reported in paddlefish eggs (Suppes and Meyer, 1975; Holloway et al., 1991). Eggs infected with P. hydriforme are enlarged and are gray in color as opposed to the normal greenish-black color. Typical stolons can be dissected from the egg and viewed microscopically; however, histological evaluations should be done to confirm identification. The coelenterate has been reported in up to 80% of the spawning female paddlefish examined in any particular study but only 1% of the total egg mass is infested (Holloway et al., 1991). Thynnascaris dollfusi Contracaecum spiculigerum, Cammallnus sp. and Hysterothylacium dollfusi, are nematodes commonly found in the digestive tract of paddlefish (Miyaziki, et al., 1988; Schmidt et al., 1974). Cestodes infestation has also been reported in paddlefish. For example, Marsipometra sp. infested nearly 100% of the paddlefish examined from the Yellowstone River, Montana (Lockard and Parson, 1975). Parasitic copepods, Ergasilus elongates, and a trematode, Diclybothrium hamulatum, were also reported to infest paddlefish from the Yellowstone River (Lockard and Parsons, 1975).

Only one study has documented a tumor in paddlefish. A large tumor at the posterior portion of the head, in a fourteen year-old paddlefish, was described as a cranial chondrosarcoma. The paddlefish was obtained from the Lake of the Ozarks, Missouri; but the authors did not speculate on the prevalence of this type of malignancy in populations of paddlefish (Bean-Knudson, et al., 1987). The most commonly noted problems of paddlefish are scars from attempts of anglers to snag fish or from propeller damage by power boats, as well as bent rostrums from the fish colliding into dam structures (Rosen and Hales, 1980). As intensive culture of paddlefish becomes more common, bacterial, viral and other parasitic problems are likely to occur.

Furunculosis Epizootic

The first known bacterial epizootic in paddlefish being reared for stocking Arkansas rivers was reported by Ford, et al., (1994). Aeromonas salmonicida, causative agent of furunculosis of fish, was isolated at the Spring River State Fish Hatchery in Arkansas. Isolates of this bacterium are gram negative, non-motile, and are homogeneous both in their biochemical and serological reactions (Griffen, 1954; Eddy, 1960; Bullock et al., 1983). This bacterium is considered to be distributed throughout the world's marine and freshwater environments, although epizootics occur more common in salmonid populations (Bullock et al., 1983). Aeromonas salmonicida has been reported in other fish including carp (Bootsma et al., 1977), goldfish (Elliott and Shotts, 1980), minnow (McFadden, 1970; Hastein et al., 1978), cod (Larsen and Jensen, 1977) and bass (Bulkley, 1969; Le Tendre et al., 1972).

Aeromonas salmonicida was isolated during acute mortality of paddlefish fingerlings (12-13 cm) reared during 1992 at the Spring River Hatchery. The Spring River, which is the main water supply for the hatchery, flooded several times during that Spring. Turbidity of water entering the hatchery increased during these flood stages. In June, overcrowding was thought to have caused fungal growths on the rostrum of these fish; therefore, they were split to reduce densities and treated with formalin. Mortality continued to rise although fungal problems were no longer evident. Two fish were necropsied and cultured for bacterial isolation. Presumptive identification of A. salmonicida was made from kidney samples. For confirmation, the bacterial cultures plus additional mortalities were shipped to the National Fish Health Research Laboratory (Leetown, WV). Gill tissue, kidney, spleen and liver were sampled from four fish and plated onto Coomassie Brilliant Blue agar according to the methods of Cipriano et al. (1992). Aeromonas salmonicida was isolated from gill tissue or kidneys of 3 out of 4 paddlefish submitted to our laboratory. Identification was confirmed based on the methods of Amos (1985) and MacFaddin (1981) and included standard biochemical reactions listed in Table 1. Isolates were also sensitive to oxytetracycline (30 g) as determined by disc-diffusion testing; therefore, remaining paddlefish were treated with terramycin for 10 days under an emergency INAD permit issued by the FDA. Treatment reduced mortality and after a 50 day withdrawal period, most fish were stocked. A subsample of paddlefish was transported from the Spring River Hatchery to Andrew Hulsey State Fish Hatchery (Hot Springs, AK). Rearing conditions were similar to those at the Spring River Hatchery. These fish were used to conduct infectivity trials in order to fulfill Koch's postulates. Unexposed stocks of juvenile paddlefish were not available; therefore, survivors of the furunculosis epizootic had to be used. From an aliquot of A. salmonicida obtained during the epizootic and frozen (-70° C), a 24 hour culture was grown on Tryptic Soy Agar and the cells were washed with sterile phosphate buffered saline (PBS). Cell suspensions were standardized with sterile PBS to 30% and 95% transmission using a spectrophotometer. An inoculum (0.1 ml) from each standardized suspension was injected, intraperitoneally, into groups of 20 fish; a control group of 20 fish were injected with PBS (0.1 ml). Only three fish died due to furunculosis during the trial. Two died from the group of fish that received the highest concentration of bacteria (30% T) and one died from the control tank.

Paddlefish were available in July, 1993 from the new year-class of juveniles. An infectivity trial was repeated, as described above, using groups of five fish. Cell suspensions of Aeromonas salmonicida were prepared from an aliquot of the original isolate frozen at -70° C. Within five days, all paddlefish injected with the highest concentration of bacteria died. Only one fish injected with the lower concentration of bacteria died, and all fish injected with PBS survived. Aeromonas salmonicida was re-isolated from each mortality in this trial.

Discussion

Purification and transport of the bacterial culture between the Arkansas hatchery and the NFHRL could have affected the virulence of the isolate and resulted in low level of mortality reported in the first infectivity trial. At the same time, a duplicate sample of the A. salmonicida isolate was used to inject brook trout maintained at NFHRL, and 80% percent (16/20) of these fish died within 10 days. Also, the isolate used in the 1993 infectivity trial remained virulent although transport of the isolate back to Arkansas occurred one-year later. Perhaps, the majority of paddlefish that received an injection of A. salmonicida in the first trial had developed some level of natural immunity or clearance ability that protected them from the subsequent exposure. Paddlefish can mount a humoral immune response against such antigens as Salmonella whole cells, sheep erythrocytes and human erythrocytes (Legler et al., 1971). Information on responses of paddlefish to A. salmonicida or other bacterial pathogens of fish has not been reported.

Susceptibility of paddlefish to A. salmonicida should be a management concern of wild and hatchery-reared stocks. McCraw (1952) reported that carriers of A. salmonicida were established among fish populations that survived furunculosis. Carriers of A. salmonicida have also been reported in rainbow trout and brook trout populations (Bullock and Stuckey, 1975). The potential for paddlefish to become a carrier of the pathogen should be examined.

Table 1. Biochemical characteristics of Aeromonas salmonicida isolated from paddlefish

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