Detailed project information for Study Plan Number 01075

Branch :	Fish Health Branch
Study Plan Number :	01075
Study Title :	Investigations on fish health in selected tributaries of the Chesapeake Bay and linkages to water quality, land use and other environmental factors
Starting Date :	04/01/1998
Completion Date :	09/30/2005
Principal Investigator(s) :	Blazer, Vicki S.; Densmore, Christine; Ottinger, Chris & Young, John
Primary PI :	Blazer, Vicki S.
Telephone Number :	(304) 724-4434
Email Address :	vicki_blazer@usgs.gov
SIS Number :	5003026
Primary Program Element :	Fisheries and Aquatic Resources
Second Program Element :	Fish and Aquatic Habitats
Status :	Completed
Abstract :	BACKGROUND During the summer and fall of 1996 and 1997, an unusually high prevalence of skin lesions in fishes from the Pocomoke River, Maryland and other tributaries of Chesapeake Bay stimulated significant public concern and scientific interest. These skin lesions ranged from small petechial hemorrhages to abrasions to deep ulcers penetrating underlying muscle and visceral organs. A variety of fish species were involved as indicated by results of surveys conducted by several state and federal agencies during this time period. In addition, two fish kills involving primarily juvenile Atlantic menhaden (Brevoortia tyrannus) occurred in the Pocomoke River during August, 1997. The fish kills as well as the variety of fish lesions were attributed to the presence of the toxic dinoflagellate, Pfiesteria piscicida or Pfiesteria-like (Pfiesteria-complex organisms) dinoflagellates. Because menhaden were the most frequent target of acute fish kills and episodes of fish lesions in the Chesapeake Bay, the penetrating ulcers so common in this species are now viewed by many as "Pfiesteria-related" and thought to be caused by exposure to Pfiesteria toxin (Burkholder and Glasgow 1997). Worldwide there has been a reported increase in frequency and severity of harmful algal blooms, HABs (Lassus et al. 1995). Dinoflagellates contribute to these HABs with over 40 species verified as producing toxins (Steidinger 1993). Pfiesteria piscicida (Steidinger et al. 1996), a recent addition to this list, was first observed in 1988 in an experimental tilapia (Tilapia aurea) culture system in North Carolina (Smith et al. 1988; Noga et al. 1993). Acute mortality was reported with moribund fish showing respiratory distress (shallow, rapid breathing), neurological signs (including hyperexcitability, lack of balance and spinning in the water column) and a cloudiness of the skin (Noga et al. 1993). Interestingly, chronic ulcerative skin lesions similar to those we observe in wild menhaden were not reported. However, subsequent to that discovery, P. piscicida was implicated as the cause of major estuarine fish kills in the Neuse and Pamlico estuaries and epidemic disease of estuarine fishes along the Atlantic Coast of the U.S. (Burkholder et al. 1992; 1995; Noga et al. 1996). In 1997 we examined histologically over 100 menhaden collected from both fish kills and areas where “significant numbers” of lesioned menhaden were observed. Consistently, these lesions are composed of a chronic inflammatory response around very invasive, penetrating fungal hyphae. The lesions we observed in the menhaden are identical to those described for epizootic ulcerative syndrome, EUS, (Vishwanath et al. 1998) in the IndoPacific, mycotic granulomatosis, MG, (Hatai et al. 1977) in Japan and red spot disease, RSD, (Callinan et al. 1989) in Australia. These ulcerative syndromes are all now known as EUS (Callinan 1994) and Aphanomyces invadans has been confirmed as the cause (Lilley et al. 1998). In Spring 1998, we began the first year of a three year study to evaluate general fish health in selected tributaries (Study Plan #01075). This research utilizes white perch as the test species because we can obtain the appropriate size specimens in all of the tributaries during the three time periods sampling occurs. This study includes a comprehensive fish health assessment, immune function evaluation and correlation of findings with land use. During the sampling trips in August 1998 we were also able to capture lesioned menhaden from the Wicomico and Pocomoke Rivers. There were no fish kills in 1998, the fish were not showing clinical signs of toxin exposure, nor were significant numbers of Pfiesteria-like dinoflagellates found in these systems. However, utilizing special techniques developed for the isolation and growth of A. invadans, we were able to isolate a fungus from lesioned menhaden in the Wicomico and Pocomoke Rivers. Recent work conducted by researchers in the Indo-Pacific has confirmed that a single species of Aphanomyces is a necessary cause of EUS - it occurs in all outbreaks and in some outbreaks it may be the only biological factor required for disease to occur (Lilley et al. 1998). The pathogenic isolate was described and initially named Aphanomyces invaderis (Willoughby et al. 1995) and is now known as A. invadans (Index of Fungi 1997). This species is a slow-growing, aquatic fungus with wide, aseptate mycelia. It grows best between 24 and 30oC, will grow at 31oC but dies at 37oC. Lilley et al. (1997a) used random amplification of polymorphic DNA (RAPD)-PCR to compare isolates of Aphanomyces from various locations in Bangladesh, Thailand, Indonesia, the Philippines, Australia and Japan. They found the isolates were conspecific and probably constitute a single clonal genotype spread throughout the Indo-Pacific area. It was speculated that both floods and massive cross-border movement of fish for aquaculture and ornamental fish industries caused this spread. A common feature of EUS is the variety of aquatic fungi (including species of Aphanomyces, Achlya and Saprolegnia) growing saprophytically on the surface of the lesions. This has led to confusion as to which fungus is responsible for the characterisitic granulomas and lesion formation (Lilley and Inglis 1997). However, recent studies have confirmed A. invadans as the cause of these lesions. Pathogenicity of isolates from wild fish was tested by inserting a small mass of mycelia under the dermis of healthy snakeheads (Channa striatus) via an incision on the left shoulder (Roberts et al. 1993). When the slow-growing, thermolabile strains of Aphanomyces were inserted, there was initially a mild, local inflammatory lesion, followed by severe myonecrosis extending deep into the muscle (within 5 days). Within 15-20 days the lesion progressed to resemble the typical chronic granulomatous response seen in wild EUS-infected fish. This experiment was carried out at 22oC. When the same infectivity studies were carried out with strains of Achlya, Saprolegnia and fast-growing Aphanomyces there was a localized foreign-body-type response and the inoculation site healed (Roberts et al. 1993). The skin lesions we observed are also similar, both grossly and histologically, to ulcerative mycosis described in the 1980's from menhaden along the east coast of the United States (reviewed by Noga 1993) and to those in menhaden collected from the Chicamacomico River in September 1997 by Kane et al. (1998). The fungal pathogen Aphanomyces was reported to be involved with ulcerative mycosis by Dykstra et al. (1986). However, isolations were made on corn meal agar (Dykstra et al. 1986; Dykstra et al. 1989). These Aphanomyces isolates were of three distinct growth and morphology patterns: 1) isolates which grew vigorously, produced zoospores copiously and formed oospores (Aphanomyces isolate ATCC 62427); 2) isolates that produced asexual zoospores abundantly and grew vigorously; and 3) isolates that produced scant mycelium and few zoospores (Dykstra et al. 1989). Aphanomyces isolate ATCC 62427 was the isolate identified with ulcerative mycosis in subsequent comparisons of this disease with EUS (Lilley and Inglis 1997; Lilley and Roberts 1997) and found to be distinct from A. invadans. Attempts to reproduce the disease by challenge with this isolate failed to induce ulcerative mycosis, however, fish challenged with lesion material did develop typical lesions (Noga 1993). Hence, based on what we now know about the difficulty in culturing A. invadans (Willoughby and Roberts 1994), lack of growth on corn meal agar and growth characteristics (Willoughby et al. 1995), perhaps the actual pathogen of ulcerative mycosis in menhaden was never isolated. Most cases of this fungal infection from our study sites were observed in menhaden although we did note it in a few other fish species (one croaker and one spot). Atlantic menhaden are found in temperate waters of the Atlantic from Nova Scotia to Florida. They are a migratory, schooling fish of commercial importance, as well as prey for other fish species and seasonally important components of estuarine fish assemblages (Rogers and Van Den Avyle 1989). Larvae migrate into estuaries from May through October in the North Atlantic region and from October to June in the Mid-Atlantic (Reintjes and Pacheco 1966). A number of studies have shown high abundance of young menhaden in portions of the estuaries with the lowest salinities. Rogers et al. (1984) provided evidence that prejuveniles select tidewater areas that are fresh or low salinity. A comparison of EUS Aphanomyces isolates showed that these organisms were killed by exposure to 20 ppt sodium chloride for 1 h while the saprophytic isolates survived (Lilley and Inglis 1997). Dykstra et al. (1986) also found a salinity preference in Oomycetes isolated from menhaden with ulcerative mycosis in the Pamlico River estuary. One isolate of Aphanomyces exhibited enhanced growth when 2-4 ppt NaCl was added to medium, but was somewhat inhibited at higher concentrations (20-26 ppt). Perhaps we see the lesions on menhaden because of their schooling and/or feeding behavior and primarily in the rivers because of low salinity preference of the causative agent. However, once the infection is established and deep in the muscle it may be somewhat protected from higher salinities, as shown for other Oomycetes securely established on suitable substrates (Padgett 1984). It has been suggested that the fungal infections seen in menhaden and other estuarine fishes in North Carolina are secondary, opportunistic infections which occur subsequent to exposure of the fish to toxins from Pfiesteria (Noga et al. 1996; Burkholder and Glasgow 1997). However, scientific evidence for the relationship of these lesions with toxic dinoflagellates is lacking. Laboratory exposures to Pfiesteria have not produced similar lesions. In the initial laboratory mortalities, respiratory distress, neurological signs and a cloudiness of the skin with acute mortalities were reported (Smith et al. 1988; Noga et al. 1993). More recently, dermatological lesions including intra- and extra-cellular edema and necrosis of the epithelium resulting in erosions extending to the basement membrane were described (Noga et al. 1996). The same study also examined fish that survived an acute sublethal exposure to P. piscicida and were placed into clean water. One of ten fish subsequently developed lesions containing fungal hyphae. However, the hyphae appeared typical of secondary, saprophytic fungi growing in necrotic tissue. The fungus did not elicit the typical granulomatous response described in wild menhaden. The remaining fish had bacterial infections (Noga et al. 1996). Burkholder and Glasgow (1997) stated that "the fungi are opportunists; their hyphae generally do not penetrate to the lesion base, suggesting they do not form the lesions; rather, they colonize lesions formed by toxin(s) from P. piscicida." This is not the manifestation we observed. In all instances the fungal hyphae and surrounding chronic inflammation extended deep into muscle, often under intact, normal skin. We believe the raised lesions caused by the massive inflammatory response and hyphae may be an earlier manifestation of the deep ulcerated lesions. As the infection progresses, necrosis results from the effects of secondary invaders colonizing the lesions and the associated intense inflammatory response. Necrotic skin and muscle ultimately slough to form the more advanced, deeply penetrating, lesions. This was previously suggested as the progression in ulcerative mycosis (Noga 1993). Epizootic ulcerative syndrome is considered a multifactorial problem in that a variety of predisposing factors may be involved - the only required cause being the presence of A. invadans. Hence, EUS has been experimentally reproduced in susceptible species by mechanical damage of epidermis, intramuscular injection of a rhabdovirus or sublethal exposure to acid sulfate soil runoff water, followed by exposure to A. invadans propagules (Lilley et al. 1998). It is possible that toxic dinoflagellates may play a role in the initiation of some menhaden skin ulcers. Sublethal levels of toxin could damage the skin or cause immunosuppression thus allowing the fungi to invade. However, it seems unlikely that the same histopathological presentation would be so consistently observed if the fungi are simply opportunistic invaders. Rather, we would expect to see a variety of infections such as acute, subacute or chronic bacterial infections, lesions with only saprophytic fungi and bacteria growing in the damaged tissue and opportunistic parasitic infections. Hence, based on our recent observations and the numerous reports of similar epizootics in other parts of the world, we believe factors other than toxic dinoflagellates need to be considered as causes or initiators of these lesions. The presence of fish, particularly menhaden, with deeply penetrating skin ulcers, is currently used in Maryland and Virginia waters of the Chesapeake Bay, as one indicator of local activity by P. piscicida or other Pfiesteria-like dinoflagellates. Hence, the observation of these lesions during 1997 led to the closure of stretches of certain tributaries in Maryland for recreational, commercial and research uses for weeks at a time. However, the chronic nature of the lesions in a migratory fish and the consistent presence of an invasive fungal pathogen raises some questions as to their utility as an indicator of local activity of toxic dinoflagellates. It is very important for fisheries managers and public officials to understand the cause(s) of these lesions to be able to intelligently manage fish kills or fish “lesion events”. A second important and emerging problem is the lesion incidence in striped bass (which will be addressed in a separate study plan). The striped bass problem has been recognized by fishery management agencies as well as professional and recreational fishermen. Again, the problem is manifested by open sores of varying size and appearance on the skin of the bass. Studies by Vogelbein (1998; personal communication) and Baya and May (1998; personal communication) have indicated the lesions in striped bass from both Virgina and Maryland tributaries are caused by Mycobacteria sp. Mycobacteria are a group of bacteria that cause both human and animal health problems. The species that generally infect fish can cause chronic, non-healing, hard-to-treat lesions on the extremities of people handling the fish. They can be a serious problem for immunosuppressed individuals. As with the fungal infections in menhaden, the response to Mycobacteria is a chronic, granulomatous reaction. All of the above findings suggest that a) the fish health issues in the Chesapeake are varied and complex and b) we need to evaluate many factors including land use, point and nonpoint pollution, interacting fish population changes, and climate for effects on the fishes as well as the potential pathogens. More specifically, the findings suggest 1) Pfiesteria may not play a role in the development of these chronic lesions; 2) although Pfiesteria may be an initiator of either epithelial damage or immunosuppression, there may be many other environmental factors which can also weaken the fish and allow opportunistic pathogens to cause lesions and 3) it is important to examine a variety of potential stressors for their effects on fish disease resistance, and microbial reproduction and virulence. The studies listed below will complement interdivisional work already ongoing in the Chesapeake Bay through the USGS Place-Based Science Program. It also responds to urgent needs to address fish mortality, fish disease problems, and human health implications. Information generated by these studies on interactions of non-point source pollution, fungal populations, and fish health (immune) response will assist and complement ongoing and proposed studies by BRD partners, including other USGS divisions, states of Maryland, Virginia, Delaware, South Carolina, Florida and Georgia, FWS, EPA, NMFS, NIH and CDC. OBJECTIVES 1) Identify and characterize the fungal pathogen isolated from menhaden. 2) Determine infectivity of fungal isolate in a number of fish species. 3) Evaluate environmental factors that may affect susceptibility of fish to fungal infections in the laboratory. 4) Determine the factors that may influence development of lesions in menhaden in the Chesapeake Bay tributaries. HYPOTHESES TO BE TESTED 1) Lesions in menhaden are caused by an Aphanomyces fungi. 2) A variety of environmental stressors can predispose fish to this infection.
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