Final Report: Ecohab: Pfiesteria Or Fungus? Etiology Of Lesions In Menhaden

EPA Grant Number: R828225
Title: Ecohab: Pfiesteria Or Fungus? Etiology Of Lesions In Menhaden
Investigators: Shields, Jeffrey , Blazer, Vicki , Haas, Larry , Kator, Howard , Kiryu, Yasunari , Vogelbein, Wolfgang K.
Institution: Virginia Institute of Marine Science , College of William and Mary-VA
EPA Project Officer: Hiscock, Michael
Project Period: June 16, 2000 through June 15, 2003
Project Amount: $508,937
RFA: Ecology and Oceanography of Harmful Algal Blooms (1999) RFA Text |  Recipients Lists
Research Category: Aquatic Ecosystems , Water Quality , Water , Ecosystems

Objective:

The overall goal of this research project was to identify the interrelationships between menhaden, Pfiesteria, and Aphanomyces invadens, and the environmental conditions that may modulate or contribute to the epidemics of ulcers on the fish. The overall objective was to determine whether Pfiesteria or the oomycete, A. invadans, was responsible for the ulcerous lesions in fish that have occurred in the Chesapeake Bay and elsewhere. The specific objectives of this research project were to conduct controlled laboratory exposure studies with menhaden to: (1) identify the causal agents responsible for the ulcerous lesions; and (2) identify the contributory environmental and biological conditions that were required for the development and progression of these lesions.

Menhaden, Brevoortia tyrannus, develop ulcerous skin lesions known as ulcerative mycosis (UM) that have been attributed to exposure to Pfiesteria toxins. The presence of these lesions in conjunction with counts of presumptive Pfiesteria-like cells in water samples have been used as the primary criteria for river closures in Maryland and Virginia because of recent Pfiesteria activity. There is controversial evidence, however, that the lesions are caused by an oomycete water mold, Aphanomyces sp., either as a primary or secondary invader. The oomycete virtually is always associated with the lesions, with hyphae penetrating the tissues and organs of the infected fish.

Summary/Accomplishments (Outputs/Outcomes):

Etiology of Ulcerous Lesions in Menhaden

We provide direct evidence that the fungus-like oomycete, A. invadans, is the primary etiological agent of UM in the Atlantic menhaden, B. tyrannus (Kiryu, et al., 2002, 2003). The fungus was capable of infecting fish without the need for other predisposing factors because 13-32 percent of fish in untraumatized bath exposures developed the typical ulcerous lesions. We demonstrated conclusively that this fungal agent readily infects menhaden and that it elicits the characteristic skin ulcers commonly observed in wild populations from Eastern U.S. estuaries. Most significantly, it does this in the absence of P. piscicida or P. shumwayae, toxic dinoflagellates recently implicated by others as the etiologic agent of this disease (e.g., Burkholder, et al., 2001).

We have fulfilled Koch's postulates using two isolates of the fungal agent and two routes of exposure. One of our strains (WIC) was isolated from naturally infected menhaden from the Chesapeake Bay. When administered as secondary zoospores to the same species of fish by sub-cutaneous injection or bath exposure, this strain elicited ulcerous skin lesions identical to those observed in the wild. Subsequent reisolation and identification of this organism (WIC) from laboratory-infected menhaden was successful. We obtained similar results with PA7, a foreign strain of A. invadans isolated from snakehead, Channa striatus, in Thailand. Interestingly, a third isolate, ATCC-62427, initially suggested as the type species for UM (Dykstra, et al., 1986), was not pathogenic to menhaden. It is not A. invadans, but rather a rapidly growing saprophytic species that has been suggested previously (Lilley and Roberts, 1997; Blazer, et al., 1999, 2002).

Contributory Factors in the Development of Lesions

A portal of entry enhanced the infectivity of A. invadans in menhaden (Kiryu, et al., 2002, 2003). Injury or stress from trauma (as net-handling) dramatically enhanced transmission of the fungus in bath exposures resulting in prevalences of 100 percent in exposed fish, but untraumatized fish also developed relatively high prevalences of disease (13-32 percent). Thus, although menhaden are susceptible to infection by A. invadans, a portal of entry, such as may be caused by other environmental factors, greatly enhances the infectivity of the fungus and the mortality of exposed fish. However, we also have demonstrated, that the oomycete can penetrate through the epidermis of the menhaden, a feature not reported for strains from Southeast Asia (Kiryu, et al., 2003).

Furthermore, the oomycete is highly infectious and highly pathogenic. In a dose-response study (Kiryu, et al., 2003), secondary zoospores of the WIC strain (an endemic isolate of A. invadans in menhaden from Maryland) were subcutaneously injected into menhaden at doses of 0, 1, 10, 50, 100, and 500 zoospores per fish. The fungus was highly pathogenic, with an LD50 of approximately 10 zoospores with a single zoospore, which was sufficient to cause frank lesions. Thus, we speculate that A. invadans is a significant factor in mass mortalities of menhaden by compromising the energetics and survivorship of infected fish subjected to hypoxia or other stressful events.

We also investigated the infectivity and role of P. piscicida in the etiology of skin ulcers in menhaden, B. tyrannus. In dose-response studies, fish were exposed to dinospores of P. shumwayae (CCMP2089, an endemic isolate from the Pamlico River, NC) at doses of 1,000, 3,000, and 5,000 dinospores per mL in 38 L aquaria. Tilapia, Oreochromis sp., also were used in similar challenge studies. None of the exposed fish developed the characteristic skin lesions that we now have confirmed are a result of A. invadans. The pathology of exposed menhaden essentially is identical to that of tilapia exposed to P. shumwayae (Vogelbein, et al., 2001). To reiterate, none of the laboratory exposed fish developed the characteristic lesions observed in lesionous menhaden from the field, the etiology of which we attribute to A. invadans.

We also explored host factors in the infectivity of the WIC strain of A. invadans. We inoculated zoospores of the oomycete into four commonly occurring host species: Atlantic menhaden, striped killifish, mummichog, and hogchoker. All four species are abundant in estuaries where UM is prevalent in menhaden; however, none of the species, other than Atlantic menhaden, have been observed with a high prevalence of lesions. Menhaden, inoculated with A. invadans, developed ulcerative lesions identical to those previously described (Kiryu, et al., 2002). Hogchokers and striped killifish developed ulcerative lesions similar to those in menhaden, but they appeared 7-10 days later and at a significantly higher prevalence (Johnson, 2003; Johnson, et al., in review). Mummichogs experienced a lower prevalence of lesions compared to the other species. All of the fish species tested inhabit similar estuarine environments, yet the Atlantic menhaden appears to be the only species that is consistently found with ulcerative lesions. This suggests that subtle hosts or environmental barriers to infection limit the development of A. invadans-induced lesions in other species.

To better understand aspects of transmission, we characterized sporulation and encystment of secondary zoospores of three isolates of A. invadans at different salinities and temperatures (Kiryu, et al., in review). Sporulation was dependent on low salinities. As a result of using "pond water," augmented with artificial-sea-salts at room temperature, the endemic strain WIC and the Thailand strain PA7 produced free-swimming secondary zoospores at salinities of 0, 1, and 2 psu, but not at 4 psu or higher. Secondary zoospores of another strain, ATCC-62427 (Aphanomyces sp.), were observed at 1, 2, 4, and 8 psu, but not at 0 and 12 psu. For both WIC and PA7, zoospores were produced within 1-3 days after the initiation of sporulation. Menhaden tissue, but not tissues of other fishes, caused rapid (2 hours) encystment of the secondary zoospores of WIC, a feature consistent with a well-established host-parasite association. Salinity and temperature requirements for sporulation indicated that juvenile menhaden must acquire infections in the relatively low salinity, oligohaline marshes of the Eastern United States.

In conclusion, our studies indicated that the oomycete water mold A. invadans was the etiological agent responsible for the development of characteristic lesions on menhaden that were formerly ascribed to Pfiesteria. The oomycete was highly invasive, could serve as a primary pathogen, and caused significant mortality in infected fish. A portal of entry enhanced its transmission.


Journal Articles on this Report : 9 Displayed | Download in RIS Format

Other project views: All 41 publications 9 publications in selected types All 9 journal articles
Type Citation Project Document Sources
Journal Article Berry JP, Reece KS, Rein KS, Baden DG, Haas LW, Ribeiro WL, Shields JD, Snyder RV, Vogelbein WK, Gawley RE. Are Pfiesteria species toxicogenic? Evidence against production of ichthyotoxins by Pfiesteria shumwayae. Proceedings of the National Academy of Sciences of the United States of America 2002;99(17):10970-10975. R828225 (2000)
R828225 (2001)
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  • Abstract: PNAS-Abstract
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  • Other: ResearchGate - Abstract & Full Text - PDF
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  • Journal Article Johnson RA, Zabrecky J, Kiryu Y, Shields JD. Infection experiments with Aphanomyces invadans in four species of estuarine fish. Journal of Fish Diseases 2004;27(5):287-295. R828225 (Final)
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  • Abstract: Wiley-Abstract
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  • Journal Article Kiryu Y, Shields JD, Vogelbein WK, Zwerner DE, Kator H, Blazer VS. Induction of skin ulcers in Atlantic menhaden by injection and aqueous exposure to the zoospores of Aphanomyces invadans. Journal of Aquatic Animal Health 2002;14(1):11-24. R828225 (2000)
    R828225 (2001)
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  • Abstract: Taylor&Francis-Abstract
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  • Journal Article Kiryu Y, Shields JD, Vogelbein WK, Kator H, Blazer VH. Infectivity and pathogenicity of the oomycete Aphanomyces invadans in Atlantic menhaden Brevoortia tyrannus. Diseases of Aquatic Organisms 2003;54(2):135-146. R828225 (2000)
    R828225 (2001)
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  • Journal Article Kiryu Y, Blazer VS, Vogelbein WK, Kator H, Shields JD. Factors influencing the sporulation and cyst formation of Aphanomyces invadans, etiological agent of ulcerative mycosis in Atlantic menhaden, Brevoortia tyrannus. Mycologia 2005;97(3):569-575. R828225 (Final)
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  • Full-text: Mycologia-Full Text - HTML
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  • Abstract: Mycologia-Abstract
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  • Journal Article Lovko VJ, Vogelbein WK, Shields JD, Haas LW, Reece KS. A new larval fish bioassay for testing the pathogenicity of Pfiesteria spp. (Dinophyceae). Journal of Phycology 2003;39(3):600-609. R828225 (Final)
    R826791 (Final)
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  • Abstract: Wiley-Abstract
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  • Journal Article Mason PL, Vogelbein WK, Haas LW, Shields JD. An improved stripping technique for lightly armored dinoflagellates. Journal of Phycology 2003;39(1):253-258. R828225 (Final)
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  • Journal Article Vogelbein WK, Lovko VJ, Shields JD, Reece KS, Mason PL, Haas LW, Walker CC. Pfiesteria shumwayae kills fish by micropredation not exotoxin secretion. Nature 2002;418(6901):967-970. R828225 (2000)
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    R826791 (Final)
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  • Abstract: Nature-Abstract
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  • Journal Article Vogelbein WK, Shields JD, Haas LW, Reece KS, Zwerner DE. Skin ulcers in estuarine fishes:a comparative pathological evaluation of wild and laboratory-exposed fish. Environmental Health Perspectives 2001;109(Suppl 5):687-694. R828225 (2000)
    R828225 (2001)
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    R826791 (Final)
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  • Other: VIMS - Full Text - PDF
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  • Supplemental Keywords:

    estuary, marine, animal, menhaden, pathology, toxicity, harmful algal blooms, fish kills, fish lesions, ecological effects, Pfiesteria, Aphanomyces., RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Chemical Mixtures - Environmental Exposure & Risk, Epidemiology, Oceanography, Ecological Effects - Environmental Exposure & Risk, algal blooms, Ecological Effects - Human Health, Ecological Risk Assessment, Ecology and Ecosystems, Ecological Indicators, Aphanomyces, marine ecosystem, dermal exposure, ecological exposure, ecological effects, pathology, dinoflagellates, fish kills, etiology of lesions, fish lesions, harmful algal blooms, algal growth, pfiesteria, ecological impacts, ECOHAB, water quality, oomycete fungus, laboratory studies

    Progress and Final Reports:

    Original Abstract
  • 2000 Progress Report
  • 2001 Progress Report