Fish and Wildlife Disease

Survey of Indigenous Bacteria in Mussels from Virginia Rivers (Starliper)

Bacteria Isolated from Fusconaia ebena Experiencing Die-Offs (Starliper)

Photo: Moribund (Fusconaia ebena) Ebonyshells. Photo credit: Clifford E. Starliper, USGS

Photo: Processing mussels for recovery of bacteria. Photo credit: Clifford E. Starliper, USGS.

Mussel die-offs have been noted in recent years in Pickwick Reservoir, Tennessee River, AL. The primary affected species was Fusconaia ebena, but also affected to lesser degrees were Ellipsaria lineolata, Quadrula pustulosa and Q. quadrula. These events were characterized by large numbers of empty shells, fresh-dead and live individuals that were presumed to be diseased, because of weak and slow valve closure responses to external stimuli. Anecdotal evidence suggested the possible involvement of an etiological agent, such as a bacterial pathogen. The die-offs have occurred within Pickwick Reservoir (river miles 236 – 256) in sequential years over the past approximately 10 years. These timeframes have coincided with reduced basin inflows and warmer water temperatures. The majority of the moribund and freshly dead F. ebena were females possibly predisposed to infection and disease from ongoing reproductive activity. Affected and healthy-cohort mussels were collected to characterize the bacterial flora prior to, during, and following a July 2006 die-off, and during a subsequent die-off in September 2008. The numbers of total bacteria from both the 2006 and 2008 die-offs were significantly greater from the diseased specimens. For example, from the September 2008 die-off the mean count from diseased F. ebena soft tissues was 9.75 × 106 cfu/g, which was more than 100 times greater (p = 0.025) than the mean from healthy cohorts (6.74 × 104 cfu/g). The predominant bacteria from affected F. ebena from July 2006 were Hafnia alvei and Aeromonas sobria, whereas from September 2008 the predominant bacteria were Enterobacter spp., A. schubertii, A. veronii bv. veronii and A. veronii bv. sobria.

For more information contact Clifford E. Starliper, Leetown Science Center.

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The soft tissues from one mussel specimen being surface disinfected prior to homogenization; this step allows recovery of bacteria from within the tissues and excludes bacteria on the surfaces. Photo credit: Clifford E. Starliper, USGS

A laboratory test to identify the bacteria from the mussels. Photo credit: Clifford E. Starliper, USGS

Freshwater mussel conservation efforts by many federal and state agencies have increased in recent years. This has led to a greater number of stream surveys, in which mussel die-offs involving high numbers of dead and moribund animals are being observed and reported with greater frequency. Typically, die-offs have been incidentally observed while research was being done for other purposes, therefore, accurate mortality data has been difficult to obtain. Specifically, seasonal die-offs were noted in localized areas of the Clinch and Holston Rivers, VA, and to lesser degrees, in neighboring rivers in this geographic region, including southeast VA. The observed mussel species affected were primarily the slabside pearlymussel (Lexingtonia dolabelloides) and to lesser extents, the pheasantshell (Actinonaias pectorosa), rainbow mussel (Villosa iris), and the endangered shiny pigtoe (Fusconaia cor). To determine if a bacterial pathogen might be involved in these recurring mussel die-offs, this study examined characteristics of the indigenous microbiota (bacteria) from healthy mussels from sites on the Clinch and Holston Rivers where die-offs were previously observed. These baseline data will allow for recognition of bacterial pathogens in future mussel die-offs. Means for total bacteria from soft tissues ranged from 1.77 × 10ˆ5 to 3.55 × 10ˆ6 cfu/g; whereas, the range in means from fluids was 2.92 × 10ˆ4 to 8.60 × 10ˆ5 cfu/mL. A diverse microbiota were recovered, including species that are common in freshwater aquatic environments. The most common bacterial groups recovered were motile Aeromonas spp. and non-fermenting bacteria. Flavobacterium columnare, a pathogen to cool- and warm-water fishes was recovered from one specimen, a Villosa iris from the Clinch River.

Publication:

Starliper, C. E., R. J. Neves, S. Hanlon, and P. Whittington. 2008. A survey of the indigenous microbiota (Bacteria) in three species of mussels from the Clinch and Holston Rivers, Virginia. Journal of Shellfish Research. 27(5): 1311-1317.

For more information contact Clifford E. Starliper, Leetown Science Center.

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Collecting hemolymph from the adductor muscle of a mussel specimen using a needle and syringe. Photo credit: Clifford E. Starliper, USGS

Two students using a ruler-board to measure and collect data from mussel specimens. The data collected include length, weight, width, depth, area inside valves, weight of soft tissues, etc. Photo credit: Clifford E. Starliper, USGS

Refugia are increasingly being employed to maintain and propagate imperiled freshwater mussels for future population augmentations. Success for this endeavor is dependent on good husbandry, including a holistic program of resource health management. A significant aspect to optimal health is the prevention or control of infectious diseases. Describing and monitoring pathogens and diseases in mussels involves examination of tissues or samples collected from an appropriate number of individuals that satisfies a certain confidence level for expected prevalences of infections. In the present study, ebonyshell mussels Fusconaia ebena were infected with a fish pathogenic bacterium, Aeromonas salmonicida, through their cohabitation with diseased brook trout Salvelinus fontinalis. At a 100 % prevalence of infection, the F. ebena were moved to clean tanks that were supplied with pathogen-free water, which initiated their depuration of A. salmonicida. Three samples (non-destructive fluid, mantle, hemolymph) collected using non-destructive procedures were compared to fluids and soft tissue homogenates collected after sacrificing the mussels for recovery of the bacterium. The results showed that the non-destructive sample collections, particularly fluid, provide a comparable alternative to sacrificing mussels to determine pathogen status.

Publication:

Starliper, C. E. 2008. Recovery of a fish pathogenic bacterium, Aeromonas salmonicida, from ebonyshell mussels Fusconaia ebena using non-destructive sample collection procedures. Journal of Shellfish Research. 27(4): 775-782.

For more information contact Clifford E. Starliper, Leetown Science Center.

See also Fish and Wildlife Disease: Fish >>

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Ebonyshell Fusconaia ebena. Photo credit: Clifford E. Starliper, USGS

Mussel die-off affected reach of the Tennessee River. Photo credit: USFWS

Dead and moribund specimens collected during the mussel die-off. Photo credit: USFWS

Pickwick Reservoir, Tennessee River, AL harbors a diverse large-river mussel fauna. Notable mussel die-offs were observed in the Reservoir in 2002, and subsequently in 2004, 2006, and 2008. Efforts to understand the cause(s) yielded observations pointing towards the involvement of an etiological agent, e.g. bacterial pathogen. The die-offs generally occurred within a similar timeframe in different years and coincided with lower basin inflows and warmer water temperatures favoring bacterial growth. Ebonyshells Fusconaia ebena were the primary affected host. Many of the moribund and fresh dead specimens were gravid, female F. ebena, perhaps related to post-spawning stress predisposing the animals to infection and disease. We examined affected mussels from the 2006 and 2008 outbreaks. Diseased ebonyshells (gaping valves, weak closure to stimuli), and healthy cohorts were assayed for bacteria from paired fluids and soft tissues. Bacteria from healthy F. ebena were typical of what we anticipated- there was a variety of bacterial species (flora) and total bacterial loads were about 1 × 10ˆ5 to 1 × 10ˆ6 cfu/mL of fluid or g of tissues. In contrast, bacterial profiles from apparently diseased F. ebena were often 100- to 1,000-fold greater. Furthermore, primary cultures from diseased specimens were often pure, and with the same bacterium from all of the specimens from that year. The predominant bacteria from 2006 and 2008 were different. Both of these bacteria are common in aquatic environments and may be isolated from healthy mussels. One scenario we surmise is that diseased F. ebena were compromised by a combination of factors and their bacterial profile criteria indicated opportunistic pathogen involvements, or secondary pathogens to another etiology.

For more information contact Clifford E. Starliper, Leetown Science Center.

Recovering bacteria from an ebonyshell Fusconaia ebena. The shells are cleaned and disinfected. Photo credit: Clifford E. Starliper, USGS

A laboratory test to identify the bacteria from the mussels. Aeromonas salmonicida bacterial colonies shown on a differential medium (CBB). Photo credit: Clifford E. Starliper, USGS

Furunculosis, caused by the bacterium  Aeromonas salmonicida, was artificially induced in brook trout (Salvelinus fontinalis) in an experimental tank. Ebonyshells Fusconaia ebena were placed with these fish to acquire the pathogen through siphoning. After 2 weeks of cohabitation, 10 of the mussels were assayed by bacterial culture and all were found to be positive for A. salmonicida. The mean cell count from soft tissue homogenates was 1.84 ×10ˆ5 cfu/g, which comprised an average 14.41 % of the total bacteria isolated from tissues. From the fluids, a mean of 2.84 × 10ˆ5 A. salmonicida cfu/mL was isolated, which comprised an average of 17.29 % of the total bacterial flora from fluids. The mussels were then removed from the cohabitation tank and distributed equally among five previously disinfected tanks. The F. ebena in each tank were allowed to depurate A. salmonicida for various durations: 1, 5, 10, 15 or 30 days. After each group had depurated for their assigned time, ten were assayed for bacteria, tank water was tested and 20 pathogen-free bioindicator brook trout were added to cohabit with the remaining mussels. Depuration was considered successful if A. salmonicida was not isolated from tank water or the mussels and there was no infection or mortality to bioindicator fish. After 1 day of depuration, A. salmonicida was not isolated from the soft tissues; however, it was isolated from one of the paired fluids (10% prevalence). The tank water was positive and the bioindicator fish became infected and died. From the 5-day depuration group, A. salmonicida was not isolated from soft tissues, but was isolated from three fluids (30 %; mean = 1.56 × 10ˆ2 cfu/mL). Tank water from the 5-day group was negative and there was no mortality among the bioindicator fish, however, A. salmonicida was isolated from 2 of 20 of the fish at the end of the 14-day observation period. One F. ebena fluid sample was positive for A. salmonicida from the 10-day depuration group, but none of the soft tissue homogenates. The pathogen was not isolated from 10-day tank water, but there was a 30 % cumulative mortality to the bioindicator fish. Aeromonas salmonicida was not isolated from any of the soft tissue homogenates, fluids or tank water from the 15 day or 30 day depuration groups and the bioindicator fish remained pathogen and disease free. Results of this study showed that the F. ebena were harboring a high A. salmonicida cell load, but after 15 days of depuration, this bacterium the mussels did not serve as pathogen vectors.

Publication:

Starliper, C.E. 2005. Quarantine of Aeromonas salmonicida-harboring ebonyshell mussels (Fusconaia ebena) prevents transmission of the pathogen to brook trout (Salvelinus fontinalis). Journal of Shellfish Research. 24(2):573-578.

For more information contact Clifford E. Starliper, Leetown Science Center.

See also Fish and Wildlife Disease: Fish >>

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