Clifford E. Starliper

Mortality of Centrarchid Fishes in the Potomac Drainage: Survey Results and Overview of Potential Contributing Factors

Skin lesions and spring mortality events of smallmouth bass Micropterus dolomieu and selected other species were first noted in the South Branch of the Potomac River in 2002. Since that year morbidity and mortality have also been observed in the Shenandoah and Monocacy rivers. Despite much research, no single pathogen, parasite, or chemical cause for the lesions and mortality has been identified. Numerous parasites, most commonly trematode metacercariae and myxozoans; the bacterial pathogens Aeromonas hydrophila, Aeromonas salmonicida, and Flavobacterium columnare; and largemouth bass virus have all been observed. None have been consistently isolated or observed at all sites, however, nor has any consistent microscopic pathology of the lesions been observed. A variety of histological changes associated with exposure to environmental contaminants or stressors, including intersex (testicular oocytes), high numbers of macrophage aggregates, oxidative damage, gill lesions, and epidermal papillomas, were observed. The findings indicate that selected sensitive species may be stressed by multiple factors and constantly close to the threshold between a sustainable (healthy) and nonsustainable (unhealthy) condition. Fish health is often used as an indicator of aquatic ecosystem health, and these findings raise concerns about environmental degradation within the Potomac River drainage. Unfortunately, while much information has been gained from the studies conducted to date, due to the multiple state jurisdictions involved, competing interests, and other issues, there has been no coordinated approach to identifying and mitigating the stressors. This synthesis emphasizes the need for multiyear, interdisciplinary, integrative research to identify the underlying stressors and possible management actions to enhance ecosystem health.

An investigation of the bactericidal activity of selected essential oils to Aeromonas spp.

Diseases of fishes caused by Aeromonas spp. are common, have broad host ranges and may cause high mortality. Treatments of captive-reared populations using antimicrobials are limited with concerns for bacterial resistance development and environmental dissemination. This study was done to determine whether selected plant-derived essential oils were bactericidal to Aeromonas spp. Initially, twelve essential oils were evaluated using a disk diffusion assay to an isolate of A. salmonicida subsp. salmonicida, cause of fish furunculosis. The greatest zones of inhibition were obtained with oils of cinnamon Cinnamomum cassia, oregano Origanum vulgare, lemongrass Cymbopogon citratus and thyme Thymus vulgaris. Minimum bactericidal concentrations (MBC’s) were determined for these four oils, Allimed® (garlic extract, Allium sativum) and colloidal silver to sixty-nine isolates representing nine Aeromonas spp. The lowest mean MBCs (0.02–0.04%) were obtained with three different sources of cinnamon oil. MBCs for three sources of oregano and lemongrass oils ranged from 0.14% to 0.30% and 0.10% to 0.65%, respectively, and for two thyme oils were 2.11% and 2.22%. The highest concentration (5%) of Allimed® tested resulted in MBCs to twelve isolates. A concentration of silver greater than 15 mg/L would be required to determine MBCs for all but one isolate.

Biochemical and Conjugation Studies of Romet-Resistant Strains of Aeromonas salmonicida from Salmonid Rearing Facilities in the Eastern United States

Abstract Strains of Aeromonas salmonicida (n = 585) were collected from covertly infected and diseased salmonid hosts from 12 hatcheries in the eastern United States. Strains and sites were selected because of their potential for harboring antimicrobial resistance, in particular, to Romet™. Resistance to Romet was displayed by 315 strains (53.8%), which were isolated from all six host species sampled at 10 of 12 sites. Thirty of the resistant strains (9.5%) from five sites had no zone of inhibition, whereas the other strains had either confluent growth or resistant colonies within a zone of inhibition. Fifty-one resistant strains, representing each of the three resistance phenotypes, were selected for biochemical and antimicrobial comparisons with Romet-sensitive strains. All were confirmed to be A. salmonicida, and no characteristic biochemical phenotypes were found to be associated with resistance to Romet. Differential resistances between resistant and sensitive strains were detected to the antimicrobi...

Bactericidal efficacy of elevated pH on fish pathogenic and environmental bacteria

Ship ballast water is a recognized medium for transfer and introductions of nonindigenous species. There is a need for new ballast water treatment methods that effectively and safely eliminate or greatly minimize movements of these species. The present study employed laboratory methods to evaluate the bactericidal efficacy of increased pH (pH 10.0–12.0) for exposure durations of up to 72 h to kill a variety of Gram-negative and Gram-positive bacteria including fish pathogens (Aeromonas spp., Yersinia ruckeri, Edwardsiella ictaluri, Serratia liquefaciens, Carnobacterium sp.), other common aquatic-inhabitant bacteria (Serratia marcescens, Pseudomonas fluorescens, Staphylococcus sp., Bacillus sp.) and indicators listed in International Maritime Organization D2 Standards; namely, Vibrio cholera (an environmental isolate from fish), Escherichia coli and Enterococcus faecalis. Volumes of 5 N NaOH were added to tryptic soy broth to obtain desired pH adjustments. Viable cells were determined after 0, 4, 12, 24, 48, and 72 h. Initial (0 h) cell numbers ranged from 3.40 × 104 cfu/mL for Bacillus sp. to 2.44 × 107 cfu/mL for E. faecalis. The effective endpoints of pH and treatment duration necessary to realize 100% bactericidal effect varied; however, all bacteria tested were killed within 72 h at pH 12.0 or lower. The lowest parameters examined, 4 h at pH 10.0, were bactericidal to V. cholera, E. ictaluri, three of four isolates of E. coli, and (three of four) Aeromonas salmonicida subsp. salmonicida. Bactericidal effect was attained at pH 10.0 within 12 h for the other A. salmonicida subsp. salmonicida, and within 24 h for P. fluorescens, and the remaining E. coli.

The Effect of Depuration on Transmission of Aeromonas salmonicida between the Freshwater Bivalve Amblema plicata and Arctic Char

Abstract A model system was used to study bacterial fish pathogen transmission between the freshwater bivalve Amblema plicata and two strains (Nauyuk and Labrador) of Arctic char Salvelinus alpinus. Aeromonas salmonicida, the cause of fish furunculosis, was readily transmitted from Arctic char to A. plicata and vice versa via simple cohabitation. Clinical furunculosis was artificially established in Nauyuk Arctic char via horizontal exposure to Labrador Arctic char that received intraperitoneal injections of A. salmonicida. After the Nauyuk Arctic char began to die, A. plicata were placed in the tank with the fish. After 33 d of cohabitation, a group of 10 A. plicata was cultured, and A. salmonicida was isolated from all 10. The remaining A. plicata were transferred to other tanks being supplied with specific-pathogen-free water. At 1, 5, 15, and 30 d after transfer, 60 uninfected Labrador Arctic char were cohabitated with the A. plicata. Transmission of A. salmonicida from A. plicata to the Arctic char w...

Mycobacterial Infections in Striped Bass from Delaware Bay

Eighty striped bass Morone saxatilis were obtained from Delaware Bay using commercial gill nets set adjacent to Woodland Beach (n = 70) and Bowers Beach (n = 10) in December 2003. Fish were examined for gross lesions. Total lengths (TLs) and eviscerated weights were determined to calculate condition factors (K). Portions of spleens were aseptically harvested for bacterial culture, and portions of spleens, kidneys (anterior and posterior), livers, and gonads were obtained for histological examination. The size distribution of the striped bass was relatively homogeneous; the mean TL was about 600 mm for all samples. Mean K exceeded 0.95 in all samples and was not significantly different (P > 0.05) among samples. Significant differences in mycobacterial infection prevalence (P < or = 0.05) were observed among samples; samples obtained at Woodland Beach (WB) on December 10 (53.8%, n = 13) and December 17 (7.1%, n = 42) exhibited the most striking differences in prevalence. Mycobacterial infection intensity ranged from 1 X 10(2) to 1 X 10(7) colony-forming units per gram of spleen. Acanthocephalan infection prevalence and intensity, non-acid-fast bacterial infection prevalence, and fish sex ratio were also significantly different among the samples (P < or = 0.05). Similar to the mycobacterial infections, differences in sex ratio, acanthocephalan infection, and non-acid-fast bacterial infection were observed between the WB samples taken on December 10 and 17. However, no significant associations (P > 0.05) were observed between sex ratio or these infections and mycobacterial infection. The differences in bacterial and parasite infection prevalence and intensity and fish sex ratio in some samples indicate that these fish had a different history and that the epizootiology of mycobacterial infection in striped bass from Delaware Bay may be relatively complex.

Efficacy of Iodine for Disinfection of Lake Sturgeon Eggs from the St. Lawrence River, New York

AbstractOptimal fish husbandry to reduce the risk of disease is particularly important when using wild fish as the source for gametes. The propagation and reestablishment of Lake Sturgeon Acipenser fulvescens in New York waters to become a viable self-sustaining population is considered a high priority by managers. While standard hatchery egg disinfection practices have been used to prevent the transmission of diseases, data on the bacterial loads present on egg surfaces following iodine disinfection is lacking. Our study investigated the bacteria present on the outer surface of Lake Sturgeon eggs and the effectiveness of an iodine disinfection treatment in eliminating bacteria that could pose a threat to egg survival and cause hatchery disease outbreaks. During the springs of 2011–2013, 12 to 41 different species of bacteria were recovered from the outer egg surfaces prior to an iodine treatment; Aeromonas, Pseudomonas, Shewanella, and Chryseobacterium were the most common genera identified. Cohort eggs ...

Efficacy of pH elevation as a bactericidal strategy for treating ballast water of freight carriers.

Treatment of ship ballast water with sodium hydroxide (NaOH) is one method currently being developed to minimize the risk to introduce aquatic invasive species. The bactericidal capability of sodium hydroxide was determined for 148 bacterial strains from ballast water collected in 2009 and 2010 from the M/V Indiana Harbor, a bulk-freight carrier plying the Laurentian Great Lakes, USA. Primary culture of bacteria was done using brain heart infusion agar and a developmental medium. Strains were characterized based on PCR amplification and sequencing of a portion of the 16S rRNA gene. Sequence similarities (99+ %) were determined by comparison with the National Center for Biotechnology Information (NCBI) GenBank catalog. Flavobacterium spp. were the most prevalent bacteria characterized in 2009, comprising 51.1% (24/47) of the total, and Pseudomonas spp. (62/101; 61.4%) and Brevundimonas spp. (22/101; 21.8%) were the predominate bacteria recovered in 2010; together, comprising 83.2% (84/101) of the total. Testing was done in tryptic soy broth (TSB) medium adjusted with 5 N NaOH. Growth of each strain was evaluated at pH 10.0, pH 11.0 and pH 12.0, and 4 h up to 72 h. The median cell count at 0 h for 148 cultures was 5.20 × 106 cfu/mL with a range 1.02 × 105–1.60 × 108 cfu/mL. The TSB adjusted to pH 10.0 and incubation for less than 24 h was bactericidal to 52 (35.1%) strains. Growth in pH 11.0 TSB for less than 4 h was bactericidal to 131 (88.5%) strains and pH 11.0 within 12 h was bactericidal to 141 (95.3%). One strain, Bacillus horikoshii, survived the harshest treatment, pH 12.0 for 72 h.

Efficacy and Toxicity of Iodine Disinfection of Atlantic Salmon Eggs

Abstract Recent interest in the restoration of Atlantic salmon Salmo salar in the Great Lakes has given rise to new culture techniques and management programs designed to reduce pathogen transmission while stabilizing and enhancing wild populations. We examined the toxicity of iodine to Atlantic salmon eggs and its effectiveness as a disinfectant against bacteria on egg surfaces. We spawned and fertilized eight gravid Atlantic salmon from Cayuga Lake, New York, and exposed their eggs to 10 concentrations of iodine (5, 10, 50, 75, 100, 500, 750, 1,000, 5,000, and 7,500 mg/L) for 30 min during water hardening. An additional subsample of unfertilized eggs was also exposed to some of the same concentrations of iodine (5, 10, 50, 75, and 100 mg/L) to determine the efficiency of disinfection. Viable eggs were only obtained from four females. Survival of eggs to the eyed stage and hatch tended to be reduced at iodine concentrations of 50 and 75 mg/L and was significantly reduced at concentrations of 100 mg/L iod...

Predominant Bacteria Isolated from Moribund Fusconaia ebena Ebonyshells Experiencing Die-Offs in Pickwick Reservoir, Tennessee River, Alabama

ABSTRACT Mussel die-offs have been noted in recent years in Pickwick Reservoir, Tennessee River, Alabama. The primary affected species was Fusconaia ebena, but also affected to lesser degrees were Ellipsaria lineolata, Quadrula pustulosa, and Quadrula 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 in Pickwick Reservoir (river miles 236–256) in sequential years during the past approximately 10 y. 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 after 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., Aeromonas schubertii, Aeromonas veronii bv. veronii, and Aeromonas veronii bv. sobria.