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Eric Leis

Publications and source records attributed to Eric Leis.

16 recordsLinked to original sources

Sampling mass mortality events to enable diagnoses: A protocol using freshwater mussels

Many taxa around the globe are threatened by often unexplained mass mortality events (MMEs), which can decimate populations and compromise key ecosystem functions. One example of a highly threatened taxon facing frequent MMEs is freshwater mussels (Unionida). There has been a recent increase in interest in understanding the causes of freshwater mussel MMEs, but standardised methodologies for how best to respond to them to facilitate diagnoses are unavailable. When an MME is observed, swift and appropriate sample collection is imperative owing to the transient nature of these phenomena. Here we provide structured guidance that will facilitate rapid and appropriate sampling of MMEs, using freshwater mussels as an example. We set out standardised procedures for sample collection, preparation and preservation. The procedures we outline will improve our capacity for diagnostic investigations of MMEs and other mortality events, not only in freshwater mussels but also across many other taxa. This, in turn, can inform appropriate management responses.

Methods in Ecology and Evolution

Bacteriological analysis of unionid hemolymph collected from freshwater mussel populations in the Pacific northwestern United States

Native freshwater mussel (Unionidae) mortality events have been occurring with increased frequency in recent decades, with few investigations into potential etiological agents. In the western United States, no surveys have been published regarding the bacteria associated with unionid mussels. Herein, we examine locations of known mussel mortality events in the Chehalis River (Washington), in the Crooked River (Oregon), and Owyhee River (Oregon). Mussel populations considered healthy were sampled in the Skookumchuck River (Washington) for comparison. A variety of bacteria were isolated from these populations, and most notably, Acinetobacter spp. were identified from 82% of moribund individuals of Gonidea angulata in the Owyhee River. Future work evaluating whether Acinetobacter spp. are pathogenic to freshwater mussels could be valuable in unraveling the factors associated with these enigmatic mortality events.

Oregon, Washington

Mussel mass mortality in the Clinch River, USA: Metabolomics detects affected pathways and biomarkers of stress

Biologists monitoring freshwater mussel (order Unionida) populations rely on behavioral, often subjective, signs to identify moribund (“sick”) or stressed mussels, such as gaping valves and slow response to probing, and they lack clinical indicators to support a diagnosis. As part of a multi-year study to investigate causes of reoccurring mortality of pheasantshell ( Ortmanniana pectorosa; synonym Actinonaias pectorosa ) in the Clinch River, Virginia and Tennessee, USA, we analyzed the hemolymph metabolome of a subset of mussels from the 2018 sampling period. Mussels at the mortality sites were diagnosed in the field as affected (case) or unaffected (control) based on behavioral and physical signs. Hemolymph was collected in the field by non-lethal methods from the anterior adductor muscle for analysis. We used ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectroscopy to detect targeted and untargeted metabolites in hemolymph and compared metabolomic profiles by field assessment of clinical status. Targeted biomarker analysis found 13 metabolites associated with field assessments of clinical status. Of these, increased gamma-linolenic acid and N -methyl- L -alanine were most indicative of case mussels, while adenine and inosine were the best indicators of control mussels. Five pathways in the targeted analysis differed by clinical status; two of these, purine metabolism and glycerophospholipid metabolism, were also indicated in the untargeted analysis. In the untargeted nalysis, 22 metabolic pathways were associated with clinical status. Many of the impacted pathways in the case group were catabolic processes, such as degradation of amino acids and fatty acids. Hierarchical clustering analysis matched clinical status in 72% (18 of 25) of mussels, with control mussels more frequently (5 of 16) not matching clinical status. Our study demonstrated that metabolomic analysis of hemolymph is suitable for assessing mussel condition and complements field-based indicators of health.

Tennessee, Virginia

Pathology and infectious agents of unionid mussels: A primer for pathologists in disease surveillance and investigation of mortality events

Freshwater mussels are one of the most imperiled groups of organisms in the world, and more than 30 species have gone extinct in the last century. While habitat alteration and destruction have contributed to the declines, the role of disease in mortality events is unclear. In an effort to involve veterinary pathologists in disease surveillance and the investigation of freshwater mussel mortality events, we provide information on the conservation status of unionids, sample collection and processing techniques, and unique and confounding anatomical and physiological differences. We review the published accounts of pathology and infectious agents described in freshwater mussels including neoplasms, viruses, bacteria, fungi, fungal-like agents, ciliated protists, Aspidogastrea, Digenea, Nematoda, Acari, Diptera, and Odonata. Of the identified infectious agents, a single viral disease, Hyriopsis cumingii plague disease, that occurs only in cultured mussels is known to cause high mortality. Parasites including ciliates, trematodes, nematodes, mites, and insects may decrease host fitness, but are not known to cause mortality. Many of the published reports identify infectious agents at the light or ultrastructural microscopy level with no lesion or molecular characterization. Although metagenomic analyses provide sequence information for infectious agents, studies often fail to link the agents to tissue changes at the light or ultrastructural level or confirm their role in disease. Pathologists can bridge this gap between identification of infectious agents and confirmation of disease, participate in disease surveillance to ensure successful propagation programs necessary to restore decimated populations, and investigate mussel mortality events to document pathology and identify causality.

Veterinary Pathology

A bacteriological comparison of the hemolymph from healthy and moribund unionid mussel populations in the upper Midwestern U.S.A. prompts the development of diagnostic assays to detect Yokenella regensburgei

Recent bacteriological investigations of freshwater mussel mortality events in the southeastern United States have identified a variety of bacteria and differences in bacterial communities between sick and healthy mussels. In particular, Yokenella regensburgei and Aeromonas spp. have been shown to be associated with moribund mussels, although it remains unclear whether these bacteria are causes or consequences of disease. To further understand the role of bacteria in mussel epizootics, we investigated mortality events that occurred in the upper Midwest in the Embarrass River (Wisconsin) and the Huron River (Michigan). For comparison, we also studied mussels from an unaffected population in the St. Croix River (Wisconsin). Diverse bacterial genera were identified from these sites, including Y. regensburgei from moribund mussels in the Embarrass River (Wisconsin). This bacterium has also been consistently isolated during ongoing mortality events in the Clinch River (Virginia). Subsequently, we developed and validated molecular assays for the detection of Yokenella to use in future investigations of mussel mortality events and to identify environmental reservoirs of this bacterium.

Michigan, Minnesota, Wisconsin

Efficacy of hydrogen peroxide to reduce Gyrodactylus species infestation density on four fish species

Objective The ability to effectively treat parasitic infestations of fish is of high importance for fish culture facilities. However, tools or approved therapies for treating infestations on fish are limited. This paper summarizes results from four separate clinical field studies that evaluated the efficacy of hydrogen peroxide (H 2 O 2 ; 35% PEROX-AID) for reducing Gyrodactylus spp. infestation density. Methods Three species of Gyrodactylus were studied ( G. salmonis , hosts: Brook Trout Salvelinus fontinalis and Lake Trout S. namaycush ; G. freemani , host: Yellow Perch Perca flavescens ; G. hoffmani , host: Fathead Minnow Pimephales promelas ) before and after the application of immersion H 2 O 2 therapy. Result Parasite density was significantly reduced for each parasite × host combination to which H 2 O 2 therapy was applied. Two clinical field studies in salmonids were found to demonstrate substantial effectiveness that enabled 35% PEROX-AID approval. Conclusion Further assessments of Gyrodactylus spp. could expand the use of H 2 O 2 for controlling these parasites in aquaculture. Specifically, H 2 O 2 was effective at all levels tested (50 or 75 mg H 2 O 2 /L for 60 min for the Yellow Perch and Fathead Minnow clinical field studies; 100 or 150 mg H 2 O 2 /L for 30 min regardless of salt pre-treatment for the Brook Trout study; and 100 mg H 2 O 2 /L for 30 min or 50 mg H 2 O 2 /L for 60 min for the Lake Trout study).

Journal of Aquatic Animal Health

Efficacy of florfenicol and oxytetracycline administered in feed to control cisco mortality associated with Aeromonas salmonicida infections

Two medications (one with florfenicol and one with oxytetracycline) that are approved in the United States to control mortality due to furunculosis associated with Aeromonas salmonicida were assessed to determine their efficacy in medicated feeds to treat A. salmonicida -infected Cisco (also known as Lake Herring) Coregonus artedi . Cisco were subjected to static infection baths containing A. salmonicida or a sham control and then were distributed to replicate test tanks within four treatment groups: (1) fish infected with A. salmonicida and treated with 15 mg florfenicol·kg body weight (BW) −1 ·d −1 , (2) fish infected with A. salmonicida and treated with 83 mg oxytetracycline·kg BW −1 ·d −1 , (3) fish infected with A. salmonicida and treated with a nonmedicated control feed, and (4) uninfected fish treated with a nonmedicated control feed. Medicated and comparative nonmedicated feed rations were administered at 2% BW/d for 10 consecutive days in accordance with the U.S. Food and Drug Administration-approved drug label, followed by a 7-d postdosing observation period using only nonmedicated feed. Cisco that were infected with A. salmonicida and treated with florfenicol (79% survival) and oxytetracycline (85% survival) had significantly higher survival than A. salmonicida -infected fish that received no medicated treatment (3% survival). No statistical difference in Cisco survival between the two medicated feed types was found. Aeromonas salmonicida was not detected in the kidney tissue of any surviving fish treated with medicated feeds at 7 d postdosing using quantitative PCR analysis. Overall, this study demonstrated that florfenicol- and oxytetracycline-medicated feeds were effective A. salmonicida treatments for Cisco. Outcomes may inform ongoing propagation efforts for Cisco restoration within the Great Lakes basin.

North American Journal of Aquaculture

Further bacteriological analysis of annual Pheasantshell (Actinonaias pectorosa) mussel mortality events in the Clinch River (Virginia/Tennessee), USA, reveals a consistent association with Yokenella Regensburgei

Pheasantshell ( Actinonaias pectorosa ) mussels in the Clinch River (Tennessee/Virginia, USA) have declined dramatically in recent years. The bacterium Yokenella regensburgei was first isolated with high prevalence from Pheasantshells during the peak of a 2017 mortality event, but it was not identified after mortality subsided a few months later. Since 2017, Pheasantshell mortality in the Clinch River has occurred each autumn. We extended the investigation of culturable bacterial communities in the Clinch River during mussel mortality events in 2018, 2019, and 2020 and examined the spatial and temporal distribution of bacterial genera among Pheasantshells, as well as among other unionid mussels. We identified Y. regensburgei each year, almost exclusively during active mortality events. The significance of Y. regensburgei remains unclear, but the continued association of this bacterium with mussel mortality events warrants further study.

Tennessee, Virginia

Freshwater mussels show elevated viral richness and intensity during a mortality event

Freshwater mussels (Unionida) are among the world’s most imperiled taxa, but the relationship between freshwater mussel mortality events and infectious disease is largely unstudied. We surveyed viromes of a widespread and abundant species (mucket, Actinonaias ligamentina ; syn: Ortmanniana ligamentina ) experiencing a mortality event of unknown etiology in the Huron River, Michigan, in 2019–2020 and compared them to viromes from mucket in a healthy population in the St. Croix River, Wisconsin and a population from the Clinch River, Virginia and Tennessee, where a mortality event was affecting the congeneric pheasantshell ( Actinonaias pectorosa ; syn: Ortmanniana pectorosa ) population. We identified 38 viruses, most of which were associated with mussels collected during the Huron River mortality event. Viral richness and cumulative viral read depths were significantly higher in moribund mussels from the Huron River than in healthy controls from each of the three populations. Our results demonstrate significant increases in the number and intensity of viral infections for freshwater mussels experiencing mortality events, whereas individuals from healthy populations have a substantially reduced virome comprising a limited number of species at low viral read depths.

Michigan, Tennessee, Virginia, Wisconsin

Mussel mass mortality and the microbiome: Evidence for shifts in the bacterial microbiome of a declining freshwater bivalve

Freshwater mussels (Unionida) are suffering mass mortality events worldwide, but the causes remain enigmatic. Here, we describe an analysis of bacterial loads, community structure, and inferred metabolic pathways in the hemolymph of pheasantshells ( Actinonaias pectorosa ) from the Clinch River, USA, during a multi-year mass mortality event. Bacterial loads were approximately 2 logs higher in moribund mussels (cases) than in apparently healthy mussels (controls). Bacterial communities also differed between cases and controls, with fewer sequence variants (SVs) and higher relative abundances of the proteobacteria Yokenella regensburgei and Aeromonas salmonicida in cases than in controls. Inferred bacterial metabolic pathways demonstrated a predominance of degradation, utilization, and assimilation pathways in cases and a predominance of biosynthesis pathways in controls. Only two SVs correlated with Clinch densovirus 1, a virus previously shown to be strongly associated with mortality in this system: Deinococcota and Actinobacteriota, which were associated with densovirus-positive and densovirus-negative mussels, respectively. Overall, our results suggest that bacterial invasion and shifts in the bacterial microbiome during unionid mass mortality events may result from primary insults such as viral infection or environmental stressors. If so, bacterial communities in mussel hemolymph may be sensitive, if generalized, indicators of declining mussel health.

Tennessee, Virginia

Mass mortality in freshwater mussels (Actinonaias pectorosa) in the Clinch River, USA, linked to a novel densovirus

Freshwater mussels (order Unionida) are among the world’s most biodiverse but imperiled taxa. Recent unionid mass mortality events around the world threaten ecosystem services such as water filtration, nutrient cycling, habitat stabilization, and food web enhancement, but causes have remained elusive. To examine potential infectious causes of these declines, we studied mussels in Clinch River, Virginia and Tennessee, USA, where the endemic and once-predominant pheasantshell ( Actinonaias pectorosa ) has suffered precipitous declines since approximately 2016. Using metagenomics, we identified 17 novel viruses in Clinch River pheasantshells. However, only one virus, a novel densovirus ( Parvoviridae ; Densovirinae ), was epidemiologically linked to morbidity. Clinch densovirus 1 was 11.2 times more likely to be found in cases (moribund mussels) than controls (apparently healthy mussels from the same or matched sites), and cases had 2.7 (log 10 ) times higher viral loads than controls. Densoviruses cause lethal epidemic disease in invertebrates, including shrimp, cockroaches, crickets, moths, crayfish, and sea stars. Viral infection warrants consideration as a factor in unionid mass mortality events either as a direct cause, an indirect consequence of physiological compromise, or a factor interacting with other biological and ecological stressors to precipitate mortality.

Virginia, Tennessee

A comparison of bacteria cultured from unionid mussel hemolymph between stable populations in the upper Mississippi River and a mortality event in the Clinch River

The diagnosis of bacterial disease in freshwater unionid mussels has been hindered by a lack of baseline information regarding the microbial communities associated with these animals. In this study, we cultured and identified bacteria from the hemolymph of stable mussel populations from the upper Mississippi River basin and compared results to those from mussel populations experiencing a mortality event in the Clinch River, VA and TN. Several bacterial genera were consistently identified across mussel species and locations, appearing to be part of the natural bacterial flora. One noteworthy bacterial species was identified from the Clinch River: Yokenella regensbergei. Y.regensbergei occurred in relatively high prevalence during the mortality event but was absent from samples acquired after the mortality event. Its role, if any, in the mortality event is unknown but deserves further investigation. We suggest that future studies of freshwater mussel health incorporate hemolymph as a sample type due to its relative separation from the aquatic environment, its role in the circulatory system and the fact that it can be collected nonlethally.

Mississippi River basin

A novel picorna-like virus in a Wabash Pigtoe (Fusconaia flava) from the upper Mississippi River, USA

Unionid mussels are threatened by multiple environmental stressors and have experienced mass mortality events over the last several decades, but the role of infectious disease in unionid health and population declines remains poorly understood. Although several microbial agents have been found in unionids, to date only one virus has been documented—Lea plague virus ( Arenaviridae ) in propagated Triangle Shell mussels ( Hyriopsis cumingii ) in China. We used next-generation DNA sequencing to screen hemolymph of seven individuals of five unionid species from the Upper Mississippi River basin, USA for viruses. We identified the complete polyprotein gene of a novel picornalike virus in one individual of the Wabash Pigtoe ( Fusconaia flava ). The virus is a member of the Nora virus clade of picornalike viruses and is most closely related to viruses from arthropods in China. We did not detect viruses in another Wabash Pigtoe or in animals of the other four species. It is premature to make inferences about the role of this virus in the health of Wabash Pigtoes or other unionid species or the origin or transmission of this virus. Nevertheless, to our knowledge, our results represent the first report of a virus in wild North American unionids. Technologies based on next-generation DNA sequencing should prove useful for identifying new viruses and investigating their role in unionid health and disease.

Minnesota, Wisconsin

Building a response network to investigate potential pathogens associated with unionid mortality events

Unexplained mortality events have confounded the mussel conservation community for over 30 years. While the effects of chemical pollutants and environmental factors have been examined, few investigations have focused on the identification of potential mussel pathogens. Consequently, very little is known regarding the impact that pathogens have on freshwater mussel health, especially as they pertain to propagation efforts. There are several barriers that have hindered progress in the study of freshwater mussel disease, one of which is lack of appropriate diagnostic tools, and another is lack of guidance for field personnel regarding sampling procedures and laboratory contacts.

Ellipsaria

Molecular characterization of a novel orthomyxovirus from rainbow and steelhead trout ( Oncorhynchus mykiss )

A novel virus, rainbow trout orthomyxovirus (RbtOV), was isolated in 1997 and again in 2000 from commercially-reared rainbow trout ( Oncorhynchus mykiss ) in Idaho, USA. The virus grew optimally in the CHSE-214 cell line at 15°C producing a diffuse cytopathic effect; however, juvenile rainbow trout exposed to cell culture-grown virus showed no mortality or gross pathology. Electron microscopy of preparations from infected cell cultures revealed the presence of typical orthomyxovirus particles. The complete genome of RbtOV is comprised of eight linear segments of single-stranded, negative-sense RNA having highly conserved 5′ and 3′-terminal nucleotide sequences. Another virus isolated in 2014 from steelhead trout (also O. mykiss ) in Wisconsin, USA, and designated SttOV was found to have eight genome segments with high amino acid sequence identities (89–99%) to the corresponding genes of RbtOV, suggesting these new viruses are isolates of the same virus species and may be more widespread than currently realized. The new isolates had the same genome segment order and the closest pairwise amino acid sequence identities of 16–42% with Infectious salmon anemia virus (ISAV), the type species and currently only member of the genus Isavirus in the family Orthomyxoviridae . However, pairwise comparisons of the predicted amino acid sequences of the 10 RbtOV and SttOV proteins with orthologs from representatives of the established orthomyxoviral genera and a phylogenetic analysis using the PB1 protein showed that while RbtOV and SttOV clustered most closely with ISAV, they diverged sufficiently to merit consideration as representatives of a novel genus. A set of PCR primers was designed using conserved regions of the PB1 gene to produce amplicons that may be sequenced for identification of similar fish orthomyxoviruses in the future.

Virus Research

Effectiveness of Aquaflor (50% florfenicol) administered in feed to control mortality associated with Streptococcus iniae in tilapia at a commercial tilapia production facility

The efficacy of Aquaflor (florfenicol; FFC) to control mortality caused by Streptococcus iniae in tilapia was evaluated under field conditions. The trial was initiated following presumptive diagnosis of S. iniae infection in a mixed group of fingerling (mean, 4.5 g) Nile Tilapia Oreochromis niloticus and a hybrid of Nile Tilapia×Blue Tilapia O. aureus . Diagnoses included mortality in source tank; examination of clinical signs and presence or absence of gram-positive cocci in brain, and collection of samples for microbiological review and disease confirmation of 60 moribund fish. Following presumptive diagnosis, tilapia (83/tank) were randomly transferred to each of 20 test tanks receiving the same water as the source tank (test tank water was not reused). Tilapia were offered either nonmedicated control feed or FFC-medicated feed (FFC at 15 mg/kg body weight/d; 10 tanks per regimen) for 10 consecutive days followed by a 14-d observation period during which only the nonmedicated control feed was offered. Streptococcus iniae was presumptively identified during pretreatment necropsy and confirmed by polymerase chain reaction assay; S. iniae was confirmed in samples taken during the dosing period but was not detected during the postdosing period. The FFC disk diffusion zone of inhibition ranged from 29 to 32 mm, while the minimum inhibitory concentration of FFC ranged from 2 to 4 μg/mL for the S. iniae isolates collected. Survival of tilapia assigned to the FFC-dose group was significantly greater at 14 d posttreatment than that of the nonmedicated controls. The odds of tilapia assigned to the FFC-dose group surviving to the end of the postdosing period were 1.34 times the odds of survival of tilapia assigned to the nonmedicated control group. There were no clinically apparent adverse effects associated with the administration of FFC-medicated feed in this study.

North American Journal of Aquaculture