Thomas P. Loch

Emerging flavobacterial infections in fish: A review

Graphical abstract

Flavobacterium spartansii sp. nov., a pathogen of fishes, and emended descriptions of Flavobacterium aquidurense and Flavobacterium araucananum.

Two strains (T16(T) and S12) of a Gram-reaction-negative, rod-shaped, yellow-pigmented bacterium were recovered from kidneys of feral spawning adult Chinook salmon (Oncorhynchus tshawytscha) and the gills of captive-reared Chinook salmon fingerlings suffering a mortality episode, respectively. Polyphasic characterization revealed that these strains possessed gliding motility, contained a flexirubin-type pigment, proteolysed multiple substrates (i.e. gelatin, casein and elastin), and had a mean DNA G+C content of 35.6 mol%. Sequencing of the 16S rRNA gene demonstrated that strains T16(T) and S12 were nearly identical to one another (>99% similarity) and were placed within the genus Flavobacterium, with Flavobacterium aquidurense CCUG 59847(T) (98.3%), Flavobaterium araucananum CCUG 61031(T) (98.2%) and Flavobacterium frigidimaris (AB183888, 98.1%) being their closest relatives. Subsequent phylogenetic analyses using neighbour-joining, maximum-parsimony and Bayesian methodologies demonstrated that strains T16(T) and S12 formed a well-supported clade within the genus Flavobacterium that was distinct from other Flavobacterium species. The major fatty acid constituents of strains T16(T) and S12 were iso-C15 : 0, C16 : 1ω6c and/or C16 : 1ω7c, iso-C17 : 0 3-OH, and iso-C15 : 0 3-OH according to fatty acid methyl ester analysis. The mean level of DNA-DNA relatedness between strain T16(T) and F. aquidurense CCUG 59847(T) and F. araucananum CCUG 61031(T) was 23 and 29%, respectively. Thus, the data accumulated in this study support the suggestion that strains T16(T) and S12 represent a novel species of the genus Flavobacterium, for which the name Flavobacterium spartansii sp. nov. is proposed. The type strain is T16(T) ( = LMG 27337(T) = ATCC BAA-2541(T)). Emended descriptions of F. aquidurense and F. araucananum are also proposed.

Chryseobacterium aahli sp. nov., isolated from lake trout (Salvelinus namaycush) and brown trout (Salmo trutta), and emended descriptions of Chryseobacterium ginsenosidimutans and Chryseobacterium gregarium.

Two strains (T68T and T62) of a Gram-reaction-negative, yellow-pigmented bacterium containing flexirubin-type pigments were recovered from the kidney of a cultured lake trout (Salvelinus namaycush) and necrotic fins of a brown trout (Salmo trutta) during disease surveillance in 2009. Both isolates possessed catalase and cytochrome oxidase activities and degraded multiple substrates (e.g. gelatin, casein, elastin and Tweens 20 and 80). The mean DNA G+C content of strain T68T was 34.1 mol%. 16S rRNA gene sequencing demonstrated that strains T68T and T62 had nearly identical sequences (≥99 % similarity) and placed the bacterium within the genus Chryseobacterium, where Chryseobacterium ginsenosidimutans THG 15T (97.8%), C. gregarium DSM 19109T (97.7%) and C. soldanellicola PSD1-4T (97.6%) were its closest relatives. Subsequent phylogenetic analyses using neighbour-joining, maximum-parsimony and Bayesian methodologies demonstrated that strains T68T and T62 formed a well-supported clade (bootstrap values of 100 and 97%; posterior probability 0.99) that was distinct from other species of the genus Chryseobacterium. The major fatty acids of strains T68T and T62 were characteristic of the genus Chryseobacterium and included iso-C15:0, summed feature 3 (C16:1ω6c and/or C16:1ω7c), iso-C17:0 3-OH, C16:0 and C16:0 3-OH. The mean DNA-DNA relatedness of strain T68T to C. ginsenosidimutans JCM 16719T and C. gregarium LMG 24952T was 24 and 21%, respectively. Based on the results from our polyphasic characterization, strains T68T and T62 represent a novel species of the genus Chryseobacterium, for which the name Chryseobacterium aahli sp. nov. is proposed. The type strain is T68T (=LMG 27338T=ATCC BAA-2540T). Emended descriptions of Chryseobacterium ginsenosidimutans and Chryseobacterium gregarium are also proposed.

Diversity of Fish-Associated Flavobacteria of Michigan

Flavobacteriosis poses a serious threat to wild and propagated fish stocks alike, accounting for more fish mortality in Michigan and its associated state fish hatcheries than all other pathogens combined. Although this consortium of fish diseases has primarily been attributed to Flavobacterium psychrophilum, F. columnare, and F. branchiophilum, herein we describe a diverse assemblage of Flavobacterium and Chryseobacterium spp. isolates recovered from diseased as well as apparently healthy wild, feral, and farmed fish of Michigan. Among 254 fish-associated flavobacterial isolates recovered from 21 fish species during 2003-2010, 211 were identified as Flavobacterium spp., whereas 43 were identified as Chryseobacterium spp. according to ribosomal RNA partial gene sequencing and phylogenetic analysis. Although F. psychrophilum and F. columnare were indeed associated with multiple fish mortality events, many previously uncharacterized flavobacteria were recovered from systemically infected fish showing overt signs of disease, and in vitro protease assays demonstrated that these isolates were highly proteolytic to multiple substrates that comprise host tissues. Indeed, the majority of the isolates either (1) were most similar to recently described fish-associated Flavobacterium and Chryseobacterium spp. that have never before been reported in North America (e.g., F. oncorhynchi, F. araucananum, C. viscerum, C. piscicola, and C. chaponense) or (2) did not cluster with any described species and most likely represent novel flavobacterial taxa. This study highlights the extreme diversity of flavobacteria that are potentially associated with flavobacteriosis in Michigan.

Isolation of a Carnobacterium maltaromaticum‐like bacterium from systemically infected lake whitefish (Coregonus clupeaformis)

Herein we report on the first isolation of a Carnobacterium maltaromaticum-like bacterium from kidneys and swim bladders of lake whitefish (Coregonus clupeaformis) caught from Lakes Michigan and Huron, Michigan. Isolates were Gram-positive, nonmotile, facultatively anaerobic, asporogenous rods that did not produce catalase, cytochrome oxidase, or H2S, and did not grow on acetate agar. Except for carbohydrate fermentation, many phenotypic characteristics of lake whitefish isolates coincided with those of C. maltaromaticum, the causative agent of pseudokidney disease. Partial sequencing of 16S and 23S rRNA genes, as well as the piscicolin 126 precursor gene, yielded 97% and 98% nucleotide matches with C. maltaromaticum, respectively (accession numbers EU546836 and EU546837; EU643471). Phylogenetic analyses showed that lake whitefish isolates of this study are highly related, yet not fully identical to C. maltaromaticum. The presence of the C. maltaromaticum-like bacterium was associated with splenomegaly, renal and splenic congestion, and thickening of the swim bladder wall with accumulation of a mucoid exudate. Examination of stained tissue sections revealed renal and splenic congestion, vacuolation and bile stasis within the liver, and hyperplasia within the epithelial lining of the swim bladder. The concurrent presence of pathological changes and the C. maltaromaticum-like bacteria suggests that this bacterium is pathogenic to lake whitefish.

Variants of a genomic island in Aeromonas salmonicida subsp. salmonicida link isolates with their geographical origins

Aeromonas salmonicida subsp. salmonicida is a fish pathogen. Analysis of its genomic characteristics is required to determine the worldwide distribution of the various populations of this bacterium. Genomic alignments between the 01-B526 pathogenic strain and the A449 reference strain have revealed a 51-kb chromosomal insertion in 01-B526. This insertion (AsaGEI1a) has been identified as a new genomic island (GEI) bearing prophage genes. PCR assays were used to detect this GEI in a collection of 139 A. salmonicida subsp. salmonicida isolates. Three forms of this GEI (AsaGEI1a, AsaGEI1b, AsaGEI2a) are now known based on this analysis and the sequencing of the genomes of seven additional isolates. A new prophage (prophage 3) associated with AsaGEI2a was also discovered. Each GEI appeared to be strongly associated with a specific geographic region. AsaGEI1a and AsaGEI2a were exclusively found in North American isolates, except for one European isolate bearing AsaGEI2a. The majority of the isolates bearing AsaGEI1b or no GEI were from Europe. Prophage 3 has also a particular geographic distribution and was found only in North American isolates. We demonstrated that A. salmonicida subsp. salmonicida possesses unsuspected elements of genomic heterogeneity that could be used as indicators to determine the geographic origins of isolates of this bacterium.

Polyphasic characterization of Aeromonas salmonicida isolates recovered from salmonid and non‐salmonid fish

Michigans fisheries rely primarily upon the hatchery propagation of salmonid fish for release in public waters. One limitation on the success of these efforts is the presence of bacterial pathogens, including Aeromonas salmonicida, the causative agent of furunculosis. This study was undertaken to determine the prevalence of A. salmonicida in Michigan fish, as well as to determine whether biochemical or gene sequence variability exists among Michigan isolates. A total of 2202 wild, feral and hatchery-propagated fish from Michigan were examined for the presence of A. salmonicida. The examined fish included Chinook salmon, Oncorhynchus tshawytscha (Walbaum), coho salmon, O. kisutcha (Walbaum), steelhead trout, O. mykiss (Walbaum), Atlantic salmon, Salmo salar L., brook trout, Salvelinus fontinalis (Mitchill), and yellow perch, Perca flavescens (Mitchill). Among these, 234 fish yielded a brown pigment-producing bacterium that was presumptively identified as A. salmonicida. Further phenotypic and phylogenetic analyses identified representative isolates as Aeromonas salmonicida subsp. salmonicida and revealed some genetic and biochemical variability. Logistic regression analyses showed that infection prevalence varied according to fish species/strain, year and gender, whereby Chinook salmon and females had the highest infection prevalence. Moreover, this pathogen was found in six fish species from eight sites, demonstrating its widespread nature within Michigan.

Assessment of Renibacterium salmoninarum infections in four lake whitefish (Coregonus clupeaformis) stocks from northern Lakes Huron and Michigan

ABSTRACT Lake whitefish (Coregonus clupeaformis) from four stocks in northern Lakes Michigan and Huron were collected seasonally from fall 2003 through summer 2006 and examined for the presence of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), using culture techniques on modified kidney disease medium (MKDM) and the quantitative enzyme-linked immunosorbent assay (Q-ELISA). R. salmoninarum was detected in 62.31% (according to Q-ELISA) of the 1284 examined lake whitefish, with some fish displaying the typical signs of BKD, such as renal congestion, swelling, and whitish nodules. Kidney cultures on MKDM yielded bacteria with morphological and biochemical characteristics identical to those of R. salmoninarum recovered from other Great Lakes fish species, as well as those from other parts of the world. Isolate identification was confirmed via nested polymerase chain reaction. Antibiograms demonstrated high sensitivity to enrofloxacin and ciprofloxacin, sensitivity to oxytetracycline, erythromycin, azithromycin, chloramphenicol, novobiocin, and carbenicillin, and resistance to polymyxin B, clindamycin, and kanamycin. Statistical analysis of R. salmoninarum prevalence and intensities revealed significant interactions among stocks, years and sampling seasons, with highest prevalence generally in fall and frequent wide variation in prevalence and intensity from one season to the next for a particular stock. It was surprising to find that the prevalence of R. salmoninarum exceeded 50% in the four stocks, much higher than originally thought. Moreover, a positive association between R. salmoninarum intensity and the abundance of the swimbladder nematode, Cystidicola farionis, was identified. Our findings suggest that Great Lakes lake whitefish are vulnerable to serious fish pathogens.

Isolation of Aeromonas salmonicida subspecies salmonicida from Lake Whitefish (Coregonus clupeaformis) inhabiting Lakes Michigan and Huron

ABSTRACT Herein we describe the first report of Aeromonas salmonicida subspecies salmonicida infections in lake whitefish (Coregonus clupeaformis) collected from four sites in lakes Michigan and Huron, Michigan, USA. The bacterium was isolated from the kidneys of four out of 1286 lake whitefish that were tested over a three-year period. The four isolates were phenotypically similar to one another and exhibited the morphological, colonial, and biochemical traits typical of A. salmonicida subspecies salmonicida. Amplification of 16S rRNA genes specific to A. salmonicida subspecies salmonicida via polymerase chain reaction and subsequent gel electrophoresis analyses confirmed the identity of the four lake whitefish isolates. Clinical signs associated with infection included extensive external hemorrhaging, exophthalmia, splenomegaly, splenic and renal congestion, fibrinous adhesions of the spleen and liver, and hemorrhagic enteritis. Histopathological examination of infected fish revealed multi-focal hemorrhage and infiltration of lymphocytes and histiocytes in subdermal adipose tissues and musculature. A low infection incidence of A. salmonicida salmonicida in Great Lakes lake whitefish does not preclude the fact that overt signs of disease were observed in infected individuals and that lake whitefish may act as a reservoir for this bacterium that is highly pathogenic to numerous fish species.

Genetic Diversity of Flavobacterium psychrophilum Isolates from Three Oncorhynchus spp. in the United States, as Revealed by Multilocus Sequence Typing.

ABSTRACT The use of a multilocus sequence typing (MLST) technique has identified the intraspecific genetic diversity of U.S. Flavobacterium psychrophilum, an important pathogen of salmonids worldwide. Prior to this analysis, little U.S. F. psychrophilum genetic information was known; this is of importance when considering targeted control strategies, including vaccine development. Herein, MLST was used to investigate the genetic diversity of 96 F. psychrophilum isolates recovered from rainbow trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch), and Chinook salmon (Oncorhynchus tshawytscha) that originated from nine U.S. states. The isolates fell into 34 distinct sequence types (STs) that clustered in 5 clonal complexes (CCs) (n = 63) or were singletons (n = 33). The distribution of STs varied spatially, by host species, and in association with mortality events. Several STs (i.e., ST9, ST10, ST30, and ST78) were found in multiple states, whereas the remaining STs were localized to single states. With the exception of ST256, which was recovered from rainbow trout and Chinook salmon, all STs were found to infect a single host species. Isolates that were collected during bacterial cold water disease outbreaks most frequently belonged to CC-ST10 (e.g., ST10 and ST78). Collectively, the results of this study clearly demonstrate the genetic diversity of F. psychrophilum within the United States and identify STs of clinical significance. Although the majority of STs described herein were novel, some (e.g., ST9, ST10, ST13, ST30, and ST31) were previously recovered on other continents, which demonstrates the transcontinental distribution of F. psychrophilum genotypes. IMPORTANCE Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) and is an important bacterial pathogen of wild and farmed salmonids worldwide. These infections are responsible for large economic losses globally, yet the genetic diversity of this pathogen remains to be fully investigated. Previous studies have identified the genetic diversity of this pathogen in other main aquaculture regions; however, little effort has been focused on the United States. In this context, this study aims to examine the genetic diversity of F. psychrophilum from the United States, as this region remains important in salmonid aquaculture.