Editor: Reggie Lo
Isolation of a Carnobacterium maltaromaticum-like bacterium from systemically infected lake whitefish (Coregonus clupeaformis)
Version of Record online: 11 SEP 2008
© 2008 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Letters
Volume 288, Issue 1, pages 76–84, November 2008
How to Cite
Loch, T. P., Xu, W., Fitzgerald, S. M. and Faisal, M. (2008), Isolation of a Carnobacterium maltaromaticum-like bacterium from systemically infected lake whitefish (Coregonus clupeaformis). FEMS Microbiology Letters, 288: 76–84. doi: 10.1111/j.1574-6968.2008.01338.x
- Issue online: 25 SEP 2008
- Version of Record online: 11 SEP 2008
- Received 21 April 2008; accepted 8 August 2008.First published online 11 September 2008
- Carnobacterium maltaromaticum;
- pseudokidney disease;
- lake whitefish;
- Laurentian great lakes
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.
Lactobacilli are a normal component of the gastrointestinal and urogenital flora of many vertebrate phyla, including fish (Ringo & Gatesoupe, 1998; Gonzalez et al., 2000; Balcazar et al., 2007). However, in fish, lactobacilli have also been associated with morbidity and mortality (Rucker et al., 1953; Ross & Toth, 1974; Cone, 1982). Based on extensive genetic studies, biochemical fermentation patterns, and association with fish lesions, Hiu et al. (1984) proposed a new name for the fish pathogenic Lactobacillus sp.; Lactobacillus pisciciola. Later studies confirmed that L. pisciciola was indeed associated with postspawning morbidity and mortality, kidney granulomas, and pseudomembrane formation (Herman et al., 1985; Michel et al., 1986; Humphrey et al., 1987). In 1987, Collins et al. (1987) renamed L. piscicola as Carnobacterium pisciola, which was again renamed by Mora et al. (2003) as Carnobacterium maltaromaticum. Despite its potential pathogenicity, this bacterium has received little attention. Therefore, in this study we describe C. maltaromaticum-like isolates that are associated with infections in lake whitefish, one of the most economically and ecologically important species within the Laurentian Great Lakes.
Materials and methods
Fish and sampling
Between the fall of 2003 and the summer of 2006, lake whitefish were collected for bacteriological analyses from four representative stocks on four sampling occasions per year (c. 30 fish per site per season, Table 1). The two Lake Michigan sites, Big Bay de Noc (latitude 4527–4548.20; longitude 8535–8722) and Naubinway (latitude 4553–4603; longitude 8513–8535), and the two Lake Huron sites, Detour Village (latitude 4555–4556.97; longitude 8350.5–8419.12) and Cheboygan (latitude 4540.24–4548; longitude 8424–8437.87), were selected for this study due to their association with large spawning aggregations and accessibility via commercial fishing (Fig. 1). A total of 1286 lake whitefish from the four sites were sampled throughout the course of this study.
|Sampling period||Collection site||Season total|
|Big Bay de Noc||Naubinway||Cheboygan||Detour Village|
|Fall 2003||0/35 (0%)||0/30 (0%)||0/30 (0%)||0/34 (0%)||0/331 (0%)|
|Fall 2004||0/26 (0%)||0/30 (0%)||0/26 (0%)||0/30 (0%)|
|Fall 2005||0/30 (0%)||0/30 (0%)||0||0/30 (0%)|
|Winter 2004||0/29 (0%)||0||0/32 (0%)||0/10 (0%)||14/275 (5.09%)|
|Winter 2005||0/16 (0%)||10/30 (33.33%)||0/15 (0%)||0/30 (0%)|
|Winter 2006||0/30 (0%)||1/23 (4.35%)||0/30 (0%)||3/30 (10.0%)|
|Spring 2004||0/30 (0%)||0/30 (0%)||0/20 (0%)||0/30 (0%)||8/350 (2.29%)|
|Spring 2005||0/30 (0%)||2/30 (6.67%)||0/30 (0%)||2/30 (6.67%)|
|Spring 2006||1/30 (3.33%)||0/30 (0%)||0/30 (0%)||2/30 (6.67%)|
|Summer 2004||0/22 (0%)||0/20 (0%)||0/30 (0%)||0/20 (0%)||1/330 (0.30%)|
|Summer 2005||0/30 (0%)||0/30 (0%)||0/28 (0%)||1/30 (3.33%)|
|Summer 2006||0/30 (0%)||0/30 (0%)||1/30 (3.33%)||0/30 (0%)|
|Site total||1/338 (0.30%)||13/313 (4.15%)||1/301 (0.33%)||8/334 (2.40%)||23/1286 (1.79%)|
Fish were captured using commercial trap nets and, when necessary, commercial gill nets, with only live fish being used for the study. The collected fish were transported alive to the Chippewa Ottawa Research Authority (CORA) Fishery Enhancement Facility near Hessel, Michigan. The fish were then transported to the Aquatic Animal Health Laboratory at Michigan State University, East Lansing, Michigan. Live fish were euthanized using an overdose of MS-222 (Tricaine methanesulfonate, Argent Chemical Laboratories, Redmond, WA) and were subjected to thorough internal and external health examinations.
Bacterial samples retrieved from lake whitefish were collected from kidneys and gross external and internal lesions. Tissue samples were streaked directly onto trypticase soy agar (TSA; Remel Inc., Lenexa, KS) and Cresol Red Thallium Acetate Sucrose Inulin (CTSI) agar (all ingredients are from Sigma Chemical Co., St. Louis, MO), a selective and differential medium for Carnobacterium spp. (Wasney et al., 2001), and incubated at 22 °C for up to 72 h. Periodic examination of bacterial growth was carried out, and individual colonies were subcultured onto TSA and then incubated for 24 h at 22 °C. A total of 23 isolates were recovered and examined (Table 2). Recovered C. maltaromaticum-like isolates were maintained at −80 °C in trypticase soy broth (TSB; Remel Inc.) supplemented with 15% glycerol.
|Whitefish isolates||Cm type strain*||Hiu et al. (1984)||Baya et al. (1991)||Starliper et al. (1992)||Toranzo et al. (1993)||Mora et al. (2003)|
|Production of H2S||−||−||−||−||−||−||−|
|Production of indole||−||−||NR||−||NR||−||NR|
|Gas production from glucose||−||−||−||−||NR||−||−|
|Utilization of malonate||−||−||NR||NR||−||NR||NR|
|Acid production from|
Isolated bacteria were initially identified using a battery of morphological and biochemical tests, including Gram reaction, cytochrome oxidase, catalase reaction (3% H2O2), motility, indole production, hydrogen sulfide production, oxidation/fermentation reaction (BD Scientific, Sparks, MD), mixed acid fermentation (methyl red test), 2,3-butanediol production from glucose (Voges–Proskauer test), nitrate reduction, citrate utilization, triple sugar iron (TSI) reaction, o-nitrophenyl-β-d-galactopyranoside (ONPG), lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, esculin hydrolysis, phenylalanine deaminase (BD Scientific), growth on acetate agar (pH 5.4), and growth on CSTI medium at pH 9.1, as described by Wasney et al. (2001). Production of acid from the following carbohydrates was examined in phenol red broth base at a final concentration of 1%: adonitol, arabinose, cellobiose, dextrose, galactose, glycerol, inositol, innulin, lactose, malonate, maltose, mannitol, mannose, melibiose, raffinose, rhamnose, salicin, sorbitol, sucrose, trehalose, and xylose. Results were recorded up to 7 days postinoculation with the following exceptions: methyl red, Voges–Proskauer, indole production, Simmons citrate, and TSI reactions were read at 2 days. All materials and reagents were purchased from Remel Inc. unless specified otherwise. For confirmation and comparative purposes, all biochemical assays described above were performed on C. maltaromaticum type strain-27865 (ATCC, Manassas, VA).
Confirmation of isolate identification with PCR and gene sequencing
Representative colonies with the typical morphological and biochemical characteristics of C. maltaromaticum, as described by Mora et al. (2003), were selected for this study. Single colonies were resuspended in TSB and incubated overnight at 22 °C. DNA was extracted using a Qiagen DNeasy Tissue Kit (Qiagen Sciences, Valencia, CA) according to the manufacturer's protocol for Gram-positive bacteria. PCR amplification was performed using the following primers: (1) 16S-4 (5′-GCT GGA TCA CCT CCT TTC T-3′) and 23S-7 (5′-GGT ACT TAG ATG TTT CAG TTC C-3′), which anneals to positions 1526–1542 of the 16S rRNA gene and positions 207–189 of the 23S rRNA gene in Escherichia coli (Kabadjova et al., 2002; Pelléet al., 2005), and (2) PisA forward (5′-GTC ACA GCA TTG ATG CGT ATC-3′) and PisA reverse (5′-GAT GTG ATA CAG TCA GCA TGT-3′), which anneal to positions 1756–1777 and 2036–2057 on each side of the pisA precursor gene for the piscicolin 126 protein produced by the C. maltaromaticum (piscicola)-JG126 strain (Pelléet al., 2005).
DNA template (4 μL) was combined with 2.5 μL of 10 × PCR buffer containing 1.5 mMol MgCl2 (Invitrogen, Carlsbad, CA), 0.5 μL dNTP (Invitrogen), 1 μL of each primer, 17.5 μL distilled water, and 0.5 μL Taq polymerase with loading buffer (Denville Scientific, Metuchen, NJ). The PCR amplification program was that of Pelléet al. (2005) with slight modifications, which included an initial denaturation at 94 °C for 10 min, 35 cycles of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C, and a final step of 10 min at 72 °C. The amplified products were subsequently run on 1.5% agarose gel for 30 min at 50 V and then stained with ethidium bromide for viewing via UV exposure. A 1-kb plus DNA Ladder (Invitrogen) was used as a molecular marker.
Amplicons were purified using the QIAquick PCR Purification kit (Qiagen). Upon purification, a portion of the product was used for ligation with pGEM-T vectors [10 μL total volume; 5 μL 2 × ligation buffer, 1 μL suspended vector (50 ng μL−1), 1 μL ligase (3 U μL−1), 3 μL PCR product; Promega, Madison, WI] and incubated at 4 °C overnight. The recombinants were then transferred into DH5-α competent cells (Invitrogen). The transformed cells were then subjected to heat shock for 45 s in a 42 °C water bath and subsequently placed on ice for 2 min. SOC (super optimal broth with catabolite repression) medium (Invitrogen) was then added to the cells (0.9 mL at room temperature) and the solution was incubated at 225 r.p.m. at 37 °C for 1 h in an Incubating Shaker (Labnet, Edison, NJ). Thirty microliters of isopropyl-β-d-thiogalactopyranoside (IPTG, Denville Scientific) was then added and 500 μL of the bacterial suspension was aliquoted to SOB (super optimal broth) + ampicillin agar plates, on which X-gal (Denville) had been applied previously (BD Scientific). Plates were incubated at 37 °C overnight. Single, white, colonies were then picked up, inoculated into 500 μL SOB, and incubated at 220 r.p.m. at 37 °C for 4 h. Clones were then submitted to Michigan State University Research Technology Support Facility for analysis.
Analysis of the sequence data
Generated sequences were analyzed using the blastn software from the National Center for Biotechnology Information, (NCBI, Bethesda, MD) to detect homologous sequences. Homology of the generated sequences with that of the database was assessed using the nucleotide database from NCBI. Additionally, blastx software (NCBI) was used to compare the translated piscicolin-126 amino acid sequences from lake whitefish isolates with those deposited within GenBank. Using the clustal w program from Molecular Evolutionary Genetics Analysis (mega; version 3.1, http://www.megasoftware.net/), a phylogenetic tree was constructed using the neighbor-joining method as the bootstrap test of phylogeny.
As C. maltaromaticum has never been reported previously from lake whitefish, tissue samples exhibiting gross clinical abnormalities were preserved in 10% buffered formalin so as to determine what pathological/degenerative changes may be occurring. The fixed samples were then embedded within paraffin, sectioned at 5 μm, stained with hematoxylin and eosin (H&E) (Prophet et al., 1992), and observed under a light microscope.
Statistical analyses were performed using the χ2 analysis of contingency tables with the help of sigma-stat software (Jandel Corporation, San Rafael, CA).
Throughout the course of this study, 23 C. maltaromaticum-like isolates, 21 from the kidneys and two from exudate within the lumen of the swim bladder, were recovered and identified based on morphological, biochemical, and molecular attributes. These isolates were subcultured onto both TSA and CTSI media. Following 24–48 h of incubation on TSA at 22 °C, the isolates produced colonies that were semi-translucent, whitish, entire, convex, and measured 1–2 mm in diameter. It is noteworthy that upon primary isolation, colonies were semi-translucent, raised, striated, and with irregular margins. Growth on CTSI agar yielded convex, entire colonies surrounded by a yellow halo with a reddish-purple center (Fig. 2). In 21 isolates, the number of colonies present upon primary isolation varied from 1 to 50/10 μL of kidney inoculum. This corresponds to the presence of c. 1 × 102–5 × 103 CFU g−1 kidney tissues in infected individuals. In the two cases that involved the swim bladder, bacterial growth was interconnected and profuse.
Presumptive Carnobacterium isolates were then subjected to morphological, cultural, and biochemical reactions. All 23 isolates were nonspore-forming, nonmotile, short, straight bacilli (1.0–1.5 μm by 0.5 μm) arranged in palisades that did not produce catalase or cytochrome oxidase (Table 2). They were facultative anaerobes that did not produce H2S, indole, phenylalanine deaminase, lysine decarboxylase, and ornithine decarboxylase, did not reduce nitrates, and gave a negative Simmons citrate reaction. They all gave an acid slant over an acid butt TSI reaction without H2S or gas being produced, were able to hydrolyze esculin, and were positive for mixed acid fermentation (MR+). Furthermore, all isolates produced acid from cellibiose, glucose, galactose, maltose, mannose, salicin, and sucrose. In addition, no acid was produced from adonitol, arabinose, rhamnose, or xylose. No growth was achieved upon inoculation of acetate or MacConkey agars. Assays for arginine dihydrolase, o-nitrophenyl-β-d-galactopyranoside, 2,3-butanediol production from glucose, and acid production from inositol, raffinose, sorbitol, melibiose, trehalose, lactose, glycerol, innulin, and mannitol yielded variable results (Table 2). Biochemical results for C. maltaromaticum type strain ATCC 27865 performed in the authors' laboratory are also reported in Table 2.
The PCR of the rRNA genes amplified 600 bp bands from the 23 lake whitefish isolates. When the PisA primers were used for amplification, 300-bp bands were generated from lake whitefish isolates. Both bands are identical in size to those generated from C. maltaromaticum as reported by Pelléet al. (2005).
Sequencing of selected portions of 16S and 23S rRNA genes (accession numbers EU546836 and EU546837) and the pisA precursor gene for the piscicolin 126 protein (EU643471) from representative lake whitefish isolates were highly homologous with the sequences deposited previously in the GenBank for C. maltaromaticum isolates worldwide. For example, the 16S/23S rRNA sequence of the lake whitefish isolates yielded an expectation value of 0.0 and a 97% nucleotide match when compared with its homologue. The pisA precursor gene sequence from lake whitefish isolates had 98% nucleotide similarity to GenBank sequences and an expectation value of e−139, corresponding to a 4 bp difference (bp #16, N to A; bp # 66, C to T; bp # 210, C to T; bp # 241, C to G). Translation of this sequence and subsequent comparison with the amino acid sequence of the pisA precursor gene contained within GenBank revealed that one amino acid substitution, a serine to leucine at 210 bp, occurred in lake whitefish isolates.
Phylogenetic analyses demonstrated that the C. maltaromaticum-like isolates recovered from lake whitefish clustered most closely with C. maltaromaticum type strain AF374295, followed by Carnobacterium gallinarum (Fig. 3). Additionally, our isolates formed a phylogenetic cluster with Tetragenococcus halophilus, Streptococcus thermophilus, and a number of Enterococcus spp.
The overall prevalence of C. maltaromaticum-like infections in the four lake whitefish stocks varied by season and site, as can be seen in Table 1. The prevalence of infections varied among sites in a statistically significant fashion (χ2=18.587; df=3; P<0.001), with Naubinway having the highest prevalence, followed by Detour Village. There were also statistically significant differences in overall C. maltaromaticum-like infections between seasons (χ2=27.740; df=3; P<0.001), with winter prevalences being the highest, followed by spring. No statistically significant differences in the overall prevalence of C. maltaromaticum-like infections between Lakes Michigan and Huron were observed (χ2=0.611; df=1; P<0.435).
In this study, we report on clinical signs and histopathology observed in fish where a C. maltaromaticum-like organism was the only pathogen detected. Among the most common clinical signs were mild to severe splenomegaly, varying degrees of hyperemia and friability within the kidney, mottling and/or pallor in the liver, and varying degrees of congestion within the testes. Additionally, of the two fish from which the C. maltaromaticum-like organism was isolated from the swim bladders, copius amounts of an opaque, mucoid exudate was present, along with hemorrhage and a general thickening and opacity of the swim bladder wall (Fig. 4).
In stained tissue sections of infected fish, there was mild to moderate hepatocyte degeneration in the form of cytoplasmic vacuolation, as well as mild bile stasis, within the livers (Fig. 5a and b). Both spleen and kidney tissues had varying degrees of congestion. In the cases where C. maltaromaticum-like isolates were isolated from fluid within the swim bladder, thickening of the swim bladder wall with fibrous connective tissue, some degree of neovascularization, as well as fibrin and cellular debris within the lumen, were evident. Additionally, the epithelial lining of the swim bladder exhibited varying degrees of hyperplasia (Fig. 5c and d).
The morphological, biochemical, and molecular assays performed in this study confirm that the 23 lake whitefish isolates were indeed Carnobacterium sp. that were closely related to C. maltaromaticum. This report is considered to be the first to document that a Carnobacterium sp. can cause systemic infections in lake whitefish. The infections were associated with both gross and microscopical lesions in infected lake whitefish tissues. This is in accordance with other published studies reporting the association between Carnobacterium infections and diseased conditions that are often fatal (Cone, 1982; Hiu et al., 1984; Herman et al., 1985; Michel et al., 1986; Baya et al., 1991; Starliper et al., 1992; Toranzo et al., 1993).
The overall prevalence of Carnobacterium sp. infections in lake whitefish was the highest in Winter samples, followed by Spring and Summer samples. When comparing the prevalence of Carnobacterium infections in the four stocks of lake whitefish, there was a statistically significant difference among the four sites. The highest prevalence was found within the Naubinway populations, but it is interesting to note that the majority of the detected infections occurred in the winter of 2005. We were unable to link other environmental factors to the high presence of C. maltaromaticum at this particular site and time.
In addition to the high prevalence of Carnobacterium infections that was observed in the winter of 2005, it is of interest to note that no infections were detected in the fall, which is the period during which whitefish spawn. Previous research has proposed a link between postspawning stress and manifestation of disease caused by C. maltaromaticum (Cone, 1982; Herman et al., 1985; Starliper et al., 1992). Our study lends credence to this fact, as lake whitefish collected during fall samplings were still gravid/ready to spawn, meaning that our winter samplings would correlate more closely with a ‘postspawning’ time period. Thus, while a multitude of factors likely play a role in the ability of Carnobacterium spp. to generate disease, spawning stress could potentially be a key predisposing and/or initiating factor.
Phylogenetic studies provided evidence that all sequenced isolates retrieved in this study are identical to each other and closely related, and yet not identical to the C. maltaromaticum type strain AF374295.1 deposited in GenBank. Indeed, based on the sequenced rRNA gene stretches, we suggest that the isolates recovered from lake whitefish comprise a unique species of Carnobacterium; however, additional studies will be carried out. Moreover, variability in a number of phenotypic characteristics typically used for definitive dichotomization of Carnobacterium spp., such as inulin and mannitol fermentation, also varied among our isolates and among C. maltaromaticum isolates recovered in other studies (Table 2). Variability in biochemical profiles has been reported by other investigators whose studies of Carnobacterium spp. characterization involved fermentation reactions (Hiu et al., 1984; Michel et al., 1986; Starliper et al., 1992; Toranzo et al., 1993) and has sometimes been attributed to the use of different basal media by different investigators (Baya et al., 1991). However, the unique phenotypic and genotypic characters observed in the Carnobacterium isolates recovered from lake whitefish may be indicative of a novel Carnobacterium sp., although further characterization and analyses are required.
While a typical pseudokidney disease was not observed in infected lake whitefish, the isolation of this bacterium from the kidneys, along with the observed clinical and histopathological changes within the tissues of infected individuals, suggests that the infection was systemic in nature. In swim bladders from which this bacterium was isolated, signs of chronic irritation, such as hyperplasia, fibrin deposition, and neovascularization, and additional pathological effects, such as fibrin and cellular debris within the lumen, again illustrate the potential for disease generation by the retrieved Carnobacterium isolates.
The sequence generated from our isolates for the pisA precursor gene were nearly identical to the sequences presently contained within GenBank, with the occurrence of only a 4-bp difference throughout the entire 300-bp sequence. Whether the observed serine to leucine amino acid substitution has any functional implications on the protein itself and whether this protein is actually being produced by lake whitefish isolates are currently unknown. The piscicolin-126 protein, a class II bacteriocin, is bactericidal to some Gram-positive bacteria, such as Listeria monocytogenes (Jack et al., 1996). Thus, in addition to the potential for disease causation in lake whitefish, the C. maltaromaticum-like isolates recovered in this study may also be inhibitive to other closely related bacterial pathogens under some conditions, although further investigation is required.
Further investigations to fulfill Koch's postulates are hampered by the inability to keep lake whitefish alive in the laboratory for an extended period of time. Although the detected prevalence throughout the course of the study was not extremely high (1.8%), infection prevalence reached 33% in one seasonal sample. This finding illustrates that Carnobacterium spp. can potentially infect large portions of resident lake whitefish populations if conditions favorable for an epizootic occur. The implications of these outbreaks of disease are currently unknown and warrant further investigation.
The studies carried out as described in this paper were funded by a grant from the Great Lakes Fishery Trust, Lansing, Michigan. We would like to thank Dr Michael Jones, Mark Ebener, Greg Wright, and Dr Michael Arts for their valuable discussion and support.
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