Article pubs.acs.org/est
Association of Toxin-Producing Clostridium botulinum with the Macroalga Cladophora in the Great Lakes Chan Lan Chun,† Urs Ochsner,† Muruleedhara N. Byappanahalli,‡ Richard L. Whitman,‡ William H. Tepp,§ Guangyun Lin,§ Eric A. Johnson,§ Julie Peller,⊥ and Michael J. Sadowsky†,∥,* †
BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108, United States Lake Michigan Ecological Research Station, U.S. Geological Survey, Porter, Indiana 46304, United States § Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706, United States ∥ Department of Soil, Water, and Climate, University of Minnesota, St. Paul, Minnesota 55108, United States ⊥ Department of Chemistry, Physics, Astronomy, Indiana University-Northwest, Gary, Indiana 46408, United States ‡
S Supporting Information *
ABSTRACT: Avian botulism, a paralytic disease of birds, often occurs on a yearly cycle and is increasingly becoming more common in the Great Lakes. Outbreaks are caused by bird ingestion of neurotoxins produced by Clostridium botulinum, a spore-forming, gram-positive, anaerobe. The nuisance, macrophytic, green alga Cladophora (Chlorophyta; mostly Cladophora glomerata L.) is a potential habitat for the growth of C. botulinum. A high incidence of botulism in shoreline birds at Sleeping Bear Dunes National Lakeshore (SLBE) in Lake Michigan coincides with increasingly massive accumulations of Cladophora in nearshore waters. In this study, free-floating algal mats were collected from SLBE and other shorelines of the Great Lakes between June and October 2011. The abundance of C. botulinum in algal mats was quantified and the type of botulism neurotoxin (bont) genes associated with this organism were determined by using most-probable-number PCR (MPN-PCR) and five distinct bont gene-specific primers (A, B, C, E, and F). The MPN-PCR results showed that 16 of 22 (73%) algal mats from the SLBE and 23 of 31(74%) algal mats from other shorelines of the Great Lakes contained the bont type E (bont/E) gene. C. botulinum was present up to 15 000 MPN per gram dried algae based on gene copies of bont/E. In addition, genes for bont/A and bont/B, which are commonly associated with human diseases, were detected in a few algal samples. Moreover, C. botulinum was present as vegetative cells rather than as dormant spores in Cladophora mats. Mouse toxin assays done using supernatants from enrichment of Cladophora containing high densities of C. botulinum (>1000 MPN/g dried algae) showed that Cladophora-borne C. botulinum were toxin-producing species (BoNT/E). Our results indicate that Cladophora provides a habitat for C. botulinum, warranting additional studies to better understand the relationship between this bacterium and the alga, and how this interaction potentially contributes to botulism outbreaks in birds.
1.0. INTRODUCTION Outbreaks of botulism, a paralytic and often fatal bacterial disease, have caused large mortalities of birds and fish in the Great Lakes.1−4 Outbreaks were first reported in this region in 19635 and have gone through episodic cycles over the last several decades. The USGS National Wildlife Heath Center estimates that there were in excess of 100 000 bird mortalities from botulism-related outbreaks between 1963 and 2007.2 These outbreaks have become increasingly more common in Lakes Michigan, Erie, Huron, and Ontario, with recent increases and expansion of the affected areas and of bird species killed. Since 1999, it has been estimated that botulism toxicity may be responsible for the deaths of more than 87 000 Great Lakes’ birds.2,3 In 2006, a large die-off of nearly 3000 native, fish-eating birds occurred in the areas of Lake Michigan within and near the Sleeping Bear Dunes National Lakeshore © 2013 American Chemical Society
(SLBE). This was not an isolated event, and bird die-offs continue to be a problem in and around SLBE.6 Clostridium botulinum, the responsible pathogen, is an obligate anaerobe that is widespread in aquatic and soil environments, mostly as dormant spores.7 The production of botulism neurotoxin (BoNT) only occurs when environmental conditions become suitable and allow for the germination of spores and the subsequent growth of vegetative cells. Seven distinct serotypes of botulism neurotoxin have been identified and classified (designated BoNT/A to/G) based on their antigenic properties.8 BoNT types C and E are primarily Received: Revised: Accepted: Published: 2587
November 28, 2012 February 19, 2013 February 20, 2013 February 20, 2013 dx.doi.org/10.1021/es304743m | Environ. Sci. Technol. 2013, 47, 2587−2594
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Figure 1. Location of sampling sites on the Great Lakes. Cladophora samples were taken from the nearshore water.
responsible for the deaths of waterfowl and fish-eating birds in the Great Lakes and BoNT type E outbreaks have recently increased in Lake Michigan.3,9 Laboratory studies10,11 have provided some insights into the abiotic factors favoring bacterial growth and some studies have attempted to correlate environmental factors with botulism outbreaks.12,13 Despite this knowledge, however, it is still unclear how ecophysiological factors in the Great Lakes lead to in situ growth, toxin production, and subsequent botulism outbreaks leading to bird mortalities. A few overlapping and hypothetical pathways have been proposed to account for bird deaths, although there may be subtle differences in the etiology among the affected avian population. One possible route is that shorebirds acquire toxin from insects or larvae that have been feeding on the carcasses of dead fish and birds, referred to as the carcass-maggot cycle.14 It has also been postulated that birds become ill due to consumption of C. botulinum spores, vegetative cells, or toxin contaminated mussels, fish, and/or algae. Aside from birds, BoNT/E can also be toxic to freshwater fish15 and has been found in wild fish living in and around Lake Erie and Lake Ontario.16 Several studies suggest that the increased botulism mortality seen in Great Lakes fish and birds may be due to consumption of contaminated invasive mussels (Dreissena spp.) and round gobies (Neogobius melanostomus).17,18 Recently, the filamentous, nuisance green alga Cladophora has also been considered as a possible vector of C. botulinum.6,19 Byappanahalli and Whitman6 demonstrated that Cladophora collected from the shoreline in Lake Michigan and incubated under laboratory conditions contained a higher frequency of C. botulinum type E (bont/E gene) than did fresh, nonincubated, samples. Likewise, C. perf ringens, which is physiologically similar to C. botulinum,20 has been found in high densities in Cladophora mats suggesting that the decomposing algal mats may provide a generally suitable niche (high nutrients, organic matter, and low Eh) for these related anaerobic bacteria.21 The potential for the association between Cladophora mats and C. botulinum has been increasingly speculated. A high incidence of botulism in shoreline birds at SLBE in Lake Michigan appears to parallel the increase in Cladophora accumulations in nearshore areas. The increased accumulation of Cladophora in water is thought to be largely due to improved
water clarity and light penetration depth as a result of the presence of invasive filter feeders such as dreissenid mussels.22 Algal exudates and thalli are rich in nutrients and can sustain a variety of organisms including epiphytes (cyanobacteria and diatoms), grazers (protozoa, mollusks, rotifers, micro and macroflora, and young crayfish),23−25 and numerous bacteria.26,27 This complex ecosystem, which includes benthic sediments, bacteria, spores, algae, and macroinvertebrates, likely play a role in the growth of C. botulinum and the transport of toxin up the food chain.28,29 Furthermore, as the algae begin to decay or accumulate in dense mats along shorelines, this leads to anaerobic conditions promoting the growth of C. botulinum. Because this bacterium lacks the ability to synthesize essential growth factors (such as some amino acids),7 decomposing algal matter (plus other aquatic vegetation) and the macroinvertebrate community most likely provide the necessary nutrients for the growth of toxin-producing C. botulinum. The resultant toxins, we believe, are then transferred to fish directly, or via several food chain intermediates, eventually to fish and then to birds. Byappanahalli and Whitman6 suggested that a likely pathway for piscivorous species is sediment − Cladophora − invertebrates − fish − birds. In this study, we examined the potential of the botulism pathogen to grow in Cladophora by determining the relative abundance and types of C. botulinum in algal mats collected from SLBE and other shorelines of the Great Lakes. Analyses were done by using the most probable number-polymerase chain reaction (MPN-PCR) technique using bont gene-specific primers. Moreover, we examined the nature of the pathogen that are present in Cladophora mats (viable/vegetative cells, spores only, or both) and determined if Cladophora-borne C. botulinum has the potential to produce botulism neurotoxin by using mouse toxin-antitoxin assays.
2.0. EXPERIMENTAL SECTION 2.1. Site Description and Sampling. Cladophora samples were collected monthly between June and October 2011 from four beaches on Lake Michigan and from a shoreline on Lake Ontario; Cladophora accumulations are common at these locations. Our principal study area was SLBE in Empire, Michigan. Other study sites included shorelines along Door County, WI, the City of Racine, WI, Porter and Cook Counties 2588
dx.doi.org/10.1021/es304743m | Environ. Sci. Technol. 2013, 47, 2587−2594
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Table 1. Clostridium botulinum Density (Most Probable Number (MPN)/g Dried Weight) in Cladophora Collected from Shorelines of the Great Lakes in 2011 location Sleeping Bear National Lakeshore, MI
site (GPS point) County Road 651 N (44.947N, 85.812W) Sunset Beach (44.931N, 85.965W) Good Harbor Bay Trail (44.938N, 85854W)
County Road 669 (44.939N, 85.870W)
Door County, WI
Esch Road (44.760N, 86.076W) Peterson Road (44.732N, 86.103W) Dimmick’s Point (44.059N, 85.965W) Europe Bay #1 (45.112N, 87.07W) Europe Bay #2 (45.258N, 86.97W)
Racine, WI
Shoop Park (42.779N, 87.764W)
Light House (42.781N, 87.758W) Samuel Meyers Park (42.443N, 87.463W)
Indiana Dunes National Lakeshore, IN, and Southern Lake Michigan, IL
Eichelman (42.344N, 87.383 W) Carre Hogle (42.711N, 87.771W) Calumet Park(41.392N, 87.399W) Ogden Dunes (41.622N, 87.191W) 63rd Street (42.344N, 87.485W) Burns Ditch (41.58N, 78.19W)
Hamlin Beach State Park, NY
HYC (43.364N, 77.951W)
date
MPN (bont Type)a
water/air temp. °C
algae conditionsb
fish/bird death
6/30/11
ND 62 ± 23 (E)
15.4/20.0
FT, FR
fish (∼1000 gobies)
8/18/11 6/30/11
ND ND 480 ± 153 (E) 310 ± 111 (E)
17.2/25.5 17.0/20.0
FT, FR FT, FR
8/18/11 8/08/11
3500 ± 1200 (E) 230 ± 75 (E) 5300 ± 1683 (E) 2250 ± 810 (E) 300 ± 96 (E) 4600 ± 1479 (E) ND 110 ± 35 (E) 210 ± 64 (E) ND ND 1000 ± 316 (E)
16.8/24.4 18.2/22.7
FT, FR FT, FR
16.8/24.4 14.0/17.0 18.2/22.7 14.0/16.0 16.4/21.2
FT, FT, FT, FT, FT,
16.4/21.2
FT, FR
NM/15.5
FT, FR
6/21/11 7/26/11 6/21/11 7/26/11 8/25/11 6/20/11
9800 ± 3600 (E) 15000 ± 5100 (E)/ 2000 ± 650 (A) 1000 ± 370 (E) 3800 ± 1300 (E) 580 ± 183 (E) 410 ± 128 (E) 80 ± 26 (E) 1400 ± 464 (E) 1000 ± 331 (E) 630 ± 355 (E)
15.0/14.6 NM/25.5 15.0/14.7 NM/25.5 NM/18.5 15.1/16.7
FT, FR AT, FR AT, FR FT, FR FT, FR FT, FR
7/26/11
1500 ± 550(E)
18.2/24.2
FT, FR
7/26/11
640 ± 240 (E)
18.2/24.2
FT, SN
8/25/11 10/10/11 6/20/11
270 ± 28 (E) 78 ± 34 (E) 190 ± 61 (E)
22.8/21.8 16.7/16.8 15.3/14.4
FT, FR FT, SN FT, FR
7/26/11 6/20/11 7/26/11 8/25/11
830 ± 260 (E) 340 ± 112 (E) 2900 ± 974 (E)/65 ± 22 (B) 490 ± 150 (E)
18.0/24.2 15.0/20.1 21.0/22.0 22.8/21.8
FT, FT, FT, FT,
6/20/11
150 ± 50 (E)
15.0/17.7
FT, SN
8/25/11
270 ± 88 (E)
21.0/21.8
FT, SN
6/21/11 7/21/11 9/6/11 6/21/11 7/21/11 9/06/11 6/21/11 7/21/11 9/6/11 9/22/11 7/21/11
12 ± 3 (E) NDc ND ND ND ND ND ND 11 ± 2.7 (E) ND 3400 ± 1100 (E) 3600 ± 1200 (E) 4800 ± 1700 (E) 12000 ± 4400 (E)
19.6/30.2 19.4/31.1 NMd/17.2 17.5/32.4 19.0/31.5 NM/17.2 17.4/30.2 19.0/31.5 NM/17.2 NM/17.6 NM/30.0
AT/SN AT/SN FT/SN AT/FR AT/FR FT/SN FT/FR FT/FR FT/SN FT/SN FT, FR
NM/21.1
FT, FR
8/18/11 10/03/11 8/08/11 10/03/11 8/15/11 8/15/11 10/04/11
8/24/11
fish (∼700 gobies)
FR FR FR FR FR
FR SN SN FR
2 birds 2 birds fish (>100 alewife) fish (>100 alewife) fish (>100 alewife) fish fish (>100 alewife)
1 fish and 1 bird
3 birds
MPN values are means ± SE on means. Numbers in parentheses refer to bont gene type detected. bAlgal condition abbreviation are as follows AT, attached; FT, detached and floating; SN, senescent (decomposing); FR, fresh (little to no decomposition). cND, not detected or below detection limit. dNM, not measured. a
along southern Lake Michigan in Indiana and Illinois (Indiana Dunes National Lakeshore, IDNL), and Hamlin Beach State Park, NY on Lake Ontario (Figure 1). GPS coordinates of each
sampling site are listed in Table 1. Three replicate samples of approximately 150 g of free-floating Cladophora algal matter (thalli) were obtained from two locations at each sampling site. 2589
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60 °C for 25 s, and elongation at 72 °C for 1 min, followed by a final extension at 72 °C for 3 min. Amplified PCR products were visualized on 1.5% agarose gels, and stained with ethidium bromide. Positive controls consisted of plasmids containing the targeted bont genes35 and no-template negative controls were used for all PCR reactions. Tubes showing positive PCR reactions for any of the tested genes, as determined by gel electrophoresis of PCR products, were counted as positive for each type of C. botulinum. Bacterial counts were expressed as MPN per gram dry weight of algae. The identity of all PCR products was determined by DNA sequence analysis done at the University of Minnesota Biomedical Genomics Center. BLAST analyses were used to identify the homology of the amplified target sequence for bont genes to those in Genbank. 2.3. Heat Treatment. A comparative study of selected Cladophora samples was done to determine if vegetative cells and/or spores of C. botulinum were present in Cladophora mats. Subsamples of Cladophora homogenates were held at 80 °C for 10 min in a water bath to kill vegetative cells prior to three-tube MPN-PCR analysis.36 The MPN counts from these samples were compared to controls with no treatment (total count). 2.4. Identification of C. botulinum Neurotoxin Serotypes Using Mouse Bioassay and Antibody Neutralization Analysis. MPN cultures from Cladophora samples with elevated MPN values were analyzed for actively growing and toxin-producing C. botulinum by using a mouse bioassay.37 Briefly, MPN cultures were diluted in 0.5 mL gelatin phosphate buffer (30 mM sodium phosphate, pH 6.3, plus 0.2% gelatin), in 1:1 ratio and injected via i.p. (intraperitoneal) into mice. Female ICR (CD-1) mice were obtained from Harlan laboratories. Each mouse within the group (2 mice) were injected with 0.5 mL of the toxin-antibody mixture and observed for 4 d for symptoms of botulism poisoning. Samples causing death indicated that neurotoxin was present. To determine the toxin serotypes produced in the samples, antibody neutralization analyses were employed. Anti-BoNT/ A (10 international unit/ml; IU/ml), anti-BoNT/B (10 IU/ ml), and/or anti-BoNT/E (100 IU/ml) antibodies used in neutralization analysis and were obtained from Centers of Disease Control and Prevention, Atlanta, GA. One IU of antiBoNT protects a mouse against a 10 000 mouse medium lethal dose units (LD50/mouse) of BoNT/A and/B or 1000 LD50/ mouse of BoNT/E. The LD50 of BoNT is estimated to be about 1 ng/kg in mice. For an initial neutralization analysis, toxicity was estimated to be about 10 LD50/mouse based on the time of death (mice died within 24 h) and toxin was diluted with gelatin phosphate to achieve the appropriate LD50 concentration. The mixtures of toxin and antibody were incubated at 37 °C for 90 min prior to injection. All procedures with mice were performed in accordance with the Institutional Animal Care and Use Committee in the Department of Bacteriology at the University of Wisconsin.
The samples consisted of detached, floating algal matter in nearshore waters but had not yet washed-up onto the dry beach. In one case, lake bottom-attached Cladophora, sand, and deep water were collected in October at Good Harbor Bay Trail in SLBE from approximately 10 m below the water surface by a diving team. Samples were collected by gloved hands, placed in Whirl-Pak bags, and transported to the laboratory on ice at 4 °C. The samples were immediately shipped to the University of Minnesota and were analyzed within 48 h of collection. Nearly axenic Pithophora spp. (a filamentous green alga) grown in a fish tank in the lab was used as the negative control because there was no contamination with clostridia. 2.2. MPN-PCR Analyses. Five-tube-MPN analyses were used to identify and quantify types of Cladophora-borne C. botulinum.30 Wet algae (50 g) from the pooled Cladophora samples (16.6 g of each original replicate) were blended with 100 mL of phosphate-buffered water (PBW; 10 mM sodium phosphate, pH 6.8). Benthic organisms attached to Cladophora were removed prior to homogenization. The blender was continually flushed with N2 gas to minimize exposure of C. botulinum to oxygen. The algal homogenate was serially diluted 10-fold, three times, in PBW. From each dilution, a five-tube MPN was set up by adding 1 mL aliquots of each dilution to 9 mL of prereduced trypticase-peptone-glucose yeast extract (TPGY) broth as described by Hielm et al.31 In addition, some of the attached benthic organisms removed from Cladophora were also incubated in TPGY broth (each organism in 9 mL broth). Anaerobic tubes were sealed using gastight rubber stoppers and aluminum caps. Immediately after the assembly, the tubes were flushed for 3 min with N2 gas, passed through 0.2 μm sterilized filter with an 18-gauge needle, to remove residual oxygen in the headspace, and tubes were incubated at 26 °C for 5 d. After incubation, 2 mL aliquots from each tube were vigorously shaken, centrifuged at 8000 rpm for 10 min, and the pelleted cells were saved at −20 °C for DNA extraction. Around 15 g triplicate samples of the remaining algal homogenates were placed overnight in an oven at 85 °C oven to measure the dried weight of algal matter. Genomic DNA from the pelleted cells prepared above was directly extracted using the UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA) as per the manufacturer’s protocol, with slight modifications: the pellets were resuspended in buffer and transferred into a microbead tube with MD1 solution (surfactant and other disruption agents for cell lysis) and tubes were incubated at 80 °C for 10 min in a dry bath before a 10 min vortexing step to increase yield and deactivate potential BoNTs that are heat labile. The non-DNA organic and inorganic materials were also precipitated with C3 solution before spin-filtering to further purify DNA. Multiplex PCR was used for the detection of genes encoding for C. botulinum toxins A, B, C, E, and F.32−34 The oligonucleotide primer sets used are listed in Table S1 of the Supporting Information. Two multiplex PCRs were conducted for toxins A, C, and F, and B and E based on the amplicons’ size. PCR was performed in 25 μL reactions containing 1 μL of template, 400 nM of each primer (Integrated DNA Technologies Inc., Coralville, Iowa), 220 μM of each deoxynucleotide triphosphate (Invitrogen, Carlsbad, CA), 1.5 mM MgSO4, 10 mM KCl, 8 mM (NH4)2 SO4, 10 mM TrisHCl, pH 9.0, 0.05% NP-40, 200 ng μL−1 BSA, and 0.08 U μL−1 of DNA Polymerase (Denville Scientific Inc.). PCR was done using 35 cycles of denaturation at 95 °C for 30 s, annealing at
3.0. RESULTS 3.1. Type and Abundance of C. botulinum Associated with Cladophora. A total of 53 Cladophora mats collected from June to October in 2011 were tested for the presence and type of C. botulinum using botulism toxin gene specific primer pairs (Table S1 of the Supporting Information). Among the five bont genes examined, C. botulinum type E strains were prevalent in Cladophora mats collected from the Great Lakes. Sixteen of 22 (73%) Cladophora mats from SLBE and 23 of 31 (74%) algal mats collected from other shorelines in the Great Lakes 2590
dx.doi.org/10.1021/es304743m | Environ. Sci. Technol. 2013, 47, 2587−2594
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Figure 2. Population density of C. botulinum type E in Cladophora samples as determined by MPN-PCR. Legend: (A) SLBE, (B) the City of Racine, (C) Door County (C), and (D) Hamlin Beach State Park. The error bars indicate standard errors.
A comparison of C. botulinum densities present in nearshore floating Cladophora versus Cladophora attached to the bottom of lake is shown in Figure 3. Water, sediment, and mussels (Dreissena spp.) near Cladophora mats were also collected when submerged Cladophora was sampled from Good Harbor Bay
contained bont/E genes (Table 1). In addition to type E toxin genes, we also detected bont types A and B genes in a few Cladophora samples from SLBE (Peterson Beach) and Racine’s Samuel Meyers Park. These BoNT types have been commonly associated with human diseases rather than birds.20 The PCR products of bont gene-positive algal samples were confirmed by DNA sequencing. The population density of C. botulinum type E strains in algal mats ranged from 100−105 MPN per gram dried algae (Table 1 and Figure 2) based on the assumption of one copy of each gene per genome and that generally C. botulinum types A, B, E, and F neurotoxin genes are located on the chromosome or plasmids, whereas in types C and D the genes are found on a bacteriophage.20,38 Whereas the population level of C. botulinum varied widely in Cladophora samples from SLBE beaches, algal samples collected in August contained the greatest number of C. botulinum, up to 15 000 MPN/g dried algae. The Cladophora samples from Hamlin Beach State Park in Lake Ontario also had a high population of C. botulinum type E (104−105 MPN/g dried algae). In contrast, most samples collected from locations along southern Lake Michigan were negative for bont/E genes, and only 2 of 10 samples (20%) contained C. botulinum type E, with counts in other samples generally below the detection limit (
