Seidler, Ramon J.

Tison, David L.

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Ecology and Virulence ofLegionnaire's Disease Bacteri ain Aquatic HabitatsNear Mt . St . Helen sb yDavid L . Tiso nRamon J . Seidle rWater Resources Research Institut eOregon State Universit yCorvallis, Orego nWRRI-84	September 1983ECOLOGY AND VIRULENCE OF LEGIONNAIRE ' S DISEASE BACTERIAIN AQUATIC HABITATS NEAR MT . ST . HELENSbyDavid L . Tison and Ramon J . Seidle rDepartment of Microbiology, Oregon State Universit yFinal Technical Completion Repor tfor Project No . A-060-OREtoUnited States Department of the InteriorWashington, D .C . 20240Project Sponsored by :Water Resources Research Institut eOregon State UniversityCorvallis, Oregon 9733 1The research upon which this report is based was financed in part by th eU .S . Department of the Interior, Washington, D .C . (Project No . A-060-ORE) ,as authorized by the Water Research and Development Act of 1978, P .L . 95-467 .Contents of this publication do not necessarily reflect the views andpolicies of the U .S . Department of the Interior, nor does mention of trad enames or commercial products constitute their endorsement or recommendationfor use by the United States Government .WRRI-84	September 1983ABSTRACTIllnesses among researchers exposed to lakes and streams i nthe Mt . St . Helens blast zone after the 18 May 1980 eruptio nprompted us to determine the occurrence and potential virulenc eof Legionella (Legionnaire's disease bacteria) in aquati chabitats near Mt . St . Helens during the summers of 198 1.and 1982 .Concentrations of L . pneumophila, L . nnicdadei, L . g©rmanii, L .dumoffii, and L . bozemanii, determined by microscopic count susing direct immunofluorescent staining, ranged from <l0 4 to 10'cells/L in lakes and rivers outside of the Mt . St . Helens blas tzone while the numbers of Legionella in aquatic habitats insid ethe blast zone were from 10 5 to 10 7 cells/L . Legionella number swere consistently highest in N . Coldwater and Spirit lakes, whic hreceived water from hydrothermal seeps . Legionella pneumophil aserogroups 4 and 6 were isolated from N . Coldwater Lake in 198 1and from S . Coldwater Creek in 1982, indicating that th epotentially virulent strains of Legionella persist in aquati chabitats in the blast zone of Mt . St . Helens .FOREWORDThe Water Resources Research Institute, located on the Orego nState University campus, serves the State of Oregon . Th eInstitute fosters, encourages and facilitates water resource sresearch and education involving all aspects of the quality an dquantity of water available for beneficial use . The Instituteadministers and coordinates statewide and regional programs o fmultidisciplinary research in water and related land resources .The Institute provides a necessary communications an dcoordination link between the agencies of local, state an dfederal government, as well as the private sector, and the broa dresearch community at universities in the state on matters o fwater-related research . The Institute also coordinates th einter-disciplinary program of graduate education in wate rresources at Oregon State University .It is Institute policy to make available the results o fsignificant water-related research conducted in Oregon' suniversities and colleges . The Institute neither endorses no rrejects the findings of the authors of such research . It doe srecommend careful consideration of the accumulated facts by thos econcerned with the solution of water-related problems .ACKNOWLEDGEMENT SWe thank Cliff Dahm (Oregon State University) and Jim Bedel l(U .S . Forest Service) for assistance in coordinating samplecollection and for helpful discussions . The U.S .. Forest Serviceis thanked for providing helicopter time . Financial support wa sprovided by the Oregon State University Department of Laborator yAnimal Resources and the U .S . Department of the Interior, Offic eof Water Policy, through the Water Resources Research Institut eat Oregon State University .11TABLE OF CONTENTSPageINTRODUCTION	 1MATERIALS AND METHODS	 3RESULTS	 7DISCUSSION	 9REFERENCES	 11LIST OF FIGURESPageFigure 1 . Mt. St . Helens and vicinity, showing river sand lakes affected by May 18, 1980 eruption	 4Figure 2 . Diagrammatic representation of a sample areain the S . Coldwater Creek drainage area 	 5Figure 3 . A portion of the S . Coldwater Creek drainag earea illustrated in Figure 2 	 6LIST OF TABLESTable 1 .	Numbers of cells per liter reacting wit hserogroup specific fluorescent antibodyconjugates to Legionella spp	 7ivINTRODUCTIONThe eruption of Mt . St . Helens on May 18, 1980 reshapednearby pre-existing lakes and streams, created new lakes as aresult of damming of drainages by debris flows, and produce dnumerous hydrothermal seeps . Tremendous quantities of pyrolize .dorganic matter from the destroyed forest and inorganic nutrient sfrom the ash and pyroclastics were added to previouslyoligotrophic sub-alpine lakes typical of those in the Cascad eRange in the Pacific Northwest U . S . These aquatic environment sprogressed from oligotrophic to hypereutrophic in a matter o fdays ; undisturbed, this progression would have taken centuries .Because of the proximity of academic research institutions t othese unique environments, characterization of the chemical an dbiological properties of these 'habitats was initiated soon afte rthe May 18, 1980 eruption (1, 7, 9) .Research personnel exposed to aquatic environments in the Mt .St . Helens blast zone reported illnesses with "flu-like" symptom sthroughout the summer of 1980 and again early in the spring o f1981 . The similarity of the reported symptoms to. the "PontiacFever" syndrome cause by Legionella'pneumophila and the nature o fthe aquatic environments produced by the cataclysmic eruption o fMt . St . Helens prompted us to initiate a study to determine th edensity and distribution of Legionella spp. (the etiologic agent sof Legionnaires'• Disease and Pontiac Fever) in the blast zone ofMt . St . Helens .MATERIALS AND METHODSThe degree of impact by the May 18, 1980 eruption on lake sand streams in the Mt . St . Helens blast zone is shown in Figur e1 . Sites sampled included spirit Lake, North Coldwater andCastle creeks, which are in the scorch zone, and Ryan Lake, whic his in the blowdown zone . South Coldwater Creek and hydrotherma lseeps in the South Coldwater drainage area and sites outside o fthe blast zone including McBride Lake, Silver Lake (not shown )and the Muddy and North Toutle rivers were also sampled . Figures2 and 3 illustrate the nature of the South Coldwater Cree kdrainage area .The physical, chemical and biological characteristics o fthese sites have been described previously (1, 7, 9) . Ingeneral, lakes in the blast zone were high in dissolved organi ccarbon (DOC), nitrogen-limited, and dominated by heterotrophi cand metal oxidizing bacteria with total direct microscopic count sranging from 10 6 to 1Q 8 bacteria per ml (1, . .7, 9) .One-to-four ]n iters of water were colleeted, concemtr-ate ;d bymembrane filtration (6), and examined microscopically usin gdirect immnn,0fluorescence (3) . Cells reacting with fluorescen tantibody conjugates (Biological Products Section, , .enters forDisease Control) specific for L . pneumophila ser®groups	L,micdadei, L . gormanii, L . dumoffii, and L . bozemanii wer equantitated . The virulence of presumptive Legionella wasassessed by guinea pig innoculation (5) .Figure 1 . Mt . St . Helens and vicinity, showing rivers and lake saffected by May 18, 1980 eruption .4UCUOO)Na)r 4-) -( 4)S- O-o a( -0a) 4-+-)CU 0 4-• CUU i ra)L +) a )a) ~ t4-) 3 -NCd3'~ C-Cl a) 0r CO•r bC)Cda)4) 4-)• C CdN 0 SU + )W N- d- =4-) rO rC C •rNbRESULTSConcentrations of Legionella species from the mostintensively sampled sites are presented in Table 1 . The numbersof Legionella spp . in Ryan and Castle Creek lakes are of the sameorder of magnitude as those found for L . pneumophila in a surveyof a number of lakes and rivers in the southeastern United State s(4) . Concentrations of Legionella spp . in North Coldwater andSpirit lakes were consistently high (10 6 to 107 cells/L) whencompared to lakes outside of the blast zone (Table 1) . Thesedata may be attributed to the high concentrations of DOC an davailable metals in these lakes . Although the concentrations o fLegionella spp . were particularly high in North Coldwater andSpirit lakes, the percentage of the total bacterial populatio nthese bacteria comprised (0 .1% to 1%) was not unusual whencompared to other aquatic habitats (4) . The enrichment of thes eaquatic environments with DOC from pyrolysis of the productiv econiferous forest and inputs of inorganic nutrients from the as hand pyroclastics have enriched for not only Legionella but thetotal bacterial population as well .Legionella pneumophila serogroups 4 and 6 were isolated o nBCYE agar inoculated with the liver, spleen and peritoneal flui dat necropsy from animals inoculated with concentrated water fromNorth Coldwater Lake collected on April 30, 1981 and from Sout hColdwater Creek collected on June 15, 19 .82 .  These isolates wer echaracterized biochemically and serologically as describe dpreviously (6) .rn•rCa,Ea )N0-CIJC0U0•rCQOs-Utoa)S-S-Q)+-,•rS-WQ.0-v) 0.r NC1 its•r4--4-OE0J•rC+,E00)v•0CO'TJUEJI-0E0CO.u7in toC) CY C)r 1	eti	r- Ix x xC) r r r r	C) r r r t r	o r r La°•	• 1•	r	'~	TZ t i	Z.1 'r	r r 1 r 1	Z 1 r 1	r$ 1 tO	1t.o	C)	C)	C)	C )	r-1	rl	ri	r--I	r- I	x	x x x 7C	0 1	1	I	•	C) I 1 1 1 1	C3 I I 1	•	I	1	M	LI•	e	Z 1 1 1 .-I	Z 1 1 1 1 1	Z I 1 I rt.)	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N.	r--1	,tcr	•	1	1	1	1	I	I	1	1	I	F	1	1	1'	1	I	r- N 1 I t	I I . l 1^	i I 1 CV 1	ri 1 1	1'	+3	I	1}i	.]G	I`	Q)C)	C)	C)	N - C) a.X r-1 .-1 r-1 r-I N 4-) r-I r-I r-I .-I r♦ r-1 Q) v -I r•I r-1 r-t r-I	r-I r-l .--I 4-1 N rO r-I .aC Cst	Cc]fa CO CO CO CO CO CoCC .CO CO CO CO CO i0000COCO00 C) CO 00 CO rt000 .-lCO rCCO Q) COJ \\\\\ \\\\\\U \\\\\ . .~C -\ \ -.J \r' \O C%J to t 0 CD O) N CO r-I .LA O 01 N tO +-L (0 CV LO r-I )) LO r- LC) C7 d-) C74-) CO CV r- M r- r- Cr) N CM r-1 C+r) r- C) M N N r-I CO J CV r-I C) r- r-l r- e--1 5- r-I	r-l•r \\\ 0 \\\\\\r	\0 \-•r \ Q) \ 0S-C - N0000toU~to1.CO00tO-1-) .1-toh. 00 CN- 000TUto 5- tO >toI- to•r	_	(0	5...N	Z	(Cl	to	 .	 0---.	-	- - a)	-	_Y	.X	QJla	fa	5	S-J	U	C.1COl0LI)U)	000	C)	ri ri v-I	ri	X X X	X.-IM CV OCI 1	CI" I	• 1 1 1	Z I 1~ CV LC)	Z l C') 1 1 1	CI U-) L)	LC) LC-) LC)	LO	Ir)CCC QC)C) 0 4ri . 1 r-i ri ri H ri rlxxx xxx x xCO N. i• 03 CO CM tO	.--IC) 1	+.r- 1V0•r4-0O8DISCUSSIONThe concentrations of Legionella spp . in hydrothermal seep sin the South Coldwater Creek drainage particularly emphasize th eenrichment potential of aquatic niches in this area . Thermalseeps eminating from the mud flow ranged in temperature from 7 50 Cto ambient (18 0 C) . Dissolved organic carbon concentration sranged from about 700 to 900 mg C/L in seeps having temperature sabove 65 0C while concentrations in thermal seeps wit htemperatures from 40 to 60 0 C were about 250 mg C/L (C . Dahm ,pers . comm .) . Microbial communities similar to those in naturalthermal springs (2) and man-made thermal habitats (8) wer eobserved . At temperatures above 40 0C these communities wer ecomprised mainly of the cyanobacteria.Phormidium.sp ., and to alesser extent, Oscillitoria sp., and associate dnon-photosynthetic bacteria . Concentrations of Legionella spp .ranged from 105 to 107 DFA positive cells per cm . It i sinteresting to note that those lakes in the Mt . St . Helens blastzone found to harbor the highest densities of Legionella, NorthColdwater and Spirit, were the only lakes receiving waters fromthermal seeps . These observations suggest that enriched therma lwaters which foster microbial communities with hig hconcentrations of biomass may act as source of Legionella wher ethese bacteria may proliferate during favorable seasona lconditions in non-thermal waters such as Spirit and Nort hColdwater lakes .9Legionella species were observed in rivers and lakes in th eMt . St . Helens blast zone in concentrations similar to thosefound in other aquatic habitats outside of the blast zone excep tat hydrothermal seeps in the South Coldwater Creek drainage ,North Coldwater Lake and Spirit Lake . The concentrations ofLegionella in those warmer waters were consistently one to twoorders of magnitude higher than those found elsewhere . Virulentstrains of L . pneumophila were isolated during both season ssampled; however, illnesses among workers exposed to aquati chabitats in the Mt . St . Helens blast zone were not confirmed a slegionnellosis. The high concentrations of Legionella in water sin the blast zone (10 7 cells/L) are diluted significantly asthese waters flow to populated areas, as shown by the low number sfound in the North Toutle River (10 4 cells/L) outside of theblast zone .10REFERENCES1. Baross, J .A., C .N . Dahm, A .R . Ward, M .D . ',they, andJ . R . Sedell . 1982 . Initial microbiological respons ein lakes to the Mt . St . Helens eruption. Nature .296 : 49-52 .2. Brock, T .D. 1978. Thermophilic Microorganisms an dLife at High Temperatures . Springer-Verlag, New York .3. Cherry, W .B ., B . Pittman, P .P . Harris, G .A . Hebert ,B .M . Thomason, L . Thacker, and R .E . Weaver . 1978 .Detection of Legionnaires' disease bacteria by direc timmunofluorescent staining . J. Clin . Microbiol . 8 :329-338 .4. Fliermans, C .B ., W .B . Cherry, L .H . Orrison, S .J . Smith ,D .L . Tison, and D .H . Pope . 1981 Ecological distributionof Legionella pneumophila . Appl . Environm . Microbiol .41 : 9-16 .5. McDade, J .E . 1979 . Primary isolation using guinea pig sand embyonated eggs . In : G .L . Jones and G .A . Hebert, eds ."Legionnaires" the disease, the bacterium and methodology .U .S . Dept . of Health, Education and Welfare, Centers forDisease Control, Atlanta, Ga .6. Orrison, L .H ., W .B . Cherry, C .B . Fliermans, S .B . Dees ,L .K . McDougal and D .J . Dodd . 1981. Characteristicsof environmental isolates of Leqionella pneumophila .Appl . Environm . Microbial . 42 : 109-115 .7. Staley, J .T ., L .G . Lehmicke, F .E . Palmer, R .W . Peet, andR .C . Wissmar . 1982 . Impact of Mt . St . Helens eruptionon bacteriology of lakes in the blast zone . Appl .Environm . Microbial . 43 : 664-670 .8. Tison, D .L ., D .H . Pope, W .B . Cherry and C .B . Fliermans .1980 . Growth of Leqionella pneumophila in associationwith blue-green algae (cyanobacteria) . Appl .. Environm .Microbial . 39 : 456-459 .9. Wissman, R .C ., A .H . Devol, J .T . Staley, and J .R . Sedell .1982 . Biological responses of lakes within the MountSt . Helens blast zone . Science . 216 : 175-181 ."1I

Ecology and virulence of Legionnaire's disease bacteria in aquatic habitats near Mt. St. Helens

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Ecology and virulence of Legionnaire's disease bacteria in aquatic habitats near Mt. St. Helens

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