parameters and the density ofA. hydrophila in Albemarle Sound and in Lake. Norman and Badin Lake, North Carolina. Since the ..... Lake Gaston. 14.3 (315).
Microb Ecol (1983) 9:137-153
MICROBIAL CCOLOGY @ 1983 Springer-Verlag
A Model for the Density of Aeromonas hydrophila in Albemarle Sound, North Carolina Terry C. Hazen Department of Biology,Facultyof Natural Sciences, Universityof Puerto Rico, Rio Piedras, Puerto Rico 00931
Abstract. The abundance ofAeromonas hydrophila was measured monthly at 29 sites in Albemarle Sound, North Carolina and its tributaries from April 1977 through July 1979. Simultaneous measurements included heterotrophic plate count bacteria, fecal coliform bacteria, and 18 physical and chemical parameters. Using only 6 water quality parameters, multiple correlation and regression analysis of the data produced a best-fit regression which explained 38% of the variation observed in A. hydrophila density. The 6 water quality parameters included dissolved oxygen, temperature, orthophosphate, chlorophyll A trichromatic, total Kjeldahl nitrogen, and ammonia. Heterotrophic plate count bacteria and fecal coliform densities were highly correlated with A. hydrophila density, but made the model very unstable. The model was successfully tested against similar data collected for 2 other North Carolina reservoirs, Lake Norman and Badin Lake. Data from 10 sites in Badin Lake over 18 months and from 7 sites on Lake Norman over 5 months were not significantly different from the Albemarle Sound model. Conditions of water quality that may give rise to "blooms" of A. hydrophila will simultaneously contribute to the probability of increased epizootics in fish in the southeastern United States.
Introduction Aeromonas hydrophila is a ubiquitous facultative pathogen. It has been reported throughout the United States in all but the most extreme habitats [20, 21]. Indeed, it has been isolated in high numbers from pristine alpine lakes [21], Louisiana bayous [21], and the aphotic zones of the Atlantic Ocean (1,000 m isolation 5 miles southeast of Puerto Rico; T. C. Hazen, unpublished observations). A wide range of poikilothermic and homeothermic animals, including man, can be infected by A. hydrophila [6, 7, 10, 15, 23, 24, 30, 33, 34]. In the southeastern United States, A. hydrophila causes extensive losses to commercial and sport fisheries as the etiological agent for red-sore disease [22]. In one documented case, 37, 500 fish were killed over a single 13-day period in one North Carolina reservoir, Badin Lake [25]. During the fall of 1976, approximately 95% of the white perch (Roccus americanus) population was killed by
138
Terry C. Hazen
r e d - s o r e d i s e a s e in A l b e m a r l e S o u n d , N o r t h C a r o l i n a ; d u r i n g t h i s e p i z o o t i c , a p p r o x i m a t e l y 5 0 % o f t h e c o m m e r c i a l fish c a t c h f o r A l b e r m a r l e S o u n d w a s discarded because of unsightly surface lesions. In view of the serious implications for the commercial and sport fishing industries in the southeastern U.S., and with inadequate information available r e g a r d i n g t h e e c o l o g y o f A. hydrophila i n N o r t h C a r o l i n a , a s t u d y w a s u n d e r taken to comprehensively examine the correlation of selected water quality p a r a m e t e r s a n d t h e d e n s i t y o f A . hydrophila i n A l b e m a r l e S o u n d a n d i n L a k e Norman and Badin Lake, North Carolina. Since the relationship between dens i t y o f A . hydrophila a n d p r e v a l e n c e o f r e d - s o r e d i s e a s e w i t h i n l a r g e m o u t h b a s s h a d b e e n p r e v i o u s l y s h o w n t o b e so s t r o n g [8], it w a s b e l i e v e d t h a t t h e p r e s e n t a p p r o a c h w o u l d b e u s e f u l in i d e n t i f y i n g t h o s e w a t e r q u a l i t y p a r a m e t e r s t h a t may increase the probability of red-sore epizootics.
Materials and M e t h o d s
Study Site The primary area of study was Albemarle Sound (76~ 36~ located in the northeast comer of North Carolina (Fig. 1). Albemarle Sound is a natural estuary with a mean depth of 3 m, a maximum depth of 20 m, and a shoreline of 600 kin. The total watershed covers 45, 695 kmL Albemarle Sound has 2 major tributaries, accounting for 83% of the total watershed: the Roanoke River (25, 123 km:) and the Chowan River (12, 872 km2). The nearest connection to the Atlantic Ocean is Oregon Inlet near Roanoke Island, site 23. The annual mean tidal range at Oregon Inlet is 0.6 m whereas tides in Albemarle Sound are less than 0.3 m. The characteristic diurnal cycle of tides is approximately 24.8 hours. Average annual rainfall in the area is 114 cm. River flow into Albemarle Sound is greatest during the winter (400 m J s -z) and lowest during the summer (>30 m 3 s-~). At times, flow can even reverse briefly during the summer [32]. In the lower Chowan River, flushing times range from more than 50 days during the summer to less than 10 days during the winter [32]. The entire basin supports a rural economy of 500,000 (estimated from the 1970 census). In 1972, commercial fishing was estimated to be producing $5 million annually [2, 4].
Sampling Water samples were collected using a 2 liter vertical lucite Kemmerer sampling bottle (Wildlife Supply Co., Saginaw, MI). The bottle was washed with 70% ethanol after each sample was taken. Each water sample was placed in a sterile 180 ml whirl-pak bag (NASCO, Ft. Wilkinson, WI) and kept on ice for transport to the lab; the time from collection site to the lab never exceeded 1 hour. Abundance and distribution ofA. hydrophilawere measured monthly. Three samples were taken at the surface and at 1 m intervals in vertical profile at each station (Fig. 1).
Bacteriological Methods Aeromonas hydrophiladensity
was estimated by viable cell count using Rimler-Shotts (R-S) medium [31]. All density estimates were made 4 times on the same sample. A specific volume of sample was filtered through a sterile, gridded, 47 mm membrane filter with a pore diameter of 0.45 ~m (Millipore Corp., Bedford, MA). The filter was then placed on R-S medium and incubated at 35~ for 20-24 hours. Following incubation, yellow colonies were counted with the aid of a
Model for A. hydrophila Density
139
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magnifying lens; each colony was assumed to represent one colony forming unit (CFU). Periodically, colonies were isolated from membrane filters and confirmed as A. hydrophila using API-20E (Analytab Products, Plainview, NY), oxidase tests, the vibriostatic agent 0/129, and A. hydrophila specific, fluorescent antibody. All techniques are as previously described by Fliermans and Hazen [12], Hazen [17] and Hazen et al. [21]. Fecal coliform estimates were obtained from 4 aliquots from each sample. A specific volume of sample water was filtered through a sterile, gridded, 47 mm membrane filter with a pore diameter o f 0.7 tzm (Millipore Corp., Bedford, MA). The filter was then placed on m-FC medium (Difco, Detroit, MI) and incubated at 44.5~ for 24 hours. Following incubation, blue colonies were counted with the aid of a magnifying lens according to APHA standard methods [1]. Heterotrophic plate count bacteria were also estimated from 4 aliquots of each sample. A specific volume of sample water was filtered through a sterile, gridded, 47 m m membrane filter with a pore diameter of 0.45 um (Millipore Corp., Bedford, MA). The filter was then placed on TGE medium (Difco, Detroit, MI) and incubated at 35~ for 24 hours. Following incubation, all colonies were counted with the aid of a magnifying lens, according to APHA standard methods [1].
Water Quality Five water quality parameters were measured simultaneously with A. hydrophila density. Dissolved oxygen, pH, conductivity, temperature, and redox potential were monitored using a Hydrolab surveyor Model 5901 (Hydrolab Corp., Austin, TX). APHA standard methods were followed for all in situ measurements. Four liters of water were collected, divided into various bottles, and small amounts of the following preservatives added: nitric acid, sulfuric acid, zinc acetate, and mercuric chloride. All samples were then placed on ice for transport to the laboratory. The ap-
140
Terry C. Hazen
propriately preserved samples were analyzed for the following parameters: ammonia, total Kjeldahl nitrogen, nitrates, nitrites (after APHA [1] except that samples were dialyzed instead of filtered); sulfates (turbidimetric method); orthophosphates, total phosphorus (ascorbic acid method and samples were dialyzed); mercury, total organic carbon (using an Oceanographic International Corporation Model 524B equipped with a Lira 303 IR detector); sulfides (methylene blue method), chlorophyll A trichromatic, chlorophyll A corrected, and pheophytin A. APHA [ 1] standard methods were used for all determinations; for more details see Esch and Hazen [9].
Data Analysis A Hewlett-Packard, 3000 series, or an IBM 370-148 computer, was used for all statistical analyses. Some data were analyzed using IDA (Interactive Data Analysis, University of Chicago), and modifications of programs by Davies [5]. Factorial analysis of variance was used to test for differences between sites and seasons. Multiple correlations were used to determine relationships of A. hydrophiladensities with water quality parameters against densities ofA. hydrophila. Parameters were then removed in a stepwise manner until all the remaining parameters showed t statistics that indicated they significantly affected the density ofA. hydrophila. Some data (bacteria counts, orthophosphates, total phosphorus, nitrate, nitrite, total organic carbon, ammonia, and chlorophyll A) were found to be heteroscedastic by determining skewness and kurtosis against a normal probability plot. Heteroscedasticity was reduced by transforming each of these measurements with Log(x + 1) or (x + 0.1) or (x + 0.01), prior to analysis [35]. Any statistical probability - l 0 s C F U m l 1); t h e s p r i n g a n d s u m m e r m o n t h s w e r e q u i t e v a r i a b l e (102-105 CFU m l 2). T h e b r a c k i s h w a t e r sites h a d m o d e r a t e
8.0 18,000 11.7 7.9 440 68 20 16 10 890 0.1 0.02 0.4 0.05 0.02 0.05 5 ND 0.9 0.19 320
Temperature a Conductivity b D i s s o l v e d oxygen ~ pH R e d o x potential d Turbidity" C A TI CAC/ PAI Sulfate ~ Sulfide c Ammonia c TKN c NO3 + NO2 ~ Phosphates~ Total p h o s p h o r u s c TOC c Mercury c AH~ FCg' HPCs
6.5 5,000 12.6 7.8 450 68 28 2| 14 220 0.1 0.05 0.7 0.02 0.02 0.06 17 ND 2.8 0 1,000
2 6.2 530 12.7 7.7 415 68 29 16 25 44 0.1 0.02 0.4 0.09 0.02 0.06 14 ND 0 0.005 160
3 6.4 3,520 12.9 7.5 430 61 23 16 16 160 0.1 0.02 0.5 0.12 0.05 0.06 23 ND , 0.43 0.026 58
7
40 0.75 3,300
ND
5.2 118 10.0 6.2 450 92 14 5 14 34 0.1 0.12 0.8 0.75 0.02 0.13 38
9 4.3 1, 162 10.3 6.5 455 68 13 5 17 33 0.1 0.22 0.8 0.95 0.02 0.10 34 ND 60 1.3 2, 400
12 8.0 1,200 12.9 6.0 420 134 15 5 21 38 0.1 0.12 0.7 1.50 0.05 0.15 28 ND 250 3.9 6,200
13 4.5 110 12.9 7.6 387 68 19 5 22 25 0.1 0.02 0.4 0.27 0.02 0.10 13 ND 8 0.2 630
15
4.0 120 11.2 7.1 300 92 13 5 22 39 0.1 0.13 0.5 0.33 0.06 0.13 14 ND 4 0.45 1,400
20
a = ~ h = u m h o cm-~; c = m g liter-~; a = m y ; e = Jackson turbidity u n i t s ; I = ug liter-~; g = C F U m l - ' ; C A T = chlorophyll A trichromatic; C A C = chlorophyll A corrected; PA = p h e o p h y t i n A; T K N = total Kjeldahl nitrogen; T O C = total organic carbon; A H = A. hydrophila; F C = fecal coliforms; H P C = heterotrophic plate count; N D = n o t d e t e r m i n e d
1
W a t e r quality in A l b e m a r l e S o u n d during J a n u a r y 1978 at 1 m d e p t h
Site
Table 1.
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v
O
28.0 94.0 8.1 7.6 340 36 17 28 5 480 0.1 0.02 0.6 0.02 0.02 0.02 5 0.2 1.5 0.06 52
Temperature a Conductivity b Dissolved oxygen c pH R e d o x potential a Turbidity"
30.5 660 10.1 8.9 340 47 45 41 5 2 0.1 0.02 1.0 0.02 0.02 0.02 10 0.5 11 0.05 240
2
See Table 1 for n o t a t i o n s a n d abbreviations
Sulfate c Sulfide c Ammonia c TKN c NO3 + NOa c Phosphates~ Total p h o s p h o r u s c TOC ~ Mercury f AHg FO HPO
PA r
CAC r
CAT r
1 30.0 1, 150 9.2 7.5 415 20 15 5 13 56 0.1 0.02 0.2 0.02 0.02 0.02 5 0.2 3.7 0.03 76
3 29.5 1,000 8.4 7.0 450 4 5 5 5 53 0.1 0.2 0.2 0.16 0.02 0.02 13 0.2 1.4 0.91 180
7
W a t e r quality in A l b e m a r l e S o u n d during A u g u s t 1978 at 1 m d e p t h
Site
Table 2.
30.0 190 4.5 6.0 515 26 20 13 12 11 0.1 0.2 0.4 0.08 0.02 0.06 5 0.2 6 1.0 430
9 29.0 2,000 7.5 6.6 475 26 5 5 5 13 0.1 0.29 1.3 0.31 0.05 0.24 37 0.2 2 1.0 370
12 32.0 145 7.8 6.6 490 12 43 27 29 13 0.1 0.02 1.1 0.15 0.02 0.09 24 0.2 4.1 0.65 390
13 29.0 80 8.6 7.0 500 36 18 14 5 ND 0.1 0.02 0.4 0.02 0.02 0.02 5 0.2 78 0.05 1,700
15
30.0 85 9.5 7.9 370 88 83 80 5 12 0.1 0.02 0.8 0.02 0.02 0.06 5 0.2 6 0.4 430
20
28.0 15,000 7.3 7.8 300 96 14 11 5 680 0.1 0.13 0.5 0.02 0.02 0.02 5 1.0 1,000 0.10 50,000
Temperature" Conductivityb Dissolved oxygenc pH Redox potential d Turbidity r CAT f CAC f PAf Sulfate ~ Sulfide c A m m o n i ar TKN c NO~ + NO2 c Phosphates~ Total phosphorusc TOC ~ Mercury f AH~ FC g HPCTM
27.5 3,660 7.1 7.8 300 94 18 16 5 150 0.1 0.02 0.5 0.02 0.02 0.02 12 0.2 1,000 ND 10,000
2
See Table 1 for notations and abbreviations
1 28.0 10,000 7.1 7.2 335 94 13 5 5 430 0.1 0.02 0.4 0.02 0.02 0.02 5 1.5 700 1.40 20,000
3
Water quality in Albemarle Sound during July 1979
Site
Table 3.
28.0 170 6.9 6.7 340 97 5 5 5 73 0.1 0.02 0.4 0.11 0.02 0.02 16 0.2 ND 0.10 30,000
7 30.0 200 4.5 5.8 340 81 21 14 12 11 0.1 0.5 1.8 0.20 0.12 0.33 82 0.6 30 1.0 5,000
9 30.0 200 8.9 6.8 300 92 44 34 16 14 0.1 0.02 0.7 0.15 0.02 0.13 26 0.2 24.3 0.20 1,900
12 26.5 210 3.7 6.0 210 99 5 5 5 13 0.1 0. ! 2 0.7 0.19 0.02 0.09 25 0.2 ND 200 100,000
13 28.0 100 7.7 6.8 350 97 16 10 12 9 0.1 0.02 0.4 0.11 0.02 0.06 12 0.2 100 0.03 15,000
15
27.5 80 8.0 7.8 290 92 59 56 5 8 0.1 0.02 0.8 0.02 0.02 0.08 14 0.9 10,000 4.83 5,000,000
20
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Fig. 2. Density ofA. hydrophilaby site; mean + 1 standard error, a = brackish sites, b = intensive agriculture runoff, c = nitrogen fertilizer factory, d = pulp mill. Fig. 3. Density ofA. hydrophila by month; mean _ 1 standard error.
densities o f heterotrophic plate count bacteria (10 4 C F U m1-1); site 29 had significantly lower densities ( < 5 X 103 C F U ml 1) than all other sites. Fecal coliform densities were not significantly different between sites; however, differences by season were significant ( F = 21.67; d f = 8 & 104; P < 0.0001). Densities o f fecal coliforms at the brackish sites were low ( < 1 C F U m1-1) whereas those sites receiving effluent were noticeably higher. Indeed, densities at site 30 (receiving pulp mill effluent) were above r e c o m m e n d e d limits [3] at all times (>10 z C F U ml-~). Aeromonas hydrophila densities (Fig. 2 and 3) were significantly different by season ( F = 5.87; d f - - 8 & 104; P < 0.01) and by site ( F = 6.60; d f = 20 & 104; P < 0.0001). Densities o f A. hydrophila were highest during the spring and fall. T h e brackish water sites had significantly lower densities than freshwater sites, whereas site 27 and site 30 had significantly higher densities o f A. hydrophila than at other stations. The A. hydrophila densities at site 29 were significantly lower (Fig. 2) than adjacent sites.
Correlation and Regression of A. h y d r o p h i l a Densities with Water Quality T h e multiple correlation half-matrix (Table 4) shows significant positive correlations between densities o f A. hydrophila and site, m o n t h , temperature, chlorophyll A trichromatic, chlorophyll A corrected, total Kjeldahl nitrogen, orthophosphate, total phosphorus, total organic carbon, heterotrophic plate count bacteria, and fecal coliform bacteria densities. Significant negative correlations were observed between densities o f A. hydrophila and dissolved oxygen, p H and a m m o n i a concentrations. T h e other parameters were not significantly correlated with A. hydrophila density. T h e best-fit regression o f the first year o f data using dissolved oxygen, temperature, orthophosphates, chlorophyll A trichromatic, total Kjeldahl nitrogen,
84
1.000 .120 .338 -.118 .016 -.037 .097 .197 .000 -.025 -.011 -.211 -.009 -.094 -.022 .050 -.113 -.074 .109.
1.000 .359 .135 -.036 -.070 .048 ,008 .081 -.494 -.415 ,045 -.217 .481 -.267 -.017 -.310 -.402
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pH
1.000 -.388 .206 .180 .117 .261 -.006 -.040 -.057 -.370 -.065 .025 -.031 -.009 -.099 -.191
See Table 1 for notations and abbreviations
1.000 .571 1,000 -.041 - , 0 4 4 -.455 - . 6 1 8 - . 0 5 7 -.055 .118 - . 0 4 2 -.056 .152 .098 .235 -.474 -.274 .040 .012 -.097 -.099 .061 ,115 .082 .106 -.037 -.231 .141 -,089 .027 .027 -.045 .068 -.131 -,301 .194 ,135 .213 .142 .05 when r >
Site M o n t h T e m p Cond
Correlation half-matrix
Site 1.000 Month .068 Temp .032 Cond -.406 DO -.276 pH -.264 Redox -.113 CAT .040 CAC .053 PA .051 SO4 -,027 SOs -.017 NH 3 .182 TKN .194 NO3,2 -.058 PO4 .106 TP .250 TOC .093 HPC .099 FC .353 AH .307 N = 322, P
