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Source of Vibrio spp. found in the hemolymph of the blue crab, Callinectes sapidus
JOURNAL
OF INVERTEBRATE
PATHOLOGY
46,
109-
110
(1985)
NOTE Source
of Vibrio spp. Found in the Hemolymph Callinectes sapidus
Hemolymph of the blue crab, Callinectes supidus, has been reported to contain bacteria in both the healthy (H. S. Tubiash, R. K. Sizemore, and R. R. Colwell, Appl. Microbial. 29, 388-392, 1975) and the diseased state (P. T. Johnson, J. Znvertebr. Pathol. 28, 25-32, 1976). We have found that the predominate bacterial types in crab hemolymph are members of the genus Vibrio, including species that are pathogenic to crabs and humans (J. W. Davis and R. K. Sizemore, Appl. Environ. Microbial. 43, 1092-1097, 1982; R. K. Sizemore, R. R. Colwell, H. S. Tubiash, and T. E. Lovelace, Appl. Microbial. 29, 393-399, 1975). Vibrio spp. are also the predominate bacterial type in the abundant crab gut flora, and we hypothesized that this gut flora may be the source of the bacteria found in the hemolymph (J. W. Davis and R. K. Sizemore, lot. cit., 1982). Because of the obvious public health problems associated with the ingestion of poorly cooked Vibriocontaining crabs and our continuing interest in the bacteriology of blue crabs, we undertook a study to determine the source of the bacteria found in crab hemolymph. During the summer of 1981, an intensive study of the bacterial flora of blue crabs from Vermilion Bay, Louisiana, was undertaken. As part of that study, 10 blue crabs were individually captured in crab traps set on a pier attached to the LUMCON’s Fearman Bayou Marine Laboratory. These crabs were examined within 2 hr of their entrance into the traps and within 10 min of the traps removal from the water. Four parts of each crab, hemolymph, gills, lower carapace, and fecal material removed from the lower digestive tract, were sampled.
of the Blue Crab,
From each site the total viable bacterial counts (using a marine salts-proteose peptone-yeast extract agar as described by G. R. Cipriani, R. S. Wheeler, and R. K. Sizemore, J. Znvertebr. Pathol., 36, 255263, 1980) and the percentage of Vibrio spp. (i.e., anaerobic growth on TCBS agar) were determined. Twelve presumptive Vibrio isolates from each site of each crab were subjected to a battery of biochemical tests which are used to identify Vibrio species. Included in the tests were sucrose, lactose, mannitol, and salicin fermentation; starch hydrolysis; citrate utilization; H,S production; arginine dihydrolase activity; and growth on 0 and 10% NaCl media. From these data, we determined certain parameters of the total bacterial flora of the crabs, and we were able to assign each of the 428 strains examined into one of 124 biotypes. Table 1 shows a summary of the bacteriological data obtained. Bacterial counts were highest in the fecal material and lowest in the hemolymph. The fecal material also had a larger percentage of Vibrio spp. and salt-requiring bacteria and fewer biotypes than other sites. Indeed, 45% of the strains isolated from the digestive tract were placed in three biotypes, and 41% of the biotypes occurred as singletons, i.e., only one strain of that biotype was isolated. In the other sites, biotype clusters were smaller, i.e., the largest clusters were less than 9% of the total strains, and contained more singletons, e.g., carapace, 58%: hemolymph 63%; gills 69%. To determine the source of the hemolymph isolates, the biotypes of strains from different sites on each crab were compared for matches. There were 18 matches (rep109 0022-2011185
$1.50
Copyright cl 1985 by Academic Press, Inc. All rights of reproduction in any form reserved
110
NOTE TABLE SUMMARY
I
OF BACTERIOLOGICAL
DATA
Sampling Site Hemolymph Average viable countsL’ Range of counts Percentage Vibrio Percentage salt requiring No. strains tested No. biotypes
5.1 x IO’ 2.0-13
x 10’
Carapace
Gills
Feces
4.5 x IO’
2.6 x IO6 0.1-86.0 x IO” 42 41 88 52
I.8 x 10s 0.3- II.0 x IOX
O.I-10.0
43 49 112
57
60 46 I13 62
x IO’
96 94
II5 32
a Counts were from 7 days growth at 25°C on marine agar plates. Hemolymph, gill, and feces counts were per miliiliter of material. Carapace counts were the number of bacteria removed by a sterile swab from t cm? of carapace surface.
resenting 30 isolates) between hemolymph isolates and isolates from the carapace, I1 matches (19 isolates) between gill and hemolymph strains, and 9 matches (12 isolates) between the digestive tract isolates and the hemolymph isolates. In this study, only 38% of the hemolymph isolates matched biotypes with an isolate from a different site on the same crab. A community similarity index [C = 2wln + b (loo)], where a = sum of the biotypes incidence at one site, b = sum of the biotypes incidence at the second site. and M’ = sum of the lower score for each biotype. (R. L. Smith, “Ecology and Field Biology,” Harper and Row, New York, 1980), was also calculated for each combination on sites on each crab. The means of these index numbers for the 10 crabs were hemolymph versus carapace, 19%; gills versus carapace, 17%; gills versus hemolymph, 12%; hemolymph versus digestive tract, 10%; carapace versus digestive tract, 7%; and gills versus digestive tract, 5%. In conclusion, we found the blue crab to contain a large number of different types of
Vibrio spp. In the fecal material, the Vibrio spp. are the normal flora and a few Vibrio biotypes predominated. These biotypes did not appear to be the common source of the hemolymph flora as had been previously hypothesized. Instead, in the crabs used in this study the most likely source of the diverse hemolymph flora appears to be the exterior of the crab. Frequent invasions of the hemolymph are likely to occur during molting, or as a result of an injury and perhaps from other unknown mechanisms. KEY WORDS: Vibrio spp.; Blue crab, Callinectes supidus; hemolymph infection; bacteremia source. RONALD
K. SIZEMORE’ JOHN
W.
DA&
Louisha Universities Marine Consortium Cocodrie. Louisiana 70344 Received September 26, 1984: accepted Jtwuary 2. I 985
’ Present address: Department of Biological Sciences, University of North Carolina at Wilmington. Wilmington, N.C. 28403-3297. ’ Present address: Department of Microbiology. University of Tennessee. Knoxville. Term. 37916.
OF INVERTEBRATE
PATHOLOGY
46,
109-
110
(1985)
NOTE Source
of Vibrio spp. Found in the Hemolymph Callinectes sapidus
Hemolymph of the blue crab, Callinectes supidus, has been reported to contain bacteria in both the healthy (H. S. Tubiash, R. K. Sizemore, and R. R. Colwell, Appl. Microbial. 29, 388-392, 1975) and the diseased state (P. T. Johnson, J. Znvertebr. Pathol. 28, 25-32, 1976). We have found that the predominate bacterial types in crab hemolymph are members of the genus Vibrio, including species that are pathogenic to crabs and humans (J. W. Davis and R. K. Sizemore, Appl. Environ. Microbial. 43, 1092-1097, 1982; R. K. Sizemore, R. R. Colwell, H. S. Tubiash, and T. E. Lovelace, Appl. Microbial. 29, 393-399, 1975). Vibrio spp. are also the predominate bacterial type in the abundant crab gut flora, and we hypothesized that this gut flora may be the source of the bacteria found in the hemolymph (J. W. Davis and R. K. Sizemore, lot. cit., 1982). Because of the obvious public health problems associated with the ingestion of poorly cooked Vibriocontaining crabs and our continuing interest in the bacteriology of blue crabs, we undertook a study to determine the source of the bacteria found in crab hemolymph. During the summer of 1981, an intensive study of the bacterial flora of blue crabs from Vermilion Bay, Louisiana, was undertaken. As part of that study, 10 blue crabs were individually captured in crab traps set on a pier attached to the LUMCON’s Fearman Bayou Marine Laboratory. These crabs were examined within 2 hr of their entrance into the traps and within 10 min of the traps removal from the water. Four parts of each crab, hemolymph, gills, lower carapace, and fecal material removed from the lower digestive tract, were sampled.
of the Blue Crab,
From each site the total viable bacterial counts (using a marine salts-proteose peptone-yeast extract agar as described by G. R. Cipriani, R. S. Wheeler, and R. K. Sizemore, J. Znvertebr. Pathol., 36, 255263, 1980) and the percentage of Vibrio spp. (i.e., anaerobic growth on TCBS agar) were determined. Twelve presumptive Vibrio isolates from each site of each crab were subjected to a battery of biochemical tests which are used to identify Vibrio species. Included in the tests were sucrose, lactose, mannitol, and salicin fermentation; starch hydrolysis; citrate utilization; H,S production; arginine dihydrolase activity; and growth on 0 and 10% NaCl media. From these data, we determined certain parameters of the total bacterial flora of the crabs, and we were able to assign each of the 428 strains examined into one of 124 biotypes. Table 1 shows a summary of the bacteriological data obtained. Bacterial counts were highest in the fecal material and lowest in the hemolymph. The fecal material also had a larger percentage of Vibrio spp. and salt-requiring bacteria and fewer biotypes than other sites. Indeed, 45% of the strains isolated from the digestive tract were placed in three biotypes, and 41% of the biotypes occurred as singletons, i.e., only one strain of that biotype was isolated. In the other sites, biotype clusters were smaller, i.e., the largest clusters were less than 9% of the total strains, and contained more singletons, e.g., carapace, 58%: hemolymph 63%; gills 69%. To determine the source of the hemolymph isolates, the biotypes of strains from different sites on each crab were compared for matches. There were 18 matches (rep109 0022-2011185
$1.50
Copyright cl 1985 by Academic Press, Inc. All rights of reproduction in any form reserved
110
NOTE TABLE SUMMARY
I
OF BACTERIOLOGICAL
DATA
Sampling Site Hemolymph Average viable countsL’ Range of counts Percentage Vibrio Percentage salt requiring No. strains tested No. biotypes
5.1 x IO’ 2.0-13
x 10’
Carapace
Gills
Feces
4.5 x IO’
2.6 x IO6 0.1-86.0 x IO” 42 41 88 52
I.8 x 10s 0.3- II.0 x IOX
O.I-10.0
43 49 112
57
60 46 I13 62
x IO’
96 94
II5 32
a Counts were from 7 days growth at 25°C on marine agar plates. Hemolymph, gill, and feces counts were per miliiliter of material. Carapace counts were the number of bacteria removed by a sterile swab from t cm? of carapace surface.
resenting 30 isolates) between hemolymph isolates and isolates from the carapace, I1 matches (19 isolates) between gill and hemolymph strains, and 9 matches (12 isolates) between the digestive tract isolates and the hemolymph isolates. In this study, only 38% of the hemolymph isolates matched biotypes with an isolate from a different site on the same crab. A community similarity index [C = 2wln + b (loo)], where a = sum of the biotypes incidence at one site, b = sum of the biotypes incidence at the second site. and M’ = sum of the lower score for each biotype. (R. L. Smith, “Ecology and Field Biology,” Harper and Row, New York, 1980), was also calculated for each combination on sites on each crab. The means of these index numbers for the 10 crabs were hemolymph versus carapace, 19%; gills versus carapace, 17%; gills versus hemolymph, 12%; hemolymph versus digestive tract, 10%; carapace versus digestive tract, 7%; and gills versus digestive tract, 5%. In conclusion, we found the blue crab to contain a large number of different types of
Vibrio spp. In the fecal material, the Vibrio spp. are the normal flora and a few Vibrio biotypes predominated. These biotypes did not appear to be the common source of the hemolymph flora as had been previously hypothesized. Instead, in the crabs used in this study the most likely source of the diverse hemolymph flora appears to be the exterior of the crab. Frequent invasions of the hemolymph are likely to occur during molting, or as a result of an injury and perhaps from other unknown mechanisms. KEY WORDS: Vibrio spp.; Blue crab, Callinectes supidus; hemolymph infection; bacteremia source. RONALD
K. SIZEMORE’ JOHN
W.
DA&
Louisha Universities Marine Consortium Cocodrie. Louisiana 70344 Received September 26, 1984: accepted Jtwuary 2. I 985
’ Present address: Department of Biological Sciences, University of North Carolina at Wilmington. Wilmington, N.C. 28403-3297. ’ Present address: Department of Microbiology. University of Tennessee. Knoxville. Term. 37916.