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Studies on gaffkemia, a bacterial disease of the American lobster, Homarus americanus (Milne-Edwards)
JOURNAL
Studies
OF
INVERTEBRATE
on
PATHOLOGY
Gaffkemia,
?,
(1965)
391-397
a Bacterial
Homarus
Disease
americanus
Biology Laboratory, School of Hygiene
the
(M&e-Edwards)
HARVEY Marine Pathobiology,
of
American
Lobster,
’
RABIN
Woods Hole, Massachusetts, and Department of and Public Health, The Johns Hopkins University. Baltimore, Maryland
Accepted
February
23, 1965
A study was made of the local distribution of Gaflkya homavi Hitchner and Snieszko and of its host-parasite relationship with the American lobster, Homarus americanzts (Milne-Edwards). Of the lobsters from the Marine Biological Laboratory supply, 11.832% were found infected and one out of ten lobsters caught off Woods Hole was infected with a Gaflkya-like organism. No isolations were made from lobsters dragged from the edge of the continental shelf nor from lobsters trapped off Martha’s Vineyard. G. homari, when inoculated into healthy lobsters, established bacteremia or septicemia on the first or second day after inoculation and induced mortality a few days later. Uninfected lobsters or those pretreated with Vibrio endotoxin did not acquire the disease on contact with the exception of one uninfected contact lobster which developed a slight, temporary bacteremia but survived. Twenty out of 21 lobster sera when tested in vitro stimulated the growth of G. homari while only one out of 21 sera stimulated the growth of Vibrio and seven actually inhibited it. Prior inoculation of heat-killed G. homari did not alter the course of the infection. Such pretreatment also had no influence on the in vitro activity of the serum. INTRODUCTION
Gaffkemia is a term applied to a fatal bacterial disease of the American lobster, Homarus americanus (Milne-Edwards) (Stewart and MacDonald, 1962). This diseaseis caused by the micrococcus Gafikya homari Hitchner and Snieszko (seeSnieszkoand Taylor, 1947). This condition was brought to our attention during the summerof 1963 in Woods Hole by workers who were studying enzymatic activity in lobster amebocytes when they noticed typical organisms on stained smears of lobster hemolymph. Little has been reported in the general literature about the distribution of this infec1 This investigation was supported Health Service Research Grant No. from National Cancer Institute.
or about the host-parasite relationships involved in the actual disease.This study was undertaken to gain information in these areas.
tion
by Public CA01230-15 391
MATERIALS
AND
METHODS
Lobsters The Iobsters
used for experiments
were all
1 lb. to 1 lb. 4 ozs. in weight. They were obtained through the Marine Biological Laboratory (M.B.L.), Woods Hole, supply department, or from a dealer on Martha’s Vineyard. Cultures on ZoBell’s agar were made of the hemolymph of all lobsters on coming into the laboratory. Only lobsters free from infection with Gajkya were used in the experiments. They were kept in a running sea water tank at about 18°C. Their diet consisted of one daily feeding of either of the clams Mya are-
392
RABIN
naria (Linnaeus) or Meycenaria mercenaria (Linnaeus). As obtained, these lobsters had one or both claws pegged (to prevent opening) with either wooden or plastic pegs. These pegswere left in place. Lobster serum was prepared from hemolymph by holding the clotted hemolymph at 4°C for 1 to 2 hours, rimming the clot, centrifuging, and drawing off the clear fluid with a needle and syringe. It was possible to obtain 40-5Orjr of the volume of hemolymph as serum. The hemolymph was obtained by aspiration from the hemocoelwith a needle and syringe. Bacteria
The strain of Gafkya homari usedwas one isolated by us from a diseasedlobster in the summer of 1963. The strain of the Vibrio species (unidentified) was one carried for several years in this laboratory and was originally isolated from a horseshoecrab (Bang, 1956). Both organismswere passagedon ZoBell’s sea-water agar (ZoBell, 1946). For the in vitro experiments, typical colonies were picked from the agar and grown in ZoBell’s broth. All cultures were routinely incubated at room temperature, 20-22”C, except for the in vitro tests with lobster serum where incubation was at 11°C. G. homari neither increased nor decreased in numbers at 11°C overnight in ZoBell’s broth, whereas the Vibrie increased four- to sixfold. The diluent used throughout was ZoBell’s broth. Heat-inactivation of G. homari was carried out by holding a 4%hour broth culture at 6070°C for 40 minutes. Endotoxin was prepared by boiling a 48hour culture of the Vibrin for 20 minutes, followed by centrifugation. The supernatant was used as the endotoxin. In vitro
tests
on growth
The procedure for measuring either stimulation or inhibition of growth has been described previously (Rabin and Bang, 196%).
It has reference to the amount of growth from a mixture of bacteria suspendedin broth plus serum to that from bacteria in broth alone. It is statistically significant to the 1 percent level, as used. RESULTS
Survey
for Gaffkya-like
of Lobsters
0Tganism.s Hemolymph was aseptically drawn and 0.05 ml was plated on ZoBell’s agar. The growth of small, white, convex colonies in 48 hours on incubation at room temperature which, on staining, showed micrococci with a tendency to grow in tetrads was taken as evidence of Gaflkya-like organisms.Two of the isolates from the M.B.L. supply group of lobsters were shown to produce typical Gaffkemia when inoculated into healthy, Gaflkyafree lobsters. Data from the survey are summarized in Table 1. Lobsters from several sourceswere tested. Those from the M.B.L. supply department were obtained by the M.B.L. from a local lobster dealer in Woods Hole. The dealer purchased lobsters locally and also from areas in New England well away from Woods Hole. These lobsters were held toTABLE ISOLATION
LOBSTER
Source
1
OF Gu~~~Yu-LIKE
of lobsters
ORGANISMS
FROM
HEMOLYMPH
tested
Number Number
Biological Laboratory supply, Woods Hole Summer 1963 Summer 1964
positivea tested
Marine
Lobsterman, Summer
Woods 1963
Lobster Hatchery, Vineyard Summer 1963
7/22 7/59
Hole l/10 Martha’s O/23
Lobster Dealer, Martha’s Vineyard Summer 1963 Summer 1964 ‘h Positive
cultures
on ZoBell’s
o/15 o/12 sea-water
agar.
GAFFKEMIA
OF
gether in running sea water tanks until sold. Lobsters were frequently added to and removed from this population. In the summer of 1963, 32% of the lobsters tested were positive for Gaffkya-like organisms. The intensity of the bacteremias in these lobsters ranged from 50 to 1000 organisms per 0.05 ml of hemolymph. In the summer of 1964, 11.870 were positive with the intensities of the bacteremias ranging from 1 to 1000 organisms per 0.05 ml of hemolymph. The lobsters from the lobsterman were animals caught in the Woods Hole area in traps. These animals were not held in impoundments and were tested soon after coming ashore. From one out of 10 of these lobsters one Gaffkya-like colony was isolated. The lobster which gave this positive culture was tested 5 and again 7 days later but no further isolations were made and the lobster survived. One other lobster from this same group was negative on the day it was brought into the laboratory and again 5 days later. However, four typical colonies were isolated from 0.05 ml of its hemolymph 2 days after it had received an inoculation of viable T/‘ibrio. The lobsters from the State Lobster Hatchery on Martha’s Vineyard were mostly eggbearing lobsters and had been caught at the edge of the continental shelf by draggers. Fifteen of these were tested on arrival at the hatchery and the other eight had been in the hatchery at least 1 month prior to testing. All 23 were negative. The dealer on Martha’s Vineyard caught his own lobsters locally in traps. He kept and sold only those lobsters which he himself had caught. No positive isolations were made from these lobsters. Experimental Infections with G. homari
THE
LOBSTER
393
These were included to test its possible role as a co-factor in diseaseproduction. This idea was based on the fact that endotoxin is known to be the major factor in the production of a diseasein another marine invertebrate (Bang, 1956) and also that as the ocean contains large numbers of gram-negative bacteria, and indeed, 48.2?, of lobsters from all sources in our series had nonGaflkya bacteremias on coming into the laboratory, it probably is available under natural circumstances. Four groups of three lobsters each were used in this experiment. Two groups were inoculated in the hemocoel with 0.2 ml of Vibrio endotoxin. Ten and one-half hours later one of these groups was also inoculated in the hemocoel with 5 X lo5 G. homari in 0.2 ml of broth. A third group was given only G. homari in the samedose and at the same time as the group previously given endotoxin. The fourth group received no treatment. All 12 animals were housed together. Table 2 shows the results of this experiment. G. homari grew in essentially the same fashion and produced mortality at about the same time in both groups into which it had been inoculated, showing that prior inoculation of endotoxin did not enhance the infection. The group receiving endotoxin alone did not becomeinfected with G. homari although one lobster died with a non-Gaflkya bacteremia. One of the lobsters in the untreated group showed a slight infection with a Gaffkya-like organism but survived. This lobster was tested again 22 days after infection and was negative for Gaflkya. With this exception, no conversion to Gaffkemia occurred in any of the contacts.
of Lobsters
The first experiment was performed to obtain information on the establishment of bacteremia or septicemia and induction of mortality by G. homari. In doing this experiment we included tests for the effects of endotoxin.
The Effect of Lobster Serum on the Growth of G. homari in vitro
The experiment above indicates that lobsters are generally unable to overcome infection with G. homari. Our next experiments were designed to determine if this failure to
3.2 x 104 1 x
6 x 103
6 X 10' 1.2 x 102
2.4 X 10'
0 0
0
10
11
12
4 x 10"
2.7 x 103
-
0
0
0
0
3.2
0
0
0
x
1.7 x 104
103
2.4 x 102
104
0
0
Titer
AND
0
WITH
2
homari
1
Gaflkya
9
BY
7 8
Number of individual lobster
OF LOBSTERS
TABLE PRETREATMENT
2 OF THE
x
104
x 104
-
>4
0
0
0
8 X 101
0
104
2.5 x loi
-
-
-
0
0
0
6
0
x
2.2 x 104 >4 x 104
>4
>4
0
0
0
4
of G. komeri in hemolymph (viable on days post-inoculation
WITHOUT
>4
cells/ml)
LOBSTERS
Vibrio
of this organism
-
-
-
-
x
0
0
>4
-
-
-
-
13
ENDOTOXIN
-F
-
-
x 104
-
-
0
0
10
BY
4 x 101
Q 0.2 ml. of Vibrio endotoxin inoculated into the hemocoel. b This lobster became bacteremic with an unidentified bacterium on day 4 and died with a pure, confluent culture c 5 x 10s G. homari in 0.2 ml of ZoBell’s broth inoculated into the hemocoel. d 0.2 ml of endotoxin was inoculated into the hemococl followed 10% hours later by 5 x 10” G. Izor$zari. c Negative for G. honzari at 22 days post-inoculation.
treatment
NO
+ G. homari __-.-~
Endotoxind
G. homa+ alone
alone
Endotoxina
Inoculation group
INFECTION
on day
104
7
4
7
13
4
3
4
7”
Survived
Survived Survived
~-
Survived
Survived
Day of death postinoculation
GAFFKEMIA
OF
inhibit the growth of G. homari would occur in vitro with lobster serum. As a control in these experiments we tested the effects of sera on the growth of the Vibrio which presumably is nonpathogenic for the lobster. Individual lobster sera were tested in parallel against both Vibrio and G. Homari by mixing equal volumes of serum and bacterial suspensionin broth, incubating at 11“C overnight, and plating in triplicate. The growth in the serum plus bacteria mixture was compared to that from bacteria diluted in an equal volume of broth. The results of two experiments are shown in Table 3. Eleven out of the 12 sera gave an increased count of G. homari, whereas none of the lobster sera inTABLE THE
EFFECT
OF Gaffkya Experiment number
SERUM
homari
AND
Number of individual serum Broth
1
3
OF LOBSTER
ON THE
Vibrio
GROWTH
in vitro
Plate count” Vibriob
G. honzaric
19
+-
4.35
1 2 3
18.3 20.6 13
k -c i
4.32 4.47 3.6
62.3 21.6 19.0
-c k k
7.89 4.58 4.3s
4 5 6
18 11.3
k & -
4.24 3.32
49 39.6 26
& & I
7 6.29 5.09
Broth 7
111.6 70.6
k 10.55 & 8.4
15.6 61.6
+IL
3.82 7.84
8 9 10
14.3 105.6 21.3
-c 3.78 2 10.25 -I 4.61
26.6 120.6 36.3
&
5.1s
11 12 13
110.3 56.3 98.3
I k k
a Average of three platings b Experiment 1: counts stimulation. counts inhibition. Experiment 2: counts stimulation. counts inhibition. C Experiment 1: counts stimulation. Experiment 2: counts stimulation.
>
10.5 7.5 9.91
8.3 k
58.6 40
2.87
k 19.98 -c 6.02 & k
on ZoBell’s agar. 30.5 were taken
7.65 6.32
as
<
7.5
were
taken
as
>
144.6
were
taken
as
<
78.6
were
taken
as
>
11.4
were
taken
as
>
27.2
were
taken
as
THE
395
LOBSTER
creasedthe growth of the Vibrio and four actually inhibited it. In all, none out of 21 sera inhibited G. homari while 20 out of 21 stimulated its growth. In contrast, seven out of 21 sera inhibited the Vibrio while only one out of 21 stimulated its growth. Efects of Prior Inoculation Heat-Killed G. homari
of
It is known that prior inoculation of certain materials can raise the levels of inhibitory substancesin the body fluids of many invertebrates (Bang, 1962; Stephens, 1963; Wagner, 1961). Three groups of three lobsters each were used in this experiment. One group was given 0.2 ml of heat-killed G. homari each, one group the same amount of ZoBell’s broth, treated in the same way, and one group was not treated. One day later, all the lobsters were challenged with 6.2 X lo5 organisms each. There was no apparent difference in the course of the infection among the groups. All lobsters were bacteremic or septicemic from the first day on and two out of three in each group were dead by the 4th day after challenge. The samepretreatment procedure was followed with three more groups of three lobsters each. On the day after treatment all lobsters were bled and the sera tested for activity against the Vibrio and G. homari. All nine sera stimulated the growth of G. horn& while one serum from each group inhibited Vibrio. All the rest had no effect on the growth of the vibrio with the exception of one serum from the untreated group which stimulated its growth. DISCIJSSION
Infection of lobsters with G. homayi is one of the few known naturally occurring bacterial infections known in marine invertebrates. In addition, the organism is gram-positive, which is unusual among marine bacteria. The commercial value of lobsters makes study of the condition a practical undertaking as well.
396
RABIN
Koch’s postulates were first fulfilled in the case of this disease by Snieszko and Taylor (1947). They reported a remarkably high mortality following inoculation with G. ho~nari which we found as well. The bacteria with which they worked were isolated from lobsters caught off the coast of Maine. Canadian workers (Stewart and MacDonald, 1962) showed that 40-60s of lobsters taken from three lobster fishing areas off the east coast of Canada were infected. It would be interesting to know just how wide the distribution of this organism really is. Stewart and MacDonald (1962) point out that their data indicate that this infection is not limited to holding impoundments, as our isolation from the recently trapped lobster would also indicate. It would seem important to test the organisms isolated from lobsters directly from fishing areas for virulence, as virulence may be a development of the conditions of the holding impoundment. In connection with the distribution of the organism, tests carried out in the summer of 1965 on lobsters caught in the Woods Hole area showed 2 out of 21 positive for G. homari. In relationship to virulence, recent experiments on dosage responseusing our strain of G. homari have shown that lobsters ars susceptible to doses as low as 9 >(: lo” organismswhich is the smallestinoculum tested so far.’ The in vitro studies suggest that some factor in lobster serum may stimulate the growth of G. homari. Further work on this point may lead to an understanding of the pathogenesis of this condition. We did one experiment to determine if Vibrio could produce diseasein lobsters. Six lobsters were inoculated with Vibrio and kept together with others inoculated at the same time with G. homari. We failed to make any positive isolations of Vibrio from any of these 12 lobsters. On the 3rd day after the original 2 Last proof.
two
sentences
of this
paragraph
added
in
inoculation, two of the Vibrio-inoculated group yielded G. homari, and all six died by the 10th day after infection. Cultures were obtained from four of these lobsters on the day of death and all four were pos:tive for G. homari. The lobsters inoculated with G. homari all had positive cultures for G. hommi on the first day after infection and all died by the 9th day after infection. Cultures were obtained from five of these lobsters on the day of death and all five were positive for G. homari. The failure to transmit diseaseto untreated or endotoxin-treated animals in contact with Gaffkemic ones suggeststhat careful examination should be made not only of the mechanics of transmission but also of the physiological and bacteriological states of the lobster when exposed, especially if it can be established that transmission regularly occurs in such experiments as describedabove in the animals treated with Vibrio. In connection with the endotoxin treatment, it may be that the effect of endotoxin is temporary and that by the time we exposed the animals to G, homari they were essentially normal again. Estimates of the amebocyte counts in the hemocoel showed a three- to fivefold drop in the number of cells 15 minutes after inoculation of endotoxin. Inoculation of ZoBell’s broth or just repeated bleeding did not produce this effect. We attempted to determine the effect of G. homari when inoculated soon after the injection of endotoxin. However, the lobsters did not survive this treatment, as six out of seven died within I day after being inoculated with G. homari 12 minutes after injection of endotoxin. It is interesting to note that there are no obvious external signs of disease even in heavily septicemic lobsters until shortly before death when they lie quietly with their chelae extended. It is also interesting that 33F8 of the lobster sera inhibited the growth of Vibrio, This
GAFFKEMIA
OF THE
adds to the number of invertebrate bloods which have been found to have antimicrobial activity. The failure to alter the course of infection or raise the antimicrobial activity of serum by prior inoculation of heat-killed G. homari by no means rules out the possibility that some form of acquired immunity does occur in lobsters, as in many other invertebrates. ACKNOWLEDGMENTS I should like to thank Mr. John Hughes of the State Lobster Hatchery, Vineyard Haven, Massachusetts, The Massachusetts Division of Natural Resources, and Dr. F. B. Bang, for their encouragement, advice, and assistance during the course of this study. REFERENCES BANG, F. B. 1956. A bacterial polyphemus. Bull. Johns 325-351.
disease Hopkins
of Limulus Hosp., 66,
LOBSTER
397
BANG, F. B. 1962. Serological aspects of immunity in invertebrates. Nature 195, 88-89. RABIN, H., and BANG. F. B. 1964. In vitro studies of the antibacterial activity of Golfing&z gouldii (PourtalCs) coelomic fluid. J. Insect Pathol. 6, 457-465. SNIESZKO, S. F., and TAYLOR, C. C. 1947. A bacterial disease of the lobster (Homarus americanus). Science 105, 500. STEPHENS, J. M. 1963. Immunity in Insects. Ziz “Insect Pathology, An Advanced Treatise.” (E. A. Steinhaus, ed.j, Vol. I, Chap. 9, pp. 273-296. Academic Press, New York. STEWART, J. E., and MACDONALD, J. F. 1962. “A Report to the Fishing Industry Regarding Lobster Disease (Gaffkaemie) Fisheries Research Board of Canada, New Series Circular No. 9. 2 PP. WAGNER, R. R. 1961. Acquired resistance to bacterial infection in insects. Bacteviol. Revs., 25, 100-I 10. ZOBELL, C. E. 1946. “Marine Microbiology (A Monograph on Hydrobacteriology) ,” Chap. IV, p. 46. Chronica Botanica Co., Waltham, Massachusetts.
Studies
OF
INVERTEBRATE
on
PATHOLOGY
Gaffkemia,
?,
(1965)
391-397
a Bacterial
Homarus
Disease
americanus
Biology Laboratory, School of Hygiene
the
(M&e-Edwards)
HARVEY Marine Pathobiology,
of
American
Lobster,
’
RABIN
Woods Hole, Massachusetts, and Department of and Public Health, The Johns Hopkins University. Baltimore, Maryland
Accepted
February
23, 1965
A study was made of the local distribution of Gaflkya homavi Hitchner and Snieszko and of its host-parasite relationship with the American lobster, Homarus americanzts (Milne-Edwards). Of the lobsters from the Marine Biological Laboratory supply, 11.832% were found infected and one out of ten lobsters caught off Woods Hole was infected with a Gaflkya-like organism. No isolations were made from lobsters dragged from the edge of the continental shelf nor from lobsters trapped off Martha’s Vineyard. G. homari, when inoculated into healthy lobsters, established bacteremia or septicemia on the first or second day after inoculation and induced mortality a few days later. Uninfected lobsters or those pretreated with Vibrio endotoxin did not acquire the disease on contact with the exception of one uninfected contact lobster which developed a slight, temporary bacteremia but survived. Twenty out of 21 lobster sera when tested in vitro stimulated the growth of G. homari while only one out of 21 sera stimulated the growth of Vibrio and seven actually inhibited it. Prior inoculation of heat-killed G. homari did not alter the course of the infection. Such pretreatment also had no influence on the in vitro activity of the serum. INTRODUCTION
Gaffkemia is a term applied to a fatal bacterial disease of the American lobster, Homarus americanus (Milne-Edwards) (Stewart and MacDonald, 1962). This diseaseis caused by the micrococcus Gafikya homari Hitchner and Snieszko (seeSnieszkoand Taylor, 1947). This condition was brought to our attention during the summerof 1963 in Woods Hole by workers who were studying enzymatic activity in lobster amebocytes when they noticed typical organisms on stained smears of lobster hemolymph. Little has been reported in the general literature about the distribution of this infec1 This investigation was supported Health Service Research Grant No. from National Cancer Institute.
or about the host-parasite relationships involved in the actual disease.This study was undertaken to gain information in these areas.
tion
by Public CA01230-15 391
MATERIALS
AND
METHODS
Lobsters The Iobsters
used for experiments
were all
1 lb. to 1 lb. 4 ozs. in weight. They were obtained through the Marine Biological Laboratory (M.B.L.), Woods Hole, supply department, or from a dealer on Martha’s Vineyard. Cultures on ZoBell’s agar were made of the hemolymph of all lobsters on coming into the laboratory. Only lobsters free from infection with Gajkya were used in the experiments. They were kept in a running sea water tank at about 18°C. Their diet consisted of one daily feeding of either of the clams Mya are-
392
RABIN
naria (Linnaeus) or Meycenaria mercenaria (Linnaeus). As obtained, these lobsters had one or both claws pegged (to prevent opening) with either wooden or plastic pegs. These pegswere left in place. Lobster serum was prepared from hemolymph by holding the clotted hemolymph at 4°C for 1 to 2 hours, rimming the clot, centrifuging, and drawing off the clear fluid with a needle and syringe. It was possible to obtain 40-5Orjr of the volume of hemolymph as serum. The hemolymph was obtained by aspiration from the hemocoelwith a needle and syringe. Bacteria
The strain of Gafkya homari usedwas one isolated by us from a diseasedlobster in the summer of 1963. The strain of the Vibrio species (unidentified) was one carried for several years in this laboratory and was originally isolated from a horseshoecrab (Bang, 1956). Both organismswere passagedon ZoBell’s sea-water agar (ZoBell, 1946). For the in vitro experiments, typical colonies were picked from the agar and grown in ZoBell’s broth. All cultures were routinely incubated at room temperature, 20-22”C, except for the in vitro tests with lobster serum where incubation was at 11°C. G. homari neither increased nor decreased in numbers at 11°C overnight in ZoBell’s broth, whereas the Vibrie increased four- to sixfold. The diluent used throughout was ZoBell’s broth. Heat-inactivation of G. homari was carried out by holding a 4%hour broth culture at 6070°C for 40 minutes. Endotoxin was prepared by boiling a 48hour culture of the Vibrin for 20 minutes, followed by centrifugation. The supernatant was used as the endotoxin. In vitro
tests
on growth
The procedure for measuring either stimulation or inhibition of growth has been described previously (Rabin and Bang, 196%).
It has reference to the amount of growth from a mixture of bacteria suspendedin broth plus serum to that from bacteria in broth alone. It is statistically significant to the 1 percent level, as used. RESULTS
Survey
for Gaffkya-like
of Lobsters
0Tganism.s Hemolymph was aseptically drawn and 0.05 ml was plated on ZoBell’s agar. The growth of small, white, convex colonies in 48 hours on incubation at room temperature which, on staining, showed micrococci with a tendency to grow in tetrads was taken as evidence of Gaflkya-like organisms.Two of the isolates from the M.B.L. supply group of lobsters were shown to produce typical Gaffkemia when inoculated into healthy, Gaflkyafree lobsters. Data from the survey are summarized in Table 1. Lobsters from several sourceswere tested. Those from the M.B.L. supply department were obtained by the M.B.L. from a local lobster dealer in Woods Hole. The dealer purchased lobsters locally and also from areas in New England well away from Woods Hole. These lobsters were held toTABLE ISOLATION
LOBSTER
Source
1
OF Gu~~~Yu-LIKE
of lobsters
ORGANISMS
FROM
HEMOLYMPH
tested
Number Number
Biological Laboratory supply, Woods Hole Summer 1963 Summer 1964
positivea tested
Marine
Lobsterman, Summer
Woods 1963
Lobster Hatchery, Vineyard Summer 1963
7/22 7/59
Hole l/10 Martha’s O/23
Lobster Dealer, Martha’s Vineyard Summer 1963 Summer 1964 ‘h Positive
cultures
on ZoBell’s
o/15 o/12 sea-water
agar.
GAFFKEMIA
OF
gether in running sea water tanks until sold. Lobsters were frequently added to and removed from this population. In the summer of 1963, 32% of the lobsters tested were positive for Gaffkya-like organisms. The intensity of the bacteremias in these lobsters ranged from 50 to 1000 organisms per 0.05 ml of hemolymph. In the summer of 1964, 11.870 were positive with the intensities of the bacteremias ranging from 1 to 1000 organisms per 0.05 ml of hemolymph. The lobsters from the lobsterman were animals caught in the Woods Hole area in traps. These animals were not held in impoundments and were tested soon after coming ashore. From one out of 10 of these lobsters one Gaffkya-like colony was isolated. The lobster which gave this positive culture was tested 5 and again 7 days later but no further isolations were made and the lobster survived. One other lobster from this same group was negative on the day it was brought into the laboratory and again 5 days later. However, four typical colonies were isolated from 0.05 ml of its hemolymph 2 days after it had received an inoculation of viable T/‘ibrio. The lobsters from the State Lobster Hatchery on Martha’s Vineyard were mostly eggbearing lobsters and had been caught at the edge of the continental shelf by draggers. Fifteen of these were tested on arrival at the hatchery and the other eight had been in the hatchery at least 1 month prior to testing. All 23 were negative. The dealer on Martha’s Vineyard caught his own lobsters locally in traps. He kept and sold only those lobsters which he himself had caught. No positive isolations were made from these lobsters. Experimental Infections with G. homari
THE
LOBSTER
393
These were included to test its possible role as a co-factor in diseaseproduction. This idea was based on the fact that endotoxin is known to be the major factor in the production of a diseasein another marine invertebrate (Bang, 1956) and also that as the ocean contains large numbers of gram-negative bacteria, and indeed, 48.2?, of lobsters from all sources in our series had nonGaflkya bacteremias on coming into the laboratory, it probably is available under natural circumstances. Four groups of three lobsters each were used in this experiment. Two groups were inoculated in the hemocoel with 0.2 ml of Vibrio endotoxin. Ten and one-half hours later one of these groups was also inoculated in the hemocoel with 5 X lo5 G. homari in 0.2 ml of broth. A third group was given only G. homari in the samedose and at the same time as the group previously given endotoxin. The fourth group received no treatment. All 12 animals were housed together. Table 2 shows the results of this experiment. G. homari grew in essentially the same fashion and produced mortality at about the same time in both groups into which it had been inoculated, showing that prior inoculation of endotoxin did not enhance the infection. The group receiving endotoxin alone did not becomeinfected with G. homari although one lobster died with a non-Gaflkya bacteremia. One of the lobsters in the untreated group showed a slight infection with a Gaffkya-like organism but survived. This lobster was tested again 22 days after infection and was negative for Gaflkya. With this exception, no conversion to Gaffkemia occurred in any of the contacts.
of Lobsters
The first experiment was performed to obtain information on the establishment of bacteremia or septicemia and induction of mortality by G. homari. In doing this experiment we included tests for the effects of endotoxin.
The Effect of Lobster Serum on the Growth of G. homari in vitro
The experiment above indicates that lobsters are generally unable to overcome infection with G. homari. Our next experiments were designed to determine if this failure to
3.2 x 104 1 x
6 x 103
6 X 10' 1.2 x 102
2.4 X 10'
0 0
0
10
11
12
4 x 10"
2.7 x 103
-
0
0
0
0
3.2
0
0
0
x
1.7 x 104
103
2.4 x 102
104
0
0
Titer
AND
0
WITH
2
homari
1
Gaflkya
9
BY
7 8
Number of individual lobster
OF LOBSTERS
TABLE PRETREATMENT
2 OF THE
x
104
x 104
-
>4
0
0
0
8 X 101
0
104
2.5 x loi
-
-
-
0
0
0
6
0
x
2.2 x 104 >4 x 104
>4
>4
0
0
0
4
of G. komeri in hemolymph (viable on days post-inoculation
WITHOUT
>4
cells/ml)
LOBSTERS
Vibrio
of this organism
-
-
-
-
x
0
0
>4
-
-
-
-
13
ENDOTOXIN
-F
-
-
x 104
-
-
0
0
10
BY
4 x 101
Q 0.2 ml. of Vibrio endotoxin inoculated into the hemocoel. b This lobster became bacteremic with an unidentified bacterium on day 4 and died with a pure, confluent culture c 5 x 10s G. homari in 0.2 ml of ZoBell’s broth inoculated into the hemocoel. d 0.2 ml of endotoxin was inoculated into the hemococl followed 10% hours later by 5 x 10” G. Izor$zari. c Negative for G. honzari at 22 days post-inoculation.
treatment
NO
+ G. homari __-.-~
Endotoxind
G. homa+ alone
alone
Endotoxina
Inoculation group
INFECTION
on day
104
7
4
7
13
4
3
4
7”
Survived
Survived Survived
~-
Survived
Survived
Day of death postinoculation
GAFFKEMIA
OF
inhibit the growth of G. homari would occur in vitro with lobster serum. As a control in these experiments we tested the effects of sera on the growth of the Vibrio which presumably is nonpathogenic for the lobster. Individual lobster sera were tested in parallel against both Vibrio and G. Homari by mixing equal volumes of serum and bacterial suspensionin broth, incubating at 11“C overnight, and plating in triplicate. The growth in the serum plus bacteria mixture was compared to that from bacteria diluted in an equal volume of broth. The results of two experiments are shown in Table 3. Eleven out of the 12 sera gave an increased count of G. homari, whereas none of the lobster sera inTABLE THE
EFFECT
OF Gaffkya Experiment number
SERUM
homari
AND
Number of individual serum Broth
1
3
OF LOBSTER
ON THE
Vibrio
GROWTH
in vitro
Plate count” Vibriob
G. honzaric
19
+-
4.35
1 2 3
18.3 20.6 13
k -c i
4.32 4.47 3.6
62.3 21.6 19.0
-c k k
7.89 4.58 4.3s
4 5 6
18 11.3
k & -
4.24 3.32
49 39.6 26
& & I
7 6.29 5.09
Broth 7
111.6 70.6
k 10.55 & 8.4
15.6 61.6
+IL
3.82 7.84
8 9 10
14.3 105.6 21.3
-c 3.78 2 10.25 -I 4.61
26.6 120.6 36.3
&
5.1s
11 12 13
110.3 56.3 98.3
I k k
a Average of three platings b Experiment 1: counts stimulation. counts inhibition. Experiment 2: counts stimulation. counts inhibition. C Experiment 1: counts stimulation. Experiment 2: counts stimulation.
>
10.5 7.5 9.91
8.3 k
58.6 40
2.87
k 19.98 -c 6.02 & k
on ZoBell’s agar. 30.5 were taken
7.65 6.32
as
<
7.5
were
taken
as
>
144.6
were
taken
as
<
78.6
were
taken
as
>
11.4
were
taken
as
>
27.2
were
taken
as
THE
395
LOBSTER
creasedthe growth of the Vibrio and four actually inhibited it. In all, none out of 21 sera inhibited G. homari while 20 out of 21 stimulated its growth. In contrast, seven out of 21 sera inhibited the Vibrio while only one out of 21 stimulated its growth. Efects of Prior Inoculation Heat-Killed G. homari
of
It is known that prior inoculation of certain materials can raise the levels of inhibitory substancesin the body fluids of many invertebrates (Bang, 1962; Stephens, 1963; Wagner, 1961). Three groups of three lobsters each were used in this experiment. One group was given 0.2 ml of heat-killed G. homari each, one group the same amount of ZoBell’s broth, treated in the same way, and one group was not treated. One day later, all the lobsters were challenged with 6.2 X lo5 organisms each. There was no apparent difference in the course of the infection among the groups. All lobsters were bacteremic or septicemic from the first day on and two out of three in each group were dead by the 4th day after challenge. The samepretreatment procedure was followed with three more groups of three lobsters each. On the day after treatment all lobsters were bled and the sera tested for activity against the Vibrio and G. homari. All nine sera stimulated the growth of G. horn& while one serum from each group inhibited Vibrio. All the rest had no effect on the growth of the vibrio with the exception of one serum from the untreated group which stimulated its growth. DISCIJSSION
Infection of lobsters with G. homayi is one of the few known naturally occurring bacterial infections known in marine invertebrates. In addition, the organism is gram-positive, which is unusual among marine bacteria. The commercial value of lobsters makes study of the condition a practical undertaking as well.
396
RABIN
Koch’s postulates were first fulfilled in the case of this disease by Snieszko and Taylor (1947). They reported a remarkably high mortality following inoculation with G. ho~nari which we found as well. The bacteria with which they worked were isolated from lobsters caught off the coast of Maine. Canadian workers (Stewart and MacDonald, 1962) showed that 40-60s of lobsters taken from three lobster fishing areas off the east coast of Canada were infected. It would be interesting to know just how wide the distribution of this organism really is. Stewart and MacDonald (1962) point out that their data indicate that this infection is not limited to holding impoundments, as our isolation from the recently trapped lobster would also indicate. It would seem important to test the organisms isolated from lobsters directly from fishing areas for virulence, as virulence may be a development of the conditions of the holding impoundment. In connection with the distribution of the organism, tests carried out in the summer of 1965 on lobsters caught in the Woods Hole area showed 2 out of 21 positive for G. homari. In relationship to virulence, recent experiments on dosage responseusing our strain of G. homari have shown that lobsters ars susceptible to doses as low as 9 >(: lo” organismswhich is the smallestinoculum tested so far.’ The in vitro studies suggest that some factor in lobster serum may stimulate the growth of G. homari. Further work on this point may lead to an understanding of the pathogenesis of this condition. We did one experiment to determine if Vibrio could produce diseasein lobsters. Six lobsters were inoculated with Vibrio and kept together with others inoculated at the same time with G. homari. We failed to make any positive isolations of Vibrio from any of these 12 lobsters. On the 3rd day after the original 2 Last proof.
two
sentences
of this
paragraph
added
in
inoculation, two of the Vibrio-inoculated group yielded G. homari, and all six died by the 10th day after infection. Cultures were obtained from four of these lobsters on the day of death and all four were pos:tive for G. homari. The lobsters inoculated with G. homari all had positive cultures for G. hommi on the first day after infection and all died by the 9th day after infection. Cultures were obtained from five of these lobsters on the day of death and all five were positive for G. homari. The failure to transmit diseaseto untreated or endotoxin-treated animals in contact with Gaffkemic ones suggeststhat careful examination should be made not only of the mechanics of transmission but also of the physiological and bacteriological states of the lobster when exposed, especially if it can be established that transmission regularly occurs in such experiments as describedabove in the animals treated with Vibrio. In connection with the endotoxin treatment, it may be that the effect of endotoxin is temporary and that by the time we exposed the animals to G, homari they were essentially normal again. Estimates of the amebocyte counts in the hemocoel showed a three- to fivefold drop in the number of cells 15 minutes after inoculation of endotoxin. Inoculation of ZoBell’s broth or just repeated bleeding did not produce this effect. We attempted to determine the effect of G. homari when inoculated soon after the injection of endotoxin. However, the lobsters did not survive this treatment, as six out of seven died within I day after being inoculated with G. homari 12 minutes after injection of endotoxin. It is interesting to note that there are no obvious external signs of disease even in heavily septicemic lobsters until shortly before death when they lie quietly with their chelae extended. It is also interesting that 33F8 of the lobster sera inhibited the growth of Vibrio, This
GAFFKEMIA
OF THE
adds to the number of invertebrate bloods which have been found to have antimicrobial activity. The failure to alter the course of infection or raise the antimicrobial activity of serum by prior inoculation of heat-killed G. homari by no means rules out the possibility that some form of acquired immunity does occur in lobsters, as in many other invertebrates. ACKNOWLEDGMENTS I should like to thank Mr. John Hughes of the State Lobster Hatchery, Vineyard Haven, Massachusetts, The Massachusetts Division of Natural Resources, and Dr. F. B. Bang, for their encouragement, advice, and assistance during the course of this study. REFERENCES BANG, F. B. 1956. A bacterial polyphemus. Bull. Johns 325-351.
disease Hopkins
of Limulus Hosp., 66,
LOBSTER
397
BANG, F. B. 1962. Serological aspects of immunity in invertebrates. Nature 195, 88-89. RABIN, H., and BANG. F. B. 1964. In vitro studies of the antibacterial activity of Golfing&z gouldii (PourtalCs) coelomic fluid. J. Insect Pathol. 6, 457-465. SNIESZKO, S. F., and TAYLOR, C. C. 1947. A bacterial disease of the lobster (Homarus americanus). Science 105, 500. STEPHENS, J. M. 1963. Immunity in Insects. Ziz “Insect Pathology, An Advanced Treatise.” (E. A. Steinhaus, ed.j, Vol. I, Chap. 9, pp. 273-296. Academic Press, New York. STEWART, J. E., and MACDONALD, J. F. 1962. “A Report to the Fishing Industry Regarding Lobster Disease (Gaffkaemie) Fisheries Research Board of Canada, New Series Circular No. 9. 2 PP. WAGNER, R. R. 1961. Acquired resistance to bacterial infection in insects. Bacteviol. Revs., 25, 100-I 10. ZOBELL, C. E. 1946. “Marine Microbiology (A Monograph on Hydrobacteriology) ,” Chap. IV, p. 46. Chronica Botanica Co., Waltham, Massachusetts.