- Email: [email protected]
A crystalliferous bacterium associated with a “blister disease” in the earthworm, Eisenia foetida (Savigny)
JOURiX-AL
OF INVERTEBRATE
PATHOLOGY
A Crystalliferous Disease”
in
8, 295-298 (1966)
Bacterium the
Associated
Earthworm,
Eisenia
A. M. Entomology
Research
with foetida
(Savigny)
HEIMPEL
Division, Agricultural Research Service, Agriculture, BeltsviUe, Maryland Accepted
a “Blister
January
6,
11. S. Department
of
1966
Several specimens of the earthworm Biscnia foetida (Savigny) afilicted with a “blister disease” were found to contain crystalliferous bacteria in all the lesions. Two strains of bacteria were isolated, identified, and typed according to their H-antigen and their vegetative cell esterases. Both strains appeared to be Bacillus thuringiensis var. thuringiensis Berliner. The association of this bacterium with the lesions suggests that it may cause this serious condition. METHODS
Upon review of the literature on the pathology of earthworms it became obvious that much remains to be accomplished. Only one reference about the effect of crystalliferous bacteria on earthworms was published, in 1961 by Smirnoff and Heimpel. They reported that large dosesof Bacillus thuringiensis Berliner invaded the body cavity of the earthworm and causedan extensive septicemia and eventual death. In 1962, a culture of Eisenia foe&a (Savigny) afflicted with an obvious pathological condition was received at Beltsvil1e.l The infected earthworms developed blisters filled with yellow cloudy material on various random locations on the body wall. Shortly after the blister formed, it broke open and caused an ulcer which eventually weakened the worm body at that point, and the afflicted animal broke in pieces and eventually died. This diseasedcondition caused serious mortality in worm farms in Tennesseeand most of the southeastern United States.
by .4cademic
Press
MATERIALS
Technique oj Diagnosis A sterile operating site was set up, and earthworms with blisters were submerged in a 5% solution of sodium hypochlorite for 3 minutes and were washed in three changes of sterile water. The externally sterilized worms were then transferred to a sterile site, and biopsies of the blisters were cut and smeared on blood-agar plates. Each blistered segment was then excised, fixed in BouinDubosque-Brazil fixative, dehydrated in a butanol series (Smith, 1943)) and imbedded in paraffin. Sections of the blister (6 p) were cut and mounted on slides,and these preparations were stained with Hertig and Wolbach’s stain for rickettsiae in tissue (Hertig and Wolbach. 1924). Isolation
and Identification
of
Bacteria
Bacterial colonies found growing on the blood-agar plates were picked and transferred to nutrient-broth tubes after slides stained with nigrosin were made. The broth cultures were plated on nutrient-agar plates after 18 hours: these plates were grown at 32°C for
1 From Mr. Edd Long, Long’s Worm Ranch, P. 0. Box 6, Savannah, Tennessee. @ 1966
AND
Inc.
295
HEIMPEL
4 days and were examined microscopically. Since sporeformers were obtained from all samples, the cultures were examined biochemically after the technique described by Heimpel and Angus (1958). Cultures of the isolates were submitted to Dr. A. Bonnefoi and Dr. H. DeBarjac of the Institute of Pasteur for serological typing and to Dr. John R. Norris, Shell Oil Co.” for vegetativecell esterasetyping. The results of the latter tests were confirmed in this laboratory. RESULTS
Histological Investigations All 19 blisters examined contained bacilli resembling Bacillus thuringiensis. The bacterial infection was localized in the area of the blister and was not detected in normal tissue; therefore, the disease was not considered systemic. In only one case had the bacteria sporulated. Unfortunately the stage of sporulation was not advanced sufficiently to permit us to clearly detect intracellular bodies; however, more than one body was detected i,n each cell. The spores in most cases lay obliquely in the cell, a characteristic of crystalforming bacteria (Smith et aZ., 1946), and the presence of one and often two diamond-shaped crystals was obvious to the experienced observer (see Fig. 1) . Identification of the Bacterium
Sporangium: Cy!indrical, not swollen and slightly pinched in the area of the spore; parasporal body formed in each cell. Vegetative rods: Stout, relatively short rods with round ends; average size 1.62 X 3.97 CL; usually found in short chains; motile, peritrichous flagellation ; gram-positive. Nutrient-agar colonies: Medium to large, flat, irregu!ar, with some whiplash outgrowths, dull. Transmitted light: creamy white; reflected; light brownish white. Nutrient bvoth: Body of broth clear, thick heavy pellicle; pellicle breaks into large flakes when shaken vigorously. Starch: Hydrolyzed. Gelaf.in: Liquefied. Sugar fermentation: Acid (with ammoniacal nitrogen) from glucose, maltose, sucrose, trehalose, cellobiose, and levulose after 48 hours. Action variable on dextrin; cellobiose positive after 12 days. No reaction with xylose, lactose, arabinose, mannitol, inulin, raffinose, melibiose, galactose, inositol, and dulcitol up to 22 days. Nitrates to nitrites: Positive. proVoges-Proskauer: Acetylmethylcarbinol duced. Phospholipase C.: Produced; no reddish pigment formed. Utilization of citrate: Positive light to medium growth. Serological type: I thuringiensis (called Berliner). Vegetative cell esterase type: 1 thuringiensis (called Berliner). Flytoxin production: Positive.
This organism is of relatively low virulence for the silkworm compared with Bacillus thuringiensis var. sotto Ishiwata and therefore may be designated as a strain of Bacillus thuringiensis var. thuringiensis Berliner.
Two slightly different strains (gross colony morphology) of crystalformers were isolated from the 19 cases.All the isolates resembled either one of these cultures. These two iso- Strain EW 2 lates were examined separately under the Spore: Oval, average culture numbers EWl and EW2. The de- lation takes place after scription of these strains follows. Sporangiz~m: Cylindrical, Strain En71 Spove: Oval, average nal ; usually sporulation incubation at 32°C.
size 1.35 X 1.72 p; subtermiis complete after 4%hour
2 Dr. John R. Norris, Milstead Chemical Enzymology, Broad Oak bourne, Kent, England.
Laboratory of Road, Sitting-
size 1.59 X 1.99 CL; sporu30 to 48 hours at 32°C. not swollen but slightly pinched in the vicinity of the spore; a parasporal diamond-shaped crystal and often two crystals formed in each cell along with the spore. Vegetative rods: Stout, short rods with round ends; average size 1.71 X 3.71 p; usually found in pairs or in short chains; motile, peritrichous flagellation; gram-positive. Nutrient-agav colonies: Medium, slightly raised flat colonies with irregular whiplash border; dull
APPARENT
BACTERIAL
DISEASE
OF
EARTHWORM
FIG. 1. (A) Four specimens of Eisenia f&i& (Savigny) with blisters showing on the anterior of the bodies. (B) A closeup photograph of the blister on the anterior end of the body on the third from the left. (C) Photomicrograph of sporeformer showing crystal-shaped body in the cell. to slightly shiny; transmitted light: grayish white; reflected; light brownish white. Nz&rient broth: Body of broth clear; medium to heavy pellicle that breaks into persistent flakes when shaken vigorously. Starch : Hydrolized.
297
ends worm
Gelatin: Liquefied. Sugar fermentation: Acid (with ammoniacal nitrogen (from glucose, maltose, sucrose, trehalose, cellobiose, levulose, and dextrin after 48 hours. Cellobiose positive after 12 days. No reaction with xylose, lactose, arabinose, mannitol, inulin, raffinose!
298 melibiose, days. lVitrute
HEIMPEL galactose,
inositol,
to nitrites:
dulcitol
up
to 22
Positive.
Voges-Proskauer: duced. Phospholipase formed.
and
pro-
Acetylmethylcarbinol C.:
Produced;
no
red
pigment
Utilizetion of citrate: Positive, light growth. Serological type: 1 thuringiensis (called Berliner). Vegetative cell esteruse type: I thuringiensis (called Berliner). Flytoxin production: Positive.
The EW2 strain resembles the EW 1 strain with reference to its toxicity for the silkworm; therefore it can also be designated as Bacillus thuringiensis var. thuringiensis. CONCLUSIONS
No claim is made here that the crystalforming bacteria described above are the causal agent of the “blister disease” found in populations of E. foetidu. It was not possible to carry out Koch’s postulates due to the lack of healthy animals. Nevertheless, the consistent presence of the bacterium in the necrotic lesions of the unhealthy worms and the presence of sporulating forms of the bacterium in these lesions imply an intimate association with the disease.
The earthworms in question were reared in pits on rotted cottonseed hulls that contain abrasive materials capable of lacerating the worms. The bacteria may have gained entrance to the worm’s body via such abrasions, which would account for the local site of infection. The bacteria will be held at this laboratory and will be made available to anyone wishing to extend the studies further. REFERENCES
A. M., AND ANGUS, T. A. 1958. The taxonomy of insect pathogens related to Bacillus cereus Frankland and Frankland. Can. J. Microbial., 4, 531-541. HERTIG, M., AND WOLBACH, S. B. 1924. Studies on rickettsia-like microorganisms in insects. JI. Med. Res., 44, 329-374. HEIMPEL,
SMIRNOFF,
W.
A.,
AND
HEIMPEL,
A.
M.
1961.
Notes on the pathogenicity of BaciUus thuringiensis var. tkuringiensis Berliner for the earthworm Lumbricus terrestris Linnaeus. J. Insect Pathol., 3, 403-408. SMITH, N. R., GORDON, R. E., 1946. Aerobic spore-forming
AND
CLARK,
F.
E.
bacteria. U.S. Dept. Agr. Misc. Publ. No. 559, 112 pp.; reissued 1952 as U.S. Dept. Agr. Monograph No. 16, 148 pp. SMITH, S. G. 1943. Techniques for the study of insect chromosomes. Can. Entomologist, 75, 2234.
OF INVERTEBRATE
PATHOLOGY
A Crystalliferous Disease”
in
8, 295-298 (1966)
Bacterium the
Associated
Earthworm,
Eisenia
A. M. Entomology
Research
with foetida
(Savigny)
HEIMPEL
Division, Agricultural Research Service, Agriculture, BeltsviUe, Maryland Accepted
a “Blister
January
6,
11. S. Department
of
1966
Several specimens of the earthworm Biscnia foetida (Savigny) afilicted with a “blister disease” were found to contain crystalliferous bacteria in all the lesions. Two strains of bacteria were isolated, identified, and typed according to their H-antigen and their vegetative cell esterases. Both strains appeared to be Bacillus thuringiensis var. thuringiensis Berliner. The association of this bacterium with the lesions suggests that it may cause this serious condition. METHODS
Upon review of the literature on the pathology of earthworms it became obvious that much remains to be accomplished. Only one reference about the effect of crystalliferous bacteria on earthworms was published, in 1961 by Smirnoff and Heimpel. They reported that large dosesof Bacillus thuringiensis Berliner invaded the body cavity of the earthworm and causedan extensive septicemia and eventual death. In 1962, a culture of Eisenia foe&a (Savigny) afflicted with an obvious pathological condition was received at Beltsvil1e.l The infected earthworms developed blisters filled with yellow cloudy material on various random locations on the body wall. Shortly after the blister formed, it broke open and caused an ulcer which eventually weakened the worm body at that point, and the afflicted animal broke in pieces and eventually died. This diseasedcondition caused serious mortality in worm farms in Tennesseeand most of the southeastern United States.
by .4cademic
Press
MATERIALS
Technique oj Diagnosis A sterile operating site was set up, and earthworms with blisters were submerged in a 5% solution of sodium hypochlorite for 3 minutes and were washed in three changes of sterile water. The externally sterilized worms were then transferred to a sterile site, and biopsies of the blisters were cut and smeared on blood-agar plates. Each blistered segment was then excised, fixed in BouinDubosque-Brazil fixative, dehydrated in a butanol series (Smith, 1943)) and imbedded in paraffin. Sections of the blister (6 p) were cut and mounted on slides,and these preparations were stained with Hertig and Wolbach’s stain for rickettsiae in tissue (Hertig and Wolbach. 1924). Isolation
and Identification
of
Bacteria
Bacterial colonies found growing on the blood-agar plates were picked and transferred to nutrient-broth tubes after slides stained with nigrosin were made. The broth cultures were plated on nutrient-agar plates after 18 hours: these plates were grown at 32°C for
1 From Mr. Edd Long, Long’s Worm Ranch, P. 0. Box 6, Savannah, Tennessee. @ 1966
AND
Inc.
295
HEIMPEL
4 days and were examined microscopically. Since sporeformers were obtained from all samples, the cultures were examined biochemically after the technique described by Heimpel and Angus (1958). Cultures of the isolates were submitted to Dr. A. Bonnefoi and Dr. H. DeBarjac of the Institute of Pasteur for serological typing and to Dr. John R. Norris, Shell Oil Co.” for vegetativecell esterasetyping. The results of the latter tests were confirmed in this laboratory. RESULTS
Histological Investigations All 19 blisters examined contained bacilli resembling Bacillus thuringiensis. The bacterial infection was localized in the area of the blister and was not detected in normal tissue; therefore, the disease was not considered systemic. In only one case had the bacteria sporulated. Unfortunately the stage of sporulation was not advanced sufficiently to permit us to clearly detect intracellular bodies; however, more than one body was detected i,n each cell. The spores in most cases lay obliquely in the cell, a characteristic of crystalforming bacteria (Smith et aZ., 1946), and the presence of one and often two diamond-shaped crystals was obvious to the experienced observer (see Fig. 1) . Identification of the Bacterium
Sporangium: Cy!indrical, not swollen and slightly pinched in the area of the spore; parasporal body formed in each cell. Vegetative rods: Stout, relatively short rods with round ends; average size 1.62 X 3.97 CL; usually found in short chains; motile, peritrichous flagellation ; gram-positive. Nutrient-agar colonies: Medium to large, flat, irregu!ar, with some whiplash outgrowths, dull. Transmitted light: creamy white; reflected; light brownish white. Nutrient bvoth: Body of broth clear, thick heavy pellicle; pellicle breaks into large flakes when shaken vigorously. Starch: Hydrolyzed. Gelaf.in: Liquefied. Sugar fermentation: Acid (with ammoniacal nitrogen) from glucose, maltose, sucrose, trehalose, cellobiose, and levulose after 48 hours. Action variable on dextrin; cellobiose positive after 12 days. No reaction with xylose, lactose, arabinose, mannitol, inulin, raffinose, melibiose, galactose, inositol, and dulcitol up to 22 days. Nitrates to nitrites: Positive. proVoges-Proskauer: Acetylmethylcarbinol duced. Phospholipase C.: Produced; no reddish pigment formed. Utilization of citrate: Positive light to medium growth. Serological type: I thuringiensis (called Berliner). Vegetative cell esterase type: 1 thuringiensis (called Berliner). Flytoxin production: Positive.
This organism is of relatively low virulence for the silkworm compared with Bacillus thuringiensis var. sotto Ishiwata and therefore may be designated as a strain of Bacillus thuringiensis var. thuringiensis Berliner.
Two slightly different strains (gross colony morphology) of crystalformers were isolated from the 19 cases.All the isolates resembled either one of these cultures. These two iso- Strain EW 2 lates were examined separately under the Spore: Oval, average culture numbers EWl and EW2. The de- lation takes place after scription of these strains follows. Sporangiz~m: Cylindrical, Strain En71 Spove: Oval, average nal ; usually sporulation incubation at 32°C.
size 1.35 X 1.72 p; subtermiis complete after 4%hour
2 Dr. John R. Norris, Milstead Chemical Enzymology, Broad Oak bourne, Kent, England.
Laboratory of Road, Sitting-
size 1.59 X 1.99 CL; sporu30 to 48 hours at 32°C. not swollen but slightly pinched in the vicinity of the spore; a parasporal diamond-shaped crystal and often two crystals formed in each cell along with the spore. Vegetative rods: Stout, short rods with round ends; average size 1.71 X 3.71 p; usually found in pairs or in short chains; motile, peritrichous flagellation; gram-positive. Nutrient-agav colonies: Medium, slightly raised flat colonies with irregular whiplash border; dull
APPARENT
BACTERIAL
DISEASE
OF
EARTHWORM
FIG. 1. (A) Four specimens of Eisenia f&i& (Savigny) with blisters showing on the anterior of the bodies. (B) A closeup photograph of the blister on the anterior end of the body on the third from the left. (C) Photomicrograph of sporeformer showing crystal-shaped body in the cell. to slightly shiny; transmitted light: grayish white; reflected; light brownish white. Nz&rient broth: Body of broth clear; medium to heavy pellicle that breaks into persistent flakes when shaken vigorously. Starch : Hydrolized.
297
ends worm
Gelatin: Liquefied. Sugar fermentation: Acid (with ammoniacal nitrogen (from glucose, maltose, sucrose, trehalose, cellobiose, levulose, and dextrin after 48 hours. Cellobiose positive after 12 days. No reaction with xylose, lactose, arabinose, mannitol, inulin, raffinose!
298 melibiose, days. lVitrute
HEIMPEL galactose,
inositol,
to nitrites:
dulcitol
up
to 22
Positive.
Voges-Proskauer: duced. Phospholipase formed.
and
pro-
Acetylmethylcarbinol C.:
Produced;
no
red
pigment
Utilizetion of citrate: Positive, light growth. Serological type: 1 thuringiensis (called Berliner). Vegetative cell esteruse type: I thuringiensis (called Berliner). Flytoxin production: Positive.
The EW2 strain resembles the EW 1 strain with reference to its toxicity for the silkworm; therefore it can also be designated as Bacillus thuringiensis var. thuringiensis. CONCLUSIONS
No claim is made here that the crystalforming bacteria described above are the causal agent of the “blister disease” found in populations of E. foetidu. It was not possible to carry out Koch’s postulates due to the lack of healthy animals. Nevertheless, the consistent presence of the bacterium in the necrotic lesions of the unhealthy worms and the presence of sporulating forms of the bacterium in these lesions imply an intimate association with the disease.
The earthworms in question were reared in pits on rotted cottonseed hulls that contain abrasive materials capable of lacerating the worms. The bacteria may have gained entrance to the worm’s body via such abrasions, which would account for the local site of infection. The bacteria will be held at this laboratory and will be made available to anyone wishing to extend the studies further. REFERENCES
A. M., AND ANGUS, T. A. 1958. The taxonomy of insect pathogens related to Bacillus cereus Frankland and Frankland. Can. J. Microbial., 4, 531-541. HERTIG, M., AND WOLBACH, S. B. 1924. Studies on rickettsia-like microorganisms in insects. JI. Med. Res., 44, 329-374. HEIMPEL,
SMIRNOFF,
W.
A.,
AND
HEIMPEL,
A.
M.
1961.
Notes on the pathogenicity of BaciUus thuringiensis var. tkuringiensis Berliner for the earthworm Lumbricus terrestris Linnaeus. J. Insect Pathol., 3, 403-408. SMITH, N. R., GORDON, R. E., 1946. Aerobic spore-forming
AND
CLARK,
F.
E.
bacteria. U.S. Dept. Agr. Misc. Publ. No. 559, 112 pp.; reissued 1952 as U.S. Dept. Agr. Monograph No. 16, 148 pp. SMITH, S. G. 1943. Techniques for the study of insect chromosomes. Can. Entomologist, 75, 2234.