WoridJournal
of Microbiology
& Biotechnology
12.5042
Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus E. Umeh” and A. Njoku-Obi A toxic factor released from disrupted cells of Vibrio paruhaemolyticus was partially purified by gel filtration after precipitation with (NH,),SO, at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed injection into mice was 6.4 pg. human erythrocytes at a concentration of 0.15 ,ug ml-‘. Its LD,, by intravenous Fluid accumulated in suckling mice force-fed with the toxic material (I to 25 ,ug). Haemolytic activity, which occurred maximally at 37°C and pH 7.0 was enhanced by Ca’+, Cu2+ and Zn’+, each at 1 mu. Anti-toxic-factor serum agglutinated V. paruhuemolyticus cells. The factor may play a role in the pathogenesis of V. purahuemolytinrs infections and in the host’s defence mechanisms against infection by the microorganism. Key words:
Immunogenicity,
pathogenicity,
toxic intracellular
Vibrio paruhuemolyticus, a Gram-negative rod, is a common cause of gastro-enteritis in many parts of the world. Although the toxin which it produces has been studied, the mechanism of pathogenicity remains unclear. The pathogenicity of V. puruhuemolyfictrs was formerly attributed to the Kanagawa phenomenon but Kanagawa-negative strains, which lack haemolysin, have been isolated from patients with gastro-enteritis caused by the vibrio (Johnson & Calia 1976). Antiserum to the thermostable direct haemolysin also failed to prevent accumulation of fluid in ligated ileal loops of rabbits, indicating that fluid accumulation is not directly related to the haemolysin (Honda et al. 1983). Likewise, the enterotoxin-like factors which have been isolated from Kanagawa-positive and Kanagawanegative strains could not be detected in culture filtrates of the microorganism isolated from patients with gastroenteritis. The symptoms associated with the infection vary from bloody stools, diarrhoea, vomiting and abdominal pains to chills, fever and headache. As a result, Sochard & Colwell (1977) suggested that the pathogenicity of V. puruhuemolyticw may be due to a combination of factors, and not to a single factor. E. Umeh was and A. University of Nigeria, with the Department Makurdi. Benue State, @ 1996 Rapid Science
Njoku-Obi is with the Department of Microbiology, Nsukka, Enugu State, Nigeria. E.U. Umeh is now of Biological Sciences, University of Agriculture, Nigeria. ‘Corresponding author. Publishers
protein,
Vibrio
puruhaemolytictls.
The present tion
and
some
study was of the isolation, physico-chemical
properties
lar toxic factor, from a V. puruhuemolyfictrs may
play
a
role
in
the
pathogenicity
partial
purifica-
of an intracelluisolate, of
the
which micro-
organism.
Materials
and Methods
Bacterial Strain and Media A marine, Kanagawa-positive V. parahaemolyficw was isolated locally and identified as described by Hughes & Sakazaki (1972). The Kanagawa phenomenon was tested on Wagatsurna blood agar (Miyamoto et al. 1980) and the isolate was maintained on brain/heart infusion (BHI) medium containing 0.3% agar and supplemented with 3% (w/v) NaCl (Iijima et al. 1981). Harvesting of Bacferial Cells Broth cultures were grown for 4 inoculated onto the same medium incubated for 24 h at 37°C. phosphate-buffered saline (PBS), 30 min, 4°C) and washed twice in
h on BHI with 3% (w/v) NaCI, solidified with 0.3% agar and Cells were washed off with pH 7.2, centrifuged (4000 X g, PBS.
Disruption of Bacterial Cells A suspension of the bacterial cells (0.7 g ml-‘), held on ice, was disrupted in a cell disrupter for 3 X 90 s. Cell-wall components and other insoluble particles were removed by centrifugation (60,000 X g, 30 min, 4°C).
Vibrio sp. intracellular toxin Ptirijicution of Protein Protein in the supematant was fractionated on an (NH,),SO, gradient. Fractions which precipitated at 20% to 100% (NH&SO, saturation were collected and eluted through a Sephadex G-150 column (2.5 X 50 cm), pre-equilibrated with PBS (pH 7.2), using 500 ml PBS at a flow rate of 15 ml h-‘. Eluates were collected in 5 ml aliquots and haemolytic fractions were pooled, concentrated by percolation and used for subsequent studies as test sample (toxic fraction). Huemdysis Assay A I% defibrinated erythrocyte suspension, 1 ml, was added to each a-fold dilution (1 ml) of test sample, to buffer solution (negative control) and to 0.1% Na,CO, (positive control) (Cherwonogrodzky & Clark 1981). After I h at 37°C with periodic shaking, unlysed erythrocytes were removed by centrifugation (110 X g, 5 min). Percent lysis was determined turbidometrically at 540 run by comparing the absorption of the sample with that of the 100% control. One median haemolytic dose (HD,,) is the amount of protein in the toxic fraction which caused 50% erythrocyte lysis. Analysis Protein was estimated by the Lowry method, with BSA as standard. Carbohydrate was assayed using the anthrone reagent, with sucrose as a standard. Lipid was determined by the methods of Iida & Takagi (1977). The haemolytic fraction (0.5 ml) was added to 2 ml chloroform/methanol/water (1:2:0.8, by vol.) and centrifuged (110 X g, 5 min). The supematant was collected, evaporated at 50°C and the weight of the residue taken as weight of the lipid. Moleculur Weight The A4, of the haemolytic fraction was determined by gel filtration using Sephadex G-150. Reference proteins were myoglobulin, trypsin, BSA, lactate dehydrogenase and pyruvate kinase. Antiserum Sub-lethal doses of the unfiltrated toxic fraction were injected intravenously into a rabbit, following a schedule for antiserum production. Thereafter, the rabbit was bled and the serum collected and used as anti-toxic-factor serum. Agglutination
Vibrio paruhaemolyticus (lo6 c.f.u. ml-‘) was added to dilutions of antiserum or to PBS (control). After I h at 37”C, agglutination of the cells was measured. Erythrocytes, sensitized antiserum dilutions and
haemagglutination Antiserum A mixture
with the toxic fraction, to PBS (control). After
were added to 2 h at 37”C,
was measured.
Neutralization of toxic fraction
and antiserum,
or of toxic
fraction
and
normal rabbit serum (control), was incubated (37”C, IS min) and assayed for toxicity. Immlmo-efectrophoresis Immune-electrophoresis was carried out by the microtechnique of Scheideger. The upper well contained the toxic fraction and the lower well the crude supematant. Antiserum was poured in the trough. Lethal Activity
Varying doses of the toxic fraction (0.6 to 20 pg) were given mice through different routes, and to embryonated the yolk sac. As a control, saline was similarly given.
eggs
through
to
Enterotoxicity Groups of suckling mice were force-fed with different doses of the toxic fraction (0.04 to 25 pg) or with PBS. The animals were sacrificed after 4 h, and fluid accumulation was quantified by determining the ratio of gut weight to remaining body weight (see Table I). Cytotoxicity
Reaction mixtures comprising mouse peritoneal macrophages (IO4 cells ml-‘), maintained in Medium 199 with 0.1% BSA, and dilutions of the toxic fraction or PBS (control) were incubated for I h at 37°C. Dead cells, marked by trypan-blue uptake, were counted in a haemacytometer.
Results
and Discussion
A toxic factor, which may be linked to the pathogenicity of V. parahaemolyficw, was isolated from the disrupted cells. Unlike the thermostable direct haemolysin, which is detected in cell-free culture filtrates (Miyamoto et al. 1980), the toxic factor was an intracellular component of the cell, precipitated from whole-cell lysates with (NH,),SO, at 40% saturation. It did not contain detectable carbohydrate or lipid and was thus different from the intracellular haemolytic factor described by Iida & Takagi (1979) and also from bacterial endotoxins, which are complexes of lipids, proteins and polysaccharides. The toxic factor had an estimated M, of 63 kDa, larger than that of the thermostable haemolysin (42 to 44 kDa) (Takeda et al. 1978; Miyamoto et a/. 1980). Unlike the thermostable direct haemolysin and the thermolabile toxin (Kreger 199x), the partially-purified toxic factor retained full toxic activity (HD,, = 0.15 pg) after heating at 60°C for 30 min and partial activity (HD,, = 2.5 pug) after 100°C for 15 min. The crude factor, on the other hand, lost activity after heating at 60°C for 30 min. Maximal toxicity of the toxin occurred between 37°C and 57°C (HD,, = 0.15 pg) and at pH 7.0 to 7.2 (HD,, = 0.15 pug). Activity (HD,,) was only 5 pg at lO”C, 1.25 ,ug at 28°C. 1.2 lug at pH 7.6 and 5 lug at pH 8.4. Absence of haemolysis at pH 5.8 and 6.4 indicated loss of toxicity under acidic conditions. Incubation of the partially-purified factor with pepsin or trypsin abolished its toxicity (no haemolysis). This sensitivity to the proteolytic enzymes may also explain the very low mortality rate of mice given the toxic fraction orally (Table 2), the substance presumably being inactivated in the gut because of the presence of such enzymes. Haemolytic activity was stimulated by CaL+, CL?+ and Zn2+, each at 1 mM concentration, as shown by the decrease of HD,, to 0.016 pg in each case. This activity was, however, totally inhibited by Fe”+ or Mgtf at 1 mM. Haemolysin, on the other hand, is stimulated by Mg2+ but is inhibited by ZnZ+ (Takeda et al. 1977). Erythrocytes were not lysed in the control solutions containing only cations.
E. Umeh and A. Njoku-Obi Table 1. Ratios of gut weight to remaining body suckling mice force-fed with graded doses of the toxic Dose
of toxin
Ratio
Fluid
weight factor.
of
accumulation”
w 25.0 5.0 1.0 0.2 0.04 * Ratios
0.12 0.10 0.09 0.08 0.06 above
0.080 were
considered
+ + + f
positive.
Table 2. Mortality rates of mice inoculated intraperitonealiy (Lp.), subcutaneously (SC.) graded doses of toxic factor. Dose
intravenously (i.v.), or orally (oral) with
Mortality
(%)*
w 18.0 9.0 4.5 2.2 1.1
I.V.
i.p.
8X.
oral
80 60 40 20 0
60 40 20 0 0
50 20 0 0 0
20 0 0 0 0
Varying doses of the toxic fraction were injected into the mice (five in each group) via the routes indicated and the animals were observed over 48 h for any signs of ill-health or death. LD,, for the factor given by i.v.. i.p., SC. or oral routes were 6.4, 11.8, 18.0 and > 18.0 pg, respectively.
l
as the results in Table 2 indicate, doses of the toxic factor given subcutaneously or orally were less lethal than those administered intravenously or intraperitoneally. The haemolytic effects of the toxic factor on mammalian erythrocytes resembled those of the thermostable haemolysin, with lysis of human, rabbit, sheep and rat erythrocytes. However, unlike haemolysin, the toxic factor affected human erythrocytes more than those of the other mammals tested, with HD,, for human, rabbit, sheep and rat erythrocytes of 0.15, 0.3, 0.7 and 5 pg, respectively. Overall, the present results indicate that the toxic factor is intracellular in V. parahaemolytictls and is distinct from the other toxins produced by and reported in the organism. This toxic factor probably contributes to the symptoms observed during infection by the organism and may be important in both the pathogenicity of V. parahaemolyticus infection and the host’s immune defence processes against the organism.
References Cherwonogrodzky, J.W. & Clark, A.G. 1981 Effect of pH on the production of the Kanagawa hemolysin by Vibrio puruhaemolyticw.
and Immunity
34, 115-119.
parahaemolyticus.
Journal
of Infectious
Diseases
147,
779-780.
Hughes, R. & Sakazaki, R. 1972 Minimal number of characters for the identification of Vibrio species, V. cholerae, and V. parahaemolyticus.
The toxic factor was antigenic and provoked antibody production on injection into adult rabbits. The antiserum to the toxic factor, which was raised in these rabbits, neutralized all the toxic activities and agglutinated cells of V. parahaemolyticw (with a titre of X28) and erythrocytes coated with the toxic factor (to a titre of 1:2048). Bacterial cells in the control tubes were not clumped. These results indicate that an antibody-mediated immune response is probably one of the host’s defence mechanisms against V. parahaemolyficw infection. Immune-electrophoretic analysis, which showed an arc of precipitin between the well containing the toxic factor and the trough containing the antiserum, indicated immunologic homogeneity of the substance. The toxic factor was cytotoxic to mouse peritoneal macrophages; cell death rates were IO%, 52% and 90% with doses of 0.03, 0.3 and 5 pg, respectively. Suckling mice force-fed with the foxic factor accumulated fluid in their gut, as evident from the ratios of gut to body weights (Table I). When the toxic factor was injected intravenously into mice, death occurred (LD,, = 6.4 ,ug). Death of chick embryos (LD,, = 1.9 ,ug) also occurred upon inoculation via the yolk sac. The cytotoxic potency of the substance depended on the route through which it was administered;
Infection
Honda, T., Takeda, Y., Miwatani, T. & Nakarana, N. 1983 Failure of an antisera to thermostable direct hemolysin and cholera enterotoxin to prevent accumulation of fluid caused by Vibrio
Public Health
Laboratory
30, 133-136.
Iida, A. & Takagi, M. 1979 Investigation hemolytic agent of Vibrio purahaemolyticus. Immunology
on
an intracellular Microbiology and
23,305-312.
Iijima, Y., Yamada, H. & Shinoda, S. 1981 Adherence of Vibrio purahaemolyticw and its relation to pathogenicity. Canadian ]ournal of Microbiology 27, 305-312. Johnson, B.E. & Calia, F.M. 1976 False-positive rabbit ileal loop reactions attributed to Vibrio parahaemolyticw broth filtrates. journal of Infectious Diseases 133, 436-440. Kreger, AS. 1991 Cytolytic toxins of pathogenic marine vibrios. In Sourcebook of Bacterial Protein Toxins, eds Alouf, J.E. & Freer, J.H. pp. 263-276. London: Academic Press. Miyamoto, Y., Obara, Y. & Nikkawa, T. 1980 Simplified purification and biophysicochemical characteristics of Kanagawa phenomenon-associated haemolysin of Vibrio paruhaemolyticus. Infection and Immunity 28, 567-576. Sochard, M.R. & Colwell, R.R. 1977 Toxin isolation from a Kanagawa phenomenon-negative strain of Vibrio parahaemojyticus. Microbiology and Immunology 21, 443-446. Takeda, Y., Ogiso, Y. & Miwatani, T. 1977 Effect of zinc ion on the hemolytic activity of thermostable direct hemolysin of Vibrio parahaemolyticw, Streptolysin 0 and Triton X-100. Infection and Immunity 17, 239-243. Takeda, Y., Taga, S. & Miwatani, I. 1978 Evidence that therrnostable direct hemolysin of Vibrio paruhaemolyticus is composed of two subunits. FEM.5 Microbiology Letters 4, 271-273.
(Received
in
September
1995)
revised
form
14
September
1995;
accepted
18
of Microbiology
& Biotechnology
12.5042
Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus E. Umeh” and A. Njoku-Obi A toxic factor released from disrupted cells of Vibrio paruhaemolyticus was partially purified by gel filtration after precipitation with (NH,),SO, at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed injection into mice was 6.4 pg. human erythrocytes at a concentration of 0.15 ,ug ml-‘. Its LD,, by intravenous Fluid accumulated in suckling mice force-fed with the toxic material (I to 25 ,ug). Haemolytic activity, which occurred maximally at 37°C and pH 7.0 was enhanced by Ca’+, Cu2+ and Zn’+, each at 1 mu. Anti-toxic-factor serum agglutinated V. paruhuemolyticus cells. The factor may play a role in the pathogenesis of V. purahuemolytinrs infections and in the host’s defence mechanisms against infection by the microorganism. Key words:
Immunogenicity,
pathogenicity,
toxic intracellular
Vibrio paruhuemolyticus, a Gram-negative rod, is a common cause of gastro-enteritis in many parts of the world. Although the toxin which it produces has been studied, the mechanism of pathogenicity remains unclear. The pathogenicity of V. puruhuemolyfictrs was formerly attributed to the Kanagawa phenomenon but Kanagawa-negative strains, which lack haemolysin, have been isolated from patients with gastro-enteritis caused by the vibrio (Johnson & Calia 1976). Antiserum to the thermostable direct haemolysin also failed to prevent accumulation of fluid in ligated ileal loops of rabbits, indicating that fluid accumulation is not directly related to the haemolysin (Honda et al. 1983). Likewise, the enterotoxin-like factors which have been isolated from Kanagawa-positive and Kanagawanegative strains could not be detected in culture filtrates of the microorganism isolated from patients with gastroenteritis. The symptoms associated with the infection vary from bloody stools, diarrhoea, vomiting and abdominal pains to chills, fever and headache. As a result, Sochard & Colwell (1977) suggested that the pathogenicity of V. puruhuemolyticw may be due to a combination of factors, and not to a single factor. E. Umeh was and A. University of Nigeria, with the Department Makurdi. Benue State, @ 1996 Rapid Science
Njoku-Obi is with the Department of Microbiology, Nsukka, Enugu State, Nigeria. E.U. Umeh is now of Biological Sciences, University of Agriculture, Nigeria. ‘Corresponding author. Publishers
protein,
Vibrio
puruhaemolytictls.
The present tion
and
some
study was of the isolation, physico-chemical
properties
lar toxic factor, from a V. puruhuemolyfictrs may
play
a
role
in
the
pathogenicity
partial
purifica-
of an intracelluisolate, of
the
which micro-
organism.
Materials
and Methods
Bacterial Strain and Media A marine, Kanagawa-positive V. parahaemolyficw was isolated locally and identified as described by Hughes & Sakazaki (1972). The Kanagawa phenomenon was tested on Wagatsurna blood agar (Miyamoto et al. 1980) and the isolate was maintained on brain/heart infusion (BHI) medium containing 0.3% agar and supplemented with 3% (w/v) NaCl (Iijima et al. 1981). Harvesting of Bacferial Cells Broth cultures were grown for 4 inoculated onto the same medium incubated for 24 h at 37°C. phosphate-buffered saline (PBS), 30 min, 4°C) and washed twice in
h on BHI with 3% (w/v) NaCI, solidified with 0.3% agar and Cells were washed off with pH 7.2, centrifuged (4000 X g, PBS.
Disruption of Bacterial Cells A suspension of the bacterial cells (0.7 g ml-‘), held on ice, was disrupted in a cell disrupter for 3 X 90 s. Cell-wall components and other insoluble particles were removed by centrifugation (60,000 X g, 30 min, 4°C).
Vibrio sp. intracellular toxin Ptirijicution of Protein Protein in the supematant was fractionated on an (NH,),SO, gradient. Fractions which precipitated at 20% to 100% (NH&SO, saturation were collected and eluted through a Sephadex G-150 column (2.5 X 50 cm), pre-equilibrated with PBS (pH 7.2), using 500 ml PBS at a flow rate of 15 ml h-‘. Eluates were collected in 5 ml aliquots and haemolytic fractions were pooled, concentrated by percolation and used for subsequent studies as test sample (toxic fraction). Huemdysis Assay A I% defibrinated erythrocyte suspension, 1 ml, was added to each a-fold dilution (1 ml) of test sample, to buffer solution (negative control) and to 0.1% Na,CO, (positive control) (Cherwonogrodzky & Clark 1981). After I h at 37°C with periodic shaking, unlysed erythrocytes were removed by centrifugation (110 X g, 5 min). Percent lysis was determined turbidometrically at 540 run by comparing the absorption of the sample with that of the 100% control. One median haemolytic dose (HD,,) is the amount of protein in the toxic fraction which caused 50% erythrocyte lysis. Analysis Protein was estimated by the Lowry method, with BSA as standard. Carbohydrate was assayed using the anthrone reagent, with sucrose as a standard. Lipid was determined by the methods of Iida & Takagi (1977). The haemolytic fraction (0.5 ml) was added to 2 ml chloroform/methanol/water (1:2:0.8, by vol.) and centrifuged (110 X g, 5 min). The supematant was collected, evaporated at 50°C and the weight of the residue taken as weight of the lipid. Moleculur Weight The A4, of the haemolytic fraction was determined by gel filtration using Sephadex G-150. Reference proteins were myoglobulin, trypsin, BSA, lactate dehydrogenase and pyruvate kinase. Antiserum Sub-lethal doses of the unfiltrated toxic fraction were injected intravenously into a rabbit, following a schedule for antiserum production. Thereafter, the rabbit was bled and the serum collected and used as anti-toxic-factor serum. Agglutination
Vibrio paruhaemolyticus (lo6 c.f.u. ml-‘) was added to dilutions of antiserum or to PBS (control). After I h at 37”C, agglutination of the cells was measured. Erythrocytes, sensitized antiserum dilutions and
haemagglutination Antiserum A mixture
with the toxic fraction, to PBS (control). After
were added to 2 h at 37”C,
was measured.
Neutralization of toxic fraction
and antiserum,
or of toxic
fraction
and
normal rabbit serum (control), was incubated (37”C, IS min) and assayed for toxicity. Immlmo-efectrophoresis Immune-electrophoresis was carried out by the microtechnique of Scheideger. The upper well contained the toxic fraction and the lower well the crude supematant. Antiserum was poured in the trough. Lethal Activity
Varying doses of the toxic fraction (0.6 to 20 pg) were given mice through different routes, and to embryonated the yolk sac. As a control, saline was similarly given.
eggs
through
to
Enterotoxicity Groups of suckling mice were force-fed with different doses of the toxic fraction (0.04 to 25 pg) or with PBS. The animals were sacrificed after 4 h, and fluid accumulation was quantified by determining the ratio of gut weight to remaining body weight (see Table I). Cytotoxicity
Reaction mixtures comprising mouse peritoneal macrophages (IO4 cells ml-‘), maintained in Medium 199 with 0.1% BSA, and dilutions of the toxic fraction or PBS (control) were incubated for I h at 37°C. Dead cells, marked by trypan-blue uptake, were counted in a haemacytometer.
Results
and Discussion
A toxic factor, which may be linked to the pathogenicity of V. parahaemolyficw, was isolated from the disrupted cells. Unlike the thermostable direct haemolysin, which is detected in cell-free culture filtrates (Miyamoto et al. 1980), the toxic factor was an intracellular component of the cell, precipitated from whole-cell lysates with (NH,),SO, at 40% saturation. It did not contain detectable carbohydrate or lipid and was thus different from the intracellular haemolytic factor described by Iida & Takagi (1979) and also from bacterial endotoxins, which are complexes of lipids, proteins and polysaccharides. The toxic factor had an estimated M, of 63 kDa, larger than that of the thermostable haemolysin (42 to 44 kDa) (Takeda et al. 1978; Miyamoto et a/. 1980). Unlike the thermostable direct haemolysin and the thermolabile toxin (Kreger 199x), the partially-purified toxic factor retained full toxic activity (HD,, = 0.15 pg) after heating at 60°C for 30 min and partial activity (HD,, = 2.5 pug) after 100°C for 15 min. The crude factor, on the other hand, lost activity after heating at 60°C for 30 min. Maximal toxicity of the toxin occurred between 37°C and 57°C (HD,, = 0.15 pg) and at pH 7.0 to 7.2 (HD,, = 0.15 pug). Activity (HD,,) was only 5 pg at lO”C, 1.25 ,ug at 28°C. 1.2 lug at pH 7.6 and 5 lug at pH 8.4. Absence of haemolysis at pH 5.8 and 6.4 indicated loss of toxicity under acidic conditions. Incubation of the partially-purified factor with pepsin or trypsin abolished its toxicity (no haemolysis). This sensitivity to the proteolytic enzymes may also explain the very low mortality rate of mice given the toxic fraction orally (Table 2), the substance presumably being inactivated in the gut because of the presence of such enzymes. Haemolytic activity was stimulated by CaL+, CL?+ and Zn2+, each at 1 mM concentration, as shown by the decrease of HD,, to 0.016 pg in each case. This activity was, however, totally inhibited by Fe”+ or Mgtf at 1 mM. Haemolysin, on the other hand, is stimulated by Mg2+ but is inhibited by ZnZ+ (Takeda et al. 1977). Erythrocytes were not lysed in the control solutions containing only cations.
E. Umeh and A. Njoku-Obi Table 1. Ratios of gut weight to remaining body suckling mice force-fed with graded doses of the toxic Dose
of toxin
Ratio
Fluid
weight factor.
of
accumulation”
w 25.0 5.0 1.0 0.2 0.04 * Ratios
0.12 0.10 0.09 0.08 0.06 above
0.080 were
considered
+ + + f
positive.
Table 2. Mortality rates of mice inoculated intraperitonealiy (Lp.), subcutaneously (SC.) graded doses of toxic factor. Dose
intravenously (i.v.), or orally (oral) with
Mortality
(%)*
w 18.0 9.0 4.5 2.2 1.1
I.V.
i.p.
8X.
oral
80 60 40 20 0
60 40 20 0 0
50 20 0 0 0
20 0 0 0 0
Varying doses of the toxic fraction were injected into the mice (five in each group) via the routes indicated and the animals were observed over 48 h for any signs of ill-health or death. LD,, for the factor given by i.v.. i.p., SC. or oral routes were 6.4, 11.8, 18.0 and > 18.0 pg, respectively.
l
as the results in Table 2 indicate, doses of the toxic factor given subcutaneously or orally were less lethal than those administered intravenously or intraperitoneally. The haemolytic effects of the toxic factor on mammalian erythrocytes resembled those of the thermostable haemolysin, with lysis of human, rabbit, sheep and rat erythrocytes. However, unlike haemolysin, the toxic factor affected human erythrocytes more than those of the other mammals tested, with HD,, for human, rabbit, sheep and rat erythrocytes of 0.15, 0.3, 0.7 and 5 pg, respectively. Overall, the present results indicate that the toxic factor is intracellular in V. parahaemolytictls and is distinct from the other toxins produced by and reported in the organism. This toxic factor probably contributes to the symptoms observed during infection by the organism and may be important in both the pathogenicity of V. parahaemolyticus infection and the host’s immune defence processes against the organism.
References Cherwonogrodzky, J.W. & Clark, A.G. 1981 Effect of pH on the production of the Kanagawa hemolysin by Vibrio puruhaemolyticw.
and Immunity
34, 115-119.
parahaemolyticus.
Journal
of Infectious
Diseases
147,
779-780.
Hughes, R. & Sakazaki, R. 1972 Minimal number of characters for the identification of Vibrio species, V. cholerae, and V. parahaemolyticus.
The toxic factor was antigenic and provoked antibody production on injection into adult rabbits. The antiserum to the toxic factor, which was raised in these rabbits, neutralized all the toxic activities and agglutinated cells of V. parahaemolyticw (with a titre of X28) and erythrocytes coated with the toxic factor (to a titre of 1:2048). Bacterial cells in the control tubes were not clumped. These results indicate that an antibody-mediated immune response is probably one of the host’s defence mechanisms against V. parahaemolyficw infection. Immune-electrophoretic analysis, which showed an arc of precipitin between the well containing the toxic factor and the trough containing the antiserum, indicated immunologic homogeneity of the substance. The toxic factor was cytotoxic to mouse peritoneal macrophages; cell death rates were IO%, 52% and 90% with doses of 0.03, 0.3 and 5 pg, respectively. Suckling mice force-fed with the foxic factor accumulated fluid in their gut, as evident from the ratios of gut to body weights (Table I). When the toxic factor was injected intravenously into mice, death occurred (LD,, = 6.4 ,ug). Death of chick embryos (LD,, = 1.9 ,ug) also occurred upon inoculation via the yolk sac. The cytotoxic potency of the substance depended on the route through which it was administered;
Infection
Honda, T., Takeda, Y., Miwatani, T. & Nakarana, N. 1983 Failure of an antisera to thermostable direct hemolysin and cholera enterotoxin to prevent accumulation of fluid caused by Vibrio
Public Health
Laboratory
30, 133-136.
Iida, A. & Takagi, M. 1979 Investigation hemolytic agent of Vibrio purahaemolyticus. Immunology
on
an intracellular Microbiology and
23,305-312.
Iijima, Y., Yamada, H. & Shinoda, S. 1981 Adherence of Vibrio purahaemolyticw and its relation to pathogenicity. Canadian ]ournal of Microbiology 27, 305-312. Johnson, B.E. & Calia, F.M. 1976 False-positive rabbit ileal loop reactions attributed to Vibrio parahaemolyticw broth filtrates. journal of Infectious Diseases 133, 436-440. Kreger, AS. 1991 Cytolytic toxins of pathogenic marine vibrios. In Sourcebook of Bacterial Protein Toxins, eds Alouf, J.E. & Freer, J.H. pp. 263-276. London: Academic Press. Miyamoto, Y., Obara, Y. & Nikkawa, T. 1980 Simplified purification and biophysicochemical characteristics of Kanagawa phenomenon-associated haemolysin of Vibrio paruhaemolyticus. Infection and Immunity 28, 567-576. Sochard, M.R. & Colwell, R.R. 1977 Toxin isolation from a Kanagawa phenomenon-negative strain of Vibrio parahaemojyticus. Microbiology and Immunology 21, 443-446. Takeda, Y., Ogiso, Y. & Miwatani, T. 1977 Effect of zinc ion on the hemolytic activity of thermostable direct hemolysin of Vibrio parahaemolyticw, Streptolysin 0 and Triton X-100. Infection and Immunity 17, 239-243. Takeda, Y., Taga, S. & Miwatani, I. 1978 Evidence that therrnostable direct hemolysin of Vibrio paruhaemolyticus is composed of two subunits. FEM.5 Microbiology Letters 4, 271-273.
(Received
in
September
1995)
revised
form
14
September
1995;
accepted
18
