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Immunity induced in rats vaccinated with toxoid prepared from heat-labile toxin produced by Pasteurella multocida serogroup D
l'eterinary Microbiology, 27 ( 1991 ) 169-174 Elsevier Science Publishers B.V., Amsterdam
169
Immunity induced in rats vaccinated with toxoid prepared from heat-labile toxin produced by Pasteurella multocida serogroup D J.R. Thurston, R.B. Rimler, M.R. Ackermann, N.F. Cheville and J.M. Sacks United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Metabolic Diseases and Immunology Research Unit, Ames. L4 50010, US.4 (Accepted 18 October 1990)
ABSTRACT Thurston, J.R., Rimler, R.B., Ackermann, M.R., Cheville, N.F. and Sacks, J.M., 1991. Immunity induced in rats vaccinated with toxoid prepared from heat-labile toxin produced by Pasteurella multocida serogroup D. Vet. Mierobiol., 27:169-174. Rats were vaccinated with a toxoid (D-toxoid) prepared from purified heat-labile toxin (D-toxin) produced by Pasteurella multocida serogroup D. Vaccination of rats with D-toxoid prevented death and other effects of D-toxin (hepatic necrosis, development of elevated leukocyte counts, lymphopenia, neutrophilia, and elevated complement titers ) that occurred in phosphate buffered saline ( PBS )vaccinated control rats.
INTRODUCTION
The heat-labile toxin (D-toxin) produced by certain isolates of Pasteurella multocida serogroup D is an important factor in the pathogenesis of atrophic rhinitis in swine (Chanter and Rutter, 1989). In addition to localized effects on nasal turbinates, D-toxin is dermonecrotic, and capable of causing systemic effects such as weight loss, hepatic necrosis, lymphopenia, increased serum complement titers, and death (Dominick and Rimler, 1986, 1988; Elling et al., 1988; Cheville et al., 1988; Cheville and Rimler, 1989; Rimler and Rhoades, 1989; Thurston et al., 1989; Williams et al., 1990). Elling et al. (1986), RiJschoff et al. (1987), and Cheville and Rimler (1989) demonstrated that systemic changes induced in rats by D-toxin are similar to those observed in pigs given D-toxin. As a consequence of these studies and reports of prevention of turbinate atrophy by immunization with toxoid prepared from toxigenic strains ofP. multocida (Foged, 1988; Foged et al., 1989), we immunized rats with a toxoid (D-toxoid) prepared from purified D-toxin to determine if immunized rats were protected against effects of D-toxin, namely
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i
death, hepatic necrosis, depressed weight gain, alterations in Icukoc:xtc numbers, and increased complement activity. MATERIALS A N D ME-IHI )I)S
Preparation qf D-lo.void Toxin from P. multocida strain P-4533 was purified as described previousl~ (Cheville and Rimler, 1989 ). For toxoid preparation, 8 mg of toxin was mixed with 72 mg of bovine serum albumin fraction V in 3.2 ml of (1.2 M acetate buffer, pH 5.0. The mixture was stirred at room temperature, and 0.32 ml of 2.5% aqueous glutaraldehyde was added. The mixture was allowed to polymerize for 3 h and then was ground with saline in a Ten-Broeck tissue grinder. The ground suspension was washed twice with saline by centrifugation and adjusted to the desired concentration in phosphate buffered saline (PBS) containing 0.01% sodium azide. Concentration of the toxoid was estimated to be approximately 280 #g per ml.
Rats Fifty-eight male Holtzman rats ( H o l t z m a n Laboratory Animals, Madison, WI ), were housed individually on ground corn cob bedding with unlimited access to food and fresh water, and were weighed daily. Average rat weight on day 0 was 118 g. They were divided into two groups: one group received 0.25 ml of D-toxoid subcutaneously on day 0 and day 10, and the other ( control ) group received 0.25 ml of PBS on the same days. On day 24, each group of rats was subdivided into four challenge groups of six rats and were given subcutaneous injections of either 0.1,0.2, 0.4, or 0.8 #g D-toxin/kg of the average rat weight on day 24 ( 320 g). The experiment was terminated on day 27 when surviving rats and the remaining ten untreated, unchallenged rats were anesthetized with CO2 and blood samples were taken by cardiac puncture. Rats were killed with CO2 and tissues were collected for histopathological study.
Serum complement Serum collected on day 27 of the experiment was tested for complement activity as previously described using sheep erythrocytes sensitized with rabbit anti-sheep hemolysin. C o m p l e m e n t titers were expressed on the log~o of the 50% hemolytic endpoint ( G a r v e y et al., 1977). An aliquot of pooled normal rat serum was included as a standard in each set of complement titrations.
Hernatoloj~y At the time blood samples were taken from the rats, smears were made tbr differential leukocyte counts, and blood was placed in tubes containing EDTA for determination of total leukocyte counts ( W B C ) by electronic counting ( Nova Celltrak, Waltham, MA ).
VACCINATION OF RATSWITH P. MULTO(TDA SEROGROUP D
171
Histopathology Samples from left and right liver lobes of rats surviving until day 27 were fixed in neutral buffered formalin and processed routinely. Sections were stained with hemolysin and eosin. RESULTS
No deaths from D-toxin occurred in any D-toxoid-vaccinated rats, and no microscopic changes typical of individual cell necrosis were observed in any of these rats (Table 1 ). Mild to marked necrosis of individual hepatocytes was seen in surviving (control) rats. Hepatocellular necrosis was most severe in portal areas but also present, to a lesser degree, throughout lobules (Fig. 1 ). The necrotizing process was generalized throughout liver sections and lesions were greatest in rats from groups challenged with 0.4 and 0.8/zg/kg of D-toxin. Rats gained weight uniformly from day 0 until day 24 when all D-toxoid and PBS rats were given subcutaneous injections of D-toxin. After injection of D-toxin, rats vaccinated with D-toxoid gained an average weight of 16 g from day 24 until day 27. Surviving control rats lost an average of 11 g weight from day 24 to day 27. Total leukocyte counts, percent of neutrophils, lymphocytes, and serum complement titers of D-toxoid and surviving control rats did not show any obvious dose-related responses to challenge with the four concentrations of D-toxin (Fig. 2) for either vaccine group by regression analysis (F-tests). Therefore, overall means and standard errors were computed for the two groups, for the ten normal rats in the current study, and for 32 normal rats from an unpublished study which served as a laboratory standard (Table 2). Rats vaccinated with D-toxoid and challenged with D-toxin had identical values to those of normal rats, which differed markedly from control rats chalTABLE 1 Death and hepatic necrosis caused by D-toxin in D-toxoid-vaccinated and PBS-vaccinated rats D-toxin (/lg/kg of body weight )
Day 27 Death per group
0.1 0.2 0.4 0.8
Necrosis per survivor
D-toxoid
PBS
D-toxoid
PBS
0/6 0/5 a 0/6 016
0/6 4/6 3/5" 5/6
0/6 0/5 0/6 0/6
2/6 1/2 2/2
aOne death occurred unrelated to D-toxin.
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Fig. 1. Section of hepatic portal area from a rat vaccirmted wilt1 PBS and challenged with 0.4/tg of D-toxin. There is necrosis of individual hcpatoc)tcs (arrm~s). Necrotic h e p a t o o t e s have p>knotic nuclei, dark staining cytoplasnl, and irregular bordcis, tlcmatoxylin and cosin stain. 4(/0 X.
40
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35
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50 0.11
11.1
0.2
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Challenge/ag/kg
11.5
0,6
D-Toxin
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-
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ILl 0.3 (?hallenge
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0.6 11.7 11.,'4 ~.tg/k~ D - T o x i n
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Fig. 2. Mean values of hematology (a, b, c) and of complement liters (d) in rats vaccinated with either D-toxoid or PBS and challenged with 0.1,0.2, 0.4, or 0.8 ~g/kg of D-toxin.
VACCINATIONOF RATSWITH P. MULTOC1DA SEROGROUP D
173
TABLE 2 Mean values of hematology and serum complement tilers for standard and normal rats and all Dtoxoid- and PBS-vaccinaled rats surviving on day 27 Variable
W B C × 103/mm 3 % neutrophils % lymphocytes C logm tiler
Day 27
Mean S.E. Mean S.E. Mean S.E. Mean S.E.
Standard (n=32)
Normal rats (n=10)
D-toxoid-vaccinated (n=23)
PBS-vaccinated (n=ll)
12 0.7 11 1.1 88 1.1 1.74 0.01
16a 1.2 11 1.7 87 2.0 ND c ND
16a 0.9 11 1.4 87 1.0 1.74 0.014
30 b 1.6 22 b 2.5 758 2.7 1.928 0.033
aDifferent from the standard P < 0.05. bDifferenl from D-toxoid P < 0.001. e N D = not done.
lenged with D-toxin, and the differences were all statistically significant (ttests; P < 0.01 ). The average total leukocyte count of the lab standard rats was somewhat lower than for normal and D-toxoid-vaccinated rats, and the difference was statistically significant ( P < 0 . 0 5 ) . Percent of neutrophils and lymphocytes for lab standard rats, normal, and D-toxoid-vaccinated rats were virtually identical, and differences were statistically nonsignificant. DISCUSSION
Rats vaccinated with D-toxoid were protected against the effects of D-toxin, including death and hepatocellular necrosis. They also had weight gain, hematologic values, and serum complement titers within limits we have observed for normal Holtzman rats. D-toxin caused an increase in total leukocyte counts of control rats. Shifts in numbers and proportions oflymphocytes and neutrophils were prevented by vaccination of rats with toxoid prepared from D-toxin. Williams et al. ( 1990 ) observed lymphopenia in pigs 24 h after they were given D-toxin (designated in their study as turbinate atrophy toxin). They also demonstrated D-toxin-stimulated activity of peripheral blood lymphocytes in vitro, but did not lyse lymphocytes or other cells even though Dtoxin in vivo causes hepatic necrosis (Cheville et al., 1988) and dermonecrosis (Rimler and Rhoades, 1989) in pigs and rats (Cheville and Rimler, 1989 ). The hematologic changes described by Williams et al. (1990) and hematological data in this study indicate a need for further investigation of the effects of D-toxin on numbers and function of peripheral blood lymphocytes and neutrophils in relationship to the as yet unresolved mode of action of D-
I~-~
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toxin. The results suggest the rat model used in the study ofahcration caused by, D-toxin (Ri,ischolt" et al.. 1987) will be useful in testing of vaccines designed to immunize against atrophic rhinitis, particularly,' vaccines purported to contain toxoid prepared from the heat-labile protein toxin of P. mtdmctda serogroup D. A('KN()WLEDGEMENTS
We thank Ms. Kim Driflmier for her excellent technical assistance.
REFERENCES Chanter, N. and RuUer, J.M.. 1989. Pasteurellosis in pigs and the determinants of xirulence of loxigenic t'asteme/la mullocidu. In: ('. Adlam and J.M. Rutter (Editors), Pastcurella and Pasteurellosis..Academic Press, New York, 340 pp. ('hex ille, N.F. and Rimler, R.B., 1989. A protein toxin from l'a,s'teurella mu/mcida t>pc I) causes acute and chronic hepatic toxicity in rats. Vet. Pathol., 26: 148-157. ("hex ille, N.F., Rimler, R.B. and Thurston. J.R., 1988. A toxin from l'~t,st~'m'~'l/amtdl¢)~'/da t3 pc D causes acute hepatic necrosis in pigs. Vet. Pathol., 2 5 : 5 1 8 - 5 2 0 . l)ominick. M.A. and Rimler, R.B., 1986. Turbinate atrophy in gnotobiotic pigs intranasally inoculated wilh protein toxin isolated from type D Pa.s'l#ure//a mtl/loCtda. Am. ,1. Vet. Res., 47: 1532-1536. Dominick, M.A. and Rimler, R.B., 1988. Turbinate osteoporosis in pigs lbllowing intranasal inoculation of purified pasteurella toxin: Histomorphometric and ultrastructural studies. Vet. pathol., 25:17-27. Elling, F.. F'edersen, K.B. and Foged, N.T., 1986. Osteoclaslic bone rcsorption induced b~ a t'~,,~lem'el/a mtdmcida produced protein. 4th Joint Meet. Vet. Pathol.. ( 'ordoba. 1986. Elling, F.. Pedersen, K.B., Hogh, P. and Foged, N.T., 1988. Characterization of the dermal Icsions induced by a purified protein from loxigenic Pa.steme//a mtdm~du. APMIS. t)6: 5055. Foged. N.T., 1988. Quantitation and purification of the l'asteurella mulmctda toxin by using monoclonal antibodies. Infect. Immun., 56:1901 - 19(]6. Foged, N.T., Nielsen, J.P. and Jorsal, S.E., 1989, Protection against progcssix c atrophic rhmitis by x accination with t'a,slemella mullocida toxin purified by monoclonal antibodies. Vet. Rec.. 125:7-11. Garvcy, J.S.. Cremer, N.E. and SussdorE D.H., 1977. Methods in Immunology, 3rd Edn. W..\. Benjamin, Reading, MA, 522 pp. Rimler, R.B. and Rhoades, K.R.. 1989. Pa,sletcrel/a muflocida. In: ('. Adlam and ,I.M. Ruttcr ( Editors ), Pasteurella and Pasteurellosis. Academic Press, NY, 340 pp. Riischoff, B., Hermanns, W. and Pelzoldt, K., 1987. Distribution of a dermonccrotic crude toxin preparation from Pasleurel/a multoctda scrotype D in rats. J. Vet. Med. B, 34:691 700. Thurston, ,I.R., ('heville, N.F., Rimler, R.B. and Sacks, ,I., l t,~8t). Serum complement activit,~ and serum enzymes in rats after a subcutaneous injection of toxin prepared flom t'a,slemv//a mldt~)cida type D. Vet. Immunol. lmmunopathol., 23: 385-388. Williams, P.P., Hall, M.R. and Rimler, R.M., 1990. Host response to t'a.sletlr{'//a i,ltt/loclda turbinate atrophy toxin in swine, Can. J. Vet. Res., 54:157-163.
169
Immunity induced in rats vaccinated with toxoid prepared from heat-labile toxin produced by Pasteurella multocida serogroup D J.R. Thurston, R.B. Rimler, M.R. Ackermann, N.F. Cheville and J.M. Sacks United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Metabolic Diseases and Immunology Research Unit, Ames. L4 50010, US.4 (Accepted 18 October 1990)
ABSTRACT Thurston, J.R., Rimler, R.B., Ackermann, M.R., Cheville, N.F. and Sacks, J.M., 1991. Immunity induced in rats vaccinated with toxoid prepared from heat-labile toxin produced by Pasteurella multocida serogroup D. Vet. Mierobiol., 27:169-174. Rats were vaccinated with a toxoid (D-toxoid) prepared from purified heat-labile toxin (D-toxin) produced by Pasteurella multocida serogroup D. Vaccination of rats with D-toxoid prevented death and other effects of D-toxin (hepatic necrosis, development of elevated leukocyte counts, lymphopenia, neutrophilia, and elevated complement titers ) that occurred in phosphate buffered saline ( PBS )vaccinated control rats.
INTRODUCTION
The heat-labile toxin (D-toxin) produced by certain isolates of Pasteurella multocida serogroup D is an important factor in the pathogenesis of atrophic rhinitis in swine (Chanter and Rutter, 1989). In addition to localized effects on nasal turbinates, D-toxin is dermonecrotic, and capable of causing systemic effects such as weight loss, hepatic necrosis, lymphopenia, increased serum complement titers, and death (Dominick and Rimler, 1986, 1988; Elling et al., 1988; Cheville et al., 1988; Cheville and Rimler, 1989; Rimler and Rhoades, 1989; Thurston et al., 1989; Williams et al., 1990). Elling et al. (1986), RiJschoff et al. (1987), and Cheville and Rimler (1989) demonstrated that systemic changes induced in rats by D-toxin are similar to those observed in pigs given D-toxin. As a consequence of these studies and reports of prevention of turbinate atrophy by immunization with toxoid prepared from toxigenic strains ofP. multocida (Foged, 1988; Foged et al., 1989), we immunized rats with a toxoid (D-toxoid) prepared from purified D-toxin to determine if immunized rats were protected against effects of D-toxin, namely
lT{l
,:
Jill
t,~" i,,>,1
i
death, hepatic necrosis, depressed weight gain, alterations in Icukoc:xtc numbers, and increased complement activity. MATERIALS A N D ME-IHI )I)S
Preparation qf D-lo.void Toxin from P. multocida strain P-4533 was purified as described previousl~ (Cheville and Rimler, 1989 ). For toxoid preparation, 8 mg of toxin was mixed with 72 mg of bovine serum albumin fraction V in 3.2 ml of (1.2 M acetate buffer, pH 5.0. The mixture was stirred at room temperature, and 0.32 ml of 2.5% aqueous glutaraldehyde was added. The mixture was allowed to polymerize for 3 h and then was ground with saline in a Ten-Broeck tissue grinder. The ground suspension was washed twice with saline by centrifugation and adjusted to the desired concentration in phosphate buffered saline (PBS) containing 0.01% sodium azide. Concentration of the toxoid was estimated to be approximately 280 #g per ml.
Rats Fifty-eight male Holtzman rats ( H o l t z m a n Laboratory Animals, Madison, WI ), were housed individually on ground corn cob bedding with unlimited access to food and fresh water, and were weighed daily. Average rat weight on day 0 was 118 g. They were divided into two groups: one group received 0.25 ml of D-toxoid subcutaneously on day 0 and day 10, and the other ( control ) group received 0.25 ml of PBS on the same days. On day 24, each group of rats was subdivided into four challenge groups of six rats and were given subcutaneous injections of either 0.1,0.2, 0.4, or 0.8 #g D-toxin/kg of the average rat weight on day 24 ( 320 g). The experiment was terminated on day 27 when surviving rats and the remaining ten untreated, unchallenged rats were anesthetized with CO2 and blood samples were taken by cardiac puncture. Rats were killed with CO2 and tissues were collected for histopathological study.
Serum complement Serum collected on day 27 of the experiment was tested for complement activity as previously described using sheep erythrocytes sensitized with rabbit anti-sheep hemolysin. C o m p l e m e n t titers were expressed on the log~o of the 50% hemolytic endpoint ( G a r v e y et al., 1977). An aliquot of pooled normal rat serum was included as a standard in each set of complement titrations.
Hernatoloj~y At the time blood samples were taken from the rats, smears were made tbr differential leukocyte counts, and blood was placed in tubes containing EDTA for determination of total leukocyte counts ( W B C ) by electronic counting ( Nova Celltrak, Waltham, MA ).
VACCINATION OF RATSWITH P. MULTO(TDA SEROGROUP D
171
Histopathology Samples from left and right liver lobes of rats surviving until day 27 were fixed in neutral buffered formalin and processed routinely. Sections were stained with hemolysin and eosin. RESULTS
No deaths from D-toxin occurred in any D-toxoid-vaccinated rats, and no microscopic changes typical of individual cell necrosis were observed in any of these rats (Table 1 ). Mild to marked necrosis of individual hepatocytes was seen in surviving (control) rats. Hepatocellular necrosis was most severe in portal areas but also present, to a lesser degree, throughout lobules (Fig. 1 ). The necrotizing process was generalized throughout liver sections and lesions were greatest in rats from groups challenged with 0.4 and 0.8/zg/kg of D-toxin. Rats gained weight uniformly from day 0 until day 24 when all D-toxoid and PBS rats were given subcutaneous injections of D-toxin. After injection of D-toxin, rats vaccinated with D-toxoid gained an average weight of 16 g from day 24 until day 27. Surviving control rats lost an average of 11 g weight from day 24 to day 27. Total leukocyte counts, percent of neutrophils, lymphocytes, and serum complement titers of D-toxoid and surviving control rats did not show any obvious dose-related responses to challenge with the four concentrations of D-toxin (Fig. 2) for either vaccine group by regression analysis (F-tests). Therefore, overall means and standard errors were computed for the two groups, for the ten normal rats in the current study, and for 32 normal rats from an unpublished study which served as a laboratory standard (Table 2). Rats vaccinated with D-toxoid and challenged with D-toxin had identical values to those of normal rats, which differed markedly from control rats chalTABLE 1 Death and hepatic necrosis caused by D-toxin in D-toxoid-vaccinated and PBS-vaccinated rats D-toxin (/lg/kg of body weight )
Day 27 Death per group
0.1 0.2 0.4 0.8
Necrosis per survivor
D-toxoid
PBS
D-toxoid
PBS
0/6 0/5 a 0/6 016
0/6 4/6 3/5" 5/6
0/6 0/5 0/6 0/6
2/6 1/2 2/2
aOne death occurred unrelated to D-toxin.
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Fig. 1. Section of hepatic portal area from a rat vaccirmted wilt1 PBS and challenged with 0.4/tg of D-toxin. There is necrosis of individual hcpatoc)tcs (arrm~s). Necrotic h e p a t o o t e s have p>knotic nuclei, dark staining cytoplasnl, and irregular bordcis, tlcmatoxylin and cosin stain. 4(/0 X.
40
•----.o--
35
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30
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2sJ
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.....................
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35
~
311 o,ooO,"
r. 25
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211 n5 =
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o
5
0
0-
1
2.2
100
c "--- 2.1
~, 90
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=_ 80 711 6o
2.11
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r,,
1.9
,~,
1.8
, o ,,,o,O ,, -, " ' ' ' " * " - . ,
- . . , -, "t)
t7 I .(i
50 0.11
11.1
0.2
(1.3
0.4
Challenge/ag/kg
11.5
0,6
D-Toxin
11.7 1).8
-
I
0,0
O. l
r
ILl 0.3 (?hallenge
r
~
0.6 11.7 11.,'4 ~.tg/k~ D - T o x i n
11.4
1).5
Fig. 2. Mean values of hematology (a, b, c) and of complement liters (d) in rats vaccinated with either D-toxoid or PBS and challenged with 0.1,0.2, 0.4, or 0.8 ~g/kg of D-toxin.
VACCINATIONOF RATSWITH P. MULTOC1DA SEROGROUP D
173
TABLE 2 Mean values of hematology and serum complement tilers for standard and normal rats and all Dtoxoid- and PBS-vaccinaled rats surviving on day 27 Variable
W B C × 103/mm 3 % neutrophils % lymphocytes C logm tiler
Day 27
Mean S.E. Mean S.E. Mean S.E. Mean S.E.
Standard (n=32)
Normal rats (n=10)
D-toxoid-vaccinated (n=23)
PBS-vaccinated (n=ll)
12 0.7 11 1.1 88 1.1 1.74 0.01
16a 1.2 11 1.7 87 2.0 ND c ND
16a 0.9 11 1.4 87 1.0 1.74 0.014
30 b 1.6 22 b 2.5 758 2.7 1.928 0.033
aDifferent from the standard P < 0.05. bDifferenl from D-toxoid P < 0.001. e N D = not done.
lenged with D-toxin, and the differences were all statistically significant (ttests; P < 0.01 ). The average total leukocyte count of the lab standard rats was somewhat lower than for normal and D-toxoid-vaccinated rats, and the difference was statistically significant ( P < 0 . 0 5 ) . Percent of neutrophils and lymphocytes for lab standard rats, normal, and D-toxoid-vaccinated rats were virtually identical, and differences were statistically nonsignificant. DISCUSSION
Rats vaccinated with D-toxoid were protected against the effects of D-toxin, including death and hepatocellular necrosis. They also had weight gain, hematologic values, and serum complement titers within limits we have observed for normal Holtzman rats. D-toxin caused an increase in total leukocyte counts of control rats. Shifts in numbers and proportions oflymphocytes and neutrophils were prevented by vaccination of rats with toxoid prepared from D-toxin. Williams et al. ( 1990 ) observed lymphopenia in pigs 24 h after they were given D-toxin (designated in their study as turbinate atrophy toxin). They also demonstrated D-toxin-stimulated activity of peripheral blood lymphocytes in vitro, but did not lyse lymphocytes or other cells even though Dtoxin in vivo causes hepatic necrosis (Cheville et al., 1988) and dermonecrosis (Rimler and Rhoades, 1989) in pigs and rats (Cheville and Rimler, 1989 ). The hematologic changes described by Williams et al. (1990) and hematological data in this study indicate a need for further investigation of the effects of D-toxin on numbers and function of peripheral blood lymphocytes and neutrophils in relationship to the as yet unresolved mode of action of D-
I~-~
;e
~111
i-vl~,\
i { ,,:
toxin. The results suggest the rat model used in the study ofahcration caused by, D-toxin (Ri,ischolt" et al.. 1987) will be useful in testing of vaccines designed to immunize against atrophic rhinitis, particularly,' vaccines purported to contain toxoid prepared from the heat-labile protein toxin of P. mtdmctda serogroup D. A('KN()WLEDGEMENTS
We thank Ms. Kim Driflmier for her excellent technical assistance.
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