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Effect of aflatoxin B1 on resistance induced by Bordetella bronchiseptica vaccine in rabbits
Veterinary Microbiology, 25 ( 1990 ) 2 0 9 - 2 1 6 Elsevier Science Publishers B.V., A m s t e r d a m
209
Effect of aflatoxin B 1 on resistance induced by Bordetella bronchiseptica vaccine in rabbits M.C. Venturini a, C.J. Perfumo a, M.A. Risso a, C.M. Gomez b, M.V. Piscopo a, M. Sala de Miguel c and H. G o d o y c alnstitute of Pathology, Faculty of VeterinarySciences, La Plata National University, C. C 296, 1900 La Plata, Argentina bDepartment of lmmunology, Faculty of VeterinarySciences, La Plata National University, C. C 296, 1900 La Plata, Argentina CDepartrnent of Microbiology, National Institute of Agricultural Technology (INTA), Castelar, Argentina (Accepted 12 March 1990)
ABSTRACT Venturini, M.C., Perfumo, C.J., Risso, M./~., Gomez, C.M., Piscopo, M.V., Sala de Miguel, M. and Godoy, H., 1990. Effect of aflatoxin B l on resistance induced by Bordetella bronchiseptica vaccine in rabbits. Vet. Microbiol., 25: 209-216. The effects of aflatoxin B 1 on the development of the immune response to oil-adjuvanted Bordetella bronchiseptica vaccine and on acquired resistance to bacterial challenge were studied in rabbits. The doses of aflatoxin used were insufficient to produce clinical intoxication. Rabbits were randomly assigned to three groups, each having six animals: control (T), vaccinated (V), and vaccinated plus aflatoxin (VA) at 0.05 mg/kg daily per os. Groups V and VA were vaccinated twice, and the three groups were subsequently challenged with virulent B. bronchiseptica. The average weight gain at weekly intervals was significantly reduced in group VA, and no statistically significant differences were found in the titers of agglutinating antibodies between groups V and VA. There were significant differences between groups V and VA in the extent and severity of the pneumonic process, group VA being most affected. Results indicated that agglutinating antibody titers were not related to the level of protection in the latter group. Other mechanisms, such as alveolar macrophage activity and cell-mediated immunity, were implicated in the impairment of the acquired resistance in rabbits subclinically intoxicated with aflatoxin.
INTRODUCTION
Aflatoxins are toxic metabolites derived from certain strains of Aspergillus flavus and A. parasiticus, which grow on several substrates used in animal feeds. Five major toxins - BI, G1, B2, G2, and M1 - have been described, and have proved to be potent hepatotoxic and carcinogenic agents. B 1 is the most toxic and the most abundant. They are low molecular weight bifururanocoumarin derivates (Edds, 1973; Newberne, 1973; Buck and Osweiler, 0378-1135/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
210
M.C. VENTURINI ET AL.
1976; Clark et al., 1980; Pier et al., 1980). The consumption of aflatoxins by birds and swine leads to increased susceptibility to diseases and impairment of acquired resistance against bacterial and parasitic infections (Pier and Heddleston, 1970; Pier, 1973; Cysewiski, 1978; Pier et al., 1980; Panagala et al., 1986). The aim of this study was to investigate the effect of aflatoxin B 1 (AFL B 1 ) on the i m m u n e response to oil-adjuvanted Bordetella bronchiseptica vaccine in chronically intoxicated rabbits, as well as to determine its effect on acquired resistance to bacterial challenge. MATERIALS AND METHODS
Animals and experimental design 18 Rabbits with an average body weight of 2.2 kg were divided into three groups: V, vaccinated; VA, vaccinated plus treatment with AFL B 1 (87.87% B 1 equivalent); and T, controls. Animals were in six blocks. One animal from each group was randomly assigned to one block ( IV, 1VA, IT; Lison, 1976 ). Animals were housed in six cages, each containing one animal from each group. Commercial food and water were available ad libitum. Animals were weighed daily.
Aflatoxin Aflatoxin produced by A. flavus, consisting of 75.74% B1 and 24.26% G 1.G 1, was considered half as toxic as B 1. Aflatoxin was prepared by dissolving it in chloroform and then in edible oil to an approximate concentration of 0.2 m g / m l . According to Clark et al. ( 1980 ), the a m o u n t of AFL B 1 required to induce chronic intoxication in rabbits was 0.05 m g / k g live weight. This dosage was administrated daily per os from day 1 until day 43 of the experiment. Bordetella bronchiseptica vaccine Groups V and VA were vaccinated on days 1 and 14 of the experiment with an experimental oil-adjuvanted B. bronchiseptica vaccine, derived from a strain isolated from an outbreak ofbronchopneumonia in rabbits. It was grown on Border Gengou Agar (Oxoid) with 10% defribrinated sheep blood, and was incubated for 48 h at 37°C, then on charcoal agar (Oxoid) for 72 h at 37 ° C. Phase control was carried out with B. bronchiseptica phase I Kitasato antiserum (Kitasato Institute, Tokyo, Japan), used as directed by the manufacturer. The culture was harvested in PBS, inactivated by adding 0.3% formalin and heating at 37 °C for 48 h, and was incorporated into a double emulsion (water-in-oil-in-water) adjuvant consisting of a mineral oil (Marcol, 86%), an emulsifier (Arlacel A, 14%) and a detergent (Tween 80, 2% in PBS). The vaccine contained 10 OIU (International Reference Preparation), equivalent to approximately 10 ~° bacteria/ml. The three groups were chal-
EFFECT OF AFLATOXINON B. BRONCHISEPTICA-VACC1NATED RABBITS
21 1
lenged intranasally with the live B. bronchiseptica used in the preparation of the vaccine (0.5 ml into each nostril, 2 × 109 bacteria/ml) on day 44.
Serology Serum samples were obtained weekly, and were examined for agglutinating antibodies using a tube agglutination test with the AR Kitasato antigen, used as directed by the manufacturer. Complete agglutination was considered positive after incubation for 2 h at 37 °C and overnight at 4°C.
Necropsy and h&topathologic examination Necropsy was carried out on all rabbits, beginning 2 days (day 46) after challenge for the first block, and on days 48, 50, 52, 54 and 56 for each remaining block. Macroscopic effects were measured by morphometric methods previously described (Reid, 1980). One photograph was taken of the dorsal and ventral aspects of each lung of each rabbit. Point-counting morphometry was done using a point test grid superimposed on the photograph. Relative percentages were obtained from the total number of points for the two sides minus the number of points counted in the affected area, multiplied by 100. Liver, lung and spleen were weighed individually; for histopathologic study they were fixed in neutral buffered 10% formalin, sectioned and stained with hematoxylin and eosin. Lung lesions were classified from grade 0 to 3 according to severity, as follows: ( 1 ) Air passages: grade 0, without microscopic lesions; grade 1, presence of exudate in the lumen of bronchi and bronchioli without changes in the walls; grade 2, presence of exudate in the lumen of bronchi and bronchioli, with epithelial desquamation and mononuclear infiltration of the walls; grade 3, obstructive bronchiolitis. (2) Alveoli: grade 0, without microscopic lesions; grade 1, mild to moderate thickening of alveolar walls due to inflammatory cell infiltration; grade 2, as grade 1 plus abundant mononuclear infiltration of the alveolar lumen in most of the fields observed; grade 3, as grade 2 plus alveolar necrosis. (3) Lymphoid tissue: grade 0, absence of lymphoid tissue; grade 1, mild lymphoid hyperplasia; grade 2, moderate lymphoid hypertrophy with bronchiolar deformity; grade 3, severe lymphoid hypertrophy with bronchiolar collapse.
Statistics Data on weekly weight gain, organ weights and serology were analyzed by Fisher's test. Gross and microscopic lesions were analyzed by the Z 2 test. A value of P < 0.05 was considered significant. RESULTS
Measurements of weekly weight gain indicated significant differences between the three groups studied. Group VA was the most affected, particularly
212
M.C. VENTURIN1 ET AL.
TABLE 1
Weekly weight gain (g). Mean _+s.d. Weeks
T
V
VA
F
145.0 (44.6) 246.7* (78.6) 155.0"* (21.7) 138.3 (42.1) 123.3 (66.2) 66.3 (75.3)
101.7 (103.6) 231.7" (42.1) 70.0* (58.9) 113.3 (45.0) 206.7* (68.9) 110.0 (37.4)
116.7 (48.0) 161.7" (62.7) 16.7"* (58.5) 113.3 (35.0) 131.7 (71.7) 90.0 (23.7)
a
- 173.3"* (117.9)
-60.0 (88.5)
-8.3 (82.1)
b
1 2 3 4 5 6
b b a b a
Challenge 7
T = controls; V = vaccinated; VA = vaccinated + aflatoxin. F = Fisher's test. a = not significant. b = significant. *P<0.05. **P<0.01.
s.d. standard deviation.
during the second and third weeks. The weight gain of group T (controls) was significantly lower than those of groups V and VA after bacterial challenge, but there were no differences between the last two groups (Table 1 ). No significant differences were observed in the agglutinating antibody titers against B. bronchiseptica expressed as geometric mean titer, between groups V and VA, though in the latter group there was a tendency towards a higher titer (Table 2). Regarding the extent of the macroscopic pneumonic lesions, there was a significant difference between the three groups (X 2 = 1003.3; P < 0.01; Fig. 1 ). The lesion percentage found in control group T (13.4%) was significantly greater than those found in groups V and VA. Likewise, the lesion percentage found in group VA (7%) was significantly greater than that in group V (2.3%; Fig. 1 ). Microscopic findings showed that the differences in the distribution of lesions in the lungs, classified from grade 0 to grade 3 according to severity, were significant (Z: = 20.2; P < 0.005 ). Group V showed a percentage of grade3 lesions (1.4%) significantly lower than group VA (17.4%) and group T (23.2%), the last two groups not differing significantly from each other. The percentage of grade-1 lesions was significantly greater in group V (50.7%)
EFFECT OF AFLATOXINON B. BRONCHISEPTICA-VACCINATED RABBITS
213
TABLE 2 Agglutinating antibody titers against B. bronchiseptica expressed as geometric mean titer Weeks
V
VA
F
1
5.6 40.4 90.6 203.4 512.8 911.4
6.3 45.3 143.8 287.6 575.4 911.4
0.1 NS 0.1 NS 2.5 NS 0.3 NS 0.2 NS 0.0NS
2 3 4 5 6
V = vaccinated; VA = vaccinated + aflatoxin. F = Fisher's test. NS = not significant. 2
0
................................................................................................ 1
15-
I
... . . . . . . . . . . . . . . . .
...."
s
71
.... I
T
V
VA
Fig. 1. Extent of macroscopic lung lesions expressed as percentages. Differences a m o n g groups V ( 2 . 3 % ) , V A ( 7 . 0 % ) a n d T ( 1 3 . 4 % ) were significant (X2= 1003.3; P < 0 . 0 1 ).
!
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.-;._
j o
i
t
-a, ,-.
J_
:3
Gr~ad~
liT
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."1) A
Fig. 2. D i s t r i b u t i o n of microscopic lung lesions classified from grade 0 to 3 according to severity. The d i s t r i b u t i o n o f lesions differed significantly a m o n g groups (Z2= 20.2; P < 0.005).
than in other groups (Fig. 2 ). Significant differences were found in the distribution of microscopic lesions in the three regions of the lung observed (X2=40.9; P < 0 . 0 0 5 ) . Alveoli presented a greater percentage of lesions of
214
M.C. V E N T U R I N I ET AL.
grades 1-3, followed in decreasing order by the air passages and lymphoid tissue. Group V showed the greatest percentage of lesions of grade 1 in the alveoli (82.6%), but showed no grade-3 lesions. This group differed significantly in the percentage of grade-3 lesions from groups VA (21.7%) and T (47.8%), the 2 last groups also differing significantly from each other. Regarding the air passages, group V showed the greatest percentage in grade 0 (56.5%), and had no grade-3 lesions. There were no differences in the distribution of lesions in lymphoid tissue. No gross or microscopic lesions in other organs were observed. There were no significant differences between groups in the weights of liver, lungs and spleen. DISCUSSION
The reduction in weight gain of rabbits receiving aflatoxin was similar to that previously reported (Richard and Thurston, 1975; Clark et al., 1980). Although the target organ in aflatoxicosis is the liver, macroscopic and microscopic changes were not observed with the dose used in the present experiment, compared with controls. These results differ from those found by Clark et al. (1980), who described a slight increase in the number of binucleate hepatocytes and an increase in karyomegaly and biliary stasis using 0.05 m g / kg of crystalline AFL B 1. It is considered that the differences could be ascribed to the type of aflatoxin used in our experiment, which was crude and contained 75.74% AFL B 1 and 24.26% G 1, the latter being less toxic than B 1. Another reason for the differences observed could be the initial weight of the animals used in this study. Significant macroscopic differences between vaccinated and control groups were observed in the extent of the morphometrically evaluated pneumonic lesions. This might be considered an objective parameter of the lung protection conferred by vaccination. Likewise, there were significant differences between the vaccinated groups that did and did not receive aflatoxin, in that the surface area of lung affected was smaller in rabbits that had not received aflatoxin than in aflatoxin-treated rabbits. These findings correspond microscopically with the greater severity of the lesions (subjectively graded 0-3 ) considered characteristic of B. bronchiseptica infection in rabbits (Perfumo et al., 1984) in the group that had received aflatoxin; no significant differences were observed for grade-3 lesions between this group and the control group. Because of the lesions found at the alveolar level it is also possible to consider that group V was better protected than group VA. It was concluded that the vaccinated group that received aflatoxin acquired a lower level of resistance to the challenge, which was not related to the titers of agglutinating antibodies developed; in both vaccinated groups the titers were very high, with a tendency to be slightly higher in group VA. Similar experiments carried out in turkeys and pigs, in which different doses of aria-
EFFECT OF AFLATOXIN ON B. BRONCHISEPTICA-VACCINATED RABBITS
215
toxin were used, also showed reduced resistance to challenge, while antibody titers did not differ from those of the control groups (Pier and Heddleston, 1970; Cysewisky, 1978; Panangala et al., 1986 ). Thus it can be generally considered that while very high doses of aflatoxin produce a reduction in antibody titer, in chronic or subclinical intoxication the mechanism that would be most severely impaired would be cell-mediated immunity (Pier et al., 1978; Pier et al., 1980). A reduction in the endocytic activity of alveolar macrophages from rabbits that received aflatoxin has been demonstrated in vitro (Richard and Thurston, 1975 ). This could explain in part the results obtained because alveolar macrophages are considered the principal defensive barrier of the lungs. Moreover, the effective participation of macrophages in the immune response is essential, as they process and present antigens to the cells that will ultimately give rise to humoral a n d / o r cellular immune responses. ACKNOWLEDGEMENTS
This work was supported in part by grants form the National Council for Scientific and Technical Research (CONICET) and the Scientific Research Commission of the Province of Buenos Aires (CIC). We thank Dr. M. C. Castellano for her help in the preparation of the manuscript. This work was presented in part at the I st Congress of Toxicology in Developing Countries, Buenos Aires, Argentina, November 1987.
REFERENCES Buck, W.B. and Osweiler, G.D., 1976. Aflatoxin. In: G.A. van Gelder, Clinical and Diagnostic Veterinary Toxicology, 2nd edn. Kendall Hunt Publishing, Debuque, IA, 380 pp. Clark, J.D., Jain, A.B., Hatch, R.C. and Mahaffey, E.A., 1980. Experimentally induced chronic aflatoxicosis in rabbits. Am. J. Vet. Res., 11: 1041-1045. Cysewiski, S.J., 1978. Effect of aflatoxins on the development of acquired immunity to swine erysipelas. Am. J. Vet. Res., 39: 445-448. Edds, G.T., 1973. Acute aflatoxicosis: a review. J. Am. Vet. Med. Assoc., 4: 304-309. Lison, L., 1976. Estadistica aplicada a la biologia experimental. Planificaci6n de experiencias y an~lisis de resultados. Ed. Univ. Bs. As, 357 pp. Newberne, P.M., 1973. Chronic aflatoxicosis. J. Am. Vet. Med. Assoc., 11: 1262-1267. Panangala, V.S., Giambrone, J.J., Diener, U.L., Davis, N.D., Hoerr F.J., Mitra, A., Shultz, R.D. and Wild, G.R., 1986. Effects of aflatoxin on the growth performance and immune response of weanling swine. Am. J. Vet. Res., 47: 2062-2067. Perfumo, C.J., Petruccelli, M.A., Brandetti, E., Menendez, N.A., Venturini, M.C. and Arce, E.J., 1984. Bronconeumonia del conejo producida por Bordetella bronchiseptica. Estudio de un caso de campo. Bol. Assoc. Esp. Cunicultura, 7: 33-39. Pier, A.C, 1973. Effects of aflatoxins on immunity. J. Am. Vet. Med. Assoc., 11: 1268-1269. Pier, A.C. and Heddleston, K.L., 1970. The effect of aflatoxin on immunity in turkey. Impairment of actively acquired resistance to bacterial challenge. Avian. Dis., 14: 797-809.
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Pier, A.C., Richard, J.L. and Thurston, J.R., 1978. The infuence of mycotoxins on resistance and immunity. In: Proceedings of the 1978 Symposium on Interaction of Mycotoxins in Animal Production. National Academy of Sciences, Washington, D.C., pp. 56-66. Pier, A.C., Richard, J.L. and Cysewiski, S.J., 1980. Implications of mycotoxins in animal diseases. J. Am. Vet. Med. Assoc., 8:719-724. Reid, M.I., 1980. Morphometric methods in veterinary pathology: a review. Vet. Pathol., 17: 552-543. Richard, J.L. and Thurston, J.R., 1975. Effect of aflatoxin on phagocytosis of Aspergillusfumigatus spores by rabbit alveolar macrophages. Appl. Microbiol., 1: 44-47.
209
Effect of aflatoxin B 1 on resistance induced by Bordetella bronchiseptica vaccine in rabbits M.C. Venturini a, C.J. Perfumo a, M.A. Risso a, C.M. Gomez b, M.V. Piscopo a, M. Sala de Miguel c and H. G o d o y c alnstitute of Pathology, Faculty of VeterinarySciences, La Plata National University, C. C 296, 1900 La Plata, Argentina bDepartment of lmmunology, Faculty of VeterinarySciences, La Plata National University, C. C 296, 1900 La Plata, Argentina CDepartrnent of Microbiology, National Institute of Agricultural Technology (INTA), Castelar, Argentina (Accepted 12 March 1990)
ABSTRACT Venturini, M.C., Perfumo, C.J., Risso, M./~., Gomez, C.M., Piscopo, M.V., Sala de Miguel, M. and Godoy, H., 1990. Effect of aflatoxin B l on resistance induced by Bordetella bronchiseptica vaccine in rabbits. Vet. Microbiol., 25: 209-216. The effects of aflatoxin B 1 on the development of the immune response to oil-adjuvanted Bordetella bronchiseptica vaccine and on acquired resistance to bacterial challenge were studied in rabbits. The doses of aflatoxin used were insufficient to produce clinical intoxication. Rabbits were randomly assigned to three groups, each having six animals: control (T), vaccinated (V), and vaccinated plus aflatoxin (VA) at 0.05 mg/kg daily per os. Groups V and VA were vaccinated twice, and the three groups were subsequently challenged with virulent B. bronchiseptica. The average weight gain at weekly intervals was significantly reduced in group VA, and no statistically significant differences were found in the titers of agglutinating antibodies between groups V and VA. There were significant differences between groups V and VA in the extent and severity of the pneumonic process, group VA being most affected. Results indicated that agglutinating antibody titers were not related to the level of protection in the latter group. Other mechanisms, such as alveolar macrophage activity and cell-mediated immunity, were implicated in the impairment of the acquired resistance in rabbits subclinically intoxicated with aflatoxin.
INTRODUCTION
Aflatoxins are toxic metabolites derived from certain strains of Aspergillus flavus and A. parasiticus, which grow on several substrates used in animal feeds. Five major toxins - BI, G1, B2, G2, and M1 - have been described, and have proved to be potent hepatotoxic and carcinogenic agents. B 1 is the most toxic and the most abundant. They are low molecular weight bifururanocoumarin derivates (Edds, 1973; Newberne, 1973; Buck and Osweiler, 0378-1135/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
210
M.C. VENTURINI ET AL.
1976; Clark et al., 1980; Pier et al., 1980). The consumption of aflatoxins by birds and swine leads to increased susceptibility to diseases and impairment of acquired resistance against bacterial and parasitic infections (Pier and Heddleston, 1970; Pier, 1973; Cysewiski, 1978; Pier et al., 1980; Panagala et al., 1986). The aim of this study was to investigate the effect of aflatoxin B 1 (AFL B 1 ) on the i m m u n e response to oil-adjuvanted Bordetella bronchiseptica vaccine in chronically intoxicated rabbits, as well as to determine its effect on acquired resistance to bacterial challenge. MATERIALS AND METHODS
Animals and experimental design 18 Rabbits with an average body weight of 2.2 kg were divided into three groups: V, vaccinated; VA, vaccinated plus treatment with AFL B 1 (87.87% B 1 equivalent); and T, controls. Animals were in six blocks. One animal from each group was randomly assigned to one block ( IV, 1VA, IT; Lison, 1976 ). Animals were housed in six cages, each containing one animal from each group. Commercial food and water were available ad libitum. Animals were weighed daily.
Aflatoxin Aflatoxin produced by A. flavus, consisting of 75.74% B1 and 24.26% G 1.G 1, was considered half as toxic as B 1. Aflatoxin was prepared by dissolving it in chloroform and then in edible oil to an approximate concentration of 0.2 m g / m l . According to Clark et al. ( 1980 ), the a m o u n t of AFL B 1 required to induce chronic intoxication in rabbits was 0.05 m g / k g live weight. This dosage was administrated daily per os from day 1 until day 43 of the experiment. Bordetella bronchiseptica vaccine Groups V and VA were vaccinated on days 1 and 14 of the experiment with an experimental oil-adjuvanted B. bronchiseptica vaccine, derived from a strain isolated from an outbreak ofbronchopneumonia in rabbits. It was grown on Border Gengou Agar (Oxoid) with 10% defribrinated sheep blood, and was incubated for 48 h at 37°C, then on charcoal agar (Oxoid) for 72 h at 37 ° C. Phase control was carried out with B. bronchiseptica phase I Kitasato antiserum (Kitasato Institute, Tokyo, Japan), used as directed by the manufacturer. The culture was harvested in PBS, inactivated by adding 0.3% formalin and heating at 37 °C for 48 h, and was incorporated into a double emulsion (water-in-oil-in-water) adjuvant consisting of a mineral oil (Marcol, 86%), an emulsifier (Arlacel A, 14%) and a detergent (Tween 80, 2% in PBS). The vaccine contained 10 OIU (International Reference Preparation), equivalent to approximately 10 ~° bacteria/ml. The three groups were chal-
EFFECT OF AFLATOXINON B. BRONCHISEPTICA-VACC1NATED RABBITS
21 1
lenged intranasally with the live B. bronchiseptica used in the preparation of the vaccine (0.5 ml into each nostril, 2 × 109 bacteria/ml) on day 44.
Serology Serum samples were obtained weekly, and were examined for agglutinating antibodies using a tube agglutination test with the AR Kitasato antigen, used as directed by the manufacturer. Complete agglutination was considered positive after incubation for 2 h at 37 °C and overnight at 4°C.
Necropsy and h&topathologic examination Necropsy was carried out on all rabbits, beginning 2 days (day 46) after challenge for the first block, and on days 48, 50, 52, 54 and 56 for each remaining block. Macroscopic effects were measured by morphometric methods previously described (Reid, 1980). One photograph was taken of the dorsal and ventral aspects of each lung of each rabbit. Point-counting morphometry was done using a point test grid superimposed on the photograph. Relative percentages were obtained from the total number of points for the two sides minus the number of points counted in the affected area, multiplied by 100. Liver, lung and spleen were weighed individually; for histopathologic study they were fixed in neutral buffered 10% formalin, sectioned and stained with hematoxylin and eosin. Lung lesions were classified from grade 0 to 3 according to severity, as follows: ( 1 ) Air passages: grade 0, without microscopic lesions; grade 1, presence of exudate in the lumen of bronchi and bronchioli without changes in the walls; grade 2, presence of exudate in the lumen of bronchi and bronchioli, with epithelial desquamation and mononuclear infiltration of the walls; grade 3, obstructive bronchiolitis. (2) Alveoli: grade 0, without microscopic lesions; grade 1, mild to moderate thickening of alveolar walls due to inflammatory cell infiltration; grade 2, as grade 1 plus abundant mononuclear infiltration of the alveolar lumen in most of the fields observed; grade 3, as grade 2 plus alveolar necrosis. (3) Lymphoid tissue: grade 0, absence of lymphoid tissue; grade 1, mild lymphoid hyperplasia; grade 2, moderate lymphoid hypertrophy with bronchiolar deformity; grade 3, severe lymphoid hypertrophy with bronchiolar collapse.
Statistics Data on weekly weight gain, organ weights and serology were analyzed by Fisher's test. Gross and microscopic lesions were analyzed by the Z 2 test. A value of P < 0.05 was considered significant. RESULTS
Measurements of weekly weight gain indicated significant differences between the three groups studied. Group VA was the most affected, particularly
212
M.C. VENTURIN1 ET AL.
TABLE 1
Weekly weight gain (g). Mean _+s.d. Weeks
T
V
VA
F
145.0 (44.6) 246.7* (78.6) 155.0"* (21.7) 138.3 (42.1) 123.3 (66.2) 66.3 (75.3)
101.7 (103.6) 231.7" (42.1) 70.0* (58.9) 113.3 (45.0) 206.7* (68.9) 110.0 (37.4)
116.7 (48.0) 161.7" (62.7) 16.7"* (58.5) 113.3 (35.0) 131.7 (71.7) 90.0 (23.7)
a
- 173.3"* (117.9)
-60.0 (88.5)
-8.3 (82.1)
b
1 2 3 4 5 6
b b a b a
Challenge 7
T = controls; V = vaccinated; VA = vaccinated + aflatoxin. F = Fisher's test. a = not significant. b = significant. *P<0.05. **P<0.01.
s.d. standard deviation.
during the second and third weeks. The weight gain of group T (controls) was significantly lower than those of groups V and VA after bacterial challenge, but there were no differences between the last two groups (Table 1 ). No significant differences were observed in the agglutinating antibody titers against B. bronchiseptica expressed as geometric mean titer, between groups V and VA, though in the latter group there was a tendency towards a higher titer (Table 2). Regarding the extent of the macroscopic pneumonic lesions, there was a significant difference between the three groups (X 2 = 1003.3; P < 0.01; Fig. 1 ). The lesion percentage found in control group T (13.4%) was significantly greater than those found in groups V and VA. Likewise, the lesion percentage found in group VA (7%) was significantly greater than that in group V (2.3%; Fig. 1 ). Microscopic findings showed that the differences in the distribution of lesions in the lungs, classified from grade 0 to grade 3 according to severity, were significant (Z: = 20.2; P < 0.005 ). Group V showed a percentage of grade3 lesions (1.4%) significantly lower than group VA (17.4%) and group T (23.2%), the last two groups not differing significantly from each other. The percentage of grade-1 lesions was significantly greater in group V (50.7%)
EFFECT OF AFLATOXINON B. BRONCHISEPTICA-VACCINATED RABBITS
213
TABLE 2 Agglutinating antibody titers against B. bronchiseptica expressed as geometric mean titer Weeks
V
VA
F
1
5.6 40.4 90.6 203.4 512.8 911.4
6.3 45.3 143.8 287.6 575.4 911.4
0.1 NS 0.1 NS 2.5 NS 0.3 NS 0.2 NS 0.0NS
2 3 4 5 6
V = vaccinated; VA = vaccinated + aflatoxin. F = Fisher's test. NS = not significant. 2
0
................................................................................................ 1
15-
I
... . . . . . . . . . . . . . . . .
...."
s
71
.... I
T
V
VA
Fig. 1. Extent of macroscopic lung lesions expressed as percentages. Differences a m o n g groups V ( 2 . 3 % ) , V A ( 7 . 0 % ) a n d T ( 1 3 . 4 % ) were significant (X2= 1003.3; P < 0 . 0 1 ).
!
-I .~ -_-.i I
303
....~ i-,71-
.-;._
j o
i
t
-a, ,-.
J_
:3
Gr~ad~
liT
I!H M
."1) A
Fig. 2. D i s t r i b u t i o n of microscopic lung lesions classified from grade 0 to 3 according to severity. The d i s t r i b u t i o n o f lesions differed significantly a m o n g groups (Z2= 20.2; P < 0.005).
than in other groups (Fig. 2 ). Significant differences were found in the distribution of microscopic lesions in the three regions of the lung observed (X2=40.9; P < 0 . 0 0 5 ) . Alveoli presented a greater percentage of lesions of
214
M.C. V E N T U R I N I ET AL.
grades 1-3, followed in decreasing order by the air passages and lymphoid tissue. Group V showed the greatest percentage of lesions of grade 1 in the alveoli (82.6%), but showed no grade-3 lesions. This group differed significantly in the percentage of grade-3 lesions from groups VA (21.7%) and T (47.8%), the 2 last groups also differing significantly from each other. Regarding the air passages, group V showed the greatest percentage in grade 0 (56.5%), and had no grade-3 lesions. There were no differences in the distribution of lesions in lymphoid tissue. No gross or microscopic lesions in other organs were observed. There were no significant differences between groups in the weights of liver, lungs and spleen. DISCUSSION
The reduction in weight gain of rabbits receiving aflatoxin was similar to that previously reported (Richard and Thurston, 1975; Clark et al., 1980). Although the target organ in aflatoxicosis is the liver, macroscopic and microscopic changes were not observed with the dose used in the present experiment, compared with controls. These results differ from those found by Clark et al. (1980), who described a slight increase in the number of binucleate hepatocytes and an increase in karyomegaly and biliary stasis using 0.05 m g / kg of crystalline AFL B 1. It is considered that the differences could be ascribed to the type of aflatoxin used in our experiment, which was crude and contained 75.74% AFL B 1 and 24.26% G 1, the latter being less toxic than B 1. Another reason for the differences observed could be the initial weight of the animals used in this study. Significant macroscopic differences between vaccinated and control groups were observed in the extent of the morphometrically evaluated pneumonic lesions. This might be considered an objective parameter of the lung protection conferred by vaccination. Likewise, there were significant differences between the vaccinated groups that did and did not receive aflatoxin, in that the surface area of lung affected was smaller in rabbits that had not received aflatoxin than in aflatoxin-treated rabbits. These findings correspond microscopically with the greater severity of the lesions (subjectively graded 0-3 ) considered characteristic of B. bronchiseptica infection in rabbits (Perfumo et al., 1984) in the group that had received aflatoxin; no significant differences were observed for grade-3 lesions between this group and the control group. Because of the lesions found at the alveolar level it is also possible to consider that group V was better protected than group VA. It was concluded that the vaccinated group that received aflatoxin acquired a lower level of resistance to the challenge, which was not related to the titers of agglutinating antibodies developed; in both vaccinated groups the titers were very high, with a tendency to be slightly higher in group VA. Similar experiments carried out in turkeys and pigs, in which different doses of aria-
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toxin were used, also showed reduced resistance to challenge, while antibody titers did not differ from those of the control groups (Pier and Heddleston, 1970; Cysewisky, 1978; Panangala et al., 1986 ). Thus it can be generally considered that while very high doses of aflatoxin produce a reduction in antibody titer, in chronic or subclinical intoxication the mechanism that would be most severely impaired would be cell-mediated immunity (Pier et al., 1978; Pier et al., 1980). A reduction in the endocytic activity of alveolar macrophages from rabbits that received aflatoxin has been demonstrated in vitro (Richard and Thurston, 1975 ). This could explain in part the results obtained because alveolar macrophages are considered the principal defensive barrier of the lungs. Moreover, the effective participation of macrophages in the immune response is essential, as they process and present antigens to the cells that will ultimately give rise to humoral a n d / o r cellular immune responses. ACKNOWLEDGEMENTS
This work was supported in part by grants form the National Council for Scientific and Technical Research (CONICET) and the Scientific Research Commission of the Province of Buenos Aires (CIC). We thank Dr. M. C. Castellano for her help in the preparation of the manuscript. This work was presented in part at the I st Congress of Toxicology in Developing Countries, Buenos Aires, Argentina, November 1987.
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Pier, A.C., Richard, J.L. and Thurston, J.R., 1978. The infuence of mycotoxins on resistance and immunity. In: Proceedings of the 1978 Symposium on Interaction of Mycotoxins in Animal Production. National Academy of Sciences, Washington, D.C., pp. 56-66. Pier, A.C., Richard, J.L. and Cysewiski, S.J., 1980. Implications of mycotoxins in animal diseases. J. Am. Vet. Med. Assoc., 8:719-724. Reid, M.I., 1980. Morphometric methods in veterinary pathology: a review. Vet. Pathol., 17: 552-543. Richard, J.L. and Thurston, J.R., 1975. Effect of aflatoxin on phagocytosis of Aspergillusfumigatus spores by rabbit alveolar macrophages. Appl. Microbiol., 1: 44-47.