Veterinary Parasitology, 42 (1992) 265-272 Elsevier Science Publishers B.V., Amsterdam
265
Renal involvement in mice experimentally infected with Toxocara canis embryonated eggs L. Casarosa, R. Papini, F. Mancianti, F. Abramo and A. Poli Dipartimento di Patologia Anirnale, Profilassi ed Igiene degli Alimenti, Facoltgt di Medicina Veterinaria, Universitgt di Pisa, Viale delle Piagge 2, Pisa, Italy (Accepted 31 October 1991 )
ABSTRACT Casarosa, L., Papini, R., Mancianti, F., Abramo, F. and Poli, A., 1992. Renal involvement in mice experimentally infected with Toxocara canis embryonated eggs. Vet. Parasitol., 42: 265-272. Histological examination of kidneys from mice experimentally infected with Toxocara canis embryonated eggs demonstrated the presence of a segmental or diffuse mesangioproliferative glomerulonephritis. Immunohistochemicalstudies established that renal alterations were associated with glomerular deposits of IgG, IgM and third component of complement (C3). These findings suggest that an immunomediated mechanism might possibly be involved in the genesis of kidney damage observed in mice infected with T. canis embryonated eggs.
INTRODUCTION
Histopathological lesions directly related to the visceral migration of Toxocara canis L2 larvae have been accurately described in humans (Beaver et al., 1984 ), rabbits and baboons (Fernando, 1968; Aljeboori et al., 1970). In particular, the mouse is considered a useful animal model for histopathological studies in visceral toxocariasis (Kayes and Oaks, 1978). Host inflammatory lesions (Zyngier, 1974) and a specific immune response against excretory/secretory products of the parasite (Nicholas et al., 1984 ), have already been documented in experimentally-infected mice. Excretory/secretory antigens derived from T. canis larvae have been demonstrated in serum and liver tissue of mice either acutely or chronically infected and play a role in granulomatous inflammation (Parsons et al., 1986 ). IgG and IgM antibodies with specific reactivity to the parasite antigens are produced and sequentially complexed to form circulating immune complexes CIC (Bowman et al., 1987). While the role of these antibodies in induction of larval trapping in the liver of infected mice and their coexistence in the granulomatous lesions with the antigens have been well documented (Akao, 1985; Akao et al., 1986 ), little is © 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
266
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known about the role of produced i m m u n e complexes in the genesis of secondary lesions in microascaridiosis. In this study, we infected mice with T. canis embryonated eggs in order to provide data on the pathological and immunological features of a possible secondary renal involvement in murine toxocariasis. MATERIALS AND METHODS
Egg cultures Toxocara canis eggs were obtained from adult female worms found at necropsy in the intestinal lumen of naturally-infected puppies. The eggs were cultured until embryonated as previously described by Bowman et al. ( 1987 ). Experimental infection The study was undertaken on ten 7-week-old BALB/c mice. Each mouse was given approximately 1000 T. canis embryonated eggs in 0.5 ml water by stomach tube. Five 7-week-old BALB/c mice were used as control. All the infected and non-infected mice were heavily anesthetized and exsanguinated via the brachial artery at 60 days post-infection.
Renal histology During necropsy of infected and non-infected mice kidneys were rapidly removed and renal tissue, which was to undergo light microscopy, was fixed in 10% buffered formalin solution and embedded in paraffin. Sections 3 / t m thick were stained with hematoxylin-eosin (H&E), periodic acid Schiff, and Jones' periodic acid silver methenamine (PASM) for histological examination. Congo red staining was performed on 5/zm thick sections.
Immunohistochemistry Localization of IgG, IgM and third c o m p o n e n t of complement (C3) deposits was performed by indirect immunofluorescence (IF) on frozen 4 p m sections fixed in acetone, and by peroxidase-anti-peroxidase (PAP) on formalin fixed and paraffin embedded sections as previously described (Poli et al., 1991 ). As primary antibodies rabbit anti-mouse IgG, IgM and C3 (Technogenetics, Turin) were used. A fluorescein-conjugated goat anti-rabbit IgG serum (Miles Italiana, Milan) and a secondary goat anti-rabbit IgG and a PAP complex (Miles Italiana, Milan ) were used for IF m e t h o d and in PAP procedure, respectively.
KIDNEY DAMAGE IN MICE INFECTED WITH T. CANIS EGGS
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Method controls involved incubation of the sections with normal rabbit serum (Dako, Glostrup, Denmark) for IF test, and omission of the primary sera for PAP procedure.
Ultrastructure For electron microscopy 1 mm 3 pieces of renal cortex were fixed in Karnowsky's solution, washed in several changes of phosphate buffered saline (PBS) (pH 7.3) and postfixed in 1% osmium tetroxide in PBS buffer (pH 7.3) for 2 h at 4-6 ° C. Pieces were then dehydrated in an increasing concentration of ethyl alcohol, immersed in two changes of propylene oxide and embedded in Epon-Araldite. Ultrathin sections, selected from 0.5/~m Blue Toluidine stained sections, were double stained with 2% uranyl acetate and lead citrate and examined with a Siemens Elmoskope 101 electron microscope (Siemens, Berlin).
Urine investigation Samples of urine were collected from infected and non-infected mice. Quantitative and qualitative proteinuria were assessed using commercial protein assay (BioRad, CA) and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970), respectively. RESULTS Sections of kidney from infected mice contained few scattered foci of inflammatory cells that included mainly neutrophils, lymphocytes, eosinophils and macrophages which surrounded helmintic larvae or their remnants. Furthermore a secondary renal involvement not directly related to the larval localization was observed in all the experimentally infected mice. In seven of the ten infected mice we observed a focal/segmental mesangio proliferative glomerulonephritis. The damaged glomeruli showed segmental areas of mesangial cell proliferation with light enhancement ofmesangial matrix (Fig. 1 ). In these mice immunohistochemical studies allowed observation of limited mesangial deposits of IgG and C3 near injured areas (Fig. 2 ), while IgM deposits were less frequently observed. The remaining three infected mice showed a generalized diffuse mesangial proliferative glomerulonephritis observed by moderate diffuse hypercellularity and mesangial matrix increase with narrowing of capillary loops (Fig. 3 ). Where mesangial proliferation was particularly evident a few granulocytes were often observed. In PASM stained sections, the basement membranes were
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Fig. 1. Infected mouse with segmental mesangio proliferative glomerulonephritis. The glomerulus showsonly segmentalmesangialwideningand mild increasein the cell number. H&E X 380.
Fig. 2. Infected mouse with segmentalmesangioproliferative glomerulonephritis.Limited mesangial IgG deposits. IFX 260. thin. These glomerular lesions were associated with the presence o f a scanty periglomerular and peritubular infiltrate made up of lymphocytes and plasmacells. By means of i m m u n o h i s t o c h e m i c a l investigations, in these mice, the most relevant finding was granular deposits o f C3 along the capillary walls and especially in mesangial areas (Fig. 4). In the same areas IgG and IgM deposits were also present. Ultrastructural studies revealed small mesangial electron-
KIDNEY DAMAGE IN MICE INFECTED WITH T. CANIS EGGS
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Fig. 3. Infected mouse with diffuse mesangio proliferative glomerulonephritis. Diffuse increase in mesangial matrix with mild hypercellularity. H&E X 380.
Fig. 4. Infected mouse with diffuse mesangio proliferative glomerulonephritis. Diffuse C3 deposits in mesangial areas and along capillary loops. IF X 260. dense deposits a n d rare subendothelial deposits (Fig. 5 ). No lesions as described above, were f o u n d in the k i d n e y s o f n o n - i n f e c t e d mice. In all infected mice a low g l o m e r u l a r selective p r o t e i n u r i a ( 3 3 0 - 3 8 0 m g l - t ) was f o u n d , whereas, in n o n - i n f e c t e d mice the p r o t e i n u r i a was lower t h a n 150 mg1-1 .
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Fig. 5. Infected mouse with diffuse mesangio proliferative glomerulonephritis. Small paramesangial electron dense deposits. Electron micrograph X 13 000. DISCUSSION
Only a few reports document a glomerular involvement in helmintic infections. Glomerulonephritis and presence of i m m u n e deposits were observed in Dirofilaria immitis infection (Abramowsky et al., 1981; Aikawa et al., 1981 ), in Angiostrongylus vasorum infection (Caruso, 1983 ) in dogs, and in Schistosoma mansoni and Schistosomajaponicum infections in humans (Andrade and Rocha, 1979). Intestinal parasites are not known to be associated with renal diseases; however, during larval migration renal involvement may Occur.
Our pathological and immunohistochemical studies demonstrated the presence of a glomerulonephropathy in mice experimentally infected with T. canis. These renal lesions might be the result of an immuno-mediated mechanism as where mesangial proliferation was evident the presence of IgG, IgM and C3 deposits could be demonstrated. Detection ofglomerular IgM deposits after a 2 m o n t h infection was unexpected. In other experimental studies,
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the apparently continual IgM response during chronic infection denies the validity of any attempt to determine the age of the infection using relative IgG and IgM levels (Bowman et al., 1987). Although the pathogenetic mechanism responsible for immune deposition is not yet understood, different mechanisms can be postulated: (a) occurrence of in situ immune complexes formation as in Arthus reaction; (b) presence of CIC and their consequent deposition at the glomerular level. In conclusion, our findings suggest that in experimental murine toxocariasis, secondary renal damage may develop, probably mediated immunologically. Further studies are needed to gain a better understanding of the role of immune complexes in the genesis of these secondary lesions.
REFERENCES Abramowsky, C.R., Powers, K.G., Aikawa, M. and Swineheart, G., 1981. Dirofilaria immitis. 5. Immunopathology of filarial nephropathy in dogs. Am. J. Pathol., 104: 1-2. Aikawa, M., Abramowsky, C.R., Powers, K.G. and Furrow, R., 1981. Dirofilariasis. IV. Glomerulonephropathy induced by Dirofilaria immitis infection. Am. J. Trop. Med. Hyg., 30: 8491. Akao, N., 1985. Immune response to excretory-secretory products of second stage larvae of Toxocara canis: humoral immune response relating to larval trapping in the liver of reinfected mice. Jpn. J. Parasitol., 34: 293-300. Akao, N., Kondo, K., Sakai, H. and Yoshimura, H., 1986. An immunopathological study of the liver of the mice infected with Toxocara canis. Jpn. J. Parasitol., 35:135-140. Aljeboori, T.I., Scott, C. and Ivey, M.H., 1970. Toxocara canis infection in baboons. II. Distribution of larvae and histopathologic response. Am. J. Trop. Med. Hyg., 19:815-820. Andrade, Z.A. and Rocha, H., 1979. Schistosomal glomerulopathy. Kidney Int., 16: 23-29. Beaver, P.C., Jung, R.C. and Cupp, E.W., 1984. Oxyuroidea and Ascaridoidea. In: Clinical Parasitology, 9th edn. Lea & Febiger, Philadelphia, PA, pp. 302-334. Bowman, D.D., Mika-Grieve, M. and Grieve, R.B., 1987. Circulating excretory-secretory antigen levels and specific antibody responses in mice infected with Toxocara canis. Am. J. Trop. Med. Hyg., 36: 75-82. Caruso, J.P., 1983. Selected aspects of the immuno-response of dogs to Angiostrongylus vasorum infection. Diss. Abstr. Int., 44(3): 1372-1373. Fernando, S.T., 1968. Immunological response of rabbit to Toxocara canis infection. Parasitology, 58: 91-103. Kayes, S.G. and Oaks, J.A., 1978. Development of the granulomatous response in murine toxocariasis. Am. J. Pathol., 93: 277-295. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophages T4. Nature, 227: 680-685. Nicholas, W.L., Stewart, A.C. and Mitchell, G.F., 1984. Antibody responses to Toxocara canis using sera from parasite-infected mice and protection from toxocariasis by immunisation with ES antigens. Aust. J. Exp. Biol. Med. Sci., 65:619-626. Parsons, J.C., Bowman, D.D. and Grieve, R.B., 1986. Tissue localization of excretory-secretory antigens of larval Toxocara canis in acute and chronic murine toxocariasis. Am. J. Trop. Med. Hyg., 35: 974-981. Poli, A., Abramo, F., Mancianti, F., Nigro, M., Pieri, S. and Bionda, A., 1991. Renal involve-
272
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ment in canine leishmaniasis. A light microscopic immunohistochemical and electronmicroscopic study. Nephron, 57: 444-452. Zyngier, F.R., 1974. Histopathology of experimental toxocariasis in mice. Ann. Trop. Med. Parasitol., 68: 225-228.
265
Renal involvement in mice experimentally infected with Toxocara canis embryonated eggs L. Casarosa, R. Papini, F. Mancianti, F. Abramo and A. Poli Dipartimento di Patologia Anirnale, Profilassi ed Igiene degli Alimenti, Facoltgt di Medicina Veterinaria, Universitgt di Pisa, Viale delle Piagge 2, Pisa, Italy (Accepted 31 October 1991 )
ABSTRACT Casarosa, L., Papini, R., Mancianti, F., Abramo, F. and Poli, A., 1992. Renal involvement in mice experimentally infected with Toxocara canis embryonated eggs. Vet. Parasitol., 42: 265-272. Histological examination of kidneys from mice experimentally infected with Toxocara canis embryonated eggs demonstrated the presence of a segmental or diffuse mesangioproliferative glomerulonephritis. Immunohistochemicalstudies established that renal alterations were associated with glomerular deposits of IgG, IgM and third component of complement (C3). These findings suggest that an immunomediated mechanism might possibly be involved in the genesis of kidney damage observed in mice infected with T. canis embryonated eggs.
INTRODUCTION
Histopathological lesions directly related to the visceral migration of Toxocara canis L2 larvae have been accurately described in humans (Beaver et al., 1984 ), rabbits and baboons (Fernando, 1968; Aljeboori et al., 1970). In particular, the mouse is considered a useful animal model for histopathological studies in visceral toxocariasis (Kayes and Oaks, 1978). Host inflammatory lesions (Zyngier, 1974) and a specific immune response against excretory/secretory products of the parasite (Nicholas et al., 1984 ), have already been documented in experimentally-infected mice. Excretory/secretory antigens derived from T. canis larvae have been demonstrated in serum and liver tissue of mice either acutely or chronically infected and play a role in granulomatous inflammation (Parsons et al., 1986 ). IgG and IgM antibodies with specific reactivity to the parasite antigens are produced and sequentially complexed to form circulating immune complexes CIC (Bowman et al., 1987). While the role of these antibodies in induction of larval trapping in the liver of infected mice and their coexistence in the granulomatous lesions with the antigens have been well documented (Akao, 1985; Akao et al., 1986 ), little is © 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
266
L. CASAROSA ET AL.
known about the role of produced i m m u n e complexes in the genesis of secondary lesions in microascaridiosis. In this study, we infected mice with T. canis embryonated eggs in order to provide data on the pathological and immunological features of a possible secondary renal involvement in murine toxocariasis. MATERIALS AND METHODS
Egg cultures Toxocara canis eggs were obtained from adult female worms found at necropsy in the intestinal lumen of naturally-infected puppies. The eggs were cultured until embryonated as previously described by Bowman et al. ( 1987 ). Experimental infection The study was undertaken on ten 7-week-old BALB/c mice. Each mouse was given approximately 1000 T. canis embryonated eggs in 0.5 ml water by stomach tube. Five 7-week-old BALB/c mice were used as control. All the infected and non-infected mice were heavily anesthetized and exsanguinated via the brachial artery at 60 days post-infection.
Renal histology During necropsy of infected and non-infected mice kidneys were rapidly removed and renal tissue, which was to undergo light microscopy, was fixed in 10% buffered formalin solution and embedded in paraffin. Sections 3 / t m thick were stained with hematoxylin-eosin (H&E), periodic acid Schiff, and Jones' periodic acid silver methenamine (PASM) for histological examination. Congo red staining was performed on 5/zm thick sections.
Immunohistochemistry Localization of IgG, IgM and third c o m p o n e n t of complement (C3) deposits was performed by indirect immunofluorescence (IF) on frozen 4 p m sections fixed in acetone, and by peroxidase-anti-peroxidase (PAP) on formalin fixed and paraffin embedded sections as previously described (Poli et al., 1991 ). As primary antibodies rabbit anti-mouse IgG, IgM and C3 (Technogenetics, Turin) were used. A fluorescein-conjugated goat anti-rabbit IgG serum (Miles Italiana, Milan) and a secondary goat anti-rabbit IgG and a PAP complex (Miles Italiana, Milan ) were used for IF m e t h o d and in PAP procedure, respectively.
KIDNEY DAMAGE IN MICE INFECTED WITH T. CANIS EGGS
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Method controls involved incubation of the sections with normal rabbit serum (Dako, Glostrup, Denmark) for IF test, and omission of the primary sera for PAP procedure.
Ultrastructure For electron microscopy 1 mm 3 pieces of renal cortex were fixed in Karnowsky's solution, washed in several changes of phosphate buffered saline (PBS) (pH 7.3) and postfixed in 1% osmium tetroxide in PBS buffer (pH 7.3) for 2 h at 4-6 ° C. Pieces were then dehydrated in an increasing concentration of ethyl alcohol, immersed in two changes of propylene oxide and embedded in Epon-Araldite. Ultrathin sections, selected from 0.5/~m Blue Toluidine stained sections, were double stained with 2% uranyl acetate and lead citrate and examined with a Siemens Elmoskope 101 electron microscope (Siemens, Berlin).
Urine investigation Samples of urine were collected from infected and non-infected mice. Quantitative and qualitative proteinuria were assessed using commercial protein assay (BioRad, CA) and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970), respectively. RESULTS Sections of kidney from infected mice contained few scattered foci of inflammatory cells that included mainly neutrophils, lymphocytes, eosinophils and macrophages which surrounded helmintic larvae or their remnants. Furthermore a secondary renal involvement not directly related to the larval localization was observed in all the experimentally infected mice. In seven of the ten infected mice we observed a focal/segmental mesangio proliferative glomerulonephritis. The damaged glomeruli showed segmental areas of mesangial cell proliferation with light enhancement ofmesangial matrix (Fig. 1 ). In these mice immunohistochemical studies allowed observation of limited mesangial deposits of IgG and C3 near injured areas (Fig. 2 ), while IgM deposits were less frequently observed. The remaining three infected mice showed a generalized diffuse mesangial proliferative glomerulonephritis observed by moderate diffuse hypercellularity and mesangial matrix increase with narrowing of capillary loops (Fig. 3 ). Where mesangial proliferation was particularly evident a few granulocytes were often observed. In PASM stained sections, the basement membranes were
268
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Fig. 1. Infected mouse with segmental mesangio proliferative glomerulonephritis. The glomerulus showsonly segmentalmesangialwideningand mild increasein the cell number. H&E X 380.
Fig. 2. Infected mouse with segmentalmesangioproliferative glomerulonephritis.Limited mesangial IgG deposits. IFX 260. thin. These glomerular lesions were associated with the presence o f a scanty periglomerular and peritubular infiltrate made up of lymphocytes and plasmacells. By means of i m m u n o h i s t o c h e m i c a l investigations, in these mice, the most relevant finding was granular deposits o f C3 along the capillary walls and especially in mesangial areas (Fig. 4). In the same areas IgG and IgM deposits were also present. Ultrastructural studies revealed small mesangial electron-
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Fig. 3. Infected mouse with diffuse mesangio proliferative glomerulonephritis. Diffuse increase in mesangial matrix with mild hypercellularity. H&E X 380.
Fig. 4. Infected mouse with diffuse mesangio proliferative glomerulonephritis. Diffuse C3 deposits in mesangial areas and along capillary loops. IF X 260. dense deposits a n d rare subendothelial deposits (Fig. 5 ). No lesions as described above, were f o u n d in the k i d n e y s o f n o n - i n f e c t e d mice. In all infected mice a low g l o m e r u l a r selective p r o t e i n u r i a ( 3 3 0 - 3 8 0 m g l - t ) was f o u n d , whereas, in n o n - i n f e c t e d mice the p r o t e i n u r i a was lower t h a n 150 mg1-1 .
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Fig. 5. Infected mouse with diffuse mesangio proliferative glomerulonephritis. Small paramesangial electron dense deposits. Electron micrograph X 13 000. DISCUSSION
Only a few reports document a glomerular involvement in helmintic infections. Glomerulonephritis and presence of i m m u n e deposits were observed in Dirofilaria immitis infection (Abramowsky et al., 1981; Aikawa et al., 1981 ), in Angiostrongylus vasorum infection (Caruso, 1983 ) in dogs, and in Schistosoma mansoni and Schistosomajaponicum infections in humans (Andrade and Rocha, 1979). Intestinal parasites are not known to be associated with renal diseases; however, during larval migration renal involvement may Occur.
Our pathological and immunohistochemical studies demonstrated the presence of a glomerulonephropathy in mice experimentally infected with T. canis. These renal lesions might be the result of an immuno-mediated mechanism as where mesangial proliferation was evident the presence of IgG, IgM and C3 deposits could be demonstrated. Detection ofglomerular IgM deposits after a 2 m o n t h infection was unexpected. In other experimental studies,
KIDNEY DAMAGE IN MICE INFECTED WITH T. CAN1S EGGS
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the apparently continual IgM response during chronic infection denies the validity of any attempt to determine the age of the infection using relative IgG and IgM levels (Bowman et al., 1987). Although the pathogenetic mechanism responsible for immune deposition is not yet understood, different mechanisms can be postulated: (a) occurrence of in situ immune complexes formation as in Arthus reaction; (b) presence of CIC and their consequent deposition at the glomerular level. In conclusion, our findings suggest that in experimental murine toxocariasis, secondary renal damage may develop, probably mediated immunologically. Further studies are needed to gain a better understanding of the role of immune complexes in the genesis of these secondary lesions.
REFERENCES Abramowsky, C.R., Powers, K.G., Aikawa, M. and Swineheart, G., 1981. Dirofilaria immitis. 5. Immunopathology of filarial nephropathy in dogs. Am. J. Pathol., 104: 1-2. Aikawa, M., Abramowsky, C.R., Powers, K.G. and Furrow, R., 1981. Dirofilariasis. IV. Glomerulonephropathy induced by Dirofilaria immitis infection. Am. J. Trop. Med. Hyg., 30: 8491. Akao, N., 1985. Immune response to excretory-secretory products of second stage larvae of Toxocara canis: humoral immune response relating to larval trapping in the liver of reinfected mice. Jpn. J. Parasitol., 34: 293-300. Akao, N., Kondo, K., Sakai, H. and Yoshimura, H., 1986. An immunopathological study of the liver of the mice infected with Toxocara canis. Jpn. J. Parasitol., 35:135-140. Aljeboori, T.I., Scott, C. and Ivey, M.H., 1970. Toxocara canis infection in baboons. II. Distribution of larvae and histopathologic response. Am. J. Trop. Med. Hyg., 19:815-820. Andrade, Z.A. and Rocha, H., 1979. Schistosomal glomerulopathy. Kidney Int., 16: 23-29. Beaver, P.C., Jung, R.C. and Cupp, E.W., 1984. Oxyuroidea and Ascaridoidea. In: Clinical Parasitology, 9th edn. Lea & Febiger, Philadelphia, PA, pp. 302-334. Bowman, D.D., Mika-Grieve, M. and Grieve, R.B., 1987. Circulating excretory-secretory antigen levels and specific antibody responses in mice infected with Toxocara canis. Am. J. Trop. Med. Hyg., 36: 75-82. Caruso, J.P., 1983. Selected aspects of the immuno-response of dogs to Angiostrongylus vasorum infection. Diss. Abstr. Int., 44(3): 1372-1373. Fernando, S.T., 1968. Immunological response of rabbit to Toxocara canis infection. Parasitology, 58: 91-103. Kayes, S.G. and Oaks, J.A., 1978. Development of the granulomatous response in murine toxocariasis. Am. J. Pathol., 93: 277-295. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophages T4. Nature, 227: 680-685. Nicholas, W.L., Stewart, A.C. and Mitchell, G.F., 1984. Antibody responses to Toxocara canis using sera from parasite-infected mice and protection from toxocariasis by immunisation with ES antigens. Aust. J. Exp. Biol. Med. Sci., 65:619-626. Parsons, J.C., Bowman, D.D. and Grieve, R.B., 1986. Tissue localization of excretory-secretory antigens of larval Toxocara canis in acute and chronic murine toxocariasis. Am. J. Trop. Med. Hyg., 35: 974-981. Poli, A., Abramo, F., Mancianti, F., Nigro, M., Pieri, S. and Bionda, A., 1991. Renal involve-
272
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ment in canine leishmaniasis. A light microscopic immunohistochemical and electronmicroscopic study. Nephron, 57: 444-452. Zyngier, F.R., 1974. Histopathology of experimental toxocariasis in mice. Ann. Trop. Med. Parasitol., 68: 225-228.
