EXPERIMENTAL
PARASITOLOGY
Ascaris
suum:
39,
69-73
( 1976)
Homocytotropic CATHERINE
Department
of Pathology,
Antibody
in Mice
A. CRANDALL
College of Medicine, Gainesville, Florida Accepted
Responses
April
Box 32610
731,
of
University
Floridu,
9, 1975
CRANDALL, CATHERINE A. 1976. Ascaris suum: Homocytotropic antibody responses in mice. ExperimentaZ Parusitology 39, 69-73. Homocytotropic antibodies in the sera of CD-l and DBA/l mice infected with larval A. suum were titered by PCA reactions. IgG, and reaginic antibody responses were similar in both strains of mice. With a dose of 8000 to 10,000 embryonated eggs, reaginic antibody was detected during the second week and IgG, antibody during the third week of infection. Doses of 1500 to 5000 eggs gave delayed antibody responses or did not induce a detectable response, although an anamnestic response followed a challenge inoculation even when no detectable antibody was observed in initial infection. Larval A. suum infections in two strains of mice did not potentiate a reaginic response to ovalbumin. INDEX DESCRIPTORS: Ascaris suum; Homocytotropic antibody; Reaginic antibody; IgG, antibody; Passive cutaneous anaphylaxis; Potentiation.
Reagin mediated hypersensitivity is a common feature in helminth infections, This type of hypersensitivity, with elevated IgE levels, has been reported to be particularly prominent in infections with Ascaris sp. (Sadun 1972). Further, it has been demonstrated that certain helminth infections can potentiate reaginic ( IgE ) antibody responses to antigens unrelated to those of the helminth (Or-r and Blair 1969, Jarrett 1972). The unique immunologic aspects of helminth infections responsible for high reagin production or potentiation of reagin responses are largely unknown. The purpose of this study was to determine if larval Ascaris suunz infection in the mouse would serve as a convenient model for study of reagin production in helminth infections. It was considered that the high allergenic properties of Ascaris, combined with the extensive information on the immunologic system of the mouse, and the ability to manipulate this system experimentally, would produce
a particularly suitable model. A previous study had shown that larval A. suum in mice induces a complex Ig and antibody response (Crandall and Crandall 1971). In this study, the response to infection was characterized by measuring homocytotropic antibodies ( IgG, and IgE) and the ability to potentiate reagin response to ovalbumin. MATERIALS
Q 1976 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Animals The animals used for ascaris infection were 2-3-month-old female CD-l mice (Charles River, Wilmington, MA) and 3-month-old female DBA/l mice (Jackson Laboratories, Bar Harbor, ME). Recipient mice for passive cutaneous anaphylaxis (PCA) reactions were 6-S-week-old female CD-l mice. To determine mouse reaginic antibodies, rats as well as mice were used as recipients for PCA reactions. Two rat strains were used, 150-g female Sprague+ 69
Copyright All rights
AND
70
CATHEFiINE
Dawley (Charles River) and 150-200-g male or female DA rats (kindly donated by Dr. Bryan Gebhardt, Department of Pathology, College of Medicine, University of Florida). Animals were maintained in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. Infection and Vaccination To measure the homocytotropic antibody responses in ascaris infection, 21 CD-l mice were inoculated with BOOO10,000 embryonated eggs as described previously (Crandall and Arean 1964) and bled at intervals after infection. Ten of these mice were challenged with 8006 eggs 120 days after initial infection and bled at intervals after challenge. To compare the homocytotropic antibody responses in CD-l mice with those of another mouse strain, a similar experiment was carried out with 30 DBA/l mice. then were repeated These experiments with 20 CD-l and 20 DBA/l mice given the same inocula. To determine the dose response in homocytotropic antibody production, 10 CD-l mice were inoculated with 1500 ascaris eggs and 10 with 5000 eggs. They were bled at intervals after inoculation and then callenged 43 days after the initial inoculation with 1500 and 5000 eggs, respectively. Hatched second-stage larvae were obtained as described previously (Crandall and Arean 1964). After hatching, the larvae suspended in distilled water were killed by placing them in a boiling water bath for 5 min. To determine the homocytotropic antibody responses to antigens of the larvae, 10 CD-l mice were injected intraperitoneally (ip) with 15,000 heatkilled hatched second-stage larvae three times with a e-week interval between each injection. The mice were bled 2 weeks after the last injection.
A.
CRANDALL
Passive
Cutaneous
Anaphylaxis
(PCA)
Assays for both classes of homocytotropic antibodies were carried out in mice (Schwartz and Levine 1973); assays for mouse reaginic antibody in rats were done as described by Mota and Wong (1969). Mice were sensitized by intradermal (id) injections of 30 ~1 of serum dilutions, and PCA reactions were elicited at 2 hr for IgGl antibody or 72 hr for reaginic antibody. Rats were sensitized by id injections of 50 ~1 of diluted serum, and PCA reactions were elicited 12 to 16 hr after sensitization. Negative and positive controls also were conducted. The ascaris antigen used to elicit PCA reactions was perienteric fluid gel filtered on Sephadex G-50 as described by Hogarth-Scott ( 1967). Mice were injected intravenously (iv) with 2.3 mg protein (Lowry et al. 1951) in 0.2 ml of 0.15 M saline containing 0.5% Evans Blue; rats were injected iv with 2.3 mg of antigen in 0.5 ml saline containing 0.5% Evans Blue. Sera were collected from mice either by puncturing the orbital sinus or by decapitation. When mice were bled from the orbital sinus, blood was collected in 100 ~1 disposable pipettes (Scientific Products, Jacksonville, FL). Then 0.1 or 0.2 ml of blood was diluted immediately in 0.2 or 0.4 ml of saline, respectively; after centrifugation and removal of the clot, the supernatant was considered a 1:5 dilution. In some cases, not enough sera were obtained to do complete titers, and these sera were tested only at a 1:5 dilution. Each serum dilution was tested in at least two recipient animals. Potentiation The methods used were as described by Orr and Blair (1969) for the rat-iV@postrongylus system. Two experiments were carried out. In the first experiment, 40 CD-l mice were injected ip with 1.0 pg ovalbumin ( OA) (Nutritional Biochemicals, Cleveland, OH) in 0.5 mg aluminum
Ascaris
stun:
HOMOCYTOTROPIC TABLE
Homocytotropic Infecting ascaris
dose of eggs
Antibody
Days after infection
I
Responses
in Mice
Reaginic
initial
8000 challenge
1500 initial 1500 challenge 5000 initial 5000 challenge
l(t12 13-15 20-25 30-60 120 3 5 8 15-20 25-30 12 15 2030 12
Infected
with Ascaris
antibody
No. positive/ No. tested 8000-10,000
71
ANTIBODY
IgGl
PCA titera
No. positive/ No. tested
antibody PCA titel”
5
PARASITOLOGY
Ascaris
suum:
39,
69-73
( 1976)
Homocytotropic CATHERINE
Department
of Pathology,
Antibody
in Mice
A. CRANDALL
College of Medicine, Gainesville, Florida Accepted
Responses
April
Box 32610
731,
of
University
Floridu,
9, 1975
CRANDALL, CATHERINE A. 1976. Ascaris suum: Homocytotropic antibody responses in mice. ExperimentaZ Parusitology 39, 69-73. Homocytotropic antibodies in the sera of CD-l and DBA/l mice infected with larval A. suum were titered by PCA reactions. IgG, and reaginic antibody responses were similar in both strains of mice. With a dose of 8000 to 10,000 embryonated eggs, reaginic antibody was detected during the second week and IgG, antibody during the third week of infection. Doses of 1500 to 5000 eggs gave delayed antibody responses or did not induce a detectable response, although an anamnestic response followed a challenge inoculation even when no detectable antibody was observed in initial infection. Larval A. suum infections in two strains of mice did not potentiate a reaginic response to ovalbumin. INDEX DESCRIPTORS: Ascaris suum; Homocytotropic antibody; Reaginic antibody; IgG, antibody; Passive cutaneous anaphylaxis; Potentiation.
Reagin mediated hypersensitivity is a common feature in helminth infections, This type of hypersensitivity, with elevated IgE levels, has been reported to be particularly prominent in infections with Ascaris sp. (Sadun 1972). Further, it has been demonstrated that certain helminth infections can potentiate reaginic ( IgE ) antibody responses to antigens unrelated to those of the helminth (Or-r and Blair 1969, Jarrett 1972). The unique immunologic aspects of helminth infections responsible for high reagin production or potentiation of reagin responses are largely unknown. The purpose of this study was to determine if larval Ascaris suunz infection in the mouse would serve as a convenient model for study of reagin production in helminth infections. It was considered that the high allergenic properties of Ascaris, combined with the extensive information on the immunologic system of the mouse, and the ability to manipulate this system experimentally, would produce
a particularly suitable model. A previous study had shown that larval A. suum in mice induces a complex Ig and antibody response (Crandall and Crandall 1971). In this study, the response to infection was characterized by measuring homocytotropic antibodies ( IgG, and IgE) and the ability to potentiate reagin response to ovalbumin. MATERIALS
Q 1976 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Animals The animals used for ascaris infection were 2-3-month-old female CD-l mice (Charles River, Wilmington, MA) and 3-month-old female DBA/l mice (Jackson Laboratories, Bar Harbor, ME). Recipient mice for passive cutaneous anaphylaxis (PCA) reactions were 6-S-week-old female CD-l mice. To determine mouse reaginic antibodies, rats as well as mice were used as recipients for PCA reactions. Two rat strains were used, 150-g female Sprague+ 69
Copyright All rights
AND
70
CATHEFiINE
Dawley (Charles River) and 150-200-g male or female DA rats (kindly donated by Dr. Bryan Gebhardt, Department of Pathology, College of Medicine, University of Florida). Animals were maintained in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. Infection and Vaccination To measure the homocytotropic antibody responses in ascaris infection, 21 CD-l mice were inoculated with BOOO10,000 embryonated eggs as described previously (Crandall and Arean 1964) and bled at intervals after infection. Ten of these mice were challenged with 8006 eggs 120 days after initial infection and bled at intervals after challenge. To compare the homocytotropic antibody responses in CD-l mice with those of another mouse strain, a similar experiment was carried out with 30 DBA/l mice. then were repeated These experiments with 20 CD-l and 20 DBA/l mice given the same inocula. To determine the dose response in homocytotropic antibody production, 10 CD-l mice were inoculated with 1500 ascaris eggs and 10 with 5000 eggs. They were bled at intervals after inoculation and then callenged 43 days after the initial inoculation with 1500 and 5000 eggs, respectively. Hatched second-stage larvae were obtained as described previously (Crandall and Arean 1964). After hatching, the larvae suspended in distilled water were killed by placing them in a boiling water bath for 5 min. To determine the homocytotropic antibody responses to antigens of the larvae, 10 CD-l mice were injected intraperitoneally (ip) with 15,000 heatkilled hatched second-stage larvae three times with a e-week interval between each injection. The mice were bled 2 weeks after the last injection.
A.
CRANDALL
Passive
Cutaneous
Anaphylaxis
(PCA)
Assays for both classes of homocytotropic antibodies were carried out in mice (Schwartz and Levine 1973); assays for mouse reaginic antibody in rats were done as described by Mota and Wong (1969). Mice were sensitized by intradermal (id) injections of 30 ~1 of serum dilutions, and PCA reactions were elicited at 2 hr for IgGl antibody or 72 hr for reaginic antibody. Rats were sensitized by id injections of 50 ~1 of diluted serum, and PCA reactions were elicited 12 to 16 hr after sensitization. Negative and positive controls also were conducted. The ascaris antigen used to elicit PCA reactions was perienteric fluid gel filtered on Sephadex G-50 as described by Hogarth-Scott ( 1967). Mice were injected intravenously (iv) with 2.3 mg protein (Lowry et al. 1951) in 0.2 ml of 0.15 M saline containing 0.5% Evans Blue; rats were injected iv with 2.3 mg of antigen in 0.5 ml saline containing 0.5% Evans Blue. Sera were collected from mice either by puncturing the orbital sinus or by decapitation. When mice were bled from the orbital sinus, blood was collected in 100 ~1 disposable pipettes (Scientific Products, Jacksonville, FL). Then 0.1 or 0.2 ml of blood was diluted immediately in 0.2 or 0.4 ml of saline, respectively; after centrifugation and removal of the clot, the supernatant was considered a 1:5 dilution. In some cases, not enough sera were obtained to do complete titers, and these sera were tested only at a 1:5 dilution. Each serum dilution was tested in at least two recipient animals. Potentiation The methods used were as described by Orr and Blair (1969) for the rat-iV@postrongylus system. Two experiments were carried out. In the first experiment, 40 CD-l mice were injected ip with 1.0 pg ovalbumin ( OA) (Nutritional Biochemicals, Cleveland, OH) in 0.5 mg aluminum
Ascaris
stun:
HOMOCYTOTROPIC TABLE
Homocytotropic Infecting ascaris
dose of eggs
Antibody
Days after infection
I
Responses
in Mice
Reaginic
initial
8000 challenge
1500 initial 1500 challenge 5000 initial 5000 challenge
l(t12 13-15 20-25 30-60 120 3 5 8 15-20 25-30 12 15 2030 12
Infected
with Ascaris
antibody
No. positive/ No. tested 8000-10,000
71
ANTIBODY
IgGl
PCA titera
No. positive/ No. tested
antibody PCA titel”
5
