THE JOURNAL OF INFECTIOUS DISEASES. VOL. 136, NO.4. OCTOBER 1977 © 1977 by the University of Chicago. All rights reserved.
Kinetics of Immunological Responses, Resistance to Reinfection, and Pathological Reactions to Infection with Trichinella spiralis From the Division of Geographic Medicine, Department of Medicine, Case Western Reserve University and University Hospitals, Cleveland, Ohio
David I. Grove, Joseph Hamburger, and Kenneth S. Warren
resistance to reinfection with T. spiralis by observing the expulsion of adult worms from the gut and the development of larvae in the muscles. Finally, inflammatory responses to the larvae in the muscle were used as a measure of host reactivity to the parasite. Each of these parameters was observed at times ranging from four to 40 weeks after the initial infection. The results confirm that chronic trichinosis is a dynamic infection with changing relationshi ps between the parasite and the host.
Most investigations of the immunology of trichinosis have been concerned only with the early stages of infection with Trichinella spiralis. Although both adult intestinal worms and clinical illness are transient [1], trichinosis must be considered a chronic infection because living worm larvae persist in the muscles for a long time. One of the few long-term studies of a chronic primary infection due to Trichinella revealed transformation of lymphocytes after exposure to specific antigens for the first few weeks after infection but a decline in the cellular responses thereafter [2]. The present investigations have attempted to delineate further the kinetics of the immunological responses in a chronic primary infection with T. spiralis. Responses were assessed by measurements of footpad reactions to antigens of Trichinella as an index of the presence of reaginic antibodies, precipitating antibodies, and cellmediated immunity. Furthermore, the ability of the T cell mitogen, concanavalin A (con A), to stimulate spleen cells was determined. We examined the influence of duration of infection on
Materials and Methods
Animals. Female Swiss albino mice (Flow Laboratories, Dublin, Va.) weighing 20-22 g were used. Infection of a colony of mice was established by a strain of T. spiralis originally supplied by Dr. W. C. Campbell (Merck Institute of Medical Research, Rahway, N.J.). Larvae were isolated by maceration of carcasses in a Waring blendor (Fisher Scientific Co., Cleveland, Ohio), incubation in I % acid-pepsin for 2-3 hr at 37 C, sedimentation in saline, and filtration through gauze [3]. Two hundred mice were infected with 150 larvae in 0.2 ml of saline by esophageal intubation with a l6-gauge needle. The infected mice and a similar number of uninfected control mice were studied at various intervals after the initial exposure to T. spiralis (figure I).
Received for publication February 24, 1977, and in revised fonn May 9, 1977. This work was supported by a grant from the Rockefeller Foundation. Please address requests for reprints to Dr. D. I. Grove, Division of Geographic Medicine, University Hospitals, Cleveland, Ohio 44106.
562
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on March 22, 2015
Changes in immune responses, resistance to reinfection, and pathological reactions were studied serially in mice that had been infected for four to 40 weeks with 150 larvae of Trichinella sp iralis. Immediate footpad hypersensitivity reactions to antigens of Trichinella were present throughout the period of observation. Delayed hypersensitivity reactions 48 hr after injection of antigen were first seen in mice infected for 14 weeks and gradually increased in size thereafter. Intestinal adult worm burdens were determined one week after challenge with 500 larvae. There was resistance to reinfection and accelerated expulsion of worms in animals challenged three weeks after the primary infection; this resistance waned at seven and 13 weeks but reappeared in mice infected for 20 weeks or longer. Counts of larvae in muscle were determined four weeks after challenge with 500 larvae. Marked resistance was present four weeks after the primary infection and was maintained for the duration of the study.
Immunology of Trichinosis
563
INFECTED MICE
/ " , . " . , " , " Iervae at u W"k'~ No further infection
Infected with 500 larvae at weeks 3,7,13,20,29 or 39
I
I
1 week 1ater
t
j
Intestinal adult worm count Peri phera1 eos i nophi1 count Small intestinal pathology
Spleen cell transformation
[7]. Worm burdens. Adult worms were recovered from the small intestines by a modification of the method of Campbell [8]. The small bowel was removed, slit longitudinally, cut into pieces (2-3 em), and digested in 1 M NaOH in 100 ml of 0.85% NaCI overnight at 4 C. The intestinal fragmen ts were shaken vigorously and poured into a petri dish; the pieces were removed, and the fluid was transferred to 50-ml centrifuge tubes. The worms were spun down at 400 g for 5 min, and the total number in the sediment was counted. A preliminary experiment was performed in previously exposed animals to determine the
Weeks 4,8,14,21,30 or 40 Muscle larval count Muscle inflammation
MICE ___________
Infected with 500 larvae at
Infected with 500 larvae at
weeks 3,7,13',0,29 or 39
weeks O,4,ln'r,26 or 36
1 week later
Weeks 4,8,14,21,30 or 40: footpad reaction
Immunological responsiveness. Footpad reactions to injected antigen were measured in infected and control mice. A saline extract of T. spiralis larvae was made [4] and sterilized with a 22-fLm Millex disposable filter unit (Millipore Corp., Bedford, Mass.), and the protein content was determined [5]. Protein (10 fLg) in 0.03 ml of 0.85% NaCI was injected into one hind footpad of each mouse, and 0.03 ml of saline was injected into the other footpad. The reactions at 15 min, 6, 24, and 48 hr were measured with a micrometer [6]. Transformation of spleen cells induced by con A was measured as previously described
t
Weeks 4,8,14,21,30 or 40:
~CONTR'1 No infection
4 weeks 1ater
t Weeks 4,8,14,21,30 or 40: Intestinal adult worm count Peripheral eosinophil count Small intestinal pathology
4 weeks later
t Weeks 4,8,14,21,30 or 40: Muscle larval count Muscle inflammation
time of expulsion of adult worms from the small intestine after a challenge infection. Fifteen mice infected with 400 larvae five months previously and 15 uninfected control mice were challenged with 500 larvae. The worm burdens in previously infected animals on days 5, 7, and 9 after challenge were 52%, 13%, and 0.4%, respectively, of those seen in the control animals. In the experiments described herein, counts of intestinal worms were therefore performed in control and previously infected mice seven days after challenge with 500 larvae. Larvae in muscle in each carcass were counted. Each animal was decapitated, skinned, eviscerated, macerated, and digested for 4 hr at 37 C in 1% acid-pepsin in a total volume of 100 ml. Each digestate was thoroughly shaken; 0.2-ml samples were taken, and the number of larvae was counted in duplicate. Muscle pathology. The right hamstring muscles were removed, fixed in 10% formalin, sectioned, and stained with hematoxylin and eosin. The areas of inflammation around 10 isolated larvae cut longitudinally, but not including the larvae themselves, were measured with a 7TMC particle measurement computer system (Millipore) [9]. Counts of eosinophils. Absolute counts of eo-
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on March 22, 2015
Figure 1. Experimental design for study of immunological responses, resistance to reinfection, and pathological reactions. in control mice and mice previously infected with 150 larvae of Trichinella spiralis.
Weeks 4,8,14,21,30 or 40: Footpad reaction Spleen cell transfonnation Muscle larval counts Muscle inflammation
Infected with 500 larvae at weeks 0,4,10,17,26 or 36
Grove, Hamburger, and Warren
564
sinophils were made from blood samples obtained in the morning from the retroorbital plexus with a microhematocrit tube with use of Discomb's fluid (one part acetone, one part 2% aqueous eosin, and eight parts distilled water) as a diluent. Results
Table 1. Footpad reactions in 10 mice infected with 150 larvae of Trichinella spiralis and in 10 control mice. Reactions were measured at 15 min, 6, 24, and 48 hr after injection of antigen or saline. Time of measurement, weeks after infection 15 min 4 8 14 21 30 40 6 hr 4 8
14 21 30 40 24 hr 4 8 14 21 30 40 48 hr 4 8 14 21 30 40
Control mice
Infected mice
Pvalue *
0.13 ± 0.06 0.18 ± 0.10 0.10 ± 0.09 0.37 ± 0.07 0.18 ± 0.14 0.03 ± 0.03
1.00 0.80 0.95 1.07 1.34 1.28
± 0.05 ± 0.07 ± 0.09 ± 0.07 ± 0.08 ± 0.07
Kinetics of Immunological Responses, Resistance to Reinfection, and Pathological Reactions to Infection with Trichinella spiralis From the Division of Geographic Medicine, Department of Medicine, Case Western Reserve University and University Hospitals, Cleveland, Ohio
David I. Grove, Joseph Hamburger, and Kenneth S. Warren
resistance to reinfection with T. spiralis by observing the expulsion of adult worms from the gut and the development of larvae in the muscles. Finally, inflammatory responses to the larvae in the muscle were used as a measure of host reactivity to the parasite. Each of these parameters was observed at times ranging from four to 40 weeks after the initial infection. The results confirm that chronic trichinosis is a dynamic infection with changing relationshi ps between the parasite and the host.
Most investigations of the immunology of trichinosis have been concerned only with the early stages of infection with Trichinella spiralis. Although both adult intestinal worms and clinical illness are transient [1], trichinosis must be considered a chronic infection because living worm larvae persist in the muscles for a long time. One of the few long-term studies of a chronic primary infection due to Trichinella revealed transformation of lymphocytes after exposure to specific antigens for the first few weeks after infection but a decline in the cellular responses thereafter [2]. The present investigations have attempted to delineate further the kinetics of the immunological responses in a chronic primary infection with T. spiralis. Responses were assessed by measurements of footpad reactions to antigens of Trichinella as an index of the presence of reaginic antibodies, precipitating antibodies, and cellmediated immunity. Furthermore, the ability of the T cell mitogen, concanavalin A (con A), to stimulate spleen cells was determined. We examined the influence of duration of infection on
Materials and Methods
Animals. Female Swiss albino mice (Flow Laboratories, Dublin, Va.) weighing 20-22 g were used. Infection of a colony of mice was established by a strain of T. spiralis originally supplied by Dr. W. C. Campbell (Merck Institute of Medical Research, Rahway, N.J.). Larvae were isolated by maceration of carcasses in a Waring blendor (Fisher Scientific Co., Cleveland, Ohio), incubation in I % acid-pepsin for 2-3 hr at 37 C, sedimentation in saline, and filtration through gauze [3]. Two hundred mice were infected with 150 larvae in 0.2 ml of saline by esophageal intubation with a l6-gauge needle. The infected mice and a similar number of uninfected control mice were studied at various intervals after the initial exposure to T. spiralis (figure I).
Received for publication February 24, 1977, and in revised fonn May 9, 1977. This work was supported by a grant from the Rockefeller Foundation. Please address requests for reprints to Dr. D. I. Grove, Division of Geographic Medicine, University Hospitals, Cleveland, Ohio 44106.
562
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on March 22, 2015
Changes in immune responses, resistance to reinfection, and pathological reactions were studied serially in mice that had been infected for four to 40 weeks with 150 larvae of Trichinella sp iralis. Immediate footpad hypersensitivity reactions to antigens of Trichinella were present throughout the period of observation. Delayed hypersensitivity reactions 48 hr after injection of antigen were first seen in mice infected for 14 weeks and gradually increased in size thereafter. Intestinal adult worm burdens were determined one week after challenge with 500 larvae. There was resistance to reinfection and accelerated expulsion of worms in animals challenged three weeks after the primary infection; this resistance waned at seven and 13 weeks but reappeared in mice infected for 20 weeks or longer. Counts of larvae in muscle were determined four weeks after challenge with 500 larvae. Marked resistance was present four weeks after the primary infection and was maintained for the duration of the study.
Immunology of Trichinosis
563
INFECTED MICE
/ " , . " . , " , " Iervae at u W"k'~ No further infection
Infected with 500 larvae at weeks 3,7,13,20,29 or 39
I
I
1 week 1ater
t
j
Intestinal adult worm count Peri phera1 eos i nophi1 count Small intestinal pathology
Spleen cell transformation
[7]. Worm burdens. Adult worms were recovered from the small intestines by a modification of the method of Campbell [8]. The small bowel was removed, slit longitudinally, cut into pieces (2-3 em), and digested in 1 M NaOH in 100 ml of 0.85% NaCI overnight at 4 C. The intestinal fragmen ts were shaken vigorously and poured into a petri dish; the pieces were removed, and the fluid was transferred to 50-ml centrifuge tubes. The worms were spun down at 400 g for 5 min, and the total number in the sediment was counted. A preliminary experiment was performed in previously exposed animals to determine the
Weeks 4,8,14,21,30 or 40 Muscle larval count Muscle inflammation
MICE ___________
Infected with 500 larvae at
Infected with 500 larvae at
weeks 3,7,13',0,29 or 39
weeks O,4,ln'r,26 or 36
1 week later
Weeks 4,8,14,21,30 or 40: footpad reaction
Immunological responsiveness. Footpad reactions to injected antigen were measured in infected and control mice. A saline extract of T. spiralis larvae was made [4] and sterilized with a 22-fLm Millex disposable filter unit (Millipore Corp., Bedford, Mass.), and the protein content was determined [5]. Protein (10 fLg) in 0.03 ml of 0.85% NaCI was injected into one hind footpad of each mouse, and 0.03 ml of saline was injected into the other footpad. The reactions at 15 min, 6, 24, and 48 hr were measured with a micrometer [6]. Transformation of spleen cells induced by con A was measured as previously described
t
Weeks 4,8,14,21,30 or 40:
~CONTR'1 No infection
4 weeks 1ater
t Weeks 4,8,14,21,30 or 40: Intestinal adult worm count Peripheral eosinophil count Small intestinal pathology
4 weeks later
t Weeks 4,8,14,21,30 or 40: Muscle larval count Muscle inflammation
time of expulsion of adult worms from the small intestine after a challenge infection. Fifteen mice infected with 400 larvae five months previously and 15 uninfected control mice were challenged with 500 larvae. The worm burdens in previously infected animals on days 5, 7, and 9 after challenge were 52%, 13%, and 0.4%, respectively, of those seen in the control animals. In the experiments described herein, counts of intestinal worms were therefore performed in control and previously infected mice seven days after challenge with 500 larvae. Larvae in muscle in each carcass were counted. Each animal was decapitated, skinned, eviscerated, macerated, and digested for 4 hr at 37 C in 1% acid-pepsin in a total volume of 100 ml. Each digestate was thoroughly shaken; 0.2-ml samples were taken, and the number of larvae was counted in duplicate. Muscle pathology. The right hamstring muscles were removed, fixed in 10% formalin, sectioned, and stained with hematoxylin and eosin. The areas of inflammation around 10 isolated larvae cut longitudinally, but not including the larvae themselves, were measured with a 7TMC particle measurement computer system (Millipore) [9]. Counts of eosinophils. Absolute counts of eo-
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on March 22, 2015
Figure 1. Experimental design for study of immunological responses, resistance to reinfection, and pathological reactions. in control mice and mice previously infected with 150 larvae of Trichinella spiralis.
Weeks 4,8,14,21,30 or 40: Footpad reaction Spleen cell transfonnation Muscle larval counts Muscle inflammation
Infected with 500 larvae at weeks 0,4,10,17,26 or 36
Grove, Hamburger, and Warren
564
sinophils were made from blood samples obtained in the morning from the retroorbital plexus with a microhematocrit tube with use of Discomb's fluid (one part acetone, one part 2% aqueous eosin, and eight parts distilled water) as a diluent. Results
Table 1. Footpad reactions in 10 mice infected with 150 larvae of Trichinella spiralis and in 10 control mice. Reactions were measured at 15 min, 6, 24, and 48 hr after injection of antigen or saline. Time of measurement, weeks after infection 15 min 4 8 14 21 30 40 6 hr 4 8
14 21 30 40 24 hr 4 8 14 21 30 40 48 hr 4 8 14 21 30 40
Control mice
Infected mice
Pvalue *
0.13 ± 0.06 0.18 ± 0.10 0.10 ± 0.09 0.37 ± 0.07 0.18 ± 0.14 0.03 ± 0.03
1.00 0.80 0.95 1.07 1.34 1.28
± 0.05 ± 0.07 ± 0.09 ± 0.07 ± 0.08 ± 0.07
