Zeitschrift far
Z. Parasitenkd. 56, 147-157 (1978)
Parasitenkunde Parasitology Research
9 by Springer-Verlag 1978
A Comparative in vitro Study of Antibody Binding to Different Stages of the Hookworm Ancylostoma caninum J.C.M. Klaver-Wesseling, J.C.M. Vetter, and W.K. Visser Laboratory of Parasitology, University of Amsterdam, Mauritskade 57, Amsterdam, The Netherlands
Summary. The Indirect Fluorescent Antibody Technique (IFAT) and the Indirect Immuno Peroxidase Technique (IIPT) have been applied to cryostat sections and intact stages of the hookworm species Aneylostoma caninum with sera from infected dogs. Especially the role of the body surface ( = cuticle (cortex, matrix, basal layer) and hypodermis) in immunity was studied. Using cryostat sections and dead intact stages as the antigen, specific antibody binding was demonstrated round the ovum membrane and the cuticle of all stages of this hookworm species. Cryostat sections of adult worms showed, that it probably is not the cuticle itself that is antigenic, but that the specific reaction that is observed consisted of a layer, covering the cortex of the cuticle. Infective and parasitic living stages, however, showed no antibody binding in contrast to the free-living stages in which specific antibody binding was demonstrated.
Index Descriptions Ancylostoma caninum; Indirect Fluorescent Antibody Technique (IFAT); Indi-
rect Immuno Peroxidase Technique (IIPT); hookworms,; cryostat sections; body surface; cuticle; dogs.
Introduction Although the evidence of immunity to Ancylostoma caninum in dogs has been well established (Otto and Kerr, 1939; Miller, 1971), no detailed data are available about immune mechanisms operating against hookworms or the eventual adaptive mechanisms which hookworms might have to ensure that they survive the immune response of their hosts. Antibody binding in hookwormshas been demonstratedusing the Indirect FluorescentAntibody Technique (IFAT) to the ovum membrane of A. caninum and Necator americanus (Zaman and
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Singh, 1965; Azevedo and Rombert, 1970) and to the body surface of intact third stage motile larvae of N. americanus (Bail and Bartlett, 1969), freshly isolated from infected soil and stimulated to exsheath with sodium hypochlorite. However, the relation between antibody binding and the time that hookworms are present in their hosts, has never been investigated. In schistosomes, for instance, it has been demonstrated that antibody binding present in serum from i m m u n e rhesus monkeys are unable to bind to the body surface of four-day schistosomula, while these antibodies can kill three-hour schistosomula in vitro by damaging the surface (Clegg and Smithers, 1972). Indeed, hookworms differ from schistosomes, but on the other hand they are also present for a certain period in similar tissues and body fluids as the schistosomes (skin, bloodstream, lungs). The
aim
the antibody including
of the present binding
those
experiment
to different
stages which
is t o g a t h e r
more
stages of the hookworm
have been present
in the host
information
about
species A. caninum, for a certain
time.
Material and Methods The h o o k w o r m species A. caninum that was used in these experiments, was originally obtained from a naturally-infected dog and was maintained in our laboratory in dogs for 14 generations of h o o k w o r m s over a period of 10 years. The different stages of this parasite were collected as follows: The ova from freshly passed feces of infected dogs were isolated using the brine floatation method (Swellengrebel and Sterman, 1961). First stage larvae were obtained by washing the isolated ova in distilled water and then placing them in a 0.05% sterile NaC1 solution at 27 ~ C for 24 h. Second stage larvae were collected by thoroughly mixing infected feces with sterilized soil. After an incubation period of three days at 27 ~ C, the mixture was wrapped in thick filter-paper and the larvae were isolated using a Baermann apparatus. Third stage larvae were similarly obtained after an incubation period of seven days. The greater portion of those larvae were still ensheathe& In order to study the cuticle of the third stage larvae itself, they were stimulated with CO2 to exsheath as outlined by Owen et al. (1969). Lung stages were isolated from the lungs of infected dogs, 1-3 days after infection. The lungs were cut into pieces and the larvae were collected using a Baermann apparatus. These larvae appeared to be third stages which had similar morphological i characteristics as the fresh exsheathed third stage larvae, except that the lung stages contain less food granules. Fourth stage larvae were collected by washing the opened small intestine of dogs infected subcutaneously seven days previously. Adult worms were similarly obtained 28 days after infection. After isolation all stages were washed twice in phosphate buffered saline (PBS) at p H 7.2. When intact stages were used as the antigen, they were placed in an excess of antiserum to a final dilution of 1:4 for 30 min. To fourth stage larvae and adults an excess of undiluted antiserum was added. The tests were performed at 27 ~ C when ova and free-living stages were used as the antigen. W h e n testing the infective and parasitic stages, the reactions were carried out in small glass tubes at 37 ~ C and incubation periods of 30 min up till 48 h were used. If the incubation period lasted longer than a few hours, antibiotics (25 U penicillin and 0.25 m g streptomycin]ml serum) and fungicides (0.01 mg pimeracin/ml serum) were added. When cryostat sections were used as the antigen, the parasites were placed in gelatin capsules filled with embedding medium. This embedding medium was prepared by adding 5 ml bovine serum albumin and 1 g gelatin (Difco) to 15 ml sterile PBS. This mixture was brought to 30 ~ C before use. These capsules were quickly frozen in a vial containing isopentane cooled with liquid nitrogen and stored at - 1 8 0 ~ until they were used. Serial sections of 8 g m were made on the cryostat microtome at - 2 0 ~ C. They were transferred to slides and withdrawn from the cryostat in order to thaw and dry. They were then immersed in acetone and dried. After this fixation the slides were stored at - 2 0 ~ until they were used. Antisera to A. caninum were obtained from eight mongrel dogs infected once subcutaneously with a high dose (5000) of larvae. Normal sera were obtained from six adult mongrel dogs which
A Comparative in vitro Study of Antibody Binding to Different Stages
149
were not infected with hookworms. Their history is the same as that of the infected dogs used. On several times after infection (from 10 days up till 6 months), blood was taken from the vena saphena. The sera were collected, pooled and divided into 0.2 ml samples, which were stored in small serum vials at - 20~ C. The IFAT was carried out as outlined by Goldman (1968), using a commercially prepared conjugate (RAD/FITC, Nordic Lab., Tilburg, Holland) mainly directed against the immunoglobulins G of the dog. The specificity of the reaction was determined by comparing the results of slides incubated in antiserum with slides incubated in 1. PBS 2. normal sera 3. antisera that were absorbed to their own antigens. Preparation of the antigen and the absorption procedure was essentially as described by Goldman (1968). 4. commericially prepared unlabeled antisera to dogs immunoglobulins (RAD/7S, Nordic Lab., Tilburg, Holland) applied after the application of the antiserum and prior to the application of the conjugate. In addition, heterologous antigens were used. A. ceylanicurn served as the related heterologous antigen and Paramecium spec. as the unrelated heterologous antigen. After performing the test, the living stages were immobilized by adding several drops of a 10% alcohol solution. They were then examined, like the cryostat sections, in PBS. The IIPT has not yet been used on hookworms before, but it has proved to be a useful technique to demonstrate human antibodies against Toxoplasma gondii (Ourth et al., 1974). This technique was carried out as outlined by Pearse (1972). A commercially prepared peroxidase labeled conjugate was used [RAD/Ig G(H/L)/PO, Nordic Lab., Tilburg, Holland], that was mainly directed against the immunoglobulins G of the dog. The antigen-antibody complex was coloured by a 20-rain incubation at room temperature in a 5 mg 3-3'-diaminobenzidine tetrahydrochloride (DAB, Sigma, St. Louis, USA) solution in a 10 ml 0.05 M Tris solution, pH 7.6, to which is added 0.l ml 1% H2Oz just before use. The specificity of this technique was determined in the same way as it was done with the IFAT. Moreover, the endogenic peroxidase activity was determined by incubating the specimens in substrate only. The different stages and cryostat sections were examined in Gum Syrup (Apathys) or they were dried at room temperature and then examined in DPX (DePex, Gurr Ldt., London, England). A Leitz Dialux microscope was employed, that could be used with or without fluorescence. The final magnification was 100, 250, 400 or 1000. Important situations were photographed using a Leica MDa camera with a Kodak Highspeed Ektachrome Daylight film (22 DIN) in the IFAT and an Ilford Pan F film (18 DIN) in the IIPT. From the IFAT coloured pictures black- and-white prints were made.
Results T h e r e s u l t s a r e r e p r e s e n t e d in F i g u r e s 1 t o 14 a n d T a b l e 1.
1. T h e I n d i r e c t F l u o r e s c e n t A n t i b o d y T e c h n i q u e ( I F A T )
1.1. Cryostat Sections When the sections were examined under the fluorescence microscope before a p p l y i n g t h e test, a b l u e - g r e e n f l u o r e s c e n c e o f d i f f e r e n t s t r u c t u r e w a s o b s e r v e d ( a u t o f l u o r e s c e n c e ) . F o r t h a t r e a s o n t h e s e c t i o n s w e r e c o u n t e r s t a i n e d w i t h different concentrations of Eriochrome Black (Difco) to reduce this autofluorescence. The lowest concentration that gave a complete reduction of the autofluorescence w a s f u r t h e r u s e d in t h e test. I n all s t a g e s o f t h e h o o k w o r m
treated with different antisera, labeled anti-
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J.C.M. Klaver-Wesseling et al.
Table 1. Specific antibody binding of the cuticle of cryostat sections and intact stages of the hookworm Ancylostoma caninum Cryostat sections
Dead stages
Living stages
Free-living stages
Ova 1~ stage 2 ~ stage
+ + +
+ +
+ + +
Infective stages
3~ stage 3 ~ stage
+ +
+ +
-
Parasitic stages
4 ~ stage 5~ stage (adult)
+ +
+ +
-
+ Specific reaction; - negative reaction
Fig. 1. IFAT applied to cryostat section of adult worm. Incubation in antiserum, e cuticle Fig. 2. IFAT applied to cryostat section of adult worm. Control reaction, e cuticle
globulins and Eriochrome black, the cuticle showed a strong yellow-green fluorescence. The internal structures appeared to be red stained. The cuticle of adult stages gave more detailed information: a weak yellow-green fluorescence w a s o b s e r v e d i n t h e c u t i c l e itself, b u t t h e o u t e r s u r f a c e s h o w e d a s t r o n g y e l l o w g r e e n f l u o r e s c e n c e ( F i g . 1). U s i n g h o m o l o g o u s a n t i g e n a n d n o r m a l s e r u m , t h e control reactions sometimes showed a faint yellow-green fluorescence of the c u t i c l e a n d t h e i n t e r n a l o r g a n s a p p e a r e d t o b e r e d s t a i n e d . All o t h e r c o n t r o l r e a c t i o n s s h o w e d n o y e l l o w - g r e e n f l u o r e s c e n c e a t all (Fig. 2).
A Comparative in vitro Study of Antibody Binding to Different Stages
151
F r o m these results it may be concluded that the reaction in the cuticle is specific.
1.2. Intact Stages 1.2.1. Stages Isolated From Feces or Soil. Ova or larvae treated with antiserum, taken after different intervals post infectionum, showed a strong specific reaction of the outer surface (Fig. 3). The intensity of the fluorescence was so high that the internal structures were not visible. The specificity was determined by the control reactions that were used, as it was done with the cryostat sections (Fig. 4). Some ova and larvae produced a strong autofluorescence of the internal structures. These ova, incubated overnight at 2 7 ~ and at a high degree of humidity, appeared to be dead, judged by an arrested development compared to the other ova and the dye exclusion test. When the larvae with the strong autofluorescence were removed and examined more carefully, these stages appeared to be dead as well, judged by motility and the dye exclusion test. 1.2.2. Infective Stages and Stages Isolated From the Host. Application of the labeled anti-globulins after incubation with the different antisera caused no specific reaction in almost all infective and parasitic stages (Figs. 5 and 7). Only a weak autofluorescence of the internal structures was observed. However, in some cases a strong specific reaction of the outer s,,rface was detected. Removing these specimens and studying them more carefully, it appeared that they were dead, judged by mobility, movements of the body fluids and the dye exclusion test (Figs. 6 and 8). Because of these results we applied the I F A T to dead intact stages, simultaneously with living stages. Similar results were obtained as described above. So, when the infective and parasitic stages were dead before the test was applied to them, they reacted specifically in contrast with the living stages.
2. Indirect I m m u n e Peroxidase Technique
2.1. Cryostat Sections There was no endogenic activity, which is an advantage compared to the IFAT, where the untreated sections showed some autofluorescence. In the control reactions sometimes a weak brown staining was visible. Incubating the sections in PBS, the reaction was completely negative. Comparing incubation in antiserum with incubation in PBS and control sera, a specific reaction was observed round the ovum m e m b r a n e and the cuticle of all h o o k w o r m stages. Cryostat sections of ova and larvae could give no details about the precise site of the reaction. However, using cryostat sections of adult worms, more information could be gathered. The different layers of the cuticle and mostly parts of the hypodermis as well, were clearly visible. A weak specific reaction
152
J.C.M. Klaver-Wesseling et al.
A Comparative in vitro Study of Antibody Binding to Different Stages
Fig. 9. IIPT applied to cryostat section of adult worm. Incubation in antiserum, c cuticle; m matrix; b basal layer; h hypodermis Fig. 10. IIPT applied to cryostat section of adult worm. Control reaction
Fig. 3. IFAT applied to living ovum. Incubation in antiserum Fig. 4. IFAT applied to living ovum. Control reaction Fig. 5. IFAT applied to living infective larva. Incubation in antiserum Fig. 6. IFAT applied to dead infective larva. Incubation in antiserum Fig. 7. IFAT applied to living adult worm. Incubation in antiserum Fig. 8. IFAT applied to dead adult worm. Incubation in antisermn
153
co
cortex;
154
J.C.M. Klaver-Wesseling et al.
Fig. 11. IIPT applied to dead infective larva. Incubation in antiserum Fig. 12. IIPT applied to living infective larva. Incubation in antiserum Fig. 13. IIPT applied to living infective larva, partly in his old cuticle (sheath). Incubation in antiserum Fig. 14. IIPT applied to living infective larva, partly in his old cuticle (sheath). Control reaction
A Comparative in vitro Study of Antibody Binding to Different Stages
155
could be observed in the cuticle itself, but a strong specific reaction was visible on the cuticle: the cortex was covered with a brown coloured layer (Figs. 9 and 10). Sometimes the hypodermis seemed to colour brown as well, but it was impossible to prove this reaction to be specific as cryostat sections most of the time only showed parts of the hypodermis.
2,2. Intact Stages Dead intact stages incubated in antiserum all showed a specific reaction: a brown colour was evenly distributed round the ovum membrane and the cuticle (Fig. 11). However, using living stages as the antigen, the cuticle of the infective larvae and the parasitic stages remained completely negative (Fig. 12), while the free-living stages were stained specifically (Figs. 13 and 14).
Discussion
In a review about the body surface of helminths Lumsden (1975) stated that the cuticles of several nematode species are thought to be antigenic and that antibody binding to the outer surface of the cuticular membrane has been demonstrated. However, data represented by Hogarth-Scott (1968) suggest the possibility that immunoglobulins adsorbed by at least certain nematode species represent cross-reacting naturally occuring antibodies rather than antibodies whose production is stimulated specifically by prior exposure of the host to nematode surface antigens. In broad outline our results indicate that the cuticle indeed is antigenic. So we do not think naturally occuring antibodies only are involved, but that the greater amount of IgG immunoglobulins which adhered to the body surface of the different hookworm stages, were produced after an infection with A. caninum. Indeed we only used a conjugate directed against the IgG - antibodies of the dog, but in our opinion this gives a reliable result. Also Seesee et al. (1976) applied the I F A T to cryostat sections of Nippostrongylus brasiliensis. They used three types of conjugate: directed against the Ig G, the IgM and the IgA immunoglobulins, and they demonstrated that antibodies to the IgG class of immunoglobulins produced intense fluorescence that could not be improved. Using cryostat sections of the different stages, the cuticle reacts specifically. Cryostat sections of adult hookworms give more detailed information: the cuticle itself does not seem to be the source of antigenic stimulation, but a substance covering the cortex of the cuticle is involved. We agree with Sinclair (1970) who stated that antigens which react on the cuticle possibly are not part of the outer layer, but secreted by the pore canals (Bird, 1958). These pore canals might be present in the cuticle of hookworms as well. In the IIPT the hypodermis was sometimes visible and then possibly reacted specifically. Maybe this structure is involved in producing the antigen that is then transported through the pore canals to the outer surface. However, definite conclusions cannot yet
156
J.C.M. Klaver-Wesseling et al.
be drawn. Therefore further investigation o f the b o d y wall is needed. Also Lee (1966) stated that not m u c h is k n o w n a b o u t the b o d y wall o f h o o k w o r m s . More information could be gathered, for instance, using electron microscope techniques as is done with Ascaris lumbricoides and combining the results o f several cytochemical techniques. Using intact stages as the antigen, the results are not so unanimous. The b o d y surface of the free-living stages were f o u n d to react specifically whether they were dead or alive. However, the b o d y surfaces of the infective and parasitic dead stages reacted specifically, while those o f the infective and parasitic living stages remained negative. So it seems that antibodies, although present, are not acting against infective and parasitic living stages o f A. caninum. This seems not to be in agreement with, for example, the results o f Ball and Bartlett (1969). These authors claimed that the cuticle of exsheathed motile infective larvae of N. americanus incubated in sera f r o m patients infected with this h o o k w o r m , showed specific fluorescence. This difference could be explained by the fact that Ball and Bartlett used a fluorescein-labelled a n t i - h u m a n serum. So the fluorescence they demonstrated does not intrinsically have to be caused by a n t i b o d y binding, but other protein fractions of the serum might be involved. O u r results with infective and parasitiC living stages seem to be m o r e in agreement with those of schistosomes. The only difference is that schistosomes are only attainable by antibodies during the first period of time they are in their host (Clegg and Smithers, 1972), while h o o k w o r m s seem to be protected against antibodies f r o m the first m o m e n t they enter the host, We do not k n o w which mechanisms protect the h o o k w o r m s against antibodies, but our results suggest that an energy-dependent process might be involved. Therefore the present experiments shall be continued by studying the relation between the metabolic activity o f the infective and parasitic stages and a n t i b o d y binding.
References Azevedo, J. Fraga de, Rombert, P. : Antigen factors that can interfere with results of immunofluorescence tests in some helminthiasis. In: Standardization in immunofluorescence. E.J. Holborow, ed., Oxford and Edingburgh: Blackwell 1970 Ball, P.A.J., Bartlett, A.: Serological reactions to infection with Necator americanus. Trans. R. Soc. Trop. Med. Hyg. 63, 362 369 (1969) Bird, A.F.: Further observations on the structure of the nematode cuticle. Parasitology 48, 32-39 (1958) Clegg, J.A., Smithers, S.R.: The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. Int. J. Parasitol. 2, 79-88 (1972) Goldman, M. : Fluorescent antibody methods. New York: Academic Press 1968 Hogarth-Scott, R.S.: Naturally occuring antibodies to the cuticle of nematodes. Parasitology 58, 221-226 (1968) Lee, D.L.: The structure and composition of the helminth cuticle. Adv. Parasitol. 4, 187 254 (1966) Lumsden, R.D. : Parasitological review. Surface ultrastrueture and chemistry of parasitic helminths. Exp. Parasitol. 37, 267-339 (1975) Miller, T.A.: Vaccination against the canine hookworm diseases. Adv. Parasitol. 9, 153 183 (1971)
A Comparative in vitro Study of Antibody Binding to Different Stages
157
Otto, G.F., Kerr, K.B.: The immunization of dogs against hookworm, Ancylostoma caninum, by subcutaneous injection, of graded doses of living larvae. Am. J. Hyg. 29, 2 5 4 5 (1939) Ourth, D.D., Matre, R., Helgeland, S.M., T6nder, O.: An indirect immuno peroxydase test for human antibodies to Toxoplasma gondii. Acta Pathol. Microbiol. Scand. 82, 145-150 (1974) Owen, J., Slocombe, D., Whitlock, J.H. : Rapid ecdysis of infective Haemonchus contortus cayugensis larvae. J. Parasitol. 55, 1102-1104 (1969) Pearse, A.G.E.: Histochemistry, Vol 2. p. 1438 Edingburgh and London: Churchill Livingstone 1972 Seesee, F.M., Wescott, R.B., Gorham, J.R.: Nippostrongylus brasiliensis: indirect fluorescent antibody studies of immunity in mice. Exp. Parasitol. 39, 214-221 (1976) Sinclair, I.J. : The relationship between circulating antibodies and immunity to helminthic infections. Adv. Parasitol. 8, 97 127 (1970) Swellengrebel, N.H., Sterman, M.M.: Animal parasites in man. Princeton, N.J.: V a n Nostrand 1961 Zaman, V., Singh, M.: Immuno-fluorescent studies with hookworms. I. Antigenic relationships of ova. Trans. R. Soc. Trop. Med. Hyg. 59, 690 693 (1965)
Received March 1, 1978
Z. Parasitenkd. 56, 147-157 (1978)
Parasitenkunde Parasitology Research
9 by Springer-Verlag 1978
A Comparative in vitro Study of Antibody Binding to Different Stages of the Hookworm Ancylostoma caninum J.C.M. Klaver-Wesseling, J.C.M. Vetter, and W.K. Visser Laboratory of Parasitology, University of Amsterdam, Mauritskade 57, Amsterdam, The Netherlands
Summary. The Indirect Fluorescent Antibody Technique (IFAT) and the Indirect Immuno Peroxidase Technique (IIPT) have been applied to cryostat sections and intact stages of the hookworm species Aneylostoma caninum with sera from infected dogs. Especially the role of the body surface ( = cuticle (cortex, matrix, basal layer) and hypodermis) in immunity was studied. Using cryostat sections and dead intact stages as the antigen, specific antibody binding was demonstrated round the ovum membrane and the cuticle of all stages of this hookworm species. Cryostat sections of adult worms showed, that it probably is not the cuticle itself that is antigenic, but that the specific reaction that is observed consisted of a layer, covering the cortex of the cuticle. Infective and parasitic living stages, however, showed no antibody binding in contrast to the free-living stages in which specific antibody binding was demonstrated.
Index Descriptions Ancylostoma caninum; Indirect Fluorescent Antibody Technique (IFAT); Indi-
rect Immuno Peroxidase Technique (IIPT); hookworms,; cryostat sections; body surface; cuticle; dogs.
Introduction Although the evidence of immunity to Ancylostoma caninum in dogs has been well established (Otto and Kerr, 1939; Miller, 1971), no detailed data are available about immune mechanisms operating against hookworms or the eventual adaptive mechanisms which hookworms might have to ensure that they survive the immune response of their hosts. Antibody binding in hookwormshas been demonstratedusing the Indirect FluorescentAntibody Technique (IFAT) to the ovum membrane of A. caninum and Necator americanus (Zaman and
0044-3255/78/0056/0147/$2.20
148
J.C.M. Klaver-Wesseling et al.
Singh, 1965; Azevedo and Rombert, 1970) and to the body surface of intact third stage motile larvae of N. americanus (Bail and Bartlett, 1969), freshly isolated from infected soil and stimulated to exsheath with sodium hypochlorite. However, the relation between antibody binding and the time that hookworms are present in their hosts, has never been investigated. In schistosomes, for instance, it has been demonstrated that antibody binding present in serum from i m m u n e rhesus monkeys are unable to bind to the body surface of four-day schistosomula, while these antibodies can kill three-hour schistosomula in vitro by damaging the surface (Clegg and Smithers, 1972). Indeed, hookworms differ from schistosomes, but on the other hand they are also present for a certain period in similar tissues and body fluids as the schistosomes (skin, bloodstream, lungs). The
aim
the antibody including
of the present binding
those
experiment
to different
stages which
is t o g a t h e r
more
stages of the hookworm
have been present
in the host
information
about
species A. caninum, for a certain
time.
Material and Methods The h o o k w o r m species A. caninum that was used in these experiments, was originally obtained from a naturally-infected dog and was maintained in our laboratory in dogs for 14 generations of h o o k w o r m s over a period of 10 years. The different stages of this parasite were collected as follows: The ova from freshly passed feces of infected dogs were isolated using the brine floatation method (Swellengrebel and Sterman, 1961). First stage larvae were obtained by washing the isolated ova in distilled water and then placing them in a 0.05% sterile NaC1 solution at 27 ~ C for 24 h. Second stage larvae were collected by thoroughly mixing infected feces with sterilized soil. After an incubation period of three days at 27 ~ C, the mixture was wrapped in thick filter-paper and the larvae were isolated using a Baermann apparatus. Third stage larvae were similarly obtained after an incubation period of seven days. The greater portion of those larvae were still ensheathe& In order to study the cuticle of the third stage larvae itself, they were stimulated with CO2 to exsheath as outlined by Owen et al. (1969). Lung stages were isolated from the lungs of infected dogs, 1-3 days after infection. The lungs were cut into pieces and the larvae were collected using a Baermann apparatus. These larvae appeared to be third stages which had similar morphological i characteristics as the fresh exsheathed third stage larvae, except that the lung stages contain less food granules. Fourth stage larvae were collected by washing the opened small intestine of dogs infected subcutaneously seven days previously. Adult worms were similarly obtained 28 days after infection. After isolation all stages were washed twice in phosphate buffered saline (PBS) at p H 7.2. When intact stages were used as the antigen, they were placed in an excess of antiserum to a final dilution of 1:4 for 30 min. To fourth stage larvae and adults an excess of undiluted antiserum was added. The tests were performed at 27 ~ C when ova and free-living stages were used as the antigen. W h e n testing the infective and parasitic stages, the reactions were carried out in small glass tubes at 37 ~ C and incubation periods of 30 min up till 48 h were used. If the incubation period lasted longer than a few hours, antibiotics (25 U penicillin and 0.25 m g streptomycin]ml serum) and fungicides (0.01 mg pimeracin/ml serum) were added. When cryostat sections were used as the antigen, the parasites were placed in gelatin capsules filled with embedding medium. This embedding medium was prepared by adding 5 ml bovine serum albumin and 1 g gelatin (Difco) to 15 ml sterile PBS. This mixture was brought to 30 ~ C before use. These capsules were quickly frozen in a vial containing isopentane cooled with liquid nitrogen and stored at - 1 8 0 ~ until they were used. Serial sections of 8 g m were made on the cryostat microtome at - 2 0 ~ C. They were transferred to slides and withdrawn from the cryostat in order to thaw and dry. They were then immersed in acetone and dried. After this fixation the slides were stored at - 2 0 ~ until they were used. Antisera to A. caninum were obtained from eight mongrel dogs infected once subcutaneously with a high dose (5000) of larvae. Normal sera were obtained from six adult mongrel dogs which
A Comparative in vitro Study of Antibody Binding to Different Stages
149
were not infected with hookworms. Their history is the same as that of the infected dogs used. On several times after infection (from 10 days up till 6 months), blood was taken from the vena saphena. The sera were collected, pooled and divided into 0.2 ml samples, which were stored in small serum vials at - 20~ C. The IFAT was carried out as outlined by Goldman (1968), using a commercially prepared conjugate (RAD/FITC, Nordic Lab., Tilburg, Holland) mainly directed against the immunoglobulins G of the dog. The specificity of the reaction was determined by comparing the results of slides incubated in antiserum with slides incubated in 1. PBS 2. normal sera 3. antisera that were absorbed to their own antigens. Preparation of the antigen and the absorption procedure was essentially as described by Goldman (1968). 4. commericially prepared unlabeled antisera to dogs immunoglobulins (RAD/7S, Nordic Lab., Tilburg, Holland) applied after the application of the antiserum and prior to the application of the conjugate. In addition, heterologous antigens were used. A. ceylanicurn served as the related heterologous antigen and Paramecium spec. as the unrelated heterologous antigen. After performing the test, the living stages were immobilized by adding several drops of a 10% alcohol solution. They were then examined, like the cryostat sections, in PBS. The IIPT has not yet been used on hookworms before, but it has proved to be a useful technique to demonstrate human antibodies against Toxoplasma gondii (Ourth et al., 1974). This technique was carried out as outlined by Pearse (1972). A commercially prepared peroxidase labeled conjugate was used [RAD/Ig G(H/L)/PO, Nordic Lab., Tilburg, Holland], that was mainly directed against the immunoglobulins G of the dog. The antigen-antibody complex was coloured by a 20-rain incubation at room temperature in a 5 mg 3-3'-diaminobenzidine tetrahydrochloride (DAB, Sigma, St. Louis, USA) solution in a 10 ml 0.05 M Tris solution, pH 7.6, to which is added 0.l ml 1% H2Oz just before use. The specificity of this technique was determined in the same way as it was done with the IFAT. Moreover, the endogenic peroxidase activity was determined by incubating the specimens in substrate only. The different stages and cryostat sections were examined in Gum Syrup (Apathys) or they were dried at room temperature and then examined in DPX (DePex, Gurr Ldt., London, England). A Leitz Dialux microscope was employed, that could be used with or without fluorescence. The final magnification was 100, 250, 400 or 1000. Important situations were photographed using a Leica MDa camera with a Kodak Highspeed Ektachrome Daylight film (22 DIN) in the IFAT and an Ilford Pan F film (18 DIN) in the IIPT. From the IFAT coloured pictures black- and-white prints were made.
Results T h e r e s u l t s a r e r e p r e s e n t e d in F i g u r e s 1 t o 14 a n d T a b l e 1.
1. T h e I n d i r e c t F l u o r e s c e n t A n t i b o d y T e c h n i q u e ( I F A T )
1.1. Cryostat Sections When the sections were examined under the fluorescence microscope before a p p l y i n g t h e test, a b l u e - g r e e n f l u o r e s c e n c e o f d i f f e r e n t s t r u c t u r e w a s o b s e r v e d ( a u t o f l u o r e s c e n c e ) . F o r t h a t r e a s o n t h e s e c t i o n s w e r e c o u n t e r s t a i n e d w i t h different concentrations of Eriochrome Black (Difco) to reduce this autofluorescence. The lowest concentration that gave a complete reduction of the autofluorescence w a s f u r t h e r u s e d in t h e test. I n all s t a g e s o f t h e h o o k w o r m
treated with different antisera, labeled anti-
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Table 1. Specific antibody binding of the cuticle of cryostat sections and intact stages of the hookworm Ancylostoma caninum Cryostat sections
Dead stages
Living stages
Free-living stages
Ova 1~ stage 2 ~ stage
+ + +
+ +
+ + +
Infective stages
3~ stage 3 ~ stage
+ +
+ +
-
Parasitic stages
4 ~ stage 5~ stage (adult)
+ +
+ +
-
+ Specific reaction; - negative reaction
Fig. 1. IFAT applied to cryostat section of adult worm. Incubation in antiserum, e cuticle Fig. 2. IFAT applied to cryostat section of adult worm. Control reaction, e cuticle
globulins and Eriochrome black, the cuticle showed a strong yellow-green fluorescence. The internal structures appeared to be red stained. The cuticle of adult stages gave more detailed information: a weak yellow-green fluorescence w a s o b s e r v e d i n t h e c u t i c l e itself, b u t t h e o u t e r s u r f a c e s h o w e d a s t r o n g y e l l o w g r e e n f l u o r e s c e n c e ( F i g . 1). U s i n g h o m o l o g o u s a n t i g e n a n d n o r m a l s e r u m , t h e control reactions sometimes showed a faint yellow-green fluorescence of the c u t i c l e a n d t h e i n t e r n a l o r g a n s a p p e a r e d t o b e r e d s t a i n e d . All o t h e r c o n t r o l r e a c t i o n s s h o w e d n o y e l l o w - g r e e n f l u o r e s c e n c e a t all (Fig. 2).
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F r o m these results it may be concluded that the reaction in the cuticle is specific.
1.2. Intact Stages 1.2.1. Stages Isolated From Feces or Soil. Ova or larvae treated with antiserum, taken after different intervals post infectionum, showed a strong specific reaction of the outer surface (Fig. 3). The intensity of the fluorescence was so high that the internal structures were not visible. The specificity was determined by the control reactions that were used, as it was done with the cryostat sections (Fig. 4). Some ova and larvae produced a strong autofluorescence of the internal structures. These ova, incubated overnight at 2 7 ~ and at a high degree of humidity, appeared to be dead, judged by an arrested development compared to the other ova and the dye exclusion test. When the larvae with the strong autofluorescence were removed and examined more carefully, these stages appeared to be dead as well, judged by motility and the dye exclusion test. 1.2.2. Infective Stages and Stages Isolated From the Host. Application of the labeled anti-globulins after incubation with the different antisera caused no specific reaction in almost all infective and parasitic stages (Figs. 5 and 7). Only a weak autofluorescence of the internal structures was observed. However, in some cases a strong specific reaction of the outer s,,rface was detected. Removing these specimens and studying them more carefully, it appeared that they were dead, judged by mobility, movements of the body fluids and the dye exclusion test (Figs. 6 and 8). Because of these results we applied the I F A T to dead intact stages, simultaneously with living stages. Similar results were obtained as described above. So, when the infective and parasitic stages were dead before the test was applied to them, they reacted specifically in contrast with the living stages.
2. Indirect I m m u n e Peroxidase Technique
2.1. Cryostat Sections There was no endogenic activity, which is an advantage compared to the IFAT, where the untreated sections showed some autofluorescence. In the control reactions sometimes a weak brown staining was visible. Incubating the sections in PBS, the reaction was completely negative. Comparing incubation in antiserum with incubation in PBS and control sera, a specific reaction was observed round the ovum m e m b r a n e and the cuticle of all h o o k w o r m stages. Cryostat sections of ova and larvae could give no details about the precise site of the reaction. However, using cryostat sections of adult worms, more information could be gathered. The different layers of the cuticle and mostly parts of the hypodermis as well, were clearly visible. A weak specific reaction
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A Comparative in vitro Study of Antibody Binding to Different Stages
Fig. 9. IIPT applied to cryostat section of adult worm. Incubation in antiserum, c cuticle; m matrix; b basal layer; h hypodermis Fig. 10. IIPT applied to cryostat section of adult worm. Control reaction
Fig. 3. IFAT applied to living ovum. Incubation in antiserum Fig. 4. IFAT applied to living ovum. Control reaction Fig. 5. IFAT applied to living infective larva. Incubation in antiserum Fig. 6. IFAT applied to dead infective larva. Incubation in antiserum Fig. 7. IFAT applied to living adult worm. Incubation in antiserum Fig. 8. IFAT applied to dead adult worm. Incubation in antisermn
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co
cortex;
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Fig. 11. IIPT applied to dead infective larva. Incubation in antiserum Fig. 12. IIPT applied to living infective larva. Incubation in antiserum Fig. 13. IIPT applied to living infective larva, partly in his old cuticle (sheath). Incubation in antiserum Fig. 14. IIPT applied to living infective larva, partly in his old cuticle (sheath). Control reaction
A Comparative in vitro Study of Antibody Binding to Different Stages
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could be observed in the cuticle itself, but a strong specific reaction was visible on the cuticle: the cortex was covered with a brown coloured layer (Figs. 9 and 10). Sometimes the hypodermis seemed to colour brown as well, but it was impossible to prove this reaction to be specific as cryostat sections most of the time only showed parts of the hypodermis.
2,2. Intact Stages Dead intact stages incubated in antiserum all showed a specific reaction: a brown colour was evenly distributed round the ovum membrane and the cuticle (Fig. 11). However, using living stages as the antigen, the cuticle of the infective larvae and the parasitic stages remained completely negative (Fig. 12), while the free-living stages were stained specifically (Figs. 13 and 14).
Discussion
In a review about the body surface of helminths Lumsden (1975) stated that the cuticles of several nematode species are thought to be antigenic and that antibody binding to the outer surface of the cuticular membrane has been demonstrated. However, data represented by Hogarth-Scott (1968) suggest the possibility that immunoglobulins adsorbed by at least certain nematode species represent cross-reacting naturally occuring antibodies rather than antibodies whose production is stimulated specifically by prior exposure of the host to nematode surface antigens. In broad outline our results indicate that the cuticle indeed is antigenic. So we do not think naturally occuring antibodies only are involved, but that the greater amount of IgG immunoglobulins which adhered to the body surface of the different hookworm stages, were produced after an infection with A. caninum. Indeed we only used a conjugate directed against the IgG - antibodies of the dog, but in our opinion this gives a reliable result. Also Seesee et al. (1976) applied the I F A T to cryostat sections of Nippostrongylus brasiliensis. They used three types of conjugate: directed against the Ig G, the IgM and the IgA immunoglobulins, and they demonstrated that antibodies to the IgG class of immunoglobulins produced intense fluorescence that could not be improved. Using cryostat sections of the different stages, the cuticle reacts specifically. Cryostat sections of adult hookworms give more detailed information: the cuticle itself does not seem to be the source of antigenic stimulation, but a substance covering the cortex of the cuticle is involved. We agree with Sinclair (1970) who stated that antigens which react on the cuticle possibly are not part of the outer layer, but secreted by the pore canals (Bird, 1958). These pore canals might be present in the cuticle of hookworms as well. In the IIPT the hypodermis was sometimes visible and then possibly reacted specifically. Maybe this structure is involved in producing the antigen that is then transported through the pore canals to the outer surface. However, definite conclusions cannot yet
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be drawn. Therefore further investigation o f the b o d y wall is needed. Also Lee (1966) stated that not m u c h is k n o w n a b o u t the b o d y wall o f h o o k w o r m s . More information could be gathered, for instance, using electron microscope techniques as is done with Ascaris lumbricoides and combining the results o f several cytochemical techniques. Using intact stages as the antigen, the results are not so unanimous. The b o d y surface of the free-living stages were f o u n d to react specifically whether they were dead or alive. However, the b o d y surfaces of the infective and parasitic dead stages reacted specifically, while those o f the infective and parasitic living stages remained negative. So it seems that antibodies, although present, are not acting against infective and parasitic living stages o f A. caninum. This seems not to be in agreement with, for example, the results o f Ball and Bartlett (1969). These authors claimed that the cuticle of exsheathed motile infective larvae of N. americanus incubated in sera f r o m patients infected with this h o o k w o r m , showed specific fluorescence. This difference could be explained by the fact that Ball and Bartlett used a fluorescein-labelled a n t i - h u m a n serum. So the fluorescence they demonstrated does not intrinsically have to be caused by a n t i b o d y binding, but other protein fractions of the serum might be involved. O u r results with infective and parasitiC living stages seem to be m o r e in agreement with those of schistosomes. The only difference is that schistosomes are only attainable by antibodies during the first period of time they are in their host (Clegg and Smithers, 1972), while h o o k w o r m s seem to be protected against antibodies f r o m the first m o m e n t they enter the host, We do not k n o w which mechanisms protect the h o o k w o r m s against antibodies, but our results suggest that an energy-dependent process might be involved. Therefore the present experiments shall be continued by studying the relation between the metabolic activity o f the infective and parasitic stages and a n t i b o d y binding.
References Azevedo, J. Fraga de, Rombert, P. : Antigen factors that can interfere with results of immunofluorescence tests in some helminthiasis. In: Standardization in immunofluorescence. E.J. Holborow, ed., Oxford and Edingburgh: Blackwell 1970 Ball, P.A.J., Bartlett, A.: Serological reactions to infection with Necator americanus. Trans. R. Soc. Trop. Med. Hyg. 63, 362 369 (1969) Bird, A.F.: Further observations on the structure of the nematode cuticle. Parasitology 48, 32-39 (1958) Clegg, J.A., Smithers, S.R.: The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. Int. J. Parasitol. 2, 79-88 (1972) Goldman, M. : Fluorescent antibody methods. New York: Academic Press 1968 Hogarth-Scott, R.S.: Naturally occuring antibodies to the cuticle of nematodes. Parasitology 58, 221-226 (1968) Lee, D.L.: The structure and composition of the helminth cuticle. Adv. Parasitol. 4, 187 254 (1966) Lumsden, R.D. : Parasitological review. Surface ultrastrueture and chemistry of parasitic helminths. Exp. Parasitol. 37, 267-339 (1975) Miller, T.A.: Vaccination against the canine hookworm diseases. Adv. Parasitol. 9, 153 183 (1971)
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Otto, G.F., Kerr, K.B.: The immunization of dogs against hookworm, Ancylostoma caninum, by subcutaneous injection, of graded doses of living larvae. Am. J. Hyg. 29, 2 5 4 5 (1939) Ourth, D.D., Matre, R., Helgeland, S.M., T6nder, O.: An indirect immuno peroxydase test for human antibodies to Toxoplasma gondii. Acta Pathol. Microbiol. Scand. 82, 145-150 (1974) Owen, J., Slocombe, D., Whitlock, J.H. : Rapid ecdysis of infective Haemonchus contortus cayugensis larvae. J. Parasitol. 55, 1102-1104 (1969) Pearse, A.G.E.: Histochemistry, Vol 2. p. 1438 Edingburgh and London: Churchill Livingstone 1972 Seesee, F.M., Wescott, R.B., Gorham, J.R.: Nippostrongylus brasiliensis: indirect fluorescent antibody studies of immunity in mice. Exp. Parasitol. 39, 214-221 (1976) Sinclair, I.J. : The relationship between circulating antibodies and immunity to helminthic infections. Adv. Parasitol. 8, 97 127 (1970) Swellengrebel, N.H., Sterman, M.M.: Animal parasites in man. Princeton, N.J.: V a n Nostrand 1961 Zaman, V., Singh, M.: Immuno-fluorescent studies with hookworms. I. Antigenic relationships of ova. Trans. R. Soc. Trop. Med. Hyg. 59, 690 693 (1965)
Received March 1, 1978
