522
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 71, No. 6, 1977.
Immunofluorescent 2. Bearing
reactions
with
on host-parasite
microfilariae relations
D. S. RIDLEY AND ELISABETH C. HEDGE Hospital jbr Tropical Diseases, London NW1 OPE
summary A number of immunofluorescent reactions involving the sheath, cuticle and cytoplasm of microfilariae were studied on the supposition that although the antibodies involved are not lethal, the mechanisms affecting immunofluorescence might nevertheless be relevant to the problem of survival of microfilariae in the blood. It is already known that in the presence of microfilaraemia specific antibodies against the sheath are almost absent. Other observations were as follows: (i) Microfilariae failed to adsorb non-specific immunoglobulin after 20 hours’ incubation, in contrast with other helminth larvae and non-blood protozoa which did adsorb immunoglobulin from normal sera. (ii) Microfilariae from five cases of loiasis and two of bancroftian filariasis were found to display the same blood group antigenicity as the blood groun of the host patient. The blood group of the micro&a&e could be -reversed by incubation in serum of another group. But mlcrofilariae of Brugia pahangi obtained from a cat failed to adsorb human blood group antigens as also did the cuticle of unsheathed human microElariae. (iii) The mlcroElaria1 cytoplasm of Lou Zoa and Wuchereria bancrofti, exposed after sonication, never gave indirect immunofluorescent titres over 1:32 with the sera of the host patient. In all cases titres were lower than those with the sera of other patients with homologous infections. This phenomenon was not seen with non-blood microfilariae, or with other parasites tested. Introduction Successful evasion of the host’s immune mechanisms presents a particular problem for the parasites of the blood. Antigenic variation, a solution adopted by the protozoa, is not available to hehninths. Although there is well documented evidence of antigenic mimicry of the host by schistosomes (SMITHERS 1972 and 1976) little is known about the host parasite relationships of microfilariae (BEAVER. 1970). The relationship between antibody levels -and the ‘number of circulating microfilariae has been briefly reviewed in the preceding paper (HEDGE & RIPLEY, 1977). Although there is no evidence that the IgG antibodies on which immunofluorescent tests are based are themselves protcctivc to the host, mechanisms that cause masking or blocking of the one might well do the same with the other. The present paper is concerned, therefore, with the further investigation by means of the indirect immunofluorescent test (IFAT), of a number of previous observations which would appear to have a bearing on hostparasite relationships in Elariasis. (i) In one case, Lou Zoa microfilariae were found to fluoresce specifically with blood group antibody of the same group as the patient
& RIDLEY, 1975). (ii) During the course of a diagnostic evaluation of the IFAT (HEDGE & RIDLEY, 1977) it was noted that autologous sera gave negative or weak reactions with the microfilariae of Loa loa when homologous or heterologous sera from other patients were almost invariably positive. (iii) HOGARTH-SCOTT (1968) noted that helminth larvae adsorbed IgG from normal human sera after prolonged incubation. This was not tried with microfilariae but we thought that it might possibly be relevant to the situation in filariasis. (HARRISON
Materials and methods In general the methods employed and the sources of material were the same as those in Part I of this paper. Antigens
MicroElariae of Lou Zoa and other human parasites were obtained from patients at the Hospital for Tropical Diseases, of Dirofilaria immitis from the uterus of an adult worm (dog host) and of Brugia pahangi from the blood of an infected cat. They were used in both intact and sonicated forms. Sera
The sera were all obtained from patients except for the anti-A and anti-B blood group sera which were obtained from a Blood Group Reference Laboratory. They were uncontaminated with dye; sera mixed with dye were found to be unusable. The agglutinating titres of each were given as 1:256 though in practice they were found to be somewhat lower than this. ZFT
Tests were carried out at screening dilutions of l/16 and l/32, and the results reported at whichever dilution seemed more significant. Tests were taken to titre when appropriate. Adsorption tesfs To test the uptake of antigen or antibody by microfilariae and a variety of other parasites, the organism was incubated at 37°C for 20 hours in the appropriate serum, either free or, as in the case of Plasmodium falciparum and Trypanosoma brucei, fixed on to slides. A comparison of the two methods with E&amoeba histolytica trophozoites showed little difference in the intensity of fluorcscence in the IFAT. After incubation, the organisms were washed thoroughly either, in the case of slides, in a magnetically stirred iar of PBS with repeated changes of the buffer- or the free parasites were- trapped on a membrane filter and 30 ml of PBS was forced nast them. This latter method has been found to be a useful way of
D. S. RIDLEY
AND
cleaning parasitic antigens. If an adsorption test was designed to test the uptake of an antigen by an organism, IFAT was subsequently carried out using the appropriate antibody. If adsorption of antibody had been performed this was followed with a direct fluorescent test using conjugated anti-human globulin. Results of antibody in vitro A number of parasites were incubated for 20 hours with normal serum. After thorough washing the IFAT was completed in the usual way. Strong surface fluorescence was shown on trophozoites of E. histolytica, cysts of Giardia lamblia, a free-living crithidia, Schistosoma cercariae, Trickinella larvae and the embryo sac of Diroflaria immitis. Fluorescence was still apparent if the normal serum used for adsorption was diluted to 1 in 20 in the case of Trichinella larvae and Schistosoma cercariae, and 1 in 40 for E. histolytica. Five different normal sera were tested in parallel with the Trichinella and E. histolytica antigens and all gave similarly positive results. The use of fluorescein labcllcd anti-human monospecific immunoglobulin showed the adsorbed antibody to be mainly IgG and, to a lesser extent, IgM. In contrast to these results, whole microfilariae of B. pakangi, L. loa, 0. volvulus and uterine D. immitis failed to show any surface fluorescence after adsorption. Similarly, Plasmodiam falcigarum and tryptomastigotes of Trypanosoma brucei in blood films showed no fluorescence. However, if the microfilariae were broken prior to the adsorption, fluorescence was seen in the exposed cytoplasm. Occasionally an irregular “speckled” fluorescence was noted after incubation of whole sheathed microfdariae but only if the microtllariae had previously been stored in the frozen state. The speckling was taken to represent damage due to freezing. Direct tests of microfilariae with conjugated antiglobulin were invariably negative. Adsorption
Blood group Sheath:
antigenicity
of microjilariae
Microfilariae of L. loa were available from four patients of known blood group and of W. bancrofti from two patients. The intact unsonicated microfilariae were tested by IFAT against anti-A and anti-B blood group sera at 1: 16 and examined for sheath fluorescence. The results are shown in Table I. Included in the table also is the one L. Zoa of blood group A previously reported (HARRISON & RIDLEY, 1975). At 1: 16 there was complete
523
E. C. HEDGE
correlation between the antigenicity of the microfilariae and the blood groups of their former hosts. Four of the five positive reactions were positive also at 1: 32. Microfilariae of B. pakangi obtained from a cat gave completely negative results. Cytoplasm: The cytoplasm of these various microfilariae, exposed after sonication, gave no reaction with anti-A or B sera. Cuticle: The non-sheathed microfilariae of Dipetalonema perstarrs, Dirojilaria immitis and Onckocerca volvulu.~, and the larvae of Strongyloides stercoralis gave no cuticular fluorescence with anti-A or anti-B sera. Conversion
of blood group
arztigenicity
Intact sheathed microfilariae, the A and B group antigenicity of which had been previously determined, were incubated for 20 hours with serum of a patient of different blood group. This serum was known to exhibit a high titre antibody against the A or B blood group of the microfilaria. After intensive washing under pressure as described, the IFAT using anti-A and anti-B sera was repeated. This was done in three cases with results as shown in Table I. In two cases microfilariae (L Zoa and W. bancrofii) which had given sheath fluorescence at 1:32 with anti-B serum -only were converted after incubation with A group serum to react at 1:32 only with anti-A. Fluorescence was strong but the titres were not taken higher. The cytoplasm of the microfilariae exposed after sonication failed to react after incubation with blood group sera. Microfilariae of B. pahangi which, as previously stated, had no blood group antigenicity after recovery from the cat host, still failed to react with either anti-A or B sera after appropriate incubation. Microfilariae of Dipetalonema perstans similarly failed to exhibit cuticular fluorescence. Inhibition
of autologous
reactions
Microfilariae of L. Zoa were available from six patients and W. bancrofti from two patients. After sonication the IFAT produced fluorescent plugs at the exposed ends of the cytonlasm. The highest IFAT titre in the cvtonlasm of any of these microfilariae tested against the patient’s own serum was a weak though positive reaction at 1: 32, and some titres were not better than 1: 16. By contrast, sera from patients with homologous infections gave full positive reactions at 1: 32 or higher in all cases and heterologous sera were positive at 1: 32 in all except a few cases. When sufficient microfilariae were available antigen-antibody pairs were crossed. This was done in
Table I - IFAT results between microlihxrial sheath and anti-A and anti-B sera at 1:16, in relation to blood group of the bost patient. And effect of subsequent incubation with serum of a diierent group Species of microiilaria L. Ioa L. loa L. loa L. loa L. loa W. bancrofti W. bancrofti
Patient’s blood group
Initial reaction with anti-A anti-B
Subsequent incubation; serum group
Subsequent reaction with anti-A anti-B
524
IMMUNOFLUORESCENT
Table II - IFAT between microfilariae and autologous or homologous serum, as crossed pair reactions
5Pe of filariasis L. loa
L. loa
W. bancrofti
Source of microfilariae and serum A x A x FxA FxF
A F
A A R R
A R A R
x x x x
Titre of reaction
3241
256+ 64t
16+ 3241
512+
64+ 324~
N
x
N
16+
N H H
x x x
H N H
32+ 32+ lb+
three instances, with the results as shown in Table II. There seemed to be good evidence of partial inhibition of the reaction with autologous serum. It was not possible to test for autologous inhibition of sheath fluorescence: if there was microfilaraemia the patient’s serum failed to give sheath fluorescence either with his own microfilariae or with those from homologous infections. Tests of inhibition of autologous reactions by crossed pairing were performed with a number of other parasitic infections, with negative results in all cases. Parasites tested included 0. volvulus (three pairs), S. stercoralis (with larvae obtained direct from the patient and also by culture), Plasmodium falciparum, E. histolytica and G. lamblia.
Immunofluorescence of the cytoplasm was due predominantly to IgG, and to a lesser extent IgM. There was no fluorescence due to IgA. It was not determined whether the inhibition of the reaction with autologous serum was associated more with IgG or IgM. Discussion On the evidence of fluorescent antibody techniques intact microfilariae and, indeed, all the blood parasites we have tested are peculiar in that their surfaces fail to adsorb immunoglobulin after prolonged incubation. By contrast with the sheath and cuticle, the exposed cytoplasm of microfilariae firmly adsorbed immunoglobulin from normal serum or from the sera of patients with other parasitic infections. Adsorption was demonstrated similarly with other helminth larvae and protozoa which have the common factor of not residing in the blood. This phenomenon of immunoglobulin adsorption by certain parasites is probably the same as that of COOMBS et al. (1965) and HOGARTH-SCOTT (1968), who demonstrated the adsorption of an immunoglobulin, predominantly IgG, by the cuticle of six species of parasitic or non-parasitic helminth larvae. This was presumed to be a naturally occurring antibody. The mixed anti-globulin reaction used by these workers was in principle the same as the IFAT used by us except that it was a non-fluorescent method. However, as far as we know there have been no previous reports of the adsorption of non-specific immunoglobulins by the nonblood protozoa.
REACTIONS
WITH
MICROFILARIAE
This evidence from in vitro experiments is supported by the fact that parasites from the circulating blood and, therefore, exposed to immunoglobulins, fail to fluoresce in the direct fluorescent antibody method. Thus there would appear to be some barrier to immunoglobulin adsorption by parasites residing in the blood. The second conclusion of this study is that, on the evidence of the IFAT, the sheath of human microfilariae (L. loa and W. bancrofti) adsorbed specific A and B blood group antigens. In all cases microfilariae on recovery from blbod displayed the blood group of their hosts, and in two cases this was reversed by incubation in vitro with a serum of a different group. It is possible that the adsorption of blood group or other host antigen may play a role in the evasion of immune mechanisms by sheathed microfilariae, but there is no direct evidence on this point. Non-sheathed microfilariae, such as D. perstans, which do not adsorb blood group antigens, survive well enough in the blood, though it is doubtful whether their numbers ever reach those sometimes seen with sheathed microfilariae. The third conclusion of this study is also hard to interpret. What is the significance of the low titre of the reaction between the microfilarial cytoplasm (L. loa and W. bancrofti) and the serum of the host, by comparison with that between the same microiilariae and the serum of any other patient with a homologous infection? Cytoplasm is not normally exposed to antibody. The mechanism is obscure. The value of the phenomenon to the microfilaria is also doubtful. We were unable to demonstrate “autologous inhibition” with non-blood microfilariae (0. volvulus) or with a number of other human parasites, including some of those that circulate in the blood. One possibility is that the immune mechanisms that we have been studying in relation to humoral factors may be of benefit to the microfilaria for the evasion of nonhumoral mechanisms. Thus PULVERTAFT & PULVERTAFT (1967) observed an anomalous failure of activation of lymphocytes by microfilariae from the same patient and also a partial or complete inhibition of activation of lymphocytes from other patients when microfilariae were present. It sound here as if there might be an inhibition of lymphocytes analogous to our own autologous inhibition of IFAT reactions. BAGAI & SUBRAHMANYAM (1970) and NELSON et al. (1976) found lymphocytes and macrophages and, occasionally, eosinophils to adhere closely and firmly to microfilariae in the thorax of albino rats with latent but not patent infections, and they suggested that this was the mechanism by which microfilariae were immobilized and perhaps eventually destroyed. They also found evidence that it was microfilariae, not adult worms, that were responsible for the induction of acquired immunity. One of the problems in studying the immuno-evasion mechanisms of microfilariae is that they are so successful. It becomes extremely difficult to demonstrate the immune process that has to be counteracted. References Bagai, R. C. & Subrahmanyam, D. (1970). Nature of acquired resistance to filarial infection in albino rats. Nature
(London),
228, 682-683.
Beaver, P. C. (1970). Filariasis without microfilaraemia. American
Journal
of Tropical
Medicine
and Hygiene,
19, 181-189. Coombs, R. A. A., Pout, D. D. & Soulsby, E. J. L. (1965). Globulin, possibly of antibody nature, combining with
D. S. RIDLEY
AND
cuticle of Turbatrix aceti. Experimental Parasitology, 16, 311-317. Harrison, J. & Ridley, D. S. (1975). Heterologous reactions involving parasites, blood group antibodies and tissue components. Transactions of the Royal Society of Tropical Medicine and Hygiene, 69, 3 12-3 17. Hedge, E. C. & Ridley, D. S. (1977). Immunofluorescent reactions with microfilariae. 1. Diagnostic evaluation. Transactions and Hygiene,
of the Royal
Society
of Tropical
Medicine
71, 304-307. Hogarth-Scott, R. S. (1968). Naturally occurring antibodies to the cuticle of nematodes. Parasitology, 58, 221-226. Nelson, D. S., Subrahmanyam, D., Rao, Y. V. B. G. & Mehta, K. (1976). Cellular morphology in pleural
525
E. C. HEDGE
exudate of albino
rats infected with Litomosoides
carinii. Transactions of the Royal Medicine and Hygiene, 70, 254-255.
Society
of Tropical
Pulvertaft, R. J. V. & Pulvertaft, 1. (1967). Activation of lymphocytes. Journalof ClinicalPathology, 20,795~805. Smithers, S. R. (1972). Recent advances in the immunologv of schistosomiasis. British Medical Bulletin, 28, 49-54 Smithers, S. R. (1976). Immunity to trematode infections with snecial reference to schistosomiasis and fascioliasis. In: Immunology of Parasitic Infections, S. Cohen & E. Sadun (Editors). Oxford: Blackwell Scientific Publications, Chapter 20. Accepted
for publication
15th July,
1977.
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 71, No. 6, 1977.
Immunofluorescent 2. Bearing
reactions
with
on host-parasite
microfilariae relations
D. S. RIDLEY AND ELISABETH C. HEDGE Hospital jbr Tropical Diseases, London NW1 OPE
summary A number of immunofluorescent reactions involving the sheath, cuticle and cytoplasm of microfilariae were studied on the supposition that although the antibodies involved are not lethal, the mechanisms affecting immunofluorescence might nevertheless be relevant to the problem of survival of microfilariae in the blood. It is already known that in the presence of microfilaraemia specific antibodies against the sheath are almost absent. Other observations were as follows: (i) Microfilariae failed to adsorb non-specific immunoglobulin after 20 hours’ incubation, in contrast with other helminth larvae and non-blood protozoa which did adsorb immunoglobulin from normal sera. (ii) Microfilariae from five cases of loiasis and two of bancroftian filariasis were found to display the same blood group antigenicity as the blood groun of the host patient. The blood group of the micro&a&e could be -reversed by incubation in serum of another group. But mlcrofilariae of Brugia pahangi obtained from a cat failed to adsorb human blood group antigens as also did the cuticle of unsheathed human microElariae. (iii) The mlcroElaria1 cytoplasm of Lou Zoa and Wuchereria bancrofti, exposed after sonication, never gave indirect immunofluorescent titres over 1:32 with the sera of the host patient. In all cases titres were lower than those with the sera of other patients with homologous infections. This phenomenon was not seen with non-blood microfilariae, or with other parasites tested. Introduction Successful evasion of the host’s immune mechanisms presents a particular problem for the parasites of the blood. Antigenic variation, a solution adopted by the protozoa, is not available to hehninths. Although there is well documented evidence of antigenic mimicry of the host by schistosomes (SMITHERS 1972 and 1976) little is known about the host parasite relationships of microfilariae (BEAVER. 1970). The relationship between antibody levels -and the ‘number of circulating microfilariae has been briefly reviewed in the preceding paper (HEDGE & RIPLEY, 1977). Although there is no evidence that the IgG antibodies on which immunofluorescent tests are based are themselves protcctivc to the host, mechanisms that cause masking or blocking of the one might well do the same with the other. The present paper is concerned, therefore, with the further investigation by means of the indirect immunofluorescent test (IFAT), of a number of previous observations which would appear to have a bearing on hostparasite relationships in Elariasis. (i) In one case, Lou Zoa microfilariae were found to fluoresce specifically with blood group antibody of the same group as the patient
& RIDLEY, 1975). (ii) During the course of a diagnostic evaluation of the IFAT (HEDGE & RIDLEY, 1977) it was noted that autologous sera gave negative or weak reactions with the microfilariae of Loa loa when homologous or heterologous sera from other patients were almost invariably positive. (iii) HOGARTH-SCOTT (1968) noted that helminth larvae adsorbed IgG from normal human sera after prolonged incubation. This was not tried with microfilariae but we thought that it might possibly be relevant to the situation in filariasis. (HARRISON
Materials and methods In general the methods employed and the sources of material were the same as those in Part I of this paper. Antigens
MicroElariae of Lou Zoa and other human parasites were obtained from patients at the Hospital for Tropical Diseases, of Dirofilaria immitis from the uterus of an adult worm (dog host) and of Brugia pahangi from the blood of an infected cat. They were used in both intact and sonicated forms. Sera
The sera were all obtained from patients except for the anti-A and anti-B blood group sera which were obtained from a Blood Group Reference Laboratory. They were uncontaminated with dye; sera mixed with dye were found to be unusable. The agglutinating titres of each were given as 1:256 though in practice they were found to be somewhat lower than this. ZFT
Tests were carried out at screening dilutions of l/16 and l/32, and the results reported at whichever dilution seemed more significant. Tests were taken to titre when appropriate. Adsorption tesfs To test the uptake of antigen or antibody by microfilariae and a variety of other parasites, the organism was incubated at 37°C for 20 hours in the appropriate serum, either free or, as in the case of Plasmodium falciparum and Trypanosoma brucei, fixed on to slides. A comparison of the two methods with E&amoeba histolytica trophozoites showed little difference in the intensity of fluorcscence in the IFAT. After incubation, the organisms were washed thoroughly either, in the case of slides, in a magnetically stirred iar of PBS with repeated changes of the buffer- or the free parasites were- trapped on a membrane filter and 30 ml of PBS was forced nast them. This latter method has been found to be a useful way of
D. S. RIDLEY
AND
cleaning parasitic antigens. If an adsorption test was designed to test the uptake of an antigen by an organism, IFAT was subsequently carried out using the appropriate antibody. If adsorption of antibody had been performed this was followed with a direct fluorescent test using conjugated anti-human globulin. Results of antibody in vitro A number of parasites were incubated for 20 hours with normal serum. After thorough washing the IFAT was completed in the usual way. Strong surface fluorescence was shown on trophozoites of E. histolytica, cysts of Giardia lamblia, a free-living crithidia, Schistosoma cercariae, Trickinella larvae and the embryo sac of Diroflaria immitis. Fluorescence was still apparent if the normal serum used for adsorption was diluted to 1 in 20 in the case of Trichinella larvae and Schistosoma cercariae, and 1 in 40 for E. histolytica. Five different normal sera were tested in parallel with the Trichinella and E. histolytica antigens and all gave similarly positive results. The use of fluorescein labcllcd anti-human monospecific immunoglobulin showed the adsorbed antibody to be mainly IgG and, to a lesser extent, IgM. In contrast to these results, whole microfilariae of B. pakangi, L. loa, 0. volvulus and uterine D. immitis failed to show any surface fluorescence after adsorption. Similarly, Plasmodiam falcigarum and tryptomastigotes of Trypanosoma brucei in blood films showed no fluorescence. However, if the microfilariae were broken prior to the adsorption, fluorescence was seen in the exposed cytoplasm. Occasionally an irregular “speckled” fluorescence was noted after incubation of whole sheathed microfdariae but only if the microtllariae had previously been stored in the frozen state. The speckling was taken to represent damage due to freezing. Direct tests of microfilariae with conjugated antiglobulin were invariably negative. Adsorption
Blood group Sheath:
antigenicity
of microjilariae
Microfilariae of L. loa were available from four patients of known blood group and of W. bancrofti from two patients. The intact unsonicated microfilariae were tested by IFAT against anti-A and anti-B blood group sera at 1: 16 and examined for sheath fluorescence. The results are shown in Table I. Included in the table also is the one L. Zoa of blood group A previously reported (HARRISON & RIDLEY, 1975). At 1: 16 there was complete
523
E. C. HEDGE
correlation between the antigenicity of the microfilariae and the blood groups of their former hosts. Four of the five positive reactions were positive also at 1: 32. Microfilariae of B. pakangi obtained from a cat gave completely negative results. Cytoplasm: The cytoplasm of these various microfilariae, exposed after sonication, gave no reaction with anti-A or B sera. Cuticle: The non-sheathed microfilariae of Dipetalonema perstarrs, Dirojilaria immitis and Onckocerca volvulu.~, and the larvae of Strongyloides stercoralis gave no cuticular fluorescence with anti-A or anti-B sera. Conversion
of blood group
arztigenicity
Intact sheathed microfilariae, the A and B group antigenicity of which had been previously determined, were incubated for 20 hours with serum of a patient of different blood group. This serum was known to exhibit a high titre antibody against the A or B blood group of the microfilaria. After intensive washing under pressure as described, the IFAT using anti-A and anti-B sera was repeated. This was done in three cases with results as shown in Table I. In two cases microfilariae (L Zoa and W. bancrofii) which had given sheath fluorescence at 1:32 with anti-B serum -only were converted after incubation with A group serum to react at 1:32 only with anti-A. Fluorescence was strong but the titres were not taken higher. The cytoplasm of the microfilariae exposed after sonication failed to react after incubation with blood group sera. Microfilariae of B. pahangi which, as previously stated, had no blood group antigenicity after recovery from the cat host, still failed to react with either anti-A or B sera after appropriate incubation. Microfilariae of Dipetalonema perstans similarly failed to exhibit cuticular fluorescence. Inhibition
of autologous
reactions
Microfilariae of L. Zoa were available from six patients and W. bancrofti from two patients. After sonication the IFAT produced fluorescent plugs at the exposed ends of the cytonlasm. The highest IFAT titre in the cvtonlasm of any of these microfilariae tested against the patient’s own serum was a weak though positive reaction at 1: 32, and some titres were not better than 1: 16. By contrast, sera from patients with homologous infections gave full positive reactions at 1: 32 or higher in all cases and heterologous sera were positive at 1: 32 in all except a few cases. When sufficient microfilariae were available antigen-antibody pairs were crossed. This was done in
Table I - IFAT results between microlihxrial sheath and anti-A and anti-B sera at 1:16, in relation to blood group of the bost patient. And effect of subsequent incubation with serum of a diierent group Species of microiilaria L. Ioa L. loa L. loa L. loa L. loa W. bancrofti W. bancrofti
Patient’s blood group
Initial reaction with anti-A anti-B
Subsequent incubation; serum group
Subsequent reaction with anti-A anti-B
524
IMMUNOFLUORESCENT
Table II - IFAT between microfilariae and autologous or homologous serum, as crossed pair reactions
5Pe of filariasis L. loa
L. loa
W. bancrofti
Source of microfilariae and serum A x A x FxA FxF
A F
A A R R
A R A R
x x x x
Titre of reaction
3241
256+ 64t
16+ 3241
512+
64+ 324~
N
x
N
16+
N H H
x x x
H N H
32+ 32+ lb+
three instances, with the results as shown in Table II. There seemed to be good evidence of partial inhibition of the reaction with autologous serum. It was not possible to test for autologous inhibition of sheath fluorescence: if there was microfilaraemia the patient’s serum failed to give sheath fluorescence either with his own microfilariae or with those from homologous infections. Tests of inhibition of autologous reactions by crossed pairing were performed with a number of other parasitic infections, with negative results in all cases. Parasites tested included 0. volvulus (three pairs), S. stercoralis (with larvae obtained direct from the patient and also by culture), Plasmodium falciparum, E. histolytica and G. lamblia.
Immunofluorescence of the cytoplasm was due predominantly to IgG, and to a lesser extent IgM. There was no fluorescence due to IgA. It was not determined whether the inhibition of the reaction with autologous serum was associated more with IgG or IgM. Discussion On the evidence of fluorescent antibody techniques intact microfilariae and, indeed, all the blood parasites we have tested are peculiar in that their surfaces fail to adsorb immunoglobulin after prolonged incubation. By contrast with the sheath and cuticle, the exposed cytoplasm of microfilariae firmly adsorbed immunoglobulin from normal serum or from the sera of patients with other parasitic infections. Adsorption was demonstrated similarly with other helminth larvae and protozoa which have the common factor of not residing in the blood. This phenomenon of immunoglobulin adsorption by certain parasites is probably the same as that of COOMBS et al. (1965) and HOGARTH-SCOTT (1968), who demonstrated the adsorption of an immunoglobulin, predominantly IgG, by the cuticle of six species of parasitic or non-parasitic helminth larvae. This was presumed to be a naturally occurring antibody. The mixed anti-globulin reaction used by these workers was in principle the same as the IFAT used by us except that it was a non-fluorescent method. However, as far as we know there have been no previous reports of the adsorption of non-specific immunoglobulins by the nonblood protozoa.
REACTIONS
WITH
MICROFILARIAE
This evidence from in vitro experiments is supported by the fact that parasites from the circulating blood and, therefore, exposed to immunoglobulins, fail to fluoresce in the direct fluorescent antibody method. Thus there would appear to be some barrier to immunoglobulin adsorption by parasites residing in the blood. The second conclusion of this study is that, on the evidence of the IFAT, the sheath of human microfilariae (L. loa and W. bancrofti) adsorbed specific A and B blood group antigens. In all cases microfilariae on recovery from blbod displayed the blood group of their hosts, and in two cases this was reversed by incubation in vitro with a serum of a different group. It is possible that the adsorption of blood group or other host antigen may play a role in the evasion of immune mechanisms by sheathed microfilariae, but there is no direct evidence on this point. Non-sheathed microfilariae, such as D. perstans, which do not adsorb blood group antigens, survive well enough in the blood, though it is doubtful whether their numbers ever reach those sometimes seen with sheathed microfilariae. The third conclusion of this study is also hard to interpret. What is the significance of the low titre of the reaction between the microfilarial cytoplasm (L. loa and W. bancrofti) and the serum of the host, by comparison with that between the same microiilariae and the serum of any other patient with a homologous infection? Cytoplasm is not normally exposed to antibody. The mechanism is obscure. The value of the phenomenon to the microfilaria is also doubtful. We were unable to demonstrate “autologous inhibition” with non-blood microfilariae (0. volvulus) or with a number of other human parasites, including some of those that circulate in the blood. One possibility is that the immune mechanisms that we have been studying in relation to humoral factors may be of benefit to the microfilaria for the evasion of nonhumoral mechanisms. Thus PULVERTAFT & PULVERTAFT (1967) observed an anomalous failure of activation of lymphocytes by microfilariae from the same patient and also a partial or complete inhibition of activation of lymphocytes from other patients when microfilariae were present. It sound here as if there might be an inhibition of lymphocytes analogous to our own autologous inhibition of IFAT reactions. BAGAI & SUBRAHMANYAM (1970) and NELSON et al. (1976) found lymphocytes and macrophages and, occasionally, eosinophils to adhere closely and firmly to microfilariae in the thorax of albino rats with latent but not patent infections, and they suggested that this was the mechanism by which microfilariae were immobilized and perhaps eventually destroyed. They also found evidence that it was microfilariae, not adult worms, that were responsible for the induction of acquired immunity. One of the problems in studying the immuno-evasion mechanisms of microfilariae is that they are so successful. It becomes extremely difficult to demonstrate the immune process that has to be counteracted. References Bagai, R. C. & Subrahmanyam, D. (1970). Nature of acquired resistance to filarial infection in albino rats. Nature
(London),
228, 682-683.
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