The specificity of immunoglobulin G and immunoglobulin M in the fluorescent-antibody test for malaria parasites in mice.

IgG AND IgM IN FLUORESCENT-ANTIBODY TEST FOR MALARIA PARASIMS IN MICE 341 The Specificity of Immunoglobulin G and Immunoglobulin M in the Fluorescent-antibody Test for Malaria Parasites in Mice * by F. E. G. Cox 1 The fluorescent-antibody technique has been widely used for the detection of malaria parasites or immune sera but the potential use of the technique for the actual identification of particular species has not been exploited because of the considerable degree of cross-reaction that occurs. Serological cross-reactions, as detected by the fluorescent-anti- body technique, have been reported between species of malaria parasites in man by Diggs & Sadun (1965), between simian and human species by Tobie et al. (1962, 1963), Coudert et al. (1965), Meuwissen (1968) and Collins, Skinner & Coifman (1967), between simian species by Collins et al. (1965), Collins, Skinner & Guinn (1966) and Voller, Gain- ham & Targett (1966), between rodent species by El-Nahal (1967) and Cox & Turner (1970c) and between avian and human species by Kielman & Weiss (1968). These results were based on observa- tions made with the parasites themselves as antigens, whole antisera and fluorescein-labelled antiglobulins. It has been shown repeatedly that both immuno- globulin M (IgM) and immunoglobulin G (IgG) are involved in the immunological response to malaria parasites. It therefore follows that these immunoglobulins might be more specific in detecting the homologous reaction than whole immuno- globulin (Ig) and thus increase the sensitivity of the fluorescent-antibody technique. IgM and IgG can be utilized in one of two ways. First, the parasites can be exposed to fractionated antisera and labelled with anti-Ig, or, secondly, the parasites can be exposed to whole antisera and labelled with specific anti-IgM or IgG. In either case the degree of reaction with IgM or IgG can be estimated. In the experiments reported in this paper the second alternative was used. Cox & Turner (1970c) have shown that there are antigens in common between four malaria parasites and two piroplasms in mice as detected by the fluorescent-antibody technique using fluorescein- * This investigation received financial support from the World Health Organization. 1 Reader in Zoology, Department of Zoology, University of London King's College, London, England. labelled anti-Ig. The degree of reaction was greatest between antisera and homologous parasites, less between the same antisera and heterologous but related parasites and least between the antisera and the most distantly related parasites. These results provide the basis for a useful model to test the possibility that fluorescein-labelled anti-IgM or anti- IgG might be more specific than anti-Ig in detecting the homologous reaction. Materials and methods The following strains of parasites were used: Plasmodium vinckei (Katanga 52), P. chabaudi (54X), P. berghei berghei (173K), P. berghei yoelii (RCA 17X), Babesia rodhaini (Antwerp) and B. microti (King's 67). The parasites were maintained in mice and used both as antigens and for the production of antisera. Further details of the methods used for obtaining immune mice are given by Cox (1970). The antisera were obtained from mice which had recovered from infection with one of the six para- sites. The sera were taken from mice immune to P. vinckei 45 days after infection, P. chabaudi 48 days after infection, P. b. berghei 34 days after infection, P. b. yoelii 36 days after infection, B. rodhaini 34 days after infection and B. microti 21 days after infection. The fluorescent-antibody technique used was an indirect one based on that of Voller (1964) and has already been described by Cox, Crandall & Turner (1969) and Cox& Turner (1970c). Briefly, thin films of infected blood were fixed in 0.3N HCI, washed, expo- sed to the appropriate antisera, washed again and finally exposed to fluorescein-labelled anti-Ig, anti- IgG or anti-IgM before being examined under ultra- violet light. Antibody titres were determined by the serial dilution of the antisera. The fluorescein-labelled immunoglobulins used were those described by Cox, Crandall & Turner (1969). Three were used: these detected Ig, IgG and IgM and were specific. The six sera from immune mice were reacted with each of the six parasites and the antibody titres determined using labelled anti-Ig, anti-IgM and 2560F 10 00 NOTES 0 o 0 0oC C 0co a O O 0 8 CD _ 0 0 0 N V" _ C 0 0O DO 0 0o0o 0 0 vco 0 (CO 0 C M CD 00 00 0eq l 00 0) C>I X _ c 0q am a ° ~~~~~OCIA > 0NX ° 0 CD 2 C0 - E Q c a co 6C ZD CD, Cg C>00N N r qr-co~~e SDeq 0 0 0 0 O 0 O0 N Q - Z CQC> Q a M - C4 %- " co O O _ o o o o o o e q X..D QC E .c- C X-0 m it 0-a 0 0.04 4 go:a anti-IgG. This produced a total of 108 titres. The reproducibility of the results was tested by compar- ing them with results previously obtained with anti-Ig (Cox & Turner, 1970c) and with other samples of anti-P. vinckei and anti-P. chabaudi sera and by using a random series of replicates. Results The results obtained are given in Table 1. These results show that the antibody titres obtained using fluorescein-labelled anti-IgG were in most cases the same as those obtained using anti-Ig or were only one dilution below. The titres obtained using anti- IgM were considerably lower. The IgG titres differed most markedly from the Ig titres in those cases where P. b. berghei was involved either as the antigen or in the production of the antiserum. In the 25 combinations in which P. b. berghei was not involved, 19 of the titres obtained using anti-IgG were the same as, or one dilution less than, the titres obtained with anti-Ig. In the 11 reactions in which P. b. berghei was involved only two were this close and one of these was the homologous reaction. The only other situations in which the IgG titres were significantly lower than the Ig titres were three in which B. microti was the antigen, two in which B. rodhaini was the antigen and one in which P. vinckei was the antigen and the antiserum was anti-B. microti. Results obtained using the same six parasites as antigens and second samples of anti-P. vinckei and anti-P. chabaudi sera are shown in Table 2. The antibody titres were comparable with those obtained using the first samples of antisera. _EC C 0 0 0 0S 0 iv C: 0 0 0 co. 04) co 0 C.P LUt: as Discussion The results obtained from the experiments out- lined in this paper show that the fluorescent-antibody technique as it is normally performed for the detec- tion of malaria parasites can be modified by using fluorescein-labelled anti-IgG or anti-IgM instead of anti-Ig. The antibody titres obtained were, in the case of anti-Ig, similar to, or, in the case of anti-IgM, lower than those obtained using anti-Ig. In all cases the highest titres were obtained with homologous antigens and antisera. These results indicate that the specificity and sensitivity of the fluorescent-anti- body technique cannot be improved by using labelled anti-IgG or anti-IgM instead of anti-Ig. In view of the difficulties inherent in the production of anti-IgG and anti-IgM sera, there seems to be no point in advocating a change in procedure. 342 0 E 'IC -0 C4D00m c LU cn z CD 0 CD 0 -J0 Ul UJ I0 z CD 0 CD 0 -J0 2 0 I z LU z - z CD z CL z 0 Ul - 0r. C 0 co P z z cD 0 ulL z LJ m bG 03 .00~ a_ 0 t '8 2 o a Q Q Q 00 le 0 v- v- IRP 4) c co c I- IgG AND IgM IN FLUORESCENT-ANTIBODY TEST FOR MALARIA PARASITES IN MICE 343 CO)~~~~~~~~LU) IL l C,)~~~~~~~~~~0C LU Mr0 I- 0U (D00 aU 00 0 0i 04~~ ~ ~ ~ ~ ~~ v C o 2 ~~~~~~ 0 0c co z 0 ~~~~~~~~) )0' 04 04 CO- -0 LU <. 040 P Ci C - 0~~~~C O~~~~~~0 ~ ~ ~ 00_CID_ 04O~~~~~ -O 040~N 4 LU S 40~~~~~~~ OIL 0~~~~~~~~~~~~~~~~C 04 0 (p 00 U.L _C Although the use of fluorescein-labelled anti-IgG and anti-IgM does not improve the specificity of the fluorescent-antibody technique, it is possible that the use of these antisera might provide information as to how recently a malaria infection had been acquired, as suggested by Collins & Skinner (1968), who drew attention to the fact that in human malaria infections both the IgM and IgG levels rose early in the infection but the IgM level then fell. The relative levels of IgG and IgM should then indicate whether or not the infection was a recent one. In fact, the information available on the occurrence of IgM and IgG in human malaria infections is limited (Abele et al., 1965; Tobie et al., 1966) and does not really lend itself to such an analysis. On the other hand, in infections in mice, recent experiments with P. cha- baudi and P. vinckei (Cox et al., 1969), P. berghei yoelii (Cox & Turner, 1970b) and the piroplasm B. microti (Cox & Turner, 1970a), did not show any significant decline in IgM levels during the course of infection and therefore the relative levels of IgM and IgG could not be used to indicate, with these parasites, how recently the infection had been acquired. In view of the reported differences between immunoglobulin responses in mice and in man it is obvious that the experiments reported in this paper will have to be repeated with malaria parasites in man, but it seems unlikely that the results obtained will be any different. ACKNOWLEDGEMENTS I should like to thank Dr C. A. Crandall for preparing the fluorescein-labelled antisera used in this study and Mrs S. A. Turner for technical assistance. REFERENCES Abele, D. C. et al. (1965) Amer. J. trop. Med. Hyg.. 14, 191 Collins, W. E. & Skinner, J. C. (1968) Fluorescent antibody techniques for malaria case detection. In: Eighth International Congresses on Tropical Medicine and Malaria, Teheran, 1968, Abstracts and Reviews, p. 1424 Collins, W. E., Skinner, J. C. & Coifman, R. E. (1967) Amer. J. trop. Med. Hyg., 16, 568 Collins, W. E., Skinner, J. C. & Guinn, E. G. (1966) Amer. J. trop. Med. Hyg., 15, 483 Collins, W. E. et al. (1965) J. Parasit., 51, 81 Coudert, J. et al. (1965) Bull. Soc. Path. exot., 59, 630 344 NOTES Cox, F. E. G. (1970) Bull. Wld Hlth Org., 43, 325 Cox, F. E. G., Crandall, C. A. & Turner, S. A. (1969) Bull. Wld Hith Org., 41, 251 Cox, F. E. G. & Turner, S. A. (1970a) Ann. trop. Med. Parasit., 64, 167 Cox, F. E. G. & Turner, S. A. (1970b) Ann. trop. Med. Parasit., 64, 175 Cox, F. E. G. & Turner, S. A. (1970c) Bull. Wld Hlth Org., 43, 337 Diggs, C. L. & Sadun, E. H. (1965) Exp. Parasit., 16, 217 El-Nahal, H. M. S. (1967) Bull. Wld Hlth Org., 36, 423 Kielman, A. & Weiss, N. (1968) Acta trop. (Basel), 25, 185 Meuwissen, J. H. E. T. (1968) Trop. geogr. Med., 20, 137 Tobie, J. E. et al. (1962) Amer. J. trop. Med. Hyg., 11, 589 Tobie, J. E. et al. (1963) J. Amer. med. Ass., 184, 945 Tobie, J. E. et al. (1966) J. Immunol., 97, 498 Voller, A. (1964) Bull. Wld Hlth Org., 30, 343 Voller, A., Garnham, P. C. C. & Targett, G. A. T. (1966) J. trop. Med. Hyg., 69, 121 Gametogenesis of Plasmodium berghei in Corticosteroid-Treated Albino Mice by NEELAM TANDON 1 & N. C. BHATTACHARYA 2 Despite the wealth of information on the life- cycle, anatomy, taxonomy, etc. of the malaria para- sites, the factors controlling their morphogenesis, particularly gametocyte production, even now remain largely unknown and speculative. It has been observed that one or more generations of schizogony in the blood appear to be a necessary preliminary to a change to sexuality (Garnham, 1966). Gameto- cytes are highly sensitive to abnormal conditions such as growth of the parasite in vitro or in unnatural hosts, and in repeated non-cyclic syringe trans- mission in the optimum host, when the sexual form becomes quite inapparent. In this last condi- tion the production of gametocytes tends to be rapidly reduced or apparently lost (Vincke & Michiels 1963). It has also been stated that a period of 4i quiescence" at subzero temperature may result in revival of gametocyte production (Bafort et al., 1965). While emphasizing the incompleteness of our knowledge of the factors regulating gametogenesis, Bishop (1955), in her review of the subject, indicated that the physiological state of the host was an important factor in the production of the sexual form. The present study was therefore undertaken to study whether gametogenesis is affected by the use of immunodepressant drugs. As a preliminary step, an attempt has been made to treat the rodent host with a known immunodepressant corticosteroid 1 Lecturer, Department of Medical Entomology, School of Tropical Medicine, Calcutta, India. 'Head, Department of Medical Entomology, School of Tropical Medicine, Calcutta, India. and to observe its effect on gametogenesis in Plasmodium berghei. Material and methods Laboratory-bred adult albino mice were inoculated intraperitoneally with 1 mg dexamethasone 21- phosphate every 12 hours throughout the period of study (12 days). P. berghei maintained in this laboratory by non-cyclic passage in albino mice were used in the experiment. This strain, as a consequence of numerous syringe passages, was observed to have considerably lost its power of gametogenesis. On the third day of corticosteroid administration, 4 mice were infected by the chosen Plasmodium strain. At the same time 4 mice of the same age not receiving the drug were infected similarly with the same strain of P. berghei to serve as control. The dosage of inoculum was the same in both cases. From the sixth day after infection two peripheral blood smears were made each day for 3 consecutive days from each animal of the two groups of mice. This was because gametocytes of this strain of P. berghei usually appeared on the sixth day after infection. The smears were stained with Leishman's stain and examined under the microscope. The num- bers of parasitized cells, gametocytes, and schizonts in 15-25 oil-immersion fields of two smears from each animal were determined daily for 3 days and the average numbers of each group per field cal- culated. The results are shown in the table. 2560OG