Annals of Tropical Medicine & Parasitology
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
Application of immunofluorescence to detection of antibody in Leishmania infections N. Behforouz, H. R. Rezai & S. Gettner To cite this article: N. Behforouz, H. R. Rezai & S. Gettner (1976) Application of immunofluorescence to detection of antibody in Leishmania infections, Annals of Tropical Medicine & Parasitology, 70:3, 293-301, DOI: 10.1080/00034983.1976.11687125 To link to this article: http://dx.doi.org/10.1080/00034983.1976.11687125
Published online: 15 Mar 2016.
Submit your article to this journal
View related articles
Citing articles: 15 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypgh19 Download by: [Australian Catholic University]
Date: 18 August 2017, At: 22:54
Annals of Tropical Medicine and Parasitology, Vol. 70, No.3 (1976)
Application of immunofluorescence to detection of antibody in Leishmania infections BY N.BEHFOROUZ, H.R.REZAI
AND
S.GEITNER
Department of Microbiology, Medical School, Pahlavi University, Shira1;., Iran Downloaded by [Australian Catholic University] at 22:54 18 August 2017
Received 25 March 1975
There are controversial reports about the presence of specific antibody in cutaneous leishmaniasis of animals and man (Adler, 1965; Bryceson et al., 1970; .Bryceson et al., 1972; Rezai, Gettner and Behforouz, 1972). In a previous communication we reported the existence of a factor in immune guinea pig serum which inhibits the growth of Leishmania enriettii in NNN media (Rezai et al., 1972). This inhibitory factor was found in immune sera of guinea pigs at titres not exceeding 1/32 and was not detectable in the sera of mice or humans infected with L. tropica. This inhibitory factor was complement dependent and heat stable. The present investigation is concerned with the establishment and application of a more sensitive technique using indirect immunofluorescence for the detection of antibody in guinea pigs as well as in mice and humans infected with Leishmania species. Our previous study showed that histological changes, generally associated with antibody production, occurred in the lymph nodes of guinea pigs 17 days after infection whereas the growth inhibitory activity could not be detected for six weeks (Rezai, Haghighi and Ardehali, 1972). The indirect immunofluorescence test developed in this laboratory was thus used for a more precise determination of the sequential development of antibody during leishmania! infections in both animals and man. Using this test, the sera from the infected mice and man was also examined for cross reactivity with the heterologous species of Leishmania. In addition, this paper presents the results of the chromatographic fractionation of immune guinea pig antibody and the immunoelectrophoretic identification of the active fraction.
MATERIALS AND METHODS Leish~nania
Parasites For infecting mice, promastigotes from the first few passages (up to the lOth) of L. tropica grown in modified NNN media were used (Lemma and Schiller, 1964). For infecting guinea pigs, amastigotes were collected by sterile swabs from the closed, secondary lesions of previously infected guinea pigs and suspended in physiological saline or Hank's Balanced Salt solution. HUIIUlD. Cases Naturally infected individuals with proven cases of active oriental sore were used as the source of sera and persons who had no known contact with Leishmania were used as a source of negative control sera.
294
IMF IN LEISHMANIAL INFEGriONS
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
IaCectecl Animals Outbred mice weighing 20-30 g from our animal breeding house were infected subcutaneously at the base of the tail with 1 X 1()8 promastigotes of L. tropica in 0 ·1 ml. Guinea pigs weighing about 250-300 g from our animal breeding house were infected on the ear with 8 X 1()6 amastigotes of L. enriettii in 0·2 ml. Fluorescent Anti-Specific Sera The anti-guinea pig, mouse and human sera were purchased from the Nordic Pharmaceutical Company. Before use the anti-guinea pig serum was diluted 1/10 in saline. Immediately following reconstitution of the fluorescent anti-mouse serum, a dilution of 1/100 was used. However,-upon standing the fluorescence apparently faded and a dilution of 1/20 was needed. Fluorescent anti-human serum was diluted 1/10 and employed throughout the experiments. Preparation of Antigen for Fluorescent Antibody Technique For all leishmania! species used, two-day old subcultures of the protozoa from NNN media were used as the source of antigen for the fluorescent antibody technique. The organisms were washed once and resuspended in saline to a concentration giving approximately 10-15 organisms per high power field of microscope. This suspension was applied to a plaque of 1·5. em diameter on glass slides and was then dried, fixed with acetone for 10 minutes and washed with saline.
Indirect Staining Sera obtained from infected humans, mice and guinea pigs were diluted from 1/161/1024. A standard method for indirect fluorescent staining was used to stain the prepared antigen slides. Fluorescent Microscopy The prepared slides were observed under a Zeiss fluorescent microscope using the darkfield condenser and the BG 12 secondary filter. The intensity of the fluorescence was graded from ± to 4+ according to the brightness and clarity of the stained antigen. Growth Inhibitory Test The test for the growth inhibitory activity of the serum of infected humans and animals was performed according to our previous communication (Rezai, Sher and Gettner, 1969). Column ChroJDatography Serum (10 ml) obtained from three guinea pigs in the convalescent stage of cutaneous leishmaniasis was treated first with saturated ammonium sulphate and then with 0·005 m phosphate buffer, pH 8 in order to obtain the pseudoglobulin fraction of the serum. This fraction was then added to a column of DEAE cellulose. Elution was carried out in a step~ wise fashion with increasing concentration of phosphate and diminishing pH as follows: 0·0051\f PO,, pH 8; 0·015M PO,, pH 7; 0·10M PO,, pH 6. Fractions of 5 ml were collected throughout the elution procedure and each fraction was analyzed for total protein content by U.V. spectroscopy at 280 mp.. The fractions corresponding to each protein peak were pooled and concentrated to approximately 2 ml each using Carbowax (Union Carbide) at 4°C. These pooled fractions were stored until use at -20°C. hnm.unoelectrophoretic Studies The immune serum fractions were submitted to immunoelectrophoresis in Noble agar
BEHFOROUZ et al.
295
for 120 minutes at 250 volts. Rabbit anti-guinea pig serum (Hoechst) was then applied to the slides. Precipitin lines were read two days later.
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
RESULTS
Detection of Antibody in Guinea Pigs by Indirect Immanoflaoresceace The fluorescent antibody of infected guinea pigs was determined at various times following infection. The results are shown in Table I. The anti-leishmania} antibody in these animals was detected at low intensities through a dilution of 1/64 as early as 12 days after infection with L. enriettii. The antibody titre and intensity- of fluorescence in guinea pigs continued to increase during the course of infection until approximately 76 days at which time the leishmaniallesion was at its height. During and after healing, the antibody titre fell one to two dilutions from the 76 day peak, and the intensity of fluorescence in low dilutions was somewhat less. The normal, control animals showed a range of antibody titre from 0-1/16 at low intensities of fluorescence. Guinea pigs having metastatic lesions consistently showed a high titre of antibody with high intensities of fluorescence at the lower dilutions. TABLE 1
Indirect immunofluorescence
of pooled sera• from guinea pigs infected with L.
enriettii
Intmsity ofjluomcencet in serum dilutions Days after i'lfection and stale of lesion
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
1/2048
±
12 (no lesion but erythema)
1+
1·5+
1+
±
±
±
±
20 (induration and erythema)
2+
1·5+
1+
1+
±
±
±
±
30 (slight open lesion)
3+
3+
1·5+
1+
±
±
±
±
45 (heavy open lesion)
3+
3+
3+
2·5+
±
±
±
±
76 (heavy lesion)
4+
4+
3+-
3+
2+
1+
±
±
90 (healing lesion)
3+
2·5+
1·5+
I+
±
±
±
±
120 (healed lesion)
3+
3+
2·5+
2+
I+
±
±
±
Metastatic lesions
3+
3+
3+
3+
I+
±
±
±
Normal
I+
±
±
±
±
±
±
±
• At each time interval, sera was obtained from at least three guinea pigs.
t 4+ =very bright, 3+ =bright, 2+ =visible, I+ =faint, ± =very faint. Detection of Antibody in Mice by Indirect Immanofl.uoresceace The anti-leishmania} antibody in infected mice was detected at a titre of 1/64 23 days after infection with L. tropica. The antibody titre and intensity of fluorescence increased during the course of infection. By the 94th day after infection, the mice sera showed an antibody titre of 1/256 with 4+ fluorescence through 1/32. Significantly, it was observed that antibody was detected even before the appearance of open lesions in these mice. Despite the early presence of antibody in mice sera, the infections continued to progress until a majority of the mice died. The normal, control mice showed a range of antibody titre from 0-1/32 at low intensities of fluorescence (Table 2).
296
IMF IN LEISHMANIAL INFEGriONS TABLE2
Indirect immunofluorescence of pooled sera* from mice injected with L. tropica
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
Intensity offluorescence in serum dilutions Days after infection
1/16
1/32
1!64
1/128
1/256
1/512
1/1024
1/2048
10
±
±
±
±
23
3+
2+
2+
1+
50
3+
1·5+
2+
1·5+
± ± ±
71
4+
3+
2-5+
2+
1·5+
94
4+
4+
2·5+
1·5+
1+
± ± ± ± ±
150
3+
3+
2+
2+
1+
1+
Normal
1+
I+
±
±
±
±
± ± ± ± ± ± ±
± ± ± ± ± ± ±
* At each time interval, sera was obtained from at least three mice.
Reactions of Mice Sera with Ho~nologous and Heterologous Antigens Upon testing the sera of mice infected with L. tropica against L. tropica, L. enriettii and L. donovani organisms, it was observed that the titres of fluorescent antibody in mice sera were the same for all three organisms. These results indicate a high degree of cross reactivity TABLE 3
Indirect immunofluorescence
of sera from human cases having leishmaniasis Intensity qffluorescence in serum dilutions
Type of leishmaniasis
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
1/2048
Cutaneous--one month duration*
2+
2+
2+
1·5+
±
±
±
±
Cutaneous-four months duration*
3+
2+
2·5+
I+
±
±
±
±
Cutaneous-five tion*
3+
months
dura2·5+
1·5+
1·5+
±
±
±
±
Cutaneous-nine months duration* 3+
3+
3+
I+
±
±
±
±
Suspected diffuse type of five years duration-two cases
4+
3+
3+
3+
3+
1·5+
±
Systemic (Kala Azar)
4+
4+
4+
3·5+
2·5+
2+
I+
± ±
Suspected systemic-(Kala Azar) Case# 1
4+
3+
2-5+
2-5+
2+
1·5+
I+
±
Suspected systemic-(Kala Azar) Case# 2
4+
3·5+
3+
3+
2+
1+
±
±
Suspected systemic-(Kala Azar) Case# 3
4+
3+
1+
I+
±
±
±
±
Suspected systemic-(Kala Azar) Case# 4
4+
3+
2+
1+
Normal*
I+
±
±
±
± ±
± ±
± ±
± ±
* In these cases at least three subjects are tested and the results were averaged.
BEHFOROUZ et al.
297
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
of the mouse serum antibodies against heterologous Leishmania organisms. Moreover, upon testing mice sera against newly isolated, low passaged Leishmania and Leishmania which had been passaged in culture for over 30 times, no difference was observed in intensity or titre of the antibody reaction.
Detection of Antibody in Haman Cases of Leiaahmani•sis Several cases of cutaneous leishmaniasis in humans were tested for antibody titre (Table 3). In individuals having lesions for less than one year (the normal course of infection is one year), the antibody titre ranges from 1/32 through 1/128 depending upon the duration of the lesion. The intensity of fluorescence in these cases was significantly greater than that of the controls. In none of these cases was the titre higher than 1/128. Two cases of cutaneous leishmaniasis of a duration longer than five years, suspected to be of the diffuse type (having spreading lesions of both arms and a negative skin test), were tested for antibody activity. It was found that the serum antibody activity in both cases exceeded a titre of 1/512 with an intensity of at least 3+ through a dilution of 1/256. One case of systemic leishmaniasis in a 20-month-old girl, who was shown to have Leishman-Donovan bodies in her bone marrow, was examined for serum antibody activity by indirect immunofluorescence. A titre of 1/1024 was observed in this patient's serum with intensities of 3-4+ through a dilution of 1/128. Four additional cases of suspected systemic leishmaniasis in young children (eight months to six years) were examined for serum antibody activity by this method. Clinically, these children demonstrated hepatosplenomegaly, fever, anaemia and in general were in poor condition. In each case, bone marrow smears were inconclusive but following treat:rnent with Glucantime (the drug of choice for Leishmania infections) all four children clinically improved. A range of antibody titres from 1/256-1/1024 were observed in these patients' sera as shown in Table 3. In all four cases, a 4+ fluorescence was observed in the 1 :16 dilution of their sera.
0.015M pH 7
Phosphate 0.005M Buffer
pH 8
O.lM pH6
1.2
} 0 00 N
.,c II
ci
c
d
.L.
\..)
.,...
:I all
A
"c
Cl
c
.L.
u
...
-Ql
:::1 all
B
0.1 oL-------------------------------------------~ FRACTIONS
Fls. 1. DEAE cellulose chromatography of immune guinea pig serum. D
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
298 IMF IN LEISHMANIAL INFECTIONS
BEHFOROUZ et al.
299
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
Reaction of HUIIUUl Serum with Homologous and Heterologou• Antigen• Upon testing the antisera of two human cases ofleishmaniasis (systemic and prolonged cutaneous) against L. tropica, L. enriettii and L. donovani organisms, it was observed that the titre of fluorescent antibody in these cases was the same against all three organisms. Again, human serum appears to have a high cross reactivity with heterologous species of Leishmania. Fractionation of lmmUDe Guinea Pig Serum The pooled fractions of immune guinea pig serum containing protein which were obtained by DEAE cellulose chromatography were tested for fluorescent antibody activity and it was observed that the two protein peaks, A and B, eluted from the column with the first buffer {0·005 M, pH 8) had antibody activity (Fig. 1). According to Oliveira et al. (1970) these two peaks correspond to the heterogeneous y 2 immunoglobulins of guinea pigs. Both fractions A and B, when tested imiilunoelectrophoretically against anti-guinea pig serum, showed one precipitin line in the region of slow-moving IgG, which, in the guinea pig, corresponds to y 2 (Fig. 2). Fractions C, D and E which contained faster moving lgG, as well as other immunoglobulins, had no antibody activity against Leishmania parasites as tested by the indirect immunofluorescent technique. DISCUSSION Our results show unequivocally that mice, guinea pigs and humans having an active leishmania! infection do produce antibody against the Leishmania parasites. In infected guinea pigs, the indirect fluorescent antibody technique is a much more sensitive test than the inhibition technique formerly developed in our laboratory. In the case of mice and humans, the indirect fluorescent antibody technique is capable of detecting antibody which was formerly undetectable in significant quantities by the inhibition culture method. The results obtained from indirect fluorescent antibOdy technique described here are measured in two parameters. One is the intensity of fluorescence of each of the various serum dilutions and the other is the titre of antibody in the serum. In general, the highest intensity of fluorescence seems to appear in the lower serum dilutions at the peak of the leishmania! infections. Using the indirect fluorescent technique for infected guinea pigs, antibody was detected in a low titre as early at 12 days after experimental infection. This result corresponds to the results previously obtained from histological studies of infected guinea pigs in which microscopic cellular changes generally associated with antibody production could be seen in the lymph nodes as early as two weeks after infection. Thus, this technique for the detection of antibody is much more sensitive than the culture inhibition technique with which antibody in guinea pigs cannot be detected until six weeks after infection. The titre and intensity of fluorescent antibody in infected guinea pigs corresponds closely with the course of clinical infection, with the peak of antibody titre and fluorescent intensity occurring at the time when the lesion is at its maximum. In experiments performed on guinea pigs by Bryceson et al. (1972) using indirect immunofluorescence, antibody was detected several weeks later than in our studies. Animals with metastatic lesions show a high level of antibody. Despite the presence of
Ftc. 2.
Immunoelectrophoresis of guinea pig serum fractions. Troughs contained anti-guinea pig serum and the wells contained: A and B = fractions eluted with 0·005M pH 8·0 PO,, C =fractions eluted with 0·015M, pH 7·0 PO, and fractions D and E =fractions eluted with O·IOM pH 6·0 PO,.
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
300
IMF IN LEISHMANIAL INFECTIONS
this antibody, these animals do not generally recover and eventually die from either their original leishmania! lesion or secondary bacterial infections. It seems, therefore, that a high level of antibody does not necessarily correspond with healing in infected guinea pigs. The chromatographic and immunoelectrophoretic studies performed on immune guinea pig serum showed that the antibody activity detected by the indirect immunofluorescent technique resided in the y 2 portion of guinea pig globulin. This finding confirms the prediction made by Turk and Bryceson (1971) that the antibody in guinea pigs against Leishmania would probably be y 2 • The Gamma 2 globulin of guinea pigs is generally accepted to be the complement fixing portion of guinea pig globulin. The leishmania! growth inhibitory substance of immune guinea pig sera described previously in our laboratory was also complement dependent, and indicates that the growth inhibitory substance is probably the same antibody as the one detected by the immunofluorescent technique. Whereas the growth inhibition test could detect no antibody activity in the sera of infected mice, the immunofluorescent technique described in this paper was able to detect antibody activity as early as 23 days after infection. This activity increased both in intensity and titre during the course of infection. As these mice usually do not survive, either due to their primary or secondary bacterial infections, the titre of antibody developed in these animals is apparently not protective. These findings are essentially in agreement with what was found by Preston et al. (1972) with CBA mice. However, the titre of fluorescent antibody in our study using outbred mice did not reach the level of the antibody titre obtained by these investigators. A possible explanation for this difference may be due to animal strain differences. CBA mice heal 12 weeks after infection whereas the strain of mice used in our investigation eventually die with active lesions. It is interesting that the antibody produced in mice against L. tropica reacts with the other Leishmania species equally well. This antibody activity therefore appears directed to an antigen or antigens common to all species of Leishmania. In human cutaneous leishmaniasis, this technique could also detect antibody activity early in the course of infection. The titre of antibody in these patients never rose above 1f 128. The cases of suspected diffuse leishmaniasis and the one case of proven systemic leishmaniasis had a higher titre of antibody at greater intensities than found in the nondisseminating cutaneous form of leishmaniasis. The higher antibody titre in these cases is probably due to greater antigenic stimulation. It is well known that active kala-azar is characterized by a massive hyperglobulinaemia. Adler (1965) and Garnham and Humphrey (1969) suggested that most of this globulin was not specific antibody. The fluorescent antibody technique described here did, however, demonstrate quantitatively a high level of leishmania-specific antibody in the patient with systemic leishmaniasis. Whether or not the leishmania-specific antibody detected in this patient accounts for the entire hyperglobulinaemia seen in kala-azar patients remains to be investigated. The fact that the human cases having widely disseminated leishmaniasis had high titres of antibody but poor prognosis is again indicative that in humans, as in mice and guinea pigs, the presence of antibody does not necessarily correspond with healing. It appears that the indirect immunofluorescent technique described here will prove to be particularly useful in the laboratory diagnosis of systemic leishmaniasis. At present, in order to diagnose systemic leishmaniasis, a bone marrow smear and culture must be performed. The fact that sera from these human cases react equally well with all three Leishmania species is an advantage in that a laboratory need not keep all species of Leishmania in culture in order to identify the leishmania! origin of questionable infections. The role which the antibody detected by this immunofluorescent technique plays in
BEHFOROUZ et al.
301
immunity to leishmaniasis is not clear. It appears in high titres even in hosts with overwhelming leishmania! infections and, in these cases, does not appear to be protective. This immunofluorescent technique does, however, provide investigators with a tool with which leishmania! infections may probably be diagnosed and in experimental animals traced from an early stage. ACKNOWLEDGEMENTS. This research was supported by grants from Pahlavi University Research Council No. 52-MD-8 and the Ministry of Higher Education No. 401-102-478.
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
SUMMARY Indirect immunofluorescence was used for determination of antibody in human cases of cutaneous leishmaniasis, mice infected with Leishmania tropica and guinea pigs infected with L. enriettii. Results indicated that antibody in mice is detectable 23 days after infection with L. tropica. The antibody titre correlates well with the extent of infection. In guinea pigs infected with L. enriettii, antibody appears much faster and is detectable 12 days after infection. Here also the antibody titre increased during the course of infection until about 76 days at which time the leishmaniallesion was at its height. Guinea pigs having metastatic lesions consistently showed a high titre of antibody. In several cases of human cutaneous leishmaniasis antibody was detectable at the titre of not more than 1/128. In five cases of suspected systemic leishmaniasis antibody titre was much greater than in cutaneous leishmania! infection. Upon characterization of anti-leishmania! serum of guinea pig with DEAE-cellulose chromatography and analysis of the fractions by immunoelectrophoresis, it was found that the antibody activity detected by the indirect immunofluorescence resided in the y 2 portion of guinea pig globulin. REFERENCES ADLER, S. ( 1965). 'Immunology of leishmaniasis.' Israel Journal of Medical Science, 1, 9-13. BRYCESON, A. D. M., BRAY, R. S., WOLSTENCROFT, R. A. & DuMONDE, D. C. (1970). 'Immunity in cutaneous leishmaniasis of the guinea pig.' Clinical and Experimental Immunig, 1, 301-341. BRYCESON, A. D. M., PRESTON, P. M., BRAY, R. S. & DuMONDE, D. C. (1972). 'Experimental cutaneous leishmaniasis. II. Effects of immunosuppression and antigenic competition on the course of infection with Leishmania enriettii in the guinea pig'. Clinical and Experimental Immunig, 10, 305-335. GARNHAM, P. C. C. & HUMPHREY, J. H. (1969). 'Problems in leishmaniasis related to immunology.' Current Topics in Microbiology and Immunology, 48, 29-42. LEMMA, A. & ScHILLER, E. L. (1964). 'Extracellular cultivation of the leishmania! bodies of species belonging to the protozoan genus Leishmania.' Experimental Parasitology, 15, 503-513. OLivEIRA, B., OsLER, A. G., SIRAGANIAN, R. P. & SANDBERG, A. L. (1969). 'The biologic activities of guinea pig antibodies I. Separation of y 1 and y 2 immunoglobulin and their participation in allergic reactions of the immediate type'. Journal of Immunology, 104, 320-328. PRESTON, P., CARTER, R., LEUCHARS, E., DAVIES, A. & DUMONDE, D. C. (1972). 'Experimental cutaneous leishmaniasis III. Effects of thymectomy on course of infection of CBA mice with Leishmania tropica'. Clinical and Experimentol/mmunology, 10, 337-357. REZAI, H. R., SHER, S. & GETTNER, S. (1969). 'Leishmania tropica, L. donovani, and L. enriettii: Immune rabbit serum inhibitory in vitro.' Experimental Parasitology, 26, 257-263. REzAI, H. R., GETTNER, S. & BEHFORouz, N. (1972). 'Antileishmanial activity of immune guinea-pig serum.' Journal of Medical Microbiology, 5, 371-375. REzAI, H. R., HAomGHI, P. & ARDEHALI, S. (1972). 'Histological appearance of the site of inoculation and lymph nodes of guinea-pigs at various times after infection with Leishmania enriettii.' Journal of Medical Microbiology, 66, 225-234. TURK, J. L. & BRYCESON, A. D. M. (1971). 'Immunological phenomena in leprosy and related diseases.' Advances in Immwwlogy, 13, 209-266.
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
Application of immunofluorescence to detection of antibody in Leishmania infections N. Behforouz, H. R. Rezai & S. Gettner To cite this article: N. Behforouz, H. R. Rezai & S. Gettner (1976) Application of immunofluorescence to detection of antibody in Leishmania infections, Annals of Tropical Medicine & Parasitology, 70:3, 293-301, DOI: 10.1080/00034983.1976.11687125 To link to this article: http://dx.doi.org/10.1080/00034983.1976.11687125
Published online: 15 Mar 2016.
Submit your article to this journal
View related articles
Citing articles: 15 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypgh19 Download by: [Australian Catholic University]
Date: 18 August 2017, At: 22:54
Annals of Tropical Medicine and Parasitology, Vol. 70, No.3 (1976)
Application of immunofluorescence to detection of antibody in Leishmania infections BY N.BEHFOROUZ, H.R.REZAI
AND
S.GEITNER
Department of Microbiology, Medical School, Pahlavi University, Shira1;., Iran Downloaded by [Australian Catholic University] at 22:54 18 August 2017
Received 25 March 1975
There are controversial reports about the presence of specific antibody in cutaneous leishmaniasis of animals and man (Adler, 1965; Bryceson et al., 1970; .Bryceson et al., 1972; Rezai, Gettner and Behforouz, 1972). In a previous communication we reported the existence of a factor in immune guinea pig serum which inhibits the growth of Leishmania enriettii in NNN media (Rezai et al., 1972). This inhibitory factor was found in immune sera of guinea pigs at titres not exceeding 1/32 and was not detectable in the sera of mice or humans infected with L. tropica. This inhibitory factor was complement dependent and heat stable. The present investigation is concerned with the establishment and application of a more sensitive technique using indirect immunofluorescence for the detection of antibody in guinea pigs as well as in mice and humans infected with Leishmania species. Our previous study showed that histological changes, generally associated with antibody production, occurred in the lymph nodes of guinea pigs 17 days after infection whereas the growth inhibitory activity could not be detected for six weeks (Rezai, Haghighi and Ardehali, 1972). The indirect immunofluorescence test developed in this laboratory was thus used for a more precise determination of the sequential development of antibody during leishmania! infections in both animals and man. Using this test, the sera from the infected mice and man was also examined for cross reactivity with the heterologous species of Leishmania. In addition, this paper presents the results of the chromatographic fractionation of immune guinea pig antibody and the immunoelectrophoretic identification of the active fraction.
MATERIALS AND METHODS Leish~nania
Parasites For infecting mice, promastigotes from the first few passages (up to the lOth) of L. tropica grown in modified NNN media were used (Lemma and Schiller, 1964). For infecting guinea pigs, amastigotes were collected by sterile swabs from the closed, secondary lesions of previously infected guinea pigs and suspended in physiological saline or Hank's Balanced Salt solution. HUIIUlD. Cases Naturally infected individuals with proven cases of active oriental sore were used as the source of sera and persons who had no known contact with Leishmania were used as a source of negative control sera.
294
IMF IN LEISHMANIAL INFEGriONS
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
IaCectecl Animals Outbred mice weighing 20-30 g from our animal breeding house were infected subcutaneously at the base of the tail with 1 X 1()8 promastigotes of L. tropica in 0 ·1 ml. Guinea pigs weighing about 250-300 g from our animal breeding house were infected on the ear with 8 X 1()6 amastigotes of L. enriettii in 0·2 ml. Fluorescent Anti-Specific Sera The anti-guinea pig, mouse and human sera were purchased from the Nordic Pharmaceutical Company. Before use the anti-guinea pig serum was diluted 1/10 in saline. Immediately following reconstitution of the fluorescent anti-mouse serum, a dilution of 1/100 was used. However,-upon standing the fluorescence apparently faded and a dilution of 1/20 was needed. Fluorescent anti-human serum was diluted 1/10 and employed throughout the experiments. Preparation of Antigen for Fluorescent Antibody Technique For all leishmania! species used, two-day old subcultures of the protozoa from NNN media were used as the source of antigen for the fluorescent antibody technique. The organisms were washed once and resuspended in saline to a concentration giving approximately 10-15 organisms per high power field of microscope. This suspension was applied to a plaque of 1·5. em diameter on glass slides and was then dried, fixed with acetone for 10 minutes and washed with saline.
Indirect Staining Sera obtained from infected humans, mice and guinea pigs were diluted from 1/161/1024. A standard method for indirect fluorescent staining was used to stain the prepared antigen slides. Fluorescent Microscopy The prepared slides were observed under a Zeiss fluorescent microscope using the darkfield condenser and the BG 12 secondary filter. The intensity of the fluorescence was graded from ± to 4+ according to the brightness and clarity of the stained antigen. Growth Inhibitory Test The test for the growth inhibitory activity of the serum of infected humans and animals was performed according to our previous communication (Rezai, Sher and Gettner, 1969). Column ChroJDatography Serum (10 ml) obtained from three guinea pigs in the convalescent stage of cutaneous leishmaniasis was treated first with saturated ammonium sulphate and then with 0·005 m phosphate buffer, pH 8 in order to obtain the pseudoglobulin fraction of the serum. This fraction was then added to a column of DEAE cellulose. Elution was carried out in a step~ wise fashion with increasing concentration of phosphate and diminishing pH as follows: 0·0051\f PO,, pH 8; 0·015M PO,, pH 7; 0·10M PO,, pH 6. Fractions of 5 ml were collected throughout the elution procedure and each fraction was analyzed for total protein content by U.V. spectroscopy at 280 mp.. The fractions corresponding to each protein peak were pooled and concentrated to approximately 2 ml each using Carbowax (Union Carbide) at 4°C. These pooled fractions were stored until use at -20°C. hnm.unoelectrophoretic Studies The immune serum fractions were submitted to immunoelectrophoresis in Noble agar
BEHFOROUZ et al.
295
for 120 minutes at 250 volts. Rabbit anti-guinea pig serum (Hoechst) was then applied to the slides. Precipitin lines were read two days later.
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
RESULTS
Detection of Antibody in Guinea Pigs by Indirect Immanoflaoresceace The fluorescent antibody of infected guinea pigs was determined at various times following infection. The results are shown in Table I. The anti-leishmania} antibody in these animals was detected at low intensities through a dilution of 1/64 as early as 12 days after infection with L. enriettii. The antibody titre and intensity- of fluorescence in guinea pigs continued to increase during the course of infection until approximately 76 days at which time the leishmaniallesion was at its height. During and after healing, the antibody titre fell one to two dilutions from the 76 day peak, and the intensity of fluorescence in low dilutions was somewhat less. The normal, control animals showed a range of antibody titre from 0-1/16 at low intensities of fluorescence. Guinea pigs having metastatic lesions consistently showed a high titre of antibody with high intensities of fluorescence at the lower dilutions. TABLE 1
Indirect immunofluorescence
of pooled sera• from guinea pigs infected with L.
enriettii
Intmsity ofjluomcencet in serum dilutions Days after i'lfection and stale of lesion
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
1/2048
±
12 (no lesion but erythema)
1+
1·5+
1+
±
±
±
±
20 (induration and erythema)
2+
1·5+
1+
1+
±
±
±
±
30 (slight open lesion)
3+
3+
1·5+
1+
±
±
±
±
45 (heavy open lesion)
3+
3+
3+
2·5+
±
±
±
±
76 (heavy lesion)
4+
4+
3+-
3+
2+
1+
±
±
90 (healing lesion)
3+
2·5+
1·5+
I+
±
±
±
±
120 (healed lesion)
3+
3+
2·5+
2+
I+
±
±
±
Metastatic lesions
3+
3+
3+
3+
I+
±
±
±
Normal
I+
±
±
±
±
±
±
±
• At each time interval, sera was obtained from at least three guinea pigs.
t 4+ =very bright, 3+ =bright, 2+ =visible, I+ =faint, ± =very faint. Detection of Antibody in Mice by Indirect Immanofl.uoresceace The anti-leishmania} antibody in infected mice was detected at a titre of 1/64 23 days after infection with L. tropica. The antibody titre and intensity of fluorescence increased during the course of infection. By the 94th day after infection, the mice sera showed an antibody titre of 1/256 with 4+ fluorescence through 1/32. Significantly, it was observed that antibody was detected even before the appearance of open lesions in these mice. Despite the early presence of antibody in mice sera, the infections continued to progress until a majority of the mice died. The normal, control mice showed a range of antibody titre from 0-1/32 at low intensities of fluorescence (Table 2).
296
IMF IN LEISHMANIAL INFEGriONS TABLE2
Indirect immunofluorescence of pooled sera* from mice injected with L. tropica
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
Intensity offluorescence in serum dilutions Days after infection
1/16
1/32
1!64
1/128
1/256
1/512
1/1024
1/2048
10
±
±
±
±
23
3+
2+
2+
1+
50
3+
1·5+
2+
1·5+
± ± ±
71
4+
3+
2-5+
2+
1·5+
94
4+
4+
2·5+
1·5+
1+
± ± ± ± ±
150
3+
3+
2+
2+
1+
1+
Normal
1+
I+
±
±
±
±
± ± ± ± ± ± ±
± ± ± ± ± ± ±
* At each time interval, sera was obtained from at least three mice.
Reactions of Mice Sera with Ho~nologous and Heterologous Antigens Upon testing the sera of mice infected with L. tropica against L. tropica, L. enriettii and L. donovani organisms, it was observed that the titres of fluorescent antibody in mice sera were the same for all three organisms. These results indicate a high degree of cross reactivity TABLE 3
Indirect immunofluorescence
of sera from human cases having leishmaniasis Intensity qffluorescence in serum dilutions
Type of leishmaniasis
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
1/2048
Cutaneous--one month duration*
2+
2+
2+
1·5+
±
±
±
±
Cutaneous-four months duration*
3+
2+
2·5+
I+
±
±
±
±
Cutaneous-five tion*
3+
months
dura2·5+
1·5+
1·5+
±
±
±
±
Cutaneous-nine months duration* 3+
3+
3+
I+
±
±
±
±
Suspected diffuse type of five years duration-two cases
4+
3+
3+
3+
3+
1·5+
±
Systemic (Kala Azar)
4+
4+
4+
3·5+
2·5+
2+
I+
± ±
Suspected systemic-(Kala Azar) Case# 1
4+
3+
2-5+
2-5+
2+
1·5+
I+
±
Suspected systemic-(Kala Azar) Case# 2
4+
3·5+
3+
3+
2+
1+
±
±
Suspected systemic-(Kala Azar) Case# 3
4+
3+
1+
I+
±
±
±
±
Suspected systemic-(Kala Azar) Case# 4
4+
3+
2+
1+
Normal*
I+
±
±
±
± ±
± ±
± ±
± ±
* In these cases at least three subjects are tested and the results were averaged.
BEHFOROUZ et al.
297
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
of the mouse serum antibodies against heterologous Leishmania organisms. Moreover, upon testing mice sera against newly isolated, low passaged Leishmania and Leishmania which had been passaged in culture for over 30 times, no difference was observed in intensity or titre of the antibody reaction.
Detection of Antibody in Haman Cases of Leiaahmani•sis Several cases of cutaneous leishmaniasis in humans were tested for antibody titre (Table 3). In individuals having lesions for less than one year (the normal course of infection is one year), the antibody titre ranges from 1/32 through 1/128 depending upon the duration of the lesion. The intensity of fluorescence in these cases was significantly greater than that of the controls. In none of these cases was the titre higher than 1/128. Two cases of cutaneous leishmaniasis of a duration longer than five years, suspected to be of the diffuse type (having spreading lesions of both arms and a negative skin test), were tested for antibody activity. It was found that the serum antibody activity in both cases exceeded a titre of 1/512 with an intensity of at least 3+ through a dilution of 1/256. One case of systemic leishmaniasis in a 20-month-old girl, who was shown to have Leishman-Donovan bodies in her bone marrow, was examined for serum antibody activity by indirect immunofluorescence. A titre of 1/1024 was observed in this patient's serum with intensities of 3-4+ through a dilution of 1/128. Four additional cases of suspected systemic leishmaniasis in young children (eight months to six years) were examined for serum antibody activity by this method. Clinically, these children demonstrated hepatosplenomegaly, fever, anaemia and in general were in poor condition. In each case, bone marrow smears were inconclusive but following treat:rnent with Glucantime (the drug of choice for Leishmania infections) all four children clinically improved. A range of antibody titres from 1/256-1/1024 were observed in these patients' sera as shown in Table 3. In all four cases, a 4+ fluorescence was observed in the 1 :16 dilution of their sera.
0.015M pH 7
Phosphate 0.005M Buffer
pH 8
O.lM pH6
1.2
} 0 00 N
.,c II
ci
c
d
.L.
\..)
.,...
:I all
A
"c
Cl
c
.L.
u
...
-Ql
:::1 all
B
0.1 oL-------------------------------------------~ FRACTIONS
Fls. 1. DEAE cellulose chromatography of immune guinea pig serum. D
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
298 IMF IN LEISHMANIAL INFECTIONS
BEHFOROUZ et al.
299
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
Reaction of HUIIUUl Serum with Homologous and Heterologou• Antigen• Upon testing the antisera of two human cases ofleishmaniasis (systemic and prolonged cutaneous) against L. tropica, L. enriettii and L. donovani organisms, it was observed that the titre of fluorescent antibody in these cases was the same against all three organisms. Again, human serum appears to have a high cross reactivity with heterologous species of Leishmania. Fractionation of lmmUDe Guinea Pig Serum The pooled fractions of immune guinea pig serum containing protein which were obtained by DEAE cellulose chromatography were tested for fluorescent antibody activity and it was observed that the two protein peaks, A and B, eluted from the column with the first buffer {0·005 M, pH 8) had antibody activity (Fig. 1). According to Oliveira et al. (1970) these two peaks correspond to the heterogeneous y 2 immunoglobulins of guinea pigs. Both fractions A and B, when tested imiilunoelectrophoretically against anti-guinea pig serum, showed one precipitin line in the region of slow-moving IgG, which, in the guinea pig, corresponds to y 2 (Fig. 2). Fractions C, D and E which contained faster moving lgG, as well as other immunoglobulins, had no antibody activity against Leishmania parasites as tested by the indirect immunofluorescent technique. DISCUSSION Our results show unequivocally that mice, guinea pigs and humans having an active leishmania! infection do produce antibody against the Leishmania parasites. In infected guinea pigs, the indirect fluorescent antibody technique is a much more sensitive test than the inhibition technique formerly developed in our laboratory. In the case of mice and humans, the indirect fluorescent antibody technique is capable of detecting antibody which was formerly undetectable in significant quantities by the inhibition culture method. The results obtained from indirect fluorescent antibOdy technique described here are measured in two parameters. One is the intensity of fluorescence of each of the various serum dilutions and the other is the titre of antibody in the serum. In general, the highest intensity of fluorescence seems to appear in the lower serum dilutions at the peak of the leishmania! infections. Using the indirect fluorescent technique for infected guinea pigs, antibody was detected in a low titre as early at 12 days after experimental infection. This result corresponds to the results previously obtained from histological studies of infected guinea pigs in which microscopic cellular changes generally associated with antibody production could be seen in the lymph nodes as early as two weeks after infection. Thus, this technique for the detection of antibody is much more sensitive than the culture inhibition technique with which antibody in guinea pigs cannot be detected until six weeks after infection. The titre and intensity of fluorescent antibody in infected guinea pigs corresponds closely with the course of clinical infection, with the peak of antibody titre and fluorescent intensity occurring at the time when the lesion is at its maximum. In experiments performed on guinea pigs by Bryceson et al. (1972) using indirect immunofluorescence, antibody was detected several weeks later than in our studies. Animals with metastatic lesions show a high level of antibody. Despite the presence of
Ftc. 2.
Immunoelectrophoresis of guinea pig serum fractions. Troughs contained anti-guinea pig serum and the wells contained: A and B = fractions eluted with 0·005M pH 8·0 PO,, C =fractions eluted with 0·015M, pH 7·0 PO, and fractions D and E =fractions eluted with O·IOM pH 6·0 PO,.
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
300
IMF IN LEISHMANIAL INFECTIONS
this antibody, these animals do not generally recover and eventually die from either their original leishmania! lesion or secondary bacterial infections. It seems, therefore, that a high level of antibody does not necessarily correspond with healing in infected guinea pigs. The chromatographic and immunoelectrophoretic studies performed on immune guinea pig serum showed that the antibody activity detected by the indirect immunofluorescent technique resided in the y 2 portion of guinea pig globulin. This finding confirms the prediction made by Turk and Bryceson (1971) that the antibody in guinea pigs against Leishmania would probably be y 2 • The Gamma 2 globulin of guinea pigs is generally accepted to be the complement fixing portion of guinea pig globulin. The leishmania! growth inhibitory substance of immune guinea pig sera described previously in our laboratory was also complement dependent, and indicates that the growth inhibitory substance is probably the same antibody as the one detected by the immunofluorescent technique. Whereas the growth inhibition test could detect no antibody activity in the sera of infected mice, the immunofluorescent technique described in this paper was able to detect antibody activity as early as 23 days after infection. This activity increased both in intensity and titre during the course of infection. As these mice usually do not survive, either due to their primary or secondary bacterial infections, the titre of antibody developed in these animals is apparently not protective. These findings are essentially in agreement with what was found by Preston et al. (1972) with CBA mice. However, the titre of fluorescent antibody in our study using outbred mice did not reach the level of the antibody titre obtained by these investigators. A possible explanation for this difference may be due to animal strain differences. CBA mice heal 12 weeks after infection whereas the strain of mice used in our investigation eventually die with active lesions. It is interesting that the antibody produced in mice against L. tropica reacts with the other Leishmania species equally well. This antibody activity therefore appears directed to an antigen or antigens common to all species of Leishmania. In human cutaneous leishmaniasis, this technique could also detect antibody activity early in the course of infection. The titre of antibody in these patients never rose above 1f 128. The cases of suspected diffuse leishmaniasis and the one case of proven systemic leishmaniasis had a higher titre of antibody at greater intensities than found in the nondisseminating cutaneous form of leishmaniasis. The higher antibody titre in these cases is probably due to greater antigenic stimulation. It is well known that active kala-azar is characterized by a massive hyperglobulinaemia. Adler (1965) and Garnham and Humphrey (1969) suggested that most of this globulin was not specific antibody. The fluorescent antibody technique described here did, however, demonstrate quantitatively a high level of leishmania-specific antibody in the patient with systemic leishmaniasis. Whether or not the leishmania-specific antibody detected in this patient accounts for the entire hyperglobulinaemia seen in kala-azar patients remains to be investigated. The fact that the human cases having widely disseminated leishmaniasis had high titres of antibody but poor prognosis is again indicative that in humans, as in mice and guinea pigs, the presence of antibody does not necessarily correspond with healing. It appears that the indirect immunofluorescent technique described here will prove to be particularly useful in the laboratory diagnosis of systemic leishmaniasis. At present, in order to diagnose systemic leishmaniasis, a bone marrow smear and culture must be performed. The fact that sera from these human cases react equally well with all three Leishmania species is an advantage in that a laboratory need not keep all species of Leishmania in culture in order to identify the leishmania! origin of questionable infections. The role which the antibody detected by this immunofluorescent technique plays in
BEHFOROUZ et al.
301
immunity to leishmaniasis is not clear. It appears in high titres even in hosts with overwhelming leishmania! infections and, in these cases, does not appear to be protective. This immunofluorescent technique does, however, provide investigators with a tool with which leishmania! infections may probably be diagnosed and in experimental animals traced from an early stage. ACKNOWLEDGEMENTS. This research was supported by grants from Pahlavi University Research Council No. 52-MD-8 and the Ministry of Higher Education No. 401-102-478.
Downloaded by [Australian Catholic University] at 22:54 18 August 2017
SUMMARY Indirect immunofluorescence was used for determination of antibody in human cases of cutaneous leishmaniasis, mice infected with Leishmania tropica and guinea pigs infected with L. enriettii. Results indicated that antibody in mice is detectable 23 days after infection with L. tropica. The antibody titre correlates well with the extent of infection. In guinea pigs infected with L. enriettii, antibody appears much faster and is detectable 12 days after infection. Here also the antibody titre increased during the course of infection until about 76 days at which time the leishmaniallesion was at its height. Guinea pigs having metastatic lesions consistently showed a high titre of antibody. In several cases of human cutaneous leishmaniasis antibody was detectable at the titre of not more than 1/128. In five cases of suspected systemic leishmaniasis antibody titre was much greater than in cutaneous leishmania! infection. Upon characterization of anti-leishmania! serum of guinea pig with DEAE-cellulose chromatography and analysis of the fractions by immunoelectrophoresis, it was found that the antibody activity detected by the indirect immunofluorescence resided in the y 2 portion of guinea pig globulin. REFERENCES ADLER, S. ( 1965). 'Immunology of leishmaniasis.' Israel Journal of Medical Science, 1, 9-13. BRYCESON, A. D. M., BRAY, R. S., WOLSTENCROFT, R. A. & DuMONDE, D. C. (1970). 'Immunity in cutaneous leishmaniasis of the guinea pig.' Clinical and Experimental Immunig, 1, 301-341. BRYCESON, A. D. M., PRESTON, P. M., BRAY, R. S. & DuMONDE, D. C. (1972). 'Experimental cutaneous leishmaniasis. II. Effects of immunosuppression and antigenic competition on the course of infection with Leishmania enriettii in the guinea pig'. Clinical and Experimental Immunig, 10, 305-335. GARNHAM, P. C. C. & HUMPHREY, J. H. (1969). 'Problems in leishmaniasis related to immunology.' Current Topics in Microbiology and Immunology, 48, 29-42. LEMMA, A. & ScHILLER, E. L. (1964). 'Extracellular cultivation of the leishmania! bodies of species belonging to the protozoan genus Leishmania.' Experimental Parasitology, 15, 503-513. OLivEIRA, B., OsLER, A. G., SIRAGANIAN, R. P. & SANDBERG, A. L. (1969). 'The biologic activities of guinea pig antibodies I. Separation of y 1 and y 2 immunoglobulin and their participation in allergic reactions of the immediate type'. Journal of Immunology, 104, 320-328. PRESTON, P., CARTER, R., LEUCHARS, E., DAVIES, A. & DUMONDE, D. C. (1972). 'Experimental cutaneous leishmaniasis III. Effects of thymectomy on course of infection of CBA mice with Leishmania tropica'. Clinical and Experimentol/mmunology, 10, 337-357. REZAI, H. R., SHER, S. & GETTNER, S. (1969). 'Leishmania tropica, L. donovani, and L. enriettii: Immune rabbit serum inhibitory in vitro.' Experimental Parasitology, 26, 257-263. REzAI, H. R., GETTNER, S. & BEHFORouz, N. (1972). 'Antileishmanial activity of immune guinea-pig serum.' Journal of Medical Microbiology, 5, 371-375. REzAI, H. R., HAomGHI, P. & ARDEHALI, S. (1972). 'Histological appearance of the site of inoculation and lymph nodes of guinea-pigs at various times after infection with Leishmania enriettii.' Journal of Medical Microbiology, 66, 225-234. TURK, J. L. & BRYCESON, A. D. M. (1971). 'Immunological phenomena in leprosy and related diseases.' Advances in Immwwlogy, 13, 209-266.
