Differentiation of Trypanosoma brucei, T. rhodesiense, and T. gambiense by the indirect fluorescent antibody test

Bull. Org. mond. Sant} 1973, 48, 401-407 Bull. Wld Hith Org. Differentiation of Trypanosoma brucei, T. rhodesiense, and T. gambiense by the indirect fluorescent antibody test B. M. A. LATIF & KATHERINE M. G. ADAM 2 Epidemiological studies, if they are to lead to appropriate preventive procedures, require knowledge of the host distribution of the parasite. Progress in the epidemiology of African trypanosomiasis is restricted by the lack of a reliable and simple method of differentiating Trypanosoma brucei, T. rhodesiense, and T. gambiense. The recently introduced blood inoculation infectivity test promises to fulfil this need by distinguishing T. brucei from T. rhodesiense, but it would not be suitable for separating T. brucei from T. gambiense, since rats and mice are frequently refractory to infection by fresh isolates of T. gambiense. Previous studies had indicated that the indirect fluorescent antibody test might differentiate not only the subgenera of the salivarian trypanosome species but also members of the subgenus Trypanozoon. A method ofperforming the test is described that enables T. brucei, T. rhodesiense, and T. gambiense to be differentiated by the titre of the sera. The method might be used in conjunction with the blood inoculation infectivity test to distinguish between new isolates of the subgenus Trypanozoon in East Africa, and also to search for possible animal reservoirs of T. gambiense in West Africa. The value of the indirect fluorescent antibody (IFA) test in the diagnosis and epidemiology of human trypanosomiasis is now well established. Cases of Gambian sleeping sickness that were undetected by the standard diagnostic procedures have been uncovered by this test. If blood samples are collected on filter paper, it can be used to screen entire populations in areas where the disease is endemic (Fife & Muschel, 1959; Sadun et al., 1963; Camargo, 1966; Souza & Camargo, 1966; Bailey et al., 1967; Lucasse, 1970; W6ry et al., 1970b). Epidemiological studies, by definition, require knowledge of the host distribution of the parasite. Such information is difficult to obtain for African trypanosomiasis since T. brucei, T. rhodesiense, and T. gambiense are morphologically indistinguish- able; only the last-named two species are infective for man. Rhodesian sleeping sickness is a zoonosis, but T. gambiense is thought to be principally a 1 Calouste Gulbenkian Scholar at the Department of Zoology, University of Edinburgh, Scotland. ' Lecturer in Protozoology, Department of Zoology University of Edinburgh, Scotland. parasite of man. The biology of these three trypano- some species has been concisely reviewed by Ash- croft (1959). The epidemiology of Rhodesian sleeping sickness has been reviewed more recently (Onyango, 1969). T. brucei and T. rhodesiense have a wide and probably similar range of animal hosts. The only way to identify a new isolate that has the character- istics of the subgenus Trypanozoon is to inoculate a human volunteer. The introduction of the blood inoculation infectivity test (BIIT) by Rickman & Robson (1970) provided a simple means of dif- ferentiating T. brucei and T. rhodesiense, although equivocal results have been reported (Mwambu & Mayende, 1971). Failure to eliminate Gambian sleeping sickness, despite the continuous efforts that have been made since the introduction of chemotherapy 60 years ago, is probably due to the presence of healthy human carriers of the causative organism. It is also possible that there is a reservoir of infection in some animal species. Van Hoof (1947) maintained T. gambiense for 4 years by cyclic transmission through 20 pigs, with 2 passages from pig to man in order to demon- 3023 401- 402 B. M. A. LATIF & KATHERINE M. G. ADAM strate that the organism had not changed its charac- ter. Van Hoof also transmitted T. gambiense cyclically through 10 goats. In both pigs and goats the infection was benign. The same author also recorded a natural infection in a dog. More recently, Yesefu (1971) infected a dog from a monkey, and 2 tsetse flies that had fed on the dog subsequently developed salivary gland infections. The need for a simple test to distinguish between pleomorphic members of the subgenus Trypanozoon prompted us to investigate the IFA test. Wery et al. (1970a) in a series of these tests were unable to distinguish T. brucei from T. gambiense; nor could Suter-Kopp & Fricker (1972) distinguish T. brucei from T. rhodesiense. However, Schindler & Sachs (1970) were able to differentiate T. brucei and T. con- golense by the titre of the sera in IFA tests. Similarly, Wilson et al. (1967) indicated that T. brucei, T. con- golense, and T. vivax could be distinguished by the IFA test and that, in applying the test to the epi- zootiology of bovine trypanosomiasis, the sera should be tested against antigen prepared from each of these species. In this paper we describe a method of performing the rFA test that makes if possible to distinguish between pleomorphic members of the subgenus Trypanozoon. MATERIALS AND METHODS Organisms The following trypanosome strains, obtained from the low temperature bank of the Centre for Tropical Veterinary Medicine (CTVM), Edinburgh, were used in the study: Trypanosoma brucei: TREU 851 (derived from EATRO 1523), TREU 852 (EATRO 1527), TREU 853 (EATRO 1529), TREU 927 (EATRO 1534), TREU 933 (EATRO 1644), TREU 958 (EATRO 1591), TREU 991 (EATRO 1589), TREU 833 (EATRO 1410)-all isolated from Glossina pallidipes caught in Kenya or Uganda by members of the EATRO/CTVM joint project 1969-71; TREU 900 (EATRO 1691) from wildebeeste in the Serengeti, 1969; TREU 1096 and 1097 from cattle in Nigeria; TREU 667, isolated from a reed buck in East Africa, 1968; and TREU 741 and 1027, old labo- ratory strains derived from EATRO 38. T. rhodesiense: TREU 35 (derived from EATRO 173, Kenya), TREU 333 (EATRO 906), TREU 789 (EATRO 165, Tanzania), and TREU 788 (EATRO 181, Botswana). T. gambiense: TREU 390 (Dakar); E 1 (Piti) and R 3 (Aliade, Nigeria) (Gray, 1972). T. evansi: TREU 379 (Colombia). T. congolense: TREU 732 (old laboratory strain), TREU 692 (East Africa, 1968), TREU 1095 (Nige- ria), and TREU 937 (old strain, origin unknown). T. lewisi (old laboratory strain from England). Sera Infected rabbits were bled from the ear; rats, from the tail. The blood was left in the refrigerator over- night. The next day, the serum was removed, inactivated, distributed into 1-ml X-Lon plastic test tubes, and stored at -20°C until required. Infections by highly pathogenic strains of trypanosome were controlled or cured with diminazene. Sera from 6 patients with Rhodesian sleeping sickness were supplied by Dr R. J. Onyango, East African Trypano- somiasis Research Organization (EATRO). These sera had been preserved with sodium azide. The EATRO code numbers of the sera are: (a) 1438 and 1474 (7); (b) 1716 and 1740 (12); (c) 2150 and 2201 (20); (d) 1880 and 1933 (12); (e) 1870 and 1872 (5); and (f) 1881 and 2209 (60).1 Sera from 3 patients from Zaire with Gambian sleeping sickness were supplied by Professor J. Jadin, Institut de Medecine tropicale Prince Leopold, Antwerp. These sera (code numbers: M 221, M 256, and M 262) had been preserved by lyophilization. Sera (RH/RRP and BH) from 2 rabbits infected by fly transmission with 2 isolates of T. gambiense from Mayir, Nigeria, were donated by Dr A.R. Gray, CTVM, Edinburgh. Fluorescein-conjugated antisera were obtained from Nordic Pharmaceuticals and Diagnostics, Tilburg, Netherlands; normal bovine and normal horse serum, from Bio-Cult Laboratories Ltd, Glasgow. Preparation of antigen Antigen was prepared from heavily infected mouse blood, except for the two Nigerian strains of T. gambiense, for which the hosts were suckling rats; T. lewisi antigen was also obtained from a rat. When the trypanosomes reached a concentration of 107-108/ml, the animals were anaesthetized with ether and bled from the heart. To separate the 1 The figure in parentheses indicates the number of days that elapsed between the last day of drug treatment and the day when the second serum sample was taken from the patient. DIFFERENTIATION OF TRYPANOSOME SPECIES 403 Table 1. Titrations of sera from rabbits a Reciprocals of serum dilutions Antigen b Ra.1 Ra.1 RH/RRP RH/RRP RH/RRP BH BH BH (12) c (24) (15) (29) (46) (14) (30) (49) T. rhodesiense 333 40 160 80 80 40 40 160 80 35 40 320 20 40 160 40 80 80 788 160 640 20 80 160 20 80 80 789 80 640 40 160 160 40 160 160 T. brucei 667 5120 20840 20 20 40 20 40 80 927 2 560 10 240 80 80 80 20 20 80 1096 2 560 10 240 40 80 80 20 40 160 933 1 280 5120 20 40 20 20 40 80 991 5120 10 240 40 160 160 40 80 160 852 2 560 10240 851 2 560 10240 853 2 560 10240 958 1 280 5120 833 1 280 5120 900 1 280 5120 1097 2560 10 240 741 2560 10 240 1027 2560 10240 T. gambiense 390 80 320 320 640 640 160 1 280 1 280 R 3 80 80 640 1 280 2 560 320 640 1 280 El 40 160 320 1 280 2 560 160 1 280 1 280 T. evansi 379 40 80 T. congolense 732 40 160 692 20 160 1095 40 80 937 20 160 T. lewisi 20 80 a Rabbit Ra.1 was infected with T. bruceiTREU 667; rabbits RH/RRP and BH, with T. gambiense. b The antigens are denoted by their TREU numbers, except for R3 and El (see under Materials and methods). t'The figures in parentheses indicate the number of days that elapsed between the day of infection and the day when the serum was obtained. B. M. A. LATIF & KATHERINE M. G. ADAM brucei-group trypanosomes, the blood was centri- fuged at 1 500 g for 3 min, the supernatant removed, and the buffy layer containing the trypanosomes transferred to another tube. The trypanosomes were resuspended in 5 volumes of normal bovine (or horse) serum. The suspension was then centrifuged at 12 000 g for 5 min and about 4/5 of the super- natant were removed to leave a concentrated sus- pension of trypanosomes at the base of the tube. With the aid of a finely drawn-out Pasteur pipette, drops of the suspension were placed within marked circles on clean, thin (<1 mm) microscope slides, and the preparations were left to dry in air. To separate T. congolense from mouse blood, the blood was quickly transferred to a centrifuge tube and mixed with 5 volumes of normal bovine serum. The suspension was allowed to stand for 15 min, after which the red cells were sedimented by gentle centrifugation and the supematant, containing about 90% of the trypanosomes, was removed. The trypanosomes were then sedimented and treated sub- sequently as described for brucei-group trypano- somes. The dried antigen was fixed by immersing the slide for 15 min in a 5% solution of formalin in distilled water. The fixative was washed off with phosphate- buffered saline (0.1 5M disodium hydrogenphosphate in physiological saline, pH 7.2). When thoroughly dry, the slides were wrapped in aluminium foil and placed in self-sealing polyethylene bags containing a few crystals of silica gel. The antigen-coated slides were stored at - 200C. When required for tests, the slides were brought to room temperature before being removed from the bag. Test procedure One drop (about 50 ,l) of the test serum was placed within each demarcated circle of the antigen- coated slides and was allowed to react for 40 min at room temperature. The serum was washed off by placing the slides for 10 min in 2 changes of phos- phate-buffered saline. The conjugated antiserum was then applied for 40 min and the washing procedure was repeated. The dried preparations were mounted in 90% glycerol in 0.5M carbonate/hydrogencarbon- Table 2. Titrations of sera from rats a Reciprocals of serum dilutions Antigen b Rt.1 Rt.1 Rt.1 Rt.2 Rt.2 Rt.2 Rt.2 Rt.3 Rt.3 Rt.3 Rt.3 (6) c (12) (18) (7) (14) (21) (28) (7) (14) (21) (28) T. rhodesiense 333 20 20 20 35 20 40 20 788 10 40 20 80 640 160 160 80 160 320 160 789 10 40 40 80 640 160 160 160 320 320 320 T. brucel 667 160 320 320 20 20 10 20 10 20 20 20 1096 80 320 320 10 20 40 20 10 40 40 40 958 160 320 320 10 10 20 10 10 10 10 10 T. gambtense 390 20 20 20 10 40 20 20 10 20 40 20 R 3 20 20 20 10 20 20 20 10 20 20 20 El 20 20 20 10 20 20 20 10 20 20 20 a Rat Rt.1 was infected with T. bruceiTREU 667; rat Rt.2, with T. rhodesiense TREU 788; and rat Rr.3, with T. rhodesiense TREU 789. b The antigens are denoted by their TREU numbers. CThe figures in parentheses indicate the number of days that elapsed between the day of infection and the day when the serum was obtained. 404 DIFFERENTIATION OF TRYPANOSOME SPECIES ate buffer, pH 9.0, and covered with a number 0 cover slip (about 0.1 mm thick). The slides were examined with a darkfield micro- scope at a magnification of 400. The light source was a 200-W high-pressure mercury vapour lamp in combination with a BV excitation filter; Y51 and Wratten 2B eyepiece filters were used. Serial dilutions of the test and control sera were prepared in phosphate-buffered saline. Control tests included incubation in normal serum or in phos- phate-buffered saline and then in conjugated serum, and incubation in conjugated serum alone. The latter was always used at a dilution of 1 in 20. Four intensities of fluorescence were recognised (+ to +++ ++) and the end point of a titration was the highest dilution that gave 1 plus (+) fluorescence. RESULTS The results of the titrations of sera from rabbits with various antigens are shown in Table 1. Rabbit Ra.1 was infected with T. brucei TREU 667; sera taken at 12 and 24 days after the infection were titrated against 14 T. brucei, 4 T. rhodesiense, 3 T. gambiense, 1 T. evansi, 4 T. congolense, and 1 T. lewisi antigen. The highest dilution of the 12-day serum to give fluorescence with any of the heterologous antigens was 1/160, whereas the lowest dilution to give fluorescence with any of the homo- logous antigens was 1/1 280. Similarly, with the 24-day serum the lowest homologous titre was 1/5 120 and the highest heterologous titre, 1/640. When the sera from rabbits RH/RRP and BH, which were infected with T. gambiense, were titrated against brucei-group antigens, the heterologous and homo- logous reactions were distinguished by the titres of the sera. Table 2 gives the results of the titrations of sera from rats infected with T. brucei TREU 667 (Rt. 1) and with T. rhodesiense TREU 788 (Rt.2) and 789 (Rt.3). In these experiments the rats were bled at 6-day or 7-day intervals and the infections were cured with diminazene, 75 mg per kg of body weight, on each of 2 consecutive days starting on the 14th day after infection. Although the antibody res- ponse of the rat was not as strong as that of the rabbit, it was possible to differentiate homologous and heterologous antigens by the titres of the sera. Drug treatment did not destroy the specificity of the sera. The results of the titrations carried out with human sera are shown in Table 3. h- a)) c E o ._ CD 0 C 0 cvi 0 ._ E 0D0 co 0 0C)C.: Ca N cD CD4 - N a) 0 N1 CN 0000 0000 ~NCDt (D CXN CO co 0000 w CDCXCr)CC)C w- 0000 __CDcco N w 0 N 0 It cD rv 0 co N 0 F- co Cc 0000 co CD cD D LO CLON N N N r 0000 0000 csntcoe 0000 Cq C4 NrN-N 0000 N N Nr- N 0000c N N N N 0000 CD (c) co CD 4) ,q) bq a 00000) 000 ooooo o-oo 00000 000 N_-SCN Ntt N_-wNN N~N N4 _ OONCM s dN 00000 000 00000 000 _N N*N 00000 000 0 00 0 NNN 00000 CDwCDwaD 00000 cDDoo 000 N N 000 N N Nq 00000 000 tcDND _.-NN 0) C-z LOOa-0CD X OnPI a) co olbcoXeDonam a)FFPIPa WM_aMa E cnG UJ 405 0000 00000 000 NNw N4It NICD (C) (0)M 0000 00000 000 _-NNw N'-NN 00 00t4w9 CO0404 0000 00000 000 *cr q '*eq No4q C14 -t 4* C1 WCD cN NVN) -NN CD D CC 0 0 c Ca ax 0 C. E 0 D D 0 -C 0 fA E -CE 0 CD ._; 3: ! CD 0 o C, U0 .2 IC V._ N 0 -C a4 m CD.' >.X -c ee 0 ED V NoD _ 0 ND 0 3-'0 . so1 B. M. A. LATIF & KATHERINE M. G. ADAM The results show that, in a total of 368 cross- titrations of antisera to trypanosomes belonging to the brucei complex, the titre of any serum was at least 4 times as great with the homologous as with the heterologous antigen. DISCUSSION In previous studies (Wilson et al., 1967; Schindler & Sachs, 1970) it was established that Trypanozoon, Nannomonas, and Duttonella could be differentiated by the IFA titres of sera. However, attempts to differentiate members of the subgenus Trypanozoon were not successful (Wery et al., 1970a; Suter-Kopp & Fricker, 1972). The greater sensitivity and specifi- city of the test in our hands may be explained by certain differences of technique. The Belgian workers prepared antigen by spreading thin films of heavily infected blood on slides, but did not use any fixative; the Swiss workers also used thin blood films which, after drying, were fixed with acetone. The East African workers added an equal volume of phos- phate-buffered saline to the infected blood, sedi- mented the red cells by gentle centrifugation, and spread the trypanosome-rich supernatant on the slides. The dried antigen was fixed by gentle heat. With each of these methods, soluble antigen and specific antibody from the blood are likely to be present in the antigen preparations. Our method has several advantages: trypanosomes adhere firmly to the slide; any antigen or antibody that was present in the blood is usually removed; many trypanosomes can be seen in each microscopic field and, finally, the removal of host cells eliminates autofluorescence. Whereas fixation by gentle heat is liable to vary, fixation with formalin can be standardized. We have found that antigen prepared as described and stored at - 20°C has remained stable for at least 18 months. A weakness of the IFA test is that the end point of titrations depends on the brightness of the image and may therefore vary with the observer and even from day to day with the same observer. For the purpose of diagnosis, Wery et al. (1970a) regarded only bright (+++) or very bright (++++) fluorescence as positive. However, Wilson & Cunningham (1971) define a positive serum as one that gives 2 plus (+ +) or brighter fluorescence at a dilution of 1/40. In our experience, a more reliable reading is obtained when 1 plus (+) is taken as the end point of a titration. In other words, the eye dis- criminates with greater certainty between the absence of fluorescence and weak fluorescence. We have shown that members of the subgenus Trypanozoon can be differentiated by this method, and we suggest that the IFA test could be used in conjunction with, or possibly instead of, the BIIT to distinguish T. brucei from T. rhodesiense. Antigen prepared from a new isolate should be tested against 3 different T. rhodesiense and three different T. brucei sera, and the means of the titrations should be compared. The present test could be usefully employed in West Africa to search for reservoir hosts of T. gam- biense. Conjugated antisera to the serum globulins of the suspected host animals would need to be pre- pared. In areas where Gambian sleeping sickness is endemic, sera from these animals could be collected and tested against T. gambiense and T. brucei anti- gens. A high titre to T. gambiense and a lower titre to T. brucei would indicate that the animal was para- sitized by T. gambiense. A systematic search for reservoir hosts could help in the eradication of human trypanosomiasis. ACKNOWLEDGEMENTS We are most grateful to Dr A. R. Gray, Professor J. Jadin, and Dr R. J. Onyango for gifts of sera. Mr Latif thanks the University of Baghdad for the award of a Gulbenkian Foundation scholarship. RIESUMI2 DIFFtRENCIATION DE TRYPANOSOMA BRUCEI, T. RHODESIENSE ET T. GAMBIENSE PAR L'tPREUVE INDIRECTE DES ANTICORPS FLUORESCENTS Les 6tudes 6pidemiologiques de la trypanosomiase afri- caine sont entrav6es par le fait que Trypanosoma brucei, T. rhodesiense et T. gambiense sont morphologiquement indiscernables. La gamme des hotes animaux de T. brucet et T. rhodesiense est etendue et probablement similaire. Pour identifier un nouvel isolat et differencier T. brucei 406 DIFFERENTIATION OF TRYPANOSOME SPECIES 407 et T. rhodesiense, on peut recourir a l'inoculation a des volontaires ou utiliser l'6preuve d'infectivit6 apres incu- bation en presence de sang humain, r6cemment mise au point. Ce dernier test ne permet cependant pas de distinguer T. gamnbiense et T. brucei car les animaux de laboratoire (rats et souris) sont habituellement refrac- taires a l'infection par des souches de T. gambiense. La valeur de l'6preuve d'immunofluorescence indirecte pour le diagnostic de la trypanosomiase est bien 6tablie. GrAce L la technique employee par les auteurs, il est possible de diff6rencier les divers membres du sous- genre TrypaMozoon en mettant en pr6sence des serums (humains, de lapins ou de rats) anti-T. brucel, anti-T. rhodesiense et anti-T. gambiense et les antigenes corres- pondants. Au cours des 368 r6actions qui ont ete prati- quees, le titre d'un antis6rum donn6 6tait au moins quatre fois plus elev6 en presence de F'antigene homologue qu'en presence d'un antigene h6t6rologue. Cette 6preuve pourrait, conjointement avec l'6preuve d'infectivite, servir a identifier les isolats du sous-genre Trypanozoon et a rechercher les r6servoirs animaux 6ventuels de T. gambiense. REFERENCES Ashcroft, M. T. (1959) Trop. Dis. Bull., 56, 1073-1093 Bailey, N. M. et al. (1967) Trans. roy. Soc. trop. Med. Hyg., 61, 696-700 Camargo, M. E. (1966) Rev. Inst. Med. trop. S. Paulo, 8, 227-234 Fife, E. 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(1967) In: East African Trypano- somiasis Research Organization Report, 1966, Nairobi, East African Common Services Organization, p. 28 Yesefu, H. M. (1971) Ann. trop. Med. Parasit., 65 341-347