Annals of Tropical Medicine & Parasitology
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
Application of a monoclonal antibody-based antigen detection enzyme-linked immunosorbent assay (antigen ELISA) for field diagnosis of bovine trypanosomiasis at Nguruman, Kenya V. M. Nantulya, K. J. Lindqvist, P. Stevenson & E. K. Mwangi To cite this article: V. M. Nantulya, K. J. Lindqvist, P. Stevenson & E. K. Mwangi (1992) Application of a monoclonal antibody-based antigen detection enzyme-linked immunosorbent assay (antigen ELISA) for field diagnosis of bovine trypanosomiasis at Nguruman, Kenya, Annals of Tropical Medicine & Parasitology, 86:3, 225-230, DOI: 10.1080/00034983.1992.11812658 To link to this article: http://dx.doi.org/10.1080/00034983.1992.11812658
Published online: 15 Nov 2016.
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Date: 11 April 2017, At: 02:53
Annals ofTropical Medicine and Parasitology, Vol. 86, No.3, 225-230 (1992)
Application of a monoclonal antibodybased antigen detection enzyme-linked immunosorbent assay (antigen ELISA) for field diagnosis ofbovine trypanosomiasis at Nguruman, Kenya* BY V. M. NANTUL YA, K. J. LINDQVIST International Laboratory for Research on Animal Diseases ( ILRAD ), P.O. Box 30709, Nairobi, Kenya
P. STEVENSON AND E. K. MWANGI Kenya Trypanosomiasis Research Institute, P.O. Box 362, Kikuyu, Kenya Received 20June 1991, Revised 17 February 1992, Accepted 17 February 1992
A monoclonal antibody-based, enzyme immunoassay (antigen ELISA) for the detection of species-specific invariant antigens of Trypanosoma congolense, T. vivax or T. brucei in the serum of infected animals was evaluated as a means of diagnosis using bovine field sera from a trypanosomiasis endemic area, Nguruman, Kenya. Circulating trypanosome antigens were detected in 126 (96·2%) of 131 serum samples from animals with parasitologically confirmed diagnosis: 74·8% were positive for antigens of two or three trypanosome species, while 21·4% tested positive for one trypanosome species. When 70 sera from animals (at Nguruman), which had tested negative for trypanosomes by the huffy coat technique, were tested, 35 (50·0%) of them were shown to be antigen-ELISA positive: 24 (34· 3%) showing infection with a single species and II (15·7%) with mixed infections. The predominant trypanosome species diagnosed in the two herds by antigen ELISA was T. vivax, which was detected in 133 (82·6%) of the 161 sera that tested positive for antigens, followed by T. congolense in 122 (75·8%) sera, with 109 (67·7%) showing evidence of mixed infections with two or three trypanosome species. In single infections, T. vivax exceeded T. congolense by a ratio of 2:1, with T. brucei accounting for less than 1·0%. Evidence for the specificity of the test was provided by analysis of field sera from 100 cattle, from a trypanosomiasis-free area, infected with other haemoparasites (anaplasmosis, babesiosis and theileriosis), which all tested negative in the assay.
The standard laboratory method for diagnosis of bovine trypanosomiasis is to demonstrate and identify trypanosomes in stained or wet smears of peripheral blood. Examination of blood smears, however, is not sensitive enough (Barnett, 194 7), as trypanosomes are often in such low numbers in peripheral circulation that *ILRAD Publication Series No. 971. 0003-4983j92j030225 + 06 $03.00/0
they cannot be detected by direct microscopic examination of blood. Hence, the introduction of trypanosome concentration methods such as the haematocrit centrifugation technique (Woo, 1969), or its variant, the buffy coat technique (Murray et al., 1977). Despite the marked improvement in sensitivity afforded by these concentration methods, many infected animals still go undetected, particularly those with © 1992 Liverpool School of Tropical Medicine
226
NANTUL YA ET AL.
chronic infections (Nantulya, 1990; Masake and Nantulya, 1991). In trypanosome infections, the organisms are sequestered in various predilection tissue sites, depending on the trypanosome species involved (Losos and Ikede, 1972), where they live and multiply. However, some parasites are destroyed by host immune responses which attempt to eliminate successive populations of trypanosome variable antigen types (VATs) arising through antigenic variation. This leads to the release of several soluble antigens in the blood and other tissue fluids of the infected host. The demonstration of trypanosome antigens in the blood of a previously untreated animal would indicate, therefore, that the animal has an active infection. Moreover, if the antigens detected were species-specific, it would be possible also to identify the trypanosome species causing the infection. Using this approach we have developed an enzyme immunoassay which can detect trypanosome species-specific invariant antigens in the serum or plasma of infected livestock (Nantulya and Lindqvist, 1989) as a means of diagnosis. This paper describes the initial evaluation of this assay (antigen ELISA) for the diagnosis of Trypanosoma congolense, T. vivax and T. brucei infections in cattle.
MATERIALS AND METHODS Negative Controls One hundred field sera were obtained from the ILRAD breeding herd ofBoran cattle at Kapiti, a trypanosomiasis-free area 50 km east of Nairobi. The sera were screened by indirect immunofluorescence (Goddeeris et al., 1982) for antibodies to several haemoprotozoan diseases endemic in the area. The common diseases identified were Anaplasmosis (90%), Babesiosis (40%), and Theileriosis due to Theileria mutans (45%) and T. taurotragi (10%). Test Sera The test field sera were from cattle at N guru man, 150 km southwest of Nairobi. Nguruman is a trypanosomiasis endemic area. Some cattle were small East African Zebu or crosses with Borans.
Others were Orma Boran which had been brought into the area the previous month. A total of 201 sera were analysed: 131 from parasitologically confirmed cases and 70 from animals grazing in an area of lower tsetse numbers with negative parasitological findings as determined by the huffy coat technique (Murray et al., 1977). All the sera were stored at - 20°C until use. Antigen-trapping Micro-plate ELISA The micro-plate ELISA was carried out as described previously (N antulya, 1989), with some modifications. Briefly, flat-bottom microELISA plates for the detection ofT. brucei antigens were coated with 0· 5 !lg/well of a partially purified IgM fraction of monoclonal antibody TR7/47.34.16 in carbonate buffer pH 9·6 with 0·02% sodium azide and left at 4°C until use. This antibody, although derived against T. rhodesiense procyclic trypomastigotes (Nantulya et al., 1987), also reacts with T. brucei. The plates used for detection of T. congolense or T. vivax antigens were coated with partially purified immunoglobulin fractions of speciesspecific monoclonal antibodies TC 39/30.38.16 or TV 27/9.45.15, respectively, and stored at 4oC. These two antibodies were derived against totallysates of bloodstream trypanosome populations following a method described previously (Nantulya et al., 1987). When plates were needed, excess coating antibody was drained off and washing buffer (0·15 M phosphate buffered saline pH 7·4 containing 0· 5% Tween 20) added to all the wells in volumes of 100 Ill/well. Each serum sample was then immediately added, in duplicate, at 5 !!1/ well for the T. brucei assay and 10 Ill/well for T. vivax and T. congolense. The plates were incubated for 15 minutes at room temperature, the excess reactants drained off and the conjugate (the horseradish peroxidase-conjugated immunoglobulin fraction of the corresponding monoclonal antibody), diluted in washing buffer containing 1% normal mouse serum or 0· 5% bovine serum albumin, added without prior washing of the plates. The plates were incubated at room temperature for 15 minutes, after which they were finally washed three times at 10 minute intervals by flooding the plates with
ANTIGEN ELISA FOR BOVINE TRYPANOSOMIASIS
227
TABLE 1 Identification of different trypanosome species in cattle at Nguruman, Kenya, using the buffy coat technique Trypanosome species identified
T. congolense
T. vivax
51 38·9
77
0
58·8
0
No. of positive cattle %Positive
washing buffer. After the last wash, the washing buffer was drained off and the substrate and chromogen (100 J..ll/well) added. The chromogen used consisted of 250 J..lg ml- 1 2, 2'-aziono bis (3-ethyl)-benzthiazoline-6-sulphonic acid (ABTS) in 50 mM citric acid buffer pH 4·0 containing 0·0 I% hydrogen peroxide as substrate. The optical densities (o.d.) were read after 30 minutes at 414 nm wavelength using a Titertek Multiskan micro-ELISA auto-reader (Type MCC340).
RESULTS Parasitological Results Parasitological results for the 131 animals with demonstrable parasitaemia are shown in Table I. Fifty-one (38·9%) of the animals had T. congolense infections only, 77 (58·8%) had T. vivax only and three animals (2·3%) had a mixed infection consisting of T. vivax and T. congotense. Trypanosoma brucei was reported not to have been detected, in any of the animals, by the buffy coat technique (Murray eta!., 1977). Antigen Detection The 100 field sera from a trypanosomiasis-free area, Kapiti (see Table 2), gave a mean o.d. value of0·018 ± 0·010. The threshold o.d. reading regarded as positive was, therefore, taken to be 0·050, which was twice the mean value plus one standard deviation. This threshold excluded all the negative control field sera. The analysis of 131 sera from parasitologically confirmed cases revealed 126 (96·2%) of them to have circulating antigens ofT. vivax, T.
T. brucei
T. congolense/ T. vivax
Total
3 2·3
100
131
congolense or T. brucei (Table 2): 98 (74·8%) had mixed infections involving two or three trypanosome species; 28 (21·4%) had single infections. Of the 70 sera from cattle in the area of lower tsetse challenge which had negative parasitological findings, 35 (50·0%) tested positive for trypanosome antigens: 24 (34·3%) showing evidence of single infections and II (15·7%) with mixed infections (Table 2). A detailed breakdown of the types of infection in the two herds is also given in Table 2. The predominant trypanosome species in the two herds was T. vivax, detected in 133/161 (82·6%) of the infections, followed by T. congotense in 122/161 (75·8%). Trypanosoma brucei antigen was detected in 90 (56·5%) of the infections. Seventy-six (47·2%) sera showed evidence of mixed infections involving all three trypanosome species, followed by 33 (20· 5%) cases of mixed infections involving two trypanosome species; hence the overlap in percentages. In the group of animals with single infections, T. vivax exceeded T. congolense by a ratio of 2:1, with T. brucei accounting for less than 1% of the infections.
DISCUSSION In the work reported here, a monoclonal antibody-based antigen detection enzyme immunoassay (Nantulya and Lindqvist, 1989) was evaluated using field sera from cattle in an endemic area. Of 131 sera from parasitologically confirmed cases, 126 (96·2%) were antigen ELISA positive. More significantly, 35 (50·0%)
228
NANTUL YA ET AL.
TABLE2 Identification ofthe various types ofinfection in cattle from a trypanosomiasis endemic area, determined by antigen ELISA
All three species
Total number ofsera antigen ELISA positive
Type ofinfection identified Origin of serum
No. of samples tested
Tc
Single infection Tv Tb
Mixed infection TvjTb Tc/Tv Tc/Tb
0
18 15 0
2 0 0
14 6 0
0 0
2 2 0
73 3 0
126 35 0
17
33
2
20
9
4
76
161
Nguruman* Ngurumant Kapiti (controls)
131 70 100
9
Total
301
8
9
*Sera from parasitologically confirmed cases. tSera from animals with negative parasitological findings as determined using the buffy coat technique (Murray eta/., 1977).
Tc, T. congolense; Tv, T. vivax; Tb, T. brucei.
of the sera from animals in a low tsetse challenge area which were aparasitaemic, as determined using the huffy coat technique, were found to have trypanosome antigens, while the control sera from 100 animals in a tsetse-free area were negative. The control sera were from an area where many common haemoparasitic diseases such as anaplasmosis, babesiosis and theileriosis due to T. mutans and T. taurotragi were prevalent, as evidenced by the serological results. These results thus demonstrate the specificity and indicate the superior sensitivity of antigen ELISA which has been estimated to be at least four times higher than that of the huffy coat method (Masake and Nantulya, 1991). In this study, a high proportion of animals was shown, by antigen ELISA, to carry mixed infections in various combinations. This was in contrast to the results obtained by the huffy coat technique. The ELISA results, however, may represent the true situation because, under natural conditions of challenge, mixed infections with T. congolense, T. vivax and T. brucei, which occur in all combinations of two or three species, are very common in cattle (Godfrey and Killick-Kendrick, 1961; Godfrey eta!., 1964; Esuruoso, 1973) and are considered to be more
InJurious than infections with single species (Metam, 1934). In studies in Nigeria, mixed infections were better diagnosed by animal inoculation rather than by direct microscopic examination of blood (Godfrey and KillickKendrick, 1961 ). Thus, the low rates of mixed infection detected by the huffy coat technique in this present study may reflect intrinsic difficulties in the application of this technique in identifying accurately the mixed infections under the pressures of field conditions. Moreover, in mixed infections the organisms belonging to the different species give rise to different levels of parasitaemia, making it difficult to identify the species that may be present in low numbers. Another interesting finding from this study relates to the role played by T. brucei. Whereas T. brucei was not detected at all in the infected animal group by the huffy coat technique, antigen ELISA did detect T. brucei antigens in 91 (45·2%) of the 201 field sera tested. It is likely that T. brucei was missed in the huffy coats because, in a subsequent study carefully executed by one of us (E. K. Mwangi, unpubl. obs.), it has been demonstrated, using the same huffy coat technique, that T. brucei does indeed
ANTIGEN ELISA FOR BOVINE TRYPANOSOMIASIS
infect cattle in these very herds and it may be responsible for up to 4% of the infections diagnosed. It was also noted that infections in less than 1% of the animals with single infections, as determined by antigen ELISA, were due to T. brucei. In mixed infections, however, T. brucei was identified in a higher proportion of the cases (Table 2). Trypanosoma brucei infections do not by themselves, usually, cause appreciable disease in cattle (Richardson, 1928; KillickKendrick, 1971 ), and infected animals can often undergo a rapid self-cure (Nantulya et al., 1984). It is suggested, therefore, that the high rate of involvement ofT. brucei in mixed infections, reported here and elsewhere (Godfrey and Killick-Kendrick, 1961; Godfrey et al., 1964), may reflect a tendency for this species to take advantage of a weakened host immune system arising from the strong immunosuppressive effect of T. congolense (Holmes et al., 1974; Rurangirwa et al., 1979), and perhaps more so the combined effect of T. congolense and T. vivax, and thereby establish more persistent infections. Sensitive as it is, however, antigen ELISA failed to detect infection in a small proportion of animals. These could have been cases of very
229
early infection, since antigens may not be detectable in the first week of infection in some animals (Nantulya and Lindqvist, 1989). The antigens detected are not secretory products: they are structural antigens which are not released in sufficient amounts until after a significant number of organisms has been destroyed by the host immune responses (Nantulya and Lindqvist, 1989). These early infections, however, can be detected readily by parasitological techniques (Nantulya and Lindqvist, 1989; Masake and Nantulya, 1991). Hence antigen detection and parasite detection are complementary. The other important advantage of antigen ELISA is that it is simple to perform and can be used for the diagnosis of the infection in several animal species, since host species-specific antisera are not required. This is convenient and it also significantly reduces the cost of reagents.
ACKNOWLEDGEMENTS. The studies at Nguruman were supported by the Overseas Development Administration (ODA) of the United Kingdom. We are grateful to Mr. James Thuo Njuguna for technical assistance and Mrs. Risper Okonji for typing the manuscript.
REFERENCES BARNETT, S. F. (1947). Bovine trypanosomiasis in Kenya with special reference to its treatment with phenanthridium. Veterinary Record, 59, 459--462. EsuRuoso, G. 0. (1973). Epizootiology, prevalence and economic aspects of bovine trypanosomiasis in Nigeria. Proceedings of the 77th Annual Congress of the U.S. Animal Health Association, 13-19 October, 1973. St. Louis, MO. GoDDEERIS, B. M., KATENDE, J. M., IRVIN, A. D. & CHUMO, R. S.C. (1982). Indirect fluorescent antibody test for experimental and epizootio1ogica1 studies on East Coast fever (Theileria parva infection in cattle). Evaluation of a cell culture schizont antigen fixed and stored in suspension. Research in Veterinary Science, 33, 360-365. GODFREY, D. G. & KILLICK-KENDRICK, R. (1961). Bovine trypanosomiasis in Nigeria. I. The inoculation of blood into rats as a method of survey in the Donga Valley, Benue Province. Annals of Tropical Medicine and Parasitology, 55, 287-297. GODFREY, D. G., LEACH, T. M. & KILLICK-KENDRICK, R. (1964). Bovine trypanosomiasis in Nigeria. III. A high incidence in a group of West African hump1ess cattle. Annals of Tropical Medicine and Parasitology, 58, 204-215. HoLMES, P. H., MAMMO, E., THOMSON, A., KNIGHT, P. A., LucKEN, R., MuRRAY, M., JENNINGS, F. W. & URQUHART, G. M. (1974). Immunosuppression in bovine trypanosomiasis. Veterinary Record, 95, 86-87. KILLICK-KENDRICK, R. (1971). The low pathogenicity of T~ypanosoma brucei to cattle. Transactions of the Royal Society of Tropical Medicine and Hygiene, 65, 104.
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Losos, G. & IKEDE, B. 0. (1972). Review of pathology of diseases in domestic and laboratory animals caused by Trypanosoma congolense, T. vivax, T. brucei, T. rhodesiense and T. gambiense. Veterinary Pathology, 9 (Suppl.), 1-71. MASAKE, R. A. & NANTULYA, V. M. (1991). Sensitivity of antigen detection enzyme-linked immunosorbent assay (antigen ELISA) for diagnosis of Trypanosoma congolense. Journal of Parasitology, 77, 231-236. METTAM, R. W. M. (1934). Annual Report of the Veterinary Department of Uganda Protectorate, pp. 26--28. MURRAY, M., MuRRAY, P. K. & MciNTYRE, W. I. M. (1977). An improved parasitological technique for the diagnosis of African trypanosomiasis. Transactions ofthe Royal Society of Tropical Medicine and Hygiene, 71, 325-326. NANTULYA, V. M. (1989). An antigen detection enzyme immunoassay for the diagnosis ofT. rhodesiense sleeping sickness. Parasite Immunology, 11, 69-75. NANTULYA, V. M. (1990). Trypanosomiasis in domestic animals: the problems of diagnosis. Review Scientifique et Technique (Office International des Epizooties), 9, 357-367. NANTULYA, V. M. & LINDQVIST, K. J. (1989). Antigen detection enzyme immunoassays for the diagnosis of Trypanosoma vivax, T. congolense and T. brucei infections in cattle. Tropical Medicine and Parasitology, 40, 263-266. NANTULYA, V. M., MusOKE, A. ]., RuRANGIRWA, F. R. & MoLoo, S. K. (1984). Resistance of cattle to tsetse-transmitted challenge with Trypanosoma brucei or Trypanosoma congelense after spontaneous recovery from syringe-passaged infections. Infection and Immunity, 43, 735-738. NANTULYA, V. M., MusOKE, A.]., RURANGIRWA, F. R., SAIGAR, N. & MINJA, S. H. (1987). Monoclonal antibodies that distinguish Trypanosoma congolense, T. vivax and T. brucei. Parasite Immunology, 9, 421-431. RICHARDSON, U. F. (1928). Notes on trypanosomiasis of cattle in Uganda. Transactions of the Royal Society of Tropical Medicine and Hygiene, 22, 137-146. RuRANGIRWA, F. R., TABEL, H., Losos, G. & TIZARD, I. R. (1979). Suppression of antibody response to Leptospira biflexa and Brucella abortus and recovery from immunodepression after Berenil treatment. Infection and Immunity, 26, 822-826. Woo, P. T. K. (1969). The haematocrit centrifuge for the detection of trypanosomes in blood. Canadian Journal of Zoology, 47, 921-923.
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
Application of a monoclonal antibody-based antigen detection enzyme-linked immunosorbent assay (antigen ELISA) for field diagnosis of bovine trypanosomiasis at Nguruman, Kenya V. M. Nantulya, K. J. Lindqvist, P. Stevenson & E. K. Mwangi To cite this article: V. M. Nantulya, K. J. Lindqvist, P. Stevenson & E. K. Mwangi (1992) Application of a monoclonal antibody-based antigen detection enzyme-linked immunosorbent assay (antigen ELISA) for field diagnosis of bovine trypanosomiasis at Nguruman, Kenya, Annals of Tropical Medicine & Parasitology, 86:3, 225-230, DOI: 10.1080/00034983.1992.11812658 To link to this article: http://dx.doi.org/10.1080/00034983.1992.11812658
Published online: 15 Nov 2016.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypgh19 Download by: [University of Tasmania]
Date: 11 April 2017, At: 02:53
Annals ofTropical Medicine and Parasitology, Vol. 86, No.3, 225-230 (1992)
Application of a monoclonal antibodybased antigen detection enzyme-linked immunosorbent assay (antigen ELISA) for field diagnosis ofbovine trypanosomiasis at Nguruman, Kenya* BY V. M. NANTUL YA, K. J. LINDQVIST International Laboratory for Research on Animal Diseases ( ILRAD ), P.O. Box 30709, Nairobi, Kenya
P. STEVENSON AND E. K. MWANGI Kenya Trypanosomiasis Research Institute, P.O. Box 362, Kikuyu, Kenya Received 20June 1991, Revised 17 February 1992, Accepted 17 February 1992
A monoclonal antibody-based, enzyme immunoassay (antigen ELISA) for the detection of species-specific invariant antigens of Trypanosoma congolense, T. vivax or T. brucei in the serum of infected animals was evaluated as a means of diagnosis using bovine field sera from a trypanosomiasis endemic area, Nguruman, Kenya. Circulating trypanosome antigens were detected in 126 (96·2%) of 131 serum samples from animals with parasitologically confirmed diagnosis: 74·8% were positive for antigens of two or three trypanosome species, while 21·4% tested positive for one trypanosome species. When 70 sera from animals (at Nguruman), which had tested negative for trypanosomes by the huffy coat technique, were tested, 35 (50·0%) of them were shown to be antigen-ELISA positive: 24 (34· 3%) showing infection with a single species and II (15·7%) with mixed infections. The predominant trypanosome species diagnosed in the two herds by antigen ELISA was T. vivax, which was detected in 133 (82·6%) of the 161 sera that tested positive for antigens, followed by T. congolense in 122 (75·8%) sera, with 109 (67·7%) showing evidence of mixed infections with two or three trypanosome species. In single infections, T. vivax exceeded T. congolense by a ratio of 2:1, with T. brucei accounting for less than 1·0%. Evidence for the specificity of the test was provided by analysis of field sera from 100 cattle, from a trypanosomiasis-free area, infected with other haemoparasites (anaplasmosis, babesiosis and theileriosis), which all tested negative in the assay.
The standard laboratory method for diagnosis of bovine trypanosomiasis is to demonstrate and identify trypanosomes in stained or wet smears of peripheral blood. Examination of blood smears, however, is not sensitive enough (Barnett, 194 7), as trypanosomes are often in such low numbers in peripheral circulation that *ILRAD Publication Series No. 971. 0003-4983j92j030225 + 06 $03.00/0
they cannot be detected by direct microscopic examination of blood. Hence, the introduction of trypanosome concentration methods such as the haematocrit centrifugation technique (Woo, 1969), or its variant, the buffy coat technique (Murray et al., 1977). Despite the marked improvement in sensitivity afforded by these concentration methods, many infected animals still go undetected, particularly those with © 1992 Liverpool School of Tropical Medicine
226
NANTUL YA ET AL.
chronic infections (Nantulya, 1990; Masake and Nantulya, 1991). In trypanosome infections, the organisms are sequestered in various predilection tissue sites, depending on the trypanosome species involved (Losos and Ikede, 1972), where they live and multiply. However, some parasites are destroyed by host immune responses which attempt to eliminate successive populations of trypanosome variable antigen types (VATs) arising through antigenic variation. This leads to the release of several soluble antigens in the blood and other tissue fluids of the infected host. The demonstration of trypanosome antigens in the blood of a previously untreated animal would indicate, therefore, that the animal has an active infection. Moreover, if the antigens detected were species-specific, it would be possible also to identify the trypanosome species causing the infection. Using this approach we have developed an enzyme immunoassay which can detect trypanosome species-specific invariant antigens in the serum or plasma of infected livestock (Nantulya and Lindqvist, 1989) as a means of diagnosis. This paper describes the initial evaluation of this assay (antigen ELISA) for the diagnosis of Trypanosoma congolense, T. vivax and T. brucei infections in cattle.
MATERIALS AND METHODS Negative Controls One hundred field sera were obtained from the ILRAD breeding herd ofBoran cattle at Kapiti, a trypanosomiasis-free area 50 km east of Nairobi. The sera were screened by indirect immunofluorescence (Goddeeris et al., 1982) for antibodies to several haemoprotozoan diseases endemic in the area. The common diseases identified were Anaplasmosis (90%), Babesiosis (40%), and Theileriosis due to Theileria mutans (45%) and T. taurotragi (10%). Test Sera The test field sera were from cattle at N guru man, 150 km southwest of Nairobi. Nguruman is a trypanosomiasis endemic area. Some cattle were small East African Zebu or crosses with Borans.
Others were Orma Boran which had been brought into the area the previous month. A total of 201 sera were analysed: 131 from parasitologically confirmed cases and 70 from animals grazing in an area of lower tsetse numbers with negative parasitological findings as determined by the huffy coat technique (Murray et al., 1977). All the sera were stored at - 20°C until use. Antigen-trapping Micro-plate ELISA The micro-plate ELISA was carried out as described previously (N antulya, 1989), with some modifications. Briefly, flat-bottom microELISA plates for the detection ofT. brucei antigens were coated with 0· 5 !lg/well of a partially purified IgM fraction of monoclonal antibody TR7/47.34.16 in carbonate buffer pH 9·6 with 0·02% sodium azide and left at 4°C until use. This antibody, although derived against T. rhodesiense procyclic trypomastigotes (Nantulya et al., 1987), also reacts with T. brucei. The plates used for detection of T. congolense or T. vivax antigens were coated with partially purified immunoglobulin fractions of speciesspecific monoclonal antibodies TC 39/30.38.16 or TV 27/9.45.15, respectively, and stored at 4oC. These two antibodies were derived against totallysates of bloodstream trypanosome populations following a method described previously (Nantulya et al., 1987). When plates were needed, excess coating antibody was drained off and washing buffer (0·15 M phosphate buffered saline pH 7·4 containing 0· 5% Tween 20) added to all the wells in volumes of 100 Ill/well. Each serum sample was then immediately added, in duplicate, at 5 !!1/ well for the T. brucei assay and 10 Ill/well for T. vivax and T. congolense. The plates were incubated for 15 minutes at room temperature, the excess reactants drained off and the conjugate (the horseradish peroxidase-conjugated immunoglobulin fraction of the corresponding monoclonal antibody), diluted in washing buffer containing 1% normal mouse serum or 0· 5% bovine serum albumin, added without prior washing of the plates. The plates were incubated at room temperature for 15 minutes, after which they were finally washed three times at 10 minute intervals by flooding the plates with
ANTIGEN ELISA FOR BOVINE TRYPANOSOMIASIS
227
TABLE 1 Identification of different trypanosome species in cattle at Nguruman, Kenya, using the buffy coat technique Trypanosome species identified
T. congolense
T. vivax
51 38·9
77
0
58·8
0
No. of positive cattle %Positive
washing buffer. After the last wash, the washing buffer was drained off and the substrate and chromogen (100 J..ll/well) added. The chromogen used consisted of 250 J..lg ml- 1 2, 2'-aziono bis (3-ethyl)-benzthiazoline-6-sulphonic acid (ABTS) in 50 mM citric acid buffer pH 4·0 containing 0·0 I% hydrogen peroxide as substrate. The optical densities (o.d.) were read after 30 minutes at 414 nm wavelength using a Titertek Multiskan micro-ELISA auto-reader (Type MCC340).
RESULTS Parasitological Results Parasitological results for the 131 animals with demonstrable parasitaemia are shown in Table I. Fifty-one (38·9%) of the animals had T. congolense infections only, 77 (58·8%) had T. vivax only and three animals (2·3%) had a mixed infection consisting of T. vivax and T. congotense. Trypanosoma brucei was reported not to have been detected, in any of the animals, by the buffy coat technique (Murray eta!., 1977). Antigen Detection The 100 field sera from a trypanosomiasis-free area, Kapiti (see Table 2), gave a mean o.d. value of0·018 ± 0·010. The threshold o.d. reading regarded as positive was, therefore, taken to be 0·050, which was twice the mean value plus one standard deviation. This threshold excluded all the negative control field sera. The analysis of 131 sera from parasitologically confirmed cases revealed 126 (96·2%) of them to have circulating antigens ofT. vivax, T.
T. brucei
T. congolense/ T. vivax
Total
3 2·3
100
131
congolense or T. brucei (Table 2): 98 (74·8%) had mixed infections involving two or three trypanosome species; 28 (21·4%) had single infections. Of the 70 sera from cattle in the area of lower tsetse challenge which had negative parasitological findings, 35 (50·0%) tested positive for trypanosome antigens: 24 (34·3%) showing evidence of single infections and II (15·7%) with mixed infections (Table 2). A detailed breakdown of the types of infection in the two herds is also given in Table 2. The predominant trypanosome species in the two herds was T. vivax, detected in 133/161 (82·6%) of the infections, followed by T. congotense in 122/161 (75·8%). Trypanosoma brucei antigen was detected in 90 (56·5%) of the infections. Seventy-six (47·2%) sera showed evidence of mixed infections involving all three trypanosome species, followed by 33 (20· 5%) cases of mixed infections involving two trypanosome species; hence the overlap in percentages. In the group of animals with single infections, T. vivax exceeded T. congolense by a ratio of 2:1, with T. brucei accounting for less than 1% of the infections.
DISCUSSION In the work reported here, a monoclonal antibody-based antigen detection enzyme immunoassay (Nantulya and Lindqvist, 1989) was evaluated using field sera from cattle in an endemic area. Of 131 sera from parasitologically confirmed cases, 126 (96·2%) were antigen ELISA positive. More significantly, 35 (50·0%)
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NANTUL YA ET AL.
TABLE2 Identification ofthe various types ofinfection in cattle from a trypanosomiasis endemic area, determined by antigen ELISA
All three species
Total number ofsera antigen ELISA positive
Type ofinfection identified Origin of serum
No. of samples tested
Tc
Single infection Tv Tb
Mixed infection TvjTb Tc/Tv Tc/Tb
0
18 15 0
2 0 0
14 6 0
0 0
2 2 0
73 3 0
126 35 0
17
33
2
20
9
4
76
161
Nguruman* Ngurumant Kapiti (controls)
131 70 100
9
Total
301
8
9
*Sera from parasitologically confirmed cases. tSera from animals with negative parasitological findings as determined using the buffy coat technique (Murray eta/., 1977).
Tc, T. congolense; Tv, T. vivax; Tb, T. brucei.
of the sera from animals in a low tsetse challenge area which were aparasitaemic, as determined using the huffy coat technique, were found to have trypanosome antigens, while the control sera from 100 animals in a tsetse-free area were negative. The control sera were from an area where many common haemoparasitic diseases such as anaplasmosis, babesiosis and theileriosis due to T. mutans and T. taurotragi were prevalent, as evidenced by the serological results. These results thus demonstrate the specificity and indicate the superior sensitivity of antigen ELISA which has been estimated to be at least four times higher than that of the huffy coat method (Masake and Nantulya, 1991). In this study, a high proportion of animals was shown, by antigen ELISA, to carry mixed infections in various combinations. This was in contrast to the results obtained by the huffy coat technique. The ELISA results, however, may represent the true situation because, under natural conditions of challenge, mixed infections with T. congolense, T. vivax and T. brucei, which occur in all combinations of two or three species, are very common in cattle (Godfrey and Killick-Kendrick, 1961; Godfrey eta!., 1964; Esuruoso, 1973) and are considered to be more
InJurious than infections with single species (Metam, 1934). In studies in Nigeria, mixed infections were better diagnosed by animal inoculation rather than by direct microscopic examination of blood (Godfrey and KillickKendrick, 1961 ). Thus, the low rates of mixed infection detected by the huffy coat technique in this present study may reflect intrinsic difficulties in the application of this technique in identifying accurately the mixed infections under the pressures of field conditions. Moreover, in mixed infections the organisms belonging to the different species give rise to different levels of parasitaemia, making it difficult to identify the species that may be present in low numbers. Another interesting finding from this study relates to the role played by T. brucei. Whereas T. brucei was not detected at all in the infected animal group by the huffy coat technique, antigen ELISA did detect T. brucei antigens in 91 (45·2%) of the 201 field sera tested. It is likely that T. brucei was missed in the huffy coats because, in a subsequent study carefully executed by one of us (E. K. Mwangi, unpubl. obs.), it has been demonstrated, using the same huffy coat technique, that T. brucei does indeed
ANTIGEN ELISA FOR BOVINE TRYPANOSOMIASIS
infect cattle in these very herds and it may be responsible for up to 4% of the infections diagnosed. It was also noted that infections in less than 1% of the animals with single infections, as determined by antigen ELISA, were due to T. brucei. In mixed infections, however, T. brucei was identified in a higher proportion of the cases (Table 2). Trypanosoma brucei infections do not by themselves, usually, cause appreciable disease in cattle (Richardson, 1928; KillickKendrick, 1971 ), and infected animals can often undergo a rapid self-cure (Nantulya et al., 1984). It is suggested, therefore, that the high rate of involvement ofT. brucei in mixed infections, reported here and elsewhere (Godfrey and Killick-Kendrick, 1961; Godfrey et al., 1964), may reflect a tendency for this species to take advantage of a weakened host immune system arising from the strong immunosuppressive effect of T. congolense (Holmes et al., 1974; Rurangirwa et al., 1979), and perhaps more so the combined effect of T. congolense and T. vivax, and thereby establish more persistent infections. Sensitive as it is, however, antigen ELISA failed to detect infection in a small proportion of animals. These could have been cases of very
229
early infection, since antigens may not be detectable in the first week of infection in some animals (Nantulya and Lindqvist, 1989). The antigens detected are not secretory products: they are structural antigens which are not released in sufficient amounts until after a significant number of organisms has been destroyed by the host immune responses (Nantulya and Lindqvist, 1989). These early infections, however, can be detected readily by parasitological techniques (Nantulya and Lindqvist, 1989; Masake and Nantulya, 1991). Hence antigen detection and parasite detection are complementary. The other important advantage of antigen ELISA is that it is simple to perform and can be used for the diagnosis of the infection in several animal species, since host species-specific antisera are not required. This is convenient and it also significantly reduces the cost of reagents.
ACKNOWLEDGEMENTS. The studies at Nguruman were supported by the Overseas Development Administration (ODA) of the United Kingdom. We are grateful to Mr. James Thuo Njuguna for technical assistance and Mrs. Risper Okonji for typing the manuscript.
REFERENCES BARNETT, S. F. (1947). Bovine trypanosomiasis in Kenya with special reference to its treatment with phenanthridium. Veterinary Record, 59, 459--462. EsuRuoso, G. 0. (1973). Epizootiology, prevalence and economic aspects of bovine trypanosomiasis in Nigeria. Proceedings of the 77th Annual Congress of the U.S. Animal Health Association, 13-19 October, 1973. St. Louis, MO. GoDDEERIS, B. M., KATENDE, J. M., IRVIN, A. D. & CHUMO, R. S.C. (1982). Indirect fluorescent antibody test for experimental and epizootio1ogica1 studies on East Coast fever (Theileria parva infection in cattle). Evaluation of a cell culture schizont antigen fixed and stored in suspension. Research in Veterinary Science, 33, 360-365. GODFREY, D. G. & KILLICK-KENDRICK, R. (1961). Bovine trypanosomiasis in Nigeria. I. The inoculation of blood into rats as a method of survey in the Donga Valley, Benue Province. Annals of Tropical Medicine and Parasitology, 55, 287-297. GODFREY, D. G., LEACH, T. M. & KILLICK-KENDRICK, R. (1964). Bovine trypanosomiasis in Nigeria. III. A high incidence in a group of West African hump1ess cattle. Annals of Tropical Medicine and Parasitology, 58, 204-215. HoLMES, P. H., MAMMO, E., THOMSON, A., KNIGHT, P. A., LucKEN, R., MuRRAY, M., JENNINGS, F. W. & URQUHART, G. M. (1974). Immunosuppression in bovine trypanosomiasis. Veterinary Record, 95, 86-87. KILLICK-KENDRICK, R. (1971). The low pathogenicity of T~ypanosoma brucei to cattle. Transactions of the Royal Society of Tropical Medicine and Hygiene, 65, 104.
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Losos, G. & IKEDE, B. 0. (1972). Review of pathology of diseases in domestic and laboratory animals caused by Trypanosoma congolense, T. vivax, T. brucei, T. rhodesiense and T. gambiense. Veterinary Pathology, 9 (Suppl.), 1-71. MASAKE, R. A. & NANTULYA, V. M. (1991). Sensitivity of antigen detection enzyme-linked immunosorbent assay (antigen ELISA) for diagnosis of Trypanosoma congolense. Journal of Parasitology, 77, 231-236. METTAM, R. W. M. (1934). Annual Report of the Veterinary Department of Uganda Protectorate, pp. 26--28. MURRAY, M., MuRRAY, P. K. & MciNTYRE, W. I. M. (1977). An improved parasitological technique for the diagnosis of African trypanosomiasis. Transactions ofthe Royal Society of Tropical Medicine and Hygiene, 71, 325-326. NANTULYA, V. M. (1989). An antigen detection enzyme immunoassay for the diagnosis ofT. rhodesiense sleeping sickness. Parasite Immunology, 11, 69-75. NANTULYA, V. M. (1990). Trypanosomiasis in domestic animals: the problems of diagnosis. Review Scientifique et Technique (Office International des Epizooties), 9, 357-367. NANTULYA, V. M. & LINDQVIST, K. J. (1989). Antigen detection enzyme immunoassays for the diagnosis of Trypanosoma vivax, T. congolense and T. brucei infections in cattle. Tropical Medicine and Parasitology, 40, 263-266. NANTULYA, V. M., MusOKE, A. ]., RuRANGIRWA, F. R. & MoLoo, S. K. (1984). Resistance of cattle to tsetse-transmitted challenge with Trypanosoma brucei or Trypanosoma congelense after spontaneous recovery from syringe-passaged infections. Infection and Immunity, 43, 735-738. NANTULYA, V. M., MusOKE, A.]., RURANGIRWA, F. R., SAIGAR, N. & MINJA, S. H. (1987). Monoclonal antibodies that distinguish Trypanosoma congolense, T. vivax and T. brucei. Parasite Immunology, 9, 421-431. RICHARDSON, U. F. (1928). Notes on trypanosomiasis of cattle in Uganda. Transactions of the Royal Society of Tropical Medicine and Hygiene, 22, 137-146. RuRANGIRWA, F. R., TABEL, H., Losos, G. & TIZARD, I. R. (1979). Suppression of antibody response to Leptospira biflexa and Brucella abortus and recovery from immunodepression after Berenil treatment. Infection and Immunity, 26, 822-826. Woo, P. T. K. (1969). The haematocrit centrifuge for the detection of trypanosomes in blood. Canadian Journal of Zoology, 47, 921-923.
