W. Olaho-Mukani1; N.M. Okuna1 and J.O. Ouma2
Antigen-ELISA was employed in the diagnosis of trypanosome infection and determination of efficacy of a trypanocidal drug, cymelarsan, in eliminating Trypanosoma evansi infection in camels in Kenya. In this first study a polyclonal rabbit anti-T. evansi antibodies (RATE) and RATE-peroxidase conjugate were employed in a sandwich ELISA. In a second study in Kenya, anti-trypanosome specific murine monoclonal antibodies were used in a sandwich ELISA to evaluate the efficacy of a commercial Cypermethrin® dip for the control of Glossina pallidipes and bovine trypanosomosis on Taita Ranch. In a third study, these monoclonal-based sandwich ELISAs were used to assess the success of the Kenya-Uganda joint border tsetse and trypanosomosis control. Both ILRI and IAEA versions of Ag-ELISA were used for detection of trypanosome antigens in naturally infected cattle in Kenya. Before these various versions of ELISA were used, they were validated and standardised for relative sensitivity and specificity. Results obtained in the camel studies in Kenya showed a relative sensitivity of >74% and a specificity of >79% in diagnosing T. evansi infection using either CTVM or KETRI diagnostic kits. The ILRI version of ELISA gave a relative sensitivity of 82 and 90% for bovine trypanosomosis in Kenya and Uganda, respectively. It was hard to determine the specificity of this test in Uganda due to lack of negative controls, but in Kenya the test was 99% specific. On the other hand, the Vienna version of bovine antigen-ELISA was 100% specific in Kenya but only 8.5% sensitive, while in Uganda the test gave specificities of 95, 97 and 75% for T. brucei, T. congolense and T. vivax, respectively, and relative sensitivity for the three trypanosome species were 75, 30 and 65%, respectively. Based on these results and others from collaborating African countries, it was recommended that the Vienna version of antigen-ELISA be readjusted to improve on its relative sensitivity. The cymelarsen treatment was instituted based on antigenaemia resulting in the reduction of the number of positive cases from 75 to 11% as revealed by Ag-ELISA. This subsequently led to total elimination of trypanosome antigens in camels.
Animal trypanosomosis is endemic in more than 90% of Uganda and is the single most important disease constraint to livestock production (COCTU 1987) in that country. In Kenya, tsetse-transmitted trypanosomosis is endemic in 60% of Kenya's rangelands, which constitute 25% of the country (KETRI 1990). Though large-scale eradication of tsetse and trypanosomosis may remain elusive for many years to come, the availability of low-cost technologies and prospects of community participation give optimism for the smaller, and more manageable tsetse and trypanosomosis programmes (Chadenga 1994). In the past, one of the major constraints to the control of trypanosomosis has been the lack of sensitive diagnostic tests. The recent development of tests which detect circulating trypanosomal antigens has given a ray of hope onto this predicament (Nantulya 1989, 1991). This paper discusses the results of studies carried out to evaluate the success of some of the smaller tsetse and trypanosomosis control programmes in Kenya and Uganda using antigen-ELISA (Nantulya and Lindqvist 1989).
RATE and RATE-peroxidase conjugate were obtained from Edinburgh, UK. The T. evansi Ag-ELISA was standardised using serum samples from known parasite-negative and parasite-positive camels. Subsequently, the test was used to study the prevalence of trypanosome infection in five camel herds comprising 641 camels and to evaluate the efficacy of cymelarsan used for treating the KETRI camel herd (61 camels) based at Athi River, Kenya. Anti-T. evansi monoclonal antibody and corresponding peroxidase conjugate were prepared at KETRI.
Reagents for bovine antigen-ELISA were either obtained from ILRI or IAEA. In Uganda, the study was conducted on sera collected from cattle herds located near the Kenya-Uganda border where tsetse and trypanosomosis control were being performed using spot-on (deltamethrin), block treatment with diminazene aceturate and deltamethrin impregnated traps or screens. In Kenya Ag-ELISA was conducted on sera collected from cattle subjected to experimental Cypermethrin® (Rhône Mérieux, France) dipping at Taita and Taru ranches. Trypanosome-negative sera were obtained from cattle residing at KETRI (Muguga) and ILRI herd (Kapiti plains), comprising 60 and 200 samples.
Buffy coat technique (BCT) was employed for parasitological diagnosis as described by Woo (1970). Trypanosomes were identified by microscopic examination of thin-stained blood smears. For the T. evansi studies mouse subinoculation was employed as described by Godfrey and Killick-Kendrick (1962).
Table 1 shows the set-up of antigen-ELISAs which have been used in Kenya and Uganda for trypanosomal studies. Table 2 summarises the specificities and sensitivities of the ELISAs determined at KETRI, Kenya and at Livestock Research Institute (LIRI), Uganda for animal trypanosomosis. The CTVM T. evansi polyclonal-based ELISA gave fairly good relative sensitivity and specificity and on that basis was employed for epidemiological studies. In the five camel herds studied, results showed prevalence rates of T. evansi infection ranging from 45.5 to 86% compared to 1 to 18% using parasitological tests. Higher ELISA OD values were observed in those herds with the highest rate of patent infection or where chemotherapeutic intervention was not being practised. However, the study revealed evidence of endemic trypanosomosis in all sampled herds and although treatment with quinapyramine sulphate suppressed parasitaemia, the presence of circulating trypanosomal antigens indicated the probable persistence of infection due to resistance to this drug. In experimental camels, the use of Ag-ELISA in evaluating the efficacy of cymelarsan revealed that more than 80% of the treated camels were apparently cured as evidenced by the disappearance of parasites and trypanosomal antigens from the blood circulation. However, relapses were observed in a few animals. Antigenaemia persisted in a number of the camels throughout the study period. Treatment on the basis of an antigenaemia reduced the proportion of ag-positive animals from 75 to 26% and parasites remained undetectable up to the end of the study.
Table 1. Types of antigen-ELISA used in Kenya and Uganda.
| Coating antibody | Blocking agent | Conjugate | Chromogen | Institution | Country of origin |
| RATE | 1% BSA | RATE-PO | TMB | CTVM | Kenya |
| T.e. MAb | None | T.e.-PO | OPD | KERTI | Kenya |
| T.b. MAb | 1% BSA | T.b.-MAb-PO | ABTS | ILRI | Uganda |
| T.c. MAb | 1% BSA | T.c.-MAb-PO | ABTS | ILRI | Uganda |
| T.v. MAb | 0.5% NMS | T.c.-MAb-PO | ABTS | ILRI | Uganda |
| T.b. MAb | 0.5% NMS | T.b.-MAb-PO | TMB | IAEA | Uganda |
| T.c. MAb | 0.5% NMS | T.c.-MAb-PO | TMB | IAEA | Uganda |
| T.v. MAb | 0.5% NMS | T.v.-MAb-PO | TMB | IAEA | Uganda |
| T.b. MAb | 0.5% NMS | T.b.-MAb-PO | TMB | ILRI | Kenya |
| T.c. MAb | 0.5% NMS | T.c.-MAb-PO | TMB | ILRI | Kenya |
| T.v. MAb | 0.5% NMS | T.v.-MAb-PO | TMB | ILRI | Kenya |
RATE = rabbit anti T. evansi; T.e. MAb = monoclonal antibody against T. evansi; T.b. = T. brucei; T.c. = T. congolence; T.v. = T. vivax; BSA = bovine serum albumin; PO = peroxidase; NMS = normal mouse serum; TMB = tetra methyl-benzidine; ABTS = 2,2'-azino di [3-ethylbenzthiazoline] sulphonate; OPD = Othophenylenediamine.
Table 2. Relative sensitivity and specificity of the antigen-ELISAs used in Kenya and Uganda.
|
Species/institution |
Relative sensitivity† (%) | Specificity (%) | Country |
| T. evansi/CTVM | 74 | 79 | Kenya |
| T. evansi/KETRI | 92 | 100 | Kenya |
| T.b.-ILRI | – | 99 | Kenya |
| T.c.-ILRI | 83 | 99 | Kenya |
| T.v.-ILRI | 80 | 99 | Kenya |
| T.b.-IAEA | 75 | 95 | Uganda |
| T.c.-IAEA | 30 | 97 | Uganda |
| T.v.-IAEA | 65 | 75 | Uganda |
| T.b.-IAEA | – | 98 | Kenya |
| T.c.-IAEA | 30 | 100 | Kenya |
| T.v.-IAEA | 18.3 | 99 | Kenya |
BCT = buffy coat technique.
In another study an IgM trypanosome-specific monoclonal antibody raised against T. evansi, MAb TEA1/23.4.6. was used to study T. evansi infection in goats and camels. Trypanosomal antigens were detected in infected goats 24 hr after intravenous inoculation of 2 × 106 trypanosomes/goat and although the levels of parasitaemia fluctuated, positivity was maintained throughout the course of infection. Following treatment, antigens concentration dropped to undetectable levels between 7 and 48 days. The same trend was observed in infected camels which were effectively treated. On the contrary there was an elevation of circulating trypanosomal antigens several weeks prior to reappearance of trypanosome in blood circulation.
The ILRI antigen-ELISA used for bovine trypanosomosis in Uganda gave a relative sensitivity of 91% based on 118 parasitologically positive cattle. In sampled herds the test revealed evidence of mixed infection and a higher prevalence of the disease than parasitological tests based on microscopic examination. In areas where 1% deltamethrin (Spot-on®, Cooper, Harare, Zimbabwe) had been applied, there was marked reduction of infection from about 10 to 0% by BCT, and 75 to 15% by antigen-ELISA, while in the areas where tsetse control was not being practised the disease incidence remained elevated.
In a subsequent study in Uganda using IAEA modified antigen-ELISA, a total of 3035 cattle (2733 from a tsetse-infested area and 302 from a tsetse-free area) were screened by both BCT and antigen-ELISA. The observed specificities of antigen-ELISA for T. brucei, T. congolense and T. vivax were 95, 97 and 75%, respectively, while sensitivities for the same trypanosome species were 75, 30 and 65%, respectively.
In Kenya, the results of the ILRI version of antigen-ELISA used to evaluate the efficacy of Cypermethrin dip for the control of Glossina pallidipes gave a specificity of 100% for the three species and sensitivities of 80% for T. vivax and 83% for T. congolense. Trypanosoma brucei infections were not detected parasitologically. The study showed a marked reduction in trypanosome infection on the ranch where Cypermethrin dip was applied (Taita Ranch) as opposed to the control ranch (Taru Ranch) where there was no fly control. The IAEA version of antigen-ELISA gave specificities of 100, 98 and 99% for T. congolense, T. brucei and T. vivax, respectively, and relative sensitivities of 1 and 18.5% for T. congolense and T. vivax, respectively. No T. brucei infections were detected parasitologically by BCT.
Studies carried out on the use of antigen-ELISA in Uganda and Kenya demonstrate the usefulness of combining serological diagnosis with tests based on parasite detection for the control of animal trypanosomosis. Fairly good relative sensitivity and specificity was achieved with most tests except for IAEA version of antigen-ELISA which suffered from low relative sensitivity.
However, in order to achieve internationally accepted standards, these tests could be improved in the following ways. Firstly, the sample sizes used for standardisation and validation were in most cases too small. This means that the sensitivities recorded cannot be relied on. Secondly there was a problem of obtaining true negatives. Given the fact that antigen-ELISA is more sensitive than the parasite detection tests (Nantulya 1989) there was the definite lack of a 'goldstandard' test for comparison. This shortcoming could be overcome by using PCR in addition to parasitological tests. Before PCR is developed, standardised and validated for routine trypanosomosis diagnosis, the problem of specificity and relative sensitivity might linger on for some time to come. It is apparent that most of the monoclonal antibody probes used in these studies are of IgM class. Since the latter tend to give non-specific reactions, attempts should be made to generate MAbs of IgG class. By paying attention to the analytical requisites, diagnostic performance, standards and quality control, the performance of trypanosomal antigen-ELISA can be greatly improved.
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