ELSEVIER Veterinary Parasitology 56 (1995) 261-267

veterinary parasitology

Polyclonal antibody-based antigen-detection immunoassay for diagnosis of Trypanosoma evansi in

buffaloes and horses

Veer Singh*, S.S. Chaudhari, S. Kumar and M.B. Chhabra Department of Veterinary Parasitology, Haryana Agricultural University, Hisar- 125 004, India

Accepted 26 April 1994

Abstract

An enzyme-linked immunosorbent assay (ELISA) was employed for the detection of Trypanosoma evansi antigens in serum samples of field cases of buffaloes and horses in northern India. In 323 naturally infected/suspected buffaloes, circulating antigenaemia was detected in 180 ( 55.72%), whereas parasitaemia by wet blood smear examination was found in 62 (19.19%) only. The antigen-ELISA was positive in 47 of the 62 parasitologi- cally proven cases and in 86 of the 116 cases with anti-trypanosome antibodies detected by ELISA. Of the 80 horses examined antigen-ELISA was positive in 45 (56.75%) sera. The antigen-detection assay was positive in 14 of the 19 parasitaemic cases whereas the antibody-detection assay was positive in l 8 of the 30 parasitaemic cases.

In the present study, antigen-ELISA was found to be more sensitive and specific com- pared with antibody-ELISA and wet blood examination, and could prove a useful tool for epidemiological studies of latent trypanosomosis in livestock.

Keywords: Trypanosoma evansi; Buffalo; Horse; Diagnosis-Protozoa

1. Introduction

Trypanosomos i s caused by the h a e m o p r o t o z o a n Trypanosoma evansi is a sig- nif icant const ra in t to l ivestock health in ma jo r areas o f Asia, including nor thern

* Paper presented at the Third Asian Congress of Parasitology held at Lucknow, India, 18-21 Feb- ruary, 1993. * Corresponding author. Present address: Department of Parasitology, Gujarat Agricultural Univer- sity, S.K. Nagar, (Dantiwada), Gujarat-385 506, India.

0304-4017/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved SSD10304-4017 ( 94 ) 00689-A

262 K Singh et al. / Veterinary Parasitology 56 (1995) 261-26 7

India where the disease is endemic. Diagnosis of the disease is largely based on the demonstration of trypanosomes in peripheral blood. This is often hampered by the paucity and fluctuating nature ofparasitaemia, especially in the more com- mon chronic phase (Mahmoud and Gray, 1980). Serological diagnoses for anti- body detection have several drawbacks, including the inability to distinguish a current infection from a past or recovered case and the lack of specificity. In re- cent years, efforts have been made to improve or develop alternative means of diagnosis (Luckins et al., 1978; Zweygarth et al., 1986). A major improvement in the diagnostic approach is based on the detection of circulating antigens in the sera of suspected/infected cases (Rae and Luckins, 1984). Nantulya et al. (1989) used an antigen-detection enzyme immunoassay based on the Trypanosoma bru- cei group specific (mAb) antibody for detecting circulating T. evansi antigens in experimental and naturally infected animal sera from several countries. The re- suits underscored the potential of antigen-ELISA (enzyme-linked immunosor- bent assay) as a tool for sero-epidemiological investigations. According to Nan- tulya and Lindqvist (1989), the antigen-ELISA is not only of diagnostic importance but is also useful as a tool for evaluation of the efficacy of treatment.

This paper reports on the application of a polyclonal antibody based on anti- gen-detection immunoassay for the diagnosis of naturally active T. evansi infec- tions among buffaloes and horses in northern India compared with an antibody- detection immunoassay and parasitological examination for the presence of try- panosome infection.

2. Materials and methods

2.1. Antigen-detection ELISA

An indirect ELISA for the detection of antigens of T. evansi in tests sera was based on the technique described by Voller et al. ( 1976 ) with slight modification.

2.1.1. Antigen Whole cell lysate (WCL) antigen was prepared from T. evansi stock originally

collected from a buffalo in the Bareilly locality of northern India. The trypano- sprees harvested from the blood of infected rats were separated through a DEAE cellulose column by the method of Lanham and Godfrey (1970), sonicated and centrifuged; the supernatant, with protein content adjusted to 1.0 mg ml- ~, con- stituted the antigen. The antigen was used as the positive control in the assay.

2.1.2. Test sera Sera were collected from 323 buffaloes and 80 horses in the Karnal region of

Haryana from July 1990 to October 1990. Simultaneously, these animals were checked for parasitaemia by the wet blood smear examination (WBE).

V. Singh et aL / Veterinary Parasitology 56 (1995) 261-267 263

2.1.3. Hyperimmune serum (HIS) Anti-trypanosome HIS raised in a batch of rabbits against the T. evansi stock

(see above) was used.

2.1.4. Enzyme antiglobulin conjugate Horseradish peroxidase (HRP) goat anti-rabbit IgG conjugate (Sigma, USA)

was used.

2.1.5. Substrate o-Phenlene diamine (OPD) dihydrochloride at a concentration of 0.4 mg m1-1,

to which was added 10/zl of 30% hydrogen peroxide, was used.

2.1.6. Standardization Optimum dilutions of rabbit anti-trypanosome HIS, as determined by chequer

board titration with known positive and known negative samples, was 1 : 100, of antigen 1 : 40 and conjugate 1 : 2000, which showed maximum colour change and maximum contrast between known positive antigen and negative samples.

2.1.7. Test procedure Flat-bottom 96 well micro-ELISA plates (Dynatech, USA) were coated with

100/zl per well in duplicate of 1:20 diluted test sera in coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6). After gently washing with buffer (0.15 M NaCI containing 0.05% Tween-20), blocking was performed with 100/tl of 3% bovine serum albumin fraction V in diluent buffer (0.01 M phosphate-buffered saline with Tween-20) for 3 h at 37 o C. After washing, an appropriate dilution of 100/zl of rabbit anti-trypanosome HIS was added and incubated at 37°C for 1 h. After further washing with washing buffer, 100/zl HRP of anti-rabbit IgG conju- gate (Sigma) optimally diluted (in diluent buffer) was added and incubated at 37°C for 1 h; 200/tl of the substrate OPD was added for washing and incubated for 30 rain at 37°C. The colour reaction was measured at 492 nm in a Titertek ELISA reader (Flow Laboratories, UK).

2.1.8. ELISA value and interpretation of results The mean absorbance of known negative sera (eight testings) and test serum

antigen (two testings) was calculated. The ELISA value for each sample was cal- culated using the formula:

Mean absorbance of test sera ELISA value = Mean absorbance of known negative sera

ELISA values greater than 1.0 were considered as positive, between 1.0 to 1.09 as doubtful and less than 1.0 as negative.

264 V. Singh et al. / Veterinary Parasitology 56 (1995) 261-267

2.2. Antibody-detection ELISA

The procedure was similar to the antigen-detection assay above, but the wells were first coated with WCL trypanosomal soluble antigen. Anti-bovine IgG and anti-horse IgG developed in rabbits (Sigma) were the conjugates. Disposable mi- croplates (Dynatech) were used and the optimum dilution of coating antigen as determined by chequer board titration was 1 : 40.

3. Results

The detection of T. evansi infection by the serological tests was predictably much higher than by parasitological examination (Table 1 ). Of the two immu- noassays, the antigen-detection test proved more sensitive than the antibody-de- tection test in buffaloes as well as in horses. The assay could even detect a few positive reactors in apparently healthy animals with no parasitaemia. However, in such animals positivity for antibody indicated past exposure to T. evansi infec- tion in both animal species. The correlations between the diagnostic methods are presented in Table 2.

Among buffaloes, out of the 62 parasitologically positive animals, 47 had cir- culating antigens in blood detectable by the antigen-detection assay, 28 were de- tected by the antibody-detection assay and 25 of these were positive by all three diagnostic tests. Of the 180 sera detectable by antigen-ELISA and the 116 sera positive by antibody-ELISA, as many as 86 were positive by both, indicating good correlation between the two assays.

Among horses, out of 19 parasitaemic animals, 14 were found to have circulat- ing antigens, eight had antibody and four were positive by all three diagnostic tests. Of the 45 sera detectable by antigen-ELISA and 30 sera by antibody-ELISA, only 18 were positive by both.

The results showed a fair degree of correlation between parasitaemia (WBE)

Table 1 Results of the diagnostic test for T. evansi in naturally infected/suspected cases

Host species Status Total Diagnostic test number of animals Wet-blood- Ag-ELISA Ab-ELISA examined examing ....

+ve - v e +ve - v e +ve - v e

Buffalo

Horse

Sick/suspected 323 Apparently healthy 25

Sick/suspected 80 Apparentlyhealthy 10

62 261 180 143 116 207 0 25 2 23 3 22

19 61 45 35 30 50 0 10 1 9 2 8

Ag, antigen; Ab, antibody.

V. Singh et al. / Veterinary Parasitology 56 (1995) 261-267

Table 2 Efficacy of diagnostic tests for T. evansi in naturally infected/suspected cases

265

Tests Host species

Buffalo (n=323) Horse (n=80)

No. positive % No. positive %

Wet blood examination (WBE) 62 19.19 19 23.75 Ag-ELISA 180 55.72 45 56.25 Ab-ELISA 116 35.91 30 37.50 WBE +Ag-ELISA 47 14.55 14 17.50 WBE + Ab-ELISA 28 8.68 8 10.00 Ag-Elisa + Ab-ELISA 86 26.62 18 22.50 WBE + Ag-ELISA + Ab-ELISA 25 7.74 4 5.00

Ag, antigen; Ab, antibody.

and circulating antigenaemia (antigen-ELISA), but poor correlation between parasitaemia and circulating antibodies in both the host species investigated. A comparison of the two ELISAs regarding sensitivity and specificity in detecting naturally infected/suspected cases of T. evansi (Table 2) showed that the anti- gen-ELISA was in general more sensitive than the antibody-ELISA. Even regard- ing the specificity, the antigen-ELISA compared favourably with the overall re- suits of the antibody-ELISA.

4. Discussion

The present results confirmed that detection by serological procedures was in- variably much higher (55.8%) than by blood examination (20.1%) in the ani- mals screened. There was a high proportion of animals without parasitaemia that tested positive for antigens, indicating the greater sensitivity of antigen-ELISA in providing the true epidemiological data. A study in Vietnam found positivity by antigen-ELISA in 13.26% of 558 buffaloes and 20.4% of 258 cattle compared with only 3.4% and 11.8%, respectively, by stained blood smears (Thuan et al., 1991 ). Abdalla (1992) reported 30% positivity in camel sera from the Sudan by serodiagnostic tests as against a maximum of 5% by wet blood examination. The lack of accompanying antigenaemia in about 27% of the cases of patent parasi- taemia in the present study could be due to an early infection (less than a week) when insufficient parasite destruction had occurred to give detectable levels of antigen in circulation. In later stages, there can also be the mopping up of antigens by excess antibody in forming immune complexes. Waitumbi and Nantulya (1993) reported that, in cases of antibody excess, antigens could exist in the form of immune complexes and would remain undetected since the epitopes would be masked by the complexing antibodies. Similarly, Nantulya et al. (1992) found

266 V. Singh et al. / Veterinary Parasitology 56 (1995) 261-267

antigens in the serum of 168 (89.4%) of 188 parasitologically confirmed cases of T. brucei gambiense sleeping sickness.

According to Shen et al. ( 198 5 ), ELISA has proved to be a satisfactory method for the diagnosis of antibodies of T. evansi in buffaloes. In a comparative study involving ELISA, indirect haemagglutination (IHA) and complement fixation test (CFT), the rates of detection were 96.2%, 78.0% and 82.3%, respectively. The results confirmed the superiority of ELISA over both CFT and IHA. The disadvantage that the presence of antibodies does not necessarily signify an active disease or even an active infection, and there are wide variations in antibody response which do not correlate with severity of disease, is overcome in the alter- native approach of ELISA for detecting circulating antigens in the sera of infected animals. It is a more reliable indication of patent infection than antibody-depen- dent assays.

In the present study antigen-ELISA was found to be highly sensitive and spe- cific. It was of special significance in detection of latent cases of surra both in buffaloes and in equines. One of its importance applications is in the follow-up of cases after treatment to determine whether the infection has been cured (Nan- tulya and Lindqvist, 1989). The potential of antigen-ELISA, not only as a tool for diagnosis but also for treatment follow-up of patients with T. brucei gam- biense sleeping sickness, has been demonstrated (Nantulya et al., 1992 ). Accord- ing to Nantulya (1990), combination of one of the more sensitive standard try- panosome detection techniques with antigen-trapping ELISA is likely to be the diagnostic strategy for the future.

Acknowledgements

The authors wish to place on record appreciation for the facilities extended by Dr. R.P. Gupta, Senior Research Officer, Parasitological Laboratory, Haryana Agricultural University, Uchani, Karnal, in collection of field samples. The grant of financial assistance in the form of a Senior Research Fellowship Award to one of us (V.S.) by the Council of Scientific and Industrial Research, New Delhi, is gratefully acknowledged.

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