Tropical Animal Health and Production, 37 (2005) 457^467

# 2005 Springer. Printed in the Netherlands

Evaluation of Diagnostic Tests for Trypanosoma evansi in

Experimentally Infected Pigs and Subsequent Use in Field
Surveys in North Vietnam and Thailand
W.G. Holland1,2*, N.G. Thanh2, T.T. Do2, S. Sangmaneedet3, B. Goddeeris1,4 and
J. Vercruysse1
1
Ghent University, Faculty of Veterinary Medicine, Department of Virology, Parasitology
and Immunology, Merelbeke, Belgium; 2National Institute of Veterinary Research, Dong
Da, Hanoi, Vietnam; 3Khon Kaen University, Faculty of Veterinary Medicine,
Department of Pathobiology, Khon Kaen, Thailand; 4Faculty of Applied Bioscience and
Engineering, Department of Animal Science, K.U. Leuven, Heverlee, Belgium
*Correspondence: Present address: Intervet International, P.O box 31, 5830 AA Boxmeer
The Netherlands
E-mail: [email protected]

Holland, W.G., Thanh, N.G., Do, T.T., Sangmaneedet, S., Goddeeris, B. and Vercruysse, J., 2005.
Evaluation of diagnostic tests for Trypanosoma evansi in experimentally infected pigs and subsequent use
in ¢eld surveys in North Vietnam and Thailand. Tropical Animal Health and Production, 37(6), 457^467

ABSTRACT

This study is concerned with the evaluation of established diagnostic tests for diagnosis of Trypanosoma
evansi in pigs. The immune trypanolysis test (TL), card agglutination test (CATT), latex agglutination
test (LATEX), enzyme-linked immunosorbent assay (ELISA), microhaematocrit centrifugation
technique (MHCT) and mouse inoculation (MI) tests were initially evaluated in experimentally
infected fattening pigs. All infected pigs were con¢rmed parasitologically positive with both MHCT
and MI. Results of the serological assays indicated that the TL could be a reference test for the presence
of RoTat 1.2 antibodies in pigs. The results of the CATT and LATEX were inconsistent with the TL
while the ELISA results correlated with the TL results. The four serological assays were subsequently
used in two ¢eld surveys in Vietnam and Thailand. Results of the two agglutination assays (CATT and
LATEX) were not consistent and did not correlate with TL results. The ELISA at percentage positivity
of 22 appeared to have good ability to discriminate between seropositive and seronegative animals. Of
the 437 samples collected at smallholder pig premises in northern Vietnam, no positive pigs were
detected with the TL test. In Thailand, 77 samples were collected from ¢ve farrowing farms with a
history of surra. Two parasitologically positive sows were found and on each farm seropositive sows
were detected.

Keywords: diagnosis, Trypanosoma evansi, pigs, serology

Abbreviations: ABTS, 2,2-azinobis(3-ethyl-benzothiazaline)-6-sulphonic acid; CATT, card

agglutination test; ELISA, enzyme-linked immunosorbent assay; LATEX, later agglutination test;
MHCT, microhaematocrit centrifugation technique; MI, mouse inoculation; OD. optical density; PI,
post infection; PP, percentage positivity; TL, immune trypanolysis; VAT, variable antigen type; VSG,
variable surface glycogen

457
458

INTRODUCTION

Trypanosoma evansi (causative agent of surra) is endemic in most countries of South-

East Asia (Luckins, 1988), but reports of disease are usually limited to water bu¡aloes
and cattle, with occasional outbreaks in horses. Although T. evansi is not highly
pathogenic for pigs (Stephen, 1986) outbreaks with abortion and death have been
observed in Thailand (Teeraprasert et al., 1984a; Bajyana Songa et al., 1987), Malaysia
(Arunusalam et al., 1995) and Indonesia (Kraneveld and Mansjoer, 1947). In India,
acute infections were observed in pigs with symptoms similar to classical swine fever
(Gill et al., 1987). As the number of reports on T. evansi infections in pigs is limited, one
might conclude that surra is of little importance. However, recently, the
immunosuppressive e¡ects of surra on pigs has been described (Holland et al., 2003)
and it could well be that vaccination failures noted in pigs in trypanosome-endemic
areas are due to chronic T. evansi infections. To further investigate the possible impact
of immunosuppression, validated diagnostic tests are a prerequisite.
Surra in pigs can be diagnosed directly by microscopy, concentration techniques or
rodent inoculation, but owing to £uctuating levels of parasitaemia, especially during
the chronic stages of infection, parasitological tests often lack sensitivity (Nantulya,
1990). Serological tests for detection of T. evansi speci¢c antibodies, based on the
predominant variable antigen type (VAT) RoTat 1.2 (Bajyana Songa and Hamers,
1988; Verloo et al., 2001) have been used extensively in water bu¡alo (Davison et al.,
1999; Verloo et al., 2000; Holland et al., 2002). A card agglutination test (CATT), a
latex agglutination test (LATEX) using serum or blood, and ELISA and immune
trypanolysis tests (TL), both using either serum or blood eluted from ¢lter paper, have
been evaluated using samples from experimentally infected bu¡alo and naturally
infected animals (Verloo et al., 2000; Holland et al., 2002).
Since validated serological assays for the diagnosis of T. evansi for pigs are not
available, an initial evaluation of LATEX, CATT, ELISA and the immune trypanolysis
(TL) tests was carried out using serum samples from pigs experimentally infected with
T. evansi. Thereafter, the tests were used in ¢eld surveys to determine the T. evansi
infection status in pig populations from northern Vietnam and on breeding farms in
Thailand.

MATERIALS AND METHODS

Experimental infections

Fourteen proven parasite-negative piglets of mixed sex, 6 weeks of age, were infected
with 106 T. evansi bloodstream trypomastigotes of the isolate WH ITMAS 101298
(Holland et al., 2001) (group A). A second group of 7 animals (group B) remained
uninfected. Animals were housed in £yproof stables. Serum and heparinized blood
were collected weekly from each animal until week 13 post infection (PI). Heparinized
blood from all pigs was examined using the microhaematocrit centrifugation technique
(MHCT) and, if it tested negative, the blood sample was inoculated into mice (mouse
 459

inoculation, MI), since MI is a more sensitive test (Paris et al., 1982). Blood for serum
collection was allowed to clot at room temperature and kept overnight at 48C. The next
day, blood samples were centrifuged and serum was removed and stored at ^208C for
serological analysis.

Field samples

Four hundred and thirty-seven serum samples were collected, mainly from fattening
pigs, on smallholder farms in Bac Can, Bac Ninh and Nghe Anh provinces in northern
Vietnam, an area with a history of surra problems in cattle and water-bu¡alo.
Seventy-seven serum and heparinized blood samples were collected from breeding
stock at ¢ve di¡erent farrowing farms around the city of Khon Kaen in north-eastern
Thailand, a T. evansi-endemic area. The heparinized blood was examined using the
MHCT. On each farm the following symptoms were observed with breeding sows to a
varying degree: abortion, a typical skin rash with erythrema and point bleedings.

Diagnostic tests

MHCT. Heparinized blood was collected into a capillary tube (7561.5 mm), which
was then sealed and centrifuged at 3000g for 10 min. The bu¡y-coat area was examined
under the microscope at 100^2006 magni¢cation) (Van Meirvenne, 1999).

MI. Heparinized blood (0.25^0.5 ml) was inoculated intraperitoneally into laboratory-
bred white mice. The inoculated mice were tail-bled and a wet blood ¢lm was prepared
and examined. Mice were checked 2^3 times over a period of 4 weeks.

CATT. The CATT/T. evansi is a rapid direct agglutination test that uses freeze-dried
trypanosomes of T. evansi VAT RoTat 1.2 ¢xed with formaldehyde and stained with
Coomassie blue (Bayjana Songa and Hamers, 1988). Testing started at a dilution of 1:4
and, if a test proved positive, additional twofold serum dilutions were tested to obtain
the ¢nal antibody titre.

LATEX. LATEX/T. evansi (Verloo et al., 1998) is a rapid, indirect agglutination test in
which the antigen consists of puri¢ed variable surface glycoprotein (VSG) of T. evansi
VAT RoTat 1.2 covalently coupled to latex particles. LATEX testing started at dilution
1:4 and, if a positive result was obtained, additional twofold serum dilutions were
tested to obtain the ¢nal antibody titre.

Immune trypanolysis. Immune trypanolysis (TL) was performed according to Van

Meirvenne and colleagues (1995) with T. evansi VAT RoTat 1.2. The sera were tested
at 1:4 dilution.
460

ELISA. The ELISA/T. evansi assay was a modi¢cation of the technique described by
Verloo and colleagues (2000). For coating, puri¢ed VSG antigen of T. evansi RoTat 1.2
was diluted in PBS (pH 7.2) to a concentration of 2 mg/ml and 150 ml was added to each
well. After overnight coating at 48C, the plates were blotted for 2 h with PBS-blotto and
control and test sera were diluted 1:400 in PBS-blotto. After incubation and subsequent
washing, rabbit anti-pig IgG peroxidase conjugate (Sigma, A-5670) diluted 1:15 000 in
PBS-Tween was added. After incubation and several washing steps, ABTS was added as
a substrate. After 45 min incubation at room temperature, the plate was read
photometrically at 415 nm. The OD of each sample tested was expressed as a
percentage of a reference positive sample to give a uniform and continuous scale of
percentage positivity (PP). Test parameters were calculated with a cut-o¡ value of PP =
22.

Statistics
Experimental infections. For each weekly sampling round, the number of positive
animals per test was determined for the infected group and the number of negative
animals for the non-infected group.

Field samples. In the absence of a reference standard, test results of CATT, LATEX
and ELISA were related to TL (Verloo et al., 2000). Concordance with the results of TL
was determined by calculating the fraction of samples within the TL-positive and TL-
negative groups that were positive in CATT/T. evansi, LATEX/T. evansi and ELISA/T.
evansi.

RESULTS

Experimental infections
Parasitological results. In total, 168 samples from 14 T. evansi-infected pigs were
examined over a 12-week period. One hundred and sixteen samples were con¢rmed
positive, 86 by MHCT, and of the 82 samples negative by MHCT, 30 were found
positive on MI. All 14 infected pigs were parasitologically positive by 2^3 weeks PI.
Afterwards, the number of positive animals decreased to 5^7 animals at weeks 9 to 12
(Figure 1). No clinical signs of disease were observed during the study.

Serological results. All samples from the infected pigs, together with 84 samples
collected from non-infected animals, were tested using the four serological assays. In
the ¢rst week PI, all infected animals tested positive with CATT/T. evansi and
LATEX/T. evansi at a dilution 1:4 and in the ELISA/T. evansi. For both
agglutination tests at a dilution of 1:4, the numbers of seropositive animals for each
sampling round were similar throughout the 12-week period, with the number of
seropositive animals varying between 10 and 14. The serum antibody titres by CATT/
LATEX declined from week 3 to week 12 (Tables I and II).
 461

Figure 1. Number of T. evansi-infected pigs (n = 14) that were parasitologically positive for
MHCT and MI when analysed on a weekly basis. MI was performed only on samples that were
negative in the MHCT

One pig was detected positive at week 2 by TL, while the last infected animal was
con¢rmed positive with TL by week 4. Once the TL was positive for an animal, it
remained positive for the duration of the experiment.
In the non-infected group, tests were mostly negative, although CATT and LATEX
gave positive results at dilution of 1:4. For the TL and the ELISA/T. evansi (cut-o¡ PP
22), all samples tested negative.

Field samples
Thailand. Of the 77 samples collected from breeding stock, two animals from one farm
were found positive by MHCT and 28 adult animals were seropositive, with the TL
indicating presence of anti-RoTat 1.2 antibodies. Each of the ¢ve farms contained
seropositive animals. The ¢ve parasitologically postive animals were seropositive by
each serological assay. To evaluate the performance of the tests, concordance between
the TL and the other assays was assessed. At a dilution of 1:4, the proportions of
CATT/T. evansi and LATEX/T. evansi negative results within the TL-negative groups
were 63% and 59% respectively (Table III). At higher serum dilutions, these
proportions increased, but the proportion of positive results within the TL-positive
group, especially for the LATEX/T. evansi test, dropped dramatically. The percentage
of ELISA-negative animals within TL-negative group at the earlier calculated PP = 17
was 57%. Shifting the PP to 22 increased this percentage to 98%, maintaining the 100%
correlation with the TL-positive samples.
462

TABLE I
Number of seropositive animals for the CATT/T. evansi and ELISA/T. evansi at di¡erent
dilutions and cut-o¡s in the infected group (n = 14) and number of seronegative animals for
these tests in the non-infected group (n = 7)

Number of seropositive animals Number of sero-negative animals

in infected group in non-infected group

CATT ELISA CATT ELISA

ööööööööö öööööö ööööööööö öööööö
Week 1:4 1:8 1:16 PP 17 PP 22 1:4 1:8 1:16 PP 17 PP 22

1 14 14 14 14 13 14 14 14 14 14
2 14 14 14 14 14 14 14 14 14 14
3 14 14 12 14 13 12 14 14 14 14
4 14 10 6 14 13 14 14 14 14 14
5 13 10 4 14 13 14 14 14 14 14
6 13 1 2 14 12 12 12 12 14 14
7 10 4 0 14 14 14 14 14 14 14
8 13 2 1 14 14 12 14 14 14 14
9 14 4 0 14 14 12 14 14 14 14
10 12 3 1 14 14 8 14 14 14 14
11 13 6 1 14 14 12 14 14 14 14
12 14 9 4 14 14 6 10 14 14 14

Vietnam. In the 437 pig sera tested, no positive TL sample was found, indicating
absence of anti-RoTat 1.2 antibodies. For both CATT/T. evansi and LATEX/T. evansi,
large numbers of apparently positive results were found at 1:8 and especially at 1:4
dilution. As with the samples originating from Thailand, raising the PP in the ELISA/
T. evansi from 17 to 22 increased the correlation with the negative samples of the TL
test from 89% to 97%.

DISCUSSION

The main objective of this study was to evaluate the usefulness of established
diagnostic tests for the diagnosis of T. evansi in pigs. Several of these serological tests
have been used successfully in water bu¡alo (Davison et al., 1999; Holland et al., 2002)
and camels (Dia et al., 1997). Except for limited ¢eld trials with CATT/T. evansi
(Bajyana Songa and Hamers, 1988; Reid and Copeman, 2000), none of these antibody
assays has been evaluated in pigs.
 463

TABLE II
Number of seropositive animals for the LATEX/T. evansi and TL at di¡erent dilutions in the
infected group (n = 14) and number of seronegative animals for these tests in the non-infected
group (n = 7)

Number of seropositive Number of seronegative in

animals in infected group animals non-infected group
ööööööööööööö ööööööööööööö
LATEX LATEX
ööööööööööööö ööööööööööööö
Week 1:4 1:8 1:16 TL 1:4 1:8 1:16 TL

1 14 14 14 0 14 14 14 14
2 14 14 14 2 14 14 14 14
3 14 14 10 11 12 14 14 14
4 10 8 5 14 14 14 14 14
5 10 9 5 14 14 14 14 14
6 13 12 3 14 12 12 12 14
7 12 9 1 14 12 12 12 14
8 13 4 1 14 12 14 14 14
9 14 5 1 14 14 14 14 14
10 14 5 1 14 6 10 14 14
11 14 8 2 14 10 12 14 14
12 14 8 4 14 6 8 12 14

TABLE III
Percentage of positive and negative animals in the CATT, LATEX and ELISA at di¡erent
dilutions or cut-o¡ levels within the groups of RoTat 1.2 TL positives and negatives

CATT LATEX ELISA

öööööööööö öööööööööö ööööööö
1:4 1:8 1:16 1:4 1:8 1:16 PP 17 PP 22

Thailand
TL-negative 63 93 100 59 96 100 57 98
TL-positive 100 97 70 97 57 20 100 100

Vietnam
TL-negative 30 78 95 48 88 97 89 97
TL-positive 0 0 0 0 0 0 0 0
464

All experimentally infected pigs were con¢rmed parasitologically positive within the
¢rst two weeks after infection. At the end of the study, fewer peaks of parasitaemia
could be detected, and it appeared that the animals were capable of controlling the
parasitaemia once the infection progressed (Reid et al., 1999). In these young pigs, no
clinical symptoms were observed, which agrees with previous observations in Thailand
(Teeraprasert et al., 1984a).
During the experimental period, the TL performed as anticipated, with all animals
testing positive within 4 weeks after infection and no positive reactions in the non-
infected group. On the basis of these results and earlier studies (Van Meirvenne et al.,
1995; Verloo et al., 2000), it was concluded that the TL is a suitable reference antibody
test for the detection of anti-RoTat 1.2 antibodies in pigs.
All animals became serologically positive by CATT, LATEX and ELISA within the
¢rst week. Despite absence of correlation during the ¢rst weeks, overall results of the
ELISA/T. evansi at cut-o¡ PP = 22 correlated well with the TL, suggesting that the
ELISA is a suitable test to con¢rm infection status.
Such early reactions in agglutination assays were also observed using the same
trypanosome isolate in experimentally infected bu¡aloes (Holland et al., 2002). Early
reactions were also found with the CATT LiTat 1.3 in pigs, a test normally used for
serodiagnosis of T. brucei gambiense in humans (Noireau, 1991; P. Bu«scher, 2000,
personal communication). It further appeared that at the end of the experiment the
CATT/T. evansi and LATEX/T. evansi lost their ability to test non-infected animals as
seronegative, as well as their capacity to test infected animals as positive. Dilution of
sera to 1:8 and 1:16 resulted in a moderate improvement to test non-infected animals
seronegative, but meanwhile it drastically decreased the ability to test infected animals
positive. It appeared that the CATT/T. evansi and LATEX/T. evansi, had limited value
for serodiagnosis as their sensitivity and speci¢city decreased with time after infection.
With the TL as a reference test, two surveys were conducted in pigs in Vietnam and
Thailand. In the Vietnam study, no pigs were detected positive by TL, despite the fact
that outbreaks of surra in bu¡alo in these areas had been reported and con¢rmed by
parasitological testing (unpublished observations) and most samples were collected at
the end of the rainy season, the season with highest transmission rates (Luckins, 1988).
The absence of any infected pigs in this endemic area is remarkable since animals are
housed close together and it could be speculated that T. evansi strains originating from
bu¡alo and cattle are not able to infect pigs. It is also possible that since smallholders
keep their pigs in dark half-open sheds, tabanidae, which prefer to feed in bright
sunlight, are prevented from biting them.
In Thailand, T. evansi serologically positive sows were detected on each farm.
Although all farms reported reproductive problems such as stillbirth and abortion, it
is unclear whether T. evansi infection accounted for these problems, although the
observed fertility problems disappeared when all sows were treated with diminazene
aceturate (S. Sangmaneedet, 2000, personal communication). Experimentally infected
sows at 1^2 months of pregnancy aborted approximately 4 days after infection
(Teeraprasert et al., 1984b). In the same areas there have been reports of abortion in
water bu¡alo induced by T. evansi infection (Lohr et al., 1986).
 465

If a cut-o¡ of PP = 22 was used on the samples of the experimentally infected pigs

and on the samples from both ¢eld surveys, it appeared that there was a good
correlation between results obtained by T.L and the ELISA/T. evansi.
In conclusion, this study indicates that agglutination assays are not suitable for the
serological diagnosis of surra in pigs. In contrast, the ELISA/T. evansi appears to be
suitable for epidemiological surveys, although more samples need to be tested to
increase the con¢dence in this test. Finally, the absence of anti-RoTat 1.2 antibodies
in pigs sampled in northern Vietnam indicates that T. evansi is not a problem in the
areas examined.

ACKNOWLEDGEMENTS
The study was carried out within the framework of the Vietnamese-Belgium project
`Strengthening the National Institute of Veterinary Research in Central- and North
Vietnam' (University of Ghent-Flemish Interuniversity Council) at the National
Institute of Veterinary Research, Hanoi, Vietnam.

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(Accepted: 13 January 2005)

Ëvaluation des tests de diagnostic du Trypanosoma evansi chez des porcs expërimentalement infestës et usage
subsëquent dans des enqueªtes faites sur le terrain dans le Nord du Vietnam et en Tha|« lande

Rësumë ^ L'ëtude est concernëe par l'ëvaluation de tests de diagnostic ëtablis pour le diagnostic de
Trypanosoma evansi chez des porcs. Le test de la trypanolyse immunitaire (TL), le test d'agglutination par
cartes (CATT), le test d'agglutination au latex (LATEX), le dosage d'immunocaptation lië aux enzymes
(ELISA), la technique de centrifugation des microhëmatocrites (MHCT) et les tests d'inoculation de souris
(MI) ont initialement ëtë ëvaluës sur des porcs d'engraissement expërimentalement infectës. Tous les porcs
infectës ont ëtë con¢rmës comme ëtant parasitologiquement positifs a© la fois a© la MHCT et au MI. Les
rësultats des dosages sërologiques ont indiquë que le test TL pourrait eªtre un test de rëfërence pour la
prësence d'anticorps RoTat 1.2 chez les cochons. Les rësultats des tests CATT et LATEX n'ont pas ëtë
compatibles avec les rësultats du test TL tandis que les rësultats du test ELISA ont correspondu aux
rësultats du test TL. Les quatre dosages sërologiques ont ensuite ëtë utilisës dans deux enqueªtes rëalisëes
sur le terrain au Vietnam et en Tha|« lande. Les rësultats des deux dosages d'agglutination (CATT et
LATEX) n'ont pas ëtë concordants et n'ont pas eu de corrëlation avec les rësultats du test TL. Le test
ELISA a© une positivitë de pourcentage de 22 a semblë avoir une bonne capacitë de discrimination entre les
animaux sëropositifs et sëronëgatifs. Sur les 437 ëchantillons recueillis dans les petites fermes d'ëlevages de
porcs dans le Nord du Vietnam, aucun porc positif n'a ëtë dëtectë avec le test TL. En Tha|« lande, 77
ëchantillons ont ëtë recueillis dans cinq fermes de mise a© bas ayant des antëcëdents de surra. Deux vaches
parasitologiquement positives ont ëtë retrouvëes et dans chaque ferme, des vaches sëropositives ont ëtë
dëtectëes.
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Evaluaciön de pruebas diagnösticas para Trypanosoma evansi en cerdos infectados experimentalmente y

utilizaciön subsiguiente en estudios de campo de Vietnam del Norte y Tailandia

Resumen ^ Este estudio centra su preocupaciön en la evaluaciön de pruebas diagnösticas establecidas para
el diagnöstico de Trypanosoma evansi en cerdos. La prueba inmune de tripanölisis (TL, en inglës), la prueba
de aglutinaciön en tarjeta (CATT), la prueba de aglutinaciön en lätex (LATEX), la prueba de
inmunoabsorciön enzimätica (ELISA), la tëcnica de centrifugaciön microhematöcrita (MHCT) y las
pruebas de inoculaciön en ratön (MI) fueron evaluadas inicialmente en cerdos de engorde infectados
experimentalmente. Se con¢rmö que todos los cerdos infectados eran positivos parasitolögicamente para
MHCT y MI. Los resultados de las pruebas serolögicas indicaban que la prueba de tripanölisis (TL) pod|¨ a
ser una prueba de referencia para detectar la presencia de anticuerpos RoTat 1.2 en cerdos. Los resultados
de las pruebas CATT y LATEX no fueron consistentes con los de TL, mientras que los resultados de
ELISA aparec|¨ an correlacionados con los resultados TL. Los cuatro ensayos serolögicos fueron
subsiguientemente utilizados en dos estudios de campo de Vietnam y Tailandia. Los resultados de las dos
pruebas de aglutinaciön (CATT y LATEX) no fueron consistentes y no se correlacionaban con los
resultados de TL. El ensayo ELISA con porcentaje de positividad de 22 parec|¨ a discriminar bien entre
animales seropositivos y seronegativos. De las 437 muestras recogidas en pequen¬as granjas de cerdos del
norte de Vietnam, no se detectaron cerdos positivos con la prueba de TL. En Tailandia, se recogieron 77
muestras de cinco granjas de cerdos con historia de surra. Se encontraron dos cerdas parasitolögicamente
positivas y en cada granja se detectaron cerdas seropositivas.