CHAPTER
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2.2.18.
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TRYPANOSOMA EVANSI INFECTIONS
(INCLUDING SURRA)
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SUMMARY
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Definition of the disease: Trypanosoma evansi causes a disease known as Trypanosomosis 1
(‘surra’). It affects a number of domesticated animals species in Asia, Africa and Central and South
America. The principal host species affected varies geographically, but buffalo, cattle, camels and
horses are particularly affected, although other animals, including wildlife, are also susceptible. It is
an arthropod-borne disease and several species of haematophagous flies, including Tabanus spp.
and Musca spp., are implicated as vectors.
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Description of the disease: The disease in susceptible animals is manifested by pyrexia, directly
associated with parasitaemia, together with a progressive anaemia, loss of condition and lassitude.
Recurrent episodes of fever and parasitaemia occur during the course of the disease. Oedema,
particularly of the lower parts of the body, urticarial plaques and petechial haemorrhages of the
serous membranes are often observed. Abortions have been reported in buffaloes and camels.
There are indications that the disease causes immunodeficiencies.
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Identification of the agent: The general clinical signs of T. evansi infection are not sufficiently
pathognomonic for diagnosis and laboratory methods for detecting the parasite are required.
Examination of the host blood is problematic as trypanosomes can be detected only when there is
a high parasitaemia. Under these circumstances examination of wet blood films, stained blood
smears or lymph node material might reveal the trypanosomes. In other, more chronic cases, such
as the carrier state, the examination of thick blood smears, as well as methods of parasite
concentration and the inoculation of laboratory animals, are required.
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Serological tests: Infection gives rise to specific antibody responses and a variety of antibody
detection tests have been introduced for laboratory use. Some have been partially validated, but
await large-scale evaluation and standardisation. Among those that are used regularly are
immunoenzyme assays, card agglutination tests and latex agglutination tests. For field use,
relatively simple tests have yet to be developed although obvious candidates might be indirect
agglutination tests using antigen-coated latex particles. Assays for detection of circulating
antibodies have high measures of validity. Estimates of predictive values of different serological
tests indicate that enzyme linked immunosorbent assays (ELISA) for detecting IgG antibodies are
more likely to classify correctly uninfected animals, and card agglutination tests (CATT) are more
likely to classify correctly truly infected animals. An IgG ELISA would thus be suitable for verifying
that animals are free from infection, prior to movement or during quarantine. In situations where
there is overt disease, CATTs can be used to target individual animals for treatment with
trypanocidal drugs. For declaring a disease-free status, serial testing – ELISA followed by retesting of suspect samples by CATT – is recommended. It must be stressed however, that there
are considerable antigenic similarities among the different species of pathogenic trypanosomes,
hence in areas where tsetse-transmitted trypanosomoses occur cross-reactions will occur with any
serological test employed.
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Requirements for vaccines and diagnostic biologicals: No vaccines are available for the
disease.
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Nomenclature of parasitic diseases: see the note in Chapter. 2.3.17. Trypanosomosis (Tsetse-borne).
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Chapter 2.2.18. – Trypanosoma evansi infections (including surra)
A.
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INTRODUCTION
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The clinical signs of surra, the disease caused by Trypansoma evansi, are indicative but are not sufficiently
pathognomonic and diagnosis must be confirmed by laboratory methods. The disease in susceptible animals,
including cattle, buffalo, camels (dromedary and bactrian), horses, pigs, sheep and goats, is manifested by
pyrexia, directly associated with parasitaemia, together with a progressive anaemia, loss of condition and
lassitude. Recurrent episodes of fever and parasitaemia occur during the course of the disease. Oedema,
particularly of the lower parts of the body, urticarial plaques and petechial haemorrhages of the serous
membranes are often observed. Abortions have been reported in buffaloes and camels (7, 15) and there are
indications that the disease causes immunodeficiency (4, 19).
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There is considerable variation in the pathogenicity of different strains and the susceptibility of different host
species to disease. Disease may manifest as an acute or chronic condition, and in the latter case may persist for
many months. The disease is often rapidly fatal in camels, buffaloes, horses, cattle, llama and dogs, but mild and
subclinical infections can also occur in these species. Wild animals such as deer and capybara can become
infected. Animals subjected to stress – malnutrition, pregnancy, work – are more susceptible to disease.
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Biologically T. evansi is very similar to T. equiperdum, the causative agent of dourine (2), and morphologically
resembles the slender forms of the tsetse-transmitted trypanosomes, T. brucei, T. gambiense and
T. rhodesiense. Molecular characterisation indicates that various strains of T. evansi isolated form Asia, Africa
and South America have a single origin. Moreover, it is also likely that T. evansi and T. equiperdum belong to the
same species. Like all pathogenic trypanosomes, T. evansi is covered by a dense protein layer consisting of a
single protein called the variable surface glycoprotein. This acts as a major immunogen and elicits the formation
of specific antibodies. The parasites are able to evade the consequences of these immune reactions by switching
the variant surface glycoprotein, the phenomenon known as antigenic variation .
B.
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DIAGNOSTIC TECHNIQUES
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1. Identification of the agent
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The classical direct parasitological methods for the diagnosis of trypanosomosis, namely examining blood or
lymph node material, are not highly sensitive. In regions where other Trypanozoon spp. occur in addition to
T. evansi, specific identification by microscopy is not possible. Specific DNA probes (17, 22) may enable
identification of trypanosome species by nonradioactive DNA hybridisation.
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a) Usual field methods
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Direct methods
i)
Blood sampling
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Trypansoma evansi is a parasite of the blood and tissues often inhabiting the deep blood vessels in
cases of low parasitaemia. For this reason, it is recommended that blood for diagnosis be obtained
from both the peripheral and deep blood vessels. However it should be realised that less than 50% of
infected animals may be identified by examination of peripheral blood.
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Peripheral blood is obtained by puncturing a small vein in the ear or tail. Deeper samples are taken
from a larger vein by syringe. Cleanse an area of the ear margin or tip of the tail with alcohol and,
when dry, puncture a vein is with a suitable instrument. Ensure that instruments are sterilised or
disposable instruments are used between individual animals, so that infection cannot be transmitted
by residual blood.
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ii)
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Wet blood films
Place a small drop of blood on to a clean glass slide and cover with a cover-slip to spread the blood as
a monolayer of cells. Examine by light microscopy (×200) to detect any motile trypanosomes.
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iii)
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Stained thick smears
Place a large drop of blood on the centre of a microscope slide and spread with a toothpick or the
corner of another slide so that an area of approximately 1.0–1.25 cm in diameter is covered. Air-dry for
1 hour or longer, while protecting the slide from insects. Stain the unfixed smear with Giemsa’s Stain
(one drop of commercial Giemsa + 1 ml of phosphate buffered saline [PBS, 2.4 g Na2HPO4.2H2O,
0.54 g NaH2PO4.2H2O, 0.34 g NaCl], pH 7.2), for 25 minutes. After washing, examine the smears by
light microscopy at high magnification (×500–1000). The advantage of the thick smear technique is
that it concentrates the drop of blood into a small area, and thus less time is required to detect the
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parasites. The disadvantage is that the trypanosomes may be damaged in the process, and the
method is therefore not suited for species identification in case of mixed infections.
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iv)
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Place a drop of blood 20 mm from one end of a clean microscope slide and draw out a thin film in the
usual way. Air-dry briefly and fix in methyl alcohol for 2 minutes and allow to dry. Stain the smears in
Giemsa (one drop Giemsa + 1 ml PBS, pH 7.2) for 25 minutes. Pour off, stain and wash the slide in
tap water and dry. Unfixed smears can be stained by covering them with May–Grünwald stain for
2 minutes, then adding an equal volume of PBS, pH 7.2, and leaving the slides for a further 3 minutes.
Pour off and add diluted Giemsa for 25 minutes. Pour off, wash the slides with tap water, and dry.
Examine at high magnification (×400–1000). This technique permits detailed morphological studies
and identification of the trypanosome species. Rapid staining techniques also exist (Field’s stain, Diff
Quick®).
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v)
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Stained thin smears
Lymph node biopsies
Samples are usually obtained from the prescapular or precrural (subiliac) lymph nodes. Select a
suitable node by palpation and cleanse the site with alcohol. Puncture the node with a suitable gauge
needle, and draw lymph node material into a syringe attached to the needle. Expel lymph on to a slide,
cover with a cover-slip and examine as for the fresh blood preparations. Fixed thin or thick smears can
also be stored for later examination.
b) Concentration methods
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In most hosts T. evansi can induce mild clinical or subclinical carrier state infections with low parasitaemia
in which it is difficult to demonstrate the parasites. In these circumstances, concentration methods become
necessary.
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i)
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Haematocrit centrifugation
Collect blood (70 µl) into at least two heparinised capillary tubes (75 × 1.5 mm). Seal at the dry end by
heat and centrifuge, sealed end down, at 3000 g for 10 minutes. Place the capillary tube between two
pieces of glass (25 × 10 × 1.2 mm) glued to a slide. Place a cover-slip on top at the level of the buffy
coat junction where the trypanosomes will be concentrated. Flood the space around this part of the
tube with water or immersion oil, and examine the buffy coat area under the microscope (×100–200).
A simpler alternative is to examine the centrifuged capillary tube by placing a drop of immersion oil on
the tube and ensuring that there is contact between the objective lens and the immersion oil.
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ii)
Dark-ground/phase-contrast buffy coat technique
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Collect blood into heparinised capillary tubes and centrifuge as above. Scratch the tube with a glasscutting diamond and break it 1 mm below the buffy coat layer – the upper part thus contains the top
layer of red blood cells (RBCs), the buffy coat (white blood cells) and some plasma.
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Partially expel the contents of this piece on to a slide, cover with a cover-slip and examine under darkground, phase-contrast or ordinary illumination.
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As an alternative to the electrically powered haematocrit centrifuge, hand-powered micro-centrifuges
have been used successfully for detection of trypanosomosis in cattle and camels (8, 13).
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iii)
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Haemolysis techniques
Sodium dodecyl sulphate (SDS) can be used as a reagent to haemolyse RBCs to facilitate detection of
motile trypanosomes in parasitised blood samples. As SDS is toxic, contact with skin, inhalation and
ingestion should be avoided. SDS solution can be stored for several months at ambient temperature.
Both the SDS solution and the blood samples should be used at a temperature above 15°C. At lower
temperatures the trypanosomes may be destroyed.
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Two general procedures, namely wet blood film clarification and haemolysis centrifugation, can be used 2.
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Wet blood film clarification method
This method uses the partial lysis of RBCs to facilitate the detection of motile trypanosomes. The
method requires an SDS solution: 1% SDS dissolved in Tris/glucose/saline, pH 7.5, inoculating loops
(10 µl), slides and cover-slips (24 × 24 mm), and a drop of fresh neat or heparinised blood. Dissolve
100 mg of SDS in 100 ml of isotonic Tris/NaCl/glucose buffer, pH 7.5 (Trizma base 14.0 g, NaCl 3.8 g,
2
All necessary materials and instructions can be obtained from the Institute of Tropical Medicine, Laboratory of Serology,
Nationalestraat 155, B-2000 Antwerp, Belgium.
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glucose 10.8 g; dissolve chemicals in 750 ml distilled H2O, add 90–100 ml 1 N HCl and adjust pH to
7.5 then make up to final volume of 1000 ml with distilled H2O). This buffer can be stored in small vials
at ambient temperature for several months. The test does not give a significantly higher sensitivity
than wet film technique. In addition, the SDS can cause problems when focusing the microscope and
the movements of trypanosomes can be severely curtailed owing to the high viscosity of the SDS.
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Put approximately 10 µl of blood on a slide. Add 10 µl of SDS solution using a dip inoculation loop and
mix gently. Apply a cover-slip and examine the preparation without delay at low magnification (×100 or
×200).
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Haemolysis centrifugation technique
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Nearly complete lysis of RBCs is required for this procedure. The materials needed include: SDS
solution (0.1% SDS dissolved in Tris/glucose/saline, pH 7.5), conical centrifuge tubes, ordinary test
tubes, large and fine plastic tapering pipettes with attached bulb, slides, cover-slips (24 × 24 mm or
24 × 32 mm), and heparinised blood.
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Using a pipette or syringe, transfer nine volumes (maximum 6.3 ml) of SDS solution into an ordinary
test tube. Aspirate one volume (maximum 0.7 ml) of heparinised blood into a bulb pipette and expel it
just above the surface of the SDS solution; mix quickly and thoroughly. Avoid foam formation, which
may result in destruction of the trypanosomes. Wait for 10 minutes so as to achieve complete
haemolysis.
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Pour the haemolysed suspension into a conical centrifuge tube and spin at approximately 500 g for
10 minutes. With a clean bulb pipette, remove as much supernatant as possible without disturbing the
sediment. Using a fine tapering bulb pipette, remove more supernatant, leaving 10–20 µl of
undisturbed sediment at the bottom.
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Very carefully collect the entire sediment and put on to a microscope slide. Apply a cover-slip and
examine the preparation without delay at low magnification (×100 or ×200).
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iv)
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Mini-anion exchange centrifugation technique
When a blood sample from animals infected with salivarian trypanosomes is passed through an
appropriate anion-exchange column, the host blood cells, being more negatively charged than
trypanosomes, are adsorbed onto the anion-exchanger, while the trypanosomes are eluted, retaining
viability and infectivity. A simplified field method for detection of low parasitaemia has been developed
(24). The sensitivity of this technique can be increased by approximately tenfold by the use of buffy
coat preparations rather than whole blood (23).
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Preparation of phosphate buffered saline glucose, pH 8
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Na2HPO4 (anhydrous) (13.48 g); NaH2PO4.2H2O (0.78 g); NaCl (4.25 g); distilled water (1 litre).
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Solutions of different ionic strength are made by diluting the stock PBS, pH 8, and adding glucose to
maintain a suitable concentration. For blood of mice, domestic and wild ruminants and dog, add four
parts PBS to six parts distilled water and adjust the final glucose concentration to 1%. For blood of
pigs and rabbits, add three parts PBS to seven parts distilled water and adjust the final glucose
concentration to 1.5%. The PBS/glucose solution (PSG) must be sterile.
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Equilibration of DEAE-cellulose
Suspend 500 g of DEAE-cellulose (diethylamino-ethylcellulose) in 2 litres of distilled water. Adjust the
pH to 8 with phosphoric acid. Allow to settle for 30 minutes. Discard the supernatant fluid containing
the fine granules. Repeat the procedure three times. Store the equilibrated concentrated suspension
of DEAE-cellulose (slurry) at 4°C or in small aliquots at –20°C.
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Packing of equilibrated DEAE-cellulose
Place a 2 ml syringe without the plunger on a test-tube rack complete with needle (20 G × 1.5 inch).
Put a disc of Whatman No. 41 filter paper at the bottom of the syringe and moisten by adding a few
drops of PSG. Pour 2–2.5 ml of the slurry of equilibrated cellulose into the syringe and allow to pack
by elution of the buffer. The height of the sediment should be approximately 3 cm. Wash and
equilibrate the column with 2 ml of PSG without disturbing the surface.
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Adsorption of blood eluate of the trypanosomes
Gently place 100–300 µl of heparinised blood above the surface of the cellulose column.Add ten drops
of PSG and discard the first ten drops of the eluate. Progressively add 1.5 ml of PSG and start
collecting the eluate into a finely tapered Pasteur pipette with a sealed end. Put the filled pipette,
protected by a conical plastic pipette tip, in a tube and centrifuge at 525 g (or up to 1000 g) for
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10 minutes. Examine the bottom of the pipette under the microscope (×100 or ×200) using a special
mounting device. Alternatively, the eluate could be collected into 50 ml plastic tubes, with conical
bottoms, centrifuged at 1000 g and the sediment examined by dark-ground microscopy.
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The cellulose column should remain wet throughout the procedure.
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c) Animal inoculation
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Laboratory animals may be used to reveal subclinical (nonpatent) infections in domesticated animals.
Trypanosoma evansi has a broad spectrum of infectivity for small rodents, and so rats and mice are often
used. Rodent inoculation is not 100% sensitive (16) but further improvement in its efficacy can be obtained
by the use of buffy coat material. Such a procedure was able to detect as few as 1.25 T. evansi/ ml blood
(23).
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Inoculate heparinised blood intraperitoneally into rats (1–2 ml) or mice (0.25–0.5 ml). Inoculate a minimum
of two animals. Bleed animals from the tail three times a week to detect parasitaemia. The incubation period
before appearance of the parasites and their virulence depends on the strain of trypanosomes, their
concentration in the inoculum, and the strain of laboratory animal used. Sensitivity of this in-vivo culture
system may perhaps be increased by use of immunosuppressed laboratory animals. Drugs such as
cyclophosphamide or hydrocortisone acetate, or X-ray irradiation or splenectomy are used for this purpose.
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d) Recombinant DNA probes
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Specific DNA probes have been used to detect trypanosomes in infected blood or tissue but are not
routinely applied (26, 28). Although molecular methods have a potentially high analytical sensitivity there
have been few convincing studies to critically evaluate the diagnostic sensitivity of these tests compared
with other techniques, such as serology.
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Indirect methods
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These methods involve tests that demonstrate the effects of the parasite on its host rather than directly detecting
the parasite itself.
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a) Haematology
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Anaemia is usually a reliable indicator of trypanosome infection, although it is not in itself pathognomonic.
However, animals with a mild subclinical infection can have parasitaemia without evidence of anaemia (3).
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Anaemia can be estimated by measuring the packed cell volume and may be used in surveys of herds at
risk. The technique is identical to that of haematocrit centrifugation. The capillary tube is examined and the
results are expressed as a percentage of packed RBCs to total blood volume.
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b) Detection of trypanosomal DNA
Detection of minute amounts of trypanosomal DNA using a polymerase chain reaction (PCR) procedure is a
possible means of identifying animals with active infections, and could have the sensitivity and specificity
required (18, 29). Experimental studies in buffalo (9) showed the diagnostic sensitivity of a PCR was only
78%, similar to that of mouse inoculation.
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2. Serological tests
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Historically, many different methods have been used to detect specific humoral antibodies to trypanosomal
antigens but, more recently, there has been a tendency to concentrate on more easily standardised techniques
such as ELISA (5, 6, 11, 20, 21, 22, 25) card agglutination tests (CATT) (1, 18, 22) and latex agglutination tests
(LAT) (10, 14). Extensive evaluation of ELISA and CATT has been carried out in buffaloes in Indonesia and
Vietnam (5, 10, 27). The collection of samples can be simplified by using filter paper blood spots for later use in
the ELISA, while for the CATT whole blood can be substituted for serum (10). Other innovative modifications that
might be developed in the future are the use of a colloidal-dye dipstick test (12) that could enable the tests to be
carried out under field conditions. It is vitally important that serological tests are validated and standardised if
they are to be suitable for correctly identifying infected animals. This means that standard criteria for interpreting
the tests might have to be developed for each animal species as well as each laboratory operating the
procedure.
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a) Enzyme-linked immunosorbent assay
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The principle of this technique is that specific antibodies to trypanosomes can be detected by enzymelinked anti-immunoglobulins using solid-phase polystyrene plates coated with soluble antigen. The enzyme
may be peroxidase, alkaline phosphatase or any other suitable enzyme. The enzyme conjugate binds to the
antigen/antibody complex and then reacts with a suitable substrate to yield a characteristic colour change
either of the substrate itself or of an added indicator (the chromogen).
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The antigen for coating the plates is derived from the blood of a heavily parasitaemic rat. The trypanosomes
are separated on a DEAE-cellulose column and washed three times by centrifugation in cold PSG, pH 8
(PBS with 1% glucose). The final pellet is suspended in cold PSG to a concentration of 3–5%, and briefly
ultrasonicated on ice for 30–120 seconds until disintegration of the organisms is complete. This preparation
is centrifuged at 4°C and 40,000 g for 60 minutes. The supernatant is diluted in water so as to obtain a
protein concentration of 1 mg/ml. The reagent thus obtained can be stored in small aliquots at –70°C for
several months. It can also be freeze-dried and stored at –20°C. Various treatments of the antigen
preparations have been applied to improve the accuracy of antibody detection (29).
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Test procedure
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i)
Dilute or reconstitute the frozen or freeze-dried antigen with freshly prepared 0.01 M
carbonate/bicarbonate buffer, pH 9.6. Add 100 µl to each well of a 96-well microtitre plate and
incubate overnight at 4°C or for 1 hour at 37°C.
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ii)
Remove excess antigen by are wash plate with 0.01 M PBS containing 0.05% Tween 20 (PBST). Add
test serum dilutions in PBST (100 µl). Include control negative and positive sera. Dilutions must be
determined empirically, but are usually between 1/100 and 1/1000.
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iii)
Incubate plates at 37°C for 30 minutes. Eject contents and wash three times with PBST.
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iv)
Add a specific peroxidase conjugated species-specific anti-globulin (100 µl) appropriately diluted in
PBST (usually between 1/1000 and 1/50,000).
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v)
Incubate the plates at 37°C for 30 minutes, eject contents and wash three times with PBST.
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vi)
For peroxidase conjugates a number of substrate/chromogen solutions can be used, consisting of
hydrogen peroxide with a chromogen, such as tetramethylbenzidene (TMB), 2,2’-azino-bis-(3ethylbenzothiazoline-6-sulphonic acid) (ABTS) and ortho-diphenylenediamine (OPD). A suitable
substrate/chromogen solution for peroxidase conjugates is 30% hydrogen peroxide (0.167 ml and
35 mg) in citrate buffer (100 ml), pH 6.0. The citrate buffer is made up as follows: Solution A
(36.85 ml): (0.1 M citric acid [21.01 g/litre]); Solution B (65.15 ml): (0.2 M, Na2HPO4 [35.59 g/litre]);
and distilled water (100 ml). Dissolve 10 mg TMB in 1 ml dimethyl suphoxide and add to 99 ml of the
citrate buffer. A number of these combinations are available commercially in ready-to-use formulations
that remain stable at 4°C for up to 1 year.
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vii)
Add the substrate chromogen (100 µl) to the plates and incubate at room temperature for 15–
20 minutes.
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viii) Stop the reaction by adding 50 µl 1 M sulphuric acid. Read the absorbance of each well at 450 nm for
TMB chromogen. Other chromogens may require the use of a different wavelength. All tests should
include known high and medium positive control sera, a negative control serum, and a buffer control.
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A large variety of other test procedures exists. For closely related animal species, cross-reacting reagents
may often be used (e.g. anti-bovine immunoglobulin for buffaloes and the use of monospecific anti-IgG
conjugates is generally recommended. There are a number of methods that can be used to determine a
cut-off point to discriminate between positive and negative results. The simplest method is to base the cutoff on visual inspection of the test results from known positive and negative populations. These results are
likely to show some overlap. The operator can choose the most appropriate point to modify the false
negative or false positive results depending on the required application of the assay. An alternative is to
base the cut-off on the mean +2 standard deviations (SD) or +3 SD values from a large sample of negative
animals. Finally, if no suitable negative/positive samples are available a cut-off can be based on the
analysis of the data from animals in an endemic situation. If a bimodal distribution separates infected from
uninfected animals, then an appropriate value can be selected. The ELISA is likely to correctly identify
uninfected animals. Equivocal results can be re-tested using CATT. Currently there are no readily available
defined antigens. The Institute for Tropical Medicine in Antwerp has a number of antigens that are still
undergoing evaluation. Diagnostic assays incorporating antigens derived from a single VAT should be
interpreted with caution and fully evaluated in different hosts and different geographical regions to
determine if their performance is consistent throughout the range of T. evansi.
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b) Card agglutination tests
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It is well known that certain predominant variable antigen types (VATs) are expressed in common in
different strains of salivarian trypanosomes from different areas. This finding was used as a basis for a test
for the diagnosis of T. evansi, the card agglutination test – CATT/T.evansi – was developed at the
Laboratory of Serology, Institute of Tropical Medicine, Antwerp. The test makes use of fixed and stained
trypanosomes of a defined VAT known as RoTat 1.2. Both variable and invariable surface antigens take
part in the agglutination reaction. The CATT is available in kit form from ITM, Antwerp. It consists of
lyophilised antigen, PBS, pH 7.4, plastic-coated cards, spatulas, positive and negative control sera and a
rotator. The lyophilised antigen can be stored at 2–8°C for up to 1 year. Reconstituted antigen can be
stored at 2–8°C for 2 days, but preferably should be used within 8 hours.
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For screening, put 25 µl of test serum diluted 1/4 or 1/8 in PBS on to circles inscribed on the plastic cards.
Add one drop (45 µl) of the prepared antigen suspension to the diluted serum sample. Mix and spread the
reagents with a spatula and rotate the card for 5 minutes using the rotator provided in the kit. Compare the
pattern of agglutination with the illustrations of different reactions provided in the kit. Blue granular deposits
reveal a positive reaction visible to the naked eye 3.
c) Latex agglutination tests
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A kit is available from ITM, Antwerp. It comprises a lyophilised latex suspension coated with T. evansi
RoTat 1.2 variable antigens, PBS, positive and negative controls, test cards, plastic spatulas and a rotator.
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Reconstitute the antigen-coated latex particles using distilled, deionised water. Mix gently. Add 20 µl of latex
suspension onto each black spot on the test cards.
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Dilute test sera with PBS (1/2 to 1/4) and add 20 µl to the latex suspension. Include appropriate controls.
Mix the reagents carefully with a plastic spatula.
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Rotate test cards at 70 rotations/minutes for 5 minutes and view cards under a good light source at the end
of the incubation. Positive sera will cause agglutination of the latex particles.
C.
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REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
No vaccines are available for this disease.
REFERENCES
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3
Experimental CATT/T. evansi kits for evaluation purposes are available at the Laboratory of Serology, Institute of Tropical
Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium. They are being evaluated in collaboration with the Institute of
Molecular Biology of the Free University of Brussels, with financial support from the International Laboratory for Research
on Animal Diseases (ILRAD), Nairobi, Kenya.
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19. ONAH D.N., HOPKINS J.A. & LUCKINS A.G. (1998). Increase in CD5 B cells and depression of immune
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20. RAE P.F., THRUSFIELD M.V., HIGGINS A., AITKEN C.G.G., JONES T.W. & LUCKINS A.G. (1989). Evaluation of
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21. REID S.A. & COPEMAN D.B. (2002). Evaluation of an antibody-ELISA using five crude antigen preparations for
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22. REID S.A. & COPEMAN D.B. (2003). The development and validation of an antibody-ELISA to detect
Trypanosoma evansi infection in cattle in Australia and Papua New Guinea. Prev. Vet. Med., 61, 195–208.
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23. REID S.A., HUSEIN A. & COPEMAN D.B. (2001). Evaluation and improvement of parasitological tests for
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24. SACHS R. (1984). Improvements in the miniature anion exchange centrifugation technique for detecting
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25. TUNTASUVAN D., CHOMPOOCHAN T., VONGPAKORN M. & MOHKAEW K. (1996). Detection of Trypanosoma evansi
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26. VENTURA R.M., TAKEDA G.F., SILVA R.A., NUNES V.L., BUCK G.A. & TEIXEIRA M.M. (2002). Genetic relatedness
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27. VERLOO D., HOLLAND W., MY L.N., THANH N.G., TAM P.T., GODDEERIS B., VERCRUYSSE J. & BUSCHER P. (2000).
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29. W UYTS N., CHODESAJJAWATEE N. & PANYIM S. (1994). A simplified and highly sensitive detection of
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*
* *
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NB: There is an OIE Reference Laboratories for Trypanosoma evansi infections (including surra) (see Table in
Part 3 of this Terrestrial Manual or consult the OIE Web site for the most up-to-date list: www.oie.int).
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