Trypanosoma Evansi Infections (Including Surra)

1 1 C H A P T E R 2 . 2 . 1 8 .

2 TRYPANOSOMA EVANSI INFECTIONS 3 (INCLUDING SURRA)

4 SUMMARY

5 Definition of the disease: Trypanosoma evansi causes a disease known as Trypanosomosis1 6 (‘surra’). It affects a number of domesticated animals species in Asia, Africa and Central and South 7 America. The principal host species affected varies geographically, but buffalo, cattle, camels and 8 horses are particularly affected, although other animals, including wildlife, are also susceptible. It is 9 an arthropod-borne disease and several species of haematophagous flies, including Tabanus spp. 10 and Musca spp., are implicated as vectors. 11 Description of the disease: The disease in susceptible animals is manifested by pyrexia, directly 12 associated with parasitaemia, together with a progressive anaemia, loss of condition and lassitude. 13 Recurrent episodes of fever and parasitaemia occur during the course of the disease. Oedema, 14 particularly of the lower parts of the body, urticarial plaques and petechial haemorrhages of the 15 serous membranes are often observed. Abortions have been reported in buffaloes and camels. 16 There are indications that the disease causes immunodeficiencies. 17 Identification of the agent: The general clinical signs of T. evansi infection are not sufficiently 18 pathognomonic for diagnosis and laboratory methods for detecting the parasite are required. 19 Examination of the host blood is problematic as trypanosomes can be detected only when there is 20 a high parasitaemia. Under these circumstances examination of wet blood films, stained blood 21 smears or lymph node material might reveal the trypanosomes. In other, more chronic cases, such 22 as the carrier state, the examination of thick blood smears, as well as methods of parasite 23 concentration and the inoculation of laboratory animals, are required. 24 Serological tests: Infection gives rise to specific antibody responses and a variety of antibody 25 detection tests have been introduced for laboratory use. Some have been partially validated, but 26 await large-scale evaluation and standardisation. Among those that are used regularly are 27 immunoenzyme assays, card agglutination tests and latex agglutination tests. For field use, 28 relatively simple tests have yet to be developed although obvious candidates might be indirect 29 agglutination tests using antigen-coated latex particles. Assays for detection of circulating 30 antibodies have high measures of validity. Estimates of predictive values of different serological 31 tests indicate that enzyme linked immunosorbent assays (ELISA) for detecting IgG antibodies are 32 more likely to classify correctly uninfected animals, and card agglutination tests (CATT) are more 33 likely to classify correctly truly infected animals. An IgG ELISA would thus be suitable for verifying 34 that animals are free from infection, prior to movement or during quarantine. In situations where 35 there is overt disease, CATTs can be used to target individual animals for treatment with 36 trypanocidal drugs. For declaring a disease-free status, serial testing – ELISA followed by re-testing 37 of suspect samples by CATT – is recommended. It must be stressed however, that there are 38 considerable antigenic similarities among the different species of pathogenic trypanosomes, hence 39 in areas where tsetse-transmitted trypanosomoses occur cross-reactions will occur with any 40 serological test employed. 41 Requirements for vaccines and diagnostic biologicals: No vaccines are available for the 42 disease.

21 Nomenclature of parasitic diseases: see the note in Chapter. 2.3.17. Trypanosomosis (Tsetse-borne).

3OIE Terrestrial Manual 2008 1 Chapter 2.2.18. – Trypanosoma evansi infections (including surra)

43 A. INTRODUCTION

44The clinical signs of surra, the disease caused by Trypansoma evansi, are indicative but are not sufficiently 45pathognomonic and diagnosis must be confirmed by laboratory methods. The disease in susceptible animals, 46including cattle, buffalo, camels (dromedary and bactrian), horses, pigs, sheep and goats, is manifested by 47pyrexia, directly associated with parasitaemia, together with a progressive anaemia, loss of condition and 48lassitude. Recurrent episodes of fever and parasitaemia occur during the course of the disease. Oedema, 49particularly of the lower parts of the body, urticarial plaques and petechial haemorrhages of the serous 50membranes are often observed. Abortions have been reported in buffaloes and camels (7, 15) and there are 51indications that the disease causes immunodeficiency (4, 19).

52There is considerable variation in the pathogenicity of different strains and the susceptibility of different host 53species to disease. Disease may manifest as an acute or chronic condition, and in the latter case may persist for 54many months. The disease is often rapidly fatal in camels, buffaloes, horses, cattle, llama and dogs, but mild and 55subclinical infections can also occur in these species. Wild animals such as deer and capybara can become 56infected. Animals subjected to stress – malnutrition, pregnancy, work – are more susceptible to disease.

57Biologically T. evansi is very similar to T. equiperdum, the causative agent of dourine (2), and morphologically 58resembles the slender forms of the tsetse-transmitted trypanosomes, T. brucei, T. gambiense and 59T. rhodesiense. Molecular characterisation indicates that various strains of T. evansi isolated form Asia, Africa 60and South America have a single origin. Moreover, it is also likely that T. evansi and T. equiperdum belong to the 61same species. Like all pathogenic trypanosomes, T. evansi is covered by a dense protein layer consisting of a 62single protein called the variable surface glycoprotein. This acts as a major immunogen and elicits the formation 63of specific antibodies. The parasites are able to evade the consequences of these immune reactions by switching 64the variant surface glycoprotein, the phenomenon known as antigenic variation.

65 B. DIAGNOSTIC TECHNIQUES

661. Identification of the agent

67The classical direct parasitological methods for the diagnosis of trypanosomosis, namely examining blood or 68lymph node material, are not highly sensitive. In regions where other Trypanozoon spp. occur in addition to 69T. evansi, specific identification by microscopy is not possible. Specific DNA probes (17, 22) may enable 70identification of trypanosome species by nonradioactive DNA hybridisation.

71• Direct methods

72a) Usual field methods 73 i) Blood sampling 74 Trypansoma evansi is a parasite of the blood and tissues often inhabiting the deep blood vessels in 75 cases of low parasitaemia. For this reason, it is recommended that blood for diagnosis be obtained 76 from both the peripheral and deep blood vessels. However it should be realised that less than 50% of 77 infected animals may be identified by examination of peripheral blood. 78 Peripheral blood is obtained by puncturing a small vein in the ear or tail. Deeper samples are taken 79 from a larger vein by syringe. Cleanse an area of the ear margin or tip of the tail with alcohol and, when 80 dry, puncture a vein is with a suitable instrument. Ensure that instruments are sterilised or disposable 81 instruments are used between individual animals, so that infection cannot be transmitted by residual 82 blood.

83 ii) Wet blood films 84 Place a small drop of blood on to a clean glass slide and cover with a cover-slip to spread the blood as 85 a monolayer of cells. Examine by light microscopy (×200) to detect any motile trypanosomes.

86 iii) Stained thick smears 87 Place a large drop of blood on the centre of a microscope slide and spread with a toothpick or the 88 corner of another slide so that an area of approximately 1.0–1.25 cm in diameter is covered. Air-dry for 89 1 hour or longer, while protecting the slide from insects. Stain the unfixed smear with Giemsa’s Stain 90 (one drop of commercial Giemsa + 1 ml of phosphate buffered saline [PBS, 2.4 g Na2HPO4.2H2O, 91 0.54 g NaH2PO4.2H2O, 0.34 g NaCl], pH 7.2), for 25 minutes. After washing, examine the smears by

52 OIE Terrestrial Manual 2008 Chapter 2.2.18. – Trypanosoma evansi infections (including surra)

92 light microscopy at high magnification (×500–1000). The advantage of the thick smear technique is that 93 it concentrates the drop of blood into a small area, and thus less time is required to detect the 94 parasites. The disadvantage is that the trypanosomes may be damaged in the process, and the 95 method is therefore not suited for species identification in case of mixed infections.

96 iv) Stained thin smears 97 Place a drop of blood 20 mm from one end of a clean microscope slide and draw out a thin film in the 98 usual way. Air-dry briefly and fix in methyl alcohol for 2 minutes and allow to dry. Stain the smears in 99 Giemsa (one drop Giemsa + 1 ml PBS, pH 7.2) for 25 minutes. Pour off, stain and wash the slide in tap 100 water and dry. Unfixed smears can be stained by covering them with May–Grünwald stain for 101 2 minutes, then adding an equal volume of PBS, pH 7.2, and leaving the slides for a further 3 minutes. 102 Pour off and add diluted Giemsa for 25 minutes. Pour off, wash the slides with tap water, and dry. 103 Examine at high magnification (×400–1000). This technique permits detailed morphological studies and 104 identification of the trypanosome species. Rapid staining techniques also exist (Field’s stain, Diff 105 Quick®).

106 v) Lymph node biopsies 107 Samples are usually obtained from the prescapular or precrural (subiliac) lymph nodes. Select a 108 suitable node by palpation and cleanse the site with alcohol. Puncture the node with a suitable gauge 109 needle, and draw lymph node material into a syringe attached to the needle. Expel lymph on to a slide, 110 cover with a cover-slip and examine as for the fresh blood preparations. Fixed thin or thick smears can 111 also be stored for later examination.

112b) Concentration methods 113 In most hosts T. evansi can induce mild clinical or subclinical carrier state infections with low parasitaemia in 114 which it is difficult to demonstrate the parasites. In these circumstances, concentration methods become 115 necessary.

116 i) Haematocrit centrifugation 117 Collect blood (70 µl) into at least two heparinised capillary tubes (75 × 1.5 mm). Seal at the dry end by 118 heat and centrifuge, sealed end down, at 3000 g for 10 minutes. Place the capillary tube between two 119 pieces of glass (25 × 10 × 1.2 mm) glued to a slide. Place a cover-slip on top at the level of the buffy 120 coat junction where the trypanosomes will be concentrated. Flood the space around this part of the 121 tube with water or immersion oil, and examine the buffy coat area under the microscope (×100–200). A 122 simpler alternative is to examine the centrifuged capillary tube by placing a drop of immersion oil on the 123 tube and ensuring that there is contact between the objective lens and the immersion oil.

124 ii) Dark-ground/phase-contrast buffy coat technique 125 Collect blood into heparinised capillary tubes and centrifuge as above. Scratch the tube with a glass- 126 cutting diamond and break it 1 mm below the buffy coat layer – the upper part thus contains the top 127 layer of red blood cells (RBCs), the buffy coat (white blood cells) and some plasma. 128 Partially expel the contents of this piece on to a slide, cover with a cover-slip and examine under dark- 129 ground, phase-contrast or ordinary illumination. 130 As an alternative to the electrically powered haematocrit centrifuge, hand-powered micro-centrifuges 131 have been used successfully for detection of trypanosomosis in cattle and camels (8, 13).

132 iii) Haemolysis techniques 133 Sodium dodecyl sulphate (SDS) can be used as a reagent to haemolyse RBCs to facilitate detection of 134 motile trypanosomes in parasitised blood samples. As SDS is toxic, contact with skin, inhalation and 135 ingestion should be avoided. SDS solution can be stored for several months at ambient temperature. 136 Both the SDS solution and the blood samples should be used at a temperature above 15°C. At lower 137 temperatures the trypanosomes may be destroyed.

138 Two general procedures, namely wet blood film clarification and haemolysis centrifugation, can be used2.

139  Wet blood film clarification method 140 This method uses the partial lysis of RBCs to facilitate the detection of motile trypanosomes. The 141 method requires an SDS solution: 1% SDS dissolved in Tris/glucose/saline, pH 7.5, inoculating loops

72 All necessary materials and instructions can be obtained from the Institute of Tropical Medicine, Laboratory of Serology, 8 Nationalestraat 155, B-2000 Antwerp, Belgium.

142 (10 µl), slides and cover-slips (24 × 24 mm), and a drop of fresh neat or heparinised blood. Dissolve 143 100 mg of SDS in 100 ml of isotonic Tris/NaCl/glucose buffer, pH 7.5 (Trizma base 14.0 g, NaCl 3.8 g, 144 glucose 10.8 g; dissolve chemicals in 750 ml distilled H2O, add 90–100 ml 1 N HCl and adjust pH to 145 7.5 then make up to final volume of 1000 ml with distilled H2O). This buffer can be stored in small vials 146 at ambient temperature for several months. The test does not give a significantly higher sensitivity than 147 wet film technique. In addition, the SDS can cause problems when focusing the microscope and the 148 movements of trypanosomes can be severely curtailed owing to the high viscosity of the SDS. 149 Put approximately 10 µl of blood on a slide. Add 10 µl of SDS solution using a dip inoculation loop and 150 mix gently. Apply a cover-slip and examine the preparation without delay at low magnification (×100 or 151 ×200).

152  Haemolysis centrifugation technique 153 Nearly complete lysis of RBCs is required for this procedure. The materials needed include: SDS 154 solution (0.1% SDS dissolved in Tris/glucose/saline, pH 7.5), conical centrifuge tubes, ordinary test 155 tubes, large and fine plastic tapering pipettes with attached bulb, slides, cover-slips (24 × 24 mm or 156 24 × 32 mm), and heparinised blood. 157 Using a pipette or syringe, transfer nine volumes (maximum 6.3 ml) of SDS solution into an ordinary 158 test tube. Aspirate one volume (maximum 0.7 ml) of heparinised blood into a bulb pipette and expel it 159 just above the surface of the SDS solution; mix quickly and thoroughly. Avoid foam formation, which 160 may result in destruction of the trypanosomes. Wait for 10 minutes so as to achieve complete 161 haemolysis. 162 Pour the haemolysed suspension into a conical centrifuge tube and spin at approximately 500 g for 163 10 minutes. With a clean bulb pipette, remove as much supernatant as possible without disturbing the 164 sediment. Using a fine tapering bulb pipette, remove more supernatant, leaving 10–20 µl of 165 undisturbed sediment at the bottom. 166 Very carefully collect the entire sediment and put on to a microscope slide. Apply a cover-slip and 167 examine the preparation without delay at low magnification (×100 or ×200).

168 iv) Mini-anion exchange centrifugation technique 169 When a blood sample from animals infected with salivarian trypanosomes is passed through an 170 appropriate anion-exchange column, the host blood cells, being more negatively charged than 171 trypanosomes, are adsorbed onto the anion-exchanger, while the trypanosomes are eluted, retaining 172 viability and infectivity. A simplified field method for detection of low parasitaemia has been developed 173 (24). The sensitivity of this technique can be increased by approximately tenfold by the use of buffy 174 coat preparations rather than whole blood (23).

175  Preparation of phosphate buffered saline glucose, pH 8 176 Na HPO (anhydrous) (13.48 g); NaH PO .2H O (0.78 g); NaCl (4.25 g); distilled water (1 litre). 2 4 2 4 2 177 Solutions of different ionic strength are made by diluting the stock PBS, pH 8, and adding glucose to 178 maintain a suitable concentration. For blood of mice, domestic and wild ruminants and dog, add four 179 parts PBS to six parts distilled water and adjust the final glucose concentration to 1%. For blood of pigs 180 and rabbits, add three parts PBS to seven parts distilled water and adjust the final glucose 181 concentration to 1.5%. The PBS/glucose solution (PSG) must be sterile.

182  Equilibration of DEAE-cellulose 183 Suspend 500 g of DEAE-cellulose (diethylamino-ethylcellulose) in 2 litres of distilled water. Adjust the 184 pH to 8 with phosphoric acid. Allow to settle for 30 minutes. Discard the supernatant fluid containing 185 the fine granules. Repeat the procedure three times. Store the equilibrated concentrated suspension of 186 DEAE-cellulose (slurry) at 4°C or in small aliquots at –20°C.

187  Packing of equilibrated DEAE-cellulose 188 Place a 2 ml syringe without the plunger on a test-tube rack complete with needle (20 G × 1.5 inch). 189 Put a disc of Whatman No. 41 filter paper at the bottom of the syringe and moisten by adding a few 190 drops of PSG. Pour 2–2.5 ml of the slurry of equilibrated cellulose into the syringe and allow to pack by 191 elution of the buffer. The height of the sediment should be approximately 3 cm. Wash and equilibrate 192 the column with 2 ml of PSG without disturbing the surface.

193  Adsorption of blood eluate of the trypanosomes 194 Gently place 100–300 µl of heparinised blood above the surface of the cellulose column.Add ten drops 195 of PSG and discard the first ten drops of the eluate. Progressively add 1.5 ml of PSG and start

196 collecting the eluate into a finely tapered Pasteur pipette with a sealed end. Put the filled pipette, 197 protected by a conical plastic pipette tip, in a tube and centrifuge at 525 g (or up to 1000 g) for 198 10 minutes. Examine the bottom of the pipette under the microscope (×100 or ×200) using a special 199 mounting device. Alternatively, the eluate could be collected into 50 ml plastic tubes, with conical 200 bottoms, centrifuged at 1000 g and the sediment examined by dark-ground microscopy. 201 The cellulose column should remain wet throughout the procedure.

202c) Animal inoculation 203 Laboratory animals may be used to reveal subclinical (nonpatent) infections in domesticated animals. 204 Trypanosoma evansi has a broad spectrum of infectivity for small rodents, and so rats and mice are often 205 used. Rodent inoculation is not 100% sensitive (16) but further improvement in its efficacy can be obtained 206 by the use of buffy coat material. Such a procedure was able to detect as few as 1.25 T. evansi/ ml blood 207 (23).

208 Inoculate heparinised blood intraperitoneally into rats (1–2 ml) or mice (0.25–0.5 ml). Inoculate a minimum of 209 two animals. Bleed animals from the tail three times a week to detect parasitaemia. The incubation period 210 before appearance of the parasites and their virulence depends on the strain of trypanosomes, their 211 concentration in the inoculum, and the strain of laboratory animal used. Sensitivity of this in-vivo culture 212 system may perhaps be increased by use of immunosuppressed laboratory animals. Drugs such as 213 cyclophosphamide or hydrocortisone acetate, or X-ray irradiation or splenectomy are used for this purpose.

214d) Recombinant DNA probes 215 Specific DNA probes have been used to detect trypanosomes in infected blood or tissue but are not 216 routinely applied (26, 28). Although molecular methods have a potentially high analytical sensitivity there 217 have been few convincing studies to critically evaluate the diagnostic sensitivity of these tests compared 218 with other techniques, such as serology.

219• Indirect methods

220These methods involve tests that demonstrate the effects of the parasite on its host rather than directly detecting 221the parasite itself.

222a) Haematology 223 Anaemia is usually a reliable indicator of trypanosome infection, although it is not in itself pathognomonic. 224 However, animals with a mild subclinical infection can have parasitaemia without evidence of anaemia (3).

225 Anaemia can be estimated by measuring the packed cell volume and may be used in surveys of herds at 226 risk. The technique is identical to that of haematocrit centrifugation. The capillary tube is examined and the 227 results are expressed as a percentage of packed RBCs to total blood volume.

228b) Detection of trypanosomal DNA 229 Detection of minute amounts of trypanosomal DNA using a polymerase chain reaction (PCR) procedure is a 230 possible means of identifying animals with active infections, and could have the sensitivity and specificity 231 required (18, 29). Experimental studies in buffalo (9) showed the diagnostic sensitivity of a PCR was only 232 78%, similar to that of mouse inoculation.

2332. Serological tests

234Historically, many different methods have been used to detect specific humoral antibodies to trypanosomal 235antigens but, more recently, there has been a tendency to concentrate on more easily standardised techniques 236such as ELISA (5, 6, 11, 20, 21, 22, 25) card agglutination tests (CATT) (1, 18, 22) and latex agglutination tests 237(LAT) (10, 14). Extensive evaluation of ELISA and CATT has been carried out in buffaloes in Indonesia and 238Vietnam (5, 10, 27). The collection of samples can be simplified by using filter paper blood spots for later use in 239the ELISA, while for the CATT whole blood can be substituted for serum (10). Other innovative modifications that 240might be developed in the future are the use of a colloidal-dye dipstick test (12) that could enable the tests to be 241carried out under field conditions. It is vitally important that serological tests are validated and standardised if they 242are to be suitable for correctly identifying infected animals. This means that standard criteria for interpreting the 243tests might have to be developed for each animal species as well as each laboratory operating the procedure.

244a) Enzyme-linked immunosorbent assay

245 The principle of this technique is that specific antibodies to trypanosomes can be detected by enzyme-linked 246 anti-immunoglobulins using solid-phase polystyrene plates coated with soluble antigen. The enzyme may be 247 peroxidase, alkaline phosphatase or any other suitable enzyme. The enzyme conjugate binds to the 248 antigen/antibody complex and then reacts with a suitable substrate to yield a characteristic colour change 249 either of the substrate itself or of an added indicator (the chromogen).

250 The antigen for coating the plates is derived from the blood of a heavily parasitaemic rat. The trypanosomes 251 are separated on a DEAE-cellulose column and washed three times by centrifugation in cold PSG, pH 8 252 (PBS with 1% glucose). The final pellet is suspended in cold PSG to a concentration of 3–5%, and briefly 253 ultrasonicated on ice for 30–120 seconds until disintegration of the organisms is complete. This preparation 254 is centrifuged at 4°C and 40,000 g for 60 minutes. The supernatant is diluted in water so as to obtain a 255 protein concentration of 1 mg/ml. The reagent thus obtained can be stored in small aliquots at –70°C for 256 several months. It can also be freeze-dried and stored at –20°C. Various treatments of the antigen 257 preparations have been applied to improve the accuracy of antibody detection (29).

258  Test procedure 259 i) Dilute or reconstitute the frozen or freeze-dried antigen with freshly prepared 0.01 M 260 carbonate/bicarbonate buffer, pH 9.6. Add 100 µl to each well of a 96-well microtitre plate and incubate 261 overnight at 4°C or for 1 hour at 37°C. 262 ii) Remove excess antigen by are wash plate with 0.01 M PBS containing 0.05% Tween 20 (PBST). Add 263 test serum dilutions in PBST (100 µl). Include control negative and positive sera. Dilutions must be 264 determined empirically, but are usually between 1/100 and 1/1000. 265 iii) Incubate plates at 37°C for 30 minutes. Eject contents and wash three times with PBST. 266 iv) Add a specific peroxidase conjugated species-specific anti-globulin (100 µl) appropriately diluted in 267 PBST (usually between 1/1000 and 1/50,000). 268 v) Incubate the plates at 37°C for 30 minutes, eject contents and wash three times with PBST. 269 vi) For peroxidase conjugates a number of substrate/chromogen solutions can be used, consisting of 270 hydrogen peroxide with a chromogen, such as tetramethylbenzidene (TMB), 2,2’-azino-bis-(3- 271 ethylbenzothiazoline-6-sulphonic acid) (ABTS) and ortho-diphenylenediamine (OPD). A suitable 272 substrate/chromogen solution for peroxidase conjugates is 30% hydrogen peroxide (0.167 ml and 273 35 mg) in citrate buffer (100 ml), pH 6.0. The citrate buffer is made up as follows: Solution A (36.85 ml): 274 (0.1 M citric acid [21.01 g/litre]); Solution B (65.15 ml): (0.2 M, Na2HPO4 [35.59 g/litre]); and distilled 275 water (100 ml). Dissolve 10 mg TMB in 1 ml dimethyl suphoxide and add to 99 ml of the citrate buffer. 276 A number of these combinations are available commercially in ready-to-use formulations that remain 277 stable at 4°C for up to 1 year. 278 vii) Add the substrate chromogen (100 µl) to the plates and incubate at room temperature for 15– 279 20 minutes. 280 viii) Stop the reaction by adding 50 µl 1 M sulphuric acid. Read the absorbance of each well at 450 nm for 281 TMB chromogen. Other chromogens may require the use of a different wavelength. All tests should 282 include known high and medium positive control sera, a negative control serum, and a buffer control.

283 A large variety of other test procedures exists. For closely related animal species, cross-reacting reagents 284 may often be used (e.g. anti-bovine immunoglobulin for buffaloes and the use of monospecific anti-IgG 285 conjugates is generally recommended. There are a number of methods that can be used to determine a cut- 286 off point to discriminate between positive and negative results. The simplest method is to base the cut-off on 287 visual inspection of the test results from known positive and negative populations. These results are likely to 288 show some overlap. The operator can choose the most appropriate point to modify the false negative or 289 false positive results depending on the required application of the assay. An alternative is to base the cut-off 290 on the mean +2 standard deviations (SD) or +3 SD values from a large sample of negative animals. Finally, 291 if no suitable negative/positive samples are available a cut-off can be based on the analysis of the data from 292 animals in an endemic situation. If a bimodal distribution separates infected from uninfected animals, then 293 an appropriate value can be selected. The ELISA is likely to correctly identify uninfected animals. Equivocal 294 results can be re-tested using CATT. Currently there are no readily available defined antigens. The Institute 295 for Tropical Medicine in Antwerp has a number of antigens that are still undergoing evaluation. Diagnostic 296 assays incorporating antigens derived from a single VAT should be interpreted with caution and fully 297 evaluated in different hosts and different geographical regions to determine if their performance is consistent 298 throughout the range of T. evansi.

299b) Card agglutination tests 300 It is well known that certain predominant variable antigen types (VATs) are expressed in common in different 301 strains of salivarian trypanosomes from different areas. This finding was used as a basis for a test for the

302 diagnosis of T. evansi, the card agglutination test – CATT/T.evansi – was developed at the Laboratory of 303 Serology, Institute of Tropical Medicine, Antwerp. The test makes use of fixed and stained trypanosomes of 304 a defined VAT known as RoTat 1.2. Both variable and invariable surface antigens take part in the 305 agglutination reaction. The CATT is available in kit form from ITM, Antwerp. It consists of lyophilised antigen, 306 PBS, pH 7.4, plastic-coated cards, spatulas, positive and negative control sera and a rotator. The lyophilised 307 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, 308 but preferably should be used within 8 hours.

309 For screening, put 25 µl of test serum diluted 1/4 or 1/8 in PBS on to circles inscribed on the plastic cards. 310 Add one drop (45 µl) of the prepared antigen suspension to the diluted serum sample. Mix and spread the 311 reagents with a spatula and rotate the card for 5 minutes using the rotator provided in the kit. Compare the 312 pattern of agglutination with the illustrations of different reactions provided in the kit. Blue granular deposits 313 reveal a positive reaction visible to the naked eye3.

314c) Latex agglutination tests 315 A kit is available from ITM, Antwerp. It comprises a lyophilised latex suspension coated with T. evansi RoTat 316 1.2 variable antigens, PBS, positive and negative controls, test cards, plastic spatulas and a rotator.

317 Reconstitute the antigen-coated latex particles using distilled, deionised water. Mix gently. Add 20 µl of latex 318 suspension onto each black spot on the test cards.

319 Dilute test sera with PBS (1/2 to 1/4) and add 20 µl to the latex suspension. Include appropriate controls. Mix 320 the reagents carefully with a plastic spatula.

321 Rotate test cards at 70 rotations/minutes for 5 minutes and view cards under a good light source at the end 322 of the incubation. Positive sera will cause agglutination of the latex particles.

323C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS

324 No vaccines are available for this disease.

325 REFERENCES

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396 * 397 * *

398NB: There is an OIE Reference Laboratories for Trypanosoma evansi infections (including surra) (see Table in 399Part 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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