Development and validation of immunochromatographic test (ict) for diagnosis of animal trypanosomosis

Development and validation of immunochromatographic test (ICT) for diagnosis of animal trypanosomosis 2015 NGUYEN, THU THUY Doctoral Program in Animal and Food Hygiene Graduate School Obihiro University of Agriculture and Veterinary Medicine 家畜トリパノソーマ病診断用イムノクロマトグラフィー法の 開発と評価に関する研究 平成 26 年 （2015） 帯広畜産大学大学院畜産学研究科 畜産衛生学専攻 グエン 博士後期課程 トゥー トゥイ Contents Abbreviations .III General introduction 1 Taxonomy and pathogenicity of trypanosome Disease impact Diagnosis importance Objective of the present study…….……………………………………………….8 Chapter Recombinant TeGM6-4r as potential diagnostic antigen for animal trypanosomosis 1-1 Introduction 1-2 Materials and methods 11 1-3 Results 13 1-4 Discussion 14 Chapter Diagnostic value of the recombinant tandem repeat antigen TeGM6-4r for surra in water buffaloes 19 2-1 Introduction 19 2-2 Materials and methods 20 2-3 Results 24 I 2-4 Discussion 26 Chapter A TeGM6-4r antigen based Immunochromatographic Test (ICT) for detection of animal trypanosome infections …………………………………… ……………………………33 3-1 Introduction 33 3-2 Materials and methods 35 3-3 Results 38 3-4 Discussion 41 Chapter Application of serological assays, ELISA, CATT and ICT for diagnosis of animal trypanosomiasis in South Africa………………… ………………………………………48 4-1 Introduction…………… ………………………………………………………….48 4-2 Materials and methods…………………………… ………………………… …50 4-3 Results……… ………………………………….………………… …… …… 51 4-4 Discussion….……………………………… ……………….…………………….53 General discussion…… ……………….……………………………………………….59 Acknowledgement 62 References 64 Abstract 76 II Abbreviations B B bigemina: Babesia bigemina B bovis: Babesia bovis BLAST: basic local alignment search tool BSA: bovine serum albumin C CATT: card agglutination test for trypanosomosis D DNA: deoxyribonucleic acid dNTP: deoxyribonucleotide triphosphate E E coli: Escherichia coli ELISA: enzyme-linked immunosorbent assay F FAO: Food and Agriculture Organization I ICT: immunochromatographic test IFAT: indirect fluorescent antibody test IgG: immunoglobulin G IgM: immunoglobulin M IPTG: isopropyl β-D-1-thiogalactopyranoside L LAMP: loop-mediated isothermal amplification O OD: optical density III OIE: Office International Des Epizooties P PBS: phosphate buffered saline PBS-T: phosphate buffered saline containing 0.05% Tween 20 PCR: polymerase chain reaction S SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis SOB: super optimal broth T T b brucei: Trypanosoma brucei brucei T cruzi: Trypanosoma cruzi T congolense: Trypanosoma congolense T equiperdum: Trypanosoma equiperdum T evansi: Trypanosoma evansi T orientalis: Theileria orientalis T simiae: Trypanosoma simiae T suis: Trypanosoma suis T theileri: Trypanosoma theileri T vivax: Trypanosoma vivax IV Unit abbreviations B bp: base pair C o H h: hour K kD: kilo dalton M µl: microliter C: degree Celsius mg: milligram min: minute ml: milliliter mM: millimolar N ng: nanogram nm: nanometer P pmol: picomol S sec: second V General introduction Taxonomy and pathogenicity of trypanosome Trypanosome is a protozoan parasite living in blood circulation of the host and cause disease to both human and animals According to the classification of Levine et al (1980), it belongs to the subkingdom PROTOZOA, phylum SARCOMATIGOPHORA, class ZOOMASTIGOPHOREA, order KINETOPLASTIDA, family TRYPANOSOMATIDAE, and genus TRYPANOSOMA Species of trypanosome infecting mammals fall into two distinct groups: (A) the Stercoraria (subgenera Schizotrypanum, Megatrypanum and Herpetosoma), in which animal infective metacyclic form (MCF) trypanosomes are typically produced in the hindgut and are then passed on by contaminative transmission from the posterior; and (B) the Salivaria (subgenera Duttonella, Nannomonas and Trypanozoon) in which transmission occurs by the anterior station and is inoculative The clinical and pathological responses to trypanosome infection in domestic animals have been well studied (Maudlin et al., 2004) Many of the clinical and pathological manifestations of the trypanosomosis are common to domestic animals, irrespective of the species of trypanosome involved None the less, the range and severity of the pathological effects are influenced by a variety of factors Distinct pathological changes may be caused by the different livestock-infective trypanosome species The range and severity of the pathological effects of the parasites are influenced by a variety of factors Pathology in tissue is associated with the relative ability of the parasites to invade extravascular spaces and organs For example, whereas T congolense remains confined to the vascular system, trypanosome species of the Trypanozoon group (T b brucei, T evansi and T equiperdum) and T vivax are distributed in both the circulation and in the tissue Furthermore, there is remarkable intra-species variation in the pathogenicity of different parasite stocks, especially stocks isolated from distinct geographical regions Some East African isolates of T vivax may cause an acute hemorrhagic disease in cattle, in contrast to a milder nonhemorrhagic disease that result from infection with most West African T vivax isolates A number of host factors also contribute to determining the severity of disease For example, the wildlife of Africa is generally more resistant than the domestic ruminants and often serves as a reservoir for human- and livestock-infective trypanosomes The physiological statuses of the host, as well as nutritional and environmental factors, also play important roles in modulating the severity of trypanosomosis There are at least three identified animal syndromes caused by pathogenic trypanosomes names nagana (animal African trypanosomosis), surra and dourine Nagana is a disease caused by T congolense, T vivax, T simiae, T b brucei or T suis to ruminants, camels, equines, swine and carnivores in Africa In South America, trypanosomosis due to infection with T vivax is predominantly a disease of cattle but sheep, goats, horses and water buffaloes can also be infected In nagana, incubation period is usually 1-3 weeks, depending on the virulence of the infecting trypanosome, the infective dose and the immune status of the host The early acute phase of the disease is characterized by the continuous presence of trypanosomes in the blood (103-108/ml) Remittent fever is parallel with parasitemia waves With the onset of parasitemia, anemia develops Lymph nodes and spleen are enlarged Weakness, lethargy and loss of condition are patent; abortion and reduced milk production are common Death of the infected animal may occur in the first few weeks or months as a result of this acute disease In some cases, the clinical condition stabilizes after 6-8 weeks and a slow recovery process begins More frequently the animals enter into the chronic phage Stunting, wasting and infertility are characteristics of cattle suffering from chronic trypanosomosis syndrome The chronic phase may last for months or years and is most often terminated by death The second type of animal trypanosomosis is surra Surra is caused by T evansi which is widely distributed in Asia, South America and Africa The disease can be acute in young animals and pregnant females, which die within few weeks, but the usual form in endemic areas is chronic one that lasts for years and leads to cachexia and death Some individuals that survive chronic infection for years may eventually self-cure Clinical signs of surra are intermittent fever, anemia, emaciation, edema, conjunctivitis and lacrymation, enlargement of the lymph nodes and spleen, impaired motor function and abortions In camels, T evansi infection is sometimes complicated by pulmonary infection Cattle are often asymptomatic carriers in South America Water buffaloes develop acute or sub-acute infections that lead to death within a few weeks in 5-15% of the cases Dourine is the last type of animal trypanosomosis It is a venereally-transmitted disease caused by T equiperdum that executively affects equines and has a wide geographically distribution: North Africa, the Middle East, Eastern Europe, South America, central and south east Asia Horses are highly susceptible to T equiperdum and usually die at the end of a chronic disease that may last for 1-2 years Occasionally, acute forms that lead to death in 2-3 months Donkeys and mules develop a mild syndrome or remain productivity of cattle in the Didessa Valley, western Ethiopia Prev Vet Med 22, 29-40 Kappmeier K., Nevill E M., and Bagnall, R J 1998 Review of tsetse flies and trypanosomosis in South Africa Onderstepoort J Vet Res 65, 195-203 Kemp D J., R L Coppel, and R F Anders., 1987 Repetitive proteins and genes of malaria Annu Rev Microbiol 41, 181-208 Kristjanson P M., Swallow B M., Rowlands G J., Kruska R L., and P N de Leeuw., 1999 Measuring the costs of African animal trypanosomosis, the potential benefits of control and returns to research Agr Syst 59, 79-98 Latif A., Ntantiso L., Majiwa P., and van Den Bossche P., 2005 Epidemiology of animal trypanosomosis in northern KwaZulu-Natal 2005-2007 J S Afr Vet Assoc 80, 126140 Lejon V., F Claes, D Verloo, M Maina, T Urakawa, P A O Majiwa, and P Büscher, 2005 Recombinant RoTat 1.2 variable surface glycoprotein as antigen for diagnosis of Trypanosoma evansi in dromedary camels Int J Parasitol 35, 455-460 Levine N D., Corliss J O., Cox F E., Deroux G., Grain J., Honigberg B M., Leedale G F., Loeblich A R 3rd, Lom J., Lynn D., Merinfeld E G., Page F C., Poljansky G., Sprague V., Vavra J., and Wallace F G., 1980 A newly revised classification of the protozoa J Protozool 27, 37-58 Li F J., Gasser R B., Zheng J Y., Claes P., Zhu X Q., and Lun Z R., 2005 Application of multiple DNA fingerprinting techniques to study the genetic relationships among three 68 members of the subgenus Trypanozoon (Protozoa: Trypanosomatidae) Mol Cell Probes 19, 400-407 Lindmark R., Thoren-Tolling K., and Sjoquist J., 1983 Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera J Immunol Methods 62, 113 Luckins A.G., 1988 Trypanosoma evansi in Asia Parasitol Today 4, 137-142 Luckins A G., and Dwinger R H., 2004 Non-tsetse-transmitted Animal Trypanosomiasis pp 269-279 In: The Trypanosomiasis (Maudlin, I Holmes, P H and Miles, M A eds.), CABI Publishing, Oxfordshire Mamabolo M V., Ntantiso L., Latif A., and Majiwa P A., 2009 Natural infection of cattle and tsetse flies in South Africa with two genotypic groups of Trypanosoma congolense Parasitol 136, 425-431 Maudlin P H Holmes, and M A Miles, 2004 The trypanosomiases CABI Publishing, UK pp 269-279, 331-341 Michelizzi V N., Dodson M V., Pan Z., Amaral M E., Michal J J., McLean D J., Womack J E., and Jiang Z., 2010 Water buffalo genome science comes of age Int J Biol Sci 6, 333-349 Motloang M., Masumu J., Mans B., Van den Bossche P., and Latif A., 2012 Vector competence of Glossina austeni and Glossina brevipalpis for Trypanosoma congolense in KwaZulu-Natal, South Africa Onderstepoort J Vet Res 79, E1-6 Müller N., Hemphill A., Imboden M., Duvallet G., Dwinger R H., and Seebeck T., 1992 69 Identification and characterization of two repetitive non-variable antigens from African trypanosomes which are recognized early during infection Parasitol 104, 110-20 My L N., Thu L T., Lan P D., Lang P S., Phuc D V., 1998 Trypanosoma evansi and trypanosomosis in Vietnam J Protozool Res 8, 171-176 Namangala B., Oparaocha E., Kajino K., Hayashida K., Moonga L., Inoue N., Suzuki Y., and Sugimoto C., 2013 Preliminary investigation of trypanosomosis in exotic dog breeds from Zambia’s Luangwa and Zambezi valleys using LAMP Am J Trop Med Hyg 89, 116-118 Natulya V M., 1994 Suratex: a simple latex agglutination antigen test for diagnosis of Trypanosoma evansi infections (surra) Trop Med Parasitol 45:9-12 Ngaira J M., B Bett S M Karanja, and E N M Njagi, 2003 Evaluation of antigen and antibody rapid detection tests for Trypanosoma evansi infection in camels in Kenya Vet Parasitol 114, 131-141 Ngaira J M., E N M Njagi, J Ngeranwa, and N K Olembo, 2004 PCR amplification of RoTat 1.2 VSG gene in Trypanosoma evansi isolated in Kenya Vet Parasitol 120, 2333 Ngaira J M., N K Olembo E N M Njagi, and J Ngeranwa, 2005 The detection of nonRoTat 1.2 Trypanosoma evansi Exp Parasitol 110, 30-38 Nguyen T T., Zhou M, Ruttayaporn N., Nguyen Q D., Nguyen V K., Goto Y., Suzuki Y., Kawazu S., and Inoue N., (2014) Diagnostic value of the recombinant tandem repeat antigen TeGM6-4r for surra in water buffaloes Vet Parasitol 201, 18-23 70 Njiru Z K., J O Ouma J C Enyaru, and A P Dargantes, 2010 Loop-mediated Isothermal Amplification (LAMP) test for detection of Trypanosoma evansi strain B Exp Parasitol 125, 196-201 OIE, 2012 Trypanosomosis Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 7th ed., OIE, Paris pp 809-818 Ozerdem D., Eroglu F., Genc A., Demirkazik M., and Koltas I S., 2009 Comparison of microscopic examination, rK39, and PCR for visceral leishmaniasis diagnosis in Turkey Parasitol Res 106, 197-200 Paek S-H., Lee S-H., Cho J-H., and Kim Y-S., 2000 Development of rapid one-step immunochromatographic assay Methods 22, 53-60 Payne R C., Sukanto I P., Graydon R., Saroso H., and Jusuf S H., 1990 An outbreak of trypansomiasis caused by Trypansoma evansi on the island of Madura, Indonesia Trop Med Parasitol 41, 445-446 Pillay D., Izotte J., Fikru R., Büscher P., Mucache H., Neves L., Boulangé A., Seck M T., Bouyer J., Napier G B., Chevtzoff C., Coustou V., and Baltz T 2013 Trypanosoma vivax GM6 antigen: a candidate antigen for diagnosis of African animal trypanosomosis in cattle PLoS One 8(10): e78565 doi:10.1371/journal.pone.0078565 Posthuma-Trumpie G A., Korf J., and van Amerongen A., 2009 Lateral flow (immuno) assay: its strengths, weaknesses, opportunities and threats A literature survey Anal Bioanal Chem 393, 569-582 Reeder J C., and G V Brown, 1996 Antigenic variation and immune evasion in 71 Plasmodium falciparum malaria Immuno Cell Biol 74, 546-554 Reid S A., and D B Copeman, 2002 Evaluation of an antibody-ELISA using five crude antigen preparations for the diagnosis of Trypanosoma evansi infection in cattle Short communication Vet Parasitol 104, 79-84 Roddy P., Goiri J., Flevaud L., Palma P P., Morote S., Loma N., Villa L., Torrico F., and Albajar-Vinas, P., 2008 Field evaluation of a rapid immunochromatographic assay for detection of Trypanosoma cruzi infection by use of whole blood J Clin Microbiol 46, 2022-2027 Rodrigues A C., Campaner M., Takata C S, Dell' Porto A., Milder R V., Takeda G F., and Teixeira M M., 2003 Brazilian isolates of Trypanosoma (Megatrypanum) theileri: diagnosis and differentiation of isolates from cattle and water buffalo based on biological characteristics and randomly amplified DNA sequences Vet Parasitol 116, 185-207 Shaw A P M., 2009 Assessing the economics of animal trypanosomosis in Africa-history and current perspectives Onderstepoort J Vet Res 76, 27-32 Simo G., Sobgwi P F., Njtchouang G R., Njiokouf F., Kuaite J R., Cuny G., and Asonganyi T., 2013 Identification and genetic characterization of Trypanosoma congolense in domestic animals of Fontem in the South-West region of Cameroon Infect Genet Evol 18, 66-73 Sivakumar T., Lan D T B., Long P T., Yoshinari T., Tattiyapong M., Guswanto A., Okubo K., Igarashi I., Inoue N., Xuan X., and Yokoyama N., 2013 PCR Detection and 72 Genetic Diversity of Bovine Hemoprotozoan Parasites in Vietnam J Vet Med Sci 75, 1455-62 Sow A., Ganaba R., Percomonah L., Sidibé I., Bengaly Z., Adam Y., Koné P., Sawadogo G J., Van Den Abbeele J., Marcotty T., and Delespaux V., 2013 Baseline survey of animal trypanosoosis in the region of the Boude du Mouhiun, Burkina Faso Res Vet Sci 94, 573-578 Sullivan L., Wall S J., Carrington M., Ferguson M A J., 2013 Proteomic Selection of Immunodiagnostic Antigens for Human African Trypanosomiasis and Generation of a Prototype Lateral Flow Immunodiagnostic Device Plos Neglected Tropical Diseases Sundar S., Reed S G., Singh V P., Kumar P C K., and Murray H W., 1998 Rapid accurate field diagnosis of Indian visceral leismaniasis The lancet 351, 563-565 Swallow B M., 2000 Impacts of Trypanosomiasis on African Agriculture Programme against African Trypanosomiasis Technical and Scientific Series FAO, Rome Tamarit A., Tejedor-Junco M T., Gonzalez M., Alberola J., and Gutierrez, C., 2011 Morphological and biometrical features of Trypanosoma evansi isolates from an outbreak in mainland Spain Vet Parasitol 177, 152-156 Tanaka R., Yuhi T., Nagatani N., Endo T., Kerman K., Takamura Y., and Tamya E., 2006 A novel enhancement assay for immunochromatographic test strips using gold nanoparticles Anal Bioanal Chem 385, 1414-1420 Thekisoe O M., Inoue N., Kuboki N., and Tuntasuvan D W B., 2005 Evaluation of loopmediated isothermal amplification (LAMP), PCR and parasitological tests for detection 73 of in experimentally infected pigs Vet Parasitol 130, 327-330 Thu L T., My L R., Lan P D., Lang P S., Phuc D V., and Doanh, P Q., 1998 Epidemiological survey on Trypanosoma evansi infection in Vietnam J Protozool Res 8, 177-181 Thuy T N., Goto Y., Lun Z-L., Kawazu S-I., and Inoue N (2012) Tandem repeat protein as potential diagnostic antigen for Trypanosoma evansi infection Parasitol Res 110,7339 Tran T., F Claes, D Verloo, H D Greve, and P Büscher, 2009 Toward a new reference test for surra in camels Clin Vaccine Immunol 16, 999-1002 Valiente-Gabioud A A., Veaute C., Perrig M., Galan-Romano F., Sferco, S J., and Marcipar I S., 2011 Effect of repetitiveness on the immunogenicity and antigenicity of Trypanosoma cruzi FRA protein Exp Parasitol 127, 672-679 Van Den Bossche P., Esterhuizen J., Nkuna R., Matjila T., Penzhorn B., Geerts S., and Marcotty T., 2006 An update of the bovine trypanosomosis situation at the edge of Hluhluwe-Imfolozi Park, Kwazulu-Natal Province, South Africa Onderstepoort J Vet Res 73, 77-79 Verloo D., Holland W., My L N., Thanh N G., Tam P T., Goddeeris B., Vercruysse J., and Buscher P., 2000 Comparison of serological tests for Trypanosoma evansi natural infections in water buffaloes from North Vietnam Vet Parasitol 92, 87-96 Viera A J., and Garrett J M., 2005 Understanding interobserver agreement: the kappa statistic Fam Med 37, 360-363 74 Villareal M V., Mingala C N., and Rivera W L., 2013 Molecular characterization of Trypanosoma evansi isolates from water buffaloes (Bubalus bubalis) in the Philippines Acta Parasit 58, 6-12 75 Abstract Trypanosome infection is the worldwide distributed disease Various species of animal including water buffaloes, horses and cattle have been affected leading to huge economic losses dues to reduction of the animal products (milk, meat, fur, etc.) Moreover, endemic areas of the disease are often located in the countryside where diagnostic laboratories are costly or inaccessible Therefore, effective and accurate field test is of great interest to the scientists and so as to the farmers My study aimed to develop and validate an immunochromatographic test (ICT), a rapid, sensitive and simple method for detection of animal trypanosomosis For that purpose, identification of a novel antigen was an important and foremost step Chapter describes the production of tandem repeat (TR) antigen TeGM6-4r as a novel diagnostic antigen for animal trypanosomosis The newly expressed TeGM6-4r demonstrated higher immune-reactivity to water buffaloes (WBs) sera, which had been experimentally infected with T evansi, compared to the previously characterized TbbGM62r, and clearly distinguished positive samples from negatives in ELISA Another advantage of TeGM6-4r was that since amino acid sequence of GM6 is highly conserved among the salivarian trypanosome, using a single universal antigen could be able to detect the animls infected with several Trypanosoma spp including T b brucei, T evansi, T congolense and T vivax Chapter describes the evaluation of the TR antigen TeGM6-4r using ELISA The antigen had 100% specificity to Theileria and Babesia spp., and 81.4% to T theileri 76 TeGM6-4r based ELISA demonstrated 100% specificity and 80% sensitivity in detection of T evansi infection in experimentally infected WBs In the field condition, TeGM64r/ELISA had a sensitivity of 86.3% and specificity of 58.3% comparing to CATT/T evansi The test detected higher number of positive sera than CATT/T evansi This might be attributed to cross-reaction with T theileri; however, existence of T theileri among WBs in Vietnam still needs to be confirmed This result indicated that TeGM6-4r-based ELISA might be an effective and useful tool for epidemiological survey of surra in WBs In Chapter 3, the on-site diagnostic test TeGM6-4r/ICT was constructed subsequently The test demonstrated remarkable performance In detection of T evansi infection from experimentally infected WBs, TeGM6-4r/ICT was comparable to TeGM6-4r/ELISA which could clearly distinguish negative and positive controls The test was able to detect T brucei, T congolense and T vivax in field derived samples obtained from cattle in Uganda and Tanzania The result was in agreement with parasitological test and showed substantial agreement with T b brucei and T congolense lysate antigen/ELISAs and TeGM64r/ELISA (kappa value 0.64, 0.72 and 0.78 respectively) Moreover, ICT could detect both IgG and IgM in the serum samples while conventional ELISA detects only IgG In Chapter 4, the TeGM6-4r based ELISA and ICT was validated for diagnosis of animal trypanosomosis among sheep, goats and cattle in Kwazulu-Natal province, South Africa This was the first time a variety of serodiagnostic tests has been evaluated for diagnosis of trypanosome infections in South Africa Both ELISA assays utilizing crude and recombinant (TeGM6-4r) trypanosome antigens were highly sensitive and efficient The TeGM6-4r ICT was less sensitive than ELISA however is relatively specific, simple 77 and rapid Remarkably, the ICT results were highly in agreement with the results obtained from PCR method, which means ICT was able to detect the truly active infections In conclusion, an ICT utilizing a novel antigen TeGM6-4r were successfully developed The antigen demonstrated high sensitivity (86.3%) and specificity (58.3-100%) in detection of T evansi infection among WBs in Northern Vietnam The TeGM6-4r/ICT was able to detect both T congolense and T vivax infections in cattle, sheep and goats in Uganda, Tanzania and South Africa The test was comparable to parasitological test, PCR and TeGM6-4r/ELISA With further improvement on sensitivity the trypanosome ICT has the potential for use in both research and on-site diagnosis in trypanosome endemic countries Beside animal trypanosomosis detection the test may also be a good candidate for detection of human African trypanosomosis 78 要旨 トリパノソーマ病は地球規模で分布しており、スイギュウ、ウマ、ウシなど 様々な動物種に感染し、家畜の生産性を低下させることで莫大な経済的損失をも たらしている。加えて、流行地が農村地帯であることから高度な診断法が利用で きないことが多い。よってトリパノソーマ病に対する効果的で正確な野外診断法 を開発する事は研究者のみならず農民においても大きな関心事である。本学位論 文で私はイムノクロマトグラフィー法（ICT）を応用して簡便、迅速かつ高感度な 家畜トリパノソーマ病診断法を開発し、評価した。 第一章では家畜のトリパノソーマ病に対する新規診断用抗原であるタンデム リピート（TR）抗原 TeGM6-4r の組換え体蛋白質調整と評価について、研究を実 施した。TeGM6-4r は Trypanosoma evansi 実験感染スイギュウ血清に対して以前の 研究で評価した TbbGM6-2r よりも強い免疫反応性を示し、同抗原を用いた ELISA 法では陽性と陰性の検体を明確に区別することができた。TbbGM6-2r と TeGM6-4r は全く同じアミノ酸配列であるが、リピートユニットの反復回数だけが異なって いる（前者は２回、後者は４回反復）。一般に TR 抗原を用いた酵素抗体法 （ELISA）では、反復回数が増えるほど OD 値が高くなることが知られている。こ の現象は反復回数を増やすことで TR 抗原中のエピトープが増加し、より多くの抗 体分子が結合できるようになるためであると説明されている。トリパノソーマ病 診断用抗原として TeGM6-4r を用いるもう一つの利点として、同抗原が病原性トリ パノソーマの種間で広く保存されていることがあげられる。これによって本研究 79 の対象である T evansi に加えて、T congolense や T vivax など、動物アフリカトリ パノソーマ病診断へ応用することも期待できる。 第二章では ELISA 法を用いて TeGM6-4r 抗原の実用性を評価した。TeGM6-4r はタイレリアおよびバベシア感染ウシ血清に対しては全く交叉反応を示さず、ウ シの非病原性トリパノソーマ T theileri 感染ウシ血清に対しては若干の交差反応性 を示し、特異性は 81.4％となった。加えて TeGM6-4r 抗原 ELISA は T evansi 実験 感染スイギュウ血清に対して 100％の特異性と 80％の感度を示した。疫学調査で 得たスイギュウ血清サンプルを用いた評価では標準血清診断法として採用した市 販の血清診断キット CATT/T evansi に対して、58.3％の特異性と 86.3％の感度を示 した。TeGM6-4r 抗原 ELISA は CATT/T evansi よりも多くの陽性例を検出したが、 この中には T theileri 感染スイギュウに対する交叉反応も含まれている可能性があ る。現在ベトナムのスイギュウに T theileri 感染がどの程度存在しているかについ ては不明であるため、今後の調査で事実を明らかにしていく必要がある。最後に、 TeGM6-4r 抗原 ELISA を用いてベトナム北部から採取したスイギュウ血清の T evansi 血清抗体陽性調査を実施した。その結果、同地域のスイギュウ群には T evansi 感染が蔓延していることが明らかとなり、血清抗体陽性率は CATT/T evansi では 27％、TeGM6-4r 抗原 ELISA では 53％であった。以上の結果から TeGM6-4r 抗原 ELISA はスイギュウのスーラ病（T evansi 感染症）血清診断法として効果的 かつ有用であることが示唆された。 80 第三章では開発途上国の獣医臨床現場で実用可能な診断法開発を目的として、 TeGM6-4r 抗原を用いた ICT 法の開発を行った。T evansi 実験感染スイギュウ血清 を用いた評価において TeGM-4r 抗原 ICT は TeGM6-4r 抗原 ELISA と同等の特異性 と感度を示し、陽性と陰性を完全に区別することができた。加えて、TeGM6-4r 抗 原 ICT は過去にウガンダおよびタンザニアで採集したウシ由来血清サンプルから T brucei, T congolense ならびに T vivax の動物アフリカトリパノソーマ症における 主要病原体感染を検出することもできた。TeGM6-4r 抗原 ICT の結果は顕微鏡検査 法、原虫細胞可溶化抗原を用いた OIE 標準 ELISA 法ならびに TeGM6-4r 抗原 ELISA と有意に一致しており、一致性の指標であるカッパ値はそれぞれ 0.64、0.72 ならびに 0.78 であった。以上の結果は動物のトリパノソーマ病血清診断に ICT 法 が有用であることを示した初めての報告である。TeGM6-4r 抗原 ICT は同抗原 ELISA よりもやや低い感度を示したが、ELISA と異なり ICT では抗原特異的 IgM と IgG が同時に検出できるという利点もある。さらに現時点で唯一の市販診断キ ットである CATT/T evansi は T evansi 感染診断専用であるが、TeGM6-4r 抗原 ICT は T evansi 感染に加えて他のアフリカトリパノソーマ感染診断にも利用できる点 が優れている。 第四章では南アフリカで採集したヤギ、ヒツジおよびウシ血清サンプルを用 いて TeGM6-4r 抗原 ICT による感染調査を実施した。原虫細胞可溶化抗原を用い た OIE 標準 ELISA 法ならびに TeGM6-4r 抗原 ELISA は極めて高感度であったが、 TeGM6-4r 抗原 ICT はそれらより感度においては劣っていた。しかし ICT は簡便性 81 において ELISA に勝っており、ELISA よりもトリパノソーマ検出 PCR との間に高 い結果の一致が認められた。この結果は ICT が治療が必要な感染動物を正確に検 出できることを示唆している。上述の様々な診断法を用いた感染調査の結果、ヒ ツジ、ヤギ、ウシの有病率はそれぞれ 0～44.4％、0～9.1％、19.9～29.0％であった。 CATT/T evansi ではヒツジ全頭が陰性で、TeGM6-4r 抗原 ICT ではヤギ全頭が陰性 を示した。過去に南アフリカで実施された疫学調査においてもウシの有病率が最 も高くなっていることから、今回得られた結果は妥当であったと考えられる。加 えて今回の感染調査の結果から、ヤギはトリパノソーマ感染に対して他の家畜よ りも感受性が低いか、またはベクターであるアブの嗜好性が低いことが示唆され た。一方で、ヤギのように感受性の低い宿主は待機宿主となる可能性もあるため、 疫学調査対象家畜としての重要性は高いと考えられる。 本学位論文を総括すると、TeGM6-4r を用いた ICT は 86.3％の感度と、58.3～ 100％の特異性を示し、ベトナム北部のスイギュウ群を用いた評価において T evansi の実用的診断法として優れていると結論付けられた。加えて TeGM6-4r 抗原 ICT はウシ、ヒツジならびにヤギのアフリカトリパノソーマ感染も診断することが 可能であり、血清診断法として汎用性が高いことも示された。今後は同 ICT の検 出感度をさらに向上させる研究を続けていくことで、実用的簡易迅速診断法が開 発できると考えられる。さらに同 ICT 法がヒトのアフリカトリパノソーマ病診断 法へ応用可能か否かについても研究を開始する必要がある。 82 ... Therefore development of a new field test is significant to supplement the current CATT and provide more tests of choice for the detection of animal trypanosomosis Immunochromatographic test (ICT). .. cattle blood and sera Handling of the experimental animals strictly accorded to the guidelines on Animal Experimentation of Department of Animal Health of Vietnam and the ethics committee of Obihiro... antigen based Immunochromatographic Test (ICT) for detection of animal trypanosome infections 3-1 Introduction Animal trypanosomosis has been considered as one of the biggest constraints for worldwide