Activities in 2009
Surra (Trypanosoma evansi)
Dr Noboru Inoue
National Research Center for Protozoan Diseases,
Obihiro University of Agriculture and Veterinary Medicine,
2-11, Inada, Obihiro, Hokkaido 080-8555, Japan
Tel.: +81 155 495647, Fax: +81 155 495643
E-mail: ircpmi@obihiro.ac.jp, Website: http://www.obihiro.ac.jp/~tryp/eng_index.html
Summary of general activities related to the disease
1.
2.
Test(s) in use/or available for the specified disease at your laboratory
Test
For
Specificity
Total
IFAT
Antibody
Salivarian trypanosome
0
ELISA
Trypanosome cell lysate
Antibody
Salivarian trypanosome
203
ELISA
Recombinant P0
Antibody
Salivarian trypanosome
0
PCR/T. evansi
DNA
Trypanosoma evansi
30
PCR/Trypanozoon
DNA
Trypanozoon
671
PCR/SRA
DNA
T. b. rhodesiense
671
PCR/TgsGP
DNA
T. b. gambiense
203
PCR/T. congolense
All subtypes
DNA
T. congolense
All subtypes
671
LAMP/Trypanozoon
DNA
Trypanozoon
671
LAMP/T. congolense
DNA
T. congolense
438
Microscopy
Parasite
Trypanosoma
491
Production and distribution of diagnostic reagents
A. Trypanosoma brucei and Trypanosoma congolense procyclic form crude antigen for use in antibody detection
ELISA.
B. Recombinant T. congolense ribosomal P0 protein (apporx. 5 mg) for antigen detection ELISA was produced.
C. LAMP Reagents (2 kits, 192 reactions per kit), Trypanozoon specific LAMP primer, and PCR primer sets
(TBR, RoTat1.2, all subtypes of T. congolense) were sent to South Africa (Dr. Oriel Thekisoe)
D. LAMP primer sets for detection of Trypanozoon (Target gene PFR), applox. 5,000 tests
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Surra (Trypanosoma evansi)
E. LAMP primer set for detection of T. congolense (approx. 5,000 tests)
F. None of those were supplied to OIE members because of no requests.
Activities specifically related to the mandate
of OIE Reference Laboratories
3.
International harmonisation and standardisation of methods for diagnostic testing or the
production and testing of vaccines
Continued from 2008, in collaboration with the Foundation for Innovative New Diagnostics (FIND, Switzerland)
and EIKEN CHEMICAL CO LTD., development of Trypanozoon specific LAMP kit was carried out. This will
be completed within a year 2010.
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines
We can provide the following trypanosome DNA and crude antigens as a standards for diagnostic tests.
However, we cannot provide live parasites due to legal restrictions in Japan.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
23
5.
Species
Trypanosoma brucei brucei
Trypanosoma brucei brucei
Trypanosoma brucei brucei
Trypanosoma brucei gambiense
Trypanosoma brucei gambiense
Trypanosoma brucei gambiense
Trypanosoma brucei gambiense
Trypanosoma brucei gambiense
Trypanosoma brucei gambiense
Trypanosoma brucei gambiense
Trypanosoma brucei gambiense
Trypanosoma brucei gambiense
Trypanosoma evansi
Trypanosoma evansi
Trypanosoma evansi
Trypanosoma evansi
Trypanosoma evansi
Trypanosoma evansi
Trypanosoma evansi
Trypanosoma congolense
Trypanosoma congolense
Trypanosoma congolense
Strain
GUTat3.1
221
427
IL1922
IL3248
IL3250
IL3253
IL3254
IL3301
IL3707
Welcome
IL2343
IL1695
IL1934
IL3354
IL3382
IL3960
IL3962
Tansui
IL1180
IL3000
IL3338
Research and development of new procedures for diagnosis and control
Trypanozoon specific LAMP kit for human African trypanosomosis will be developed within a year (2010) in
collaboration with FIND and EIKEN CHEMICAL CO LTD. Validation based on OIE protocol of the kit will be
carried out after completion of the development.
Financed by Japan Society for the Promotion of Science (JSPS): Development of Trypanozoon specific
immunochromatographic test (ICT) will start in 2010.
Financed by JSPS: Epidemiological study on animal trypaosomoses were carried out in Northern Province in
Zambia and in Northern region of Tanzania, and Southern Uganda.
2
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
Surra (Trypanosoma evansi)
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
(1) Molecular parasitology
1) Li, S. Q., Reid, S. A., Fung, M. C., Inoue, N. and Lun, Z. R. (2009) Analysis of gene expression profiles in the
liver and spleen of mice infected with Trypanosoma evansi by using a cDNA microarray. Parasitology Research
104 (2) 385-397.
2) Mekata, H., Konnai, S., Witola, W. H., Inoue, N., Onuma, M. and Ohashi, K. (2009) Molecular detection of
trypanosomes in cattle in South America and genetic diversity of Trypanosoma evansi based on expression-siteassociated gene 6. Infection, Genetics and Evolution 9 (6) 1301-1305.
In South American countries, trypanosomiasis as a result of Trypanosoma evansi and Trypanosoma vivax
infections causes significant economic losses in livestock. The objectives of this study were to characterize the
epidemiology of bovine trypanosomiasis in South America and to draw a comparison between South American
and Asian T. evansi isolates based on the polymorphisms in their transferrin receptor encoding gene 6. We
assessed the prevalence rates of T. evansi and T. vivax infections in cattle in different regions of Peru and Bolivia
using the polymerase chain reaction (PCR) and found that, in Lima and Pucallpa in the Republic of Peru, T. evansi
infection rates were 5.8% (6/104) and 2.5% (5/195), respectively, while in Santa Cruz, Republic of Bolivia, the
infection rate for T. evansi was 11.5% (59/510). The prevalence rates of T. vivax in Lima and Santa Cruz were
3.8% (4/104) and 0.9% (5/510), respectively. In T. evansi, uptake of host transferrin is mediated by a receptor
derived from the two expression site-associated genes 6 and 7 (ESAG6 and ESAG7). We previously showed that
the ESAG6 depicts genetic diversity among different isolates of T. evansi in Asia. In this study, we cloned and
sequenced the ESAG6 genes from T. evansi isolates from South America, and found, in addition to some of the
previously observed variants, 20 novel variants of ESAG6 genes which could be categorized into three new clades
among the various isolates. To conclude, the results obtained in this study suggest that T. evansi isolates from
South America are more diverse than the Asian isolates.
(2) Epidemiology and development of diagnostics
1) Konnai, S., Mekata, H., Mingala, C. N., Abes, N. S., Gutierrez, C. A., Herrera, J. R. V., Dargantes, A. P.,
Witola, W. H., Cruz, L. C., Inoue, N., Onuma, M. and Ohashi, K. (2009) Development and application of
quantitative real-time PCR for the diagnosis of Surra in water buffaloes. Infection, Genetics and Evolution 9 (4)
449-452.
Trypanosoma evansi (T. evansi) causes the disease called Surra in domestic animals, which is of great economic
importance in South Asian countries. In order to improve the diagnosis of Surra, we endeavored to develop a realtime PCR assay for the detection and quantification of parasites in water buffaloes using specific primers for the T.
evansi antigen type (RoTat) 1.2 Variable Surface Glycoprotein (VSG) gene, which is a known diverse DNA
region in trypanosomes. The quantitative detection limit of the assay was 10 2 trypanosomes per mL of blood, and
the identity of the amplicon was confirmed in all assays by melting curve analysis. To evaluate the clinical
applicability of this procedure, detection and estimation of parasitemia in blood samples obtained from water
buffaloes and horses were conducted. T. evansi was detected in 17/607 (2.8%) blood samples, with parasitemia
levels ranging from >10 to 107 parasites per mL of blood. Interestingly, out of the 17 PCR positive animals, 3 had
previously received trypanocidal treatment and 1 had abortion history. These data indicate that real-time PCR for
the estimation of putative parasitemia levels is a quantitatively and objectively applicable technique for clinical
diagnosis of Surra, and could help to understand disease stage and risk of transmission of T. evansi.
2) Jing, Z., Magona, J. W., Sakurai, T., Thekisoe, O. M. M., Otim, C. P., Sugimoto, C. and Inoue, N. (2009) A
field study to estimate the prevalence of bovine African trypanosomosis in Butaleja district, Uganda. The Journal
of Veterinary Medical Science 71 (4) 525-527.
Prevalence of bovine trypanosomosis was determined from a total of 203 blood samples collected from Butaleja
district, eastern Uganda. All samples were examined by microhematocrit centrifuge test (MHC), PCR and ELISA.
ELISA was performed in accordance with the OIE standard procedures using Trypanosoma brucei gambiense
procyclic form crude antigens. PCR were utilized to identify the species and the subspecies of trypanosome. The
overall prevalence of bovine African trypanosomosis was 8.9% by MHC, and 45.3% by the ELISA. Since
substantial number (12 out of 18) of MHC positive samples were negative in the PCR tests, we could not conclude
the most epidemic trypanosome species in the studied area. Nevertheless, the PCR results suggests that the most
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
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Surra (Trypanosoma evansi)
prevalent trypanosome was T. b. brucei (31/203), followed by T. congolense (6/203). In addition, only a few
(3/203) mixed infections of T. b. brucei and T. congolense was detected by the PCR. Results obtained from this
study indicates that bovine trypanosomosis is endemic in Butaleja district, Uganda.
3) Thekisoe, O. M. M., Bazie, R. S., Coronel-Servian, A. M., Sugimoto, C., Kawazu, S. and Inoue, N. (2009)
Stability of loop-mediated isothermal amplification (LAMP) reagents and its amplification efficiency on crude
trypanosome DNA templates. The Journal of Veterinary Medical Science 71 (4) 471-475.
This study evaluated the stability of LAMP reagents when stored at 25 degrees C and 37 degrees C, and also
assessed its detection efficiency on different DNA template preparations. Accordingly, LAMP using reagents
stored at 25 degrees C and 37 degrees C amplified DNA of in vitro cultured T. b. brucei (GUTat 3.1) from day 1 to
day 15 of reagent storage. There were no significant differences (P>0.05) in detection sensitivity of LAMP among
the reagents stored at 25 degrees C, 37 degrees C and -20 degrees C (recommended storage temperature). LAMP
using the reagents stored at above-mentioned temperatures amplified serially diluted DNAs (genomic DNA
extracted by phenol-chloroform method, FTA card and hemolysed blood) of T. b. gambiense (IL2343) with high
sensitivity. Reactions were conducted on the reagents stored from 1 day to 30 days. LAMP detection sensitivity
was poor when fresh blood as DNA template was added directly into reactive solution. Results of this study
demonstrated that LAMP has the potential to be used in field conditions for diagnosis of trypanosome infections
without being affected by ambient temperatures of tropical and sub-tropical countries where trypanosomosis is
endemic.
7.
Provision of consultant expertise to OIE or to OIE Members
Assistance to Dr. Louis Touratier, Secretary-general of the OIE ad hoc group on non-tsetse transmitted animal
trypanosomoses (T. evansi and T. equiperdum).
The Ad hoc Group meeting on Diagnostic Tests for Trypanosomoses was held from 30 March to 1 April 2009 at
the OIE Headquarters, Paris. We have done revision of the diagnostic manual for Surra.
8.
Provision of scientific and technical training to personnel from other OIE Members
Supported by Japan International Cooperation Agency (JICA): Trainees from China and Zimbabwe were trained
for improvement of their basic molecular diagnostic techniques for 10 months.
Financed by Japan Society for the Promotion of Science (JSPS), and National Research Foundation (NRF) in
South Africa: Tanzanian, and South African scientists were trained for improvement of their basic molecular
parasitology skills for 2 weeks.
9.
Provision of diagnostic testing facilities to other OIE Members
None
10. Organisation of international scientific meetings on behalf of OIE or other international bodies
None
11. Participation in international scientific collaborative studies
Financed by JSPS: Study of the animal trypanosomoses in Zambia and Tanzania. Molecular epidemiological
survey of animal trypanosomoses was carried out. Counterpart: Dr. Boniface Namangala, University of Zambia,
and Prof. Chihiro Sugimoto, Hokkaido University. Total blood DNA from 402 cattle and 36 goats were subjected
to PCR or LAMP tests for Trypanozoon, T. b. rhodesiense, T. congolense and T. vivax. As a results, 4
(Trypanozoon), 23 (T. congolense), 1 (T. b. rhodesiense) and 6 (T. vivax) positive animals were detected either
PCR or LAMP tests.
4
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
Surra (Trypanosoma evansi)
Financed by JSPS: Study of the animal trypanosomoses in Uganda. Molecular epidemiological survey of animal
trypanosomoses was carried out. Counterpart: Dr. Magona, J.W., NaLIRRI, Uganda, and Prof. Chihiro Sugimoto,
Hokkaido University. Details are shown above in Chapter 6.
12. Publication and dissemination of information relevant to the work of OIE (including list of
scientific publications, internet publishing activities, presentations at international conferences)

Scientific publications in peer-reviewed journals
1) Helm, J. R., Hertz-Fowler, C., Aslett, M., Berriman, M., Sanders, M., Quail, M. A., Soares, M. B., Bonaldo, M.
F., Sakurai, T., Inoue, N. and Donelson, J. E. (2009) Analysis of expressed sequence tags from the four main
developmental stages of Trypanosoma congolense. Molecular and Biochemical Parasitology 168 (1) 34-42.
2) Mekata, H., Konnai, S., Witola, W. H., Inoue, N., Onuma, M. and Ohashi, K. (2009) Molecular detection of
trypanosomes in cattle in South America and genetic diversity of Trypanosoma evansi based on expression-siteassociated gene 6. Infection, Genetics and Evolution 9 (6) 1301-1305.
3) Konnai, S., Mekata, H., Mingala, C. N., Abes, N. S., Gutierrez, C. A., Herrera, J. R. V., Dargantes, A. P.,
Witola, W. H., Cruz, L. C., Inoue, N., Onuma, M. and Ohashi, K. (2009) Development and application of
quantitative real-time PCR for the diagnosis of Surra in water buffaloes. Infection, Genetics and Evolution 9 (4)
449-452.
4) Li, S. Q., Reid, S. A., Fung, M. C., Inoue, N. and Lun, Z. R. (2009) Analysis of gene expression profiles in the
liver and spleen of mice infected with Trypanosoma evansi by using a cDNA microarray. Parasitology Research
104 (2) 385-397.
5) Jing, Z., Magona, J. W., Sakurai, T., Thekisoe, O. M. M., Otim, C. P., Sugimoto, C. and Inoue, N. (2009) A
field study to estimate the prevalence of bovine African trypanosomosis in Butaleja district, Uganda. The Journal
of Veterinary Medical Science 71 (4) 525-527.
6) Sakurai, T., Tanaka, M., Kawazu, S. and Inoue, N. (2009) Establishment of an in vitro transgene expression
system in epimastigotes of Trypanosoma congolense. Parasitology International 58 (1) 110-113.
7) Thekisoe, O. M. M., Bazie, R. S., Coronel-Servian, A. M., Sugimoto, C., Kawazu, S. and Inoue, N. (2009)
Stability of loop-mediated isothermal amplification (LAMP) reagents and its amplification efficiency on crude
trypanosome DNA templates. The Journal of Veterinary Medical Science 71 (4) 471-475.
13. Inscription of diagnostic kits on the OIE Register
i)
Did you participate in expert panels for the validation of candidate kits for inscription on the
OIE Register? If yes, for which kits?
None
ii)
Did you submit to the OIE candidate kits for inscription on the OIE Register? If yes, for
which kits?
None
_______________
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
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