BOVINE TRYPANOSOMIASIS  IN TSETSE-INFESTED AND TSETSE-FREE  AREAS OF THE AMHARA REGION, NORTH-WEST ETHIOPIA

TRYPANOSOMOSE BOVINE DANS UNE ZONE INFESTEE DE TSETSE ET DANS UNE AUTRE ZONE INDEMNE DE TSETSE DE LA REGION AMHARA AU NORD-OUEST DE L’ETHIOPIE

T. Cherenet¹, R.A. Sani¹, N. Speybroeck³, J.M. Panandam² , S. Nadzr¹ &   P. Van den Bossche³, ⁴,*

¹Bahir Dar Regional Veterinary Laboratory (Ethiopia)
²Faculty of Veterinary Medicine, Addis Abeba University, P.O.Box 34,  Debre Zeit , Ethiopia
³Faculty of Veterinary Medicine, University Putra Malaysia, 43400 Serdang, Malaysia
⁴Institute of Tropical Medicine, Veterinary Department, Nationalestraat 155, 2000 Antwerp, Belgium thomascherenet@yahoo.com

Résumé

            Entre octobre 2001 et juin 2002, l’épidémiologie de la trypanosomose bovine a été étudiée dans une zone infestée de tsétsé (Glossina tachinoides et Glossina submorsitans) et dans une autre zone exempte de glossine de la région Amhara au nord-ouest de l’Ethiopie. On a collecté chaque mois des prélèvements sanguins de 120 bovins choisis au hasard pour détecter des trypanosomes à l’aide de la technique Buffy coat. On a aussi effectué le Buffy coat sur papiers  filtre aux fins d’analyse par PCR. Dans chaque zone d’étude,des enquêtes entomologiques mensuelles ont été faites en utilisant des pièges biconiques non-appâtés et des pièges NGU.

            Au total, 7.079 prélèvements sanguins recueillis de la zone infestée de tsétsé et de celle exempte de glossine étaient examinés. La prévalence mensuelle moyenne des infections trypanosomiennes était de 6,6% et 7,3% dans la zone infestée de tsétsé et dans celle indemne de glossine respectivement. L’espèce de trypanosome prédominante était T. vivax dans la zone exempte de glossine et T. congolense dans la zone infestée de tsétsé.

            Au total, 3.751 glossines et autres mouches piqueuses étaient capturées. L’abondance mensuelle des mouches piqueuses a montré une nette tendance saisonnière et avait une corrélation significative avec la prévalence mensuelle des infections trypanosomiennes.

Summary

            Between October 2001 and June 2002, the epidemiology of bovine trypanosomosis was studied in tsetse-infested (Glossina tachinoides and G. morsitans submorsitans) and tsetse-free areas of the Amhara Region in North-west Ethiopia. Blood samples were collected monthly from 120 randomly selected cattle to detect trypanosomes using the buffy coat method.  Buffy coat spots on filter papers were made for PCR analysis.  In each study area, monthly entomological surveys were conducted using unbaited biconical and NGU traps. 

            A total of 7079 blood samples from the tsetse-infested and tsetse-free areas were examined.  The average monthly prevalence of trypanosome infections was 6.6% and 7.3% in the tsetse-free and tsetse-infested area respectively.  The dominant trypanosome species was T. vivax in the tsetse-free and T. congolense in the tsetse-infested area.

            A total of 3751 tsetse and other biting flies were captured. The monthly abundance of biting flies showed a clear seasonal trend and was significantly correlated with the monthly prevalence of trypanosome infections.

Introduction

            Trypanosomosis is one of the major diseases which contribute to the direct and indirect economic losses to crop-livestock production. The most important species of trypanosome affecting livestock production in Ethiopia are T.congolense, T.vivax and T.brucei in cattle, T.evansi in camels and T.equiperdum in horses.

            A recent report bySolomon (1997) and Thomas (1999) indicated a prevalence rate of 15-21.6% of trypanosomosis in some tsetse-infested districts of the Amhara region Abebe and Jobre (1996) indicated that an 8.71% trypanosome prevalence rate was recorded in the highlands (tsetse-free areas) of which 99% was due to T. vivax.

            This study was designed for  the first time for a better understanding of the epidemiology of trypanosomosis in the Amhara Region North west Ethiopia using different diagnostic methods with different sensitivity and specificity, molecular (PCR), serological (Ab-ELISA) and parasitological (thin and wet blood film),  to asses the prevalence of the disease in both tsetse-infested and tsetse-free areas and to compare the infection rate on the basis of tsetse-infested and tsetse-free areas of the region and seasonal variation of the disease. Trypanosome infections in tsetse and other biting flies were also analyzed from tsetse-infested and tsetse-free areas of the area.

Materials And Methods

The study area

            The study was conducted in two districts of the Amhara Regional State, North western Ethiopia. The districts were tsetse-free area Bahir Dar Zuria with an altitude of 1800 m.a.s.l, surface area of 1511.19 Km² and tsetse-infested Dangla (Jawi) with an altitude of 1200 m.a.s.l, surface area of 4191.68 Km². The location of the study areas are between 9ºN -13º45’N and 36ºE- 40º35’E. Cultivation and grazing land make up 30% of the total area in each district. Forest shrub, bush and woodland, bodies of water and wasteland make up 17%, 4% and 17% and 16% of the total area, respectively, and the remaining 3% is taken by settlement, around the Beles River valley in tsetse-infested area (Jawi). A multistage random sampling method was applied to select  districts - farmers association (villages) and small-holder farms for the study (Putt et al, 1988).

Diagnostic techniques used during the study

            Every month for eight consecutive months from October/2002 to May/2003, the monitoring of all experimental animals were conducted using parasitological, molecular (PCR) and serological methods. The stained thin smears and wet films were examined for the parasitological diagnostic tests (Paris et al., 1982). For Ab-ELISA sample were collected as dried blood spot on filter paper (240) and serum (240) making a total 480 samples collected from tsetse-infested and tsetse-free areas for the first three months of the study, for screening for the trypanosome antibody levels. Every month blood sample were collected form the same selected animals for PCR analysis. The cattle blood was collected with mirohaematocrit centrifuge capillary tubes.  Blood was centrifuged in a microhaematocrit centrifuge and the micro-tube cut just above the buffy coat with a diamond pencil. The buffy coat was collected on filter paper Whatman no. 3 by bringing the tube in contact with the filter paper.  Filter papers were stored in sealed plastic bags with silica gel at -18 °C. PCR analysis was carried out at the Genetics Laboratory, Department of animal Science, Universiti Putra Malaysia.  DNA extraction and Standard PCR amplifications were  performed following a modified protocol of de Almeida et al., (1998).

            Parasitological prevalence was estimated by two techniques wet blood smears and thin blood smears. A total of 795 samples were examined by each technique.

Entomological surveys

            The population of biting flies were monitored using unbaited biconical (Challier, et al 1977), NGU traps (Brightwell, et al 1987) and Monoconical or Pyramidal trap (Gouteux and Lancien, 1986).   Traps were deployed in representative habitats in the tsetse-infested and tsetse-free study sites for three days each month. Also hand nets were used to capture from the animal body.  Records were kept of the fly species and number and sex of each species captured in each trap during each month. The monthly mean index of apparent abundance (IAA) of biting flies in the tsetse-infested and tsetse-free study sites was calculated as the mean number of biting flies (males and females) captured per trap per month. Standard PCR amplifications were carried out to identify fly infection Penchenier et al. 1996).

Statistical analyses

            Statistical analyses were carried out in SPSS 11.5 and EpiInfo. Univariate analysis provided an overview of the data. Seasonality, disease trends and patterns, and relative proportion of trypanosome were examined from cross-tabulations and graphs. Use was made of logistic regression and regression analysis. The index of apparent abundance and the infection rate of flies, was performed to investigate the relationship between these two variables. Proportions were statistically analyzed with a classical chi-square  tests. A correlation test was also applied when necessary.

Results

Diagnostic results from cattle

            In wet blood smear 107 (13.5%) were found positive for trypanosome. In thin blood smear 124 (15.6%) were found positive (Table 1) In PCR analysis the overall prevalence obtained from both tsetse-infested and tsetse-free areas reached 143(18 %) (Table1).

Table 1.  Prevalence of Trypanosomosis based on different diagnostic tests during the study period in tsetse -infested and tsetse free areas.

DBS = Dried Blood Spot, PCR= Polymer Chain Reaction

Table 2 Relative prevalence of trypanosome in tsetse infested and tsetse-free areas by PCR in Amhara

Region,  North west Ethiopia.

P<0.05

Diagnostic results from flies

            PCR amplification analyses for trypanosome identification was carried out on 3751 flies, with primer sets specific for Trypanosoma (Duttonella) vivax, T. (Nannomonas) congolense and T. (Trypanozoon) brucei. Out of the 3751 flies; 699 (18.64%) were positive in PCR analysis (Table 3) with 132 (12.13%) from tsetse-free areas and 567 (21.29%) from tsetse-infested areas (P<0.01). Within 1314 tsetse flies (Glossina morsitans submorsitans and Glossina tachinoides) 366 (27.85%) were positive and within 2437 other biting flies which belong to the family Tabanidae (Tabanus and Chrysops) and Stomoxyinae (Stomoxys calcitrans) 333 (13.66 %) were found positive (P<0.01). Trypanosomes of the Duttonella subgenus was the most abundant parasite among trypanosome species (Table 3) These results showed that T.vivax was the frequent parasite both in the tsetse (14.7 %) and other biting flies (9.1%) respectively. Relationship between cattle infection rate and Fly infection rate is shown in Figure 1

Discussion

            The study has demonstrated that the prevalence rate of trypanosomosis differed between different diagnostic methodes. Combination of these different tests therefore increased certainty in diagnosis. This study indicated that trypanosomosis was one of the major diseases of cattle.  Infection rate observed using Polymers Chain Reaction (PCR) in the tsetse-infested area was 20.51 % and tsetse-free area 15.5 % and with an overall prevalence rate of 18 %. Trypanosomosis caused by T.vivax is a major animal health proplem in both tsetse-infested and tsetse-free areas, followed by T.congolense. Langridge, (1976); Roeder, et al., (1984); Jordan, (1986); ILRAD, (1990) have reported that as the distance from recognized edge of tsetse belt areas increase, the species of trypanosome most encountered and diagnosed is T. vivax because T. vivax has the ability to adapt and establish itself in the absence of tsetse flies and is transmitted by other biting flies. The reason why T. congolense were less encountered in tsetse-free areas in the present study was attributed to the fact that these trypanosome species establish themselves more in cyclical transmission in tsetse-infested areas than tsetse-free. This has also been reported by Leak, (1999) that greater proportion of infections are transmitted mechanically rather than cyclically in such areas (tsetse-free) and T.vivax is more readily transmitted in this manner than other trypanosome specis.

Table 3 Infection rate of biting flies with trypanosome in  tsetse-infested and tsetse-free areas using PCR of the study

            This is the first study in Ethiopia that  an analysis  on the dried blood meal residue of the biting flies and the tsetse flies using a molecular technique. This study further demonstrated how PCR can be used to determine with accuracy the distribution of different kinds of trypanosomes in tsetse and other biting flies. As was indicated in tsetse-free areas 12% of the biting flies were found infected by trypanosome. In case of tsetse infested areas 21% of tsetse flies and biting flies were found positive for trypanosome. T.vivax was the highest trypanosome detached  with 8.3% in tsetse-free area and 12% in tsetse-infested areas.

            This result agreed with the suggestion given by Leak, (1999) It is easier to infect any species of flies with T.vivax than with other trypanosomes and very high infection rates may be obtained. It normally develops in the proboscis and  matures within 5 days in case of cyclical transmission (Desowitz and Fairbairm, 1955), T.congolense develops cyclically within  19 – 53 days, T.brucei can take 17 – 45 days (Hoare, 1970). T. vivax generally has the highest infection rates (usually 5-15%) in tsetse flies and often also in the host (about 5%).  It occurs throughout the tsetse belts and can adapt to non-tsetse transmission (Leak, 1999). PCR had identified infections in tsetse-flies and other biting flies. The results of this study are sufficiently encouraging for the technique to merit further development and application, particularly with primers specific for other trypanosomes.

            The result indicates that T.vivax and T.congolense has been identified in tsetse-free areas. This also showed the role of mechanical vector in the transmission of African livestock trypanosomes has always been controversial relative to tsetse flies, their cyclical vectors. As reported by Wilson and Stevenson, (1985) Trypanosoma vivax was transmitted both cyclically by tsetse flies and mechanically by other arthropod vectors; T.evansi is transmitted only mechanically. Solano et al, (1995) detected T.congolense savanna type from tabanids in Burkina Faso.  

            T. evansi does not normally occur within the tsetse belts: its main vectors are tabanid flies.  T. evansi infections in camels have been classified into five types and detailed surveillance is required as an apparently enzootically stable situation can change rapidly with the introduction of new parasite strains, bad management, increased vector activity or chemotherapy.  T. vivax mainly affects cattle, goats and horses.

Figure 1 Relationship between cattle infection rate and Fly infection rate diagnosed using PCR against apparent density of fly every month from  both  tsetse-infested and tsetse-free areas.

Acknowledgments

            The work presented in this paper was funded by the Ethiopian Agricultural Research Organization and the Amhara regional Agricultural Research Institute and Bureau of Agriculture of the Region. The authors acknowledge Prof. Dr. Getachew Abebe of the Addis Abeba University, Faculty of Veterinary Medicine for his field supervision and assistance. All staff of the Genetics Laboratory of the University Putra Malaysia and   The Bahir Dar Regional veterinary Laboratory staffs are thanked for their assistance

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