Regassa Fikru

Ghibe river basin in Ethiopia: present situation of trypanocidal drug resistance in Trypanosoma congolense using tests in mice and PCR-RFLP.

A cross-sectional study was carried out in the Ghibe valley from August to October 2010. 411 head of cattle were sampled in eight villages for buffy coat examination (BCE) and blood spots were collected from each animal for trypanosomose diagnosis by 18S-PCR-RFLP and diminazene aceturate (DA) resistance by Ade2-PCR-RFLP. Three villages were selected in a zone where trypanosomosis control operations are currently on-going whereas the other 5 villages were located outside these control operations. Twenty-four samples (5.84%) were diagnosed positive for Trypanosoma congolense by BCE and injected in mice for further characterization. Twelve of those isolates successfully multiplied in mice and were tested by an in vivo mouse test for diminazene (DA) (10 and 20mg/kg B.W.) and isometamidium (ISM) (1mg/kg B.W.) resistance. All were shown to be resistant to both drugs at all doses. The use of the Ade2-PCR-RFLP on these isolates confirmed their DA-resistance profile. Seventy-three of the collected blood spots (17.8%) were diagnosed positive for T. congolense by 18S-PCR-RFLP of which 37 (50.7%) gave amplification products with the Ade2-PCR-RFLP. Here, 35 (94.6%) showed a resistant profile, 1 (2.7%) a sensitive profile and 1 (2.7%) a mixed profile. The data were analysed by logistic regression model and the relapsing time in mice tests was assessed using the Cox regression model. There was no significant intervention effect (P=0.83) with odds ratio equal to 1.21 when using the BCE data. 18S-PCR-RFLP test also showed no significant intervention effect (P=0.60) with odds ratio equal to 1.43. The hazard ratio of getting parasitaemic after treatment with DA at 20mg/kg B.W. compared to the control group was 0.38 which differs significantly from one (P<0.001). Relapsing time after treatment with DA 10mg/kg B.W. or ISM 1mg/kg B.W. was also significantly longer than the prepatent period of the control group. The situation of drug resistance in the Ghibe valley is further discussed.

Epidemiology of Trypanosoma evansi and Trypanosoma vivax in domestic animals from selected districts of Tigray and Afar regions, Northern Ethiopia

BackgroundAfrican animal trypanosomosis, transmitted cyclically by tsetse flies or mechanically by other biting flies, causes serious inflictions to livestock health. This study investigates the extent of non-tsetse transmitted animal trypanosomosis (NTTAT) by Trypanosoma (T.) evansi and T. vivax in domestic animals in the tsetse-free regions of Northern Ethiopia, Afar and Tigray.MethodsA cross sectional study was conducted on 754 dromedary camels, 493 cattle, 264 goats, 181 sheep, 84 donkeys, 25 horses and 10 mules. The microhaematocrit centrifugation technique was used as parasitological test. Plasma was collected for serodiagnosis with CATT/T.evansi and RoTat 1.2 immune trypanolysis (ITL) while buffy coat specimens were collected for molecular diagnosis with T. evansi type A specific RoTat 1.2 PCR, T. evansi type B specific EVAB PCR and T. vivax specific TvPRAC PCR.ResultsThe parasitological prevalence was 4.7% in Tigray and 2.7% in Afar and significantly higher (z = 2.53, p = 0.011) in cattle (7.3%) than in the other hosts. Seroprevalence in CATT/T.evansi was 24.6% in Tigray and 13.9% in Afar and was significantly higher (z = 9.39, p < 0.001) in cattle (37.3%) than in the other hosts. On the other hand, seroprevalence assessed by ITL was only 1.9% suggesting cross reaction of CATT/T.evansi with T. vivax or other trypanosome infections. Molecular prevalence of T. evansi type A was 8.0% in Tigray and in Afar and varied from 28.0% in horses to 2.2% in sheep. It was also significantly higher (p < 0.001) in camel (11.7%) than in cattle (6.1%), donkey (6%), goat (3.8%), and sheep (2.2%). Four camels were positive for T. evansi type B. Molecular prevalence of T. vivax was 3.0% and was similar in Tigray and Afar. It didn’t differ significantly among the host species except that it was not detected in horses and mules.ConclusionsNTTAT caused by T. vivax and T. evansi, is an important threat to animal health in Tigray and Afar. For the first time, we confirm the presence of T. evansi type B in Ethiopian camels. Unexplained results obtained with the current diagnostic tests in bovines warrant particular efforts to isolate and characterise trypanosome strains that circulate in Northern Ethiopia.

Widespread occurrence of Trypanosoma vivax in bovines of tsetse- as well as non-tsetse-infested regions of Ethiopia: A reason for concern?

A cross-sectional study was undertaken to assess the prevalence of bovine trypanosomosis in some tsetse-infested and tsetse-free areas of Ethiopia. From August 2010 till April 2011, a total of 1524 animals were parasitologically examined and compared by the haematocrit centrifugation technique (Woo test) and polymerase chain reaction (ITS-1 PCR). The ITS-1 PCR was more sensitive and more accurate in species identification than the Woo test. In ITS-1 PCR, an overall trypanosome prevalence of 31.0% was observed that is significantly (P<0.001) higher than in the Woo test (5.3%). Trypanosoma vivax was the predominant taxon (24.9%), followed by T. theileri (6.0%), T. congolense (2.9%) and Trypanozoon (1.6%). Mixed infections were quite common (14% of all infections). The overall prevalence of trypanosome infections in tsetse area (32.4%) was not different from non-tsetse area (30.5%) neither were the prevalences of T. vivax in both areas (respectively 22.6% and 25.7%). With these high prevalences, bovine trypanosomosis continues to hinder animal production and productivity in Ethiopia, both in tsetse-infested and non-infested parts of the country. Attempts to control African trypanosomosis should also pay attention to mechanically transmitted pathogenic trypanosomes and should adopt the most advanced molecular tests for species identification.

Trypanosoma vivax GM6 Antigen: A Candidate Antigen for Diagnosis of African Animal Trypanosomosis in Cattle

Background Diagnosis of African animal trypanosomosis is vital to controlling this severe disease which hampers development across 10 million km2 of Africa endemic to tsetse flies. Diagnosis at the point of treatment is currently dependent on parasite detection which is unreliable, and on clinical signs, which are common to several other prevalent bovine diseases. Methodology/Principle Findings the repeat sequence of the GM6 antigen of Trypanosoma vivax (TvGM6), a flagellar-associated protein, was analysed from several isolates of T. vivax and found to be almost identical despite the fact that T. vivax is known to have high genetic variation. The TvGM6 repeat was recombinantly expressed in E. coli and purified. An indirect ELISA for bovine sera based on this antigen was developed. The TvGM6 indirect ELISA had a sensitivity of 91.4% (95% CI: 91.3 to 91.6) in the period following 10 days post experimental infection with T. vivax, which decreased ten-fold to 9.1% (95% CI: 7.3 to 10.9) one month post treatment. With field sera from cattle infected with T. vivax from two locations in East and West Africa, 91.5% (95% CI: 83.2 to 99.5) sensitivity and 91.3% (95% CI: 78.9 to 93.1) specificity was obtained for the TvGM6 ELISA using the whole trypanosome lysate ELISA as a reference. For heterologous T. congolense field infections, the TvGM6 ELISA had a sensitivity of 85.1% (95% CI: 76.8 to 94.4). Conclusion/Significance this study is the first to analyse the GM6 antigen of T. vivax and the first to test the GM6 antigen on a large collection of sera from experimentally and naturally infected cattle. This study demonstrates that the TvGM6 is an excellent candidate antigen for the development of a point-of-treatment test for diagnosis of T. vivax, and to a lesser extent T. congolense, African animal trypanosomosis in cattle.

Efficacy of Cymelarsan® and Diminasan® against Trypanosoma equiperdum infections in mice and horses

Trypanocidal sensitivity studies were conducted to assess the efficacy of Diminazene diaceturate (Diminasan) and Bis (aminoethylthio) 4-melaminophenylarsine dihydrochloride (Cymelarsan) against Trypanosoma equiperdum (isolated from two mares with chronic cases of dourine) 713/943 and 834/940 Dodola strains in experimentally infected mice and horses. Diminasan at doses from 3.5 mg/kg to 28 mg/kg and Cymelarsan at doses of 0.25 mg/kg and 0.5 mg/kg body weight failed to cure any of the mice, indicating a clear dose dependent relationship in the mean time of relapse observed in mice. Indeed, mice treated with lower doses relapsed after a shorter time than mice treated with higher doses. However, mice treated with Cymelarsan at doses of 1.0 mg/kg and 2.0 mg/kg body weight were cured and no parasitemia was observed for 60 days. The efficacy of Cymelarsan was also tested in horses. Two groups of horses containing two animals each were infected with T. equiperdum 834/940 Dodola strain and treated with Cymelarsan at a dose rate of 0.25 mg/kg and 0.5 mg/kg, respectively. Cymelarsan at 0.25 mg/kg and 0.5 mg/kg body weight cleared parasitemia within 24 h post treatment and none of the animals were found to show relapse throughout the 320 days of observation. The sensitivity of the particular trypanosome strain to Cymelarsan was also supported by the relative improvement in the mean PCV levels of horses following treatment. A statistically significant difference (p<0.01) in the mean PCV levels of horses treated with Cymelarsan was observed between day 20 at peak parasitemia and days 40 as well as 60 of observation. The mean PCV levels of horses in the control group progressively decreased within the first 60 days of post infection. Two of the horses in the control group developed chronic form of dourine manifested by genital as well as nervous signs with progressive loss of body condition within 320 days post infection. The efficacy of Cymelarsan against the chronic form of dourine was confirmed after treatment of one of the control horses with Cymelarsan at a dose rate of 0.25 mg/kg body weight at day 282 post infection. It was noted that the treated horse improved overall body condition and clinical signs such as incoordination of hind legs, weakness and ventral oedema disappeared within 10 days of treatment. Thus, Cymelarsan was found to be quite effective in curing horses in acute as well as chronic form of dourine. The results obtained from the present study will be important for designing effective control measures against dourine.

A Proline Racemase Based PCR for Identification of Trypanosoma vivax in Cattle Blood

A study was conducted to develop a Trypanosoma vivax (T. vivax) specific PCR based on the T. vivax proline racemase (TvPRAC) gene. Forward and reverse primers were designed that bind at 764–783 bp and 983–1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide.

Trypanosome infection in dromedary camels in Eastern Ethiopia: Prevalence, relative performance of diagnostic tools and host related risk factors.

A cross-sectional study was conducted in Chifra and Dewe districts of Afar region, Eastern Ethiopia, to determine the prevalence, agreement between diagnostic tests and host related risk factors of trypanosome infection in camel. An overall prevalence of 2%, 24.1%, 21.3%, 9.5% and 7.8% was recorded with respectively Giemsa stained thin blood smear, CATT/T. evansi, RoTat1.2 PCR, 18S PCR and ITS-1PCR in a cohort of 399 animals. Only one T. vivax infection was confirmed by TvPRAC PCR indicating T. evansi as the predominant species affecting camels in the study area. No single animal was positive when tested with T. evansi type B specific EVAB PCR. There was slight agreement between the CATT/T. evansi and the molecular tests. Among the PCR methods, RoTat 1.2 PCR yielded a significantly higher positivity rate compared to 18S PCR and ITS-1 PCR. There was no significant difference in the positivity rate observed in each gender of camels (p>0.05). The positivity rate was significantly higher in camels with poor body condition and in older animals when tested using the CATT/T.evansi or RoTat 1.2 PCR (p>0.05). Camels that tested positive with all tests had significantly lower PCVs (p<0.05). This study provides further evidence that T. evansi is endemic in the Afar region of Ethiopia. The latent class analysis indicated an estimate overall prevalence of 19% (95% CI: 13-28). Moreover, the model indicated low sensitivity of CATT/T. evansi (43%) and the PCR tests (39-53%) but higher specificity of the PCR tests (86-99%) and low specificity of CATT/T. evansi (80%). This study suggests that improved sensitivity and reliability of the tests would help diagnosis of trypanosomosis. Further studies are required to determine the prevalence of clinical disease and losses due to trypanosomosis.

Detection of African animal trypanosomes: The haematocrit centrifugation technique compared to PCR with samples stored on filter paper or in DNA protecting buffer

The present study aimed at comparing the trypanosome specific 18S-PCR-RFLP using samples stored either on Whatman filter papers (PCR-RFLP-fp) or in a commercial cell lysis and DNA protecting buffer (PCR-RFLP-pb) with the haematocrit centrifugation technique (HCT), a method widely used for the diagnosis of African Animal Trypanosomosis. Out of 411 head of cattle, 49 (11.92%) (CI=8.95-15.45) scored positive for the presence of trypanosomes by HCT whereas 75 (18.25%) (CI=14.63-22.33) and 124 (30.17%) (CI=25.77-34.86) scored positive using PCR-RFLP-fp and PCR-RFLP-pb, respectively. Out of the 49 positives by HCT, 14 (28.57%) (CI=16.58-43.26) and 28 (57.14%) (CI=42.21-71.18) were concordant by PCR-RFLP-fp and PCR-RFLP-pb, respectively. None of the PCR techniques detected parasites from the Trypanozoon group. Although HCT detected more cases of Trypanosoma vivax (33), species identification using PCR-RFLP-fp and PCR-RFLP-pb were significantly different (p<0.001) from the HCT technique. The use of DNA protective buffer is thus recommended as the output of the PCR-RFLP-pb is improved and the risk of contamination between samples is reduced.

Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis

Animal trypanosomosis caused by Trypanosoma vivax (T. vivax) is a devastating disease causing serious economic losses. Most molecular diagnostics for T. vivax infection target the ribosomal DNA locus (rDNA) but are challenged by the heterogeneity among T. vivax strains. In this study, we investigated the rDNA heterogeneity of Ethiopian T. vivax strains in relation to their presence in tsetse-infested and tsetse-free areas and its effect on molecular diagnosis. We sequenced the rDNA loci of six Ethiopian (three from tsetse-infested and three from tsetse-free areas) and one Nigerian T. vivax strain. We analysed the obtained sequences in silico for primer-mismatches of some commonly used diagnostic PCR assays and for GC content. With these data, we selected some rDNA diagnostic PCR assays for evaluation of their diagnostic accuracy. Furthermore we constructed two phylogenetic networks based on sequences within the smaller subunit (SSU) of 18S and within the 5.8S and internal transcribed spacer 2 (ITS2) to assess the relatedness of Ethiopian T. vivax strains to strains from other African countries and from South America. In silico analysis of the rDNA sequence showed important mismatches of some published diagnostic PCR primers and high GC content of T. vivax rDNA. The evaluation of selected diagnostic PCR assays with specimens from cattle under natural T. vivax challenge showed that this high GC content interferes with the diagnostic accuracy of PCR, especially in cases of mixed infections with T. congolense. Adding betain to the PCR reaction mixture can enhance the amplification of T. vivax rDNA but decreases the sensitivity for T. congolense and Trypanozoon. The networks illustrated that Ethiopian T. vivax strains are considerably heterogeneous and two strains (one from tsetse-infested and one from tsetse-free area) are more related to the West African and South American strains than to the East African strains. The rDNA locus sequence of six Ethiopian T. vivax strains showed important differences and higher GC content compared to other animal trypanosomes but could not be related to their origin from tsetse-infested or tsetse-free area. The high GC content of T. vivax DNA renders accurate diagnosis of all pathogenic animal trypanosomes with one single PCR problematic.