Trypanosoma evansi and the three subspecies of T. brucei are indistinguishable from each other morphologically and are thought to be very closely related evolutionarily. The most notable molecular difference between T. evansi and the T. brucei subspecies is the structure of the DNA molecules found in their kinetoplast, a subcellular organelle located near the flagellar pocket. We have exploited this difference in kDNA sequence and organization to develop a polymerase chain reaction (PCR)-based assay that can distinguish between T. evansi and T. brucei. Oligonucleotide primers spaced about 370 bp apart on the T. evansi kDNA sequence generate a PCR-amplification product when total T. evansi DNA is used as the template, whereas no amplification product occurs when total DNA from T. brucei or Leishmania is the template. In contrast, when PCR primers specific for three different nuclear DNA sequences of T. brucei -a 177-bp repeat, the procyclin gene or the spliced leader gene - are used in similar PCR amplifications, identical PCR products are obtained from the T. brucei and T. evansi template DNAs. Thus, the nuclear DNA sequences of T. evansi and T. brucei are very similar, but their kDNAs are sufficiently different to serve as the basis for PCR-based diagnosis of the two trypanosome species.
Molecular genetic studies on the African trypanosome have in the past focused on only a few genes or gene products; consequently, a significant number of genes of this organism remain unknown. We are sequencing random directional cDNAs clones of Trypanosoma brucei rhodesiense to generate Expressed Sequence Tags (ESTs). Analysis of 2128 clones sequenced so far showed significant similarities with proteins already described in diverse organisms including man, rodents, kinetoplastids, yeasts and plants. A number of the ESTs encode homologues of proteins involved in: signal reception and transduction, cell division, gene regulation, DNA repair and replication, general metabolism, and structural integrity. A large proportion, 562 individual ESTs (26%, representing 299 different transcripts), had significant homology with genes described in organisms other than the African trypanosomes; however, 14% of the ESTs were from genes already described in trypanosomes. We expect that a number of the genes will be present in the other species of the African trypanosomes. This prediction has been tested by hybridization of the random ESTs with total cDNAs of other trypanosome species including T. evansi. The data will be discussed in the context of their potential contribution to understanding the biology of these and other parasitic protozoa, and the diseases they cause.
We have induced the loss of kinetoplast DNA in Trypanosoma evansi by successive treatment of infected mice with a basic fuchsin dye, pararosaniline.
The “akinetoplastic” clones induced from a “kinetoplastic” strain were propagated by single cell inoculation into mice. In the course of assessing akinetoplastidy by DAPI staining after pararosaniline treatment, various fragmentation patterns and smaller–sized kDNA were detected. Semi-quantitative measurement of kDNA fluorescence indicated that pararosaniline causes a gradual decrease of the minicircle DNA. We attempted to obtain the parasites with intermediate kDNA, however these were unstable and tending to become akinetoplastic.
We then compared the growth rate of the akinetoplastic parasites with the original kinetoplastic strain. The growth of the kinetoplastics was relatively constant in the presence or absence of pararosaniline. On the other hand, the kDNA-deficient mutants showed a delayed growth rate and it was retarded when they were subjected to increasing dosage of the dye. According to these results we speculate that minicircle DNA of Trypanosoma evansi is not essential for the parasite growth, however it may facilitate the division and segregation of the mitochondrion and the genome during cell division.
An attempt to the development of a simple in vitro method to detect the drug sensitivity of Trypanosoma evansi was made. Bloodstream forms (BSFs) of kinetoplastic T. evensi, T. evansi IL3354 and T. evensi IL3960 populations, and akinetoplastic T. evensi AK, T. evensi IL1934 and T. evensi IL3960-AK populations were propagated in vitro in an axenic culture system, in which bloodstream forms of T. evansi were cultured in the absence of feeder layer cells using HMI-9 culture medium following the procedure described earlier (Hirumi and Hirumi 1994).
With a minor modification of the original drug sensitivity test (Hirumi et al. 1993), the minimum effective concentration of diminazene aceturate (DA) (Berenil ® and TRYPAN®) was tested for theT. evensi populations. Five hundred μl aliquots of trypanosome suspension in the medium, containing 2 × 104 BSFs, were placed in each well, added an equal volume of drug solution containing various concentrations of DA in culture medium and maintained at 37℃ with 5% CO2 in air for 10 days without medium change. Effects of the drug were examined by phase-contrast microscopy every 24 hrs. In the control group with no drug, trypanosomes increased in number during the initial 3 days and reached the maximum cell density (1-2 ×106 BSFs/ml). Trypanosomes, thereafter, rapidly decreased during the following 2 days and died by day 7 due to overpopulation. In contrast, trypanosomes maintained in the medium contained ineffective doses of DA, rapidly decreased in number and died by days 3-5. While, in the medium contained lower doses, numbers of BSFs gradually decreased to the levels of 10-103 BSFs/well during the initial 3-5 days. Thereafter, the survived parasites increased in number reaching the maximum density during the following 3-5 days and then died by days 7-10.
Although no significant difference was observed between the kinetoplastic and chemically induced akinetoplastic populations tested, the results demonstrated that the minimum effective concentration of the drug tested can be readily detected by means of phase-contrast microscopy without aids of sophisticated equipments, including a freeze-dryer which was used in the original method. The method may be simple enough to apply in ordinary research laboratories which are equipped with standard cell culture equipments including a CO2 incubator and an inverted phase-contrast microscope. The results were highly reproducible.
A study was conducted to assess the efficacy of three trypanocidal drugs, viz. melarosamine, diminazine and quinapyramine, in the treatment of experimental Trypanosoma evansi infection. Sixteen camels were divided into four groups (four camels per group). Groups Ⅰ, Ⅱ and Ⅲ were treated with melarosamine, diminazine and quinapyramine, respectively. Group Ⅳ served as non-infected and non-treated control. Within each groupⅠ, Ⅱand Ⅲ, one camel was kept as infected non-treated control. Cure rates of 66.66％, 66.66％ and 33.33％ were recorded, for melarosamine, diminazine and quinapyramine treated camels, respectively. Some hematological and blood biochemistry parameters were also recorded on control and trypanosome infected camels. The mean Packed Cell Volume (PCV) of infected animals dropped from 29±3 to 21.35±6.5 before treatment. The mean value of neutrophils, eosinophils, basophils，lymphocytes and monocytes were 32.7％, 1.80％, l.06％, 62.4％ and 1.30％, respectively. Value of mean total protein was 8.92g/dl while Albumin and Globulin were 2.62g/dl and 6.20g/dl respectively.
A study was conducted in Uaso area on the border of Isiolo and Laikipia districts in Kenya to determine the cause(s) of treatment failure in camel trypanosomosis. Ten trypanosome isolates from infected camels were characterized by morphology and procyclic transformation test. Their sensitivity to quinapyramine sulphate and melarsomine were then determined using an in vitro assay system. Six isolates were Trypanosoma evansi, three T. congolense and one had both T. congolense and T. evansi. All the T. congolense isolates were resistant to melarsomine at 1.2 mg/kg bwt whereas one was resistant to the quinapyramine sulphate at 7.4 mg/kg bwt. On the other hand one of the six T. evansi isolates, one was resistant to melarsomine at 1.2 mg/kg bwt and three to quinapyramine sulphate. Camel owners got little veterinary advice leading to wrong models administration of trypanocides, underdosing and use of unrecommended drugs. The efficacy of trypanocides in the area appears to be hampered by drug resistance and inappropriate drug use. Uaso falls within a tsetse belt and as such camels are susceptible to infection with tsetse transmitted trypanosomes．A drug effective against T. evansi，T. congolense and T. brucei brucei should therefore be used in the treatment of trypanosomosis in camels in this area. They should be educated on the proper use of trypanocidal drugs.
In the Soudano-guinean zone of Madina-Diassa Ranch in Mali a comparative study was carried out to assess attractiveness of Biconical, Cubical(F3), Pyramidal and “Vavoua” traps and/or odour associations: acetone/1-octen-3-o1, acetone/1-octen-3-ol/meta-cresol, 1-octen-3-ol/meta-cresol and acetone/1-octen-3-ol/cow urine.
In the woody savannah, Cubical, “Vavoua” and Pyramidal traps were found to be efficient against tabanidae than the Biconical trap. They attracted respectively 6.4, 5.9 and 4.2 times more than the Biconical trap. The “Vavoua” trap was most efficient against Stomoxys. It caught 15.6 tines more than the Cubical one, 15.4 times more than the Biconical trap and 6.2 tines more than the Pyramidal one.
In the forest gallery. the Cubical trap attracted 2.0 times more tabanidae than the Pyramidal trap and 4.4 times more than the Biconical trap. The “Vavoua” trap was 3 times more efficient against tabanidae than the Biconical trap. It caught 2.4, 3.2 and 12.2 times more Stomoxys than respectively the Cubical, Pyramidal and Biconical traps.
As far as odours are concerned, tabanidae were more attracted by the associations of acetone/1-octen-3-ol/cow urine, acetone/1-octen-3-ol/metal cresol and 1-octen-3-ol/meta- cresol. In the woody savannah, Stomoxys were more attracted by the combination acetone/1-octen-3-ol/meta-cresol and 1-octen-3-ol/meta-cresol ; in the forest gallery they are more attracted by acetone/1-octen-3-ol and acetone/1-octen-3-ol/cow urine associations than the others.
The Biconical trap is more effective against tsetse flies, but less effective against tabanidae. The “Vavoua” trap is very attractive for tsetse flies and Stomoxys. Therefore we recommend the use of “Vavoua” traps for vectors control. However, it appears necessary to integrate the control of all the vectors of African Trypanosomiasis, including tsetse flies, tabanidae and Stomoxys.