Molecular characterisation of Trypanosoma Evansi stocks from Indonesia

Abstract

The work described in this thesis used the techniques of karyotype analysis, random amplified polymorphic DMA analysis (RAPD), Riboprinting and simple sequence repeat polymerase chain reaction (SSR-PCR) to examine genetic variation in stocks of Indonesian Trypanosoma evansi. The 80 T. evansi stocks investigated were isolated from natural infections in cattle, buffaloes and horses in 10 widely separated areas, including Aceh, North Sumatra, Lampung, West, central and East Java, Madura, South Kalimantan, North and South Sulawesi.Transverse alternating field electrophoresis (TAFE) was used to determine the polymorphisms present in the karyotype patterns of the 80 T. evansi stocks. Electrophoretic conditions were developed to give good resolution of chromosome bands in the size range 50 kb to 5.7 Mb. The total number of chromosome bands in the T.evansi genome varied from 17-27 depending on the stock. A high degree of polymorphism was detected in the karyotype patterns of T. evansi stocks from Indonesia by TAFE. In the 80 stocks studied, 46 different karyotype patterns were detected. Cluster analysis with a grouping level of 10% fitted the patterns into seven main karyotype groups which correlated with the locality from which the stocks had been isolated. The stability of the karyotype patterns within stocks was not related to differences between the surface variant antigenic types (VAT) in that relapse populations derived from a single infection with a cloned stock of T. evansi showed the identical karyotype pattern to the original infecting stock. In relapse populations collected from an uncloned T.evansi stock different karyotype patterns were found indicating that more than one population was present in the original isolate. Attempts to isolate VAT specific chromosomal probes were not successful. The results from karyotype analysis of T. evansi were shown to be sufficiently stable with regard to the number, sizes, and intensities of the chromosomal bands seen 1) among different cryopreserved preparations of the same strain or clone; 2) after passage through laboratory animals; 3) among different agarose embedded DNA preparation of the same stock or clone; 4) among relapse populations collected from a single infection with a cloned stock; 5) between stocks sensitive and resistant to trypanocidal drugs such as suramin and cymelarsan.Karyotype analysis of T. evansi stocks isolated from buffaloes transported from central Java to North Sumatra indicated that T. evansi infection had been transferred from the local to the transported buffaloes, not vice-versa, during a period of 13 months after transportation. A high degree of similarity in karyotypes was observed in T. evansi stocks isolated from a group of Bali cattle kept together in a feedlot in Lampung indicating that a single T. evansi stock of a particular pattern was responsible for the infection in this outbreak. Stocks collected from West and central Java during a six-year period from 1988-1994 showed a high degree of karyotype patterns stability.patterns stability. The potential polymorphisms with regard to chromosomal locations of several genetic markers including Phospholipase C, Cysteine Proteinase, T. brucei ribosomal RNA coding region, T. brucei tubulin, Aldolase and Glucose 6-phosphate isomerase were determined in T. evansi, T. brucei and T. congolense. The hybridisation patterns correlated with the karyotype pattern shown by each stock of T.evansi. The hybridisation patterns shown by T. evansi stocks were different to those of T. brucei and T. congolense. Random amplified polymorphic DNA (RAPD) analysis of the 80 T. evansi stocks studied yielded only four RAPD patterns. Trypanosoma evansi stocks isolated from a group of buffaloes transported from central Java to North Sumatra showed RAPD patterns that indicated an introduction of T. evansi infection in the central Java buffaloes by North Sumatra stocks during a 13 months period after the transportation. Polymorphism in the RAPD patterns was also detected in T. evansi stocks isolated from a group of Bali cattle kept in a feedlot in Lampung, a particular RAPD pattern was detected in most of the stocks collected. Trypanosoma evansi stocks isolated from relapse populations, however, showed identical RAPD pattern.This study showed that RAPD analysis is useful for characterising T. evansi stocks and could be used to analyse a large number of samples collected from widely distributed areas. The sample preparation for RAPD analysis can be simple, the DNA can be extracted directly from cryopreserved stabilates and the results obtained in the same day. The riboprinting applied in the study did not detect variations among T. ewnsi stocks. Variation in riboprint banding patterns was shown between the T. evansi, T. brucei and T. congolense stocks. The simple sequence repeat PCR (SSR-PCR) assay showed the least specificity. The analysis generated complex banding patterns and polymorphisms in the banding pattern were not easily detected.detected. A number of molecular techniques were used to compare morphologically identical stocks of T. evansi in Indonesia. The results demonstrated greater heterogeneity in T. evansi than previously reported by other workers with stocks of T.cvansi from other areas of the world. This study showed that of the methods used , karyotype analysis by TAFE detected stock variations at a finer level than RAPD analysis, riboprinting and the SSR-PCR assay. The RAPD analysis is, however, preferable for epidemiological studies due to the technical simplicity of performing the assay and the ease of sample preparation.