Molekular-phylogenetische Differenzierung von Babesien des Rindes

Molecular phylogenetic differentiation of bovine Babesia In the present study, Babesia bovis, B. bigemina, B. divergens and B. ovata were differentiated with molecular biological techniques. These are four babesia species of cattle which are important in veterinary medicine. In addition to that, relationships within species were investigated. Therefore, two ribosomal DNA (rDNA) genes from babesia of different geographical origin were amplified and sequenced and then analyzed with regard of their phylogenetic correlation. Altogether 50 blood samples were examined, deriving from naturally infected animals or vaccine strains. 11 isolates originated from Europe, 22 from North- und South America, 5 from Africa, 4 from Asia and 8 from Australia. Previously, the Babesia contained in the samples had been assigned to the different species, using phenotypic characteristics. For the PCR amplifications of the 18S gene using the primers RIB-19 and RIB-20 and of the ITS region with the primers RIB-3 and RIB-13 PCR protocols developed for babesia of dogs were used. These were directly applied for B. divergens and, after optimization of MgCl2, DMSO, BSA concentrations and cycle number, with modifications also for B. bovis, B. bigemina and B. ovata. The length of the directly sequenced PCR products of the 18S rDNA was 1448 to 1552 base pairs for B. bovis, 1481 to 1485 bp for B. bigemina and 1516 to 1519 bp for B. divergens, exept one isolate which originated from a human infection and had a length of 1535 bp. For B. ovata, only a partial sequence of 889 bp was obtained. Compared with corresponding genebank sequences, the declared babesia species was confirmed with exception of one isolate. The PCR products of the ITS region were sequenced after cloning and their length resulted in 654 to 729 bp for B. bovis, 883 bp for B. ovata, 923 to 943 bp for B. bigemina and 995 to 997 bp for B. divergens. Despite the variability of the ITS sequence length of the B. bovis und B. bigemina isolates, this feature indicates characteristic values for each species. In the present study the ITS region of bovine Babesia was sequenced and analysed for the first time. With the phylogenetic analysis of the 18S sequences the four examined babesia species of the cattle could unequivocally be distinguished. Different phylogenetic trees (dendrograms) generated with the algorithms Distance Matrix, Maximum Parsimony and Maximum Likelihood showed all isolates in separated species groups. High bootstrap values (Distance Matrix and Maximum Parsimony) of 100 % for B. divergens, for B. bovis and B. bigemina, and 94 % resp. 70 % for B. ovata support the classification in four species. An additional phylogenetic analysis including also piroplasms of other hosts demonstrated a separation of bovine babesia species without overlap. Thereby, the present taxonomy and validity of the species B. divergens, B. bovis, B. bigemina and probably B. ovata were confirmed for the isolates examined in this study. It was not possible to differentiate between the B. divergens isolate collected from a human and the other B. divergens isolates which were very homogeneous. An additional intraspecific analysis for B. bovis and B. bigemina represented the heterogeneity of these two species in different dendrograms. The analysis of the ITS region was based on 95 clones, produced from 50 Babesia bovis, B. bigemina, B. divergens and B. ovata isolates of different geographic origin. Within the B. bovis and B. bigemina isolates an enormous variability was detected. This finding was indicated by intraisolate identities from 93,7 to 100 % for B. bovis and 95,8 to 100 % for B. bigemina. In contrast, the intraisolate identities of B. divergens clones (99,4 to 100 %) and the two B. ovata sequences (100 %) reached high values. On a species level, all clones within each species were compared. This procedure indicated the variability between isolates within each species. Thereby, the homogeneity of the B. divergens clones was confirmed by the identity of at least 99,1 % between two sequences. The corresponding values of B. bigemina (92,7 %) and B. bovis (82,7 %) turned out to be lower. The heterogeneity within the two species was confirmed by these identity scores and by the fact that clones from different isolates exhibited higher identities than clones from the same isolate. In the phylogenetic analysis of ITS sequences dendrograms for each species were generated with the algorithms Distance Matrix, Maximum Parsimony und Maximum Likelihood. The heterogeneity within B. bovis and B. bigemina was reflected by the fact that separated groups of isolates occurred in the phylogenetic trees. In addition, these groups correlated with their geographical origin. The phylogenetic relationship between isolates from Southern Africa and Australia was remarkably high. B. bigemina isolates were separated into two groups: one included the isolates from Australia und South Africa, the other isolates from Latin America. Within B. bovis four isolate groups of different geographic origin were detected. The first included isolates from Israel, Turkey and Morocco, the second isolates from Australia and South Africa. The Brazilian isolates appeared in the third group and the remaining isolates from the Americas in the fourth. There was no arrangement of isolates in phylogenetic trees of B. divergens.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Ludwig-Maximilians-Universität

Year

2004

Author dc:creator

• Vogl, Sigrid

Repository record source_url

https://edoc.ub.uni-muenchen.de/1810/

OAI identifier oai:identifier

oai:edoc.ub.uni-muenchen.de:1810