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.