Abstract
A wide range of animal reservoir hosts occurs for the two trypanosome subspecies infective
to
man, Trypanosoma brucei rhodesiense and T. b. gambiense. Morphologically, these two
subspecies are not distinguishable from a third one, T. b. brucei, which does solely infect
animals. Only a partial differentiation is possible by biological, biochemical or molecular
biological methods. Therefore, quick and reliable test systems are required, which identify
animals as carriers of human infectious trypanosomes.
In this study the PFGE karyotype patterns of 16 T. brucei reference stocks/clones (5 T. b.
brucei, 9 T. b. gambiense und 2 T. b. rhodesiense), as well as 18 field isolates and 7 clones
from animals and 4 field isolates from Rhodesian sleeping sickness patients were compared
regarding their human serum sensitivity/resistance. The field isolates have been isolated
between February 1990 and July 1992 in Bulutwe, Mukono district, south-eastern Uganda,
where Rhodesian sleeping sickness is known to be endemic.
The in vitro HSRT as well as the in vivo BIIT (8 selected field isolates) were used to
determine the resistance of Trypanosoma brucei bloodstream forms to human serum. The
reference stocks showed human serum sensitivity (T. b. brucei) or resistance (T. b.
gambiense) in the HSRT as expected. Of the T. b. rhodesiense reference stocks, one was
strongly resistant but the other one sensitive. However, unequivocal HSRT results of
rhodesiense stocks can be explained by loss of human serum resistance after subpassaging
or
cultivation in absence of human serum. Of the 18 field isolates from pigs and cattle in
Bulutwe, 3 were resistant, 6 subresistant and 9 sensitive in the HSRT. The 7 clones showed
human serum sensitivity like their origins. Of the 5 animal isolates selected for BII-testing, 3
were resistant and 2 subresistant. These results were only partially identical with earlier
BIIT-investigations,
supporting the fact that T. b. rhodesiense can loose human serum resistance.
The 4 stocks isolated from Rhodesian sleeping sickness patients were human serum
resistant,
either in the HSRT or the BIIT (3 selected ones), as expected.
To figure all trypanosome chromosomes in a range from 95kbp to 3Mbp, a Biometra
Rotaphor ® type V PFGE unit (Biometra, Göttingen, D) was used applying four different
PFGE running conditions (0-III). Good PFGE results were achieved by using 2x10^9
trypanosomes/ml PSG buffer to prepare agarose blocks and lysing them in situ in the blocks
with 1mg proteinase K/ml NDS buffer for 48h. Chromosome bands were visualized by
staining with 1 µg/ml ethidium bromide (EtBr) for 30 min and destaining over night. Gels
were photographed on a UV light box, the pictures edited in the documentational program
BioDoc ® (Biometra, Göttingen, D) and the banding patterns analysed by ScanPack 3.0 ®
cluster analysis program (Biometra, Göttingen, D).
The stability and reproduceability of PFGE karyotype patterns were confirmed by analysing
clones and their origins as well as bloodstream forms and procyclics or
different VATs. A
100% similarity was observed. These findings implicate that trypanosomes do not change
their karyotype during cyclical or variant antigen type development and that clones exhibit
the
same karyotype as their origin.
The reference stocks and clones exhibited extremely diverse banding patterns both in terms
of
size and numbers of chromosome-sized DNA molecules. In the region of mini- (MC) and
mega-base (MBC) chromosomes all reference stocks displayed almost the same number of
bands (4-7 MC, 4-6 MBC). Only the number of intermediate chromosomes (IC) varied, with
T. b. gambiense showing less (0-4 IC) than non-gambiense stocks (3-6 IC). However, none
of
the T. brucei subspecies formed a separately clustered group within the dendrogram. The
reference stocks were distributed among the different branches with low similarities between
them, implicating that no subspecies differentiation is possible according to the PFGE
results.
The banding patterns of the Bulutwe field isolates from pigs and cattle appeared to be more
homogeneous, some of them even identical, as far as analysed. They showed a higher level
of
similarity than the human field isolates, whose banding patterns were as different as those of
the references. However, similar or identical karyotypes of different stocks could only be
related to an identical isolation date or animal. The grouping of the trypanosome stocks and
clones analysed (I-V) according to their PFGE banding patterns does not allow a
differentiation into human serum sensitive or resistant stocks, with the exception of group IV
containing only human serum resistant T. b. gambiense stocks. Additionally, no single band
correlated with the human serum sensitivity/resistance of a stock could be determined.
The PFGE method is easy to learn and carry out. It can specifically characterize
trypanosome
stocks and is able to detect even minimal changes in their genotype. Nevertheless, no
differentiation into human serum sensitive or resistant trypanosome stocks, nor into the 3
subspecies was possible with respect to the karyotypes. The reason for this is the sensitivity
of PFGE lying at the level of chromosome size changes and therefore being relatively low. In
trypanosomes, those size changes appear very often due to spontaneous
gene-rearrangements.
To enlarge the sensitivity of the method, restriction enzyme digestion of chromosomes is
suggested. Even more sensitive are the PCR methods, although they contain a high risk of
contamination. |
