Untersuchungen zur Piroplasmose bei Hauskatzen in Südafrika

Investigations on piroplasmosis in domestic cats in South Africa
Domestic cats originating from an area in South Africa known to be
endemic for babesiosis were included in an investigation on feline
piroplasmosis. In order to receive some information about the range
of species involved, molecular biological methods were used to
characterize the causal agents genotypically. Two segments of the
rDNA gene, the regions of the 18S and of the first and second
internal transcribed spacers (ITS) were sequenced and phylogenetic
analyses were performed. Based on these sequence data a modern and
sensitive diagnostic test for a specific detection of B. felis, a
real-time PCR, was developed and validated. Using this test, 206
blood samples of cats belonging to the patients of a small animal
surgery in Port Elizabeth were screened for infections with B.
felis and the test results were compared with results of
examinations of stained blood smears and an indirect
immunofluorescent antibody test (IFAT). Sequence analysis of the
18S rDNA and comparison with available sequence data from the
Genbank NCBI demonstrated that at least 2, possibly 3 species are
responsible for piroplasm infections in domestic cats in Port
Elizabeth. The species B. felis was diagnosed in 11 of the 13
isolates. In one isolate, K 8, the parasites were identified as B.
leo, a piroplasm species which had been documented to occur in
lions before. This represents the first detection of B. leo in a
naturally infected domestic cat. The sequence of the isolate K 68
could not be assigned to any known species, but it was closely
related to the other feline piroplasms. The genetic relationship
was slightly closer to B. leo with an identity of 98,3 % than to B.
felis where an identity of 97,8 % was found. Compared to two other
feline piroplasms isolated from a caracal, Babesia sp. Caracal
Strain A and B, the isolate K 68 shared identities of 96,8 % and
96,7 % respectively. Phylogenetic trees clearly separated the
isolate K 68 from the other feline piroplasms indicating a separate
species status. Furthermore the results of the IFAT support a
separation at least from B. felis. The isolate did not show any
cross reactions with B. felis antigen. However, the parasites could
not be differentiated from B. felis or B. leo in stained blood
smears. In phylogenetic analyses including several Babesia and
Theileria species all the feline piroplasm species of the present
study were grouped together with B. rodhaini, B. microti and T.
annae and seperated from the group of the `typical´ babesia and the
classical theileria. When sequence analyses of the ITS rDNA of the
13 selected isolates were performed, the 11 isolates diagnosed as
B. felis showed a polymorphism rate of 2,6 % and high pairwise
identities between 98,6 % and 99,9 %. The sequences of the isolate
K 8, already identified as B. leo, and of the isolate K 68 were
less homologous with identities of 77,0 % and 76,0 % respectively
compared with B. felis. In the phylogenetic tree the isolates K 8
and K 68 were clearly separated from each other and from the B.
felis – isolates. This supports the existence of three distinct
species. For one isolate, the isolate K 60, the intraisolate
variation was determined in more detail. 7 different genotypes were
found. 6 of them were very similar to B. felis and showed sequence
identities between 97,2 % and 99,6 %. The sequence of the genotype
7, clone K 60 E.2, clearly differed from the other genotypes. With
an identity of 76,9 % to B. felis the sequence could not be
assigned to any hitherto described species. In phylogenetic
analyses this clone K 60 E.2 was more closely related to B. leo and
K 68 than to the B. felis isolates. Based on the sequence data of
the ITS rDNA, a real-time PCR method was developed for the specific
detection of B. felis. This test was proven to be sensitive and
very specific in a blinded, externally controlled evaluation. The
specifity was shown to be 100 %. The sensitivity of the test was 75
%. The positive prediction value reached 100 % and the negative
prediction value was 82,1 %. A detection limit of 7 to 77
parasites/µl blood was determined. With this real-time PCR a
modern, sensitive and highly specific method for the detection of
B. felis is now available. The performance of the real-time PCR
were compared with those of stained blood smears and the IFAT by
investigating 206 blood samples of domestic cats of a small animal
surgery in Port Elizabeth.When the results of the blood smears were
compared with the results of the real-time PCR an agreement was
found in 94,6 % (192/203) of the results. Identical results of the
blood smears and IFAT were demonstrated in 91,2 % (187/205) of the
cases. The results of the PCR and the IFAT corresponded in 96,1 %
(196/204) of the investigations. The isolates K 8 and K 68, which
had been diagnosed as B. leo and a closely related species by 18S
rDNA sequence analyses and which had revealed babesia in stained
blood smears, showed a negative test result in the PCR as well as
in the IFAT. 9 isolates that were found to be babesia negative in
blood smears gave positive results in the PCR as well as in the
IFAT. 8 isolates were positive in the IFAT but negative in the PCR.
7 out of these isolates were also proven negative in the blood
smears whereas in the remainig one case the blood smear was not
examined. Infections with piroplasms were diagnosed in a high
portion of the cats examined. The prevalence of babesia determined
by stained blood smear was 32,2 % (66/205). DNA of B. felis was
detected in 35,3 % (72/204) of the cats by real-time PCR. B. felis
specific antibodies were found in 39,3 % (81/206) of the examined
cats in the IFAT. No specific breed predilection was evident. The
results of blood smears and PCR did not correlate with the sex or
age of the cats. However B. felis specific antibodies were detected
more frequently in male than in female animals and in a higher
portion of the cats older than 2 years. Concerning the housing
conditions, significantly more “indoor/outdoor” cats were affected.
DNA of B. felis and specific antibodies were found in a portion
above average of the cats coming from the “Walmer” area. Typical
clinical signs attributed to feline babesiosis were observed in
28,8 % (19/66) of the cats showing babesia in the blood smear, in
25 % (18/72) of the cats with DNA of B. felis detected by PCR and
in 22,2 % (18/81) of the cats that showed a positive test result in
the IFAT. The part of latently infected was thus very high. No
specific breed or sex predilection for clinical affection was
evident. However a correlation of the age of the cats with the
manifestation of a clinical babesiosis is assumed. In the age group
between 0,5 and 2 years the percentage of infected cats showing
clinical signs was above average.