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.
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