Babesia pisicii n. sp. and Babesia canis Infect European Wild Cats, Felis silvestris, in Romania

Abstract

Haemoparasites of the genus Babesia infect a wide range of domestic and wild animals. Feline babesiosis is considered endemic in South Africa, while data on Babesia spp. infection in felids in Europe is scarce. Using samples from 51 wild felids, 44 Felis silvestris and 7 Lynx lynx, the study aimed to determine the presence and genetic diversity of Babesia spp. in wild felids in Romania by analyzing the 18S rDNA and two mitochondrial markers, cytochrome b (Cytb) and cytochrome c oxidase subunit I (COI) genes. By 18S rDNA analyses, Babesia spp. DNA was detected in 20 European wild felids. All sequences showed 100% similarity to B. canis by BLAST analysis. Conversely, Cytb and COI analyses revealed the presence of two Babesia spp., B. pisicii n. sp., which we herein describe, and B. canis. The pairwise comparison of both mitochondrial genes of B. pisicii n. sp. showed a genetic distance of at least 10.3% from the most closely related species, B. rossi. Phylogenetic analyses of Cytb and COI genes revealed that B. pisicii n. sp. is related to the so-called “large” canid-associated Babesia species forming a separate subclade in a sister position to B. rossi.

Apicomplexan haemoparasites in domestic cats in Romania

“…In Europe, epidemiological data on Babesia infection in domestic cats are limited to a few molecular studies targeting the 18S rDNA , with B. canis reported in Spain and Portugal [ 14 , 15 ], B. vogeli in Portugal [ 15 , 16 ], and B. microti in Italy [ 12 , 13 ]. Babesia canis was also recently reported in wild cats from Romania [ 18 ].…”

“…The 18S rDNA , a highly conserved region, is the primary PCR target used in studies addressing the diagnosis of piroplasmids [ 71 , 72 ]. However, several studies have questioned its ability to differentiate between closely related species [ 2 , 18 , 64 , 73 , 74 ]. In the present study, BLAST analyses of the 18S rDNA sequences showed 99–100% identity with different sequences of B. canis , although the presence of this species was confirmed in only six samples by Cytb gene amplification and analyses.…”

“…Babesia canis was previously reported in domestic cats in Europe, the data being supported exclusively by relatively short 18S rDNA fragments [ 14 , 15 ]. Therefore, as previously highlighted [ 18 ], our recommendation remains to avoid using protocols targeting the 18S rDNA for piroplasmid species differentiation, and samples amplified by these protocols should be considered as Babesia sp. PCR protocols based on mitochondrial gene detection can be successfully used for species confirmation, but have shown lower sensitivity than 18S rDNA amplification protocols (100 to 1000 times lower sensitivity than the protocol targeting the 376-bp fragment of B. canis 18S rDNA ) [ 18 , 74 ].…”

“…Amplification of the second round was carried out in a 25 μl reaction mixture consisting of 12.5 μl of 2× PCRBIO Taq Mix Red (PCR Biosystems, London, UK), 400 nM of each primer, and 1 μl of the first PCR round as the template. One positive control, consisting of DNA from carnivores previously confirmed as positive for the targeted pathogens [ 18 , 64 , 68 ], and a negative control represented by sterile water were included in each reaction set. Amplicons were visualized on 1.5% agarose gels stained with ECO Safe Nucleic Acid Staining Solution (PacificImage Electronics, New Taipei City, Taiwan).…”

“…Nevertheless, the European cases were rarely associated with clinical manifestations. Recently, Babesia pisicii was described in European wild cats, Felis silvestris in Romania [ 18 ], but its presence in domestic cats has not been documented yet.…”