Molecular detection of Piroplasmida's multiple infection in Burmese ferret badger (Malogale personata)

Manakorn Sukmak1,2*, Worawidh Wajjwalku1,3, Benchapol Lorsunyaluck4, Nongnid Kaolim2, Waradee Buddhakosai3

ABSTRACT Small carnivora, e.g., ferrets, raccoon, mongoose, and civet, most of them were fed as a domestic pet but some of them were bred in farm system for essential oil production. Burmese ferret- badger has been listed as least concern by IUCN (International Union for Conservation of Nature) and declared as a protected wildlife according to Wildlife Preservation and Protection Act, B.E.2535 (1992) of Thailand. Several genera of blood parasites are transmitted by tick. Tick-borne parasites, including Babesia spp., Theileria spp., and Cytauxzoon spp., affected health in various mammal species by multiplication themselves intraerythrocyte, lead to anemia. Many infections have been reported across human, domestic and wildlife animals. In this study, we amplified entired 18s rRNA gene to identify the blood parasites. The generated fragments were ligated to the vector by using TA cloning kit before transformed to E. coli strain DH10β. The transformed colonies were randomly sent for sequencing. Sequences analysis revealed the multiple co-infection of four blood parasites in this animal. The phylogenetic tree showed different four distinct clades i) Babesia gibsoni, ii) raccoons’ Babesia spp, iii) unidentify Babesia spp. and iv) Cytauxzoon spp. However, more colonies of transformed E. coli should be subjected to sequence for checking the repeatability of the results. For more clarifier on speciation of blood parasites which found in this study, sequencing of other informative regions e.g. ITS 1, 5.8s rRNA, ITS 2 and 28s rRNA, are needed.

Key words: multiple infection, piroplasmida, ferret badger * Corresponding author; email-address: [email protected] 1Department of Farm Resources and Production Medicine, Faculty of Veterinary Medicine, Kasetsart University, Kampaengsaen campus, Nakhon Pathom, Thailand. 2Kampaengsaen Veterinary Diagnosis Center, Faculty of Veterinary Medicine, Kasetsart University, Kampaengsaen campus, Nakhon Pathom, Thailand. 3Interdisciplinary Program in Genetic Engineering, Graduate School, Kasetsart University, Bangkhen campus, Bangkok, Thailand. 4Wildlife Unit, Veterinary Teaching Hospital, Kasetsart University, Kampaengsaen campus, Nakhon Pathom, Thailand.

INTRODUCTION Blood parasite infection causing a vital health problem has been reported from several domestic and wild animals, both herbivore and carnivore. Several genera of blood parasites are transmitted by tick. Tick-borne parasites, including Babesia spp., Theileria spp., and Cytauxzoon spp., affected health in various mammal species by multiplication themselves intraerythrocyte, lead to anemia. Many infections have been reported across human and domestic animals, and showed some evidences of multiple genotypes co-infection especially in high endemic area (Homer et al., 2000). Normally, blood parasites are identified by their morphology, e.g., their length, size. However, identify by their characteristic need personally parasitologists’ experience and sometimes could not conclude their species due to morphology variation. The molecular tools are more precise and revealing informative genetic data. Genetic data reveals the detail on species identification, phylogenetic analysis, molecular epidermiology and host-parasite interaction. Since the small subunit ribosomal rRNA gene (SSU rRNA) has mostly been studied as a genetic marker in various species of tick-borne parasites, this gene clarified the phylogenetic relationship within and between them. Moreover, the SSU rRNA gene has been used to enable finer molecular distinction among trypanosome isolates by using entire and/or partial part of 18s rRNA gene, internal transcribed spacer 1 ( ITS 1), 5.8s ribosomal RNA gene ( 5.8s rRNA), internal transcribed spacer 2 (ITS 2) and 28s ribisomal rRNA gene (28s rRNA). Recently, we used the molecular technique to identify the tick-borne Piroplasmida in Burmese ferret-badger (Sukmak et al., 2011). Two genotypes of Babesia spp. were detected based on partial 18s rRNA. In this study, we sequenced entired 18s rRNA gene to reveal the multiple co- infection of blood parasites in this animal. The obtained sequences were compared with available sequences of tick-borne parasites to construct the phylogenetic tree.

MATERIALS AND METHODS The male ferret badger was anesthesia and collected blood sample for blood morphology and biochemistry in order to annual health check. Microscopic study on blood smear revealed blood parasite infection. According to morphology, they were suspected as Babesia spp. The genomic DNA from blood sample was further extracted by phenol-chloroform method. The entired 18S rRNA fragment was amplified by using two new design primers which specific to tick-borne parasites including BAB1w (5’-GAA CCT GGT TGA TCC TGC CAG T-3’) and BAB2w (5’-GAT CCT TCT GCA GGT TCA CCT A-3’). The PCR condition was performed by Phusion Hotstart High-felidity II. The PCR step were consisted of 98°C for 5 minutes pre-denaturation following by 40 cycles of 98°C for 30 seconds denaturation, 58°C 30 seconds annealing and 72°C 45 seconds extension, and final extension step at 72°C for 5 minutes. The generated fragment was ligated to the vector by using TA cloning kit before transformed to E. coli strain DH10β. We randomly selected 4 colonies of each PCR product to extract inserted gene for sequencing by First Base Laboratory, Shah Alam, Malaysia. The sequences were identified by BLAST with the database in GenBank (ncbi.nlm.nih.gov). Sequences alignment was done by Bioedit software (Copyright ©Tom Hall 1997-2007). Phylogenetic analysis was constructed by MEGA6 (Tamura et al., 2013).

RESULTS AND DISCUSSION About 1,600 base pairs of 18S rRNA gene were successfully generated by PCR using this new design primer. Among 4 colonies, the Blasts’ results divided our sequences into two distinct groups; (i) first group was similar to Babesia spp., and (ii) second group was similar to Cytauxzoon spp. To deeper verify the species of parasites, we also constructed phylogenetic tree by comparison the sequences of entired 18S rRNA gene of Babesia spp., Theileria spp., Cytauxzoon spp., from GenBank. Neighbor-joining phylogenetic tree with 10,000 bootstraps was constructed by MEGA6 (Figure 1) and 18s rRNA sequence of Cryptosporidium felis was used as an outgroup. The phylogenetic tree showed complete separation between Babesia spp., Theileria spp., Cytauxzoon spp., Babesia microti, and Cryptosporidium felis. Our sequences were divided into four clades. Clone no. 26 was clustered with Cytauxzoon spp., while others clustered with Babesia spp. similar to Blasts’ results. Among Babesia spp., clone no. 5 was clustered with Babesia gibsoni group, clone no. 27 was clustered with new Babesia spp. which commonly found in raccoon and American mink from Japan(Jinnai et al., 2009; Hirata et al., 2013). Interestingly, clone no. 25 was not clustered with any reported Babesia spp. or Cytauxzoon spp., and showed distinctly branching from other sequences with high bootstrapping score (99). It was considered that the host animal had not shown any clinical symptom suppose to be the new reservoir host. However, it was kept in captive circumstance and able to contact with other species. The clinical sign should be observed along with health checking of other surrounding animals. Not only the host infection but also the tick infestation should be survey. We can conclude that the method could be preliminary applied to survey the infection in other host species as well. In this study, only one animal was molecularly detected the incidence of multiple infection by Piroplasmida parasites. More samples should be collected not only ferret badgers but also other carnivores and their vectors to understand the transmission of these parasites.

Figure 1: Neighbor-joining phylogenetic tree (10,000 bootstraps) of 18S rRNA gene (about 1,600 bp.) was calculated by MEGA6. Asterisk refers to sequence obtained in this study. The phylogenetic tree showed complete separation between Babesia spp., Theileria spp., Cytauxzoon spp. The black baskets showed the group of Babesia spp. reported in raccoon, B. gibsoni, Cytauxzoon spp. and Theileria spp.

CONCLUSION This study is the first report of multiple infections of tick-borne parasites including Babesia gibsoni, raccoons’ Babesia spp., unidentify Babesia spp. and Cytauxzoon spp. infection in Burmese ferret badger. However, more colonies of transformed E. coli should be subjected to sequence for checking the repeatability of the results. For further studies, sequencing of other informative regions e.g. ITS 1, 5.8s rRNA, ITS 2 and 28s rRNA, can help to clarify the parasite linage.

ACKNOWLEDGEMENTS We thank Benchapol Lorsunyaluck from Wildlife Unit, Kasetsart University Veterinary Teaching Hospital, Kampaeng Saen Campus and Private Zoo for helping us with sample collection and we thank the Faculty of Veterinary Medicine, Kasetsart University,Thailand for laboratory supports.

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