Malayopython reticulatus (Reptilia: Pythonidae) is a non-venomous snake commonly found in Indonesia. This snake is also one of the wild animals susceptible to infestations by various ectoparasites, including ticks. Five tick species have been separately reported to infest M. reticulatus snakes, which are Amblyomma helvolum ((Anastos, 1950); (Anderson & Tzianabos, 1989); (Andoh et al., 2015)),

A. varanense (Anastos, 1950); (Andoh et al., 2015), A. cordiferum (Auffenberg, 1988), and Rhipicephalus sanguineus (Chao et al., 2013). Amblyomma latum was also reported to infest Python regius snakes in Ghana and Togo (Pandit et al., 2011); (Mariana et al., 2011); (Sumrandee et al., 2014); (Andoh et al., 2015).

Ticks known to be associated with pathogens include Ixodes spp., Rhipicephalus spp., Haemaphysalis spp., Amblyomma spp., and Dermacentor spp. ((Estrada-Peña & Jongejan, 1999); (Raoult et al., 2002); (Takano et al., 2014)). Several pathogens are associated with ticks, a few of which are Borrelia sp. and Rickettsia sp. The major groups of Borrelia impacting animal and human health are relapsing fever Borrelia, Lyme disease Borrelia, and reptile-associated (REP) Borrelia ((Bunikis & Barbour, 2005); (Takano et al., 2010); (Franke et al., 2013)). Borrelia spp. was previously detected in A. varanense infesting P. reticulatus in Thailand (Trinachartvanit et al., 2016). Meanwhile, Rickettsia sp. belonging to the spotted fever group (SFG) was also detected from A. transversale and A. trimaculatum ticks infesting P. regius and Boiga forsteni snakes, respectively (Andoh et al., 2015). This research aimed to determine the presence of Rickettsia spp. and Borrelia sp. in ticks that infest wild snakes in Indonesia. In addition, phylogenetic analyses of detected pathogens were also presented.

Ticks were collected within the period of 2021- 2022 from three wild-caught M. reticulatus snakes in Bogor (6°35’42.1368’’ S and 106°48’59.8860’’ E) and Jakarta (20°58’50.736’’ N and 89°40’45.876’’ W) and one P. bivittatus snake found in Jakarta (20°58’50.736’’ N and 89°40’45.876’’ W). The snakes were handled in accordance with good animal welfare practices and released to their original habitats upon examination.

From the skin beneath their scales, ticks were collected using forceps and stored in 70% ethanol. Tick species, stage, and sex were identified based on morphologic features following taxonomic keys and molecular analysis (Anastos, 1950)(Kohls, 1957).

The collected ticks were washed individually and homogenized in 200 μL 10x PBS solution. Tick DNA was individually extracted using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Oligonucleotide primer pairs used in this study were 16SrDNA (mt-rrs 1 5’-CTGCTCAATGATTTTTTAAATTGCTGTGG-3’; mt-rrs 2 5’-CCGGTCTGAACTCAGATCAAGTA-3’) for tick identification, 17-kDa antigen (R1 5’-TCAATTCACAACTTGCCATT- 3’,R2 5’-TTTACAAAATTCTAAAAACC-3’) for Rickettsia detection, and flaB (PAD 5’-GATCARGCWCAAYATAACCAWATGCA-3’, PDU 5’-AGATTCA AGTCTGTTTTGGAAAGC-3’) for Borrelia detection ((Anderson & Tzianabos, 1989); (Takano et al., 2010)).

Amplifications were performed with the following conditions: 95 °C for 5 minutes, 94 °C for 45 seconds, 50-52 °C for 30 seconds, 72 °C for 45 seconds, and 72 °C for 10 minutes. PCR products were visualized using electrophoresis on 1.2% agarose gel stained with ethidium bromide in 1X TAE buffer. Electrophoresis was carried out at 100 V for 25 minutes. DNA from positive samples then amplified again as much as 50 μL for sequencing and the sample was sent to PT. Genetika Science Indonesia, Tangerang.

The sequences of positive samples were compared with sequences in the NCBI GenBank database by nucleotide BLAST. Phylogenetic analyses were performed using the MEGA7 software (www.megasoftware.net) (Tamura et al., 2007). The phylogenetic trees were constructed by the neighbor-joining method. Bootstrap analyses (1,000 replicates) were carried out according to the Kimura 2-parameter model. All sequences were deposited in GenBank (Accession numbers: Rickettsia sp. ST9 (OQ164644), Rickettsia sp. ST10 (OQ164645), Rickettsia sp. ST13 (OQ054254), and Borrelia sp. ST10 (OQ187772)).

From the three M. reticulatus snakes, a total of 38 ticks were collected which consisted of 13 individuals of A. helvolum (11 males, 2 females) and 25 individuals of A. varanense (20 males, 5 females) which were confirmed by morphological examination. There were no ticks found infesting the P. bivittatus.

Amblyomma helvolum had 3/3 dentition, long and narrow palps, and oval porose areas on the rectangular basis capituli. The male ticks had an ovoid scutum with metallic and yellowish patches of ornamentation. The female ticks, on the other hand, had no scutum ornamentations. The eyes were flat and located at the lateral margins of the scutum. Coxa I bore a pair of triangular spurs, with the external spur about twice as long as internal one. Coxae II-IV each bore a single, triangular spur.

Amblyomma varanense had 3/3 dentition, long and narrow palps, and oval porose areas on the rectangular basis capituli. The male ticks had a reddish-brown round scutum, nearly as broad as long, with five metallic-green spots of variable thickness, shape, and intensity. The female ticks had a reddish-brown cordiform scutum with three greenish metallic spots. Coxa I bore two short, distinct, and separated spurs; the external spur was slightly longer than the internal. Coxae II-IV each bore a single, blunt spur about as wide as long. PCR was used to confirm the identification of ticks.

Amblyomma helvolum has a natural distribution that extends from the Nicobar Islands of India eastward through parts of Thailand, Laos, Malaysia, Singapore, Vietnam, Indonesia, the Philippines, and Taiwan ((Auffenberg, 1988); (Kolonin, 1995); (Petney & Keirans, 1995); (Chao et al., 2013)). Amblyomma helvolum has been reported to infest various reptiles, such as Python spp., Ptyas korros (Zamensis), and Naja naja (Kohls) (Imaoka et al., 2011)in Malaysia (Kohls, 1957). Quite similarly, A. varanense is distributed in India, Thailand, Myanmar, Singapore, and Indonesia (Anastos, 1950)(Supriyono et al., 2019). Amblyomma varanense commonly infests reptiles, but is also known to feed on various mammals (Burridge, 2001).

All collected ticks were individually examined by PCR for Rickettsia spp. and Borrelia spp. The results showed that three male A. helvolum ticks infested on M. reticulatus were positive for Rickettsia spp. and one male A. helvolum tick infested on M. reticulatus was positive for Borrelia sp. The detected Rickettsia spp. and Borrelia sp. were compared with sequences in the NCBI GenBank database by nucleotide BLAST. There were no pathogens detected in A. varanense in this study.

The 17-kDa gene sequences of two detected Rickettsia, Rickettsia sp. ST10 and Rickettsia sp. ST13, showed 100% (500/500) and 99.4% (497/500) similarity, respectively, to Rickettsia sp. RT2 (GenBank: LC428381) detected from A. varanense ticks infesting an Asian water monitor (Varanus salvator) in Indonesia. However, Rickettsia sp. ST9 was 99.2% (499/503) in similarity to Rickettsia sp. (GenBank: AB795164) detected from A. sparsum ticks infesting tortoises (Geochelone pardalis) from Zambia (Figure 1). The detected Rickettsia spp. belonged to the spotted fever group (SFG), which are potential human pathogens.

Several studies have reported the detection of SFG in ticks from various countries in Southeast Asia, including Japan, Thailand, Malaysia, and Indonesia (Imaoka et al., 2011); (Doornbos et al., 2013); (Kho et al., 2015); (Supriyono et al., 2019). Rickettsia sp. RT2, closely related to R. tamurae, was detected in A. varanense ticks infesting an Asian water monitor (V. salvator) in Indonesia (Supriyono et al., 2019). Although there have been no reports of SFG infection in humans due to tick bites in Indonesia thus far, tick infestations pose a potential risk to human health. Infection caused by R. tamurae, a member of the SFG Rickettsia, has been reported in Japan and Laos (Phongmany et al., 2006); (Imaoka et al., 2011). Additionally, antibodies against SFG infection in humans have been reported in rural areas in Malaysia (Tay et al., 2000). The results of this study indicated that there was a natural circulation of SFG in ticks infesting snakes.

Analysis of the flaB gene showed that the detected Borrelia sp. was 99.3% (296/298) in similarity to Borrelia sp. BF16 (GenBank: AB473488) detected from A. trimaculatum ticks infesting B. forsteni snakes. The detected Borrelia sp. was also 99.2% (370/373) in similarity to uncultured Borrelia sp. (GenBank: KT758064) detected from A. varanense ticks infesting P. reticulatus in Thailand (Trinachartvanit et al., 2016).

The constructed phylogenetic tree details that the detected Borrelia sp. in this study belongs to the REP Borrelia group and differs from the Lyme disease-associated Borrelia and the relapsing fever- associated Borrelia ( Figure 2).

REP Borrelia has been detected in ticks infesting various reptiles, including snakes, turtles, and monitor lizards in Thailand, Indonesia, and Japan (Takano et al., 2010); (Trinachartvanit et al., 2016); (Supriyono et al., 2019). REP Borrelia has also been detected in ticks infesting imported captive-bred Geochelone sulcata tortoises and wild- caught Chelonoidis carbonarius and C. denticulatus tortoises in Indonesia (Sophia et al., 2023).

Malayopython reticulatus has a habitat distribution in rainforests, woodlands, and nearby grasslands. It is also associated with rivers and is found in areas with nearby streams and lakes (Murray-Dickson et al., 2017). The snakes in this study were wild-caught in rural areas and thus, have potential to transmit diseases via hematophagous arthropods which include ticks.

This data is essential for vector surveillance to predict the risk of emerging diseases and zoonoses, primarily caused by tick-borne bacteria. Although there are no reports of A. helvolum and A. varanense infestations in humans in Indonesia, the detected tick pathogens can pose risks to wildlife and human health. It is not uncommon that many reptiles, including snakes, are kept as pets that live close to humans or other animals.

Rickettsia spp. and Borrelia sp. were detected in

A. helvolum which belong to spotted fever group and reptile-associated borrelia, respectively. The prevalence of spotted fever group Rickettsia spp. in these ticks was 7.89%, and the prevalence of reptile-associated Borrelia sp. was 2.63%. These findings provide new insights into the parasite-host dynamics within the tick and tick-borne pathogen system in wild snakes in urban areas of Indonesia, enhancing our understanding of the ecology and interactions among wildlife, ticks, and tick- borne pathogens in the region. Monitoring the circulation patterns of tick-borne bacteria is crucial for assessing infection risks to wildlife and humans, as well as for developing strategies for disease control, mitigation, and early warning systems in case of outbreaks. Further studies are required to assess the prevalence of tick-borne bacteria in other animal species across Indonesia.