TICK-BORNE PATHOGENS DETECTION FROM TICKS INFESTING Malayopython reticulatus (REPTILIA: PYTHONIDAE) SNAKES IN INDONESIA

Article Highlights:
- A total of 38 ticks were collected from M. reticulatus, comprising 13 A. helvolum and 25 A. varanense.
- Spotted fever group Rickettsia spp. (7.89%) and reptile-associated Borrelia sp. (2.63%) were detected in male Amblyomma helvolum ticks collected from Malayopython reticulatus snakes in Indonesia.
- Snake-associated ticks may harbor emerging pathogens, underscoring the importance of tick surveillance in reptiles for early disease detection and zoonotic prevention.

Abstract:
Ticks are important arthropod vectors of numerous diseases in humans and animals. Furthermore, ticks are also established vectors and reservoirs of pathogens important to wildlife and human health. Rickettsia and Borrelia are two genera of bacteria that may be transmitted by ticks, and some pathogenic species are zoonosis. This research investigated the prevalence of Rickettsia spp. and Borrelia sp. in Amblyomma helvolum and Amblyomma varanense ticks fed on Malayopython reticulatus and Python bivittatus snakes in Indonesia. A total of 38 ticks were collected from three M. reticulatus snakes, while no ticks were found on the P. bivittatus snake. The 38 ticks consisted of 13 individuals A. helvolum and 25 individuals A. varanense. PCR analysis revealed that three (3/38; 7.89%) male A. helvolum ticks were positive for spotted fever group Rickettsia spp. and one (1/38; 2.63%) male A. helvolum tick was positive for a reptile-associated group Borrelia sp. Although the overall prevalence of tick-borne pathogens was low, this study underscores the importance of monitoring the prevalence and prevention of tick-borne diseases. Surveillance of ticks infesting reptiles can facilitate the early detection of disease transmission to both animals and humans. These findings also suggested that snake-associated ticks may harbor emerging tick-borne pathogens

INTRODUCTION

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.

MATERIALS AND METHODS

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)).

RESULTS AND DISCUSSION

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);

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