MOLECULAR IDENTIFICATION OF BLASTOCYSTIS SP. IN LONG-TAILED MACAQUE AT ALAS PURWO PARK (Identifikasi Molekuler Blastocystis sp . pada Monyet Ekor panjang di Taman Nasional Alas Purwo)

Zoonotic diseases can be transmitted through close interactions between long-tailed monkeys. Blastocystis sp. is one of the parasites that can attack mammals and is most commonly found in the intestinal tract. This study aims to analyze the presence of infection and the phylogenetic tree of Blastocystis sp . in long-tailed monkeys in Alas Purwo National Park, Banyuwangi, East Java. Identification of Blastocystis sp . in this study using morphological and molecular methods. A total of 100 stools were examined microscopically using the floating method, showing that 61 samples were positive, followed by a PCR test with a target of 600bp. PCR results obtained three positive samples followed by squencing. Sequences processed in BLAST isolate samples TNAP2 and TNAP9 having homology with Blastocystis sp . Subtype 3 was 98-99%, while the TNAP7 isolate had a lower homology level of 78-79% and the level of phylogenetic analysis of the TNAP2 and TNAP9 isolates was related to Blastocystis sp. from the Philippines (KY610153.1) and Egypt (OP942294.1) and the TNAP7 isolate is related to Blastocystis sp . from Thailand (MH197670.1, MH197668.1). Isolate Blastocystis sp . from Alas Purwo National Park has high homology analysis results to Blastocystis type hominis from Rep. Czech and Chinese by 80-99%, it is possible to have a connection with the zoonotic problem.


INTRODUCTION
Blastocystis is a parasite that can attack mammals and is most commonly found in the intestinal tract in various vertebrae including humans (Zhu et al., 2017). Transmission of Blastocystis sp through direct contact with infected animals is a zoonotic risk that can occur (Osman et al., 2015). The interaction between visitors and long-tailed monkeys can cause changes in behavior to become aggressive. This causes the risk of disease transmission between long-tailed monkeys and humans to increase (Friishansen et al., 2015). In developing countries the prevalence distribution of blastocystis infection is up to 100%, while in developed countries it is up to 56% (El Safadi et al., 2014;Scanlan et al., 2014). Blastocystis sp. found in children and monkeys in an area in Khatmandu India (Yoshikawa et al. (2009). Blastocystis sp. identified based on analysis of the small subunit ribosomal RNA (SSU rRNA) gene has 17 subtypes (Alfellani et al., 2013). In research on Blastocystis sp., there are similar subtypes between humans and Non-Human Primates (NHP), ST1, ST2, ST3, and ST4 (Yoshikawa et al., 2009;Alfellani et al., 2013;Ramírez et al., 2016) In determining subtypes and phylogenetic trees to analyze the presence of Blastocystis sp. infection in long-tailed macaques in Alas Purwo National Park, Banyuwangi, East Java, it is necessary to carry out morphological and molecular examinations.

Sample Collection
100 samples of long-tailed monkey feces in Alas Purwo National Park, Banyuwangi, East Java were taken 5 grams and then collected and put in pots. sample containing 2% potassium dichromate. Each pot was given a sample code, then put in an ice box and then stored at -4OC until DNA extraction was carried out.

Sample Inspection
Microscopic Examination of Blastocystis sp. Stool samples were examined at the Laboratory of the Institute of Tropical Diseases, Airlangga University, Surabaya. Samples were examined to observe each phase of Blastocystis namely vacuolar, granular and cyst. Identification for this protozoa using native and floating methods and observed using a microscope with a magnification of 400 and 1000 times. Microscopic identification of Blastocystis is based on vacuolar and granular forms. The vacuolar form has two nuclei located at opposite poles, while the granular form shows small granules that fill the cytoplasm (Arpitha et al., 2018).

Molecular Examination by PCR
Stool samples that have been confirmed through morphological examination are then followed by microscopic examination. There were three samples which were continued at the stage using the PCR method. A sample of 100 µl was put in a 1.5 ml microtube and DNA was extracted using the QIAamp DNA stool mini kit (QIAGEN, Hilden, Germany) according to the protocol in the kit. Furthermore, the samples were stored at -20°C until further examination. This research was conducted at the Institute of Tropical Disease (ITD) Airlangga University, Surabaya. PCR amplification used the primers BhRDr (GAGCTTTTTAACTGCA-ACAACG) and RD5 (ATCTGGTTGATC-CTGCCAGT) (Kurniawati et al., 2020). Initial denaturation was carried out at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute and elongation at 72°C for 1 minute, and additional elongation for 5 minutes. The PCR product was then read on 2% agarose gel electrophoresis and showed the band at the 600 bp position using Ultraviolet light (Mahendra et al., 2020). Samples that have been confirmed through morphological and molecular examinations, samples that are positive will continue sequencing.

Data analysis
Data from the sequencing results from the samples obtained will be processed in the BLAST NCBI Genbank database and forwarded using a phylogenetic tree with the genetyx ver.10 application.

Results of Morphological Examination of Blastocystis sp
Microscopic morphological analysis of 100 stool samples examined microscopically showed that 61 samples (61%) were positive for Blastocystis sp. The results of microscopic examination using the floating method showed that the parasite was morphologically similar to Blastocystis sp. (Figure 1). According to Ahmed and Karanis (2018) the morphological form of Blastocystis sp has characteristics, namely the nucleus is located on the edge of the cytoplasm, there is sizable cytoplasm inside the cell, has 1 central vacuole or there is a vacuole in the middle (vacuolar) (Figure 1 A), No there are vacuoles or vacuoles are replaced by granules (granular) (Figure 1 B) and there is no central vacuole (cyst) (Figure 1 C).
PCR results obtained three positive samples followed by squencing. RD5 is a primer that amplifies eukaryotic species widely and BhRDr is a primer with high Blastocystis specificity. False positives can occur, for that confirmation through sequencing is done to confirm the results are positive (Stensvold et al., 2013). Sequences processed in BLAST isolate samples TNAP2 and TNAP9 having homology with Blastocystis sp. Subtype 3 was 98-99%, while the TNAP7 isolate had a lower homology level of 78-79% and the level of phylogenetic analysis of the TNAP2 and TNAP9 isolates was related to Blastocystis sp. from the Philippines (KY610153.1) and Egypt (OP942294.1) and the TNAP7 isolate is related to Blastocystis sp. from Thailand (MH197670.1, MH197668.1). Subtypes 1 and 3 are subtypes that are commonly found in humans and non-human primates (NHP) in the world (Ramírez et al., 2016). Isolate Blastocystis sp. from Alas Purwo National Park has high homology analysis results to Blastocystis type hominis from Rep. Czech and Chinese by 80-99% which can be seen in the phylogenetic tree ( Figure 3).

Conclusion
Long-tailed macaques in Alas Purwo National Park, Banyuwangi, East Java, as many as 61 samples (61%) found Blastocystis sp on microscopic examination, while molecularly there were three samples that were positive for Blastocystis sp. Long-tailed macaques in Alas Purwo National Park were infected with Blastocystis Subtype 3, at the level of phylogenetic analysis the isolates TNAP2 and TNAP9 were related to Blastocystis sp. from the Philippines and Egypt and the TNAP7 isolate is related to Blastocystis sp. from Thailand. High homology analysis to Blastocystis type hominis from Rep. Czech and Chinese by 80-99%, it is possible to have a connection with the zoonotic problem.

Suggestion
Further research is needed regarding the molecular characters and phylogenetic analysis of Blastocystis sp. on other hosts in order to know the zoonotic status in other areas of Indonesia.