Human Plasmodium in Domestic Animals in West Sumba and Fakfak, Indonesia

Background: Although maximum efforts have been made, malaria in several areas in Indonesia is still high. This study aims to detect the possibility of a Plasmodium reservoir in domestic animals in endemic malaria areas. Methods: Blood from the domestic animal was collected by EDTA tube, smeared and stained by Giemsa for detecting Plasmodium microscopically. Ten µl blood from EDTA tube dripped into lter paper for Plasmodium DNA capture. Nested PCR was used for the molecular detection of parasites, and DNA was sequenced from PCR products to ascertain Plasmodium species. Result: A total of 208 and 62 animal blood samples were collected from Gaura and Fakfak villages. Thirty-two of 270 animals contained P. falciparum or P. vivax, and all are from Gaura village. The percentage of Plasmodium in buffalo, horse, goat, and dog is 20.7%, 14.3%, 5.8%, 16.7%, respectively. Neither P. knowlesi found in all samples, nor parasite detected in 18 pig blood samples. Conclusion: Human Plasmodium exists in domestic animals in Indonesia. This nding may partly explain the persistence of the high prevalence of malaria in some endemic areas in Indonesia and may affect public health and malaria control strategy.


Study area and population
This research was conducted in Gaura village, West Sumba Regency in 2018, and Fakfak, West Papua Province, in 2019 ( Fig. 1). Gaura's area is 29.96 km2 wide and inhabited by 9,584 people (15). Fakfak has 11,036 km2 in size and inhabited by 84,692 people (16). The main occupation of residents in both locations was farmers, and almost every family has livestock such as goats, horses, cows, pigs, and buffalos, whose cages are located around the residential. The residents also have pets such as dogs and cats that freely in and out of the house.

Sampling collection
Sampling collection was done with the help of a veterinarian and staff from the Animal Husbandry Department. The blood was drawn using a 5 ml EDTA tube. A vacutainer needle [16][17][18] in size was used to collect blood from buffaloes, goats, pigs, and horses from the jugular vein located in the neck's ventrolateral part. The dog's blood was drawn using a 21 size vacutainer needle and bleed from the cephalic antebrachial located in the leg. For the Plasmodium microscopic identi cation, approximately 10 ul of EDTA blood was dropped onto a glass object, smeared and stained with Giemsa. The remaining blood was dripped onto lter paper (Whatman CAT No. 1442-090) until the blood was absorbed with approximately 1.5 cm in diameter. The dry lter paper was put in a sterile plastic clip and stored at room temperature for a maximum of 10 days.

DNA extraction
A dried blood spot (DBS) isolation kit (Cat. 36000) from Norgen Biotec was used for lter paper DNA extraction. A 6 × 3 mm lter paper containing blood was put into a 1.5 ml tube containing 100 µl of digestion buffer B. The tube was then vortexed and incubated at 85 °C. Twenty µl of proteinase K and 300 µl of lysis buffer B was added to the tube and then vortexed before incubated at 56 °C. All incubation processes were carried out for 10 minutes. Two hundred and fty µl of 95% ethanol was added to the tube and then vortexed. The tube's DNA was washed by adding 500 µl of WN wash solution and then centrifuged for 1 minute at a speed of 8000 rpm. The second washing was carried out twice with 500 µl washed with wash solution and centrifuged at 14000 rpm. For DNA elution, 90 µl of elution buffer B was put into the tube and centrifuged at 8000 rpm for 1 minute. The puri ed DNA was stored at -20ºC.

DNA Ampli cation and electrophoresis
DNA ampli cation and electrophoresis were performed by following procedures as directed by Tiangen.
For details, Plasmodium DNA ampli cation was carried out used the Nested PCR method with a 2x Tag Plus PCR mix (Tiangen) enzyme. The nal volume of each sample was 12.5 µl, which contained 6.25 µl enzyme, 2.25 µl ddH2O, 1 µl forward-, 1 µl reverse-primers, and 2 µl DNA samples. For sequencing, the volume of mixtures was double, with the nal volume of 25 ul. In table 1, the primer sequences can be seen.
For Nested 1 DNA ampli cation, the temperature was set up as follows: Denaturation 94ºC (1 minute), annealing 55ºC (1 minute), extension 72ºC (1 minute) in 35 cycles. For Nested 2, denaturation was carried out at 94ºC (30 seconds) and extension at 72ºC (30 seconds) in 35 cycles. There was a difference in annealing temperature for each species in Nested 2; 55 °C (1 minute) for PCR multiplex P. falciparum and P. vivax, while for P. knowlesi 56 °C (1 minute). Nested one products were used as templates for Nested two. Nested one and Nested two products were run by 1.5% and 2%, respectively, for electrophoresis. We did not perform molecular work for P. ovale and P. malariae since we have di culty nding the positive control, and according to the local health o ce, these species have never been reported from Sumba and Fakfak.

Sequencing and alignment
To determine Plasmodium's species, Nested two products that had positive bands were sent to Genetics Science in Singapore to be requested. Results of DNA sequences adjusted using multiple alignments found in the Bioedit application (17). The results were then read by the BLAST program from the NCBI website (18).

Results
A total of 208 and 62 animal blood samples were collected from Gaura and Fakfak villages, respectively. respectively. The forms of trophozoites, schizonts, and gametocytes microscopically at 100 times magni cation can be seen in Fig. 3. Apart from dogs, the erythrocytes in animal samples appeared to be smaller than the human erythrocytes. All blood that was containing Plasmodium were collected from Gaura. The distribution of Plasmodium species in animals' blood samples from Gaura and Fakfak can be seen in table 2.

Discussion
The suspicion of Plasmodium presence in domestic animals arose because malaria cases in those two villages remained high even though many efforts, including insecticide-treated bed nets, have been applied. This study identi ed 32 of 270 animal blood (11.9%) contained Plasmodium, known as human parasites.
The previous studies found Plasmodium relictum in avian (19), P. cephalophi in ungulate (20), P. traguli in mousedeer (21), P. brucei in gray duiker (22,23), P. bubalis in water buffalo (24), and P. odocoilei in whitetailed deer (25,26). In ruminant, P. caprae was found in goat (27), in Rodentia was P. bergei (28), and in primates were found P. cynomolgi, P. inui, and P. fragile (29). The ve Plasmodium that infect humans were originally parasites in primates (1,2,6,7,8,9,30). In this study, we found P. falciparum in buffalo, goats, dogs, P. vivax in buffalo, goats, dogs, and horses. At rst, we were not so sure about the presence of Plasmodium in these animal erythrocytes. However, the Nested PCR showed the same results for all positive samples. The sequencing results of the positive bands in Nested 2 showed that the bands were P. falciparum and P. vivax (Fig. 7). This is the rst investigation reporting human Plasmodium in domestic animals (ruminant, ungulate, and carnivore).
We have considered the possibility of contamination. Therefore, we re-extracted DNA from the same lter paper's blood, and the results were no different from the rst Nested PCR. We also performed PCR using other primers, namely rPF1, rPF2, and primers rPV1, rPV2 (31) to detect P. falciparum and P. vivax, and the results were still positive.
The discovery of Plasmodium in buffaloes, goats, horses, and dogs in malaria-endemic areas raises the following questions. How did P. falciparum and P. vivax live in these pets? Are they intermediate hosts for this parasite? Did these Plasmodium species evolve to live in ruminants, ungulates, and carnivores? As a result of repeated exposure, have these animals become more permissive to Plasmodium, which generally lives in humans? Is this parasite pathogenic in animals? As is known, P. knowlesi is a commensal microbe in primates but is pathogenic in humans (1, 2, 3). In P. knowlesi, migration from primates to humans can be caused by loss of forests or human invasion of primate habitat (11). It might be possible that the proximity between animals and humans makes it easier for mosquitoes to transfer human parasites to animals and vice versa.
Although both Fakfak and West Sumba have high API, only animals in West Sumba (Gaura village) have human Plasmodium. This difference may be due to the distance between the human house and animal cages. In Fakfak, the cages are separated approximately 50-500 meters from the main house, while in Gaura, people generally live in a stilt house where the ground oor functions as animal sheds. This condition allows microbial to be transferred by mosquitoes between humans and animals. Unlike in Gaura, the sampling locations in Fakfak are far from each other, and the steep geographical conditions in Fakfak made it di cult for us to collect as many samples as we got from Gaura. This may indirectly lead to the absence of Plasmodium found in Fakfak domestic animals.
Although the use of a microscope can detect Plasmodium's presence well, due to the size of erythrocytes, which are generally smaller in animals than humans, the molecular method becomes signi cant in detecting the presence of Plasmodium. In this study, Nested PCR was used to detect Plasmodium because its sensitivity was as high as Real-Time PCR and its relatively lower cost (32, 33). For further studies, the recommended microscopic method of using a double uorescent dye with Giemsa's stain may need to be considered (34).

Conclusion
This study found Plasmodium species in domestic-animals (ruminants, ungulates, and carnivores) previously known as human parasites. These results may partly answer the question of why malaria is challenging to eliminate in an area. It is hoped that these ndings can be applied to improve public health and become a reference in formulating malaria prevention strategies.