Macaca fascicularis and Macaca nemestrina infected with zoonotic malaria parasites are widely distributed in Sarawak, Malaysian Borneo

Human infections with Plasmodium knowlesi, a malaria parasite of Macaca fascicularis and Macaca nemestrina (long-tailed and pig-tailed macaques respectively), occur throughout Southeast Asia, especially Malaysian Borneo. Other naturally-acquired human infections with malaria parasites from macaques in Southeast Asia are P. cynomolgi, P. inui-like, P. coatneyi and P. simiovale. In Sarawak, Malaysian Borneo, M. fascicularis and M. nemestrina from only the Kapit Division have been examined previously for malaria parasites. In order to determine the distribution of P. knowlesi and other zoonotic malaria parasites, 73 macaque blood samples derived from 7 other administrative divisions in Sarawak were studied. Of 45 blood samples from M. fascicularis and 28 from M. nemestrina tested by nested PCR assays, 23 (51.1%) M. fascicularis and 15 (53.6%) M. nemestrina samples were positive for Plasmodium DNA. Thirty-two of these macaques from 7 divisions sampled, harboured either single (n = 12), double (n = 9), triple (n = 7) or quadruple (n = 4) infections of P. knowlesi, P. inui, P. cynomolgi and P. coatneyi, while the infecting species of Plasmodium could not be identified for 6 samples. P. knowlesi was detected in 15.5% (7/45) M. fascicularis and in 7.1% (2/28) M. nemestrina sampled. Despite the small number of samples analysed from each administrative division, the current study indicates that macaques infected with the zoonotic malaria parasites P. knowlesi, P. cynomolgi, P. inui and P. coatneyi are widely distributed throughout Sarawak, Malaysian Borneo. Travelers to forested areas in Sarawak should be made aware of the potential risk of acquiring zoonotic malaria.

www.nature.com/scientificreports/ of natural infections of P. knowlesi in a Presbytis melalophos (banded leaf monkey) in Peninsular Malaysia 27 , and in Trachypithecus obscurus (dusky leaf monkey) and Macaca arctoides (stump-tailed macaque) in Thailand 26,28 . Apart from P. knowlesi, there are other malaria parasites of M. fascicularis and M. nemestrina which can infect humans. A case of naturally-acquired P. cynomolgi was reported in Peninsular Malaysia in 2014 29 . This was followed by descriptions of other human P. cynomolgi infections in Cambodia, Malaysia and Thailand [30][31][32][33] . More recently, human infections with P. inui, P. coatneyi, P. simiovale and P. inui-like parasites have been described in Malaysia 34,35 . In South America, zoonotic malaria cases originating from other non-human primates have been reported; P. brasilianum acquired from Alouatta seniculus (red howler monkey) 36 and P. simium from Alouatta guariba (brown howler monkey) 37,38 . Previous studies on malaria parasites of M. fascicularis and M. nemestrina in Sarawak State were confined to the Kapit Division 21,39 . In order to determine the distribution of P. knowlesi and other zoonotic malaria parasites, macaque blood samples collected from 7 other administrative divisions in Sarawak were examined with molecular detection assays.

Methods
Ethics statement and collection of blood samples. In Sarawak, permits are required from the Sarawak Forestry Department and the Sarawak Biodiversity Centre for the collection of samples from animals and for the use of these samples for research. The protocols for capture, collection of blood samples and release of wild macaques, together with collection of blood samples from captive macaques were approved by the Sarawak Forestry Department ( ). In addition, a permit to access and collect animal blood samples was obtained from the Sarawak Biodiversity Centre (Permit Number: SBC-RP-0081-BS). All methods were performed in accordance with relevant guidelines and regulations.
A veterinarian collected blood samples from the femoral vein of each wild macaque following anaesthesia by intramuscular injection of tiletamine and zolazepam (Zoletil® 100, Virbac, France) at a dose of 5 mg/kg. The blood collection was performed using 21G × 1" eedle attached to a 5 ml syringe (Terumo, Japan), where not more than 5 ml blood was drawn from each macaque for the study purposes. Blood samples were collected at the trap sites and the animals were released after they had recovered from anaesthesia. Similar procedures were employed when venous blood samples were obtained from captive macaques at Matang Wildlife Centre and Semenggoh Wildlife Rehabilitation Centre in the Kuching division. For macaques kept as pets, blood spots on filter paper were collected by pricking the end of the tails of the animals by staff of the Sarawak Health Department during their visits to malaria affected communities. This study was approved by the Medical Ethics Committee of Uni- . The macaques at the wildlife centres were not born in captivity and were kept in enclosures that were not netted. No information was collected regarding the period of captivity of the macaques at the wildlife centres or at the rural communities. Each of the wild and captive macaques was only sampled once. The macaques were identified by the veterinarians and by the staff of the Sarawak Health Department based on the length of the tails of the macaques; M. fascicularis have long tails (32-75 cm) and M. nemestrina have very short tails (< 25 cm). The veterinarians at the wildlife centres were informed which macaques were malaria-positive and were advised to treat them with antimalarial drugs. None of the wild macaques that were trapped nor those kept as pets were treated with antimalarial drugs. DNA extraction. DNA was extracted from dried blood spots on filter paper using InstaGene Matrix (Bio-Rad Laboratories) and from frozen blood samples using the QIAamp DNA Micro Kit (QIAGEN) as described previously 41 .
Nested PCR assays. DNA samples were initially analysed using nested-PCR assays for detecting Plasmodium DNA 42 . All Plasmodium-positive samples were then analysed by nested PCR assays with PCR primers specific for P. knowlesi, P. cynomolgi, P. inui, P. fieldi and P. coatneyi as described previously 21 .

Results
Of the 73 macaque blood samples examined with nested PCR assays, 51 www.nature.com/scientificreports/ 15 macaques from Limbang, Kuching, and Miri divisions harboured malaria parasites while 5 from Sibu and Samarahan were found to be Plasmodium-negative.

Discussion
Molecular detection methods such as nested PCR assays have proved to be more sensitive and specific than microscopy for the detection of malaria parasites in humans, non-human primates and mosquitoes 5,7,23,39,43-46 . In the current study, for 6 macaque samples that were Plasmodium-positive in nested PCR assays, the species of Plasmodium could not be identified with nested PCR assays using species-specific primers for P. knowlesi, P. cynomolgi, P. inui, P. fieldi and P. coatneyi. Similar observations have been reported in studies of blood samples from macaques 28,47 and humans 34 , and during entomological studies to incriminate vectors of malaria [45][46][47] . For  www.nature.com/scientificreports/ these 6 samples the approximately 240 bp fragments that were amplified by PCR with the Plasmodium-specific primers, rPLU3 and rPLU4, were not sequenced. Previously, we sequenced these fragments from a number of samples that were Plasmodium-positive but negative with the species-specific PCR primers and found through the Basic Local Alignment Search Tool (BLAST) searches that they had high similarity to species of Plasmodium, including P. inui and P. knowlesi. However, it was not possible to confirm the identity of the infecting species by phylogenetic analyses of these and referral sequences since the bootstrap values were low and it was not possible to differentiate between the various species of Plasmodium. It is possible that the parasite counts in these 6 macaques were low but were sufficiently amplified by the genus-specific primers which target all the copies of both the A-and S-forms of the small subunit (SSU) rRNA genes 41,42 . However, with the species-specific primers that target either the S-or the A-type SSU rRNA genes, sufficient PCR amplification was probably not attained and no amplicons were detected by gel electrophoresis. An alternative explanation is that these macaques may have been infected by P. simiovale, which has been demonstrated in M. fascicularis in Sarawak 39 , and/or by one or more species of Plasmodium for which PCR primers are currently unavailable. The complex nature of infections with Plasmodium was noted in the current study where 84.2% (32/38) of the Plasmodium-positive macaques had either double, triple or quadruple infections with P. knowlesi, P. inui, P. cynomolgi and P. coatneyi. Similar observations were made in our previous study in the Kapit Division of Sarawak where 101 of 108 wild macaques were Plasmodium-positive and 91% (91/101) had mixed species infections 21 . Multiple species of Plasmodium in each macaque host, as reported in that 21 and other studies 28,39 , may not accurately reflect the actual number of species of Plasmodium harboured by each macaque, since blood samples at only a single time point were obtained and analysed. It has been noted that parasite counts fluctuate and can even change daily in non-human primates that have been kept in captivity 4 . In addition, erythrocytes containing late asexual stages of certain parasites like P. coatneyi sequester 4 , so the infection would not be detected by PCR analysis of blood samples if the sample was collected when the majority of the parasites were late stages. Sequential samples would provide more accurate information on the prevalence of each species of Plasmodium in a host in nature but it would not be possible to undertake these investigations on wild macaques due to ethical and logistic reasons.
It is important to determine the geographical range of M. fascicularis and M. nemestrina infected with zoonotic malaria parasites to inform the public of the risks of acquiring zoonotic malaria. Previous studies in Sarawak were confined to the Kapit Division and in the current study macaques from another 7 divisions were examined 21,39 . A total of 32 macaques infected with either single or multiple species of the zoonotic malaria parasites P. knowlesi, P. cynomolgi, P. inui and P. coatneyi were found in all the 7 administrative divisions of Sarawak studied, from the southernmost division of Kuching to the northernmost division of Limbang. A limitation of the current study is the relatively small number of samples examined, involving between one to 14 macaques from each administrative division. There is a need for more extensive studies, involving a larger number of samples from varied locations at each administrative division to determine accurately the prevalence of malaria infection among macaques at these locations. Furthermore, these studies should focus on sampling wild macaques rather than captive ones. In the current study, the prevalence of malaria was much higher in wild than in captive macaques. This difference is primarily due to the mobility of the macaques, greater abundance of the mosquito vectors of P. knowlesi and other simian malaria parasites in the forests rather than close to human habitats, and to the feeding behaviour of the vectors. The vectors of P. knowlesi in Sarawak, Sabah, Peninsular Malaysia and Vietnam are forest-dwelling mosquitoes and a study in Sarawak, found that the vector, Anopheles latens, feeds predominantly in the forest and the forest fringe, rather than in longhouse communities 48,49 . Due to the forest habitats and feeding behaviour of the vectors, wild macaques would be at risk of acquiring malaria while foraging for food or while resting at night on trees in the forest. In contrast, some of the captive macaques in the current study were housed in wildlife centres situated away from malaria transmission areas, and those kept as pets by residents of rural communities would have limited exposure to mosquito vectors as they were tethered. Therefore, examination of blood samples from wild as opposed to captive macaques would provide more accurate information on zoonotic malaria parasites that are present at areas surrounding each sampling site. Such information would be essential for informing the local population and visitors to rural areas of Sarawak of the risks of acquiring zoonotic malaria. Combined with data from concurrent entomological and community-based studies, they would provide valuable information on malaria transmission and assist in the implementation of appropriate prevention and control measures for zoonotic malaria.
In conclusion, despite the small number of macaques sampled at each administrative division, the current study indicates that that the zoonotic malaria parasites P. knowlesi, P. cynomolgi, P. inui and P. coatneyi, are found in M. fascicularis and M. nemestrina that are widely distributed throughout Sarawak, Malaysian Borneo. P. knowlesi is the main cause of malaria in Sarawak and has the potential to cause fatalities 5,15,44,50,51 so the local rural population and travelers to habitats of these macaques in Sarawak should be made aware of the potential risk of acquiring P. knowlesi and other zoonotic malaria parasites.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/