Leptospiral agglutinins in captive and free ranging non-human primates in Sarawak , Malaysia

Aim: Materials and Methods:


Macaca fascicularis Trachypithecus cristatus Nasalis
have been detected from wildlife in many countries, however their role as reservoirs is still poorly understood [1][2][3].Leptospirosis can result in economic losses in domesticated animals and has the potential to be an important zoonotic disease of humans [4].Leptospires were first isolated from rats in 1917 and it is widely acknowledged that rodents are a key source of infection for humans [5].However, recently Australian and Peruvian researchers have reported that bats can also carry pathogenic , [1][2]6], although their role as carriers is not fully understood.Other wildlife, including primates, can also act as potential carriers of these pathogens [7][8][9][10].However to date there has been little research conducted on free ranging wildlife.Leptospirosis in wildlife can affect biodiversity, human and livestock health, animal welfare and consequently the national economy [4].
Recently leptospirosis has been recognised as a re-emerging public health problem in Malaysia [11].At present Malaysian wildlife disease surveillance is poorly coordinated and emerging zoonotic infectious diseases represent a growing threat.Tourism is a major contributor to the economy of Malaysia with 24.6 million tourists visiting the country annually.It has been estimated that approximately one million tourists are involved in eco-tourism activities and this group is particularly at increased risk of exposure to infectious diseases [12][13].
The current research reports on the carriage of by opportunistically sampled non-human primates in Sarawak.
All procedures were performed with the approval of the Animal Ethics Committee of the Murdoch University (W2376/10) and Sarawak Forestry cooperation (NCCD.907.4.4 (V)-235).In recent years outbreaks of leptospirosis in Malaysia have been documented around the wildlife reserves and parks resulting in confirmation of a high number of confirmed cases and associated mortalities.Wildlife tourism is an important source of revenue in Malaysia, particularly in the state of Sarawak and leptospirosis has the potential to impact on this.Trapping of non-human primates was carried out opportunistically around the Bako National Park and the Matang Wildlife Center in the vicinity of human settlements and tourism areas of Sarawak.Blood samples were obtained from the saphenous vein to determine the presence of antibodies by the Microscopic Agglutination Test (MAT) to 17 serovars of commonly found in Malaysia.

Leptospiral agglutinins in captive and
This study reports the screening of twelve primates (eight captive and four free ranging) for leptospirosis.Eight of the 12 monkeys (66.6%; 95% CI 34.9-90.1)reacted against one or two serovars of (Lai and Lepto175).The serovar Lai is considered pathogenic for different mammals, including humans.
Lepto 175 has been identified as an intermediate strain and further studies are being undertaken on this serovar.These results are important as primates may act as reservoirs of spp.for humans, which may potentially affect tourism (economic loss), conservation efforts and public health.In this study eight captive primates from Matang (three pig/short-tailed macaques, two Bornean gibbons, a long tailed macaque and two orangutans), and four free ranging primates from Bako (one silver-leaf monkey and three proboscis monkeys) were tested for the presence of leptospiral antibodies.Animals were tranquilised with Zoletil (5mg/kg; 100mg/ml).Five ml of blood was collected from the saphenous vein into a plain tube from each animal.The tubes were maintained at room temperature for 15 minutes and then centrifuged at 15,000 RPM for 5 minutes.The serum was then separated and stored at -20 C until it was analysed.

Acknowledgements
Competing interests

References
Positive antibodies to leptospires were detected in captive primates from the Matang Wildlife Centre.These infections may have arisen from poor sanitation or ineffective rodent control and adopting programs to improve these would likely minimise the spread of leptospires to other animals, including humans.Specifically the highest antibody titre (1:800) was detected against .Lepto 175 (Sarawak) in a short-tailed macaque.This single high antibody titre may indicate active infection [10], however confirmation of its significance requires testing of serially collected samples.
In Bako National Park several short and longtailed macaques and wild boars roam freely on the ground eating leftover food and garbage.The two species of macaque have the potential to transmit leptospires to proboscis and silver-leaf monkeys because these later species have an arboreal lifestyle.Besides contact with rats, ingestion of water contaminated with leptospires could be a potential source of infection as silver-leaf and proboscis monkeys drink from freshwater streams and rivers [17].Proboscis monkeys are also competent swimmers [18][19] and are known to have the most aquatic lifestyle of primates rarely ranging more than one kilometre from water sources.
In this study a positive titre in a proboscis monkey was reported for possibly the first time in Malaysia.Information on Lepto 175 is deficient although it would appear to be endemic in Sarawak, although its pathogenicity has yet to be confirmed [16].The serovars has close genetic similarities with wolffii, [16] which has been isolated from humans and wildlife species, including non-human primates, from Thailand, India, Iran and Sabah [10,[20][21].Serovar Lai is pathogenic and is a major cause of zoonotic spread to humans involved in ecotourism adventure activities associated with water [22].Studies by Kilbourn [10] on freeranging orangutans in Sabah found a high prevalence of antibodies against serovars and , as well as the novel strain wolffii.Experimental infection of marmoset monkeys ( ) with serovar Copenhageni resulted in the presence of histological evidence of kidney damage [23].
According to the results of previous researchers, natural infection of monkeys by leptospires is an unusual event, however it has been observed in serological surveys or under exceptional conditions in captivity [23].Lilenbaum [24] detected anti-Leptospira agglutinins in (lion tamarins) in Rio de Janeiro, Brazil, even though these animals showed no clinical signs or prior history of disease.Romero [25] reported the potential transmission of leptospires from zoo animals, in particular neotropical monkeys, to staff in Colombian zoos.
From this study, evidence of exposure of silverleaf and proboscis monkeys to leptospires was found and there is an urgent need to undertake more investi-gations on free ranging non-human primates in Malaysia to understand their role in the transmission of leptospires.Although leptospires are endemic in Sarawak this is the first reported evidence of infection in non-human primates.The possibility of non-human primates acting as reservoir hosts for leptospires needs further investigation.
ST carried out the study and drafted the article.IDR and MTA participated in the scientific discussion, ST assisted in conducting MAT.All authors read and approved the final manuscript.
The authors would like to thank Murdoch University and SOS rhino (US) for providing the financial support to conduct this research in Sarawak, Malaysia.The Sarawak Forestry cooperation is acknowledged for providing the necessary permits for this research to be conducted.Primate blood samples were collected as part of the Proboscis Monkey Genome Project lead by M.T. Abdullah.Special thanks go to staff from the Institute for Medical Research, Kuala Lumpur for their technical support.
The authors declare that they have no competing interests.

Figure- 1 :
Figure-1: Location of research area in the Sarawak state of East-Malaysia

Table 1 :
-MAT results obtained from sera of 12 primates in Sarawak.