Molecular Characterization of Leptospira Species Isolated from Urban Rats in Peninsular Malaysia Assoc. Prof. Dr Siti Nursheena Mohd Zain Institute of Biological Sciences, University of Malaya nsheena@um.edu.my

Introduction Leptospirosis is a common infectious disease that affects humans and animals worldwide. It has been is recognized as a re-emerging infectious disease particularly in tropical and sub-tropical countries. Genus: Leptospira Family: Leptospiraceae Order: Spirochaetales. Pathogenic (domestic and wild animals) Non-Pathogenic (environment)

Epidemiology of leptospirosis http://healthyinmalaysia.blogspot.com/2010/10/of-leptospirosis-and-melioidosis-in.html

Clinical manifestations Pathogenesis of Leptospirosis Levett, 2001

MAINTENANCE HOSTS ENVIRONMENT Dogs Rats Pigs Cattle (Canicola) (34 serovars) (Pomona) (Hardjo) ENVIRONMENT

Number of leptospirosis cases in Malaysia (2004-2010)

Leptospirosis outbreaks in Malaysia 2010 1984 1999 2000 2006/07 2011 2012 2013 2015

Objectives of the study There is a paucity of scientific information on the reservoirs of Leptospira spp. and the genetic diversity of the strains circulating in Malaysia. Therefore, the research aims are: To isolate and determine the main serovar of Leptospira spp. circulating in urban rats. To determine the genetic diversity of Leptospira isolated from urban rat hosts in Peninsular Malaysia.

Materials and methods Sample collection and isolation Identification of the Leptospira genus and the pathogenic species Leptospira genus (Merien et al., 1992): GF: 5'-GGCGGCGCGTCTTAAACATG-3’ GR: 5'-TTCCCCCCATTGAGCAAGATT-3’ Pathogenic Leptospira species: G1: 5’-CTGAATCGCTGTATAAAAGT-3’ G2: 5’-GGAAAACAA ATGGTCGGAAG-3’ (Gravekamp et al., 1993)

Microscopic agglutination test (MAT). -To identify Leptospiral serogroups -The antisera used in this study were raised against serovars; Sejroe (M84), Javanica (Veldrat Batavia 46), Canicola (Hond Ultrecht IV), Hebdomadis (Hebdomadis), Pomona (Pomona), Hardjo (Hardjoprajitno), Australis (Ballico), Bataviae (Swart), Pyrogenes (Salinem), Icterohaemorrhagiae (RGA), Autumnalis (Akiyami A), Grippotyphosa (Mandemakers).

2. Randomly Amplified Polymorphic DNA (RAPD) To characterize the isolates and to determine genetic relatedness, 2 methods were used and compared 1. Pulsed-Field Gel Electrophoresis (PFGE) According to Galloway and Levett, 2008 with minor modifications DNA plugs restriction digestion with NotI enzyme Electrophoresis conditions: pulse time: 2.2s-35s, run time: 24 hours in 0.5X TBE at 14°C 2. Randomly Amplified Polymorphic DNA (RAPD) According to Ramdass et al. (1997) with minor modifications. Primers used: B1: 5`-CCGGAAGAAGGGGCGCCAT-3` B2: 5`-CGATTTAGAAGGACTTGCACAC-3`

Results A total of 357 rats were captured from 5 states States Rat Species Result   RR RN RE Negative Positive Selangor 32 26 4 59 3 Penang 11 10 5 Pahang 49 15 62 12 Malacca 92 6 Perak 108 95 13 Total 285 (24) 53 (8) 19 (7) 318 39 RR: Rattus rattus RN: Rattus norvegicus RE: Rattus exulans

Infections were more prevalent in R. exulans (38%) compared to R Infections were more prevalent in R. exulans (38%) compared to R. norvegicus (18%) and R. rattus (7%). According to host-sex, infections were similar between females (20) compared to male rats (19). According to host-ages, more adults rats (38) infected compare to juvenile (1). More rats infected during the wet season recorded in the States of Pahang, Penang and Perak.

Confirmation using Dark Field Microscope.

PCR confirmation PCR confirmation for Leptospira genus (GR/GF) 331 bp PCR confirmation for pathogenic Leptospira (G1/G2) 285 bp All the 39 isolates confirmed as pathogenic, using the primers G1/G2.

Confirmation and identification using MAT Using MAT, 2 serogroups were determined: L. interrogans serogroup Javanica (16 strains) L. borgpetersenii serogroup Bataviae (23 strains)

RAPD using a pair of primers (B11, B12) subtyped the 39 isolates into 14 RAPD profiles. PFGE of NotI-digested chromosomal DNA subtyped the 39 isolates into 2 pulso-types (PFPs).

Conclusion The prevalence of the Leptospira spp. in the urban rat population of Peninsular Malaysia is low. L. interrogans serovar Javanica and L. borgpetersenii serovar Bataviae are the two major pathogenic serovars circulating in the rat population. Identification of the Leptospira serovars using the MAT is time consuming and laborious as it requires large collection of hyperimmune rabbit antisera. PFGE is less labor, more rapid and able to differentiate the closely related serovars of Leptospira genus. This finding suggests that the RAPD-PCR method may be an alternative tool in subtyping the Leptospira isolates as it is easier and could generate results more rapidly than PFGE.

References Levett, P. N. (2001). Leptospirosis. Clin Microbiol Rev 14, 296–326. Benacer, D., Mohd Zain, S.N., Sim, S.Z., Mohd Khalid, M.K., Galloway, R.L., Souris, M., Thong, K.L. (2016). Determination of Leptospira borgpetersenii serovar Javanica and Leptospira interrogans serovar Bataviae as the persistent Leptospira serovars circulating in the urban rat populations in Peninsular Malaysia. Parasit Vectors 9, 117. Gravekamp, C., van de Kemp, H., Franzen, M., Carrington, D., Schoone, G. J., van Eys, G. J. J. M., Everard, C. O. R., Hartskeerl, R. A. & Terpstra, W. J. (1993). Detection of seven species of pathogenic leptospires by PCR using two sets of primers. J Gen Microbiol 139, 1691–1700 Merien F, Amouriaux P, Perolat P, Baranton G, Saint Girons I. (1992). Polymerase chain reaction for the detection of Leptospira spp. in clinical samples. J Clin Microbiol 30, 2219-2224. Galloway, R.L. and Levett, P.N. (2008) Evaluation of a modified pulsed-field gel electrophoresis approach for the identification of leptospira serovars. Am J Trop Med Hyg. 78, 628–632. Ramadass, P., Meerarani, S., Venkatesha, M.D., Senthilkumar, A., Nachimuthu, K. (1997). Characterization of leptospiral serovars by randomly amplified polymorphic DNA fingerprinting. Int J Syst Bacteriol 42: 215- 219.

Acknowledgements This study was funded by the University of Malaya Research Grant (RG053/11BIO & RP016B-14AFR) and the Malaya High Impact Research Grant (Reference UM.C/625/1HIR/MOHE/-02 [A000002-50001]).

Thank You