Leptospirosis: an update of potential reservoirs in Thailand COL Duangporn Phulsuksombati D.V.M., M.Sc. Deputy Director, Analysis Division Armed Forces Research Institutes of Medical Sciences Bangkok, Thailand Good morning, Chair, Co chair, Ladies and gentlemen. First of all I would like to thank the scientific committee to give me an opportunity to give a presentation here. My topic to present is: Leptospirosis : an update of potential reservoirs in Thailand.

Outlines of Presentation Background Leptospirosis Etiology Diagnostic Method Epidemiological Pattern Objective Study Methods Results Discussions Potential reservoirs Association to human Leptospirosis Conclusions Outlines of presentation will follow as shown in this slide. Background, Objective & methods, results, discussions, conclusions and acknowledgements.

Leptospirosis Etiology Caused by the pathogenic spirochetes of the genus Leptospira Leptospires are thin, helically coiled, motile bacteria. Serologically classified into more than 200 pathogenic serovars. As you all know very well that the cause of Leptospirosis is the pathogenic spirochetes Leptospira interrogans. They are thin, helically coiled, motile bacteria and serologically classified into more than 200 serovars.

Factors Associated with Leptospirosis Agent: Leptospires Harbor in animal’s renal tubule, excrete in urine. Animal reservoirs and carriers: Domestic animals, Wild animals, Rodents Man: Occupation, Activities, Behaviors  Factors associated with Leptospirosis include Leptospires, Man and Environment. Leptospires are adapted to specific mammalian reservoir hosts and harbor in their renal tubules and shed in their urine to environment. Transmission to humans occurs through direct or indirect contact with infected urine or exposure to contaminated soil or water by their occupation, activities or behaviors. Environment: Suitable environment for growth: Damp soil and water pH 7.2-7.6 Temp. 28-32 oC

Diagnostic tools MAT PCR Semisolid Solid(MORU) Serological detection Available and practical False positive (titer >100) Molecular detection PCR LipL32 False positive with certain gram negative bacteria 16SrDNA Indistinguishable between non-pathogenic and pathogenic leptospira Nested 16SrDNA Most validity Isolation Semisolid Indistinguishable multi-serovars Solid(MORU) Pure isolation Among the diagnostic tools, Serological detection by MAT is often used for Diagnosis of Leptospirosis

Animal leptospirosis researches Serotyping is a useful epidemiological tool because establishing the causal serogroup or serovar is the first step towards identifying reservoirs and generating control strategies. Serological detection by MAT is essential tool for serotyping. Serotyping is useful for establishing the causal serogroup or serovar which is the first step towards identifying reservoirs and generating control strategies.

Antibodies detection by MAT Microscopic Agglutination Test (MAT) development: Reading result: on slide to micro plate Period of time: 2 days to 1 day to 3 hrs Monitoring microscope: using program computer reading Panel antigen: serovars 12 – 15 – 24 - ? Leptospirosis center of National Institute of Animal Heath has developed micro agglutination test by using micro plate instead of glass slide which take less time than ever from 1 or 2 days to 3 hours. This is very useful tool for national sero-surveillance in animals that will show the result later on.

Situation of Leptospirosis in Thailand What is the situation of Leptospirosis in Thailand now? During 2003-2013, Average Morbidity rate was 5.6 per 100,000 populations Average Mortality rate was 0.11 per 100,000 populations Average Case fatality rate was 1.38 % Source: Bureau of Epidemiology, Ministry of Public Health

Epidemiologic patterns Monthly reported cases of Leptospirosis 2003-2013 This graph shows monthly reported cases. Mostly were found in July –October, raining season Source: Bureau of Epidemiology, Ministry of Public Health

Epidemiologic patterns Source: Bureau of Epidemiology, Ministry of Public Health 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 Morbidity rate is highest in Northeast and then in North Since 2006, More cases were reported in South This slide shows the distribution of Leptospirosis cases. Morbidity rate washighest in Northeast and then in North. Obviously, Since 2006, More cases were reported in South

Epidemiologic patterns Serovar identification by NIH of 146 sera of Leptospirosis cases No Positive Shermani (70%) Australis (20%) Autumnalis (10%) Shermani (30.4%) Tarasovi (52.2%) Australis (4.4%) Grippotyphosa (4.4%) Department of Medical Sciences, Ministry of Public Health, investigated 146 sera of leptospirosis cases to identify antibodies against L. interrogans serovars. Serovars Shermani showed highest percentage in South, central and northeast region following by Tarrasovi and Australis Pomona (4.4%) Sarmin (4.4%) Shermani (100%)

Objectives Review the relevant publications and reports related to animal reservoirs of leptospires in Thailand Unveil the potential of animal sources of Leptospira serovars in different parts of Thailand Due to the animals’ role as reservoir hosts and carriers of this organism and also serve as a source of human infection. Thus, knowledge of the prevalent serovars and their maintenance hosts are essential to understanding the epidemiology of the disease. Then, the objective of this study is to present the relevant data from publications and reports related to animal reservoirs of leptospires in Thailand and unveil the potential of animal sources of Leptospira serovars in different parts of Thailand

Study Methods Secondary data of leptospirosis prevalence in animals of 10 publications and reports during 2005-2015 were reviewed. The potential of animal sources of Leptospirosis associated to human leptospirosis was collected and discussed. Study Methods : Secondary data of leptospirosis prevalence in animals of 10 publications and reports during 2005-2015 were reviewed. The potential of animal sources of Leptospirosis associated to human leptospirosis was collected and discussed.

? Results Result from reviewing the publication and reports of sero prevalence studies in animals were found in dogs pig cattle Buffaloes elephant and leptopira isolation in rodents

Studies of prevalence in dogs Authors, Years Wimol et al., 2005 Meeyam et al., 2006 Jittapalapong et al., 2009 et al., 2014 # Dogs 211 210 230 255 Places 36 provinces Chiangmai province Bangkok 4 provinces in 3 regions with high prevalence Method MAT Sero +ve 12.8% 11% 83.5% 7.8% L. Interrogans serovar: Most found Bataviae 24.4% Bataviae 5.2% 20.3% Shermani 6.9% (then Bataviae 5.2%) From the review, there were 4 studies of prevalence of Dogs in Thailand using MAT for serological detection. The study in Bangkok found seropositive 83.5% which is much greater than other studies. Bataviae was mostly found in the first 3 studies. For the last study, the most common was Shermani then Bataviae.

This report was the largest study in animal leptospirosis This report was the largest study in animal leptospirosis. Dr Suwancharoen and her colleagues from Thailand National Institute of Animal Health, Department of Livestock Development have done the serological survey of leptospirosis in livestock in Thailand. Suwancharoen, et al. 2013

Prevalence in ruminants Seroprevalence from cross sectional survey in 36 provinces in Thailand, 2001 N > 14,188 MAT titer > 50 Region 30.4% +VE in Northeast 22.2% +VE in Central Prevalence 9.9% for cattle (918/9,288) 30.5% for buffaloes (419/1,376) 4.7% for sheep (52/1,110) 7.9% for goat (41/516) More than Fourteen thousand of livestock sera were collected from 36 provinces in all part of the country. The highest prevalence was found in the northeast region with 30.4%. Seroprevalences recorded for cattle, buffaloes, sheep and goats were 9.9, 30.5, 4.7 and 7.9 percent. Seropositivity of Buffaloes were 3.1 times more likely than cattle. Suwancharoen, et al. 2013

Factors associated sero-prevalence of leptospira in ruminants This slide shows factors of age and sex associated of seropositivity. For age, it was found that, Seroprevalences in cattle and buffaloes trended upwards with increasing age. So, chronic carriers could be shedders of leptospires into environment.. Chronic carriers could be shedders of leptospires into environment!!!

Distribution of leptospira serovar in Thai ruminants Cattle Ranarum, Sejroe, and Mini Buffaloes Mini, Sejroe, Bratislava and Ranarum Sheep Mini, Shermani, and Ranarum Goat Mini, Shermani, Ranarum and Sejroe The most commonly detected antibodies were against L. interrogans serovars Ranarum, Sejroe, and Mini in cattle, Mini, Sejroe, and Bratislava in buffaloes, and Mini, Shermani, and Ranarum in sheep and goats. Zimbabwe and Malaysia: serogroup Sejroe (serovars Hardjo and Sejroe) Portugal: serogroups Sejroe (serovar Sejroe) and serogroups Ranarum (serovar Ranarum)

Seroprevalence in pigs Suwancharoen, et al. 2013 10.8% (205 of 1898) Ranarum > Pomona > Bratislava Veterinary Practitioner Notices Infective rate may be relevant to abortion rate High incidence during over population of rodent in farm Vaccine is not routinely used in Thailand Hypersensitivity could be observed. Seropositive in pigs was also studied. 10.8 % was recorded positive and the most commonly detected antibodies were against L. interrogans serovars Ranarum, Pomona and Bratislava. Leptospirosis infection in pig effected to the reproduction and related to abortion. High incidence of abortion was found during over population of rodents in farm. Combination vaccine: Five strains of lepto, and Parvo Shield E, and erisypelas

Prevalence in pigs 10% sero-positive (>100) grippotyphosa (55% of positive number) canicola patoc Surveillance at central area: Ratchaburi, Ayutthaya, Nakhorn Pratom and Saraburi by MAT (n= 400) Australia pomona Vietnam autumnalis > bratislava > grippotyphosa > icterohaemorrhagiae > pomona > tarassovi Japan copenhageni > icterohaemorrhagiae > canicola Niwetpathomwat, et al., 2006 Another study reported the Leptospirosisi surveillance in 400 pigs which was done in central area of Thailand. 10% of sample gave a positive reaction and Grippothosa was the most common serovar with 55% IgM at 5-10 days PI Max IgG at 3 weeks PI Piglet is the most susceptible period

Prevalence in elephants Oni et al, 2007 Northern Thailand (Chiang Mai, Lampang) 58.0% (Elephas maximus) Sejroe, Tarassovi, Ranarum, and Shermani Western Thailand (Kanchanaburi) 57% Tarassovi, Sejroe, Ranarum, Bataviae, and Shermani In elephants Oni and colleagues reported their investigation of Leptospirosis antibodies by MAT. In northern Thailand the seoprevalence was 58 percent and prevalent serovars were Sejroe, Tarassovi, Ranarum and Shermani. In westhern Thailand, the seoprevalence was 57 percent and prevalent serovars were Tarassovi, Sejroe, Ranarum, Bataviae and Shermani.

1,310 trapped Rodents from Northeast 15% +ve of Leptospires isolation Southeast Asia J Trop Med 2005 1,310 trapped Rodents from Northeast 15% +ve of Leptospires isolation Pyrogenes 49% > Bataviae 31% > Autumnalis 13% >Australis 4% and Javanica 3% From my own study, My colleagues and I conducted a survey of Leptospirosis in 1,310 trapped rodents from Northeastern provinces by leptospira isolation from kidney tissue. The result showed that 15 percent were presence of leptosira and react to antibodies of L. interrogans serovar Pyrogenes which is the most commom following by Bataviae, Autumnalis and Javanica

Discussion Discussion, from reviewing of those studies, The results showed that these kind of animals carried pathogenic leptospira and played role as the reservoir host

Shermani Bataviae Canicola Sejroe Grippotyphosa Ranarum Pomona Bratislava Tarassovi Pyrogenes Bataviae Autumnalis Australis Javanica These kinds of animals carried pathogenic leptospira and played role as the reservoir host. Some notices were found that predominant Leptospira serovar in dogs has shifted from Bataviae and canicola to shermani. Leptospira interrogans serovar Ranarum, Sejroe, Tarassovi and Shermani were observed mainly in ruminants. Pyrogenes Bataviae Autumnalis Austrails were most common in Rodents

Leptospirosis outbreak investigation Kanchanaburi province Results of MAT Sample Leptospira serovar Number (%) Cattle (30) Shermani Bratislava Sejroe Ranarum (96.6) 11 (36.6) (13.3) 29 (96.6) Dogs (9) 4 (44) 7 (77.7) 2 (22.2) Human (45) (22.2) (31.1) 6 (13.3) This was the interesting report of Leptospirosis outbreak investigation. Serovar identification by MAT in cattle, dogs and patients was done to reveal the source of infection. In this table, cattle were predominant reservoir host of Leptospira serovar Shermani which was associated to Leptospira serovar Shermani found in patient. Rochana Wattanarangsan, et al. 2008 FETP and DLD

Association to Human Leptospirosis Serovars Human (n) Shermani 24 Bratislava 22 Ranarum 9 Hebdomadis Sejroe 5 Autumnalis 3 Bataviae 2 Icterohemorrhagiae Djasiman 1 Grippotyphosa Javanica Unpublished data from Duangchawee in the slide showed that Leptospira serovar Shermani and Bratislava were found most frequently in patient Doungchawee’s unpublished data 2015

Serovar identification by NIH of 146 sera of Leptospirosis cases Bataviae Canicola Shermani Sejroe Grippotyphosa Ranarum Pomona Pyrogenes Bataviae Autumnalis Australis Javanica Bratislava Tarassovi No Positive Shermani (70%) Australis (20%) Autumnalis (10%) Shermani (30.4%) Tarasovi (52.2%) Australis (4.4%) Grippotyphosa (4.4%) From previous slide, Department of Medical Sciences, Ministry of Public Health, reported the results of serovar identification of 146 sera of leptospirosis cases. Leptospira serovar Shermani showed highest prevalence in different part of the country. This implys that ruminants were more likely to be the source of infection rather than rodents which had played an important role as a source of infection in the past. Pomona (4.4%) Sarmin (4.4%) Shermani (100%)

Conclusions Stray dogs, cattle, buffaloes, pigs, elephants and rodents play roles as the reservoir hosts of Leptospira in Thailand. Mostly common Leptospiral serovars in animal reservoirs are Shermani, Bratislava, Ranarum, Hebdomadis, Sejroe, Autumnalis, Bataviae, Pyrogenes. Ruminants are more likely to be a potential reservoirs and disseminate Leptospira to environment and cause the disease. Leptospirosis vaccination program in ruminants would be beneficial to reduce the number of leptospirosis cases Leptospirosis is one of a disease that should be aware during military exercises in Thailand. Stray dogs, cattle, buffaloes, pigs, elephants and rodents play roles as the reservoir hosts of Leptospira in Thailand. Mostly common Leptospiral serovars in animal reservoirs are Shermani, Bratislava, Ranarum, Hebdomadis, Sejroe, Autumnalis, Bataviae, Pyrogenes. Ruminants are more likely to be a potential reservoirs and disseminate Leptospira to environment and cause the disease. Leptospirosis vaccination program in ruminants would be beneficial to reduce the number of leptospirosis cases Leptospirosis is one of diseases that we should be aware during military exercises in Thailand.

Acknowledgements I would like to thank to my colleagues working for this institute shown on this slide who providing me the information of my presentation today. Bureau of Epidemiology, Department of Disease Control, MoPH for providing annual surveillance data of Leptospirosis in Thailand. National institute of animal health, Thai national institute of Health Faculty of veterinary science, Chulalongkorn University and Mahidol University Faculty of Science, Mahidol University Finally, I’m very grateful to the 2015 APMHE Organizing Committee for kind support and funding me to participate this conferences. Thank you.

Thank you for your attention

SAWASDEE Ka