1. 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.

2. 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.

3. 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.

4. 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
Temp oC

5. 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

6. 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.

7. 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.

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

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

10. 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

11. 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%)

12. 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

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

14. ?
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

15. 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.

16. 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

17. 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

18. 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!!!

19. 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)

20. 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 % 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

21. 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

22. 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.

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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%)

29. 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.

30. 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.

31. Thank you for your attention

32. SAWASDEE Ka