A SERO-EPIDEMIOLOGICAL STUDY OF LEPTOSPIROSIS IN SARAWAK, MALAYSIA.
By SIVAPIRAGASAM THAYAPARAN BVSc, MSc, Postgrad Certificate in Conservation Medicine.
College of Veterinary Medicine School of Veterinary and Life Sciences Murdoch University Western Australia
This thesis is presented for the degree of Doctor in Philosophy Murdoch University 2014
DECLARATION
I declare this thesis is my own account of my research and contains as its main
content work, which has not been submitted for a degree at any tertiary education
institution.
------Sivapiragasam Thayaparan
ii
ABSTRACT
Several recent outbreaks of leptospirosis involving human deaths have alarmed
health professionals in Malaysia. The study outlined in this thesis was conducted to
increase the understanding of the involvement of wildlife in the disease in Malaysia.
A strain of Leptospira (designated Lepto 175 Sarawak) was isolated from water in
Sarawak, Malaysia. This strain did not produce any titres towards other known
Leptospira sera, and thus represents a novel serovar. This serovar had 99.1% 16S
rRNA gene sequence similarity with Leptospira wolffii and was the dominant strain
present in the region.
In this study eight of the 12 non-human primates sampled (66.6%; 95% CI 34.9-
90.1) and 73 of 155 wild small mammals (47.1%: 95% CI 39.0-55.3) were
seropositive to leptospires. The seroprevalence was slightly higher in rats than in
squirrels or bats. Seropositive animals were detected in all localities sampled, with
the highest prevalence at Mount Singai (64.7%; 95%CI 38.3-85.8). Antibodies were
detected to two different serovars in non-human primates, eight serovars were
detected in rats, six serovars in bats and five in squirrels. Of 155 kidney samples
from individuals, 17 were positive for Leptospira on PCR analysis (11%; 95% CI
6.5-17).
A cross-sectional serological survey of 198 humans was conducted in four villages
around Kuching, Sarawak with 35.9% (95%CI 29.2-43.0) testing positive on the
MAT. Antibodies to serovar Lepto 175 Sarawak were most commonly detected
(31.3%; 95%CI 24.9-38.3) and were detected in individuals at all four locations. The
presence of skin wounds (OR 3.1), farm animals (OR 2.5) and rats (OR 11.2) were
iii all significantly associated with seropositivity in a multivariable logistic regression
model.
The results of the current study are important as wildlife may act as reservoirs of
leptospires for humans. Health authorities should expand disease control measures
to minimise the spill-over from wildlife to humans visiting, living or working in the
sampled locations. The pathogenic status of serovar Lepto 175 Sarawak also
requires further investigation.
iv ACKNOWLEDGEMENTS
First of all I would like to thank Almighty God Shiv Baba who I believe to be the source
of knowledge, wisdom and understanding. Secondly I would like to express my
sincere gratitude to the following people who have helped me make the presentation
of this thesis possible:
I am very grateful to my primary supervisors Ian Robertson and Mohd Tajuddin
Abdullah. I offer my sincerest gratitude for your support, ideas, constructive
criticism and scientific acumen I have valued during the project.
To Professor Ian Robertson, for giving me the honour and the privilege of accepting
to be my scientific supervisor. Professor Ian is an acknowledged world expert on
Epidemiology with admirable vast knowledge, published work and experience in
many aspects of this field and it has been not only a privilege but also a wonderful
opportunity to work under his direction and guidance and to benefit from his
experienced advice and sharp insight.
To Professor Mohd Tajuddin Abdullah for co-supervising this work, especially in
what concerns the fieldwork on animals and sharing his animal sample collection
permit for his participation as principal investigator of the primate project, under
which part of the work presented in this thesis was done.
I am grateful to thank Dr. Fairuz Amaran, Institute for Medical Research, Kuala
Lumpur for her invaluable advice and help in culturing and techniques in performing
the Microscopic Agglutination Test (MAT) and culturing the leptospires for testing
antigen. Her analytic and positive spirit, her genuine interest and incentive to the
general development of this work have further provided useful discussions and
v fundamental support and encouragement which greatly helped me in the progression
and conclusion of this work. Professor Lela Suut has a recognised experience of
relevant work in bacteriology, and her help and advice on human sample collection
from the community in Sarawak.
Dr. Nan Schaffer (President SOS rhino) and Dr. Rajasingham (Jay) Jayendran for
supporting financially to carry out my research successfully and also Murdoch
University for finance support for the molecular work.
I wish to thank Universiti Malaysia Sarawak (UNIMAS) School of Science, Medical
Faculty and Institute for Medical Research, Kuala Lumpur. Also thankful to the
Malaysia and Sarawak Economic Planning Unit (EPU) (UPE: 40/200/19/2669),
National Medical Research Register (NMRR ID-10-879-7153), Sarawak Forestry
Department (NCCD.907.4.4 (V)-235) for issuing the relevant permits to make this
research successful.
I am also thankful to all those at UNIMAS Medical faculty laboratory staff
Albert Jugah AK Paon and Joseph Tau AK Katip for collecting human blood
samples and students especially Isham, Kishen, Madinah, Aida, Ridzuan, Ikhwan
and Ridwan Rahman for helping me to carry out my field work, they are numerous
to list. Finally to my family, for support, with special thanks to: My mother, Peria
Mama & Mami and brothers and sisters for love and support, to my brother-in-law
M.Sivaparan, for being like a personal adviser during my research, finally to my
lovely wife Maithil for much patience, advice and support for making me happy,
also provided her unreserved trust in me when I doubted my capability of finishing
this thesis, which enabled me to finish this thesis.
May God bless you all for your loving contributions to my achievement.
vi TABLE OF CONTENTS
DECLARATION ...... ii
ABSTRACT ...... iii
ACKNOWLEDGEMENTS ...... v
LIST OF TABLES ...... xiii
LIST OF FIGURES ...... xv
PUBLICATIONS ...... xvi
CHAPTER 1: GENERAL INTRODUCTION ...... 1
1.1 Background ...... 2
1.2 Study hypothesis ...... 4
1.3 Objectives ...... 4
1.4 Anticipated outcomes ...... 5
1.5 Study constraints and limitations ...... 6
1.6 Thesis structure ...... 7
CHAPTER 2: LITERATURE REVIEW ...... 9
2.1 Introduction ...... 9
2.2 History of leptospirosis ...... 10
2.1.1 World wide ...... 10
2.1.2 Southeast Asia ...... 11
2.1.3 Malaysia ...... 13
2.1.4 Sabah and Sarawak ...... 19
2.3 Taxonomy and classification ...... 21
2.3.1 Serological classification ...... 22
2.3.2 Genotypic classification ...... 23
vii 2.4 Biology of leptospires ...... 25
2.4.1 Morphology ...... 25
2.4.2 Genomic organization ...... 26
2.5 Pathogenesis ...... 27
2.5.1 Entry ...... 27
2.5.2 Spread and growth ...... 27
2.5.3 Persistence and carrier sites ...... 28
2.5.4 Toxin production and virulence factors ...... 28
2.6 Clinical features of leptospirosis ...... 29
2.6.1 Domestic animals ...... 29
2.6.2 Wild animals ...... 30
2.6.3 Humans ...... 31
2.7 Pathology ...... 32
2.8 Epidemiology of leptospirosis ...... 32
2.8.1 Geographical distribution of leptospirosis worldwide and in Southeast Asia
34
2.8.2 Geographical distribution of leptospirosis in Malaysia ...... 35
2.8.3 Sources and mode of transmission of leptospirosis ...... 36
2.8.4 Cycle of host infection ...... 37
2.8.5 Survival of leptospirosis in the environment ...... 38
2.8.6 Major leptospirosis outbreaks in Malaysia ...... 38
2.8.7 Leptospirosis and ecotourism ...... 39
2.8.8 Deforestation and wildlife-human conflict ...... 41
2.8.9 Ecotourism and wildlife contact ...... 41
2.9 Economic impact of leptospirosis ...... 42
viii 2.10 Laboratory Diagnostic Methods ...... 42
2.10.1 Microscopic demonstration of Leptospira ...... 43
2.10.2 Cultural methods ...... 44
2.10.3 Microscopic Agglutination Test (MAT) ...... 46
2.10.4 ELISA ...... 46
2.10.5 Polymerase Chain Reaction (PCR) ...... 46
2.10.6 Multi-Locus Sequence Typing (MLST) ...... 47
2.10.7 Modern rapid tests ...... 48
2.11 Diagnostic techniques used in Malaysia ...... 50
2.12 Treatment, control and prevention of leptospirosis ...... 51
CHAPTER 3: LEPTOSPIRA, LEPTO 175 SARAWAK SP. NOV., ISOLATED
FROM AN ENVIRONMENTAL SAMPLE IN SARAWAK, MALAYSIA...... 53
3.1 Introduction ...... 53
3.2 Materials and Methods ...... 55
3.2.1 Morphological identification of Leptospira, Lepto 175 Sarawak ...... 55
3.2.2 Serological identification of Leptospira, Lepto 175 Sarawak ...... 56
3.2.3 Analysis of PCR products ...... 56
3.2.4 Polymerase chain reaction using conventional PCR ...... 58
3.2.5 DNA sequencing ...... 58
3.2.6 Analysis of sequence results ...... 58
3.3 Results ...... 59
3.3.1 Morphological Features of Leptospira Lepto 175 Sarawak ...... 59
3.3.2 Serological identification of Leptospira Lepto 175 Sarawak ...... 59
3.3.3 Molecular characterization of Leptospira, Lepto 175 Sarawak ...... 61
3.4 Discussion ...... 62
ix 3.5 Description of Leptospira, Lepto 175 Sarawak sp. nov...... 65
CHAPTER 4: LEPTOSPIRAL AGGLUTININS IN CAPTIVE AND FREE
RANGING NON-HUMAN PRIMATES IN SARAWAK, MALAYSIA ...... 67
4.1 Introduction ...... 67
4.2 Materials and methods ...... 69
4.2.1 Study Area ...... 69
4.2.2 Sampling procedure ...... 73
4.2.3 Microscopic Agglutination Test (MAT) ...... 74
4.3 Results ...... 77
4.4 Discussion ...... 79
4.5 Conclusions ...... 83
CHAPTER 5: SEROLOGICAL AND MOLECULAR STUDY OF LEPTOSPIRA
IN SMALL WILDLIFE AROUND WILDLIFE RESERVES AND DISTURBED
FOREST REGIONS IN KUCHING, SARAWAK, MALAYSIA...... 85
5.1 Introduction ...... 85
5.1.1 Objectives ...... 86
5.1.2 Rats and leptospirosis ...... 87
5.1.3 Bats and leptospirosis ...... 88
5.2 Materials and Methods ...... 89
5.2.1 Study/sampling sites ...... 89
5.2.2 Animal capturing ...... 96
5.2.3 Collection and preservation of samples from animals ...... 99
5.2.4 Culture and isolation of leptospirosis ...... 101
5.2.5 Microscopic Agglutination Test (MAT) ...... 102
5.2.6 Molecular analysis ...... 102
x 5.3 Results ...... 103
5.3.1 Results of capturing small mammals (Rats, Squirrels, Tree shrew and Bats)
103
5.3.2 Results of serological analysis of trapped rats,squirrels, treeshrews and bats
106
5.3.3 Influence of locality on seroprevalence...... 110
5.4 Discussion ...... 113
CHAPTER 6: PREVALENCE AND RISK FACTORS OF LEPTOSPIRA
EXPOSURES IN HUMAN LIVING IN FOREST BORDER AREAS AND
DISTURBED JUNGLE ENVIORNMENTS IN SARAWAK MALAYSIA...... 117
6.1 Introduction ...... 117
6.1.1 General Introduction ...... 117
6.1.2 Risk factors for leptospirosis in humans ...... 118
6.2 Materials and methods ...... 119
6.2.1 Study areas ...... 120
6.2.2 Study design ...... 120
6.2.3 Laboratory assays ...... 122
6.2.4 Statistical analyses ...... 125
6.3 Results ...... 125
6.3.1 Serological results ...... 126
6.3.2 Descriptive epidemiology ...... 130
6.4 Discussion ...... 137
6.5 Potential causes of bias and confounding factors in the study ...... 141
6.6 Conclusions ...... 141
6.7 Future research ...... 142
xi CHAPTER 7: GENERAL DISCUSSION AND CONCLUSIONS ...... 143
7.1 Introduction ...... 143
7.2 Discussion ...... 144
7.2.1 General Aims and Chapter Summaries ...... 144
7.2.2 Epidemiology ...... 146
7.2.3 Leptospirosis status in Sarawak ...... 148
7.2.4 Risk factors in the spread of leptospirosis ...... 149
7.3 Need for further studies ...... 150
7.4 Conclusions ...... 150
REFERENCES ...... 153
APPENDIX 1- Leptospirosis Risk Assessment Survey in Malaysian Patients and
Participants Questionnaire ...... 175
APPENDIX 2-Consent Form - Sero-epidemiological Study Of Leptospirosis In
Communities In Sarawak, Malaysia...... 181
xii LIST OF TABLES
Table 2.1:Status of leptospirosis in humans in Malaysia from 2004 to 2009...... 18
Table 2.2:Serogroups and serovars of clinical importance in L. interrogans...... 23
Table 2.3:Genomo-species of Leptospira and distribution of serogroups...... 24
Table 2.4:Wildlife and their seroprevalence to leptospirosis...... 30
Table 3.1:Serological groups tested against Lepto175...... 57
Table 3.2:Results of serological groups tested against Lepto175...... 60
Table 3.3:Pair wise distance and sequence similarities of Leptospira strains against
Lepto 175 Sarawak...... 64
Table 4.1:Serovars of Leptospira commonly found in Malaysia and used as antigens in the MAT panel...... 77
Table 4.2:MAT results obtained from 12 non-human primates...... 77
Table 4.3:Results of serology for Leptospira serovars...... 79
Table 5.1:Species, location and MAT results for animals other than bats caught. ... 104
Table 5.2:Species, location and MAT results for bats caught...... 105
Table 5.3:Seroprevalence to leptospires in small mammals trapped in different localities...... 107
Table 5.4:Seroprevalence to leptospires in different orders of small mammals...... 107
Table 5.5:Number of animals belonging to the order Rats, Squirrels and Treeshrews seropositive to different leptospiral serovars...... 108
Table 5.6:Number of Bats seropositive to different leptospiral serovars...... 109
Table 5.7:Titres of antibodies to different serovars found in small mammals belonging to the Rats, Squirrels, Treeshrews and Bats...... 110
Table 5.8:Seroprevalence to leptospires in mammals belonging to the Rats, Squirrels and Treeshrews in different localities...... 111
xiii Table 5.9:Seroprevalence to leptospires in bats in different localities...... 112
Table 5.10:PCR results from the kidneys of small mammals and their potential PCR using Neighbouring method...... 112
Table 6.1:Gender distribution of humans sampled at the four localities...... 126
Table 6.2:Age distribution of participants in the study...... 126
Table 6.3:Seroprevalence (MAT) to leptospirosis in humans from different localities.
...... 127
Table 6.4:Seroprevalence (ELISA) of leptospirosis in humans according to their locality...... 127
Table 6.5:Seroprevalence (MAT) to Lepto 175 Sarawak in humans according to their locality...... 128
Table 6.6:Distribution of seropositive (MAT) reactions to Leptospira spp. in humans according to serovar and their respective titres...... 129
Table 6.7:Relationship between seropositivity (MAT) to Leptospira spp. and host and environmental factors...... 133
Table 6.8:Final logistic regression model...... Error! Bookmark not defined.
xiv LIST OF FIGURES
Figure 2.1:Case fatality rate (CFR) of leptospirosis in Malaysia from 2004 to 2010 . 17
Figure 2.2:The CFR of leptospirosis in different Malaysian states (2004 to 2010). ... 17
Figure 2.3: Number of confirmed cases of leptospirosis in humans in Sarawak...... 21
Figure 3.1:Leptospira, Lepto 175 Sarawak under dark field microscopy (x200)...... 59
Figure 3.2:The phylogenetic tree of Lepto 175 Sarawak with other strain 16 s rRNA partial sequences...... 62
Figure 4.1:Location of Bako National Park ...... 71
Figure 4.2:Location of Matang wildlife Centr ...... 72
Figure 4.3: Silvered leaf monkey and Proboscis monkey being prepared for blood collection...... 73
Figure 5.1:Sampling sites of small wildlife...... 91
Figure 5.2:UNIMAS and surroundings where samples were collected...... 92
Figure 5.3:Location of Wind Cave and Fairy Cave...... 94
Figure 5.4:Location of Mount Singai Conservation Area...... 95
Figure 5.5:Wire mesh trap used for trapping small mammals ...... 97
Figure 5.6:A fruit bat caught in a mist net...... 98
Figure 5.7:Illustrated diagram of the setup of a mist net .
(http://www.ilmb.gov.bc.ca/risc/pubs/tebiodiv/bats/batsml20-04.htm) ...... 98
Figure 5.8:Harp Net set along the flight path of bats...... 99
Figure 5.9:Collection of kidney samples...... 100
Figure 5.10:Examining a fruit bat prior to the collection of blood...... 101
xv
PUBLICATIONS
In the course of the undertaken research work, the following publication and
posters were done:
A) PEER – REVIEWED JOURNALS
Thayaparan S., Robertson ID., and Abdullah MT- (2013) Leptospiral Agglutinins
in Captive and Free Ranging Non-Human Primates in Sarawak, Malaysia,
Veterinary World, 7(6): 428-431.
Thayaparan S., Robertson ID., Amaran F., Lela S. and Abdullah MT. (2013)
Leptospirosis, An Emerging Zoonotic Disease in Malaysia: A Review, The
Malaysian Journal of Pathology, 35 (2):123-132.
Thayaparan S., Robertson ID., Amaran F., Lela S. and Abdullah MT. (2013)
Serological Prevalence of Leptospiral Infection in Wildlife in Sarawak, Malaysia,
Borneo Journal of Resource of Science and Technology, 2013:2:79-82.
Thayaparan S., Robertson ID., and Abdullah MT., Serology and molecular
detection of Leptospira spp. from small mammals captured in Kuching, Sarawak,
Malaysia, Malaysian Journal of Microbiology, 2015: 11(1):93.101.
MANUSCRIPT UNDER REVIEW
Thayaparan S., Robertson ID., Amaran F., Lela S. and Abdullah MT. Sero-
epidemiological and risk factor analysis on Leptospira infection in communities
living around wildlife habitats in Kuching, Sarawak, Malaysia.
xvi B) CONFERENCE PAPERS
Thayaparan S., Amaran F., Robertson ID., Abdullah ID, Preliminary results on noval strain of Leptospira, Lepto 175 (Sarawak), in Sarawak Malaysia, 8th
Scientific Meeting of International Leptospirosis Society, Fukuoka, Japan, During
8-11 October 2013.
Thayaparan S., Robertson ID., Fairuz A., Su'ut L and Abdullah MT. Deforestation and Wildlife Tourism could be a Potential threat due to Leptospirosis Outbreak in
Sarawak, Malaysia, The 2nd International Symposium on Zoonoses and Emerging
Infectious Diseases (Malaysia, Universiti Malaysia Sarawak), during 15-16
December 2011.
THE POSTER PRESENTATION:
Thayaparan S., Robertson I D., and Abdullah M T., 2013. A Seroepidemiological
Study on novel strain of Leptospira, Lepto 175 Sarawak in Sarawak, Malaysia.
Thayaparan S., Robertson I D., and Abdullah M T., 2012. Preliminary Results of the Seroprevalence of leptospiral infection in wildlife in Sarawak, Malaysia.
Thayaparan S., Robertson I D., and Abdullah M T., 2012. A Seroepidemiological study of Leptospirosis in Kuching Sarawak, Malaysia.
xvii
CHAPTER 1 : GENERAL INTRODUCTION
Leptospirosis in Malaysia can be defined as an emerging or re-emerging endemic
disease. The disease can affect both humans and other animals throughout Malaysia,
and can result in serious socio-economic and public health consequences (Arief,
2013). Due to the abundance of wildlife and the tropical climate, infection in
wildlife can lead to economic loss and pose a potential source of infection for
communities in the country. This disease is caused by pathogenic or intermediate
Leptospira, which belong to the family Leptospiraceae (Adler and de la Peña
Moctezuma, 2010; Zakeri et al., 2010). Humans contract the disease through direct
contact with infected blood, tissues, organs or urine of infected hosts. Transmission
can also occur by direct penetration of the leptospiral organisms through the
conjunctiva or surface epithelium (Faine, 1982; Russ et al., 2003). Indirect contact
with the environment, including soil, mud, fresh water, vegetation, foodstuffs and
workplaces infested with rodents, can also result in infection of humans (Turner,
1973). Tsai and Fresh (1975) reported the first human case of leptospirosis in Asia
through contact or exposure to wild animals in Vietnam. In recent years outbreaks of
human leptospirosis in Malaysia have been documented around wildlife reserves and
parks (Lim et al., 2011; Thayaparan et al., 2013a) resulting in 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 (Yasak, 2000), and
leptospirosis has the potential to impact on this important source of income.
The proposed study was carried out to determine the extent of exposure to
leptospirosis in human communities on the periphery of wildlife reserves and in an
urban area. In addition, small wildlife (bats, rodents, squirrels, treeshrew and non
1 human primates) were trapped in the forest habitats around the reserves and in the
urban area and blood samples screened for the presence of leptospiral antibodies.
This provided information on the possible zoonotic importance of wild mammals in
maintaining this disease in communities in Sarawak. Serum samples were also
collected from villagers who were at risk of having contact with small wildlife. The
results will be used to develop suitable preventive and control measures for
leptospirosis in the future. The results of this study will give a clear indication of the
disease prevalence, reservoir host and the mode of transmission in the region and
will also further understanding of the potential relationship between wildlife and
humans in rural areas.
1.1 Background
Leptospirosis is a zoonosis of ubiquitous distribution, caused by the spirochaete
Leptospira, of which there are two forms: the pathogenic species; and the non-
pathogenic species (Bharti et al., 2003; Morey et al., 2006; Treml et al., 2002).
Leptospirosis in humans was first reported 100 years ago by Adolf Weil in
Heidelberg (Levett, 2001). The importance of occupation as a risk factor for
infection was recognized early in the history of the disease (Turner, 1973). The role
of rats as a source of human infection was discovered in 1917 (Levett, 2001) and
subsequently some researchers have identified that flying foxes can carry pathogenic
Leptospira in Australia (Cox et al., 2005; Smythe et al., 2002b; Tulsiani et al.,
2011). In humans, leptospirosis can cause headaches, fever, chills and sweating
(Edwards, 1960; Levett, 2001; Magill, 1998; Turner, 1973), with some highly
pathogenic serovars causing pulmonary haemorrhage and even death. Leptospirosis
2 is increasing in importance as an occupational disease as intensive farming practices
and adventure tourism activities become more widely adopted in Malaysia.
Diagnosis of leptospirosis by the presenting clinical signs is not definitive and the
disease can be confused with several other diseases including influenza, hepatitis
and various haemorrhagic fevers (Dutta and Christopher, 2005; Enria and Pinheiro,
2000). Early diagnosis in humans can be confirmed by culture of blood samples
during the initial fever and culture of urine samples during recovery from the fever.
One week after the onset of symptoms has passed, serological assays, such as the
Microscopic Agglutination Test (MAT) or Enzyme Linked Immune Sorbent Assay
(ELISA), may be used to detect antibodies in the blood. Only the pathogenic and
intermediate pathogenic Leptospira were investigated in the research reported in this
thesis.
In Malaysia isolation of leptospires from black rats (Rattus rattus) was first reported
in 1928 by Fletcher (Russ et al., 2003). From 1953 to 1955, 30 pathogenic serovars
were identified by researchers (El Jalii et al., 2002) from both military personnel and
civilians. Kennedy and Robinson, (1956) reported 31 cases of leptospirosis among
British army personnel in Malaysia. In a study highlighting the endemic nature of
leptospirosis in Malaysia, a high prevalence of antibodies to leptospires was
demonstrated in humans and animals throughout Malaysia (Lim et al., 2011;
Thayaparan et al., 2013a). The highest seroprevalence was reported in labourers
working in rubber estates and those working in the sewage, drainage, forestry and
cleaning industries (Bahaman and Ibrahim, 1988). Many more investigations on
human leptospirosis in Malaysia have revealed a high prevalence of infection
(Bahaman and Ibrahim, 1988; El Jalii and Bahaman, 2004; El Jalii et al., 2002).
However since 1986 no major investigations have been undertaken on human
3 leptospirosis in Peninsular Malaysia.
Studies carried out in Sabah in Malaysian Borneo, have highlighted the possible
zoonotic importance of wild mammals in the maintenance of the disease within the
community. One study (Russ et al., 2003) showed that a significant percentage
(25.75%) of people living within the periphery of a wildlife park had been exposed
to leptospiral antigens. Leptospirosis has been reported among British cavers in
Sarawak (Self et al., 1987) and another incident occurred in an American caver
returning from Sarawak, Malaysia in 1994 (Mortimer, 2005). Most recently in 2000
there was an outbreak of leptospirosis in international participants in an “Eco-
Challenge” adventure race in Sabah, Malaysia (Haake et al., 2002; Sejvar et al.,
2003).
1.2 Study hypothesis
For the purpose of this study, it is hypothesised that local communities (people)
living around forested habitats that are in potential contact with wildlife (bats,
rodents, tree shrews and non-human primates) will be more significant to infection
with Leptospira.
1.3 Objectives
The research described in this thesis was designed to collect detailed information
about the prevalence and pattern of Leptospira in wildlife and in humans adjacent to
wildlife reserves in Kuching, Sarawak, Malaysia. This information will help in
identifying risk factors for human infection and allow development of suitable
4 measures for disease control. The outcome of this study will give a clear indication
of the carrier status of pathogenic Leptospira by small wildlife, the disease
prevalence, possible reservoir hosts, and provide potential information on the mode
of transmission within the region.The specific objectives were:
a. To determine the cause of recent human outbreaks around forested areas.
b. To determine the role of wild small mammals in the lifecycle of Leptospira.
c. To document animal hosts, and their seroprevalence around wildlife reserves and
human settlements.
d. To investigate the epidemiology of human leptospirosis, including risk factors for
infection.
1.4 Anticipated outcomes
The anticipated outcomes of this study are:
1. To identify the leptospiral serovars in wildlife and humans.
2. To provide information about the epidemiology of human leptospirosis, including
the prevalence of the disease and potential risk factors for disease.
3. To provide information upon which prevention measures could be developed and
applied to reduce the potential for disease.
4. To provide data on the distribution of infection in wildlife to medical authorities
to enable prediction of future outbreaks of disease.
5. To publish results in high impact journals.
5
1.5 Study constraints and limitations
The Malaysian government has classified all species of flying foxes as protected,
especially in Sarawak, and all studies involving these mammals have been
prohibited. However, it was possible to obtain results from other species including
small bats, rodents and squirrels, as well as non-human primates. As the role played
by flying foxes in the spread of leptospirosis needs to be investigated further, this
places challenges on undertaking research on these species in Malaysia.
With leptospirosis being a major health concern in Malaysia, it would be desirable
to undertake a study on wildlife and at-risk humans across the country, however, due
to funding constraints; this study focused on Sarawak. This state was selected, as it
appeared to be having the most serious problem in keeping the disease in check. The
success of this particular research would possibly facilitate similar studies being
conducted in other areas of Malaysia. This study was possible because of the
collaboration with the Zoology Department of the University Malaysia Sarawak
(UNIMAS), the medical faculty of UNIMAS and the Institute of Medical Research
(IMR), Kuala Lumpur. Such relationships will certainly be helpful in future
endeavours to investigate leptospirosis in Malaysia.
6 1.6 Thesis structure
In order to achieve these aims, this thesis is presented in seven chapters.
Ø Chapter 1 General introduction. This provides a brief introduction on the
status and the reason for initiation of research into leptospirosis in wildlife and
humans in Sarawak, Malaysia. It also provides the aims and structure of the thesis.
Ø Chapter 2 Literature review. In this chapter the history and nature of
leptospirosis worldwide and in Malaysia is reviewed. The epidemiology, clinical
features, diagnostic facilities in Malaysia, treatment, control and prevention of
leptospirosis are also discussed in this chapter.
Ø Chapter 3 Leptospira, Lepto 175 Sarawak Nov., isolated from an
environmental sample in Sarawak. This chapter describes the isolation of the
organism, including the molecular and serological methods used to identify the
strain and to confirm it as a new strain of Leptospira.
Ø Chapter 4 Serological study on leptospirosis in captive and free ranging
non-human primates in Sarawak, Malaysia. In this chapter the prevalence of
leptospirosis is estimated in non-human primates using a MAT.
Ø Chapter 5 Serological and molecular study of Leptospira in Rats,
Squirrels, Treeshrews and Bats around wildlife reserves and disturbed forest in
Kuching, Sarawak, Malaysia. In this chapter the results of testing of samples
collected from small wild mammals is reported. The seroprevalence of Leptospira is
estimated using the MAT, the pathogen is cultured and some organisms isolated.
Ø Chapter 6 Serological study and analysis of risk factors that may be
involved in the introduction of leptospirosis to a human community located
around wildlife reserves in Kuching, Sarawak, Malaysia. In this chapter the
seroprevalence to Leptospira in humans is reported through examining the results of
7 the MAT and ELISA tests. Also a risk assessment on the likelihood of Leptospira
entering a community from wild small mammals is reported. Data are collected
using questionnaires applied during the blood collection.
Ø Chapter 7 General discussion and conclusions. In this chapter the results of
the thesis are discussed. The prevalence in animals and humans is discussed and the
possible risk offered by wild animals to humans evaluated.
8
CHAPTER 2 : LITERATURE REVIEW
2.1 Introduction
Leptospirosis is a tropical disease of major concern to countries such as Malaysia
and has been linked to exposure to tropical jungles (MacKay-Dick, 1971). Many
species of wildlife can be found in jungle environments, and some of these species
may be vectors of pathogenic leptospires. In 1917 rats were identified as carriers of
leptospires (Levett, 2001; Monahan et al., 2009), and subsequently flying foxes have
been found to also carry pathogenic leptospires in Australia and South America
(Cox et al., 2005; Matthias et al., 2005; Smythe et al., 2002a; Tulsiani et al., 2011).
Although the role of flying foxes as carriers of pathogenic Leptospira is not fully
understood, it is known that several other species of wildlife can also act as potential
carriers (Cox et al., 2005; Hartskeerl and Terpstra, 1996). The bacteria can cause
polymorphic disease conditions in wild and domestic animals, as well as in humans
(Cox et al., 2005). However, to date there has been little research undertaken on the
role of wildlife in outbreaks. Due to the current significant level of deforestation
occurring and the involvement of humans in tropical jungles, there is the potential
for exposure of humans to new serovars of Leptospira. Leptospirosis in wildlife can
have negative consequences for biodiversity, livestock health, animal welfare and
the economy of a country, as well as human health (Caley and Ramsey, 2002; Russ
et al., 2003). In this thesis, the focus was on the disturbed jungle habitat surrounding
the capital city of Sarawak, Kuching, where pockets of human inhabitation can be
found.
Zoonotic diseases represent key biological threats to human health. Members of the
9 genus Leptospira can infect a broad range of animal species, including humans.
Wildlife can also play a major role in the organism’s transmission and
understanding the role of wildlife in the disease’s epidemiology is important when
addressing disease in domestic animals and humans. Leptospirosis in wildlife is also
important in its own right, with impacts on biodiversity as well as animal welfare.
The proximity of wildlife to potential livestock carriers, the amount of infective
agents in the environment, geographical, climatic, and socio-cultural factors
affecting animal husbandry and agricultural practices may result in cross-over of
infection from domesticated animals to wildlife species.
Dramatic societal and environmental changes, particularly involving deforestation
and development of large scale agricultural industries (Colchester, 1994; Persoon,
2004), have caused the spectrum of infectious disease to change rapidly (McMichael
et al., 1996). Across the world, explosive growth in the human population and the
associated expanding poverty and urban migration, along with increased
international travel and commerce, increase the risk of humans to exposure to
infectious agents such as Leptospira. In this chapter the impacts of leptospires on
wildlife, and the current status of surveillance and the options to strengthen control
policies for leptospirosis in Malaysia are reviewed.
2.2 History of leptospirosis
2.1.1 World wide
Leptospirosis has been documented worldwide, but formal reporting systems vary
widely between countries (Lau et al., 2010a; Lau et al., 2010b; Pappas et al., 2008).
10 Frequently the disease gains public attention when outbreaks occur in association
with natural disasters, such as flooding in Nicaragua in 1995 or among foreign
travellers and extreme athletes (Mortimer, 2005). Southeast Asia is an endemic area
for leptospirosis, and infection in humans has been reported throughout the region
with 70% of the major pathogenic serovars having been isolated from Asia (Laras et
al., 2002). Although leptospirosis is likely to have been present for millions of years
(Levett, 2001), it is only in relatively recent times that the bacterial cause of the
disease was confirmed. In 1886 Adolf Weil published an account detailing the
icteric form of leptospirosis, and the disease was subsequently named after this
discoverer. However, his observations had already been postulated by others,
including Hippocrates (Levett, 2001). It has been hypothesised that leptospirosis
was responsible for an epidemic among the natives of the Massachusetts coastal
region, USA just before the arrival of the Pilgrims in 1620 (Marr and Cathey, 2010).
What Weil described was the icteric form of leptospirosis with jaundice as the key
clinical sign. In contrast the milder forms were harder to diagnose at that time due to
a lack of advanced bacteriological techniques. Spirochaetes continued to cause
problems for the later part of the 19th century, and it was only in 1907 that Stimson
managed to isolate a leptospire from a patient (Heath et al., 1965). His subsequent
research highlighted that the bacteria were concentrated in the renal tubules and
were shaped like question marks. This gave rise to the name “Spirocheta
interrogans”, and this name has been retained (Heath et al., 1965).
2.1.2 Southeast Asia
Leptospirosis is emerging as a serious concern to public health in Southeast Asia
(Bengis et al., 2004; Bhatia and Narain, 2010; Flynn, 1999). The disease has been
recognised in patients from Indonesia with clinical jaundice and non-malarial fever,
11 patients with clinical jaundice from Laos and Vietnam, and patients with
haemorrhagic fever in Cambodia (Laras et al., 2002; Vijayachari et al., 2008). In
addition, a cross-sectional community-based study was conducted in Laos to obtain
estimates of the background seroprevalence. These findings suggested that
leptospirosis was under-reported in Southeast Asia due to the wide range of clinical
symptoms associated with acute leptospiral infection (Vijayachari et al., 2008).
During the Indochina War, the French experienced three epidemics of leptospirosis
in 1950, 1951 and 1952, as well as having one quarter of their men incapacitated
during one operation (MacKay-Dick, 1971). Easton, (1999) reported a spike in
infection in Manila following flash floods during the monsoon season. The bacteria
would most likely have entered the patients’ bodies through cuts on the skin when
they waded through the floodwaters. Residents in low-lying areas and rice farmers
were identified as a high-risk group for infection. One possible source of the
problem was the increasing rat population with farmers from one province culling
approximately 800 rats per night in their rice fields during the dry season (Easton,
1999). Studies in various locations in Thailand found that, after rats, dogs were the
most important reservoir for leptospirosis with a seroprevalence of 11%, with the
Bataviae serogroup being the most prevalent (5.2%) (Meeyam et al., 2006). Contact
with sewage, staying outdoors more than half the time and consuming raw meat
increased the likelihood of seropositivity in the sampled dogs. It was possible that
the dogs acquired their infection from rats, which were responsible for an outbreak
in north-eastern Thailand between 1999 and 2003 (Meeyam et al., 2006;
Thaipadungpanit et al., 2007). During that period Thaipadungpanit et al., (2007)
developed a scheme of multi-locus sequence typing of pathogenic Leptospira and
12 they confirmed that serovar Autumnalis was responsible for the outbreak and was
being maintained by bandicoot rats (Bandicota bengalensis).
Besides infecting local residents, Leptospira can also cause illness in visitors to
tropical regions, particularly those associated with eco-tourism and adventure travel
(van Crevel et al., 1994). van Crevel et al., (1994) investigated leptospirosis in 32
Dutch travellers between 1987 and 1991 and found 28 of them had returned from
Southeast Asia with the majority visiting Thailand and 21 of these had taken a
rafting tour. From this research it was recommended that doctors should consider
leptospirosis in their differential diagnosis whenever a patient with fever returned
from the tropics. Due to the increasing number of reported cases of leptospirosis in
the western world, it has been recommended that travellers try to avoid high-risk
aquatic activity in the tropics and undertake chemoprophylaxis if they do take part in
water-sports in Southeast Asia (Haake et al., 2002; Monahan et al., 2009).
Saunders, (1979) examined 78 cases of leptospirosis in Pahang and reported the lack
of specificity of the clinical syndrome, highlighting the challenges in diagnosing the
disease purely on presenting clinical symptoms.
In Thailand, researchers conducted a serological survey of workers who had been
involved in cleaning a pond in Khumuang, Buriram Province and analysis by
multivariable logistic regression indicated that wearing long pants or skirts was
protective against leptospiral infection, while the presence of more than two wounds
on the body was associated with infection (Phraisuwan et al., 2002).
2.1.3 Malaysia
In Malaya, Fletcher isolated Leptospira from black rats (Rattus rattus) in 1928
(Bahaman and Ibrahim, 1988; Russ et al., 2003). Subsequently, many investigations
13 have demonstrated a high prevalence of infection in humans (El Jalii and Bahaman,
2004) with Kennedy and Robinson, (1956) reporting 31 cases among British army
personnel in Malaysia. Also, 35% of febrile illness among British forces in Malaysia
was attributed to leptospirosis (MacKay-Dick, 1971). Between 1953 and 1955, 30
pathogenic leptospiral serovars were identified by Alexander and his colleagues
(Bahaman et al., 1987) from both military personnel and civilians. Their studies
demonstrated a high seroprevalence in humans throughout Malaysia. The highest
distribution was found in labourers working in rubber estates and those working
with the sewage, drainage, forestry and town cleaning industries (Bahaman et al.,
1987). In the 1950s and 1960’s a comprehensive study of leptospirosis in Malaysia
was undertaken that included testing various mammals from a range of
environments, as well as assessing occupational risks to humans. The results
suggested that rats were the main maintenance hosts for leptospirosis, despite the
presence of infection across many animal species. Over one hundred (104) strains
were isolated and identified (Smith and Turner, 1961). Bahaman et al., (1987)
conducted a cross-sectional serological survey of domestic animals in West
Malaysia and found that approximately one quarter of the animals examined had
agglutinating antibodies to L. interrogans. Cattle, buffaloes and pigs were all
observed to have a high seroprevalence with temperate cattle breeds appearing more
susceptible to infection than local breeds. A subsequent study found that the
seroprevalence in cattle and buffaloes was 14.4% (Bahaman and Ibrahim, 1988). A
new serovar was isolated from a bovine kidney, while six other serovars were
isolated for the first time from Malaysian cattle. Serovar Hardjo was shown to be
maintained in Malaysian cattle (Bahaman et al., 1987). Recently leptospirosis has
been reported in detention centres for refugees in Malaysia (Jones, 2011; Wright,
14 2011). Health authorities managed to contain an outbreak at the Juru Detention
Centre, but the death of six Burmese at an undisclosed detention centre in September
2009 raised fresh concerns for several Malaysian NGOs. In July 2010, cases of
leptospirosis were reported nation-wide and eight people who took part in a search
and rescue operation in Lubok Yu, Maran, Pahang died from the disease, causing the
picnic spot to be closed to the public (Hin et al., 2012; Sapian et al., 2012). Other
cases were reported in Kedah during July 2011, with one fatality at Lata Bayu (Lim
et al., 2011). As recent as March 2012, a national service trainee died of suspected
leptospirosis at Sungai Siput in Perak, Malaysia (Thayaparan et al., 2013b). This
resulted in a suspension of water-based activities at all National Service Training
army camps.
According to data from the Ministry of Health the number of leptospirosis cases
increased dramatically from 2004 to 2009 (Table 2.1), however the number of
deaths from leptospirosis did not change from 2004 to 2007, although it increased
markedly to 47 and 62 deaths in 2008 and 2009, respectively. The highest numbers
of cases were reported in the state of Perak during 2005, 2006, 2008 and 2009 with
71, 93, 289 and 280 cases, respectively (Table 2.1). In 2004, more cases (32) were
reported in Sarawak than in other years and in 2007 the highest numbers of cases
(184) were reported in Pahang. The states of Sarawak, Selangor and Terengganu
showed a gradual increase in the number of cases over this period. No cases were
reported in WP Labuan owing to its small geographical area with no forest habitat
and the fact that it is surrounded by seawater.
Case fatality rates (CFR) over the period from 2004 to 2009 varied from 1.8% to
7.6% (Figure 2.1) with an average of 4.44%. During this period a total of 5,267
cases of leptospirosis were reported nationwide with 234 known fatalities. Perak had
15 the highest CFR for this period (6.81%), followed by Sarawak (6.42%) and Perlis
(6.25%). Approximately one-fifth of all cases of leptospirosis for this period were
documented in Perak, of which 71 proved fatal (Figure 2.2). The almost threefold
increase in the number of cases since 2007 may be due to improved diagnostic
techniques or a greater awareness of the disease. This may also have contributed to
reducing the CFR from 2004. It is also possible that climate change in the last four
years of this period played a part in influencing the pattern of infection, as outbreaks
of leptospirosis usually follow after a major flood during monsoon seasons (Lau et
al., 2010).
16
Figure 2.1: Case fatality rate (CFR) of leptospirosis in Malaysia from 2004 to 2010 Source: (Hakim, 2011).
Figure 2.2: The CFR of leptospirosis in different Malaysian states (2004 to 2009) Source: (Hakim, 2011).
17 18
Table 2.1: Status of leptospirosis in humans in Malaysia from 2004 to 2009.
State Year 2004 2005 2006 2007 2008 2009 Total Cases Deaths Cases Deaths Cases Deaths Cases Deaths Cases Deaths Cases Deaths Cases Deaths Sarawak 32 2 71 2 37 3 70 4 58 5 70 4 309 20 Sabah 13 1 12 1 19 0 35 1 34 2 35 1 148 6 Perak 29 4 71 4 93 9 280 19 289 16 280 19 1042 71 Selangor 16 5 20 3 37 0 208 7 97 2 208 7 586 24 Pahang 29 1 24 0 51 3 184 5 198 7 184 5 670 21 Kelantan 15 1 38 1 17 1 138 4 180 4 138 4 526 15 Terengganu 7 1 17 0 42 1 126 9 107 4 126 9 425 25 Kedah 15 1 27 0 31 0 106 9 52 2 106 9 337 21 Negri-Sembilan 27 1 41 0 24 0 91 0 59 0 91 0 333 1 Johor 30 1 29 6 31 2 59 3 87 3 59 3 295 18 Kuala Lumpur 31 0 20 1 27 1 54 0 45 0 54 0 231 2 Penang 9 0 25 1 28 0 32 0 25 2 32 0 151 3 Melaka 7 1 9 1 79 2 20 1 25 0 20 1 160 6 WP Putrajaya 1 0 1 0 7 0 14 0 1 0 14 0 38 0 Perlis 2 1 2 0 4 0 1 0 6 0 1 0 16 1 WP Labuan 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total 263 20 378 20 527 22 1418 62 1263 47 1418 62 5267 234 Source: (Hakim, 2011)
18 2.1.4 Sabah and Sarawak
As well as Peninsular Malaysia, scientific research has also focused on Malaysian
Borneo. There have been reports of leptospirosis in humans in Sabah and Sarawak,
notably an outbreak following the 2000 Eco-challenge in Sabah (Lam and Kan,
2005; Sejvar et al., 2003). This highlighted the risks of eco-tourism and adventure
travel, as most of the victims were athletes from western countries. In the Eco-
challenge event, participants competed in jungle trekking, swimming and kayaking
in freshwater and caving. A team of researchers from the CDC Atlanta contacted
189 athletes from 27 countries following the outbreak (Sejvar et al., 2003). Eighty
athletes met their case definition, with 29 cases being hospitalised, although no
fatalities were reported. Another study in Sabah revealed that swimming in the
Segama River identified as a risk factor for infection and 20 athletes reported taking
doxycycline prophylactically and this seemed to have a protective effect, with a
preventive efficacy of 55% (Haake et al., 2002; Ricaldi and Vinetz, 2006).
Studies carried out in Sabah reported a high seroprevalence (25.75%) in people
living within the periphery of a national park, presumably due to exposure/contact
with wild mammals (Russ et al., 2003). Leptospirosis has also been reported in a
caver from the USA after he returned from Gunung Mulu National Park in Sarawak
(Mortimer, 2005). Recently researchers from Universiti Malaysia Sarawak
(UNIMAS) and the Sarawak Health Department have conducted research in the
Rejang Basin area, Sarawak finding that 31% of humans sampled were seropositive
to leptospires and antibodies were associated with farming and/or water activities
(Stoye, 2012; Suut et al., 2011). In Bakun in Sarawak, a Chinese national working
on the hydro-electric dam project site was hospitalised with a serious condition
suspected to be leptospirosis, and the subsequent death of 10 workers involved in
19 the translocation of animals from the Bakun Dam water catchment area to higher
ground were speculated to be due to leptospirosis or melioidosis (Baer, 2009; Sibon,
2011).
In 2011, 186 cases of leptospirosis were reported in humans in Sarawak including
13 deaths, compared with 49 cases in the previous year (Koh, 2011). The CFR
associated with this outbreak (6.9%) was higher than that in previous years. On the
12th December 2011, an outbreak occurred at RSAT Army camp at Penrissen Batu
8, Kuching, Sarawak with five army recruits showing clinical signs of leptospirosis
and the disease was subsequently confirmed serologically. The source of infection
was identified as drinking and bathing activities in the small river near the camp
(Safii, Downloaded from jknsarawak.moh.gov.my/en/ on 18th March 2012a). The
Kuching Divisional Health Office was notified of another suspected leptospiral
outbreak on the 30th December 2011 by the Sarawak General Hospital. This incident
involved two army recruits from Blok G10, Kem Semenggok, Kuching and both
were serologically positive for leptospirosis (Safii, Downloaded from
jknsarawak.moh.gov.my/en/ on 7th Dec 2012). The health authority of Sarawak
confirmed an outbreak of leptospirosis in residents of Tiong Hua Road and
leptospires were isolated from drain water in Sibu during June 2012 (Safii,
Downloaded from jknsarawak.moh.gov.my/en/ on 18th March 2012b). In the state
of Sarawak, the number of cases of leptospirosis was similar from 2004 to 2010,
however in 2011 and 2012 there was a large increase in the number (Figure 2.3).
20
Figure 2.3: Number of confirmed cases of leptospirosis in humans in Sarawak Source: (Hakim, 2011).
The four-fold spike in the number of cases in Sarawak is a valid cause for concern
for health authorities in both Kuching and Kuala Lumpur. For 2011 alone, the CFR
was 6.9%. The number of cases (186) reported in 2011 alone was already equivalent
to 60% of the combined total from 2004 to 2009. The record high of 271
documented cases the following year may be attributed to the implementation of
mandatory reporting procedures or increased awareness of the symptoms of
leptospirosis by medical practitioners. This epidemic may also reflect the living
conditions of residents in various areas of Sarawak.
2.3 Taxonomy and classification
The genus Leptospira is characterized by Gram-negative flexuous, helical
organisms. The classification of leptospires has been unclear and problematic.
Currently there are two methods that can be used for classification of the genus
Leptospira: serological and molecular biological based methods.
21 2.3.1 Serological classification
Leptospires are spirochaetes in the order Spirochaetales, family of Leptospiraceae
and include two genera, Leptospira and Leptonema (Adler and de la Pena
Moctezuma, 2010). Leptospira are obligate aerobes with an optimum growth
temperature ranging from 28°C to 30°C (Chang, 1947; Johnson et al., 1969). The
genus Leptospira was divided into two species based on serological classification:
Leptospira interrogans, which comprises all the pathogenic strains and Leptospira
biflexa, the saprophytic strains, which are present in soil, fresh water or marine
environments (Bharti et al., 2003; Cerqueira and Picardeau, 2009; Dikken and
Kmety, 1978). Each species is divided into serogroups on the basis of serological
cross reactivity and these are further divided into serovars. According to this
classification, leptospires are classified into over 250 serovars by the Microscopic
Agglutination Test (MAT), that uses specific antisera to identify the distinct
serovars (Dikken and Kmety, 1978; Faine, 1994). Serovars that show 10% cross-
agglutination are amalgamated into a serogroup (Faine, 1994). Classification and
identification of leptospires using serology is a difficult and time-consuming
procedure. The serogroups of L. interrogans and their common serovars are shown
in Table 2.2.
22 Table 2.2: Serogroups and serovars of clinical importance in L. interrogans.
Serogroups Serovars Icterohaemorrhagiae Icterohaemorrhagiae, Copenhageni, Lai Hebdomadis Kremastos, Hebdomadis, Jules Autumnalis Autumnalis, Fort-bragg, Bim Pyrogenes Pyrogenes, Zanoni Bataviae Bataviae Sejroe Hardjo, Sejroe, Saxkoebing Grippotyphosa Grippotyphosa Pomona Pomona Canicola Canicola, Portlandvere Tarassovi Tarassovi Australis Australis, Bratislava Javanica Javanica Ballum Ballum, Arborea Djasiman Djasiman Source: ( Wai'in, 2007)
2.3.2 Genotypic classification
With the development of tools in molecular biology, classification and taxonomy of
leptospires has changed. Genetic taxonomy involves DNA/DNA hybridization and
guanine-plus-cytosine mol percentage (G+C mol%) content of the bacteria’s DNA
(Wai'in, 2007). This has given rise to a number of genomo-species, which include
serovars of both L. interrogans and L. biflexa. However unfortunately, genomo-
species of Leptospira do not correspond to the previous two species and pathogenic
and non-pathogenic serovars can be classified within the same species (Table 2.3).
23 Table 2.3: Genomo-species of Leptospira and distribution of serogroups.
Genomo-species Serogroup
L. interrogans Icterohaemorrhagiae, Canicola, Pomona, Australis, Autumnalis, Pyrogenes, Grippotyphosa, Djasiman, Hebdomadis, Sejroe, Bataviae, Ranarum, Louisiana, Mini, Sarmin
L. noguchii Panama, Autumnalis, Pyrogenes, Louisiana, Bataviae, Tarassovi, Australis, Shermani, Djasiman, Pomona
L. santarosai Shermani, Hebdomadis, Tarassovi, Pyrogenes, Autumnalis, Bataviae, Mini, Grippotyphosa, Sejroe, Pomona, Javanica, Sarmin, Cynopteri
Ranarum, Semaranga, Sejroe, Mini, Javanica L. meyeri
Hurstbridge L. fainei
Semaranga, Andamana L. biflexa
Javanica, Ballum, Hebdomadis, Sejroe, Tarassovi, Mini, Celledoni, L. borgpetersenii Pyrogenes, Bataviae, Australis, Autumnalis
L. kirschneri Grippotyphosa, Autumnalis, Cynopteri, Hebdomadis, Australis, Pomona, Djasiman, Canicola, Icterohaemorrhagiae, Bataviae
L. weilii Celledoni, Icterohaemorrhagiae, Sarmin, Javanica, Mini, Tarassovi, Hebdomadis, Pyrogenes, Manhao, Sejroe
L. inadai Lyme, Shermani, Icterohaemorrhagiae, Tarassovi, Manhao, Canicola, Panama, Javanica
Manhao, Hebdomadis, Javanica, Mini L. alexanderi
Source: (Wai'in, 2007)
24 2.4 Biology of leptospires
2.4.1 Morphology
Leptospires are tightly-coiled spirochaetes, typically measuring 10 to 20 µm in
length, although cultures occasionally contain longer cells. Their helical amplitude is
about 0.1 to 0.15 µm, with a wavelength in the region of 0.5 µm (Faine et al., 1999).
The cells have pointed ends, at least one which is bent like a question mark, hence
the name “Interrogans”. In the periplasmic space are two axial filaments with polar
insertions (Swain, 1957). Leptospires move in two distinct ways, translational and
rotational (Faine et al., 1999). Although all leptospires are morphologically
indistinguishable, the morphology of individual isolates may vary with subculture in
vitro, although this can be restored with passage through hamsters (Ellis et al.,
1983).
Like other spirochaetes, leptospires have a distinctive double membrane structure.
The cytoplasm and peptidoglycan cell wall are closely associated and overlain by an
outer membrane (Haake et al., 2000). The outer membrane appears fluid and is
porous, allowing the exchange of solutes between the periplasmic space and the
environment. Salt water and desiccation can cause disorganisation of the envelope.
The composition of leptospiral lipopolysaccharide is similar to that of other Gram-
negative bacteria, albeit with lower endotoxic activity (Vinh et al., 1986).
Leptospires are obligate aerobes that thrive at temperatures between 28ºC to 30ºC.
They lack the ability to synthesise fatty acids, only reproducing in nature within
animal hosts (Plank and Dean, 2000). They can grow in simple media enriched with
vitamins, notably Vitamins B2 and B12, long-chain fatty acids and ammonium.
25 Long-chain fatty acids are utilised as the sole source of carbon and are metabolised
by beta oxidation salts (Levett, 2001).
2.4.2 Genomic organization
Leptospires have complex genomes and it was only recently that the entire sequence for the serovar Lai was established (Ren et al., 2003). In comparison to the genomes of other spirochaetes, the leptospiral genome is large. Hence it is capable of surviving in various habitats including living freely in the environment, as well as in animals (Bharti et al., 2003). Both pathogenic and saprophytic leptospires have genomes of about
5000kb in size (Baril and Girons, 1990), although smaller genomes of 2000kb have been reported (Taylor et al., 1997). The genome consists of two sections: a 4400kb chromosome; and a smaller 350kb chromosome (Wai'in, 2007). In the L. biflexa genome has 3,590 protein-coding genes distributed across three circular replicons: a major one; a smaller one, and a third 74-kb replicon (Adler and de la Peña Moctezuma,
2010; Tulsiani, 2010). Pathogenic leptospires possess two sets of 16S and 23S ribosomal rRNA genes but only one 5S rRNA gene, with each rRNA gene located far from the others on the genome (Baril et al., 1992; Fukunaga and Mifuchi, 1989). In pathogenic serovars, but not in saprophytic species, copies of several insertion- sequence-like elements coding for transposases have been identified (Boursaux-Eude et al., 1995; Kalambaheti et al., 1999).
26 2.5 Pathogenesis
The most adverse clinical and pathological signs usually occur in young animals,
especially when their maternally-derived immunity is waning (Boulanger et al.,
1958). However, the common signs of infection with Leptospira in farm animals are
abortions, stillbirths, decreased milk production and a failure to thrive (Alston et al.,
1958; LaGrange et al., 1953; Rocha, 1998). In all species congenital infection and its
sequelae are well reported. The most important difference between infection of
animals and humans is the presence of chronic carriers in animals through reservoirs
in the kidneys and genital tract (Acha and Szyfres, 2001; Alston et al., 1958; Wai'in,
2007; Sullivan, 1974).
2.5.1 Entry
Leptospires are capable of entering the lymphatics or bloodstream via a number of
sites. In some animals, they enter through the eyes, genital tract and mucous
membranes. In many human cases, the bacterium has also been found to have
entered the body through abraded skin. Evidence has also pointed to transplacental
infection during pregnancy (Wai'in, 2007). Transmission of disease to accidental
hosts occurs through indirect contact with a maintenance host (Richardson and
Gauthier, 2003).
2.5.2 Spread and growth
The ability of leptospires to thrive in tissues is a major factor for their virulence.
They gain immediate exposure to the non-specific factors like pH, redox potential,
27 electrolytes, fatty acids and other organic compounds, some of which may be
nutrients affecting the ability of them to survive and grow (Faine et al., 1999). Their
resistance to innate (non specific) immunoglobulins in tissue fluids mediates their
survival in the host body. When present in tissues, leptospires do not cause acute
inflammatory responses (Arimitsu et al., 1989). They can spread rapidly following
entry via lymphatics to the bloodstream, circulating to all tissues of the body.
Leptospires are first found in the lungs and subsequently in the liver and spleen
(Faine, 1964). In the renal tubules, leptospires move through the interstitial space
and attach to the renal epithelial cells. The time taken for lesions to develop is
dependent on the size of the infecting dose, the rate of organism growth in the host,
their level of toxicity and the opsonic immunity development rate. Toxicity is
usually a function of the serovars of leptospires in a given host (Faine et al., 1999).
2.5.3 Persistence and carrier sites
Following clearance from the bloodstream, leptospires may persist and multiply in
certain tissues in immunologically privileged sites. These sites include the proximal
renal tubules, brain, anterior chamber of the eye and genital tract (Faine et al., 1999).
Growth of leptospires in the kidneys continues exponentially and reaches a
maximum concentration about 21 to 28 days after infection (Faine, 1962b).
2.5.4 Toxin production and virulence factors
Several leptospiral serovars have been reported to produce endotoxins (Levett,
2001). In biological assays, leptospiral lipopolysaccharide preparations exhibit
28 endotoxic activity, but at much lower potencies than in the host (Masuzawa et al.,
1990). In strains of Pomona and Copenhageni, a protein cytotoxin has been
demonstrated (Miller et al., 1970) and cytotoxic activity has been found in the
plasma of infected animals (Knight et al., 1973). Leptospires virulence factors such
as hemolysins, lipopolysaccharide, glycolipoprotein, peptidoglycan, heat shock
proteins, flagellin, KatE and Htp6 may contribute to this (Picardeau et al., 2008).
This toxin has been shown to induce a histopathological effect with infiltration of
macrophages, polymorphonuclear cells, also virulent factors can activate apoptosis
in macrophages (Yam et al., 1970).
2.6 Clinical features of leptospirosis
2.6.1 Domestic animals
In domestic species clinical signs are influenced by several factors including the
species affected, the inoculation dose, the immune status and the age of the animal.
Clinical signs may vary from high fever, pulmonary congestion, jaundice, weight
loss, abnormal milk secretion, haemoglobinuria, abortion, stillbirths or persistent
infection in newborns, and death. Leptospirosis can cause serious economic loss to
the livestock industries (Bennett, 1993; Lewis, 2003) and is a major cause of
abortions in cattle (Prescott et al., 1988; Shapiro et al., 1999). Chronic infection can
also lead to infertility and reproductive failure in cattle, goats and horses (Hanson,
1982; Ngbede et al., 2013; Prescott et al., 1988; Shapiro et al., 1999). As the disease
is common in domestic animals, humans and wildlife, control in domestic animals
can have a positive impact on the level of disease in wildlife and humans.
29 2.6.2 Wild animals
Knowledge on naturally occurring leptospiral infections in wildlife is limited,
although experimental challenge of captive wild animals has resulted in clinical
signs ranging from an inapparent infection to a febrile response, abortion, and death
in Odocolileus virginianus and Eumetopias jubatus (white tailed deer and Steller sea
lion, respectively) (Gulland et al., 1996; Twigg et al., 1969). The full impact of
leptospirosis in wildlife is not known and studies on the impact of the disease on
wildlife are required. Data on the seroprevalence in wildlife around the world is
presented in Table 2.4.
Table 2.4: Reported Leptospira seroprevalence in wildlife.
Animal Location Seroprevalence Reference Small rodents, Shrews Zurich, Switzerland 12.6% (Adler et al., 2002)
Mammals Peruvian Amazon 20% (Bunnell et al., 2000)
Rodents Peruvian Amazon 20% (Bunnell et al., 2000)
Marsupials Peruvian Amazon 35% (Bunnell et al., 2000)
Chiropteran (Bats) Peruvian Amazon 35% (Bunnell et al., 2000)
Carnivores Peruvian Amazon 89% (Cirone et al., 1978)
Rodents Peruvian Amazon 60% (Cirone et al., 1978)
Flying fox Australia 11-39% (Cox et al., 2005)
Possums Australia 35% (Durfee and Presidente, 1979) Californian sea lion California 33-71% (Gulland et al., 1996) Black rats New Zealand 34% (Hathaway and Blackmore, 1981)
30 Animal Location Seroprevalence Reference Brown rats New Zealand 26% (Hathaway and Blackmore, 1981) Lynx Spain 32% (Millán et al., 2009) Fox Spain 47% (Millán et al., 2009) Mongoose Spain 20% (Millán et al., 2009) Genet Spain 12% (Millán et al., 2009) Badger Spain 50% (Millán et al., 2009) Cat Spain 20% (Millán et al., 2009) Dog Spain 36% (Millán et al., 2009) Feral Pigs NSW Australia 20% (Mason et al., 1998) Caribou Alaska 2% (Zarnke, 1983) Moose Alaska 3% (Zarnke, 1983) Grizzly bears Alaska 5% (Zarnke, 1983) Black bears Alaska 4% (Zarnke, 1983)
2.6.3 Humans
More than 184 distinct serovars of L. interrogans belonging to 20 serogroups have
been identified across the world (Nascimento et al., 2004). In Southeast Asia, the
most common serovars associated with disease of domestic animals and humans are
Icterohaemorrhagiae, Autumnalis, Canicola, Pomona, Patoc, Grippotyphosa,
Australis and Poi (Srivastava, 2006; Victoriano et al., 2009). The first three are the
most important serovars with respect to veterinary and public health perspectives.
Fever is an important and common presentation of tropical diseases and may
sometimes be the only manifestation of a serious illness. Diagnosis in humans is
often difficult because of the many diagnostic possibilities, symptoms which may
often be non-specific, and because many medical doctors are unfamiliar with the
spectrum of tropical diseases. Treating the disease appropriately will reduce the risk
to public health, and this can only come following a correct diagnosis. With the
31 recent popularity of adventure travel and triathlete competitions, there is an
increased awareness of the potential for leptospiral infection amongst those who
partake in such activities (Cachay and Vinetz, 2005; Morgan et al., 2002).
2.7 Pathology
Symptoms can vary from a mild non-specific influenza-like infection to Weil’s
disease, where serious complications can occur (Esen et al., 2004). The primary
histological lesion observed in clinical leptospirosis is the damage to the endothelial
membrane of the capillaries, caused by leptospiral toxin (Wai'in, 2007). This has the
effect of loosening intercellular junctions, allowing both fluid and leptospires to
migrate into extravascular spaces, followed by red blood cells, wherever the damage
is severe. Secondary effects of ischaemic change, anoxia and increased tissue
pressure reinforce damage, resulting in cellular functional disintegration and death of
the cell (Ellis, 1994). In the kidneys, the main finding is interstitial nephritis. This is
accompanied by intense cellular infiltration composed of neutrophils and monocytes
(Penna et al., 1963), but renal disease is uncommon.
2.8 Epidemiology of leptospirosis
Leptospires are widespread and their abundance is due to their ability to infect a
range of animal species, including humans, as well as the ability to survive outside
the host, if environmental conditions are favourable (Duncan et al., 2011; Hathaway
and Blackmore, 1981; Smith et al., 1961). The main sources of infection are urine of
infected or carrier animals and contaminated surface water, mud and soil (Monahan
32 et al., 2009). Transmission can happen as a result of direct or indirect contact with
infected animals or their secretions (Monahan et al., 2009). The major route of
infection is via mucous membranes, however carnivores can be infected through the
ingestion of leptospiral-infected carcasses (Murray et al., 2006). Usually leptospires
appear in the blood four to 10 hours after infection and can be detectable in the
blood for a few hours up to seven to 10 days (Plank and Dean, 2000; Smith et al.,
1994; Tsai and Fresh, 1975). Clinical signs may not occur in every case but severe
fever is an important sign of acute leptospirosis. Animals that have recovered from
leptospirosis may become carriers, with the organism present in the renal tubules for
periods of days to years (Babudieri, 1958; Bahaman et al., 1987) with subsequent
shedding of leptospires via urine into the environment (Babudieri, 1958).
Infectious diseases are transmitted globally through animal and human movements
due to eco-tourism, wildlife research, reintroduction, rehabilitation, hunting, pet
trade, laboratory and food industry demands and farming (Beatty et al., 2008;
Osofsky, 2005). These movements and activities are major contributing factors for
the transfer of leptospirosis to animals and humans and the spread of the disease to
new areas.
Research on leptospirosis has highlighted that rodents (20%), marsupials (35%) and
bats (35%) are most likely to spread the disease (Bunnell et al., 2000). Marsupials
and chiropterans have recently been implicated as more significant reservoir hosts of
leptospires pathogenic to humans than previously was recognized (Bunnell et al.,
2000). Studies on leptospirosis involving bats have produced equivocal results (Cox
et al., 2005), however the fact that leptospires can potentially be spread through bats
is a major concern due to their abundance, their ability to travel long distances and
their potential to expose humans to infected urine. Ground-dwelling species, such as
33 rodents foraging under bat roosts, could also encounter Leptospira contaminated
urine and potentially spread the organism to urban areas (Tulsiani et al., 2011). From
the limited research on wildlife, several pathogenic serovars have been isolated with
the most prominent serovars including Grippotyphosa and Pomona from white-
tailed deer (Odocoileus virginianus) in North America (Cantu et al., 2008).
Surveillance of leptospirosis is important to determine the emergence of new strains,
which have the potential to cause an outbreak. Although it is not economically
viable to survey all wildlife species, it is important to regularly screen particular
species which live on the periphery of forests and have the potential to interact with
humans (eg: wild rats, carnivores and bats). Screening these ‘sentinels’ provides
useful cost-effective information on the health status of a broader range of species.
2.8.1 Geographical distribution of leptospirosis worldwide and in
Southeast Asia
Leptospirosis is a major health concern globally, more so in tropical regions such as
South America, South Asia and Southeast Asia. Major outbreaks have been reported
recently in India, Indonesia, Sri Lanka and Thailand (Ciuchi-Nicolau, 2010). In
Sarawak, East Malaysia, leptospirosis has been reported to have occurred at
numerous locations, sometimes involving Western tourists. Given the regional
peculiarities of climate, population density and the degree of contact between
humans (accidental hosts) and animals (maintenance hosts), Southeast Asia is
conducive for maintaining a high level of transmission of leptospirosis (Ciuchi-
Nicolau, 2010). Numerous pathogenic serovars circulate and there are abundant
34 reservoir host species including rodents, farm animals and dogs present in this
region.
Transmission is not only occupational as is observed in temperate climates, but is
the consequence of exposure to a contaminated environment, in particular during the
wet season when outbreaks can occur configuring an epidemic-endemic pattern of
disease (Levett, 2001). According to Pappas et al., (2008), the Caribbean and Latin
America, the Indian subcontinent, Southeast Asia, Oceania and to a lesser extent
Eastern Europe, are the most significant foci of the disease, including areas that are
popular travel destinations.
Between 1987 and 1991, researchers surveyed 32 Dutch travellers with leptospirosis
who had mainly been to Thailand and other Southeast Asian countries. Their studies
found that the main infective agents belonged to the serogroups Australis and Sejroe
(van Crevel et al., 1994).
2.8.2 Geographical distribution of leptospirosis in Malaysia
At least 37 serovars have been isolated from humans and animals in Malaysia (El
Jalii and Bahaman, 2004) and serovars from the Pyrogenes, Canicola, Autumnalis
and Pomona serogroups have been isolated from human patients (Tan, 1964).
During the 1950’s a group of researchers successfully isolated leptospiral serovars
from serogroups Hebdomadis, Grippotyphosa and Canicola from three species of
Malaysian rodents and a serogroup of Icterohaemorrhagiae from Rattus whiteheadi
from Sabah (Wisseman et al., 1955). In 2004, researchers discovered a new
pathogenic serovar, Lai, from a patient who had returned from Langkawi Island in
West Malaysia (Wagenaar et al., 2004). As late as 2009, a new serovar, L. Kmetyi
35 Bejo-Iso 9, was isolated from a soil sample in the southern state of Johor (Slack et
al., 2009). In the next chapter, the discovery of a novel serovar in Sarawak
Leptospira, Lepto 175 Sarawak is discussed.
2.8.3 Sources and mode of transmission of leptospirosis
Domestic and wild mammals, reptiles and amphibians are maintenance hosts for
different leptospiral serovars (Wai'in, 2007), however rodents and cattle are
considered the main source of human infection (Levett, 2001; Smythe et al., 2000),
although dogs may also be an important source (Meeyam et al., 2006). In addition,
bats have recently also been suggested as a possible source of exposure to
Leptospira for humans (Cox et al., 2005; Matthias et al., 2005), particularly because
of their size, abundance, spatial distribution, and interrelationship with domestic
animals (Kunz, 1982; Matthias et al., 2005; Vashi et al., 2010). Leptospires colonise
the kidneys of carrier animals and are shed in the urine, which is the primary source
of environmental contamination. It is estimated that between 10,000 and 1,000,000
leptospires are shed by carriers in every ml of urine passed (Faine et al., 1999).
Humans and other animals are usually infected by exposure to this urine from the
infected animals (Ellis and Michna, 1976).
Transmission modes can be direct or indirect. Direct transmission happens when
animals come into contact with the urine of chronically infected animals (Ellis et al.,
1986; Ellis and Thiermann, 1986). However in cattle and pigs, evidence exists of
leptospires crossing directly from the genital tract to the foetus (Goldenberg and
Thompson, 2003). Indirect transmission happens when animals or humans acquire
infection from the environment via contamination of the conjunctivae, oral mucosa,
36 and respiratory tract mucous membrane or via skin cuts (Levett, 2001). Also human-
to-human transmission has been postulated through sexual transmission (Harrison
and Fitzgerald, 1988) and breastfeeding (Bolin and Koellner, 1988).
2.8.4 Cycle of host infection
The epidemiology of human leptospirosis reflects the ecological relationship
between humans and chronically-infected mammalian hosts (Wai'in, 2007). Humans
are regarded as incidental dead-end hosts from which further transmission has not
been demonstrated. Two natural cycles of leptospiral infection have been identified:
a sylvatic cycle and a domestic cycle (Faine et al., 1999). In the former case,
leptospirosis is accidentally transmitted to livestock and humans from numerous
species of rodents and chiropterans. The main mode of spread and continuity of
infection in rodents is by direct transmission from mother to the young (Wai'in,
2007). Humans can pick up infection through contact with an environment
contaminated with urine, particularly that of rodents or bats. Bats forage in fruit
orchards and forest clearings created by humans, and roost in buildings, water
cisterns, culverts, abandoned structures and bridges (Kunz, 1982). In contributing to
a sylvatic cycle of leptospiral transmission, ground-dwelling species, such as rodents
or marsupials, that reside or forage under bat roosts could be exposed to Leptospira-
contaminated urine (Matthias et al., 2005).
Animals that do not exhibit signs of clinical infection but shed leptospires for long
periods of time are regarded as maintenance (or reservoir) hosts. Different animal
species may be reservoirs for distinct serovars. The domestic cycle of leptospiral
37 transmission involves cattle, swine, sheep, buffalo, goats and dogs. These animals
are known to be maintenance hosts of specific serovars (Voronina et al., 2014).
2.8.5 Survival of leptospirosis in the environment
The extent to which leptospires are transmitted depends on the survival of them in
the environment which is influenced by temperature, climate, soil pH and soil
moisture (Ringen and Okazaki, 1956). The optimal survival condition for leptospires
is a warm, wet environment with neutral to slightly alkaline water (Farrar, 1995).
Like other spirochaetes, leptospires are well-adapted to viscous environments, where
they show greater translational motility than other bacteria (Kaiser and Doetsch,
1975). They can survive for approximately two weeks in soil contaminated with
urine from an infected host (Karaseva et al., 1977).
2.8.6 Major leptospirosis outbreaks in Malaysia
Records of leptospiral human infections in Malaysia date back to 1925 (El Jalii and
Bahaman, 2004; Lim et al., 2011; Thayaparan et al., 2013b). Cases of febrile illness
among both military and civilian personnel were frequent during the 1950’s,
drawing attention to leptospirosis as an emerging disease in both Peninsula and East
Malaysia (Broom, 1953). According to Lam and Kan, (2005), there have been two
known outbreaks in recent years. The first involved a group of schoolboys who had
been swimming in a river. The patients reported fever, headache, giddiness,
vomiting, body ache, cough, chest pain and conjunctivitis. Tragically, one of them
succumbed to the disease, which was believed to be leptospirosis. The second
instance involved the widely-publicised Eco-Challenge in Sabah in 2000, in which
38 many participants, most from Western countries, became ill when they returned
home. Serological studies carried out in Sabah on people who lived near national
parks in the early 2000s reported a high seroprevalence to leptospirosis, possibly
from exposure to infected wildlife (Russ et al., 2003). Leptospiral infection of
inmates at various immigration detention centres all over the Malay Peninsula have
also been reported, with six confirmed fatalities, causing major concern among
human rights groups (Jones, 2011; Wright, 2011). In Terengganu, there were
outbreaks reported in Hulu Terengganu and Sungai Berua, as well as in an Orang
Asli settlement (Lim et al., 2011). Leptospirosis has also been implicated in the
death of a Universiti Malaysia Terengganu staff member in June 2013 (Thayaparan
et al., 2013b). Patterns of infection in Malaysia had appeared to be sporadic up until
2010, with the disease concentrated mostly in one or a few regions at any time (Lim
et al., 2011) and occurring during the monsoon seasons. However, with climate
changes, disease has been reported throughout the year, as has been demonstrated in
Sarawak during the last two years (Thayaparan et al., 2013b). Although human-to-
human transmission has not been reported, the severity of the disease raises
significant concerns when outbreaks occur. MacKay-Dick, (1971) suggested that the
severity of leptospirosis within a community was co-related to the length of time its
members spent in the jungle. With leptospirosis being prevalent in wildlife,
containment of the disease is difficult due to a lack of a suitable and acceptable
approach for wildlife management (Thayaparan et al., 2013a).
2.8.7 Leptospirosis and ecotourism
Leptospirosis is a zoonosis with a wide geographical distribution, primarily because
the pathogen can be harboured by a wide range of wild and domestic animals.
39 Although leptospires cannot survive in dry environmental conditions, the presence
of moist soil, standing water or surface waters allows them to maintain their
virulence and persistence outside their animal hosts (Faine, 1982). Contact with
these contaminated surfaces poses an occupational risk to humans, although recently
more people are exposed to the spirochaete during recreational activities. One such
activity would be adventure travel. Southeast Asia is a popular destination for such
travellers, many of whom hail from the Western world. The region’s tropical climate
provides a suitable environment for leptospires to thrive, which puts such tourists at
risk of disease when they visit. However, leptospirosis is often misdiagnosed, even
though it is endemic, because of the non-specific symptoms associated with
infection and the difficulty in confirming a diagnosis (Sejvar et al., 2003).
Eco-tourism comprises a range of adventure activities including four-wheel driving,
flying microlight planes and white water rafting and in Malaysia there are large
numbers of tour operators involved in this sector of the tourism industry. It is
believed that 7% of tourist activity could be nature-related with over 500,000
international eco-tourists visiting Malaysia each year (Yasak, 2000). Furthermore it
is estimated that 7 to 10% of all overseas tourists undertake some form of
ecotourism activities, and a further 14% are interested in walking, hiking or
trekking. This amounts to potentially over one million international tourists each
year (Yasak, 2000). Statistics from Tourism Malaysia (2011)
(http://corporate.tourism.gov.my/) reveal that 24.6 million tourists visit Malaysia
each year, contributing to a major part of the country’s income. Most of these
tourists also want to see Malaysia’s native wildlife (Thayaparan et al., 2013a).
Although ecotourism is a major industry in Malaysia and other Southeast Asian
countries, its potential impact on wildlife and the people raises significant concerns.
40 Furthermore the threat of diseases such as leptospirosis could result in bad publicity
and loss of an important source of foreign revenue for the country.
2.8.8 Deforestation and wildlife-human conflict
Urbanisation inevitably causes deforestation, which can result in displacement of
wildlife and extinction of specific species. The loss of habitat can disrupt animal’s
food supply, and many species take shelter near human settlements where there
exists a potential food source. Some species, such as bats, have spatial and temporal
dynamics that are particularly sensitive to anthropogenic activity (Matthias et al.,
2005). In the Iquitos region in Peru, bats are believed to be a major player in the
sylvatic cycle of leptospiral infection. Deforestation not only results in a loss of
habitat and food for wildlife but also results in increased contact with humans,
increasing the risk of transmitting zoonotic diseases.
2.8.9 Ecotourism and wildlife contact
Although little physical contact with wildlife has been documented by eco-tourists,
it is virtually impossible to avoid contact with contaminated water and soil. The
majority of infections would appear to result from exposure to contaminated
freshwater rather than exposure directly to animal urine. Most tourists come from
more temperate climates and do not wear suitable protective clothing due to the high
heat and humidity of the tropics (Mortimer, 2005), putting themselves at risk of
exposure and subsequent infection. The Malaysian jungle is home to numerous
wildlife species, including bats; whose numbers populate many cave formations in
Borneo. Other species that may be potential reservoirs of Leptospira are primates,
41 and the role these play in leptospirosis will be discussed in Chapter 4. With forest
areas shrinking it is likely there will be even greater interaction between humans and
wildlife in the future and the potential for diseases, such as leptospirosis, to reach
epidemic proportions (Thayaparan et al., 2013a).
2.9 Economic impact of leptospirosis
The economic impact of leptospirosis is usually felt by those who are involved in
the livestock industries. Primarily, it has had a significant impact on the cattle
industry through abortions, stillbirths and mastitis. However with the disease
becoming endemic and the zoonotic aspect becoming more important, the
consequences are now greater for the wider community and general economy.
As with other zoonotic pandemics, fear and anxiety can grip the communities most
vulnerable to infection. The economic impact of the disease includes loss of income
to the affected individuals, loss of export products from decreased productivity of
diseased livestock, loss of income from reduced tourism over concerns about the
disease, costs associated with the investigation and control of an outbreak and
compensation to farmers for the loss of their animals (Arief, 2013).
2.10 Laboratory Diagnostic Methods
Diagnosis of leptospirosis requires use of laboratory methods as the clinical
presentation is not specific. The diagnostic method selected depends on the available
samples and the purpose of testing. Identification of the infecting serovar is
42 important both epidemiologically and clinically, since this may assist in determining
the source and likely outcome of infection. Different assays have been developed in
an attempt to diagnose leptospirosis accurately, however the majority are unsuitable
for use in developing countries due to their requirement for the maintenance of
multiple strains and the need for expensive equipment. Several methods exist for the
diagnosis of leptospirosis. These include simple microscopic demonstration of the
organism to more advanced tests such as the Polymerase Chain Reaction (PCR).
2.10.1 Microscopic demonstration of Leptospira
Leptospires may be visualized in clinical material through dark-field microscopy,
immunofluorescence or light microscopy after appropriate staining (Wai'in, 2007).
Where laboratory resources are limited, dark-field microscopic examination of body
fluids has been used to rapidly detect the presence of leptospires. However the
technique lacks sensitivity (Faine et al., 1999) with a detection limit of 104
leptospires/ml (Turner, 1970). Microscopic examination of blood samples needs to
take place within the first few days of acute illness during which time leptospiraemia
is present (Levett, 2001). Serum protein and fibrin strands and other cell debris in
the blood can resemble leptospires, while the concentration of organisms in the urine
of humans and animals is frequently too low to be detectable by this method (World
Health Organization, 2003). This method calls for high operator skill and no
information on the infecting serovar can be gained (Smith et al., 1994).
For post-mortem diagnosis and to increase the sensitivity of direct microscopic
examination, specific staining methods have been deployed. Standard stains for
leptospires have been silver impregnation techniques, strong carbol fuchsin and
43 methylene blue, or Gram stain using a carbol fuchsin counterstain. However, like
dark-field microscopy, staining methods also present a high risk of false-positive or
false-negative results (World Health Organization, 2003). Immunofluorescent
staining is also used for demonstrating leptospires in clinical and environmental
specimens, such as urine, body fluids, frozen kidney samples, soil and water,
because of the ease of identifying leptospires and the serovars can be presumptively
determined (Ellis et al., 1982; Faine et al., 1999). Recently the more sensitive
immune-histochemical methods have been applied, notably the immunogold silver
staining method. This method produces a permanent image that does not fade with
time. Furthermore a fluorescent microscope is not required.
2.10.2 Cultural methods
Leptospires can be cultured from the blood, body fluids and tissues of infected
subjects. These bacteria grow in culture media containing dilute animal (usually
rabbit) serum or bovine serum albumin (Faine et al., 1999; World Health
Organization, 2003). The Ellinghausen-McCullough-Johnson-Harris (EMJH)
formula is currently most commonly used. Its base is a serum-free oleic acid-
albumin medium with derivatives containing Tween 80 as the source of fatty acids
and BSA as a detoxifier (Ellinghausen and McCullough, 1965a; Johnson and Harris,
1967). The anti-microbial agent, 5-fluorouracil, is sometimes added to media to
inhibit the growth of contaminants present in clinical specimens (Johnson and
Rogers, 1964). To make EMJH media semi-solid or solid, sugar at respective
concentrations of 0.1-0.2% and 0.8-1.5% is added (Faine et al., 1999).
44 Unfortunately, culture is a slow process requiring several weeks of incubation, and
has low sensitivity (Bharti et al., 2003). Palmer and Zochowski, (2000)
recommended that samples be inoculated into media within 24 hours of collection.
Growth may occasionally be detectable after one week culture, however it often
takes longer. The culture medium should therefore be checked for growth of
leptospires at regular intervals for a period up to 4 months (World Health
Organization, 2003). In semi-solid media, growth reaches maximum density beneath
the surface of the medium, which becomes increasingly turbid as incubation
proceeds (Palmer and Zochowski, 2000; Wai'in, 2007).
Leptospiral cultures are maintained by repeated subculture, though this repeated
process makes the organisms less virulent (Adler and de la Pena Moctezuma, 2010).
Isolates can be grown to reasonable density in semisolid EMJH medium at 30ºC,
and can subsequently be stored in the dark at room temperature for up to three years.
Long-term storage at -70ºC in glycerol is also used (Palit et al., 1986).
During acute and chronic infections, isolation of leptospires is frequently attempted
from various clinical specimens. Suitable specimens, including blood (whole and
clotted), serum, urine, cerebrospinal fluid and tissue (particularly kidney) samples,
can be inoculated into EMJH medium containing 5-fluorouracil (Wai'in, 2007).
Cultures should be incubated at 29 ± 1°C for at least 16 weeks, and preferably for 26
weeks and examined by dark-field microscopy every 1 to 2 weeks (OIE, 2008).
Identifying isolates to the serovar level is usually performed at reference laboratories
and involves time-consuming cross-absorption agglutination procedures with panels
of monoclonal antibodies (Levett, 2004; Smith et al., 1994).
45 2.10.3 Microscopic Agglutination Test (MAT)
The MAT relies on a panel of live cultures as diagnostic antigens, making the
quality assurance of this test particularly difficult (Chappel et al., 2004). This is the
standard diagnostic test for leptospirosis but requires sophisticated equipment and is
time consuming.
2.10.4 ELISA
Although the ELISA can detect different classes of antibody, it may be subject to
false positive reactions and requires confirmation of these results by the MAT
(Smythe et al., 2002b). In contrast to the MAT, ELISAs can be performed rapidly
and without the need for very sophisticated equipment. Two commercially available
ELISA kits – the INDX Dip-S-Ticks and the PanBio ELISA were evaluated against
the MAT, using sera from the University Hospital, Kuala Lumpur, between 1991
and 1997 (Sekhar et al., 2000). Results from this research suggested that the INDX
Dip-S-Ticks test was a practical alternative to the MAT.
2.10.5 Polymerase Chain Reaction (PCR)
The PCR has been evaluated by several groups for its usefulness in detecting
leptospiral DNA from both humans and animals. Although PCR technology is now
widely used for the diagnosis of many diseases, its general value for the rapid
diagnosis of leptospirosis has not been evaluated worldwide and it is not yet widely
used in tropical and subtropical countries (World Health Organization, 2003). Over
the years, the PCR has evolved from a gel-based method to a real-time one,
incorporating specific probes and primers and can be very sensitive, detecting as few
46 as 2 to 3 organisms per sample. However, the test requires selection of specific
primers to allow for amplification of the DNA. A number of primer pairs have been
described based on specific gene targets (Renesto et al., 2000), including the 16S
and 23S ribosomal RNA genes found in all pathogenic leptospires (Merien et al.,
1992). Other primers have also been constructed from genomic libraries
(Gravekamp et al., 1993).
There is evidence that PCR assays display greater sensitivity than conventional
diagnostic methods, such as culture or dark-field microscopy, although the
sensitivity of culture may vary between laboratories (Brown et al., 1995; Heinemann
et al., 2000; Merien et al., 1992). The PCR is very useful for obtaining an early
diagnosis of leptospirosis, when bacteria may be present but before antibody titres
are at detectable levels (World Health Organization, 2003). The ability of PCR
assays to identify specific serovars is limited, with authors often describing
genotypic groupings of serovars instead of serovar-specific groupings (O'Keefe,
2002).
2.10.6 Multi-Locus Sequence Typing (MLST)
The Multi-Locus Sequence Typing (MLST) database has information on numerous
bacterial species, including leptospires. It currently has information on over 200
isolates, most of which have been isolated from Thailand between 2000 and 2005
(Thaipadungpanit et al., 2007; Boonslip et al 2013). Ahmed et al., (2006) developed
a robust MLST method for genotyping Leptospira. This method of typing is
comparably more reproducible, robust, consistent and portable than other methods.
The MLST approach allows a better study of the genetic relatedness of leptospires
47 and can be used for molecular epidemiological studies and population genetics
(Ahmed et al., 2006). It is a simple PCR-based technique using DNA sequences to
assign and characterise alleles present in different target genes (Romero et al.,
2011a). However, Romero et al., (2011a) reported that a major drawback of the
MLST was its lack of discriminatory power when applied on uncommonly occurring
isolates.
2.10.7 Modern rapid tests
A more recent method of detecting leptospires is the Lepto Dipstick test (Sehgal et
al., 2000). This uses a broadly reactive antigen for detecting IgM antibodies. This
method was evaluated on 867 sera collected from patients confirmed with
leptospirosis and controls from the Andaman Islands between 1993 and 1997. Using
these samples the Lepto Dipstick had a sensitivity of 78.7%, a specificity of 88.3%
and a positive predictive value of 91.0%. The test had a good level of agreement
with the standard criteria for diagnosis using paired MAT results (K = 0.64). The
sensitivity of the Lepto Dipstick was high between the second and fourth weeks of
the disease, however lower, but still acceptable, sensitivities were recorded before
and after that period. This test is easy to perform as it does not involve sophisticated
equipment. The reagents and dipsticks can also be stored for long periods, even at
room temperature, and therefore it is suitable for use as a rapid screening test for the
diagnosis of leptospirosis.
Sehgal et al., (2003) evaluated the Lepto lateral flow assay on samples from the
Andaman Islands between October 1999 and December 2000. Like the Lepto
Dipstick, it was developed at the Dutch Royal Tropical Institute. This test can be
48 performed at the patient’s bedside, as whole blood can be used. One hundred and
seventeen patients suspected to have leptospirosis were included in the study with
acute serum samples collected from all of them and convalescent samples from 104.
The results of the lateral flow test were compared with the standard criteria for
diagnosing leptospirosis, and the test was found to have a sensitivity of 52.9%
(37/70) in the first week of illness and 86% (49/57) during the second to fourth
weeks of illness. Corresponding specificities were 93·6% (44/47) and 89·4%
(42/47), respectively. Agreement of the results with the standard criteria was low
during the first week, but high during weeks 2 to 4. All indices of validity and utility
of the lateral flow test were similar to those of the IgM ELISA and Lepto dipstick
(Sehgal et al., 2003).
Samples from inhabitants of the Andamans were again selected for the Lepto Dri
Dot card agglutination test (Vijayachari et al., 2002). The results of this test were
compared with blood culture and MAT tests on paired serum samples. Based on
these criteria, 74 of 124 patients were diagnosed with leptospirosis. The Lepto Dri
Dot had a sensitivity of 67.6 % (50/74) and a specificity of 66.0% (33/50) during the
first week. During weeks 2 to 4 the values increased to 85.5% (47/55) and 80%
(40/50), respectively. An IgM ELISA was also performed on the serum samples for
comparison and this was marginally less sensitive, but more specific, during the first
week of illness. The Lepto Dri Dot test does not require special storage or
sophisticated equipment and can be performed by relatively low skilled personnel.
In Hawaii, eight different IgM detection methods were compared using 379 serum
samples obtained in 1998 and 1999 from 236 patients, of which 33 were confirmed
infected (Effler et al., 2002). The median time between the onset of illness and the
collection of samples was 9 days. This is the most comprehensive field evaluation of
49 screening tests for leptospirosis reported to date. The sensitivity of the tests ranged
from 29 to 86% and specificity from 85 to 100% for seven of the eight tests
evaluated. The sensitivity was particularly low (<25%) on samples collected during
the first week of illness for seven of the tests. The data indicated that IgM detection
tests have limited use for diagnosing leptospirosis during the initial stage of disease,
a time when important therapeutic decisions are usually made (Effler et al., 2002).
2.11 Diagnostic techniques used in Malaysia
Several methods are used in the diagnosis of leptospirosis in Malaysia. These
include the simple MAT, PCR and ELISA’s. The MAT is inexpensive, but time
consuming and laborious, and is only undertaken at the Institute for Medical
Research (IMR) Kuala Lumpur, Makmal Kesihatan Awam Kebangsaan (MKAK)
Sungai Buloh Malaysia for community outbreaks and at universities undertaking
research on leptospirosis (Hakim, 2011). ELISA’s are performed at hospitals with
the necessary facilities as well as the IMR and MKAK. Currently culture of
Leptospira is only performed at the IMR (Hakim, 2011). Diagnosis of animal
leptospirosis is only undertaken at the Veterinary Research Institute, Ipoh and the
Wildlife Department, Kuala Lumpur (PERHILITAN). Environmental samples can
be tested at the MKAK, Sungai Buloh, Malaysia (Hakim, 2011).
Diagnosis of leptospirosis in wildlife requires evaluation of presenting clinical signs
and serological test results. There are three methods used to diagnose leptospirosis
in mammals: serological, demonstration and histological methods. The serological
methods consist of fluorescent antibody and microscopic and macroscopic
agglutination tests, which detect antibodies to leptospires. Depending on the severity
or stage of infection, an animal may react serologically to one or all antigens used to
50 determine the serological profile. The demonstration methods involve examining
body fluids and tissues under dark field microscopy, and histological examination
involves examination of biopsy material with specific silver stains (Warthin-Starry)
or a Giemsa stain.
2.12 Treatment, control and prevention of leptospirosis
A range of antibiotics are used to treat hosts with leptospirosis, with intravenous C-
penicillin (2M units 6 hourly for 5-7 days) being commonly used in severe adult
human cases (Ganoza et al., 2006; Hakim, 2011). Less severe cases are usually
treated with oral antibiotics such as doxycycline, tetracycline, ampicillin or
amoxicillin (Hakim, 2011). A trial on the penicillin derivative, doxycycline, on the
prevention of infection and clinical disease was conducted in the North Andaman
Islands in 1999 (Sehgal et al., 2000). Although the findings indicated that
doxycycline prophylaxis did not prevent leptospiral infection in an endemic area,
such treatment did have a significant protective effect in reducing the morbidity and
mortality during outbreaks.
It is important to investigate the nature of leptospirosis, and identifying the endemic
strain in an area will largely dictate the direction of any studies on the organism and
the disease in wildlife and humans. This will determine the resources required to be
invested into control and prevention strategies. After several outbreaks of
leptospirosis around wildlife reserves, the IMR, Malaysia undertook an investigation
to determine the cause of the disease in Sarawak. During the initial surveillance
project, Leptospira-like organisms were collected around the study area for the
research described in this thesis. In the next chapter a novel strain of Leptospira
collected in the study area near Kuching, Sarawak is described.
51
52
CHAPTER 3 : LEPTOSPIRA, LEPTO 175 SARAWAK SP.
NOV., ISOLATED FROM AN ENVIRONMENTAL
SAMPLE IN SARAWAK, MALAYSIA.
3.1 Introduction
Leptospirosis is emerging as a grave public health concern in Malaysia and other
Southeast Asian countries (Arief, 2013; Bhatia and Narain, 2010; Coker et al., 2011;
Victoriano et al., 2009). There are many animal hosts for the bacteria, and these
hosts can shed leptospires in their urine for many years (Monahan et al., 2009). Soil
and water contaminated by this urine can result in severe illness when humans and
other animals come into contact with it. Leptospirosis is a widespread zoonotic
disease resulting in morbidity and mortality of humans and animals, and the disease
is endemic in Malaysia (Antony, 1996). Outbreaks of leptospirosis have recently
been reported in and around wildlife reserves/parks and mortalities have previously
been reported in humans in Malaysia as reported in the previous chapter (Koay et
al., 2004; Lim et al., 2011; Thayaparan et al., 2013b).
Although leptospires survive best in moist conditions, flourishing in pools of
stagnant water for up to several days, they cannot thrive in salt water (Dutta and
Christopher, 2005) surviving for only a few hours in this medium. Ecuadorian
researchers have shown that leptospires can remain motile in pure water for up to
110 days without nutrients (Trueba et al., 2004). However they are easily destroyed
by desiccation, exposure to disinfectants and detergents, and heating to 50°C for five
53 minutes (Ferguson, 1991). Although reports on the isolation of leptospires from
environmental samples are numerous, little direct information is available on their
actual distribution in various bodies of water or in the soil in Malaysia.
In 2009 a novel strain, L. kmetyi Bejo-Iso 9, was identified from a soil sample
obtained from the southern Malaysian state of Johor (Slack et al., 2009). More
recently in June 2012, the Borneo Post reported that contamination from rat urine
with stagnant water was responsible for the widespread distribution of leptospires
around Sibu City in Sarawak (Thayaparan et al., 2013b). Health authorities warned
that children should stay away from stagnant ponds to avoid “untoward incidents
such as coming into contact with leptospirosis or other water-borne diseases” (Boon,
2012).
The genus Leptospira is divided into pathogenic, intermediate and non-pathogenic
leptospires. Members of the pathogenic group are associated with disease outbreaks
and some intermediate strains have also been isolated from humans and other animal
species displaying signs of disease (Slack et al., 2008; Zakeri et al., 2010). The
isolation of members of pathogenic leptospires from water and soil has been well
documented (Ganoza et al., 2006; Henry and Johnson, 1978; Henry et al., 1971).
One of the problems with the diagnosis of leptospirosis in humans in Malaysia has
been the confusing nature of the symptoms associated with the disease. Since it is a
febrile illness and several tropical diseases also manifest with fever in the early
stages, the disease is easily misdiagnosed (Saunders, 1979), thus delaying the
commencement of effective treatment. However, recent improvements in
environmental sampling and isolation and identification of Leptospira strains have
been reported (Hakim, 2011). To gain additional information on the extent of the
54 distribution of leptospires in wildlife and humans around wildlife reserves/park in
Sarawak, environmental samples were collected and the results are described in this
chapter. Initially, strain Lepto 175 Sarawak was isolated from a sample of water
collected from Sarawak by researchers from the IMR who were undertaking a
survey around a suspected leptospirosis outbreak area. The morphological,
serological and molecular characterization of a novel species of the genus
Leptospira is reported in this Chapter.
3.2 Materials and Methods
Several 50 mL of surface stagnant water was collected from different spots in sterile
polypropylene centrifuge tubes and filtered through Whatman no. 1 filter paper to
eliminate large particles prior to filtering through a 0.45 µm nitrocellulose filter
membrane, then a 0.22 µm filter was used to filter only leptospires (Ganoza et al.,
2006; Henry and Johnson, 1978). 1 mL of filtered water was inoculated, in
duplicate, into EMJH (Difco) media and Fletcher media, which were incubated at
30°C. The media were examined weekly using dark-field microscopy for the
presence of leptospires. If leptospires were detected under dark field microscopy, 1
mL of the media was inoculated into EMJH and Fletcher media for subculture at 2
weekly intervals.
3.2.1 Morphological identification of Leptospira, Lepto 175 Sarawak
Dark field microscopy was used to demonstrate leptospires in the media.
Microscopy was undertaken at 200 and 400x after four weeks of culture in the
EMJH media.
55
3.2.2 Serological identification of Leptospira, Lepto 175 Sarawak
Strain Lepto 175 Sarawak was isolated and forwarded to the WHO/FAO/OIE
Collaborating Centre for Reference and Research on Leptospirosis, Brisbane,
Australia, for further identification. The strain was maintained in EMJH and
Fletcher medium at 30°C. Serological identification of the isolate was performed
with the Cross Agglutination Absorption Test (CAAT) carried out with the
recommended techniques (Babudieri, 1971) using hyper-immune serum from rabbits
against the known serotypes in the reference laboratory (Table 3.1).
3.2.3 Analysis of PCR products
Cultures were prepared for isolation of DNA by centrifugation as described
previously (Thaipadungpanit et al., 2007), followed by genomic DNA extraction
using a High Pure Viral Nucleic Acid kit (Roche, Germany). Amplification of the
16S rRNA was performed as described by others (Slack et al., 2008; Slack et al.,
2006; Thaipadungpanit et al., 2007) with the following modifications: PCR
amplification was performed in 25 µL volumes containing 1x Taq PCR buffer, 2.0
mM MgCl2, 200 uM dNTPs, 10.0 pmol Forward primer ( 5’- GTT TGA TCC TGG
CTC AG 3’) and 10.0 pmol Reverse primer (5’- CCG CAC CTT CCG ATAC-3’)
(Thaipadungpanit et al., 2007), 1U Taq Polymerase (Fermatas, Cat#-EP0405), 2.5
µL template DNA and nuclease free water to make up the final volume of 25 µL.
56 Table 3.1: Serological groups tested against Lepto175.
Serogroup Serovar Strain Pomona Pomona Pomona Sejroe Hardjo Hardjoprajitno Tarassovi Tarassovi Perepelitsin Grippotyphosa Grippotyphosa Moskva V Celledoni Celledoni Celledoni Icterohaemorrhagiae Copenhageni M 20 Australis Australis Ballico Pyrogenes Pyrogenes Salinem Canicola Canicola Hond Utrecht IV Hebdomadis Hebdomadis Hebdomadis Mini Mini Sari Sarmin Sarmin Sarmin Autumnalis Autumnalis Akiyami A Cynopteri Cynopteri 3522C Ballum Ballum Mus 127 Bataviae Bataviae Swart Djasiman Djasiman Djasiman Javanica Javanica Veldrat Bataviae 46 Panama Panama CZ 214 Shermani Shermani 1342 K Australis Pohnpei Mwalok Sejroe Saxkoebing Mus 24 Semarang Patoc Patoc 1 Wolffii Khorat Khorat-H2 Inadai spp. - - Ranarum Ranarum ICF Louisiana Louisiana LSU 1945 Manhao Lichuan Manhao 4 - Fainei Hurstbridge - Bejo ISO 9 - Licerasiae VAR 10
57
3.2.4 Polymerase chain reaction using conventional PCR
The DNA was amplified by using the following thermal-cycling profile: 95°C for 5
minutes, followed by 30 cycles at 94°C for 30 seconds, 58°C for 30 seconds and
72°C for 1 minute, and a final extension for 7 minutes at 72°C. DNA sequencing was
performed using the Big Dye Terminator (BDT) sequencing kit version 3.1 (Applied
Biosystems) using the original primer as described previously (Thaipadungpanit et
al., 2007). The PCR products were confirmed by agarose gel electrophoresis
(1.5%w/v) of 5µL PCR products for 45 minutes at 116V.
3.2.5 DNA sequencing
The cycle-sequencing products were purified by using sodium acetate/alcohol
precipitation as per the manufacturer’s instructions (Applied Biosystems) and the
purified products were forwarded to the First Base Laboratories Sdn Bhd, DNA
sequencing facility, Selangor, Malaysia, for capillary electrophoresis using an ABI
3130XL instrument.
3.2.6 Analysis of sequence results
The sequences were assembled and trimmed to a minimum of two contiguous
sequences using Bio Edit software (Invitrogen). Sequences from strain Lepto 175
Sarawak and representative 16S rRNA (1331 bp) gene sequences from members of
the genus Leptospira were aligned with CLUSTAL W (Thompson et al., 1997). By
using MEGA 5 (Tamura et al., 2011), distances of aligned 16S rRNA gene
sequences were estimated by the Jukes–Cantor method (Jukes and Cantor, 1969),
58 bootstrapped 1000 times and the tree topology was determined by the neighbour-
joining method (Slack et al., 2008; Slack et al., 2009; Slack et al., 2006). The final
phylogenetic tree was rooted by using Turneriella parva serovar Parva strain H as
an out-group and bootstrap values (1000) were displayed as percentages
(Thaipadungpanit et al., 2007).
3.3 Results
3.3.1 Morphological Features of Leptospira Lepto 175 Sarawak
The bacterial cells were thin, finely coiled and actively motile. The size of the cells
ranged from 10–13 µm in length and 0.2 µm in diameter. Under low power
magnification (x200) cells appeared as a small piece of thread with hooked ends and
were visible as dense, dot like structures (Figure 3.1).
Figure 3.1: Leptospira, Lepto 175 Sarawak under dark field microscopy (x200).
3.3.2 Serological identification of Leptospira Lepto 175 Sarawak
When strain Lepto 175 Sarawak was tested against hyper-immune antiserum
representing the major Leptospiral serogroups, there was no significant titre to any
59 leptospiral group (Table 3.2). This suggests that strain Lepto 175 Sarawak is
serologically distinctive to other leptospires maintained by the reference laboratory.
Table 3.2: Results of serological groups tested against Lepto 175 Sarawak.
A/S Lepto Serogroup Serovar Strain titre 175 Pomona Pomona Pomona 6400 <50 Sejroe Hardjo Hardjoprajitno 3200 <50 Tarassovi Tarassovi Perepelitsin 3200 <50 Grippotyphosa Grippotyphosa Moskva V 6400 <50 Celledoni Celledoni Celledoni 6400 <50 Icterohaemorrhagiae Copenhageni M 20 3200 <50 Australis Australis Ballico 6400 <50 Pyrogenes Pyrogenes Salinem 6400 <50 Canicola Canicola Hond Utrecht IV 6400 <50 Hebdomadis Hebdomadis Hebdomadis 3200 <50 Mini Mini Sari 3200 <50 Sarmin Sarmin Sarmin 1600 <50 Autumnalis Autumnalis Akiyami A 6400 <50 Cynopteri Cynopteri 3522C 3200 <50 Ballum Ballum Mus 127 1600 <50 Bataviae Bataviae Swart 6400 <50 Djasiman Djasiman Djasiman 6400 <50 Veldrat Bataviae Javanica Javanica 46 6400 <50 Panama Panama CZ 214 3200 <50 Shermani Shermani 1342 K 3200 <50 Australis Pohnpei Mwalok 6400 <50 Sejroe Saxkoebing Mus 24 6400 <50 Semarang Patoc Patoc 1 800 <50 Wolffii Khorat Khorat-H2 800 <50 Inadai spp. 1600 <50
60 A/S Lepto Serogroup Serovar Strain titre 175 Ranarum Ranarum ICF 1600 <50 Louisiana Louisiana LSU 1945 3200 <50 Manhao Lichuan Manhao 4 3200 <50 Fanei Hurstbridge 1600 <50 Bejo ISO 9 1600 <50 Licerasiae VAR 10 800 <50
3.3.3 Molecular characterization of Leptospira, Lepto 175 Sarawak
The 16S rRNA gene sequence similarity between strain Lepto 175 Sarawak and the
21 previously described species in the genus Leptospira was in the range of 87.19-
99.10% (Figure 3.2; Table 3.3). Leptospira licerasiae, Leptospira fainei and
Leptospira wolffii sv. Khorat strain Khorat-H2 (99.1 %) showed the highest levels of
16S rRNA gene sequence similarity to strain Lepto 175 Sarawak. Phylogenetic
analysis using 16S rRNA gene sequences showed that strain Lepto 175 Sarawak was
placed within the radiation of the genus Leptospira and was found to cluster with the
intermediate Leptospira species.
61
Figure 3.2: The phylogenetic tree of Lepto 175 Sarawak with other strain 16s rRNA partial sequences. A - Pathogenic strains; B - Intermediate strains; C- Non-Pathogenic strains; D - Outgroup
3.4 Discussion
Strain Lepto 175 Sarawak was identified as an intermediate strain of Leptospira.
This means that it is considered to be generally non-lethal, however it could be
potentially fatal under suitable environmental and host situations. Thus it could be
placed in the same group as Leptospira wolffii, a strain recently isolated from the
urine of a patient from Nakhon Rachasima, Thailand (Slack et al., 2008) and from
humans, sheep, buffalo, cattle, rodents and dogs from Iran and India (Balamurugan
et al., 2013; Zakeri et al., 2010). Intermediate strains of leptospires present a new
dimension in the fight against this disease. They have the possibility of becoming
62 pandemic and causing mortality in victims if not properly treated. Since the majority
of patients infected with intermediate strains are likely to make a full recovery,
scientists may overlook the public health significance of these strains (Balamurugan
et al., 2013; Zakeri et al., 2010). However, any new strains of Leptospira that may
potentially result in fatalities should be taken seriously and efforts made to minimize
the risk of exposure and improve the level of medical treatment. By studying
intermediate leptospiral strains in detail, the scientific community may gain more
insight into how they should tackle these bacterial strains. It is imperative that Lepto
175 Sarawak and similar strains be studied in greater detail so as to combat this
potentially new threat.
So far, several known serovars of Leptospira, notably L. interrogans
Icterohaemorrhagiae have been shown to be highly lethal (Viriyakosol et al., 2006).
A handful of others are intermediate and saprophytic and fatalities attributed to them
are uncommon. Treatments for the different categories of leptospires already exist
and these are administered according to the magnitude of infection (Sehgal et al.,
2000). Newly discovered intermediate strains of Leptospira thus pose a challenge in
the treatment of this illness. As fatalities may arise in patients infected with
intermediate strains, administering the correct treatment is important.
63 Table 3.3: Pair wise distance and sequence similarities of Leptospira strains against Lepto 175 Sarawak.
Pair wise Sequence Leptospiral strains distance similarities (%) L.wolffii sv Khorat Khorat-H2 0.009 99.10 L.licerasiae sv Varillal VAR010 0.013 98.74 L.broomii 5399 0.021 97.85 L.fainei sv Hurstbridge BUT6 0.022 97.76 L.inadai 0.036 96.41 L.kmetyi sv Malaysia. 0.046 95.43 L.borgpetersenii str Veldrat 0.047 95.34 L.santarosai sv Shermani LT 0.047 95.34 L.noguchii sv Panama CZ 214 0.047 95.25 L.alexanderi sv Nanding_str M 0.048 95.16 L.interrogans str RGA 0.048 95.16 L.kirschneri 0.048 95.16 L.santarosai 0.048 95.16 L.alexanderi sv Manha3L60 0.048 95.16 L.interrogans 0.048 95.16 L.sv.Sichuan str 79601 0.050 94.98 L.noguchii Fort Bragg 0.050 94.98 L.weilii Sarmin 0.052 94.80 L.biflexa Patoc I 0.126 87.37 L.wolbachii sv Codice CDC 0.126 87.37 L.meyeri sv Ranarum Iowa City 0.128 87.19 T.parva sv Parva H 0.195 80.48
Medical practitioners should be more alert if a patient presents with febrile
symptoms. Probing questions should be asked, especially if the patient is a returned
traveller from the tropics as early diagnosis of leptospirosis is important to prevent
the disease becoming established in the patients home country (Ricaldi and Vinetz,
2006).
64
3.5 Description of Leptospira, Lepto 175 Sarawak sp. nov.
The morphology and motility under dark-field microscopy of the new strain were
consistent with those for the genus Leptospira. Cells were 10–13 µm in length and
0.2 µm in diameter, with a wavelength of 0.5 um and amplitude of approximately
0.2 µm. No serologically titres were produced against the currently recognised
members of Leptospira. Phylogenetically the isolate was placed within the radiation
of the genus Leptospira (based on 16S rRNA gene sequence analysis).
Identifying host animals and reservoirs for intermediate leptospiral strains, such as
L. Lepto 175 Sarawak, will help in containing the disease it may induce.
Understanding the animal reservoirs for serovars and strains is critical in controlling
leptospirosis in both the human and animal (domestic and non-domestic) community
(Nascimento et al., 2004). In the following chapter the results of a serological study
in non-human primates from Sarawak are described.
65
66 CHAPTER 4 : LEPTOSPIRAL AGGLUTININS IN
CAPTIVE AND FREE RANGING NON-HUMAN
PRIMATES IN SARAWAK, MALAYSIA
4.1 Introduction
The role of wildlife as reservoirs of leptospirosis has been widely documented from
many countries; however it is still poorly understand (Matthias et al., 2005; Smythe
et al., 2002a; Tulsiani, 2010; Tulsiani et al., 2011). The disease can result in
economic losses in domesticated animals and has the potential to be an important
zoonotic disease of humans (Russ et al., 2003), as well as an infectious agent for
non-human primate species, which represent potential reservoir hosts. Leptospires
were first isolated from rats in 1917 in Malaysia and it is widely acknowledged that
rats are a key source of infection for humans (Levett, 2001). However, recently
Australian and Peruvian researchers have reported that bats can also carry
pathogenic Leptospira (Cox et al., 2005; Matthias et al., 2005; Smythe et al., 2002a),
although their role as carriers is not fully understood. Other wildlife, such as
primates, wild boar, and deer, also can act as potential carriers of these pathogens
(Bengis et al., 2004; Cantu et al., 2008; Cole and Landres, 1995; Diesch et al., 1970;
Hathaway et al., 1981; Kilbourn et al., 2003). However to date there has been little
research conducted on free ranging wildlife. Due to heavy deforestation and human
involvement in the jungles of Malaysia, there is the potential for exposure of
humans to leptospires from wildlife and the opportunity to be exposed to yet to be
recognised leptospiral serovars. This is particularly relevant to tourists in Malaysia
with 24.7 million tourists visiting the country annually, many of whom are keen to
67 see native wildlife (Jayaraman et al., 2010). Leptospirosis in wildlife can have
negative consequences on biodiversity, human and livestock health, animal welfare
and the national economy (Russ et al., 2003). At present Malaysian surveillance of
disease in wildlife is poorly coordinated and emerging zoonotic infectious diseases
represent a growing threat. Ecotourism is a major industry in Malaysia, especially in
the state of Sarawak (Chin et al., 2000; Musa, 2000; Yasak, 2000; Yea, 2002);
however its impact on wildlife and the local people is controversial. Ecotourism can
have a direct effect on wildlife species, communities and populations by influencing
their feeding, and reproductive and social behaviours, as well as indirect effects
through loss of vegetation, pollution, intra/inter-specific competition, close contact
with humans, and introduction of disease (Cole and Landres, 1995). Knowledge on
the leptospires naturally infecting wildlife is limited, although experimental
infections in captive wild animals have resulted in clinical signs ranging from
inapparent infections to febrile responses, abortions and deaths (Gulland et al., 1996;
Twigg et al., 1969).
The research reported in this study arose from the need to investigate the role of
wildlife in outbreaks of leptospirosis in humans (Thayaparan et al., 2013b) in
Sarawak. Previous studies have mainly focused on rodents and bats as carrier of
leptospires, as well as the effects on domestic animals and livestock. Few detailed
investigations on primates have been performed and few have concentrated on
animals from south-east Asia (Lilenbaum et al., 2002; McClure et al., 1986; Minette,
1966; Perolat et al., 1992; Pinna et al., 2012; Stasilevich et al., 2000). The study
outlined in this chapter was designed to evaluate the seroprevalence to Leptospira in
non-human primates in Sarawak.
68 4.2 Materials and methods
4.2.1 Study Area
Trapping of non-human primates was carried out around the Bako National Park and
the Matang Wildlife Centre.
4.2.1.1 Bako National Park
Bako National Park is located 37 km from Kuching, Sarawak, East Malaysia (Figure
4.1). It is Sarawak’s oldest national park, covering an area of 2,727 hectares and is
located at the tip of the Muara Tebas Peninsula (Chin et al., 2000; Ngui, 1991). It is
one of the smallest national parks in Sarawak, yet one of the most interesting, as it
contains almost every type of vegetation found in Borneo. The well-maintained
network of nature trails, from easy forest strolls to full-day jungle treks, allows
visitors to get the most out of this unique environment. Long-tailed macaques
(Macaca fascicularis), silvered langur (Trachypithecus cristatus/Presbytis cristata),
proboscis monkey (Nasalis larvatus), common water monitors lizards (Varanus
salvator), plantain squirrels (Collosciurus notatus), wild boar (Sus scrofa) and
mouse deer (Tragulus kanchil) are commonly found around the reserve. The major
attraction of this place is the proboscis monkey and many visitors visit this park
solely to view this species in their natural setting (Chin et al., 2000).
4.2.1.2 Matang Wildlife Centre
The Matang Wildlife Centre is situated at the western corner of the Kubah National
Park in Sarawak (Ngui, 1991), East Malaysia, and covers 180 hectares of lowland
forest (Figure 4.2). It is dedicated to education, research, conservation and
69 recreational activities (Ngui, 1991). In this park there are several species of
confiscated wildlife for public viewing and research is also conducted by local and
international scientists. There are long tailed macaques, pig/short-tailed macaques
(Macaca nemestrina), Bornean gibbons (Hylobates muelleri), orangutans (Pongo
pygmaeus) and sun bears (Helarctos malayanus) housed for various purposes.
70
Figure 4.1: Location of Bako National Park.
71 72
Figure 4.2: Location of Matang Wildlife Centre
72 4.2.2 Sampling procedure
In this study eight captive primates (three pig/short-tailed macaques, two Bornean
Gibbons, a long-tailed macaque and two orangutans) and four free ranging primates
(one silvered langur and three proboscis monkeys) were tested for the presence of leptospiral antibodies. The use of wildlife was approved by the Murdoch University
Animal Ethics Committee (Animal ethics No: W2376/10) and Sarawak Forestry
Department, Sarawak Malaysia (Permit No: NCCD.907.4.4 (V)-235). Free-range animals were first tracked down and tranquilised with Zoletil (5 mg/kg; 100 mg/mL) whilst captive animals were sedated with Zoletil (5 mg/kg). Five ml of blood was collected from the saphenous vein in 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 -20oC until it was analysed.
Figure 4.3: Silvered langur and proboscis monkey being prepared for blood collection.
73 4.2.3 Microscopic Agglutination Test (MAT)
The MAT was performed at the IMR according to the methods of Faine and
Organization, (1982) to check for Leptospira-specific antibodies to 17 serovars
endemic in Malaysia (Table 4.1).
4.2.3.1 Preparation of leptospiral antigens
Subcultures of the 17 serovars were prepared by inoculating 200-300 µL of well-
grown culture from previous subcultures maintained by the IMR into fresh EMJH
media. The serovars were subcultured in triplicate and incubated at 30oC for 5 days
prior to testing. During incubation the cultures were checked daily for growth and
for the presence of contamination. Once there was confluent growth, the cultures
were used in the antigen panel.
4.2.3.2 Dilution of sample sera
64 µL of serum from sampled animals was mixed with 1536 µL of phosphate
buffered saline (PBS) to make a 1:25 dilution.
4.2.3.3 MAT screening procedure
Two micro-titre plates were used to screen 11 serum samples. These plates were
labelled with the respective antigens in the columns and the serum reference number
in the rows. The details of the screening procedure were:
74 1. 50 µL of PBS was added to wells in column 1.
2. 50 µL of the 1:25 diluted serum of each animal sample was added to the respective column.
3. 50µL of leptospiral cultures were then added to the wells in the respective rows as labelled.
4. The plate was then shaken for 1 minute.
5. The plate was covered and incubated at 30oC for 2 hours.
6. After the incubation a loopful of the suspension was transferred via a wire loop to each well of a slide and the density of the leptospires observed under dark field microscopy at x100 magnification. The agglutination in wells containing the animal’ sera were compared with the controls in column 1.
7. Positive agglutination was considered when the approximate number of free leptospires was ≤ 50% of the number in the control wells.
8. Results were recorded on the result sheet.
9. For positive sera, full titration of the sera was performed and agglutination was observed with the 17 specific serovars common to Malaysia as reported below.
4.2.3.4 MAT full titration
1. The microtitre plates were labelled.
2. 50 µL of PBS were added to wells in columns 1, and 3 until 12 for the relevant rows.
3. 50 µL of the 1:25 diluted sera were added to wells 2 and 3 according to their respective rows.
75 4. The sera from wells 3 to 12 were titrated using a multi-channelled pipette. The
sera were mixed with PBS in well 3 and then 50 µL of this mixture was transferred
into well 4 and the procedure repeated until well 12.
5. 50 µL of the antigen was added into all of the wells according to the respective
rows.
6. The plate was incubated at 30oC for 2 hours.
7. Agglutination was detected by observing free leptospires in each well and this
was compared with the control wells. Positive agglutination was considered when
the approximate number of free leptospires was <50% of the number in control
wells.
8. The titre for each sample was recorded for each serovar as the last dilution that
showed <50% of free leptospires compared to the control wells. Sera were
considered to be positive if the titre was ≥ 1:100 by MAT.
76 Table 4.1: Serovars of Leptospira commonly found in Malaysia and used as antigens in the MAT panel.
Serogroup Serovar Reference Strain Australis Australis Ballico Autumnalis Autumnalis Akiyami A Bataviae Bataviae Swart Canicola Canicola Hond Utrecht IV Icterohaemorrhagiae Celledoni Celledoni Celledoni Grippotyphosa Grippotyphosa Moskva V Javanica Javanica Veldrat batavia 46 Pomona Pomona Pomona Pyrogenes Pyrogenes Salinem Sejroe Hardjo Hardjoprajitno Semaranga Patoc Patoc 1 Djasiman Djasiman Djasiman - Lai - - Copenhageni - - Hebdomadis - Lepto175 Sarawak
4.3 Results
Antibodies to leptospires were detected in 8 (66.6%; 95% CI 34.9-90.1) of the 12
animals sampled (Table 4.2). All tested primates were negative to all serovars tested
except Lepto 175 Sarawak and L.interrogans Lai. Of the eight captive non-human
primates six (three short-tailed macaque, two Müller's Bornean gibbon and one
orangutan) were seropositive (75%; 95%CI 34.9-96.8). Two of the four free ranging
non-human primates (one proboscis monkey and one silvered langur) also were
seropositive (50%, 95%CI 6.8-93.2).
77 78
Table 4.2: MAT results from the 12 positive non-human primates for L.Lepto 175 Sarawak and L. interrogans Lai. Common name Species Location L. Lepto 175 L. interrogans Sarawak Lai Short-tailed macaque Macaca nemestrina Matang (C) 800 400 Short-tailed macaque Macaca nemestrina Matang (C) 400 200 Short-tailed macaque Macaca nemestrina Matang (C) 400 <50 Proboscis monkey Nasalis larvatus Bako (F) <50 <50 Proboscis monkey Nasalis larvatus Bako (F) <50 <50 Proboscis monkey Nasalis larvatus Bako (F) 200 100 Müller's Bornean gibbon Hylobates muelleri Matang (C) 400 200 Müller's Bornean gibbon Hylobates muelleri Matang (C) 200 <50 Orangutan Pongo pygmaeus Matang (C) 200 <50 Orangutan Pongo pygmaeus Matang (C) <50 <50 Long-tailed macaque Macaca fascicularis Matang (C) <50 <50 Silvered langur Trachypithecus cristatus Bako (F) 400 <50 (C) – Captive animal; (F) – Free ranging animals
78
The antibody titres for seropositive animals varied from 1:100 to 1:800. One of the
sera from a captive short-tailed macaque had a titre of 1:800 to serovar Leptospira
Lepto 175 Sarawak (Table 4.2). Two thirds of the primates were positive (n = 8) for
serovar Lepto 175 Sarawak and one third (n = 4) for Lai (Table 4.3). Four primates
were positive for both serovars Lai and Lepto 175 Sarawak (Table 4.2).
Table 4.3: Results of serology for Leptospira serovars in 12 primates. Number positive Serovar (%, 95% CI) Lai 4 (33.3%; 9.9-65.1) Lepto 175 Sarawak 8 (67.6%; 34.9-90.1)
4.4 Discussion
Antibodies to leptospires were detected in captive primates from the Matang
Wildlife Centre. These infections may have arisen from poor sanitation or
inadequate rodent control and adopting a program to improve these may result in
reducing the spread of leptospires to other animals, including humans. In this study
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
(Kilbourn et al., 2003), but assessment of its significance requires testing of serial
samples which was not possible in the current study.
Szonyi et al., (2011) described an outbreak of leptospirosis in Capuchin (Cebus
capucinus) monkeys and advised of the risk of disease from these animals.
According to the EAZWV transmissible Disease Fact Sheet (Hartskeerl and Ellis,
79 2003) the Macaca spp., Cercopithecus aethiops (Green monkey), Pongo pygmaeus,
Saimiri sciureus (common squirrel monkey), Saguinus oedipus (Tamarins) and
Galago senegalensis (Bush babies) are susceptible to infection with serovars of L.
interrogans. In primates leptospirosis is mostly asymptomatic; however in fatal
cases anaemia, facial oedema, vomiting, jaundice, weakness, lethargy and fever may
occur (Hartskeerl and Ellis, 2003; Kessler and Everard, 1988). Palmer et al., (1987)
experimentally challenged Grivet/Green monkeys with the Hardjo serovar and found
these animals to be a suitable model for infection in humans.
In this study a positive titre in a proboscis monkey was possibly first published
report in the world. Information on Lepto 175 Sarawak is deficient, although it
would appear to be endemic in Sarawak. As reported from the results of the previous
chapter, Lepto 175 Sarawak has close genetic similarities with Leptospira wolffii.
This latter species has been isolated from humans and wildlife species, including
non-human primates, from Thailand, India, Iran and Sabah (Balamurugan et al.,
2013; Kilbourn et al., 2003; Slack et al., 2008; Zakeri et al., 2010). Although the
pathogenicity status of Lepto 175 Sarawak is not yet confirmed, it is important that
the public health authorities are aware of these preliminary seroprevalence results
and the potential for an outbreak in humans visiting Bako National Park should be
evaluated and monitored. In contrast it is known that the serovar Lai is pathogenic
and is a major cause of zoonotic spread to humans involved in adventure activities
(Sejvar et al., 2003). Studies by Kilbourn et al., (2003) on free-ranging orangutans in
Sabah found a high prevalence of antibodies against L. interrogans serovars, notably
Grippotyphosa and Autumnalis, as well as the novel species Leptospira wolffii.
Experimental infection of marmoset monkeys (Callithrix jacchus) with serovar
Copenhageni resulted in histological tissue reactions including intra-alveolar
80 haemorrhage (Pereira et al., 2005). Most animals had microscopic changes to the kidneys when they were euthanased 6 and 12 days after challenge.
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 (Pereira et al., 2005). Baulu et al.,
(1987) studied Vervet monkeys (Chlorocebus pygerythrus) in Barbados and found that they retained naturally acquired antibodies to Leptospira for at least 2.5 years.
The primates were transmitting the organism between themselves independently of other animals and were not a major source of infection for humans. Kessler and
Everard, (1988) surveyed free-ranging Rhesus monkeys (Macaca mulatta) in Puerto
Rico and found evidence of seropositivity. However these primates were apparently healthy, despite their contact with rats and ingestion of stagnant water. Lilenbaum et al., (2002) found anti-Leptospira agglutinins in lion tamarins (Leontopithecus rosalia) in Rio de Janeiro, Brazil, even though these animals showed no clinical signs or prior history of disease. Pinna et al., (2012) suggested that captive New
World monkeys should be screened for leptospirosis before they are released into the wild, as there is a danger they may have been exposed to serovars originating from urban areas prior to release. Following the death of an endangered Colobus monkey (Colobus guereza) at the National Zoo in Washington DC (Hutchins, 2006),
William Foster of the American Zoo and Aquarium Association reported that leptospirosis was not often diagnosed in zoo animals. He advocated tougher pest- control policies to tackle the rodents that carry the bacterium. Romero et al., (2011b) also reported on a potential risk of transmission of leptospires from zoo animals to staff in Zoo Colombia.
81 This study has found evidence of exposure of langur leaf and proboscis monkeys to
leptospires and there is an urgent need to undertake more investigations on free
ranging non-human primates in Malaysia. Macaques are scavengers that search for
food in garbage heaps, which mean they may ingest food possibly contaminated by
rodent urine or have direct contact with it through damaged skin. This results in
leptospires entering their bodies and infecting them, potentially resulting in them
becoming maintenance hosts for the bacteria. When the macaques urinate onto the
ground, there is danger that leptospires shed could be transferred to silvered leaf
langur and proboscis monkeys through contact with the organisms in the
environment. Besides contact with rats, ingestion of stagnant water contaminated
with leptospires could be a potential source of infection for these non-human
primates. The latter possibility was highlighted by Famatiga, (1973) who observed
silvered langur and proboscis monkeys drinking from freshwater streams and rivers.
These water bodies are known to be suitable habitats for leptospires and when these
two predominantly arboreal primate species drink from such places, they may
become infected. Proboscis monkeys are also competent swimmers (Jalil, 2009) and
are known to have the most aquatic lifestyle of primates. They usually live near
water bodies, rarely ranging more than one kilometre from it. Such activities would
increase the opportunity for exposure to leptospires.
In Bako National Park several short and long-tailed macaques and wild boars roam
freely eating leftover food and garbage (Personal observation). Based on the
previous discussions it is possible that these two species of macaque may transmit
leptospires to proboscis and silvered langur, as well as to the many tourists who visit
this national park. Humans are also at risk through contact with contaminated soil or
water. It is important to educate visitors to national parks not to leave food out
82 which may attract rodents or other scavengers. Additionally, animals in
rehabilitation centres should be thoroughly screened for leptospirosis prior to their
release to the natural environment to prevent them from spreading the pathogen on
release. The results of this study are important as they indicate that macaques
possibly form a key node in the chain of leptospiral transmission.
4.5 Conclusions
Although leptospires are endemic in Sarawak this is the first reported evidence of
the organisms in non-human primates with the proboscis monkey having the highest
seroprevalence. As mentioned previously, rodent control is a vital step in containing
and controlling leptospirosis. Litter prevention in and around national parks is an
important component of this to reduce the likelihood of macaques getting infected
and passing on the leptospires to humans or other animals. The possibility of non-
human primates acting as reservoir hosts for leptospires requires further
investigation.
Other mammals, including bats, play a key role in the distribution of Leptospira.
Infection of native mammals is potentially deleterious to the animals themselves but
also to the fledgling nature tourism industry in Sarawak, as these animals may
inadvertently result in infection of foreign or domestic visitors. In the following
chapter the results of a serological survey of other wildlife (small mammals) in
Sarawak are reported.
83
84
CHAPTER 5 : SEROLOGICAL AND MOLECULAR
STUDY OF LEPTOSPIRA IN SMALL WILDLIFE
AROUND WILDLIFE RESERVES AND DISTURBED
FOREST REGIONS IN KUCHING, SARAWAK,
MALAYSIA.
5.1 Introduction
Leptospirosis is endemic to tropical regions of the world and is re-emerging as a
new danger to public health in Southeast Asia, including Malaysia. Although studies
have been conducted in Malaysia for more than 70 years (Hanson, 1982),
leptospirosis and the threat it poses is still not well understood. Malaysian jungles
are home to numerous species of wildlife and peri-domestic animals. These include
bats, squirrels and rats, as well as primates. Bats and rats are known to harbour
leptospiral serovars and pass them to other species including humans (Faine et al.,
1999; Matthias et al., 2005; Richardson and Gauthier, 2003; Roth, 1964; Vashi et
al., 2010).
With deforestation becoming more commonplace in many tropical environments,
including Malaysia, there is increased likelihood of contact between humans and
wildlife due to the disturbance of natural habitats. Bats are known to respond to
habitat modification, loss and fragmentation at the population level, so their spatial
and temporal dynamics are particularly sensitive to anthropogenic activities. They
85 forage in fruit orchards and forest clearings, and roost in buildings, water cisterns,
culverts, abandoned structures and bridges (Matthias et al., 2005). This often results
in humans coming into contact with bat urine or surfaces contaminated with it. Also,
ground-dwelling species, such as rodents or marsupials, that reside or forage under
bat roosts, could encounter Leptospira-contaminated urine (Zarnke, 1983).
Sarawak has seen an increase in human cases of leptospirosis in recent years. As
summarised in Chapter 2, leptospirosis is suspected to have made a Chinese national
working at Bakun Dam seriously ill, and contamination by rat urine was believed to
have been the source of infection in Sibu city in June 2012. The increasing
popularity of eco-tourism raises another risk for the disease. Such tourism brings
visitors to Malaysia close to nature and native wildlife, some of which may be
reservoir hosts for leptospirosis, as well as potentially interfering with an already-
fragile ecosystem.
5.1.1 Objectives
The objectives of the study reported in this chapter were to:
a. Determine the role of bats, rats, squirrels and treeshrews in the lifecycle of
Leptospira in and around wildlife reserves and disturbed forest areas in Sarawak.
b. Determine the seroprevalence to Leptospira in these species
c. Determine the carrier stage of animals by performing culture and molecular
studies.
86 5.1.2 Rats and leptospirosis
Rats have been shown to be carriers of leptospires throughout the world and are important reservoirs of infection for animals and humans (Priya et al., 2007; Roth,
1964). Sarawak, East Malaysia is a tropical region with diverse animal species including 61 different species of rats and mice (Santa Rosa et al., 1975). The
Müller’s rat (Sundamys muelleri), the rice field rat (Rattus argentiventer) and the brown spiny rat (Maxomys rajah) have a broad-based distribution between the border of forested areas and human settlements and many different leptospiral serovars have been isolated from these species in Sarawak and other countries
(Smith et al., 1961). There has been a higher seroprevalence reported in rice field rats compared to other species of rats (Smith et al., 1961). The brown spiny rat is generally regarded as the maintenance host for leptospires of the
Icterohaemorrhagiae serogroup and the species can be an occasional carrier of other serovars.
Researchers have indicated a striped field mouse (Apodemus agrarius), a forest vole
(Clethrionomys glareolus), Apodemus spp. and common voles (Microtus arvalis) as the prevailing species infected by leptospires (Slavica et al., 2008; Treml et al.,
2002). Treml et al., (2002) found striped field mice and forest voles in the Czech
Republic and reported a seroprevalence of 20.6% to sv. Grippotyphosa. Similar findings in voles were described in the Netherlands by other researchers (Kuiken et al., 1991). Rodents have long been associated with leptospirosis as reservoir or maintenance hosts and verminous rodents are considered a key source for the distribution of leptospires in an urban setting (Emanuel et al., 1964; Faine et al.,
1999; Hathaway et al., 1981; Lau et al., 2010a; Lau et al., 2010b; Mohan et al.,
2009; Slack et al., 2006; Turner, 1970). The bacteria colonise the renal tubules of
87 rodents, who may then shed leptospires continuously or intermittently throughout
their life (Farrar, 1995). Although leptospires are endemic in rodents, clinical
disease is not reported and serovar-specificity is observable (Levett, 2001). Rodents
may also present low titres to serovars of Leptospira that are not endemic to their
species (Webster et al., 2009). The higher seroprevalence in rodents is also
influenced by seasonal effects with a higher population of rats in the wet than in the
dry season (Kasso et al., 2010). This population growth also coincides with
outbreaks of leptospirosis in humans (Pappas et al., 2008).
5.1.3 Bats and leptospirosis
Bats have been implicated in the spread of leptospirosis to humans (Mortimer, 2005;
Vashi et al., 2010). Mortimer, (2005) reported the case of an American caver who
may have had contact with bat (and possibly rat) urine-contaminated water while
carrying out a reconnaissance project at Gunung Buda, Sarawak.
Despite the negative reputation that bats pose to public health, these creatures play a
major role in the earth’s fragile ecosystem. Bats are a critical component of
Southeast Asia’s threatened fauna; they constitute approximately 30% of the
region’s mammal species, and can comprise as many as half of all mammal species
in tropical rainforest regions (Kingston et al., 2006). Moreover, the region is pivotal
for international bat conservation as it supports nearly 30% of the world’s bat fauna
with 330 species listed (Kingston, 2010; Simmons, 2005). Habitat loss is the main
threat to the survival of bats, which provide essential ecosystem services as
pollinators (Corlett, 2005; Whittaker and Jones, 1994) and seed dispersers to more
than 300 paleotropical plant species (Kingston, 2010). It is believed that without
88 bats both the regeneration of the forest ecosystem (Cox et al., 1991; Gumal, 2001;
Marshall, 1985; McConkey and Drake, 2006; Rainey et al., 1995) and the success of
bat-dependent fruit crops will be severely compromised. Other bats feed on insects
and are responsible for eliminating pests of rice crops, reducing the need for
insecticides (Cleveland et al., 2006) and the consequent impacts on local
biodiversity that result from their use. Large bat colonies are also a source of guano,
which is widely used in Southeast Asia as an organic fertilizer (Kingston, 2010).
The purpose of this particular study was to determine the common leptospiral
serovars present in small wild mammals living around wildlife reserves/disturbed
forest habitats and human communities.
5.2 Materials and Methods
From January 2011 to March 2012 blood and kidneys were collected from bats, ro-
dents and squirrels trapped in Sarawak and tested to determine exposure to
leptospires.
5.2.1 Study/sampling sites
The sampling of animals was undertaken in the wider range of Kuching, and Kota
Samarahan area included Universiti Malaysia Sarawak (UNIMAS) area, Matang &
Kuba National Park, Bako National Park, Wind and Fairy Cave National Reserves
and Mount Singai Conservation Area (Figure 5.1). Localities were chosen in such a
way that different environments were represented (disturbed jungle habitats around
Kuching and secondary forest located around human settlements) to gain an accurate
89 picture of the possible transmission of leptospires in the particular localities. During
the investigation 155 small mammals (rats, squirrels,treeshrews and bats) were
sampled.
5.2.1.1 UNIMAS area (UNIMAS, Kampung Sebayor and Kampung
Etingan)
Universiti Malaysia Sarawak (UNIMAS) is located in Kota Samarahan,
approximately 30 kilometres east of the state capital Kuching (Figure 5.2). The
university is surrounded by forested and swampy areas, providing an ideal
environment for leptospires to thrive (www.unimas.my). The eastern (old) campus
contains two residential colleges while the western (main) campus has five colleges.
The campus grounds have a disturbed habitat or deforested area. Kampung (Village)
Sebayor is located inside the campus and Kampung (Kg) Etingan is located close to
UNIMAS campus.
The locations and descriptions of Bako National Park and Matang Wildlife Centre
were outlined in Chapter 4.
90
Figure 5.1: Sampling sites of small wildlife.
91 UNIMAS Area
Figure 5.2: UNIMAS and surroundings where samples were collected.
92 5.2.1.2 Wind and Fairy Caves
The Wind and Fairy caves are part of the Bau Formation, a narrow belt of limestone covering approximately 150 sq km in south-west Sarawak. Most caves in this network are remote and inaccessible; however Wind Cave is within easy reach of
Kuching and is a popular day trip and picnic destination
(www.sarawakforestry.com). The Wind Cave Nature Reserve covers 6.16 hectares and includes the cave itself and the surrounding forest. Squirrels, shrews and a variety of birds can be found along the river and the limestone hill (Ngui, 1991).
Black nest swiftlets (Aerodramus maximus) can be seen and heard inside the cave, as well as 14 species of bat (Barapoi, 2004). Immediately to the south of Wind Cave lies Fairy Cave (Wong and Chung, 2001) (Figure 5.3).
5.2.1.3 Mount Singai Conservation Area
Mount Singai is located midway between the town of Bau and the Matang wildlife reserve (Figure 5.4). This 333.3 metre (1,000-ft) high mountain can be reached by a bitumen road via Batu Kawa, while its flat top can be accessed via the same jungle trail used by village people to travel to their lowland farms (Ngui, 1991; Wong and
Chung, 2001). Once occupied by members of the Bisingai community, the mountain is now overgrown with secondary vegetation and in recent times has been occupied by the Catholic Memorial Pilgrimage Centre. This mountain holds significant socio- ecological value as well as attracting many devout Catholics. Twenty-two species of mammals have been recorded at Mount Singai, including 10 species of bats, seven species of rodents and four species of tree shrews (Ngui, 1991; Wong and Chung,
2001).
93 94
Figure 5.3: Location of Wind Cave and Fairy Cave.
94
Mount Singai Conservation Area
Figure 5.4: Location of Mount Singai Conservation Area.
95
5.2.2 Animal capturing
The species sampled in this study included small mammals (rats, squirrels, tree
shrew and bats) that were captured in the selected areas over a one-year period from
January 2011 to March 2012. The small mammals were trapped around bat caves,
forest areas of the national parks and disturbed habitats. Trapping of wildlife was
approved by the Murdoch University Animal Ethics Committee (Animal ethics No:
W2376/10) and the Sarawak Forestry Department, Sarawak Malaysia (Permit No:
NCCD.907.4.4 (V)-235).
5.2.2.1 Rats, squirrels and treeshrews
In Malaysia wild rats are mainly nocturnal and occur in a variety of habitats
including coastal forests (especially mangroves), secondary forests and grasslands
and have also adapted to rubber and oil palm plantations. One hundred and two
species of bats and 61 species of rodents have been identified in Borneo (Santa Rosa
et al., 1975).
The rats, squirrels and treeshrews were trapped using cage traps (Figure 5.5)
(Baeumler and Brunner, 1988). A maximum of 50 cage traps were set at each
sampling time. A mixture of one-part raisins and two parts peanut butter with rolled
oats, banana and dried fish was used as bait and placed in each trap (Hickie and
Harrison, 1930; Wood, 1984). Traps were opened at 10 pm and checked the
following morning. Any by-catch was immediately released at the site of trapping. If
pregnant or lactating small mammals were captured these were also released
immediately at the captured site. Any trapped rats, squirrels and tree shrews were
collected and taken to the UNIMAS laboratory. The cage containing the trapped
96 mammal was covered with cloth and kept in the animal house until processing. Each small mammal was housed in individually ventilated cages. During transportation the animals were fed sunflower seeds, cereals, cooked corn kernels and cheese and provided with water ad libitum.
Figure 5.5: Wire mesh trap used for trapping rats, squirrels and treeshrews
5.2.2.2 Trapping and sampling of bats
All the bats were wild caught either using a mist or harp net. A mist net (Figures 5.6 and 5.7) is a lightweight net that was placed in the flight path of the bats; for example across trails, over streams, adjacent to fruiting trees and at the mouths of caves. Bats that were caught in the nets were carefully removed from the net to ensure their delicate wings were not injured. Harp traps (Figure 5.8) were also set across trails or over small streams. Harp traps are made of vertical sets of fine fishing line strung under tension in a frame. The bats are not able to see or detect
97 them, and consequently get caught between the sets of line and slide down into a bag
beneath the frame. They then remained in the bag until they were removed. Three
harp traps and five mist nets were placed along bat flight paths for a maximum of
six hours between 5 pm and 11 pm. Nets were checked every 30 minutes and any
bats caught were removed from the traps. After removal each bat was placed in an
individual cloth bag, which was tied and placed in a wicker basket. The basket was
covered with a cloth and placed in a safe quiet place to minimise disturbance. If any
birds were trapped these were removed and released immediately.
Figure 5.6: A fruit bat caught in a mist net.
Figure 5.7: Illustrated diagram of the setup of a mist net . (http://www.ilmb.gov.bc.ca/risc/pubs/tebiodiv/bats/batsml20-04.htm)
98
Figure 5.8: Harp Net set along the flight path of bats.
5.2.3 Collection and preservation of samples from animals
5.2.3.1 Rats, squirrels and treeshrews
The captured animals were anaesthetised using Ketamine + Xylazine (50–75 mg/kg
+ 10 mg/kg IP, respectively) (Karwacki et al., 2001). Once the animsl were anaesthetised 1-3 ml of blood was collected via cardiac puncture using a 25 G needle and a 5 mL syringe. After blood collection, the rats were euthanased with barbiturate (sodium pentobarbitone at 150-200 mg/kg) via either an intracardiac or intravenous (IV) injection. After euthanasia the animals were necropsied and kidney samples collected (Figure 5.9). Blood samples were left at room temperature for 30 minutes to clot and were then centrifuged at 3,000 RPM for 1 minute. The serum was removed and stored at -20oC until testing in the laboratory.
99
Figure 5.9: Collection of kidney samples.
5.2.3.2 Bats
Bats were anaesthetised to reduce the stress of handling and to minimise the risk of
the handler being bitten (Figure 5.10). Each animal was anaesthetised with an
intramuscular injection of ketamine (6–7 mg/ Kg) and medetomidine (60–70 µg/Kg)
(Plowright et al., 2008). A blood sample (1 to 3 mL) was collected by cardiac
puncture from each bat using a 5 mL syringe and a 25-gauge needle. Prior to
collection the hair was sterilized with cotton wool dipped in 70% alcohol. Data were
collected on the age, gender, location and general health status of each sampled
animal.
The bats were then placed in a fabric pouch and placed in a plastic airtight container.
Cotton balls were thoroughly saturated with isoflurane in the barrel of a six ml
syringe (after removing the plunger). The syringe barrel containing the isoflurane
100 saturated cotton balls were then placed into the container and the lid closed. Bats were usually anaesthetised within seconds and death was confirmed by auscultation with a stethoscope. Kidney samples were removed for culturing and molecular analysis. Carcasses were stored in 99% ethanol for future use by UNIMAS students.
All personnel who handled bats had been vaccinated against rabies and all samples were treated as potentially infectious.
Figure 5.10: Examining a fruit bat prior to the collection of blood.
5.2.4 Culture and isolation of leptospirosis
Kidney samples from all animals were cultured for leptospires. A cross-section of the cortex of one kidney was removed using a sterile scalpel and this section was cut into small pieces and inoculated into two EMJH and two Fletcher media tubes for
101 culture. The remaining part was deposited in 100% pure PCR grade alcohol for
future PCR work. The tubes with the inoculated material were incubated at 30°C and
checked twice weekly under dark field microscopy for 12 weeks. If there was no
growth after 12 weeks, or if any fungal growth was detected, the specimen was
discarded.
5.2.5 Microscopic Agglutination Test (MAT)
All serum samples were tested against a panel of 17 live antigens in the Microscopic
Agglutination Test (MAT). The protocol followed was that described in Chapter 4.
In this study an agglutination titre ≥ 50 was indicative of seropositivity to
Leptospira.
5.2.6 Molecular analysis
5.2.6.1 Preparation of genomic DNA from kidney samples
Approximately 30 mg of kidney was placed into a sterile Petri dish. Using a sterile
scalpel and a needle the tissue was dissected into small pieces. The tissue was then
transferred into a micro centrifuge tube and mixed with 50 µL protein kinase K
(Qiagen) and 200 µL lysis buffer and incubated at 55°C overnight or until the tissue
was completely digested.
5.2.6.2 Analysis of PCR products
The methods for DNA isolation, sequencing and analysis of the PCR have been
described in Chapter 3.
102
5.3 Results
5.3.1 Results of capturing small mammals (Rats, Squirrels, Tree shrew
and Bats)
In total 155 animals were sampled including 70 bats, 57 rodents, 20 squirrels and
eight tree shrew. The rats trapped were Müller’s rat (Sundamys muelleri) (n = 29),
ricefield rat (Rattus argentiventer) (n = 20), brown spiny rat (Maxomys rajah) (n =
7), and Whitehead’s rat (Maxomys whiteheadi) (n = 1). All squirrels were either
Low’s squirrels (Sundasciurus lowii) (n=1) or plantain squirrels (Callosciurus
notatus) (n=19) and the tree shrew were Tupaia tana (n=8). The bats trapped were
dusky fruit bat (Penthetor lucasi) (n = 20), short-nosed fruit bat (Cynopterus
brachyotis) (n = 22), spotted-winged fruit bat (Balionycteris maculata) (n = 13),
fawn roundleaf bat (Hipposideros cervinus) (n = 5), Bornean horseshoe bat
(Rhinolophus borneensis) (n = 2), bicolored roundleaf bat (Hipposideros bicolor) (n
= 2), Malaysian slit-faced bat (Nycteris tragata) (n = 1), intermediate horseshoe bat
(Rhinolophus affinis) (n = 1), dusky roundleaf bat (Hipposideros ater) (n = 1), lesser
woolly horseshoe bat (Rhinolophus sedulous) (n = 1), papillose woolly bat
(Kerivoula papillosa) (n = 1) and dayak roundleaf bat (Hipposideros dyacorum) (n =
1). The largest number of animals was caught in the locality of UNIMAS (40)
followed by Wind Cave and Fairy Cave areas (36), Bako National park (31), Matang
and Kubah (30) and Mount Singai (18) (Tables 5.1 and 5.2).
103 104
Table 5.1: Species, location and MAT results for animals other than bats caught.
Mt. Wind & Fairy Matang & Total # % Positive Species UNIMAS Bako % MAT (+) Singai Caves Kubah trapped (95% CI) Sundamys muelleri 10 6 3 4 6 29 34.1 20 68.9 (42.9, 84.7) Rattus argentiventer 6 4 4 2 4 20 23.5 9 45.0 (23.1, 68.5) Maxomys rajah 0 1 4 2 0 7 8.2 4 57.1 (18.4, 90.1) Maxomys whiteheadi 0 0 1 0 0 1 1.2 0 0.0 (0, 97.5) Sundasciurus lowii 0 0 0 1 0 1 1.2 1 100.0 (2.5, 100) Callosciurus notatus 4 2 4 3 6 19 22.4 10 52.6 (28.9, 75.6) Tupaia tana 0 0 1 4 3 8 9.4 1 12.5 (0.3, 52.7) Total 20 13 17 16 19 85 100.0 45 52.9 (41.8, 63.9) % 23.5 15.3 20.0 18.8 22.4 100.0
104
Table 5.2: Species, location and MAT results for bats caught. Mt. Wind & Fairy Matang & Total % Positive Species UNIMAS Bako % MAT (+) Singai Caves Kubah trapped (95% CI) Penthetor lucasi 5 2 11 0 2 20 28.5 12 60.0 (36.1, 80.9) Cynopterus brachotis 10 3 0 4 5 22 31.4 9 40.9 (20.7, 63.6) Balionycteris maculate 5 0 0 4 4 13 18.5 3 23.1 (5.0, 53.8) Hipposideros cervinus 0 0 2 3 0 5 7.1 2 40.0 (5.3, 85.3) Other bat species 0 0 6 3 1 10 14.2 2 20.0 (2.5, 55.6) Total 20 5 19 14 12 70 100.0 28 40.0 (28.5, 52.4) % 23.5 15.3 20.0 18.8 22.4 100
105 5.3.2 Results of serological analysis of trapped rats,squirrels, treeshrews and bats
Antibodies to leptospires were detected in 73 of the 155 animals tested (seroprevalence of 47.0%: 95% CI 39.0-55.3%). The seroprevalence for rats (57.9%; 95% CI 44.1-70.9) was slightly higher than that for squirrels (42.9%; 95% CI 24.5-62.8) and bats (40%;
95% CI 28.5-52.4), however this difference was not significant (χ2= 4.28; dƒ=2;
P=0.117).
The highest seroprevalence was found in Müller’s rats (68.9%; 95% CI 42.9-84.7) followed by the brown spiny rat (57.1%; 95% CI 18.4-90.1), plantain squirrels (52.6%;
95% CI 28.9-75.6) and ricefield rats (45%; 95% CI 23.1-68.5). The highest seroprevalence in bats was observed in the dusky fruit bat (60%; 95% CI 36.1-80.9), followed by the short-nosed fruit bat (40.9%; 95% CI 20.7-63.6) and spotted-winged fruit bat (23.1%; 95% CI 5.0-53.8) (Tables 5.1 and 5.2). There was no significant difference in the seroprevalence between the different animal species (χ2 = 24.9; dƒ=18;
P=0.126).
Some seropositive animals were detected in all of the localities sampled. The highest prevalence was found at Mount Singai (64.7%; 95%CI 38.3-85.8) and the lowest at the
UNIMAS area (35%; 95%CI 20.6-51.7) (Table 5.3). There was no significant difference overall in the seroprevalence among the five sampling locations (χ2= 4.28; dƒ=4;
P=0.117); however significantly more seropositive animals were detected at Mount
Singai than at UNIMAS (OR 3.4; 95% CI 1.04, 11.17).
Antibodies to 10 different serovars were detected in rodents and six different serovars in bats. Antibodies to serovar Lepto 175 Sarawak were detected in 30 (24.7%) rats, 11
(9.0%) squirrels and 27 (52.9%) bats (Table 5.4). Antibodies to serovar
Icterohaemorrhagiae were detected in 20 (16.5 %) rodents, squirrels and treeshrew;
106 serovar Australis in 13 (10.6 %) blood samples and serovar Autumnalis in 12 (9.6 %) blood samples (Tables 5.5 and 5.6). Serovars Australis and Lai were detected in one dusky fruit bat and one short-nosed fruit bat respectively. Antibodies to serovar
Pyrogenes were detected in 10 (19.6%) samples from dusky fruit bats and short-nosed fruit bats.
Table 5.3: Seroprevalence to leptospires in small mammals trapped in different localities.
No. of Locality Seroprevalence (95% CI) OR (95% CI) seropositive mammals UNIMAS 14 35.0 (20.6, 51.7) 1.0
Mt. Singai 11 64.7 (38.3, 85.8) 3.4 (1.04, 11.17)
Wind & Fairy Caves 17 47.2 (30.4, 64.5) 1.7 (0.66, 4.18)
Matang & Kubah 17 56.7 (37.4, 74.5) 2.4 (0.92, 6.42)
Bako NP 14 45.2 (27.3, 64.0) 1.5 (0.59, 4.0)
Table 5.4: Seroprevalence to leptospires in different orders of small mammals.
Animals MAT Positive Seroprevalence (95% CI) OR (95% CI)
Rats 33 57.9 (44.1, 70.9) 1.0
Squirrels &Tree shrew 12 42.9 (24.5, 62.8) 0.55 (0.24, 0.99)
Bats 28 40.0 (28.5, 52.4) 0.48 (0.22, 1.36)
107 108
Table 5.5: Number of animals belonging to the rats, squirrels and treeshrews seropositive to different leptospiral serovars.
Serovar
+ (%) Lepto 175 Australis Autumnalis Ictero Javanica Patoc Copenhageni Pomona Lai
Sundamys muelleri 66 (55.0) 17 13 9 14 0 8 3 0 2
Rattus argentiventer 23 (19.1) 9 0 3 3 4 2 2 0 0
Maxomys rajah 9 (7.5) 4 0 0 2 0 3 0 0 0
Sundasciurus lowii 1 (0.8) 1 0 0 0 0 0 0 0 0
Callosciurus notatus 19 (15.8) 10 0 0 1 1 2 0 5 0
Tupaia tana 2 (1.7) 0 0 0 0 0 0 1 0 1
Total 120 (100) 41(34.1) 13(10.7) 12(10) 20(16) 5(5) 15(12.5) 6(5.0) 5(4.1) 3(2.5)
108
Table 5.6: Number of bats seropositive to different leptospiral serovars.
Serovars
Species + (%) Lepto 175 Australis Icterohaemorrhagiae Pyrogenes Patoc Lai
Penthetor lucasi 21 (41.1) 12 1 0 8 0 0
Cynopterus brachyotis 17 (33.3) 9 1 0 2 4 1
Balionycteris maculate 7 (13.7) 3 0 1 0 3 0
Hipposideros cervinus 3 (5.8) 2 0 0 0 1 0
Rhinolophus borneensis 2 (3.9) 1 0 0 0 1 0
Nycteris tragata 1 (1.9) 0 0 0 0 0 1
Total 51 (100) 27 (52.9) 2(3.9) 1 (1.9) 10 (19.6) 9 (17.6) 2 (3.9)
109 The antibody titres for seropositive animals varied from 1:50 to 1:800. More animals had a serum titre of 1:100 (n = 65) than other titer (1:50 in 20 serum samples; 1:200 in
30 serum samples and 1:400 in 8 samples, while only one squirrel had a titer of 1: 800)
(Table 5.7).
Table 5.7: Titres of antibodies to different serovars found in small mammals.
Titres
Serovar + ve 1:50 1:100 1:200 1:400 1:800
Lepto 175 Sarawak 68 23 27 12 5 1
Australis 15 3 5 4 3 0
Autumnalis 12 2 6 4 0 0
Icterohaemorrhagiae 21 5 13 3 0 0
Javanica 5 1 3 1 0 0
Pyrogenes 10 8 2 0 0 0
Patoc 24 8 14 3 0 0
Copenhageni 6 0 4 2 0 0
Pomona 5 2 2 1 0 0
Lai 5 4 1 0 0 0
Total/% 171/100 56/32.5 77/44.7 30/17.4 8/4.6 1/0.6
5.3.3 Influence of locality on seroprevalence.
Small mammals from Mount Singai were 3.4 times (95% CI 1.04-11.17) more likely to have leptospiral antibodies than animals captured from the UNIMAS area (Table 5.3).
The prevalence in animals sampled from Matang and Kubah, Wind Cave and Fairy
Caves and Bako National Park were similar to that of UNIMAS (all OR 95% CI
110 included the value 1.0) (Table 5.3). Representatives of rats and squirrels & treeshrew from Matang and Kubah were more likely to be seropositive than representatives from
UNIMAS (OR 4.5; 95% CI 1.1, 19.1) (Table 5.8). All other locations had a similar seroprevalence to UNIMAS.
There was no significant difference among the seroprevalence in bats and the five different sampling sites (Table 5.9).
The squirrels and treeshrew were less likely to be seropositive than rats (OR 0.55:
95%CI 0.24, 0.99). Although the bats were less likely to be seropositive than animals from the order rats (0.48: 95% CI 0.22, 1.36), this difference was not significant.
Table 5.8: Seroprevalence to leptospires in mammals belonging to the order rats, squirrels and treeshrew from different localities.
Number Locality Seroprevalence (95% CI) OR (95% CI) Seropositive UNIMAS 8 40.0 (19.1, 63.9) 1.0
Mt. Singai 7 53.9 (25.1, 88.8) 2.1 (0.49, 9.0)
Wind & Fairy Caves 10 58.8 (32.9, 81.6) 2.14 (0.57, 7.9)
Matang & Kubah 12 75.0 (47.6, 92.7) 4.5 (1.06, 19.1)
Bako National Park 8 42.1 (20.3, 66.5) 1.1 (0.30, 3.9)
111 Table 5.9: Seroprevalence to leptospires in bats from different localities.
Number Locality Seroprevalence (95% CI) OR (95% CI) seropositive UNIMAS 6 30.0 (11.9, 54.3) 1.0
Mt. Singai 4 80.0 (28.4, 99.5) 9.3 (0.85, 10.9)
Wind & Fairy Caves 7 36.9 (16.3, 61.6) 1.4 (0.36, 5.2)
Matang & Kubah 5 35.7 (12.8, 64.9) 1.3 (0.30, 5.5)
Bako National Park 6 50.0 (21.1, 78.9) 2.3 (0.53, 10.3)
Table 5.10: Leptospira serovar PCR results from all positive kidneys of sampled small mammals.
Place Species Serovar Mt Singai Callosciurus notatus Pomona Sundamys muelleri Icterohaemorrhagiae UNIMAS Sundamys muelleri Lai Wind & Fairy Caves Callosciurus notatus Pomona Callosciurus notatus Pomona Maxomys rajah Icterohaemorrhagiae Maxomys rajah Icterohaemorrhagiae Sundamys muelleri Lai Callosciurus notatus Pomona Sundamys muelleri Icterohaemorrhagiae Matang & Kubah Sundamys muelleri Icterohaemorrhagiae Callosciurus notatus Pomona Bako National Park Callosciurus notatus Pomona Callosciurus notatus Pomona Callosciurus notatus Pomona Sundamys muelleri Icterohaemorrhagiae Callosciurus notatus Pomona
112 Of 155 kidney samples from individual animals only 17 were positive for
Leptospira on the molecular study (10.97%; 95% CI 6.5, 17) (Table 5.10). The
majority of the positive results were from plantain squirrels (53%; 95%CI 27.8, 77),
Müller’s rat (35%; 95%CI 14.2, 61.7) and brown spiny rats (12%; 95%CI 1.5, 36.4).
No other animals were positive on the PCR. Nine samples were positive to Pomona,
six to Icterohaemorrhagiae and two to serovar Lai (Table 5.10).
5.4 Discussion
All locations sampled in the current study were within 30 kilometres of Kuching, the
capital city of Sarawak, East Malaysia. Two of these, Matang-Kubah and Bako, are
situated within the confines of national parks while Mount Singai is a small
mountain located between Matang and Bau. UNIMAS is an educational facility
based in Kota Samarahan District. The Wind and Fairy Caves are located close to
the town of Bau. A common trend in most of the human leptospirosis outbreaks in
the Kuching region have been exposure to wildlife and contact with water
contaminated with leptospires. Bako National Park, Matang-Kubah Wildlife Centre,
Mount Singai and the Wind and Fairy Caves are known to contain several wildlife
species that may be responsible for transmitting leptospires. UNIMAS is located in
an area where the natural environment has been disturbed, similar to other areas of
Sarawak. Certain parts of the UNIMAS campus are also known to be waterlogged
and have patches of forest environment, increasing the possibility of exposure of
students at the campus to leptospires.
Studies have shown that rodents and bats are reservoirs for leptospirosis (Matthias et
al., 2005; Vashi et al., 2010), notably the pathogenic serovar Icterohaemorrhagiae
113 that causes fatalities in humans. These two wildlife groups are believed to be
potential sources of infection for humans, and contact with them is highly likely
when visiting the five sampled locations. The results indicate that small mammals
from Mount Singai were 3.4 times more likely to have leptospiral antibodies than
animals at UNIMAS. A greater proportion of rats were shown to be seropositive
than bats, squirrels and tree shrews, highlighting the importance of these species as
carriers of leptospires. These findings highlight potential risk for acquiring
leptospirosis to villagers living around Mount Singai, as well as tourists visiting the
mountain.
Deforestation, which is becoming commonplace across Malaysia (Aiken and Leigh,
1992; Jomo et al., 2004), can promote the emergence of infectious diseases, such as
leptospirosis, by placing humans into contact with novel reservoirs or infectious
agents (Arief, 2013; Matthias et al., 2005). Bats respond to the destruction of their
habitat at the level of populations and communities, making their spatial and
temporal dynamics particularly sensitive to anthropogenic activity (Calisher et al.,
2006). Matthias et al., (2005) detected Leptospira serovar Icterohaemorrhagiae in
one bat from the Peruvian Amazon region and proposed a rodent-bat cycle of
infection (Vashi et al., 2010), adding further support to the growing awareness of the
role played by bats in the transmission of this pathogen to humans and other
animals.
Forests in Southeast Asia, including Malaysia, are characterised by flowering and
fruit production, substantially contributing to the abundance of rodents (Nakagawa
et al., 2007). A ricefield rat captured by Mohamed-Hassan et al., (2012) at a military
training camp in West Malaysia was identified as a carrier of leptospires. Rats are
also reservoirs of a number of other parasites and infectious pathogens, including the
114 agent of plague, Yersinia pestis (Zahedi et al., 1984), and are key sources of infection for humans.
Culture of leptospires or a positive PCR result from kidney samples was considered to be indicative of a carrier status of this pathogen. In contrast animals positive on the MAT are not necessarily carriers as Leptospira-specific antibodies can be detected in convalescent sera and positivity to an MAT indicates past or current infection but not necessarily renal shedding (Faine et al., 1999). In the current study,
Leptospira serovar Pomona was most prevalent among the plantain squirrels, being detected in kidneys collected from animals from four out of the five regions sampled. Müller’s rats were identified as carriers of both serovars Lai and
Icterohaemorrhagiae, with the latter also found in brown spiny rats. Many small mammals also displayed high levels of antibodies to the newly discovered strain serovar Lepto 175 Sarawak, with plantain squirrels and Müller’s rats being more commonly affected with this serovar. Lepto 175 Sarawak was also found in a large number of dusky fruit bats tested. At present Lepto 175 Sarawak is best considered an intermediate strain, as its lethal capacity is unknown. In Chapter 3 Lepto 175
Sarawak was shown to have a close similarity to Leptospira wolffii, which has been isolated from Thailand, Iran and India (Slack et al., 2008; Zakeri et al., 2010).
Evidence of infection with Icterohaemorrhagiae, a well-known pathogen implicated in many fatal cases of leptospirosis in humans (Viriyakosol et al., 2006), was evident in this study being slightly less common than Australis and Autumnalis.
Pyrogenes appeared to be more common in bats while Autumnalis was found mainly in rodents. Rats tested positive for at least two known pathogenic serovars
(Icterohaemorrhagiae and Lai).
115 This particular study should generate concerns and lead to the health authorities
expanding disease control measures as there are significant levels of human activity
at all five locations where the animals were sampled. Visitors should be advised to
protect themselves and avoid direct contact with contaminated soil or water.
Wherever possible, pest control measures should be implemented to contain the rat
population, particularly considering that these rodents appear to be the main source
of infection in these locations.
Wildlife that is undergoing rehabilitation should be screened for Leptospira
periodically before their eventual re-introduction to the natural environment. This
helps prevent them from transmitting the disease to other species in the wild or to
humans involved in their release (Birtles, 2012).
The pathogenesis of serovar Lepto 175 Sarawak also needs to be monitored closely
considering its similarities to serogroup Wolffii. It remains to be seen if serovar
Lepto 175 Sarawak will be as virulent as Wolffii and hence extra vigilance is
required and the Malaysian health authorities need to implement disease control
measures to prevent the occurrence of a potential epidemic.
In the following chapter risk factors for infection in humans are investigated so that
risk factor modification can be implemented to reduce the number of cases of
leptospirosis in humans.
116 CHAPTER 6 : PREVALENCE AND RISK FACTORS OF
LEPTOSPIRA EXPOSURES IN HUMAN LIVING IN
FOREST BORDER AREAS AND DISTURBED JUNGLE
ENVIORNMENTS IN SARAWAK MALAYSIA.
6.1 Introduction
6.1.1 General Introduction
Determining the seroprevalence of Leptospira spp., especially the novel Leptospira
Lepto 175 Sarawak, is an important step in understanding its role in infection and
will largely influence the nature of future work on leptospirosis in Sarawak,
Malaysia. Across the world researchers have focussed on the more pathogenic
strains and their zoonotic significance, epidemiology, control and prevention;
however future studies on intermediate pathogenic strains are important to prevent
new disease outbreaks in communities. There are many technical challenges in
confirming the virulence factors of intermediate strains, although genotyping and
proteomic methods have been developed to investigate these factors (Ganoza et al.,
2006; Plank and Dean, 2000). The pathogenicity of a Leptospire serovar for humans
can only be established after it is isolated from a patient presenting with the clinical
symptoms of leptospirosis.
Leptospirosis is known worldwide for its harmful effects on both humans and
animals, resulting in morbidity and mortality and it is classed as a direct
anthropozoonosis (Sanders et al., 1999; Schwabe, 1969). Infection in domestic
animals can result in severe economic losses due to decreased milk production,
117 abortions, stillbirths and infertility, although fatal infection is rare (Sullivan, 1974).
Pathogenic leptospires thrive in the kidneys of mostly mammals and are excreted
through the urine into the environment, where they survive for up to several months
if conditions are favourable. When humans come into direct contact with infected
animals, their urine or urine-contaminated environments, there is the risk of
infection. The bacteria enter the body via open wounds or through the mucous
membranes. Symptoms of illness in humans are varied and easy to misdiagnose as
other diseases. In extreme cases, a fatal condition characterised by organ failure and
gross haemorrhage can result (Daher et al., 2003). In Malaysia, leptospirosis is a
disease that is subject to mandatory reporting on humnan due to its severity and the
difficulty in bringing it under control (Hakim, 2011). In Sarawak alone, the number
of cases rose almost fourfold from 49 in 2010 to 186 in 2011, as discussed in
Chapter 2. It is widely believed that bats and rodents may be responsible for the
spread of leptospirosis in Sarawak. With increased urbanisation and rapid
deforestation happening across the state, the possibility of people coming into
contact more frequently with wildlife becomes all the more inevitable. This can put
them at risk of contracting diseases such as leptospirosis. Furthermore, domestic
animals can be exposed to a large number of leptospiral serovars and may act as
vital maintenance hosts for the bacterium (Bahaman et al., 1988).
6.1.2 Risk factors for leptospirosis in humans
It is well established that leptospirosis is an occupational disease (Waitkins, 1986)
through contact with infected animals or urine-contaminated surfaces, such as soil or
water. Shafei et al., (2012) investigated municipal workers of Kota Bharu, the
capital of Kelantan state in northern Malaysia, and found that garbage collectors and
118 street cleaners had a high seroprevalence of leptospirosis. Earlier, Russ et al., (2003)
reported that a significant number of inhabitants of settlements surrounding Sabah’s
Crocker Range had been exposed to leptospiral antigens if they were involved in
agricultural activities or used water sourced from rivers for their household use.
Unsanitary living conditions that allowed rats to thrive also increased the risk of
leptospiral infection in humans, as did residing in flood-prone areas (Easton, 1999).
Poor knowledge of disease control can also be considered a potential risk factor for
infection of humans (Wiwanitkit and Wiwanitkit, 2006). Rafizah et al., (2013)
studied 999 febrile patients originating from 10 hospitals in north-eastern Malaysia
and found that those who were seropositive for leptospires (n=84) had all been
exposed to the organism through their occupation or through involvement in
recreational activities.
The study described in the current chapter was conducted to assess the extent of
exposure to leptospires in humans living on the fringes of forested areas and
disturbed jungle environments. The major objective of this study was to determine
which strains had led to antibody development in the sampled humans.
6.2 Materials and methods
A cross-sectional serological survey was conducted in four different locations
around Kuching, Sarawak, between January 2011 and March 2012. A total of 198
individuals over the age of 18 were sampled from four villages. The villages were
selected based on their location adjacent to areas where wildlife inhabited. A single
venous blood sample (5 mL) was collected from each participant. At the same time
119 as the blood was collected, a questionnaire survey was administered to gather
information about exposure, contact with animals, general knowledge of
leptospirosis and socio-economic status (Appendix 1). Households were randomly
selected for this study using the detailed information provided by the village chief of
the respective village. If the selected household member was not interested in
participating in the study the next household member was selected. A minimum of
10% of village people >18 years old were targeted for sampling from each village.
This research was approved by the Murdoch University Human Ethics Committee
(Permit number: 2010/240) and the Malaysian Research Ethics Committee (NMRR-
10-879-7153). A case was defined as a person who participated in the survey and
who was positive for anti-leptospiral antibodies by an IgG and IgM ELISA kit (CTK
Biotech cat: E0331) indicating current or past leptospiral infection. At the same time
the MAT was performed on all samples to identify the serovar responsible for the
infection. A titre ≥1:100 on the MAT was considered as evidence of recent or past
infection with Leptospira.
6.2.1 Study areas
Four locations were chosen for this particular study: Kampung (Kg) Sebayor, Kg
Etingan, Mount Singai and Wind and Fairy Caves (described in Chapter 5).
6.2.2 Study design
6.2.2.1 Blood collection
Blood samples (5 mL) were collected from the cephalic vein from apparently
healthy participants of either gender over the age of 18 years. Disposable needles
120 and vacuum tubes were used to collect blood from each participant by a qualified
Medical Laboratory Assistant (MLT) of the Universiti Malaysia Sarawak Medical
Faculty. Samples were maintained at 20°C for 6 hours to allow coagulation and then were centrifuged at 3,000 RPM for 1 minute to separate the sera. The serum was then removed and transferred to vials and stored at -70°C until testing was performed.
6.2.2.2 Information about potential risk factors (Questionnaire design)
The main objective of the questionnaire was to establish whether seropositivity to
Leptospira was associated with any risk factors (eg: age, sex, occupation and exposure to animals).
A structured questionnaire was developed, based on validated questionnaires performed by others, and used in this study (Kawaguchi et al., 2008; Sarkar et al.,
2002; Sasaki et al., 1993). The questions were categorised under six broad categories: general (age, sex, education and occupation); knowledge and symptoms of leptospirosis; exposure to animals; outdoor activities; potential sources of infection; and socio-economic status. All the questions, except for the source of drinking water and socio-economic status, were categorised as either a yes or no answer.
6.2.2.3 Questionnaire implementation
In each village, researchers contacted the household with the help of the village chief and requested permission for their participation in the study. On the day of blood collection a small introduction session was run to provide brief information
121 about the research and the disease. UNIMAS postgraduate students were selected
and trained to administer the questionnaire. After the blood was collected the
questionnaire was administered, which took approximately 20 to 30 minutes. All
participants completed a consent form to agree to be involved in the study
(Appendix 2).
6.2.3 Laboratory assays
6.2.3.1 MAT
Serum samples were tested by the MAT in the Leptospirosis Laboratory at the
Institute for Medical Research, Kuala Lumpur, Malaysia. The MAT was performed
using antigen from a collection of 17 live serovars of Leptospira that are common in
Malaysia (Table 4.1). The MAT procedure followed was as described in Chapter 4.
Initially sera were tested at a 1/100 dilution and the antigens that presented
agglutination at that dilution were then titrated with the serovar reagents using a
series of dilutions from 1/100 to 1/1600. Persons with a titre of 1:100 or more were
considered as positive (cases).
6.2.3.2 Enzyme linked Immunosorbent assay (ELISA)
The ELISA was performed at the IMR according to the standard procedure provided
by the CTK Biotech Data sheet (Cat No: E0331) to check for Leptospira-specific
antibodies, IgG and IgM.
122 6.2.3.2.1 Preparation of the reagents
All reagents and control samples were brought to room temperature (28°C). The concentrated washing buffer was then diluted approximately 30 fold with water using the following methods:
20 mL of concentrated washing buffer was added to 580 mL of water and the solution mixed well.
Next, the washing buffer was warmed to 37°C to dissolve the precipitates that had appeared.
Subsequently, each reagent was mixed well before adding to the test wells. In the final step, the number of microwells needed was determined and appropriate information was marked on the ELISA working sheet. Positive and negative controls were run in duplicate on each plate.
6.2.3.2.2 Assay procedure
The first procedure was removal of the desired number of strips, which were secured in the microwell frame. Specimens were then added according to the designation on the ELISA working sheet. No reagents were added to the blank well. For the control wells, 100 µL of positive or negative control were added into the designated wells.
For the test wells, 100 µL of sample diluent was added to each well along with 10
µL of the test specimen. Subsequently, the plates were gently rocked for 20 seconds and covered with sealant. The wells were then incubated at 37°C for 30 minutes. The incubation mixtures were carefully removed by emptying the solution into a waste container. Each well was then filled with diluted wash buffer and shaken gently for
123 20-30 seconds. The wash solution was then completely discarded by tapping the
plate on absorbent paper. The above procedure was repeated a further four times.
Then 100 µL of HRP-Protein-A conjugates was added to each well, except for the
blank well, the plate covered and incubated at 37°C for 20 minutes. The plate was
then washed five times as described previously. Next, 50 µL of TMB substrate A
and 50 µL of TMB substrate B were added to each well, including the blank well.
The wells were incubated at 37°C in the dark for 10 minutes. The reaction was
stopped by adding 50 µL of stop buffer to each well and gently mixing for 30
seconds. During the process, it was important to make sure that all blue colour
changed to yellow. Lastly, the absorbance (OD) of each well was measured in a
microplate reader at a wavelength of 450 nm against the blank well within 15
minutes after the addition of the Stop Solution. For optimization of the assay result,
a filter of 620-690 nm was used as a reference wavelength.
6.2.3.2.3 Interpretation of results
The cut-off value for a positive was set at 0.15 + N where N was the mean OD of
the negative control wells. If the mean OD of the negatives was less than 0.05 then
0.05 was used as the cut-off value. The OD ratio was calculated for each specimen
by dividing its OD value by the cut-off value.
The assay validations were carried out when the positive controls were ≥ 0.50 and
when the negative controls were ≤ 0.10. A positive result was assigned when the
specimen OD ratio was greater than 1.0 and a negative result if the OD ratio was
lesser than 0.1. A negative result indicated that there was no detectable IgG/IgM
antibody to L. interrogans in the specimen.
124
6.2.4 Statistical analyses
Data were entered into Microsoft Excel and statistical analyses were performed
using SPSS version 22. The seroprevalence on the MAT test and their 95%
confidence intervals (95%CI) were calculated. Categorical variables were
summarised using percentages and compared using the Chi-square test. A
multivariable logistic regression was performed to identify factors associated with
seropositivity to leptospires. Odd ratios (ORs) and their 95% confidence intervals
(CIs) were calculated for factors in the reduced subset model. Model building was
performed by considering variables that were statistically significant (p values
<0.20) in the univariable analyses, and including them in the preliminary
multivariate logistic regression model. Once the preliminary main effects model was
obtained (fully saturated model) a manual backward stepwise approach was used to
remove non-significant variables and only variables with p values<0.05 were
retained in the final model. Overall fit of the final logistic model was assessed with
the use of the Hosmer-Lemeshow goodness-of-fit statistic. To determine possible
interactions between the final set of factors interaction factors between each
remaining variable were added one at a time to determine the impact of the
interaction factor on the model. Findings were presented with Odds ratio (OR),
95%CI and p-value.
6.3 Results
Samples were collected from Kg. Sebayor (n=27), Kg. Etingan (n=40), Mt. Singai
(n=70) and Wind and Fairy Caves (n=61) for this study. Of the 198 participants, 126
125 (63.64%) were women and 72 (36.36%) men (Table 6.1). The majority of the
respondents were over the age of 55 years (Table 6.2).
Table 6.1: Gender distribution of humans sampled at the four localities.
Locality Gender Total Female Male Kg. Sebayor 17 (63.0%) 10 (36.0%) 27 Kg. Etingan 21 (52.5%) 19 (47.5%) 40 Mt. Singai 47 (67.1%) 23 (32.9%) 70 Wind & Fairy Caves 41 (67.2%) 20 (32.8%) 61 Total 126 (63.6%) 72 (36.4%) 198
Table 6.2: Age distribution of participants in the study.
Locality Age Group (years) 18-25 26-35 36-45 46-55 >55
Kg. Sebayor 0 6 3 6 12 Kg. Etingan 5 0 5 14 16 Mt. Singai 7 6 9 22 26 Wind &Fairy Caves 3 9 11 14 24
Total 15 21 28 56 78
6.3.1 Serological results
An overall seroprevalence of 35.9% (95%CI 29.2-43.0) was obtained on the MAT.
There was no significant difference (χ2=3.04; dƒ=3; P=0.38) in the seroprevalence
among the four sampled locations, with the highest seroprevalence (41%) (95%CI
126 28.6-54.3) obtained in people from the Wind and Fairy Caves. Individuals from
Mount Singai had a seroprevalence of 38.6% (95%CI 27.2-51.0) (Table 6.3).
People from the Wind and Fairy Caves were 2.08 (95% CI 0.87-5.02) times more
likely to have leptospiral antibodies than people residing in Kg. Etingan. Similarly
people from Mt. Singai were 1.88 (95% CI 0.80-4.46) times more likely to have
antibodies than people from Kg. Etingan. Kg. Sebayor had an odds of seropositivity
of 1.5 (95%CI 0.51-4.39) compared to Kg. Etingan.
Table 6.3: Seroprevalence (MAT) to leptospirosis in humans from different localities.
Locality Number Total Seroprevalence (%) 95% CI Positive Kg. Sebayor 9 27 33.3 16.5-54.0 Kg. Etingan 10 40 25.0 12.7-41.2 Mount Singai 27 70 38.6 27.2-51.0 Wind & Fairy Caves 25 61 41.0 28.6-54.3 Total 71 198 35.9 29.2-43.0
Based on the results from the ELISA 33.3% (95%CI 26.8-40.4) of samples were
seropositive to leptospires (Table 6.4). The highest percentage (41%; 95%CI 28.6-
54.3) was again in residents from the Wind and Fairy Caves area. Only 5 of 40
respondents from Kampung Etingan (12.5%; 95%CI 4.2-26.8) were seropositive
(Table 6.4). The seroprevalence in Kg Etingan was significantly less than that in Mt
Singai and the Wind and Fairy Caves (p < 0.05).
Table 6.4: Seroprevalence (ELISA) of leptospirosis in humans according to their locality.
127 Locality Number Total Seroprevalence (%) 95% CI Positive Kg. Sebayor 9 27 33.3 16.5-54.0 Kg. Etingan 5 40 12.5 4.2-26.8 Mt. Singai 27 70 38.6 27.2-51.0 Wind & Fairy Caves 25 61 41.0 28.6-54.3 Total 66 198 33.3 26.8-40.4
Table 6.5: Seroprevalence (MAT) to Lepto 175 Sarawak in humans according to their locality.
Locality Number Total Seroprevalence (%) 95% CI Positive Kg. Sebayor 9 27 33.3 16.5-54.0 Kg. Etingan 4 40 10.0 2.8-23.7 Mt. Singai 24 70 34.3 23.3-46.6 Wind & Fairy Cave 25 61 41.0 28.6-54.3 Total 62 198 31.3 24.9-38.3
Reactivity to serovar Lepto175 (Sarawak) was found in humans residing at all four
locations (Table 6.5). Most of the serum samples collected from people residing at
Kg. Sebayor, Mt. Singai and Wind &Fairy Caves that tested positive on the MAT
analysis (Table 6.3) were positive to serovar Lepto175 (Sarawak) (62/71, 87.3%)
(Table 6.5). Overall the seroprevalence to serovar Lepto175 (Sarawak) was
significantly different among locations (χ2=11.43; dƒ=3; P=0.010), being
significantly lower in Kg. Etingan than in the other locations (Table 6.5).
Antibodies to 17 different serovars were detected in the serum samples collected
from humans from the four locations sampled. Antibodies to serovar Lepto 175
Sarawak were most commonly detected being found in 62 (31.31%; 95%CI 24.9-
38.3) of all samples. Thirty-three samples (16.7%; 95%CI 11.8-22.6) showed
128 reactivity to Djasiman, 15.7% (95%CI 10.9-21.5) were positive to Autumnalis and a
similar percentage were positive to Canicola and 14.6% (95%CI 10.0-20.4) were
positive to Australis (Table 6.6). The antibody titres of the seropositive samples
varied from 1:100 to 1:400. More samples had a serum dilution of 1:100 (239)
compared to other dilutions (1:200 in 70 samples and 1:400 in 18 samples).
Table 6.6: Distribution of seropositive (MAT) reactions to Leptospira spp. in humans according to serovar and their respective titres.
Serovars 1:100 1:200 1:400 Total % (95%CI)
Lepto 175 42 17 3 62 31.3 (24.9-38.3)
Djasiman 28 4 1 33 16.7 (11.8-22.6)
Autumnalis 24 4 3 31 15.7 (10.9-21.5)
Canicola 23 8 - 31 15.7 (10.9-21.5)
Australis 18 9 2 29 14.6 (10.0-20.4)
Lai 13 5 3 21 10.6 (6.7-15.8)
Patoc 9 4 3 16 8.0 (4.7-12.8)
Grippotyphosa 15 - - 15 7.6 (4.3-12.2)
Hebdomadis 14 1 - 15 7.6 (4.3-12.2)
Javanica 13 2 - 15 7.6 (4.3-12.2)
Bataviae 9 2 - 11 5.6 (2.8-9.7)
Hardjo-bovis 10 - - 10 5.0 (2.4-9.1)
Pomona 3 7 - 10 5.0 (2.4-9.1)
Celledoni 7 1 - 8 1.8 (1.8-7.8)
129 Serovars 1:100 1:200 1:400 Total % (95%CI)
Icterohaemorrhagiae 3 4 1 8 1.8 (1.8-7.8)
Pyrogenes 5 1 1 7 1.4 (1.4-7.1)
Copenhageni 3 1 1 5 0.8 (0.8-5.8)
6.3.2 Descriptive epidemiology
6.3.2.1 Location
Seropositive humans were detected from all localities sampled (Section 6.3.1).
6.3.2.2 Gender
Males had a slightly higher seroprevalence (MAT) (38.4%; 95%CI 27.2-50.5) than
females (34.4%; 95%CI 26.1-43.4), although this difference was not significant (χ2=
0.31; dƒ=1; P=0.57; OR 1.12, 95%CI 0.61, 2.04).
6.3.2.3 Age
Overall the seroprevalence (MAT) varied between age groups (χ2= 9.38; dƒ=4;
P=0.052). Results were not significantly different but in regards to age as a rick
factor (P=0.052), while this is approaching significance. People in the 36-45 year
age group had the highest seroprevalence (53.6; 95%CI 33.9-72.5) followed by the
18-25 age group (46.7; 95%CI 21.3-73.4) (Table 6.7). People 34 to 45 years old
were 2.45 times (95%CI 1.01-5.91) more likely to have leptospiral antibodies than
those older than 55 years of age.
130 6.3.2.4 Educational status
The seroprevalences based on the results of the MAT were not significantly different among the different educational levels (χ2= 0.38; dƒ=3; P=0.95).
6.3.2.5 Occupation
Overall the seroprevalence (MAT) was not significantly different among the different occupations (χ2= 4.12; dƒ=3; P=0.25). However people who were working around the forest (54.5%; 95%CI 23.4-83.3) and national service participants (50%;
95%CI 27.2-72.8) had the highest seroprevalence (Table 6.7).
6.3.2.6 Symptoms of clinical disease
People with a fever were significantly more likely to be seropositive (OR 3.83,
95%CI 2.07-7.07; χ2= 19.2; dƒ=1; P=0.001) than those without fever. People who had been treated for fever were also significantly more likely to be seropositive than those not treated (OR 3.39, 95%CI 1.84-6.27; χ2= 7.52; dƒ=1; P=0.006).
Patients with skin wounds also had a significantly higher seroprevalence (58.8%;
95%CI 40.7-75.4) than those without skin wounds (31.1%; 95%CI 24.1-38.8) (OR
5; 95%CI 2.34-10.68; χ2= 9.41; dƒ=1; P=0.002) (Table 6.7).
6.3.2.7 Owning pets or farm animal contact
Owning a pet animal, such as a dog or cat, did not increase the likelihood of a seropositive reaction (χ2= 1.27; dƒ=1; P=0.25). In contrast contact with farm
131 animals (OR 2.2, 95%CI 1.22-3.99; χ2= 6.89; dƒ=1; P=0.009) increased the
probability of leptospiral infection (Table 6.7).
6.3.2.8 Presence of rats
The presence of rats in or near the participant’s residences (χ2= 60.6; dƒ=1;
P=0.001) or the sighting of rats more than 3 times within a day of the survey (χ2=
53.0; dƒ=1; P=0.001) increased the probability of seropositivity in the residents
(Table 6.7).
132
133 134
134
135 136
136 In the final multivariable logistic regression model the presence of skin wounds (OR
3.05), farm animals (OR 2.51) and rats (OR 11.2) were all significantly associated
with seropositivity. The logistic regression model for Leptospira antibodies
exhibited a good overall fit (Hosmer and Lemeshow Chi-square value 2.484,
P=0.648).
6.4 Discussion
In this study over one third of the sampled individuals were seropositive to
leptospires (35.9%; 95%CI 29.2-43.0). Other serological surveys of the general
population have reported a seroprevalence up to 25% (Poeppl et al., 2013). However
the current study targeted a population where it could be expected that the
seroprevalence was higher than the overall general population. Another targeted
study of residents in the Rajang Basin of Sarawak similarly reported an elevated
seroprevalence of 30.6% (Suut et al., 2011). Studies in other tropical countries,
including Barbados, have reported a seroprevalence of 29.8% in various
occupational groups and in 1987 a seroprevalence as high as 25% was reported in
patients hospitalised in Pakistan (Rao et al., 2003). From a global perspective a
seroprevalence that is this high is unusual, even in areas where leptospirosis is
rampant, although a seroprevalence of 30 to 50% has been reported in high risk
groups (Rao et al., 2003).
In the current study inhabitants residing in the vicinity of the Wind and Fairy Caves
had a higher, although not significant, risk of seroconversion to leptospires than
residents from the other surveyed locations. Wind and Fairy Caves are surrounded
by forest and residents would be expected to more likely to come into contact with
137 wildlife (rats, squirrels and bats) or leptospire-contaminated water or soil than
residents from urban areas. In Chapter 5 a high proportion of rats, squirrels and bats
from these regions were shown to have antibodies to leptospires. These could be the
result of a current infection but also could be the result of a previous infection as
titres can remain for up to 20 years (Blackmore et al., 1984).
In this study Serovar Lepto 175 was the dominant strain (31.3%) affecting people.
Although its virulence has not been confirmed and it is currently classified as an
intermediate strain, it has a high level of 16S rRNA gene sequence similarity to
Leptospira wolffii sv. Khorat strain Khorat-H2 (99.1%) (Chapter 3). Leptospira
wolffii has been linked to clinical leptospirosis and has been isolated from humans
and other animals in Thailand, India and Iran (Balamurugan et al., 2013; Zakeri et
al., 2010) and this serovar could potentially cause an outbreak in the near future.
Serovars Autumnalis (15.7%), Canicola (15.7%) and Lai (10.6%) were also
commonly found in this study with evidence of infection to most serovars present in
all four locations.
In this study the seroprevalence in males (37.5%; 95%CI 26.4-49.7) was slightly
higher than that in females (34.9%; 95%CI 26.6-43.9). This could be attributed to
the fact that most of the work done outside of the home environment in this region
was performed by men. Furthermore, jobs that revolve around the forest, such as
woodcutting and hunting, tend to be male-dominated positions because of the harsh
environment involved.
Although in the univariable analyses people between the ages of 36 and 45 and 18 to
25 years were at a greater likelihood of being seropositive compared to people older
than 55, age did not remain in the final multivariable logistic regression model. The
138 greater risk found in the univariable analysis for people between 18 and 25 years of age may be associated with this group be dominated by National Service
Participants and researchers who would be expected to have greater exposure to the forest and hence wild animals than other groups.
Of 90 respondents who reported to have suffered from a fever within three months of sampling, approximately half (52.2%; 95%CI41.4-62.9) were seropositive to leptospires. Similarly just under half (49.23%) of those who received treatment for fever were also diagnosed with leptospirosis, while 20 of 34 (58.82%; 95%CI 40.7-
75.4) who had skin wounds were seropositive. This supports the importance of infection entering via skin lesions and the presence of fever as a common symptom of leptospiral infection (Bovet et al., 1999; Sethi et al., 2010). Individuals with wounds have the potential to be infected if they are involved in activities likely to expose them to leptospires such as through agricultural pursuits, forestry businesses or wildlife and rodent interactions. In a previous case control study in the
Seychelles, leptospirosis was positively associated with activities in forests, which may be related to an increase in environmental exposure to the pathogen (Bovet et al., 1999).
The presence of domesticated pets did not increase the risk of infection significantly in this study. In contrast exposure to farm animals and rodents presented a major risk of infection. These findings highlight the role of peri-urban rats as the principal source of leptospiral transmission as reported by others (Collares-Pereira et al.,
2000; Felt et al., 2011; Matthias et al., 2008; Szyfres, 1976). However, surprisingly rodent-associated serovars, such as Icterohaemorrhagiae (1.8% positive) and
Copenhageni (0.8%), did not appear to be of major importance in this study. In
139 contrast, Canicola (15.7%), which is mostly carried by dogs (Schreiber et al., 2005),
presented itself as a major threat in this study.
Overseas travel, involvement in water-related activities and camping around forests
(data not shown) were not linked to seropositivity, however the number of residents
engaging in these pursuits was small. As has been documented in previous studies, a
number of outbreaks in Borneo and around the world have occurred after patients
have been exposed to jungle environments or been involved in aquatic sports
(Ashford et al., 2000; Barcellos and Sabroza, 2001; Hathaway and Blackmore, 1981;
Meng et al., 2009).
The key findings of this study indicate that residents of forested areas having skin
wounds, contact with farm animals and the presence of rats are at a greater risk of
leptospirosis. This is further compounded if the subjects work with animals or
depend on the forest for their livelihood. Skin wounds appear to be the primary
mode of infection, with fever as the main symptom, meaning anyone with skin-cuts
who complains of subsequent fever should be considered as a potential case of
leptospirosis. This research also identified the potential role of rats as a source of
leptospiral infection for residents, highlighting the need to make rodent control a
priority in disease management. In the multivariable logistic model the findings that
owning farm animals or the presence of rats in the vicinity of the participant’s
house, together with the results of other studies (Alonso-Andicoberry et al., 2001;
Waitkins, 1986), highlight the importance of these species in leptospiral infection of
humans. In contrast, as water-related activities did not increase the risk of
seropositivity, it suggests that the local waterways may not be contaminated with
leptospires at a level likely to cause infection in humans.
140
6.5 Potential causes of bias and confounding factors in the study
This study was designed to gain information on the seroprevalence to leptospires
from a population living and working around wildlife inhabitants. The population
was biased, as individuals younger than 18 years of age were not sampled, which
may have resulted in overestimation of the seroprevalence by targeting individuals
more likely to have exposure to the pathogen through occupational exposure. It is
possible that there was also a degree of selection bias with participants who were
more concerned about leptospirosis, fever or rats being more likely to participate. It
was not possible to quantify the impact of this potential selection bias.
6.6 Conclusions
Although the findings of this study indicate that the intermediate strain Lepto 175
Sarawak might be responsible for asymptomatic infections in Sarawak, there is a
need for increased awareness of the risks associated with living close to places
where wildlife inhabit and measures should be developed to minimise exposure to
these animals. The presence of rodents appears to be a strong risk factor in this
instance, and it would be pertinent to keep the population of these animals under
control in urban and peri-urban areas. While pets may currently not pose a major
threat to the health of local villagers, these animals may become hosts of leptospires
acquired from peri-urban wildlife such as rats and bats.
141 6.7 Future research
Information obtained from this study should be used in the future to design more
focused and specific research. Further blood sampling, serology and isolation of the
strains from the community and hospital based leptospirosis confirmed cases should
be performed to determine whether the infections are current or indicative of
previous infection. By doing so, it is also possible to establish, albeit roughly, which
particular strains are consistently active in the vicinity. Ideally a prospective cohort
study should be conducted with the screening of participants over a period of five to
ten years to determine the incidence of infection. If possible, children less than 18
years should be included in a future study as they sometimes accompany their
parents into the forest and Everard et al., (1989) suggested that children as young as
seven may be infected with leptospirosis. A prospective study should also include a
control group from the same villages so as to enable more thorough epidemiological
analyses to be conducted.
142 CHAPTER 7 : GENERAL DISCUSSION AND
CONCLUSIONS
7.1 Introduction
The focus of this study was to determine the seroprevalence of leptospirosis in the
community living around several wildlife habitats and in small wildlife (mammals)
located in these wildlife reserves. The impetus for this work came from anecdotal
evidence of outbreaks of leptospirosis in and around wildlife reserves in Malaysia
and a high seroprevalence to Leptospira in humans and wildlife as discussed in
Chapter 2.
Leptospirosis is a major zoonosis that has serious effects on the livestock industry,
as well as implications for public health (Bharti et al., 2003). It is endemic in
tropical regions and has been associated with the monsoonal seasons and flooding.
With the recent growth in eco-tourism, it is pertinent to identify those species of
wildlife which may act as potential carriers of the pathogen and to develop strategies
to minimise contact between human communities and these wildlife. In Malaysia,
there is a potential for an outbreak in Sarawak due to the risk factors prevalent there.
However the available data on the transmission of leptospirosis between wild
animals and humans is limited (Caley and Ramsey, 2001), despite the significance
of the disease in humans. This thesis was designed to further the current knowledge
on the likely transmission of leptospires between small wildlife and primates and
people who were living in or were associated with wildlife habitats. To investigate
143 this aim, the focus of the thesis was to survey rodents, bats and primates, including
humans, to quantify the seroprevalence of different leptospiral serovars.
7.2 Discussion
7.2.1 General Aims and Chapter Summaries
This thesis is made up of seven parts. The opening chapter and the second chapter
provided an overview of the current situation of leptospirosis in the world and gave
a brief history of the international research that had been conducted to combat this
disease and also explored the link between leptospirosis and eco-tourism in
Malaysia, and discussed the possible economic impacts of the disease.
The following chapter discussed the novel strain of Leptospira, Serovar 175
Sarawak. This new serovar was found to be genetically similar to Leptospira wolffii
and was classified as an intermediate leptospire, although its pathogenicity has yet to
be confirmed. In Malaysia, several other researchers have isolated and identified
serovars of Leptospira from humans and animals (El Jalii and Bahaman, 2004).
During the 1950’s a group of researchers successfully isolated leptospiral serovars
Hebdomadis, Grippotyphosa and Canicola from three species of Malaysian rodents
and a serogroup of Icterohaemorrhagiae from Rattus whiteheadi from Sabah
(Wisseman et al., 1955). In 2004, researchers discovered a new pathogenic serovar,
Lai, from a patient who had returned from Langkawi Island in West Malaysia
(Wagenaar et al., 2004). As late as 2009, a new serovar, L. kmetyi Bejo-Iso 9, was
isolated from a soil sample in the southern state of Johor (Slack et al., 2009).
In Chapter 4 the tests performed on a small number of captive and free-ranging
primate species found in the region surrounding Kuching, Sarawak were described.
144 Few researchers had previously studied Asian primates, although anti-leptospiral agglutinins had been reported in various primate species conducted in other countries (Bengis et al., 2004; Cantu et al., 2008; Cole and Landres, 1995; Diesch et al., 1970; Hathaway et al., 1981; Kilbourn et al., 2003). Studies by Kilbourn et al.,
(2003) on free-ranging orangutans in Sabah found a high prevalence of antibodies against L. interrogans serovars, notably Grippotyphosa and Autumnalis, as well as
Leptospira wolffii. In the current work a positive titre of Lepto 175 Sarawak was demonstrated in a proboscis monkey – the first such report in this species.
The results of screening from various small mammal species from the same area for leptospiral antibodies were reported in Chapter 5. The animals screened included rats, squirrels and bats, which have often been implicated as reservoir hosts for the disease.
The strong presence of Lepto 175 Sarawak in many of these animals was confirmed.
A higher seroprevalence of Leptospira in rats and bats was found in this research, confirming the results of other researchers (Cox et al., 2005; Matthias et al., 2005;
Smythe et al., 2002a; Tulsiani et al., 2011).
The focus of Chapter 6 shifted to the human population residing either in forested areas or in established communities within the vicinity. The results highlighted the presence of antibodies to Lepto 175 Sarawak in the residents and it was hypothesised that rats were most likely responsible for the spread of this serovar. Studies carried out in Sabah Malaysia, reported a high seroprevalence (25.75%) in people living within the periphery of a national park, presumably due to exposure/contact with wild mammals (Russ et al., 2003). Recently researchers from Universiti Malaysia Sarawak
(UNIMAS) and the Sarawak Health Department have conducted research in the
Rejang Basin area, Sarawak reporting that 31% of humans sampled were seropositive
145 for leptospirosis and infection was associated with farming and/or water activities
(Stoye, 2012; Suut et al., 2011).
7.2.2 Epidemiology
Leptospires are known to infect various mammals and colonise their kidneys (Vashi
et al., 2010) with rats and bats appearing to be the main reservoir hosts for the
disease shedding leptospires into the environment via their urine. Some mammals,
however, have been shown to be susceptible to specific serovars (Faine, 1962a). As
the main routes of infection are through mucous membranes, abraded skin or the
genital tract, infection usually results from contact with urine from an infected
animal or leptospire-contaminated soil and water (Faine, 1994). In this study in at-
risk communities in Sarawak, it was found that many patients had cuts on their skin
highlighting this route of infection. Furthermore many were identified to have had
received treatment for fever. One of the major clinical symptoms of leptospirosis is
fever, and the non-specific nature of this symptom is one reason why the disease can
be difficult to diagnose. Blood (serum) needs to be collected from patients suspected
of having leptospirosis and tested using the MAT and ELISA. In Malaysia, only the
Institute of Medical Research has the facilities for performing the MAT due to this
test’s laborious nature. In contrast ELISAs can be performed at most major hospitals
and it is recommended that diagnostic facilities for leptospirosis be expanded in
Malaysia, particularly in higher risk areas such as Sarawak. Cross-referencing of
results from the MAT and ELISA are needed to determine the infecting serovar of
Leptospira, which in turn can help identify the potential reservoir host for the
bacterium.
146 Several measures can be adopted to restrict the spread of leptospirosis, with pest control being one of the most important (Mohamed-Hassan et al., 2012). As rats are critical in the spread of the disease (Collares-Pereira et al., 2000), it is vital that communities be educated about the role played by these animals. In areas of human settlement, rats are often seen close to rubbish or food waste. This poses an occupational risk for cleaners and garbage collectors. Reducing the rat population and stopping indiscriminate dumping is one way to minimise the risk of leptospirosis. Poor pest management was reported to be a key factor in the 2012 leptospirosis outbreak in Sibu city, Sarawak (Thayaparan et al., 2013b).
Contact with bats should also be avoided as these have been shown in this study, and in the studies of others (Tulsiani, 2010), to be carriers of leptospires. Most bats live in jungles and seldom come into contact with people, but with increased deforestation and the rise of eco-tourism, humans are increasingly likely to have contact with them. With land clearing bats can now be found on the fringes of human settlements with some roosting in buildings. Herbivorous bats are also commonly associated with fruit trees in people’s homes. The bat’ urine contaminates the ground, which allows leptospires to pass into the bodies of other animals, such as rats, perpetuating a sylvatic cycle of infection (Tulsiani, 2010).
The Malaysian government has implemented restrictions on bat hunting (Epstein et al., 2009), making it difficult for bats to be culled as a pre-emptive measure for control of the disease. However the culling of such mammals, although potentially beneficial for restricting the spread of leptospires to humans, would impact negatively on the ecosystem (Chivian, 2002; Horan et al., 2010).
147 The rise of ecotourism in East Malaysia has increased the potential for tourists to
have direct contact with wildlife infected with leptospires or soil and groundwater
contaminated with the bacterium. In the current study eight of 12 captive and free-
ranging primates at Matang Wildlife Centre and Bako National Park were
seropositive to leptospires and could be potential sources of infection for visitors to
these establishments. Quite often, the symptoms of leptospirosis manifest after the
tourists have returned to their country due to the incubation period (4-14 days)
(Mortimer, 2005; Reisberg et al., 1997). As leptospirosis is not really well-
understood by many western physicians, misdiagnosis may result and inappropriate
treatment be prescribed.
Wildlife species that are undergoing rehabilitation should also be screened annually
for leptospires to avoid introducing the disease into the wild populations when they
are eventually released.
7.2.3 Leptospirosis status in Sarawak
Leptospirosis is subjected to mandatory reporting in Sarawak due to its pathogenic
nature. In fact, almost all states in Malaysia have had problems keeping leptospirosis
in check (Hakim, 2011). Figures obtained from the country’s health department
show a recent increase in the number of confirmed cases of leptospirosis in Sarawak,
as well as a high case fatality rate (Hakim, 2011) and several well-documented
outbreaks have been reported (Thayaparan et al., 2013b). Access to better diagnostic
methods may help to reduce the case fatality rate of leptospirosis in Malaysia,
however conversely it may also result in increased reporting. The recent increase in
the number of cases in Sarawak could be due to the implementation of mandatory
reporting for the disease in 2010 (Hakim, 2011) as well as increased awareness by
medical practitioners, rather than a real increase in the incidence of disease. With the
148 discovery of the new serovar Lepto 175 Sarawak in a sample of water collected from
Sarawak, there will be increased pressure on the State health authorities to control the disease. Although, serovar Lepto 175 Sarawak is currently considered to be an intermediate strain, its characteristics are similar to that of Leptospira wolffii, which has been identified as the main cause of recent outbreaks in the neighbouring country of Thailand as well as in India and Iran (Slack et al., 2008; Zakeri et al.,
2010).
7.2.4 Risk factors in the spread of leptospirosis
Leptospirosis is often regarded as an occupational disease. Previous research has identified farmers, meat industry workers, cleaners and veterinarians as being at high-risk of acquiring the disease due to the nature of their work (Waitkins, 1986).
In recent years the popularity of adventure travel and eco-tourism in Asia has meant that Western tourists are now considered to also have a higher risk of acquiring leptospirosis. Poor knowledge about disease, control measures and prevention could also be considered a factor in cases of human infection (Waitkins, 1986).
Communities with poor sanitation have also been shown to have a higher risk of infection, primarily due to the presence of rodents (Costa et al., 2014). In the current study in Sarawak, a high seroprevalence was observed in communities living near forested areas (Chapter 6) and the presence of livestock or rats near the participant’s property increased the likelihood of infection. The seroprevalence was higher in males than in females and this is likely to be associated with occupation and personal hygiene habits rather than an innate higher susceptibility in males (Ashford et al., 2000; Ciceroni et al., 2000; Kawaguchi et al., 2008).
149 7.3 Need for further studies
This research has uncovered evidence of a high leptospiral seroprevalence in
proboscis monkeys (Chapter 4), although only few animals were sampled. This is
despite their arboreal lifestyle and the fact that they only feed on vegetation. Other
primates, such as macaques, were found to have high titres to the novel serovar
Lepto 175 Sarawak. It is known that macaques are scavengers and may have been
exposed to the bacterium after rummaging through leftovers and garbage. Further
study as to whether macaques may be perpetuating a sylvatic cycle of infection
needs to be undertaken. The possibility of non-human primates acting as reservoir
hosts also needs to be explored.
In Chapter 6, the survey of communities living near forested areas has produced
some interesting, albeit inconclusive results. It seems likely that many of the people
surveyed may have acquired the infection several years ago and a prospective cohort
study is required to determine the incidence of infection. Follow-up studies should
also determine which serovars induce antibodies that remain the longest in humans
to help confirm the extent of leptospirosis in humans residing in Sarawak.
7.4 Conclusions
This study has highlighted that leptospires are a potential threat to public health in
Sarawak, and a number of risk factors associated with human infection were
identified.
Educating local communities about the disease and methods to control it,
particularly the role of rodents in the disease, is critical. More also needs to be done
150 to prevent exposure to wildlife so as to reduce the infection rates in these animals.
At present, there is a significant potential for a major outbreak of leptospirosis on the outskirts of Kuching and measures should be adopted to minimise this risk.
151
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168 Sehgal, S., Vijayachari, P., Sugunan, A., Umapathi, T., 2003, Field application of Lepto lateral flow for rapid diagnosis of leptospirosis. Journal of Medical Microbiology 52, 897-901. Sejvar, J., Bancroft, E., Winthrop, K., Bettinger, J., Bajani, M., Bragg, S., Shutt, K., Kaiser, R., Marano, N., Popovic, T., 2003, Leptospirosis in “eco-challenge” athletes, Malaysian Borneo, 2000. Emerging Infectious Diseases 9, 702. Sekhar, W., Soo, E., Gopalakrishnan, V., Devi, S., 2000, Leptospirosis in Kuala Lumpur and the comparative evaluation oftwo rapid commercial diagnostic kits against the MAT Test for the detection of antibodies to Leptospira interrogans. Singapore Medical Journal 41, 370-375. Self, C., Iskrzynska, W., Waitkins, S., Whicher, J., Whicher, J., 1987, Leptospirosis among British cavers. Cave Science 14, 131-134. Sethi, S., Sharma, N., Kakkar, N., Taneja, J., Chatterjee, S.S., Banga, S.S., Sharma, M., 2010, Increasing trends of leptospirosis in northern India: A clinico- epidemiological study. PLoS neglected tropical diseases 4, e579. Shafei, M.N., Sulong, M.R., Yaacob, N.A., Hassan, H., Wan Mohd Zahiruddin, W., Daud, A., Ismail, Z., Abdullah, M.R., 2012, Seroprevalence of leptospirosis among town service workers on northeastern State of Malaysia. International Journal of Collaborative Research on Internal Medicine & Public Health 4, 395- 403. Shapiro, J., Prescott, J., Henry, G., 1999, Equine abortions in eastern Ontario due to leptospirosis. The Canadian Veterinary Journal 40, 350. Sibon, P. 2011. No mystery in Deaths. In The Borneo Post. Simmons, N.B., 2005, An Eocene big bang for bats. Science 307, 527-528. Slack, A.T., Kalambaheti, T., Symonds, M.L., Dohnt, M.F., Galloway, R.L., Steigerwalt, A.G., Chaicumpa, W., Bunyaraksyotin, G., Craig, S., Harrower, B.J., 2008, Leptospira wolffii sp. nov., isolated from a human with suspected leptospirosis in Thailand. International Journal of Systematic and Evolutionary Microbiology 58, 2305-2308. Slack, A.T., Khairani-Bejo, S., Symonds, M.L., Dohnt, M.F., Galloway, R.L., Steigerwalt, A.G., Bahaman, A.R., Craig, S., Harrower, B.J., Smythe, L.D., 2009, Leptospira kmetyi sp. nov., isolated from an environmental source in Malaysia. International Journal of Systematic and Evolutionary Microbiology 59, 705-708. Slack, A.T., Symonds, M.L., Dohnt, M.F., Smythe, L.D., 2006, Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene. BMC Microbiology 6, 95. Slavica, A., Cvetnić, Ž ., Milas, Z., Janicki, Z., Turk, N., Konjević, D., Severin, K., Tončić, J., Lipej, Z., 2008, Incidence of leptospiral antibodies in different game species over a 10-year period (1996–2005) in Croatia. European Journal of Wildlife Research 54, 305-311.
169 Smith, C., Ketterer, P., McGowan, M., Corney, B., 1994, A review of laboratory techniques and their use in the diagnosis of Leptospira interrogans serovar Hardjo infection in cattle. Australian Veterinary Journal 71, 290-294. Smith, C., Turner, L., Harrison, J., Broom, J., 1961, Animal leptospirosis in Malaya: 1. Methods, zoogeographical background, and broad analysis of results. Bulletin of the World Health Organization 24, 5. Smith, C.E.G., Turner, L., 1961, The effect of pH on the survival of leptospires in water. Bulletin of the World Health Organization 24, 35. Smythe, L., Dohnt, M., Symonds, M., Bamett, L., Moore, M., Brookes, D., Vallanjon, M., 2000, Review of leptospirosis notifications in Queensland and Australia: January 1998-June 1999. Communicable Diseases Intelligence 24, 153-156. Smythe, L., Field, H., Barnett, L., Smith, C., Dohnt, M., Symonds, M., Moore, M., Rolfe, P., 2002a, Leptospiral antibodies in flying foxes in Australia. Journal of Wildlife Diseases 38, 182. Smythe, L., Smith, I., Smith, G., Dohnt, M., Symonds, M., Barnett, L., McKay, D., 2002b, A quantitative PCR(TaqMan) assay for pathogenic Leptospira spp. BMC Infectious Diseases 2, 13. Srivastava, S., 2006, Prospects of developing leptospiral vaccines for animals. Indian Journal of Medical Microbiology 24, 331. Stasilevich, Z., Dzhikidze, E., Anisimova, T., Krylova, R., 2000, Serological investigations for leptospirosis in monkeys of the Adler monkey colony. Baltic Journal of Laboratory Animal Science 10, 33-38. Stoye, E., 2012, Leptospirosis in Rajang Basin. Sceince & Technology, Society & Culture. Asiaresearchnews.com/leptospirosis-in-the-rejang-basin, Accessed on 18th May 2012. Sullivan, N., 1974, Leptospirosis in animals and man. Australian Veterinary Journal 50, 216-223. Suut, L., Yusof, H., Arif, M.T., Rahim, N.A.A., Katip, J.T., Suhaili, M.R., 2011, The Prevalence of Leptospirosis in the Rejang Basin of Sarawak. Research update 7(2), 1-5.
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170 evolutionary distance, and maximum parsimony methods. Molecular biology and evolution 28, 2731-2739. Tan, D., 1964, The Importance of Leptospirosis in Malaya. The Medical Journal of Malaysia 18, 164. Taylor, M., Ellis, W., Montgomery, J., Yan, K.-T., McDowell, S., Mackie, D., 1997, Magnetic immuno capture PCR assay (MIPA): Detection of Leptospira borgpetersenii serovar Hardjo. Veterinary Microbiology 56, 135-145. Thaipadungpanit, J., Wuthiekanun, V., Chierakul, W., Smythe, L.D., Petkanchanapong, W., Limpaiboon, R., Apiwatanaporn, A., Slack, A.T., Suputtamongkol, Y., White, N.J., 2007, A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand. PLoS Neglected Tropical Diseases 1, e56. Thayaparan, S., Robertson, I., Amraan, F., Su'ut, L., Abdullah, M.T., 2013a, Serological Prevalence of Leptospiral Infection in Wildlife in Sarawak, Malaysia. Borneo Journal of Resource Science and Technology 2, 79-82. Thayaparan, S., Robertson, I., Fairuz, A., Suut, L., Abdullah, M., 2013b, Leptospirosis, an emerging zoonotic disease in Malaysia. The Malaysian Journal of Pathology 35, 123-132. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997, The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25, 4876- 4882. Treml, F., Pejcoch, M., Holesovska, Z., 2002, Small mammals-natural reservoir of pathogenic leptospires. Veterinarni Medicina-Praha- 47, 309-314. Trueba, G., Zapata, S., Madrid, K., Cullen, P., Haake, D., 2004, Cell aggregation: a mechanism of pathogenic Leptospira to survive in fresh water. International Microbiology 7, 35-40. Tsai, C., Fresh, J., 1975, Leptospira canalzonae in man: A case report. The Southeast Asian journal of Tropical Medicine and Public Health 6, 133-135. Tulsiani, S., 2010, Transmission of Emerging Pathogenic Leptospira Species from Fruit Bats in Northern Australia. PhD Thesis, University of Queensland www. espace.library.uq.edu.au. Tulsiani, S., Cobbold, R., Graham, G., Dohnt, M., Burns, M.A., Leung, L.K.P., Field, H., Smythe, L., Craig, S., 2011, The role of fruit bats in the transmission of pathogenic leptospires in Australia. Annals of Tropical Medicine and Parasitology 105, 71-84. Turner, L., 1970, Leptospirosis III: Maintenance, isolation and demonstration of leptospires. Transactions of the Royal Society of Tropical Medicine and Hygiene 64, 623-646. Turner, L., 1973, A new look at infectious diseases. Leptospirosis. British Medical Journal 1, 537-540.
171 Twigg, G., Cuerden, C., Hughes, D., 1969. Leptospirosis in British wild mammals. In Symposia of the Zoological Society of London 24, 75-98. Van Crevel, R., Speelman, P., Gravekamp, C., Terpstra, W.J., 1994, Leptospirosis in travelers. Clinical Infectious Diseases 19, 132-134. Vashi, N., Reddy, P., Wayne, D., Sabin, B., 2010, Bat-Associated Leptospirosis. Journal of General Internal Medicine 25, 162-164. Victoriano, A., Smythe, L., Gloriani-Barzaga, N., Cavinta, L., Kasai, T., Limpakarnjanarat, K., Ong, B., Gongal, G., Hall, J., Coulombe, C., 2009, Leptospirosis in the Asia Pacific region. BMC Infectious Diseases 9, 147. Vijayachari, P., Sugunan, A., Sehgal, S., 2002, Evaluation of Lepto Dri Dot as a rapid test for the diagnosis of leptospirosis. Epidemiology and Infection 129, 617-621. Vijayachari, P., Sugunan, A., Shriram, A., 2008, Leptospirosis: an emerging global public health problem. Journal of Biosciences 33, 557-569. Vinh, T., Adler, B., Faine, S., 1986, Ultrastructure and chemical composition of lipopolysaccharide extracted from Leptospira interrogans serovar Copenhageni. Journal of General Microbiology 132, 103-109. Viriyakosol, S., Matthias, M.A., Swancutt, M.A., Kirkland, T.N., Vinetz, J.M., 2006, Toll-like receptor 4 protects against lethal Leptospira interrogans serovar Icterohaemorrhagiae infection and contributes to in vivo control of leptospiral burden. Infection and Immunity 74, 887-895. Voronina, O.L., Kunda, M.S., Aksenova, E.I., Ryzhova, N.N., Semenov, A.N., Petrov, E.M., Didenko, L.V., Lunin, V.G., Ananyina, Y.V., Gintsburg, A.L., 2014, The Characteristics of Ubiquitous and Unique Leptospira Strains from the Collection of Russian Centre for Leptospirosis. BioMedical Research International, 1-15. Wagenaar, J.F., Vries, P.J., Rudy Hartskeerl, A., 2004, Leptospirosis with pulmonary hemorrhage, caused by a new strain of serovar Lai: Langkawi. Journal of Travel Medicine 11, 379-382. Wai'in, P.M., 2007. Epidemiology of infection with Leptospira species in livestock in Papua New Guinea. Murdoch University, Waitkins, S., 1986, Leptospirosis as an occupational disease. British Journal of Industrial Medicine 43, 721. Webster, J., Ellis, W., Macdonald, D., 2009, Prevalence of Leptospira spp. in wild brown rats (Rattus norvegicus) on UK farms. Epidemiology and Infection 114, 195-201. Whittaker, R.J., Jones, S.H., 1994, The role of frugivorous bats and birds in the rebuilding of a tropical forest ecosystem, Krakatau, Indonesia. Journal of Biogeography, 245-258. Wisseman, C., Traub, R., Gochenour Jr, W., Smadel, J., Lancaster, W., 1955, Leptospirosis of man and animals in urban, rural and jungle areas of southeast Asia. The American Journal of Tropical Medicine and Hygiene 4, 29.
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173
174 APPENDIX 1- Leptospirosis Risk Assessment Survey in
Malaysian Patients and Participants Questionnaire
(Confidential Data)
Instructions: Please tick or circle the most appropriate options and/or fill in the necessary information as required
A. Interviewer details i. Interviewer’s Name
:…………………………………………………………. ii. Mobile # :………………………………………………………….
B. Respondent’s details i. Participant/Patient
:…………………………………………………………. ii. Ethnic group: iii. City: :…………………………………………………………………. iv. State /Sub district
:…………………………………………………. vi. Contact details: ………………………………………………….
1. Participant/Patient Sex:
a. Female b. Male
2. Participant/Patient Age
175 a. 18-25 b. 26- 35 c. 36-45 d. 46-55 e. > 56
3. What level of education did you complete?
a. No schooling
b. Primary school
c. Secondary school
d. Tertiary education
4. What is your main occupation?
a. Farmer
b. Wildlife researcher/ work around forest
c. Veterinarian
d. Military personnel/ National service participant
e. Cleaner
Others please specify……………………….
SYMPTOMS AND SIGNS OF LEPTOSPIRAL INFECTION
5. Have you had a fever within the last 3 months?
a. Yes b. No
If yes which hospital/clinic did you visit? …………………………………
6. Do you have/had any skin wounds on your hands or legs within the last 3
months
176 a. Yes b. No
7. Have you received any treatment for leptospirosis in the past year: a. Yes b. No
If yes please provide the name of the hospital/clinic…………………………...
EXPOSURE TO ANIMALS OR PETS
8. Do you have any pet animal/s at home? a. Yes b. No
If yes what type of pets do you have? ……..………………………….
9. Do you have any farm animal/s at home/farm? a. Yes b. No
If yes what type of farm animal/s? ……………………………………..
10. Do you have a problem with rats around your house/farm? a. Yes b. No
11. Have you seen rats around your house or farm more than three times in one day? a. Yes b. No
OUTDOOR ACTIVITIES AND POTENTIAL EXPOSURE TO
LEPTOSPIRA
177 12. Have you traveled overseas within the last month?
a. Yes c. No
If yes which country/countries did you visit?………………………………...
13. Have you undertaken any water sports (eco-challenge activities like boating,
canoeing, running and cycling along stagnated water areas) within the last
three months?
a. Yes c. No
If yes what sport? …………………………………………………...
In which area? ……………………………………………………
14. Have you been camping within the last three months?
a. Yes b. No
If yes in which area? ……………………………………………………
15. Have you been swimming in a lake within the last 3 months?
a. Yes b. No
If yes in which lake/area? ……………………………………………
16. Have you done any water rafting activities in the last 3 months?
a. Yes b. No
If yes in which area? ……………………………………………………
POTENTIAL SOURCE OF INFECTION
178 17. What is your usual source of drinking water? a. Stream water b. Tube well c. Municipal chlorinated tap water d. Rainwater e. Other please specify………………………………………………..
18. Do you usually drink treated/boiled water? a. Yes b. No
Thank you for completing this survey. Leptospirosis is a zoonotic disease found in wildlife, domestic animals and humans. This disease is caused by a bacterium called a Leptospira. With the rise of ecotourism and related activities, recreational exposure to leptospira has emerged as a concern to global health. This research is attempting to identify those risk factors that can lead to infection of humans with leptospirosis in Malaysia. Your participation is greatly appreciated to allow this research to be completed successfully.
179
180 APPENDIX 2-Consent Form - Sero-epidemiological Study Of
Leptospirosis In Communities In Sarawak, Malaysia.
Leptospirosis is a disease found in wildlife, domestic animals and also humans. It is caused by a type of bacteria or germ called a Leptospira. This disease is believed to be common in Malaysia but little work has been done in this country to identify factors that lead to the disease. This research is designed to confirm if there is a link between wildlife and disease in humans in Malaysia. Your participation in this research is greatly appreciated.
1. I confirm that I meet the criteria for participation in this study, namely: I am over the age of 18 years.
2. I agree voluntarily to take part in this study.
3. I have read the Leptospirosis Information Sheet provided and have been given a full explanation of the purpose of this study, of the procedures involved and of what is expected of me. The researcher has answered all my questions and has explained the possible problems that may arise as a result of my participation in this study.
4. I understand I am free to withdraw from the study at any time without needing to give any reason.
5. I understand I will not be identified in any publication arising out of this study.
6. I understand that my name and identity will be stored separately from the data, and these are accessible only to the investigators. All data provided by me will be analysed anonymously using code numbers.
7. I understand that all information provided by me is treated as confidential and will not be released by the researcher to a third party unless required to do so by law.
8. I agree to be contacted by phone two weeks from now for a brief follow up survey. I would like to be contacted at the following phone number ______(daytime / evening) for the purpose of this survey.
9. I would like to receive a copy of the feedback from the study. Please contact me at ______
181 10. I consent to be photographed during the sample collection for presentation purposes. I am not willing for this session to be photographed.
Signature of Participant: ______Date: …..../..…../……. (Name)
Signature of Investigator: ______Date: ..…../…..../…….
182
Borneo J. Resour. Sci. Tech. (2013) 2(2): 79-82
SHORT COMMUNICATION
Serological Prevalence of Leptospiral Infection in Wildlife in Sarawak, Malaysia
SIVA THAYAPARAN*1, IAN ROBERTSON1, FAIRUZ AMRAAN2, LELA SU’UT3 & MOHD TAJUDDIN ABDULLAH4
1School of Veterinary Medicine and Biomedical Science, Faculty of Health Science, Murdoch University, 6150 Murdoch, Western Australia; 2Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia; 3Lot 77, Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak, Jalan Tun Ahmad Zaidi Adruce, 93150, Kuching, Sarawak, Malaysia; 4Department of Zoology, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak, Malaysia
ABSTRACT
Leptospirosis is a zoonotic disease caused by pathogenic leptospiral bacteria, which are transmitted directly or indirectly from animals to humans or animal to animal. The first phase of this proposed study was carried out to determine the extent of exposure to leptospirosis in wild mammals surrounded by human settlements around wildlife or tourism area (Wind Cave, Fairy Cave, Bako National Park and Matang Wildlife Center). This study reports an incident of leptospirosis among primates (three captive and two free ranging), rats, bats, squirrels and mongoose around Kuching, Sarawak area, which has been screened for Leptospirosis. Blood samples were obtained to determine the presence of antibodies through the microscopic agglutination test (MAT) using eighteen serovars of Leptospira commonly found in Malaysia as antigens. It was observed that four out of the five monkeys (80%), rats (9/4) (44%), bats (20/5) (20.8%), squirrels 4/4 (100%) and mongoose (1) (100%) reacted against one or more serovars of Leptospira. In this study antibody of five serovars of Leptospira interrrogans Copenheni, Leptospira interrrogans Lai, Leptospira interrrogans Pomona, Leptospira interrrogans Pyrogenes, Lepto 175* were detected. Serovars Copenhegeni, Lai, Pomona and Pyrogenes were considered pathogenic for different mammals including human beings. No information about serovars lepto 175 and further studies going on. This is providing information on the possible zoonotic importance of mammalian species in maintaining this disease in Sarawak. The transmission of leptospires in rats reported several incidents and between primates, bats, squirrels, mongoose and human is not reported elsewhere but this could create new reservoir and transmission routes and may affect the tourism, conservation effort and public health. Keywords: Leptospirosis, wildlife, mammals, Sararawak, Borneo
Leptospirosis can affect both humans and through the conjunctiva or surface epithelium animals throughout the world resulting in (Russ et al. 2003). The role of rats as a source morbidity and mortality (Russ et al. 2003; of human infection was discovered in 1917 WHO Headquarters, Geneva, 2006). The (Levett 2001) and subsequently some important epidemiological feature of researchers have identified that flying foxes can leptospirosis in domestic animals and wildlife carry pathogenic leptospires in Australia (Cox, can lead to economic loss and potentially Smythe, & Leung 2005; Smythe et al. 2002). spread to the human communities. The bacteria can cause polymorphic disease conditions in wild, domestic animals and in Leptospirosis can be transmitted in human human (Terpstra 2003). However, to date there by direct contact with infected blood, tissues, has been little research on the role of wildlife in organs or urine of infected hosts or through outbreak throughout the world. Due to the indirect contact with contaminated formites, current significant levels of reforestation soil, mud, fresh water, vegetation and food occurring and the involvement of humans in stuffs or working in places infested with the jungle, there is the potential for exposure of rodents (Terpstra 2003; Zavitsanou & humans to new serovars of leptospires. Babatsikou 2008). Transmission can also occur via the direct penetration of the leptospires Trapping of monkeys, rats, bats, squirrels *Corresponding authors: [email protected]; [email protected] and mongoose were carried out around Wind
80 THAYAPARAN et al. 2013
Cave, Fairy Cave, Bako National Park and Copenhageni, Celledoni, Shermani, Djasiman, Matang Wildlife Center. Rats, squirrels and Grippotyphosa, Hebdomadis, bats were trapped alive, anaesthetized and Icterohaemorrhagiae, Javanica, Patoc, Pomona, blood extracted by cardiac puncture. Serum Pyrogenes, Sejroe, Lai and Lepto 175 samples were kept at -20oC until perform the (Sarawak) commonly found in Malaysia. Microscopic Aggulutination Test (MAT). To Serum positive at titre 1:50 was further titrated screen the status of Leptospira, three captive until 1:1600. Sera were considered to be primates (Macaca nemestrina, Hylobates positive if the titre was 1:50 by MAT. muelleri, Macaca fascicularis) and two free A total of five primates, nine rats, 20 bats, ranging primates (Presbytis cristata, Nasalis four squirrels, and one mongoose were caught larvatus) have chosen for this study. 5 ml of during the first phase of sample collection blood collected in plain tube for the serum. around wildlife area. Four out of the five After 15 min plain tube centrifuged at 15,000 monkeys (80%) Macaca nemestrina, Hylobates RPM for 5 minutes and serum separated and o muelleri, Presbytis cristata, Nasalis larvatus stored under -20 C for MAT. The mongoose showed the positive titre and Macaca caught accidently in cage trap included into this fascicularis did not show any titre. Rats (9/4) screening and collected 5ml blood from (44%), bats (20/5) (20.8%), squirrels cephalic vein and serum separated and stored o (Callosciurus notatus) (4/4) (100%) and under -20 C before screen for leptospirosis mongoose (Herpestes brachyurus) (1) (100%), antibodies. were positive for leptospiral antibodies from The Microscopic Agglutination Test was their serum. From the positive cases 72% were performed according to Faine (1982) to check showed the positive titer with Lepto 175, 33% for Leptospira-specific antibodies from non- with Lai, 17% with Pomona, 11% with human primates, rats, bats, squirrels and Copenhageni and 11% with Pyrogenes. mongoose. Serum was tested against 18 Detailed information about antibodies serovars using the following serovars: indentified in different species shown below. Australis, Autumnalis, Bataviae, Canicola,
Table 1. Findings of antibodies of different leptospira serovars in particular species. Order/Species sv.lep175*. sv.copen. sv.lai. sv.pom. sv.pyro. Macaca nemestrina + - + - - Hylobates muelleri + - - - - Trachypithecus cristatus + - - - - Nasalis larvatus + - + - - Sundamys muelleri + - + - - Ratus argentiventer - + - - - Cynopterus brachyotis + - - - - Penthetor lucasi + - - - + Nycteris tragata - - + - - Hipposideros cervinus + - - - - Callosciurus notatus + - - + - Herpestes brachyurus + - + - + Sv.copen- copenhegeni, sv.lai.- lai, sv.pom.-pomona, sv.pyro.-pyrogenes, svlep175.- lepto175. *Leptospirosis Lepto 175 (Sarawak) strain was isolated from Sarawak environmental source and no further information on this strain and further studies is going on in Institute for Medical research (IMR) Kuala Lumpur.
Malaysia is endemic to Leptospira (Sejvar et Screening the wildlife around the wildlife area al. 2003). In recent decade Malaysia is clearly showing there is a source of infection becoming famous for Eco-tourism and many around those areas and it could cause direct tourist from various countries are visiting for transmission to humans or it could transmit Eco-challenge activities and wildlife tourism. from animals to human. Rats are well known
SEROLOGICAL PREVALENCE OF LEPTOSPIRAL INFECTION 81
carrier of leptospirosis and spread among other REFERENCES animals and human. But bats, squirrels, Antony, S.J. (1996). Leptospirosis–an mongoose and monkeys are not known about emerging pathogen in travel medicine: a their carrier status and need more studies to review of its clinical manifestations and explore their carrier status. A positive result on management. Journal of Travel Medicine, proboscis monkey is alarming the situation and 3(2): 113-118. its first time reported this case in Malaysia. The information on Lepto 175 (Sarawak) is Cox, T.E., Smythe, L.D., & Leung, L.K.P. unknown and its endemic in Sarawak (2005). Flying foxes as carriers of Malaysia. The status of its pathogenic or not is pathogenic Leptospira species. Journal of not known and further studies is going on by wildlife diseases, 41(4): 753. Institute for Medical Research Kuala Lumpur, El Jalii, I.M. & Bahaman, A.R. (2004). A but the serovars Lai is pathogenic and its major review of human leptospirosis in Malaysia. cause of zoonotic spread from adventure Tropical biomedicine, 21(2): 113. activities, such as rafting, cannoning, and swimming in river (Sejvar et al. 2003). Past Faine, S. (1982). Guidelines for the control of history of the human cases such as in 1984 Leptospirosis, World Health Organization, there were two teams of cave explorers from Geneva. WHO offset Publication No. 67. British confirmed with Leptospira infection Informal Consultation on Global Burden of after their return from Mulu cave, Sarawak Leptospirosis: Methods of Assessment. (Mortimer 2005). Again in 1985 another two WHO Headquarters, Geneva, 25-27 October British tourist confirmed with Leptospira after 2006. their return from Sarawak. After the 2000 Eco- challenge in Sabah, several participants from Levett, P.N. (2001). Leptospirosis. Clinical USA, UK, Australia and New Zealand are Microbiol. Rev., 14(2): 296-326. confirmed with Leptospira infection (Sejvar et Mortimer, R.B. (2005). Leptospirosis in a caver al. 2003). This all incidents clearly indicating returned from Sarawak, Malaysia. that Leptospira infection might be going to Wilderness & Environmental Medicine, affect the tourism industry in future. It is best 16(3): 129-131. to find out the source of infection and take the preventive measures from Leptospira infection Sejvar, J., Bancroft, E., Winthrop, K., before getting any major outbreak. Bettinger, J., Bajani, M., Bragg, S., Shutt, K., Kaiser, R., Marano, N., Papovic, T., Tappero, J., Ashford, D., Mascola, L., Vugia, ACKNOWLEDGEMENTS D., Perkins, B., Rosentein, N., & Eco- The authors would like to thank Murdoch Challenge Investigation Team. (2003). University and SOS rhino (US) to provide the Leptospirosis in “Eco-Challenge” Athletes, financial support to conduct the research in Malaysian Borneo, Emerging Infectious Sarawak Malaysia. Especially thankful for Diseases, 9: 702-707. Sarawak Forestry cooperation for providing Smythe, L.D, Field, H.E., Barnett, L.J., Smith, permit (Permit No: NCCD.907.4.4 (V)-235) to C.S., Dohnt, M.F., Symonds, M.L., Moore, capture rats and bats; the primate blood M.R., & Rolfe, P.F. (2002). Leptospiral samples were collected using the Proboscis antibodies in flying foxes in Australia. Monkey Genome Project lead by M.T. Journal of wildlife diseases, 38(1): 182. Abdullah. Thanks for the masters and undergraduate students from the Department of Russ, A, Jali, I., Bahaman, A., Tuen, A., & Zoology, Universiti Malaysia Sarawak, for Ismail, G. (2003). Seroepidemiological study helping to capture small mammals and of leptospirosis among the indigenous identification. Special thanks go to staffs from communities living in the periphery of Institute for Medical Research (IMR), Kuala Crocker Range Park Sabah, Malaysia. Lumpur for technical support. This research is http://www.arbec.com.my/pdf/ conducted according to the Murdoch Animal art10janmar03.pdf. Accessed March 10, ethics (W2376/10). 2010.
82 THAYAPARAN et al. 2013
Terpstra, W.J. (2003). Human leptospirosis: guidance for diagnosis, surveillance and
control, World Health Organization, International Leptospirosis Society.
Malaysian J Pathol 2013; 35(2) : 123 – 132
REVIEW ARTICLE Leptospirosis, an emerging zoonotic disease in Malaysia
S THAYAPARAN BVSc, MSc, I D ROBERTSON PhD, A FAIRUZ* MD, L SUUT** PhD and M T ABDULLAH*** PhD
College of Veterinary Medicine, School of Veterinary and Life Sciences, Murdoch University, Western Australia, *Institute for Medical Research, Kuala Lumpur, Malaysia, **Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak (UNIMAS) and***Department of Zoology, UNIMAS, Sarawak
Abstract
Leptospirosis is an endemic disease in Malaysia and recently has received increasing attention mainly due to several recent incidents that have resulted in human mortality which have alarmed health professionals in Malaysia. The increasing incidence of leptospirosis in forested regions is associated with the bacteria infecting small wild mammals other than rats. Infection in wildlife could result in the introduction of new serovars to humans and domesticated animals. More research on leptospirosis and the screening of wildlife and humans near wildlife habitats is required to have a better understanding of the involvement of wildlife in the disease.
Key words: infectious disease, leptospirosis, zoonotic disease, wildlife
INTRODUCTION include two genera, Leptospira and Leptonema.11 Leptospira are obligate aerobes with an optimum Leptospirosis, caused by infection with growth temperature ranging from 28oC to Leptospira interrogans, is a worldwide disease 30oC.12,13 The genus Leptospira was divided into affecting both humans and animals resulting two species based on serological classification: in morbidity and mortality.1 The agent can be 2,3 Leptospira interrogans, which comprises all the transmitted both directly and indirectly. In pathogenic strains and Leptospira biflexa, the 1917, the role of rats as a source of human 14-16 4,5 environmental saprophytic strains. Based infection was first discovered, and subsequently on the microscopic agglutination test (MAT), some previous studies have demonstrated that leptospires can be further divided into over other wild mammals can also act as potential 6,7 7,8 250 serovars. Serovars that are antigenically carriers, including flying foxes. However, similar have been aggregated into serogroups.14 to date there has been little research conducted Furthermore both pathogenic and non-pathogenic on the role of wildlife in outbreaks. As a result serovars can be classified within the same species. of the current significant level of deforestation A combination of methods is now used to confirm and the increasing anthropogenic activities in the species of Leptospira. the forested habitats and jungle, humans are at a greater risk of being exposed to new serovars Pathogenesis of leptospires. Leptospirosis in wildlife can have negative consequences for biodiversity, The most adverse clinical and pathological signs human and livestock health, animal welfare and usually occur in young animals, especially when the economy of a country.9,10 This review will their maternally-derived immunity is waning.17 examine the impact of leptospirosis on wildlife, However, the common signs of Leptospira the current status of surveillance and the options infection in farm animals are abortions, stillbirths, to strengthen policies in Malaysia. decreased milk production and a failure to thrive.18-20 In all species, congenital infection Morphology and taxonomic classification and its sequelae are well reported. The most important difference between infection of animals Leptospires are spirochaetes in the order and humans is the presence of chronic carriers Spirochaetales, family of Leptospiraceae and
Address for correspondence and reprint requests: S.Thayaparan, College of Veterinary Medicine, School of Veterinary and Life Sciences, Murdoch University, 6150 Murdoch, Western Australia. Tel: +61468462223. Email: [email protected]; [email protected]
123 Malaysian J Pathol December 2013 in animals through reservoirs in the kidneys and research on wildlife, several pathogenic serovars genital tract.20-23 have been isolated with the most prominent serovars including grippotyphosa and pomona Epidemiology of leptospirosis from white-tailed deer (Odocoileus virginianus) in North America.37 Surveillance of leptospirosis Leptospires are widespread and their abundance is important to determine the emergence of new is due to their ability to infect a range of animal strains, which have the potential to cause an species, including humans, as well as the ability outbreak. Although it is not economically viable to survive outside the host, if environmental to survey all wildlife species, it is important to conditions are favourable.24-26 The main sources regularly screen particular species, which live on of infection are urine of infected or carrier the periphery of forests and have the potential to animals, contaminated surface water, mud and interact with humans (e.g. wild rats, carnivores soil.5 Transmission can happen as a result of and bats). Screening these ‘sentinels’ provides direct or indirect contact with infected animals or useful cost-effective information on the health their secretions.5 The major route of infection is status of a broader range of species. via mucous membranes, however carnivores can Leptospirosis can cause serious economic loss be infected through the ingestion of leptospiral- to the livestock industries,38,39 and is a major infected carcasses.27 Usually leptospires appear cause of abortions in cattle.40,41 As the disease in the blood four to 10 hours after infection and is common in domestic animals and wildlife, can be detectable in the blood from a few hours control in domestic animals can also have a to seven days.28-30 Clinical signs may not occur in positive impact on the disease in wildlife. every case but severe fever is an important sign of Worldwide there have been more than 184 acute leptospirosis. Animals that have recovered distinct serovars of L. interrogans belonging to from leptospirosis may become carriers with the 20 serogroups identified.42 In Southeast Asia, organism present in the renal tubules for periods the most common serovars associated with of days to years,31,32 and subsequently leptospires disease of domestic animals and humans are are shed in urine into the environment.32 icterohaemorrhagiae, autumnalis, canicola, pomona, patoc, grippotyphosa, australis and Impact on humans, domestic animals, wildlife poi.43,44 The first three are the most important and ecotourism serovars with respect to veterinary and public Infectious diseases are transmitted globally health perspectives. Fever is an important and through animal and human movements due to common presentation of tropical diseases and eco-tourism, wildlife research, reintroduction, may sometimes be the only manifestation of rehabilitation, hunting, pet trade, laboratory and a serious illness. Diagnosis is often difficult food industry demands and farming.33,34 These because of the many diagnostic possibilities, movements and activities are major contributing and symptoms, which may often be non-specific, factors for the transfer of leptospirosis to animals and because of many doctors’ unfamiliarity with and humans and the spread of the disease to the spectrum of tropical diseases. Treating the new areas. disease appropriately will reduce the risk to Research on leptospirosis has highlighted that public health, and this can only come following rodents (20%), marsupials (35%) and bats (35%) a correct diagnosis. With the recent popularity are most likely to spread the disease.35 Marsupials of adventure travel and triathlete competitions, and chiropterans have been implicated as there is an increased awareness of the potential more significant reservoir hosts of leptospires for leptospiral infection amongst those who pathogenic to humans than previously was partake in such activities.45,46 recognized.35 Studies on leptospirosis involving bats have produced equivocal results,7 however Laboratory diagnostic methods used in the fact that leptospires can potentially be spread Malaysia to diagnose leptospirosis through bats is a major concern due to their Diagnosis of leptospirosis depends on adoption abundance, their ability to travel long distances of appropriate laboratory methods as the and their potential to expose humans to infected clinical presentation can vary greatly. The urine. Ground-dwelling species, such as rodents diagnostic method selected depends on the foraging under bat roosts, could also encounter available samples and the purpose of testing. Leptospira-contaminated urine and spread the Identification of the infecting serovar is important organism into urban areas.36 From the limited
124 LEPTOSPIROSIS both epidemiologically and clinically, since Malaysia.3 These animals are capable of being this may assist in determining the source and infected with one or more leptospiral serovars likely outcome of infection. Different assays and can then serve as reservoir hosts for these have been developed in an attempt to diagnose serovars. These animals have an asymptomatic leptospirosis accurately, but the majority are and transient infection with no obvious clinical unsuitable for use in developing countries due to signs of disease. their requirement for the maintenance of multiple strains or expensive equipment. Several methods History of leptospirosis and geographical are used in the diagnosis of leptospirosis in distribution Malaysia. These include the simple Microscopic Leptospirosis has been documented worldwide, Agglutination Test (MAT); Polymerase Chain but formal reporting systems vary widely.2,50,51 Reaction (PCR) test, as well as the Enzyme- Frequently the disease gains public attention linked Immunosorbent Assay (ELISA). The when outbreaks occur in association with natural MAT is inexpensive, but time consuming and disasters, such as flooding in Nicaragua in laborious, and is only carried out at the Institute 1995 or among foreign travellers and extreme for Medical Research (IMR) Kuala Lumpur, athletes.52 Southeast Asia is an endemic area MKAK (Makmal Kesihatan Awam Kebangsaan) for leptospirosis, and infection in humans has Sungai Buloh Malaysia for community outbreaks been reported throughout the region. Seventy and also at some universities undertaking percent of the major pathogenic serovars have research on leptospirosis.47 ELISAs are been isolated from Asia.53 Although leptospirosis performed at hospitals with the necessary has been around for millions of years,54 it is facilities and the IMR and MKAK. Currently only in recent times that the bacterial cause of culture of Leptospira is only performed at the this disease has been identified. In 1886 Adolf IMR.47 Diagnosis of animal leptospirosis is only Weil published an account detailing the icteric undertaken at the Veterinary Research Institute, form of leptospirosis, which has since taken Ipoh and the Wildlife Department, Kuala Lumpur on his name.54 However, his observations had (PERHILITAN). Environmental samples can be already been postulated by others, including tested in MKAK, Sungai Buloh, Malaysia.47 Hippocrates.54 It has been hypothesised that leptospirosis was responsible for an epidemic Treatment, control and prevention of among the natives of the Massachusetts coast leptospirosis just before the arrival of the Pilgrims in 1620.55 A range of antibiotics are used to treat hosts with What Weil described was the icteric form of leptospirosis, with IV C-Penicillin (2M units 6 leptospirosis with jaundice. In contrast the milder hourly for 5-7 days) being commonly used in forms were hard to diagnose due to a lack of severe adult human cases.47,48 The less severe advanced bacteriology. Spirochaetes continued cases are usually treated orally with antibiotics to cause problems for the later part of the 19th such as doxycycline, tetracycline, ampicillin or century, and it was only in 1907 that Stimson amoxicillin.47 A trial on the penicillin derivative, managed to isolate a leptospire from a patient.56 doxycycline, on the prevention of infection and His subsequent research highlighted that the clinical disease was conducted in the North bacteria were concentrated in the renal tubules Andaman Islands in 1999.49 Although the findings and were shaped like question marks. This gave indicated that doxycycline prophylaxis did not rise to the name “Spirocheta interrogans”, which prevent leptospiral infection in an endemic area, has remained.56 such treatment did have a significant protective effect in reducing the morbidity and mortality Status of leptospirosis in South East Asia during outbreaks. Leptospirosis is emerging as a serious concern to Cases of naturally occurring leptospirosis in public health in Southeast Asia.57-59 The disease wildlife have not been documented and little is has been recognised in patients from Indonesia known about the immunological features of the with clinical jaundice and non-malarial fever, disease in wild mammals. However researchers patients with clinical jaundice from Laos and have demonstrated antibodies to several serovars Vietnam, and patients with haemorrhagic fever that have been found in a range of wildlife in Cambodia.53,60 In addition, a cross-sectional species (non-human primates, bats, rats, community-based study was conducted in squirrels and mongoose) in Kuching Sarawak, Laos to obtain estimates of the background
125 Malaysian J Pathol December 2013 sero-prevalence. These findings suggested that involved in cleaning a pond in Khumuang, leptospirosis was under-reported in Southeast Buriram Province. Multivariable logistic Asia due to the wide range of clinical symptoms regression indicated that wearing long pants or associated with acute leptospiral infection.60 skirts was protective against leptospiral infection, Easton,61 reported a spike in infection while the presence of more than two wounds on in Manila following flash floods during the the body was associated with infection.67 monsoon season. The bacteria would most likely In Malaysia isolation of Leptospira from have entered the patients’ bodies through cuts black rats (Rattus rattus) was reported in 1928 on the skin when they waded barefoot in the by Fletcher.9,68 Many subsequent investigations floodwaters. Residents in low-lying areas and rice have demonstrated a high prevalence of infection farmers were identified as a high-risk group for in humans in Malaysia,69 with Robinson and infection. One possible source of the problem was Kennedy,70 reporting 31 cases among British the increasing rat population with farmers from army personnel in Malaysia. From 1953 to 1955, one province culling approximately 800 rats per 30 pathogenic leptospiral serovars were identified night in their rice fields during the dry season.61 by Alexander,31 from both military personnel Studies in various locations in Thailand found and civilians. Their studies demonstrated a high that, after rats, dogs were the most important sero-prevalence in humans throughout Malaysia. reservoir for leptospirosis with a seroprevalence The highest distribution was found in labourers of 11%, with the Bataviae serogroup being the working in rubber estates and those working most prevalent (5.2%).62 Playing in sewage, with the sewage, drainage, forestry and town staying outdoors more than half the time and cleaning industries.31 In the 1950s and 1960’s a consuming raw meat increased the likelihood comprehensive study of leptospirosis in Malaya of sero-positivity in the sampled dogs. It was was undertaken that included testing various possible that the dogs acquired their infection mammals from a range of environments, as from rats, which were responsible for an outbreak well as assessing occupational risks to humans. in north-eastern Thailand between 1999 and The results suggested that rats were the main 2003.62,63 During that period, Thaipadungpanit maintenance hosts for leptospirosis, despite et al,63 developed a scheme of multi-locus the presence of infection across many animal sequence typing of pathogenic Leptospira and species. Over one hundred (104) strains were confirmed serovar Autumnalis was responsible isolated and identified.71 Bahaman et al,31 for the outbreak and was being maintained by conducted a cross-sectional serological survey Bandicota bengalensis (bandicoot rats).63 of domestic animals in West Malaysia and found Besides infecting local residents, Leptospira that approximately one quarter of the animals can also cause illness in visitors to tropical examined had agglutinating antibodies to L. regions, particularly those associated with interrogans. Cattle, buffaloes and pigs were eco-tourism and adventure travel.64 Van Crevel all observed to have a high seroprevalence et al,64 investigated leptospirosis in 32 Dutch with temperate cattle breeds appearing more travellers between 1987 and 1991 and found 28 susceptible to infection than local breeds. A of them had returned from Southeast Asia with subsequent study found that the bacteriological the majority visiting Thailand and 21 of these seroprevalence in cattle and buffaloes was had taken a rafting tour. From this research it 14.4%.68 A new serovar was isolated from a was recommended that doctors should consider bovine kidney, while six other serovars were leptospirosis in their differential diagnosis isolated for the first time from Malaysian cattle. whenever a patient with fever returned from Serovar hardjo was shown to be maintained in the tropics. Due to the increasing number of Malaysian cattle.31 Recently leptospirosis has reported cases of leptospirosis in the western been reported in detention centres for refugees world, it has been recommended that travellers in Malaysia.72,73 An outbreak at Juru Detention try to avoid high-risk aquatic activity in the Centre was contained by the health authorities, tropics and undertake chemoprophylaxis if they but the death of six Burmese at an undisclosed do take part in water-sports in Southeast Asia.5,65 detention centre in September 2009 has raised Saunders66 examined 78 cases of leptospirosis fresh concerns for several Malaysian NGOs. In in Pahang and reported the lack of specificity July 2010, cases of leptospirosis were reported of the clinical syndrome. nation-wide and eight people who took part In Thailand, researchers conducted a in a search and rescue operation in Lubok Yu, serological survey of workers who had been Maran, Pahang died from the disease, causing
126 LEPTOSPIROSIS
FIG. 1: Case Fatality Rate (CFR) of leptospirosis in Malaysia from 2004 to 2010. the picnic spot to be closed to the public.74,75 cases (184) were reported in Pahang. The states Other cases were reported in Kedah during July of Sarawak, Selangor and Terengganu showed a 2011, with one fatality at Lata Bayu.76 As recent gradual increase in the number of cases over this as March 2012, a national service trainee died period. No cases were reported in WP Labuan of suspected leptospirosis at Sungai Siput in owing to its small geographical area with no Perak, Malaysia.77 This resulted in a suspension forest habitat and the fact that it is surrounded of water based activities at all National Service by sea-water. Training camps. Case fatality rates (CFR) over the period According to data from the Ministry of from 2004 to 2009 varied from 1.8% to 7.6% Health the prevalence of leptospirosis increased (Figure 1) with an average of 4.44%. During dramatically from 2004 to 2009 (Table 1), this period a total of 5,267 cases of leptospirosis however the number of deaths from leptospirosis were reported nationwide with 234 known did not change from 2004 to 2007, although it fatalities. Perak had the highest CFR for this increased markedly to 47 and 62 deaths in 2008 period (6.81%), followed by Sarawak (6.42%) and 2009, respectively. The highest numbers of and Perlis (6.25%). Approximately one-fifth of cases were reported in the state of Perak during all cases of leptospirosis for this period were 2005, 2006, 2008 and 2009 with 71, 93, 289 documented in Perak state, of which 71 proved and 280 cases, respectively (Table 1). In 2004, fatal (Figure 2). The almost threefold increase more cases32 were reported in Sarawak than in in the number of cases in 2007 and subsequently other years and in 2007 the highest numbers of may be due to improved diagnostic techniques
FIG. 2: Leptospirosis CFR in different Malaysian states from 2004 to 2009.
127 Malaysian J Pathol December 2013 Total 2009 2008 2007 Year 2006 2005 Deaths Cases Deaths Cases Deaths Cases Deaths Cases Deaths Cases Deaths Cases Deaths 2004
2 1 2 0 4 0 1 0 6 0 1 0 16 1 7 1 9 1 79 2 20 1 25 0 20 1 160 6 9 0 25 1 28 0 32 0 25 2 32 0 151 3 30 1 29 6 31 2 59 3 87 3 59 3 295 18 15 1 27 0 31 0 106 9 52 2 106 9 337 21 29 1 24 0 51 3 184 5 198 7 184 5 670 21 29 4 71 4 93 9 280 19 289 16 280 19 1042 71 13 1 12 1 19 0 35 1 34 2 35 1 148 6 32 2 71 2 37 3 70 4 58 5 70 4 309 20 263 20 378 20 527 22 1418 62 1263 47 1418 62 5267 234 Cases Total WP LabuanWP 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Perlis WP PutrajayaWP 1 0 1 0 7 0 14 0 1 0 14 0 38 0 Melaka Penang Kuala Lumpur 31 0 20 1 27 1 54 0 45 0 54 0 231 2 Johor Negri-Sembilan 27 1 41 0 24 0 91 0 59 0 91 0 333 1 Kedah Terengganu 7 1 17 0 42 1 126 9 107 4 126 9 425 25 Kelantan 15 1 38 1 17 1 138 4 180 4 138 4 526 15 Pahang Selangor 16 5 20 3 37 0 208 7 97 2 208 7 586 24 Perak Sabah Sarawak State Sources: (3) Table 1: Table Status of leptospirosis in humans in Malaysia from 2004 to 2009
128 LEPTOSPIROSIS or a greater awareness of the disease. This may reported in humans in Sarawak including 13 also have contributed to reducing the CFR from deaths, compared with 49 cases in the previous 2004. It is also possible that climate change in year.83 The CFR associated with this outbreak the last four years of this period played a part in (6.9%) was higher than that in previous years. On influencing the pattern of infection, as outbreaks the 12th December 2011, an outbreak occurred at of leptospirosis usually follow after a major flood RSAT Army camp at Penrissen Batu 8, Kuching, during monsoon seasons. Sarawak with five army recruits showing symptoms of leptospirosis and the disease was Status of leptospirosis in Sabah and subsequently confirmed serologically. The source Sarawak of infection was identified as drinking and bathing activities in the small river near the camp.84 The As well as Peninsular Malaysia, scientific Kuching Divisional Health Office was notified research has also focused on Malaysian Borneo. of another suspected leptospiral outbreak on the There have been reports of leptospirosis in 30th December 2011 by the Sarawak General humans in Sabah and Sarawak, notably an Hospital. The incident involved two army outbreak following the 2000 Eco-challenge recruits from Blok G10, Kem Semenggok, event in Sabah.78,79 This highlighted the risks Kuching and both were serologically positive for of eco-tourism and adventure travel, as most leptospirosis.85 The health authority of Sarawak of the victims were western athletes. In the confirmed a leptospirosis outbreak in the drains Eco-challenge event, participants competed in along the Tiong Hua Road in Sibu during June jungle trekking, swimming and kayaking in 2012.86 In the state of Sarawak, the number of freshwater and caving. A team of researchers cases of leptospirosis was similar from 2004 to from the CDC Atlanta contacted 189 athletes 2010, however in 2011 and 2012 there was a from 27 countries following the outbreak.79 large increase in the number (Figure 3). Eighty athletes met their case definition, with 29 The four-fold spike in the number of cases cases being hospitalized, although no fatalities in Sarawak is a valid cause for concern for were reported. Another study in Sabah revealed health authorities in both Kuching and Kuala that swimming in the Segama River appeared Lumpur. For 2011 alone, the CFR was 6.9%, to be a risk factor for infection and 20 athletes above the six-year average of 6.42% (2004 and reported taking doxycycline prophylactically and 2009). The number of cases (186) reported in this seemed to have a protective effect, with a 2011 alone was already equivalent to 60% of the preventive efficacy of 55%. combined total from 2004 to 2009. The record Studies carried out in Sabah reported a high high of 271 documented cases the following seroprevalence (25.75%) in people living within year may be attributed to mandatory reporting the periphery of a national park, presumably procedures being implemented, or increased due to exposure/contact with wild mammals.9 awareness of the symptoms of leptospirosis by Leptospirosis has also been reported in a caver medical practitioners. These also reflect on the from the USA after he returned from Gunung living conditions of residents in various areas Mulu National Park, in Sarawak.52 Recently of Sarawak. researchers from Universiti Malaysia Sarawak (UNIMAS) and Sarawak Health Department Conclusions have conducted research in the Rejang Basin area, Sarawak. They found that 30.6% of humans Over the years, leptospirosis has presented sampled were seropositive for leptospirosis itself intermittently to the Malaysian health and infection was associated with farming and/ authorities and further research has consequently or water activities.80 In Bakun in Sarawak, a been conducted. The biggest issue for doctors Chinese national working on the hydro-electric has been the confusing nature of symptoms dam project site was hospitalised with a serious presented by affected humans. Since leptospirosis condition suspected to be leptospirosis, and is a febrile illness and several tropical diseases the subsequent death of 10 workers involved manifest in their early stages with fever, it is in the translocation of animals from the Bakun easy to misdiagnose the disease unless further Hydroelectric Dam water catchment area to laboratory testing is undertaken. This can higher ground were speculated to be due to result in delayed commencement of appropriate either leptospirosis or meliodosis.81,82 treatment (single dose doxycycline therapy). In 2011, 186 cases of leptospirosis were Fortunately with advances in medical technology,
129 Malaysian J Pathol December 2013
FIG. 3: Number of confirmed cases of leptospirosis in humans in Sarawak diagnosis of leptospirosis has become more carriers of pathogenic Leptospira species. J Wildl accurate. Currently there is much research being Dis. 2005; 41(4): 753-7. undertaken on leptospirosis, however due to poor 8. Smythe LD, Field HE, Barnett LJ, et al. Leptospiral antibodies in flying foxes in Australia. J Wildl Dis. coordination of this research the actual incidence 2002; 38(1): 182-6. of disease is not truly reflected in Malaysia. There 9. Russ A, Jali I, Bahaman A, Tuen A, Ismail G. is a need for cooperation between researchers Seroepidemiological study of leptospirosis among from veterinary and medical backgrounds to the indigenous communities living in the periphery determine the real status of leptospirosis. Loss of Crocker Range Park Sabah, Malaysia. ARBEC. of biodiversity is a possible outcome of this 2003; 1-5. disease as wildlife comes under constant threat 10. Caley P, Ramsey D. Estimating disease transmission in wildlife, with emphasis on leptospirosis and and increasing pressure to survive. Emerging bovine tuberculosis in possums, and effects of zoonotic infectious diseases represent a growing fertility control. J Appl Ecol. 2001; 38(6):1362- threat and danger to wildlife as well as humans. 70. Real-time surveillance through integrated human, 11. Adler B, de la Peña Moctezuma A. Leptospira and veterinary and wildlife disease systems will leptospirosis. Vet Microbiol. 2010; 140(3-4): 287-96. reduce the time taken to recognise disease and 12. Johnson RC, Harris VG, Walby JK. Characterization implement suitable disease control practices. of leptospires according to fatty acid requirements. J Gen Microbiol. 1969; 55(3): 399-407. 13. Chang SL. Studies on L e p t o s p i r a REFERENCES icterohaemorrhagiae; the growth rate of, and some biochemical observations on Leptospira 1. Antony SJ. Leptospirosis—An Emerging Pathogen icterohaemorrhagiae in culture. J Infect Dis. 1947; in Travel Medicine: A Review of its Clinical 81(1): 35-47. Manifestations and Management. J Travel Med. 14. Dikken H, Kmety E. Serological typing methods of 1996; 3(2): 113-8. leptospires. Methods Microbiol. 1978; 11: 259-307. 2. Lau CL, Smythe LD, Craig SB, Weinstein P. Climate 15. Bharti AR, Nally JE, Ricaldi JN, et al. Leptospirosis: change, flooding, urbanisation and leptospirosis: a zoonotic disease of global importance. Lancet fuelling the fire? Trans R Soc Trop Med Hyg. 2010; Infect Dis. 2003; 3(12): 757-71. 104(10): 631-8. 16. Cerqueira GM, Picardeau M. A century of 3. Thayaparan S, Robertson I, Amraan F, Su’ut L, Leptospira strain typing. Infect Genet Evol. 2009; Abdullah MT. Serological Prevalence of Leptospiral 9(5): 760-8. Infection in Wildlife in Sarawak, Malaysia. Borneo 17. Boulanger P, Mitchell D, Smith AN, Rice CE. J Resour Sci Tech. 2013; 2(2): 79-82. Leptospirosis in Canada. III. A study of the 4. Levett PN. Leptospirosis. Clin Microbiol Rev. 2001; importance of the disease in cattle as shown in 14(2): 296-326. combined serological, clinical and bacteriological 5. Monahan AM, Miller IS, Nally JE. Leptospirosis: investigations. Can J Comp Med Vet Sci. 1958; risks during recreational activities. J Appl Microbiol. 22(4): 127-43. 2009; 107(3): 707-16. 18. LaGrange WE, McCahon JV, Little RB. Leptospirosis 6. Hartskeerl PA, Terpstra WJ. Leptospirosis in wild in farm animals. Proc 89th Ann Meet Amer vet med animals. Vet Q. 1996; 18(sup3): 149-50. Ass June 23-26th 1952. 1953: 90-4. 7. Cox TE, Smythe LD, Leung LK. Flying foxes as
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132 Veterinary World, EISSN: 2231-0916 RESEARCH ARTICLE Available at www.veterinaryworld.org/Vol.7/June-2014/13.pdf Open Access
Leptospiral agglutinins in captive and free ranging non-human primates in Sarawak, Malaysia
11 2 S. Thayaparan , I. D. Robertson and M. T. Abdullah
1. College of Veterinary Medicine, School of Veterinary and Life Sciences, Murdoch University, 6150 Murdoch, Western Australia; 2. Department of Zoology, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak, Malaysia Corresponding author: S. Thayaparan, email:[email protected] IDR:[email protected], MTA: [email protected] Received:16-03-2014, Revised: 19-05-2014, Accepted: 24-05-2014, Published online: 25-06-2014 doi: 10.14202/vetworld.2014.428-431 How to cite this article: Thayaparan S, Robertson ID and Abdullah MT (2014) Leptospiral agglutinins in captive and free ranging non-human primates in Sarawak, Malaysia,Veterinary World 7(6): 428-431.
Abstract Aim: The proposed study was carried out to determine the extent of exposure to leptospirosis in non-human primates. Materials and Methods: 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 MicroscopicAgglutination Test (MAT) to 17 serovars ofLeptospira commonly found in Malaysia. Results: 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 ofLeptospira (Lai and Leptospira Lepto175). The serovar Lai is considered pathogenic for different mammals, including humans.Leptospira Lepto 175 has been identified as an intermediate strain and further studies are being undertaken on this serovar. Conclusion: These results are important as primates may act as reservoirs ofLeptospira spp. for humans, which may potentially affect tourism (economic loss), conservation efforts and public health. Keywords: leptospirosis, MAT,non-human primates, seroprevalence, wildlife, zoonotic disease.
Introduction are involved in eco-tourism activities and this group is Leptospira have been detected from wildlife in particularly at increased risk of exposure to infectious many countries, however their role as reservoirs is still diseases [12-13]. poorly understood [1-3]. Leptospirosis can result in In recent years outbreaks of leptospirosis in economic losses in domesticated animals and has the Malaysia have been documented around the wildlife potential to be an important zoonotic disease of reserves and parks resulting in confirmation of a high humans [4]. Leptospires were first isolated from rats in number of confirmed cases and associated mortalities. 1917 and it is widely acknowledged that rodents are a Wildlife tourism is an important source of revenue in key source of infection for humans [5]. However, Malaysia, particularly in the state of Sarawak and leptospirosis has the potential to impact on this. The recently Australian and Peruvian researchers have current research reports on the carriage of Leptospira reported that bats can also carry pathogenicLeptospira , by opportunistically sampled non-human primates in [1-2, 6], although their role as carriers is not fully Sarawak. understood. Other wildlife, including primates, can also act as potential carriers of these pathogens [7-10]. Materials and Methods However to date there has been little research conducted Ethical approval: All procedures were performed with on free ranging wildlife. Leptospirosis in wildlife can the approval of the Animal Ethics Committee of the affect biodiversity, human and livestock health, animal Murdoch University (W2376/10) and Sarawak welfare and consequently the national economy [4]. Forestry cooperation (NCCD.907.4.4 (V)-235). Recently leptospirosis has been recognised as a re-emerging public health problem in Malaysia [11].At Study area: Trapping of monkeys was carried out present Malaysian wildlife disease surveillance is around Bako National Park and Matang Wildlife poorly coordinated and emerging zoonotic infectious Centre. Bako National Park is located 37 km from diseases represent a growing threat. Tourism is a major Kuching, Sarawak, East Malaysia (Figure-1). It is contributor to the economy of Malaysia with 24.6 Sarawak's oldest national park, covering an area of million tourists visiting the country annually. It has 2,727 hectares and is located at the tip of the Muara been estimated that approximately one million tourists Tebas Peninsula [14]. Although it is one of the smallest national parks in Sarawak, it contains almost most Copyright: The authors. This article is an open access article licensed under the terms of the Creative Commons Attribution License types of vegetation found in Borneo along with long-tailed (http://creativecommons.org/licenses/by/2.0) which permits macaques (Macaca fascicularis ), silver-leaf monkeys unrestricted use, distribution and reproduction in any medium, provided the work is properly cited. (Trachypithecus cristatus ), proboscis monkey ( Nasalis Veterinary World, EISSN: 2231-0916 428 Malaysian Journal of Microbiology, Vol 11(1) 2015, pp. 93-101 Malaysian Journal of Microbiology Published by Malaysian Society of Microbiology (In since 2011)
Serological and molecular detection of Leptospira spp. from small wild mammals captured in Sarawak, Malaysia
S Thayaparan1*, Ian D Robertson1 and Mohd Tajuddin Abdullah2
1College of Veterinary Medicine, School of Veterinary and Life Sciences, Murdoch University, 6150 Murdoch, Western Australia. 2Centre for Kenyir Ecosystems Research, Institute for Kenyir Research, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia. Email: [email protected]
Received 2 September 2014; Received in revised form 23 December 2014; Accepted 1 January 2015
ABSTRACT
Aims: Leptospirosis is endemic to tropical regions of the world and is re-emerging as a new danger to public health in Southeast Asia, including Malaysia. The purpose of this particular study was to determine the common leptospiral serovars present in small wild mammals living around wildlife reserves and disturbed forest habitats and human communities. Methodology and results: The samples of blood and kidneys of small rodents, bats and squirrels were analyzed. Antibodies to different serovars of leptospires were detected in 73 of 155 wild small mammals captured (47.0%: 95% CI 39.0-55.3%). The seroprevalence for rats (57.9%; 95% CI 44.1-70.9) was slightly higher than that for squirrels (42.9%; 95% CI 24.5-62.8) and bats (40%; 95% CI 28.5-52.4). Seropositive animals were detected in all 5 localities sampled. Antibodies to serovar Lepto 175 Sarawak were detected in 30 (24.7%) rats, 11 (9.0%) squirrels and 27 (52.9%) bats. Of 155 kidney samples from individual animals only 17 were positive for Leptospira on a molecular study (10.97%, 95% CI 6.5-17). The majority of the positive results were from plantain squirrels (53%; 95% CI 27.8, 77), Müller’s rat (35%; 95% CI 14.2, 61.7) and brown spiny rats (12%; 95% CI 1.5, 36.4). Conclusion, significance and impact of study: This particular study should generate concerns and lead to the health authorities expanding disease control measures in the region as there are significant levels of human activity at all five locations where the animals were sampled. The pathogenesis of serovar Lepto 175 Sarawak also needs to be monitored closely, considering its similarities to the pathogenic Leptospira wolffii.
Keywords: Leptospirosis, zoonotic disease, bats, rodents, wildlife
INTRODUCTION 2010a; Lau et al., 2010b; Tulsiani, 2010; Costa et al., 2014). Rats have been shown to be carriers of leptospires Leptospirosis is endemic to tropical regions of the world throughout the world and are important reservoirs of and is re-emerging as a new threat to public health in infection for animals and humans (Roth, 1964; Priya et Southeast Asia, including Malaysia. Although studies al., 2007). Recently bats have also been implicated in the have been conducted in Malaysia for more than 70 years spread of leptospirosis to humans (Mortimer, 2005; Vashi (Hanson, 1982), the threat leptospirosis poses is still not et al., 2010). well understood. Malaysian jungles are home to With deforestation becoming more commonplace in numerous species of wildlife and peri-domestic animals. many tropical environments, including Sarawak, Malaysia, These include bats, squirrels and rats, as well as there is increased likelihood of contact between humans primates. Bats and rats are known to harbour leptospiral and wildlife due to the disturbance of natural habitats. The serovars in their bodies and pass them to other species increasing popularity of eco-tourism raises another risk for including humans (Roth, 1964; Faine et al., 1999; the disease. Such tourism brings visitors to Malaysia Richardson and Gauthier, 2003; Matthias et al., 2005; close to nature and native wildlife, some of which may be Vashi et al., 2010). reservoir hosts for leptospirosis, as well as potentially Rodents have long been associated with leptospirosis interfering with an already-fragile ecosystem. as reservoir or maintenance hosts and verminous rodents The purpose of this study was to determine the are considered a key source for the distribution of common leptospiral serovars present in small wild leptospires in an urban setting (Faine et al., 1999; Slack mammals living around wildlife reserves, disturbed forest et al., 2006; Priya et al., 2007; Harris, 2009; Lau et al.,
*Corresponding author 93 ISSN (print): 1823-8262, ISSN (online): 2231-7538
Mal. J. Microbiol. Vol 11(1) 2015, pp. 94-102 habitats and human communities around Kuching, Animal capturing and ethical aspects Sarawak, Malaysia. During the investigation 155 individual small mammals were captured and sampled. The species sampled in this MATERIALS AND METHODS study included any small mammals (rats, squirrels, tree shrew and bats) were captured in the selected areas over Study and sampling sites a one-year period from January 2011 to March 2012. The rats, squirrels and tree shrews were trapped The sampling of animals was undertaken in the wider using cage traps (Baeumler and Brunner, 1988). A ecological ranges of Kuching, and Kota Samarahan area, maximum of 50 cage traps were set around the border of including Universiti Malaysia Sarawak (UNIMAS) area, a wildlife area and human settlement, with the intention of Matang Wildlife Centre, Kubah National Park, Bako trapping small mammals at one sampling time. A mixture National Park, Wind and Fairy Cave Reserves and Mount of one-part raisins and two parts peanut butter with rolled Singai Conservation Area (Figure 1). Sampling sites were oats, banana and dry fish was used as bait and placed in chosen to cover different ecological environments (e.g. each trap (Hickie and Harrison, 1930; Wood, 1984). Traps disturbed habitats and secondary forests located around were opened from 6 am and checked once the afternoon human settlements) to gain an accurate representation of and following morning. Any by-catch was immediately the possible transmission of leptospires. All locations released at the site of trapping. If pregnant or lactating sampled in the current study were within approximately small mammals were captured these were also released 30 kilometres of Kuching, the capital city of Sarawak. A immediately at the captured site. Any trapped rats, common trend in most of the human leptospirosis squirrels and tree shrews were collected and taken to the outbreaks in the Kuching region have been exposure to UNIMAS laboratory for processing. The cage was wildlife and contact with water contaminated by covered with cloth and kept in the animal house until leptospires (Thayaparan et al., 2013a). Bako National processing. Each small mammal was housed in Park, Matang-Kubah Wildlife Centre, Mount Singai and individually ventilated cages. During the period of the Wind and Fairy caves are known to contain several transportation small mammals were fed sunflower seeds, wildlife species (Hall et al., 2004; Khan et al., 2007; cereals, cooked corn kernels and cheese and provided Thayaparan et al., 2013a) that may be potentially with water ad libitum. accountable for transmitting leptospires (Abdullah, 2011). The rodents were anaesthetised using Ketamine + UNIMAS is located in an area where the natural Xylazine (50–75 mg/kg + 10 mg/kg IP) (Karwacki et al., environment has been disturbed, similar to other areas of 2001). Once the rats were anaesthetised 1-3 mL of blood Sarawak. Certain parts of the UNIMAS campus are also was collected via cardiac puncture using a 25G needle known to be waterlogged and have patches of forested and a 5 mL syringe. After blood collection, the rats were environment, increasing the possibility of exposure of euthanased with barbiturate (sodium pentobarbitone at students at the campus to leptospires. 150-200 mg/kg) via either an intracardiac or intravenous (IV) injection. After euthanasia the animals were necropsied and kidney samples collected. Blood samples were left at room temperature for 30 min to clot and were then centrifuged at 3,000 RPM for 1 min. The serum was removed and stored at ‒20 °C until testing in the laboratory. All the bats were wild-caught either using mist nets and harp traps and terrestrial small mammals using cage traps. Mist net is a lightweight net that was placed in the flight path of the bats, for example across walking or animal trails, over streams, adjacent to fruiting trees and at the mouths of caves. Bats that were caught in the nets were carefully removed from the net to ensure their delicate wings were not injured. Harp traps were also set across trails or over small streams. Three harp traps and five mist nets were placed along bat flight paths for a maximum of six hours between 6 pm and 11 pm. Nets were checked every 30 min and any bats caught were removed from the traps. After removal each bat was placed in an individual cloth bag. The cloth bags were hung and tied in a handmade wicker basket. The basket was covered with a cloth and placed in a safe quiet place
to minimise disturbance. If any birds were trapped these Figure 1: Sampling sites of small wildlife in Kuching and were removed and released immediately. Kota Samarahan, Sarawak. Bats or small mammals were anaesthetised to reduce the
stress of handling and to minimise the risk of the handler
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Mal. J. Microbiol. Vol 11(1) 2015, pp. 94-102 being bitten. Each animal was anaesthetized with an 200 µL lysis buffer and incubated at 55 °C overnight or intramuscular injection of ketamine (6–7 mg/kg) and until the tissue was completely digested. medetomidine (60–70 μg/kg) (Plowright et al., 2008). A blood sample (1 to 3 mL) was collected by cardiac Analysis of PCR products puncture from each bat using a 5 mL syringe and a 25G needle. Prior to collection the hair was sterilized with Cultures were prepared for DNA isolation by cotton wool dipped in 70% alcohol. The bats were placed centrifugation as described previously (Thaipadungpanit in a fabric pouch and placed in a plastic airtight container. et al., 2007), followed by genomic DNA extraction using a Cotton balls were thoroughly saturated with isoflurane in High Pure Viral Nucleic Acid kit (Roche, Germany). the barrel of a six ml syringe (after removing the plunger). Amplification of the 16S rRNA was performed as The syringe barrel containing the isoflurane saturated described by others (Slack et al., 2006; Thaipadungpanit cotton balls were then placed into the container and the et al., 2007; Slack et al., 2008;) with the following lid closed. Bats were usually anaesthetised within modifications: PCR amplification was performed in 25 µL seconds and death was confirmed by auscultation with a volumes containing 1x Taq PCR buffer, 2.0 mM MgCl2, stethoscope. Kidney samples were removed for culturing 200 uM dNTPs, 10.0 pmol forward primer (5’- GTT TGA and molecular analysis. Carcasses were stored in 99% TCC TGG CTC AG 3’) and 10.0 pmol reverse primer (5’- ethanol for future use by UNIMAS students and kept as CCG CAC CTT CCG ATAC-3’) (Thaipadungpanit et al., museum voucher specimens following Abdullah et al. 2007), 1U Taq Polymerase (Fermatas, Cat#-EP0405), 2.5 (2010). All personnel who handled bats had been µL template DNA and nuclease free water to make up the vaccinated against rabies and all samples were treated as final volume of 25 µL. potentially infectious. Trapping of wildlife was approved by the Murdoch Polymerase chain reaction University Animal Ethics Committee (Animal ethics No: W2376/10) and Sarawak Forestry Department, Sarawak The DNA was amplified by using the following thermal- Malaysia (Permit No: NCCD.907.4.4 (V)-235). cycling profile: 95 °C for 5 min, followed by 30 cycles at 94 °C for 30 s, 58 °C for 30 sec and 72 °C for 1 min, and Diagnostic methods a final extension for 7 min at 72 °C. DNA sequencing was performed using the Big Dye Terminator (BDT) Microscopic Agglutination Test (MAT) sequencing kit version 3.1 (Applied Biosystems) using the original primer as described previously (Thaipadungpanit The Microscopic Agglutination Test (MAT) was performed et al., 2007). The PCR products were confirmed by according to Faine (1982) to check for Leptospira-specific agarose gel electrophoresis (1.5% w/v) of 5 µL PCR antibodies to 17 serovars commonly found in Malaysia: products for 45 min at 116V. Australis, Autumnalis, Bataviae, Canicola, Icterohaemorrhagiae, Celledoni, Grippotyphosa, DNA sequencing Javanica, Pomona, Pyrogenes, Hardjo, Tarassovi, Patoc, Djasiman, Lai, Copenhegeni, Lepto 175 Sarawak. The The cycle-sequencing products were purified by using preliminary results for Lepto 175 Sarawak have been sodium acetate-alcohol precipitation as per the presented previously (Thayaparan et al., 2013b). Serum manufacturer’s instructions (Applied Biosystems) and the positive at a titre of 1:50 was further titrated until 1:1600. purified products were forwarded to the First Base Laboratories Sdn Bhd, DNA sequencing facility, Selangor, Molecular analysis Malaysia, for capillary electrophoresis using an ABI 3130XL instrument. Culture and isolation and preparation of genomic DNA from kidney samples Analysis of sequence results
Kidney samples from all animals were cultured for The sequences were assembled and trimmed to a leptospires. A cross-section of the cortex of one kidney minimum of two contiguous sequences using Bio Edit was removed using a sterile scalpel and this section was software (Invitrogen). Sequences from identified strains cut into small pieces and inoculated into two EMJH and and representative 16S rRNA (1331 bp) gene sequences two Fletcher media tubes for culture. The remaining part from members of the genus Leptospira were aligned with was deposited in 100% pure PCR grade alcohol for future CLUSTAL W (Thompson et al., 1997). By using MEGA 5 PCR work. The tubes with the inoculated material were (Tamura et al., 2011), distances of aligned 16S rRNA incubated at 30 °C and checked twice weekly under dark gene sequences were estimated by the Jukes-Cantor field microscopy for 12 weeks. If there was no growth method (Jukes and Cantor, 1969), bootstrapped 1000 after 12 weeks, or if any fungal growth was detected, the times and the tree topology was determined by the specimen was discarded. Approximately 30 mg of kidney neighbour-joining method (Slack et al., 2006; Slack et al., was placed into a sterile Petri dish. Using a sterile scalpel 2008; Slack et al., 2009). The final phylogenetic tree was and a needle the tissue was dissected into small pieces. rooted by using Turneriella parva serovar Parva strain H The tissue was then transferred into a micro-centrifuge as an out-group and bootstrap values (1000) were tube and mixed with 50 µL protein kinase K (Qiagen) and displayed as percentages (Thaipadungpanit et al., 2007).
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(Tupaia tana) (n=8). The bats netted and trapped were RESULTS dusky fruit bats (Penthetor lucasi) (n=20), short-nosed fruit bats (Cynopterus brachyotis) (n=22), spotted-winged In total 155 animals were sampled including 70 bats, 57 fruit bats (Balionycteris maculata) (n=13), fawn roundleaf rodents, 20 squirrels, and eight treeshrew. The rats bats (Hipposideros cervinus) (n=5), Bornean horseshoe trapped were Müller’s rat (Sundamys muelleri) (n=29), bats (Rhinolophus borneensis) (n=2), bicolored roundleaf ricefield rat (Rattus argentiventer) (n=20), brown spiny rat bats (Hipposideros bicolor) (n=2), hollow-faced bat (Maxomys rajah) (n=7), and whitehead’s rat (Maxomys (Nycteris tragata) (n=1), intermediate horseshoe bat whiteheadi) (n=1). All squirrels were either Low’s squirrels (Rhinolophus affinis) (n=1), dusky roundleaf bat (Sundasciurus lowii) (n=1) or plantain squirrels (Hipposideros ater) (n=1), lesser woolly horseshoe bat (Callosciurus notatus) (n=19) and captured treeshrew ( Rhinolophus sedul us) ( n = 1), p a p i l l o s e
Table 1: Species, location and MAT results for animals other than bats caught.
Species UNIMAS Mt. Wind & Matang & Bako Total % MAT % Positive Singai Fairy Kubah (+) (95% CI)
Sundamys muelleri 10 6 3 4 6 29 34.1 20 68.9 (42.9, 84.7)
Rattus argentiventer 6 4 4 2 4 20 23.5 9 45.0 (23.1, 68.5)
Maxomys rajah 0 1 4 2 0 7 8.2 4 57.1 (18.4, 90.1)
Maxomys whiteheadi 0 0 1 0 0 1 1.2 0 0.0 (0, 97.5)
Sundasciurus lowii 0 0 0 1 0 1 1.2 1 100.0 (2.5, 100)
Callosciurus notatus 4 2 4 3 6 19 22.4 10 52.6 (28.9, 75.6)
Tupaia tana 0 0 1 4 3 8 9.4 1 12.5 (0.3, 52.7)
Total 20 13 17 16 19 85 100.0 45 52.9 (41.8, 63.9)
% 23.5 15.3 20.0 18.8 22.4 100.0
Table 2: Species, location and MAT results for bats caught.
Species UNIMAS Mt. Wind & Matang & Bako Total % MAT % Positive Singai Fairy Kubah (+) (95% CI)
Penthetor lucasi 5 2 11 0 2 20 28.5 12 60.0 (36.1, 80.9)
Cynopterus brachotis 10 3 0 4 5 22 31.4 9 40.9 (20.7, 63.6)
Balionycteris maculate 5 0 0 4 4 13 18.5 3 23.1 (5.0, 53.8)
Hipposideros cervinus 0 0 2 3 0 5 7.1 2 40.0 (5.3, 85.3)
Other bat species 0 0 6 3 1 10 14.2 2 20.0 (2.5, 55.6)
Total 20 5 19 14 12 70 100.0 28 40.0 (28.5, 52.4)
% 23.5 15.3 20.0 18.8 22.4 100 woolly bat (Kerivoula papillosa) (n=1) and dayak Serological and descriptive analysis roundleaf bat (Hipposideros dyacorum) (n=1). The largest number of animals was caught in the locality of UNIMAS The seroprevalence for rats (57.9%; 95% CI 44.1-70.9) (40) followed by Wind Cave and Fairy Cave areas (36), was slightly higher than that for squirrels (42.9%; 95% CI Bako National park (31), Matang and Kubah (30) and 24.5-62.8) and bats (40%; 95% CI 28.5-52.4), however Mount Singai (18) (Tables 1 and 2). these differences were not significant (χ2= 4.28; dƒ=2; P=0.117). The highest seroprevalence was found in Müller’s rat (68.9%; 95% CI 42.9-84.7) followed by brown spiny rat (57.1%; 95% CI 18.4-90.1), plantain squirrel (52.6%; 95%
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CI 28.9-75.6) and ricefield rats (45%; 95% CI 23.1-68.5). P=0.117), however significantly more seropositive The highest seroprevalence in bats was observed in the animals were detected at Mount Singai than at UNIMAS dusky fruit bat (60%; 95% CI 36.1-80.9), followed by the (OR 3.4; 95% CI 1.04, 11.17). short-nosed fruit bat (40.9%; 95% CI 20.7-63.6) and For the analysis of locality UNIMAS was selected as spotted-winged fruit bat (23.1%; 95% CI 5.0-53.8) (Tables the referent to ensure that all OR were greater than 1. 1 and 2). There was no significant difference in the Small mammals from Mount Singai were 3.4 times (95% seroprevalence between the different animal species (χ2= CI 1.04-11.17) more likely to have leptospiral antibodies 24.9; dƒ=18; P=0.126). than animals captured from the UNIMAS area. Animals Some seropositive animals were detected in all of the from Matang and Kubah were 2.4 times (95% CI 0.92, localities sampled. The highest prevalence was found at 6.42) more likely to have antibodies than those from the Mount Singai (64.7%; 95%CI 38.3-85.8) followed by UNIMAS area (not significant). Wind Cave & Fairy Cave Matang and Kubah (56.7%; 95%CI 37.4-74.5), Wind and Bako National Park had odd ratios of 1.7 and 1.5 Cave and Fairy cave (47.4%; 95%CI 30.4-64.5), followed respectively (95% CI 0.66, 4.18; 0.59, 4.0). There was by Bako National Park (45.2%; 95%CI 27.3-64.0) and little difference between the risk of animals from UNIMAS UNIMAS area (35%; 95%CI 20.6-51.7) (Table 3). There and those from Matang and Bako being seropositive was no significant difference in the seroprevalence (95% confidence intervals including the value 1.0) (Table between the five sampling location (χ2= 4.28; dƒ=4; 3).
Table 3: Seroprevalence to leptospires in small mammals according to their locality.
Locality No of seropositive Seroprevalence (95% CI) OR (95% CI) UNIMAS 14 35.0 (20.6, 51.7) 1.0 Mt. Singai 11 64.7 (38.3, 85.8) 3.4 (1.04, 11.17) Wind & Fairy Caves 17 47.2 (30.4, 64.5) 1.7 (0.66, 4.18) Matang & Kubah 17 56.7 (37.4, 74.5) 2.4 (0.92, 6.42) Bako NP 14 45.2 (27.3, 64.0) 1.5 (0.59, 4.0)
Table 4: Seroprevalence to leptospires in small mammals and their odd ratios.
Animals MAT Positive Seroprevalence (95% CI) OR (95% CI) Rats 33 57.9 (44.1, 70.9) 1.0 Squirrels & Treeshrew 12 42.9 (24.5, 62.8) 0.55 (0.24, 0.99) Bats 28 40.0 (28.5, 52.4) 0.48 (0.22, 1.36)
Among the Rodentia (rats), Scandentia (Squirrel, fruit bats. The antibody titres for seropositive animals treeshrew) and Chiroptera (bats), rats were selected as varied from 1:50 to 1:800. More animals had a serum the referent animal group. The odds of disease in dilution of 1:100 (n=65) than other dilutions (1:50 in 20 Scandetia and Chiroptera compared to Rodentia were serum samples; 1:200 in 30 serum samples and 1:400 in 0.55 (95%CI 0.24, 0.99) and 0.48 (95% CI 0.22, 1.36), 8 samples, while only one squirrel had a dilution of 1: indicating these animals were less likely to be 800) (Table 5). seropositive than rats; although the Chiroptera result was not significant (Table 4). Culture and molecular analysis Antibodies to 10 different serovars were detected in rodents and six different serovars in bats. Antibodies to None of the culture sample maintained in the lab serovar Lepto 175 Sarawak were detected in 30 (24.7%) produced positive results and majority of the specimens rats, 11 (9.0%) squirrels and 27 (52.9%) bats. Antibodies were contaminated and after four weeks all the samples to serovar Icterohaemorrhagiae were detected in 20 (16.5 were discarded. Out of 155 kidney samples from %) rodentia and scandetia; sv. Australis in 13 (10.6 %) individual animals, only 17 were positive for Leptospira on blood samples and sv. Autumnalis in 12 (9.6 %) samples. the molecular study (10.97%, 95% CI 6.5, 17). The Serovars Australis and Lai were detected in one dusky majority of the positive results were from plantain fruit bat and one short-nosed fruit bat respectively. squirrels (53%; 95% CI 27.8, 77), Müller’s rat (35%; 95% Antibodies to serovar Pyrogenes were detected in 10 CI 14.2, 61.7) and brown spiny rats (12%; 95% CI 1.5, (19.6%) samples from dusky fruit bats and short-nosed 36.4). No other animals were positive on the PCR (Table 6).
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Table 5: Titres of antibodies to different serovars found in small mammals.
Titres Serovar Positive 1:50 1:100 1:200 1:400 1:800 Lepto 175 Sarawak 68 23 27 12 5 1 Australis 15 3 5 4 3 0 Automnalis 12 2 6 4 0 0 Icterohaemorrhagiae 21 5 13 3 0 0 Javanica 5 1 3 1 0 0 Pyrogenes 10 8 2 0 0 0 Patoc 25 8 14 3 0 0 Copenhageni 6 0 4 2 0 0 Pomoma 5 2 2 1 0 0 Lai 5 4 1 0 0 0 Total/% 172/100 56/32.5 77/44.7 30/17.4 8/4.6 1/0.6
Table 6: PCR results from the kidneys of small mammals and their potential PCR using Neighbouring method.
Place Species PCR Serovar Mt. Singai Callosciurus notatus Positive L. pomoma Sundamys muelleri Positive L. icterohaemorrhagiae UNIMAS Sundamys muelleri Positive L. lai Wind Cave & Fairy Caves Callosciurus notatus Positive L. pomoma Callosciurus notatus Positive L. pomoma Maxomys rajah Positive L. icterohaemorrhagiae Maxomys rajah Positive L. icterohaemorrhagiae Sundamys muelleri Positive L. lai Callosciurus notatus Positive L. pomoma Sundamys muelleri Positive L. icterohaemorrhagiae Matang & Kubah Sundamys muelleri Positive L. icterohaemorrhagiae Callosciurus notatus Positive L. pomoma Bako National Park Callosciurus notatus Positive L. pomoma Callosciurus notatus Positive L. pomoma Callosciurus notatus Positive L. pomoma Sundamys muelleri Positive L. icterohaemorrhagiae Callosciurus notatus Positive L. pomoma
DISCUSSION and recreationists visiting the forested mountain. The studies undertaken indicated a greater proportion of rats The results indicate that small mammals from Mount were seropositive than bats, squirrels and tree shrews, Singai were 3.4 times more likely to have leptospiral highlighting the importance of these species as carriers or antibodies than animals from the UNIMAS area. These reservoirs of leptospires. findings highlight potential risk for acquiring leptospirosis Deforestation, which is becoming commonplace to villagers living around Mount Singai and eco-tourists across Malaysia (Aiken and Leigh, 1992; Jomo et al., 2004; Abdullah, 2011), can promote the emergence of
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Mal. J. Microbiol. Vol 11(1) 2015, pp. 94-102 infectious diseases, such as leptospirosis, by placing state health department in 2011 in the Rejang basin found humans into contact with novel reservoirs or infectious that 31% of humans sampled were seropositive for agents (Arief, 2013; Matthias et al., 2005). Bats respond leptospirosis and infection was associated with farming to the destruction of their habitat at the level of and/or water activities (Suut et al., 2011). populations and communities, making their spatial and temporal dynamics particularly sensitive to anthropogenic CONCLUSIONS AND RECOMMENDATIONS activity (Calisher et al., 2006). Matthias et al. (2005) detected Leptospira serovar Icterohaemorrhagiae from This particular study has generate concerns that should one bat in the Peruvian Amazon region and proposed a lead to the health authorities to expand their disease rodent-bat cycle of infection. (Vashi et al., 2010) added control measures as there are significant levels of human further support to the growing awareness of the role activity at all five locations where the animals were played by bats in the transmission of this pathogen to sampled. Visitors and eco-tourists should be advised to humans and other animals. protect themselves and avoid direct contact with Forests in Southeast Asia, including Malaysia, are contaminated soil or water. Local nearby villagers who characterized by flowering and fruit production, are workers at the Universiti Malaysia Sarawak and substantially contributing to the abundance of rodents Universiti Teknologi MARA in Kota Samarahan should be (Nakagawa et al., 2007). A ricefield rat captured by screened to avoid major health risks among the students Mohamed-Hassan et al. (2012) at a military training camp and academic staff. Wherever possible, pest control in West Malaysia was identified as a carrier of leptospires. measures should be implemented to contain the rat Rats are also reservoirs of a number of other parasites population, particularly considering that these rodents and infectious pathogens, including the agent of plague, appear to be the main source of infection in these Yersinia pestis (Zahedi et al., 1984). locations. Studies have shown that rodents and bats are Wildlife that is undergoing rehabilitation at captive reservoirs for leptospirosis (Matthias et al., 2005; Vashi et facilities should be screened for Leptospira periodically al., 2010). In the current study the positive PCR result before their eventual re-introduction to the natural from kidney samples are considered to be indicative of a environment. This helps prevent them from transmitting carrier status of this pathogen in squirrels and rodents. In the disease to other species in the wild or to humans contrast animals positive on the MAT are not necessarily involved in their release. carriers as Leptospira-specific antibodies can be detected The pathogenesis of serovar Lepto 175 Sarawak also in convalescent sera and positivity to an MAT indicates needs to be monitored closely considering its similarities past or current infection but not necessarily renal to Leptopsira wolffii. The latter has been isolated from shedding (Faine et al., 1999). In the current study, both humans and animals by scientists in Thailand, Iran serovar Pomona was most prevalent among the plantain and India. It remains to be seen if serovar Lepto 175 squirrels, being detected in kidneys collected from Sarawak will be as virulent as Leptospira wolffii and animals from four out of the five regions sampled. Müller’s hence extra vigilance is required and the Malaysian rats were identified as carriers of both serovar Lai and health authorities need to implement disease control Icterohaemorrhagiae, with the latter also found in brown measures to prevent the occurrence of a potential spiny rats. Many small mammals also displayed high epidemic. levels of antibodies to the newly discovered strain sv Lepto 175 Sarawak, with plantain squirrels and Müller’s CONFLICT OF INTEREST rats being more commonly affected with this serovar. This Lepto 175 Sarawak was also found in a large number of The authors declare that there are no conflicts of interest. dusky fruit bats tested. At present serovar Lepto 175 Sarawak is best considered an intermediate strain, as its ACKNOWLEDGEMENTS lethal capacity is unknown. According to (Thayaparan et al., 2013b) Lepto 175 The authors would like to thank Murdoch University and was shown to have a close similarity to Leptospira wolffii SOS Rhino (US) and Eco-zoonosis grant of UNIMAS to group, which has been isolated from Thailand, Iran and provide the financial support to conduct the research in India (Zakeri et al., 2010; Balamurugan et al., 2013). Sarawak Malaysia. Especially thankful for Sarawak Antibodies to serovar Icterohaemorrhagiae, a well-known Forestry cooperation for providing permit (Permit No: pathogen implicated in many fatal cases of leptospirosis NCCD.907.4.4 (V)-235). Special thanks go to staffs from in humans, was the fourth-most common serovar Institute for Medical Research (IMR), Kuala Lumpur and detected after Australis and Autumnalis. Pyrogenes Universiti Malaysia Sarawak for technical and field seemed to be more common in bats while Autumnalis supports. This research is conducted according to the was found mainly in rodents. 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