Leptospirosis is a zoonotic disease with a worldwide distribution. Leptospira weilii serovar (sv.) Topaz is a newly described serovar first isolated in the far north of Queensland, Australia. The epidemiology of L. weilii sv. Topaz infections in Australia was characterised through the use of surveillance questionnaires and molecular studies. There have been 24 human and 2 animal (bovine and bandicoot) L. weilii sv. Topaz infections diagnosed since 1991. The majority of these infections have occurred in Far North Queensland, with the remaining infections occurring in South East Queensland and in Western Australia. The majority of patients with L. weilii sv. Topaz infections presented with classical leptospirosis symptoms including; fever, headaches, sweats, chills and myalgia. The occupations of human cases of L. weilii sv. Topaz infection included banana farming, dairy and beef cattle production and tourist related activities. Fluorescent amplified fragment length polymorphism (FAFLP) was performed on 15 L. weilii sv. Topaz isolates including 2 animal isolates. Clustering analysis grouped the 15 isolates into 5 main clades with 13 unique FAFLP profiles. A high level of relatedness was demonstrated between 2 animal and 2 human isolates. Commun Dis Intell 2007;31:216–222.

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Introduction

Leptospirosis is caused by infection with spirochaetes of the genus Leptospira.¹ Leptospira are motile helical spirochaetes that metabolise long chain fatty acids as their sole carbon source.¹ The genus Leptospira contains 17 species as delineated by DNA-DNA hybridisation.^2–5 Leptospira are divided serologically into serovars of which there have been over 200 described. Leptospirosis is one of the world’s most widespread zoonotic diseases with outbreaks reported worldwide in both humans and animals.^6,7,8 The organism enters humans through contact of abrasions or mucus membranes with urine or body fluids from an infected animal. This may occur directly or indirectly through contact with contaminated water or soil. Leptospirosis is more prevalent in tropical countries than temperate countries as the higher humidity, rainfall and temperature promote the survival of the organism in the environment.⁹

Leptospirosis was first reported in Australia in 1933. Since 1991, leptospirosis infections have been notified to the National Notifiable Diseases Surveillance System. From 1991 to 2005, there have been 7,629 cases notified in Australia.¹⁰ The majority of cases have occurred in Queensland situated on the eastern seaboard of Australia.¹¹ Twenty-three Leptospira serovars have been identified in Australia including several which were first discovered in Australia.¹¹ L. weilii sv. Topaz was first isolated by Corney, et al. from a bovine source near the township of Topaz in Far North Queensland and is the second member of the L. weilii species found in Australia.¹² It was found to be a unique serovar by cross agglutinin absorption test (CAAT). The aim of this study is to present the descriptive and molecular epidemiology of L. weilii sv. Topaz in Australia.

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Materials and methods

Serological identification of L. weilii sv. Topaz

Leptospira IgM-specific enzyme linked immunosorbent assay (ELISA, Panbio) was performed as a screening test by the submitting private and hospital laboratories. Positive ELISA samples were forwarded to the WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis, Brisbane (WHO Reference Centre) for confirmation and serovar identification using the microscopic agglutination test (MAT). L. weilii sv. Topaz strain 94-79979/3 was used as the reference culture in the MAT for the detection of human antibodies against L. weilii sv. Topaz. Patients with a positive IgM ELISA supported by a single MAT titre of greater than or equal to 400, or a demonstrated fourfold rise or fall in MAT titres over paired specimens, were considered to meet the notification criteria for leptospirosis.

Culture identification

Leptospira culture from humans was performed as follows: 2–5 drops of uncoagulated whole blood was inoculated into Ellinghausen McCullough Johnson Harris (EMJH) media containing 0.5% agar. This was performed by the submitting laboratories and forwarded at room temperature to the WHO Reference Centre. Once at the WHO Reference Centre, the cultures were sub-cultured into EMJH media (containing no agarose) and incubated at 30°C for 6 weeks. The cultures would be inspected weekly for the growth of Leptospira using dark field microscopy. Positive cultures were identified to a species level by sequencing of a partial fragment of the DNA gyrase sub-unit B gene (gyrB).¹³ Serological identification of the isolates was performed by MAT using reference antisera covering the major serogroups of the genus Leptospira. Hyper-immune antiserum was prepared in rabbits using standard techniques.¹⁴ Definitive serovar identification was performed by CAAT¹⁵ using the L. weilii sv. Topaz strain 94-79979/3.

Surveillance questionnaires and descriptive epidemiology

Information regarding infection was collected from the patient’s doctor using enhanced surveillance questionnaires. This provided information on symptoms, recreational activities, animal contacts, occupational data and hospitalisation. The data were entered into a Microsoft Access database and Microsoft Excel was used to conduct statistical analysis of these data.

Fluorescent amplified fragment length polymorphism

Cultures were prepared for DNA extraction by centrifugation of 1 mL of an actively growing Leptospira culture in EMJH at 12,500 g for 5 minutes. Genomic DNA was then extracted from the pellet using the ChargeSwitch gDNA mini bacterial kit (Invitrogen) as per manufacturers’ instruction. FAFLP restriction digestion, ligation and amplification reactions was performed as previously described.^16,17

Fragment sizing and data analysis

One mL of the 6 selective polymerase chain reaction products was mixed with 18.5 mL of HiDi formamide (Applied Biosystems) and 0.5 mL of Geneflo-625 (Chimerx) and denatured at 95°C for 5 minutes. The products were then loaded onto the ABI-310 capillary sequencer (Applied Biosystems) and were injected into a 47 cm capillary filled with performance-optimised polymer 4 (Applied Biosystems) at 15 kV for 12 seconds. The fragments were separated at 1 3kV for 35 minutes. The resulting electropherograms were manipulated using the Genotyper v2.5 software (Applied Biosystems) and the combined allele sizes were exported into an Excel spreadsheet. A previously described Excel macro¹⁸ was used to convert the alleles into a binary sequence suitable for analysis using Bionumerics software (Applied maths). Each unique FAFLP binary pattern was assigned a letter code (e.g. A, B, C) to allow for easier referencing of the data.

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Results

Descriptive epidemiology of Leptospira weilii sv. Topaz

Since the initial isolation of L. weilii sv. Topaz in 1994,¹² there have been 26 additional cases of this serovar identified: 24 from human and 2 from animal sources (bovine and bandicoot). The first isolation of L. weilii sv. Topaz predates the identification of the type strain; 94-79970/3 having been isolated in 1991 and was identified at the time as a member of the Tarrasovi serogroup. Subsequently, examination of the isolate by CAAT using L. weilii sv. Topaz strain 94-7997/03, identified the isolate as serovar Topaz. Fifteen cases were diagnosed from Leptospira cultures taken from blood (human), urine (bovine) or from kidney tissue (bandicoot). The remaining 11 cases were diagnosed by MAT with titres ranging from 1 in 800 to 1 in 6,400 (Table 1).

Table 1. Leptospira weilii serovar Topaz cases in Australia, 1991 to 2006

Year	Source	Laboratory details	Age	Sex	Serum MAT titre (against strain 94-79970/3)	Culture
1991	Human	LT 596*	Unknown	F	Not performed	Detected
1994	Bovine	94-79970/3* (type strain)	N/A	N/A	N/A	Detected
1996	Human	LT 762*	30	M	1600	Detected
1998	Human	LT 925*	34	M	Not performed	Detected
1999	Human	LT 952*	22	M	800	Detected
1999	Human	LT 969*	33	M	Not performed	Detected
1999	Human	LT 974*	21	M	Not performed	Detected
1999	Human	LT 981*	39	M	400	Detected
2000	Human	LT 1060*	58	M	1600	Detected
2000	Human	LT 1055*	37	M	Not performed	Detected
2001	Human	LT 1187*	14	M	Not performed	Detected
2001	Human	LT 1188*	33	M	800	Detected
2001	Human	LT 1191*	28	M	1600	Detected
2002	Bandicoot	LT 1412*	N/A	N/A	N/A	Detected
2002	Human	LT 1414*	48	M	Not performed	Detected
2004	Human	TSI 1†	62	M	800	Not performed
2004	Human	TSI 2†	23	M	800	Not performed
2004	Human	TSI 3†	40	M	800	Not performed
2005	Human	TSI 4†	28	M	3200	Not performed
2005	Human	TSI 5†	64	M	1,600	Not performed
2005	Human	TSI 6†	54	M	800	Not performed
2005	Human	TSI 7†	47	M	800	Not performed
2006	Human	TSI 8†	56	F	800	Not performed
2006	Human	TSI 9†	22	M	6400	Not performed
2006	Human	TSI 10†	24	M	800	Not performed
2006	Human	TSI 11†	21	M	1,600	Not performed

*  Diagnosis of Leptospira weilli sv. Topaz made by identification of the Leptospira isolate from the blood culture

†    Diagnosis of Leptospira weilli sv. Topaz made by serology using the microscopic agglutination test (MAT).

N/A  Not applicable.

Since 1994, L. weilii sv. Topaz infections in humans have been reported every year except in 1997 and 2003. Four cases were reported in 1999, 2005 and 2006 (up to August) (Figure 1). The majority of isolations occurred between January and June, with a single case reported in October, this is consistent with the seasonal trend of Leptospira infections noted by Slack, et al.¹¹

Figure 1. Distribution of Leptospira weilii sv. Topaz cases from 1996 to August 2006 compared with the total Queensland leptospirosis notifications for the same period

The geographical distribution of L. weilii sv. Topaz in Australia has been dominantly in the far north of Queensland around the leptospirosis endemic areas extending south from Cairns to Innisfail and Tully, and west onto the eastern side of the Atherton Tablelands (Figure 2). Twenty cases (77%) of L. weilii sv. Topaz have originated from this area; an area that has accounted for approximately 66% of Queensland leptospirosis notifications from 1998 to 2004 (Figure 2).¹¹ Five cases of L. weilii sv. Topaz were detected in South East Queensland and 1 case in northern New South Wales. The first detection of L. weilii sv. Topaz away from the eastern seaboard of Australia was a case in 2005 from Western Australia in which the patient had no history of recent travel to the eastern seaboard.

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Figure 2. Geographical distribution of Leptospira weilli sv. Topaz cases, Australia, 1991 to 2006

Of the 24 L. weilii sv. Topaz cases in humans, there was a male:female ratio of 11:1, which is consistent with the sex distribution of leptospirosis in previous studies.^11,20 The age of the patients ranged from 14 years to 64 years with a median age of 33 years. Twelve (50%) cases were reported to have been hospitalised with an average stay of 3 days. Patients with L. weilii sv. Topaz infections presented with classical symptoms of leptospirosis including; fever, headaches, sweats, chills and myalgia. Two patients presented with the more severe leptospirosis complications of pulmonary haemorrhage and aseptic meningitis (Table 2). The occupations of cases of L. weilii sv. Topaz infection included banana farming, dairy and beef cattle production and tourist related activities (Table 2). Contact with animals before infection was reported in the majority of cases, which is consistent with the rural occupations and/or recreational exposure generally associated with Leptospira infections. The major animal contacts reported include rats or mice (this included native rodents such Rattus fuscipes or R. sordidus as well as the imported R. rattus and Mus domesticus), dogs, beef and dairy cattle (Table 2).

Table 2. Clinical and epidemiological characteristics of Leptospira weilli sv. Topaz infections

Clinical characteristics	Positive responses n=18	Percentage of positive responses
Fever	14	78
Headache	12	67
Arthralgia	10	56
Sweats	10	56
Chills	9	50
Myalgia	9	50
Nausea	7	39
Vomiting	6	33
Back pain	4	22
Respiratory symptoms	2	11
Asceptic meningitis	1	6
Conjunctival suffusion	1	6
Diarrhoea	1	6
Liver involvement	1	6
Pulmonary haemorrhage	1	6
Rash	1	6
Vision disturbance	1	6
Occupation	Positive responses n=24	Percentage of positive response
Banana farmer	4	17
Tourist operator/tourist	4	17
Dairy	3	13
Grazier	3	13
Labourer/tradesperson	2	8
Veterinarian	1	4
Not reported or unemployed	7	29
Animal	Positive responses n=20	Percentage of positive responses
Rats and mice	9	45
Dogs	8	40
Beef cattle	5	25
Dairy cattle	3	15
Feral pigs	3	15
Cats	2	10
Domestic pigs	1	5
Bandicoot	1	5
Sheep	1	5
No contact	1	5

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Molecular epidemiology of Leptospria weilii sv. Topaz

FALFP was performed on all 15 L. weilii sv. Topaz isolates including the 2 animal isolates (94-79970/3 and LT1412). There were 13 unique FAFLP profiles (designated A to M) amongst the 15 isolates tested. Clustering analysis of the FAFLP data was performed using the Jaccard coefficient (>50% mean) and unweighted pair group method with arithmetic mean (UPGMA) algorithm (Figure 3). There were 5 identifiable clades (designated i to v) within the dataset each containing between 2 and 4 isolates. Each clade contained isolates found over multiple years and showed clustering around the geographic area of isolation. For example clade ii and clade v contained isolates from the Cairns or Atherton tableland section of Far North Queensland whilst clade i and iv contained isolates from the more southern areas of Tully and Innisfail. An isolate from a native bandicoot species (LT1412) showed a high level of similarity to the human isolate; LT952. The type strain 94-7997/03 isolated from cattle, was found to share an identical FAFLP pattern with the human isolate; LT974.

Figure 3. Clustering analysis of Leptospira weilii sv. Topaz constructed from the FAFLP data using the Bionumerics software package

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Discussion

L. weilii sv. Topaz has been identified in both animal and human sources since its initial isolation in 1994 using both culture and serological methods (IgM ELISA combined with MAT) (Table 1). By utilising both methods initially, we were able to validate the specificity of the MAT in the diagnosis of L. weilii sv. Topaz infections. Diagnosis by MAT alone has been possible since the inclusion of L. weilii sv. Topaz in the WHO Reference Centre MAT panel since 2000. Initially, this testing was conducted by screening samples that had low L. borgpetersenii sv. Tarassovi titres (L. weilii sv. Topaz produces a serological cross-reaction with members of the Tarassovi serogroup, data not shown) for the presence of L. weilii sv. Topaz antibodies and in 2004 was added to the panel as a standalone serovar. Whilst the MAT has been criticised for a lack of specificity when determining the infecting Leptospira serovar,19 we believe that its use in a well-defined leptospirosis environment such as Australia means that it can be sufficiently relied upon to determine the individual serovar involved.

The geographical distribution of L. weilii sv. Topaz in Australia suggests that there are 2 distinct pockets of this serovar in Queensland and northern New South Wales. However, the single Western Australia case of L. weilii sv. Topaz may indicate that this serovar may be much more widespread in Australia compared to the more geographical isolated Leptospira serovars such as L. interrogans sv. Zanoni.¹¹

Since L. weilii sv. Topaz infection has been isolated from a native animal (bandicoot) this serovar may be indigenous to Australia.¹¹ Several Leptospira serovars in Australia are not found elsewhere in the world.¹¹ A recent unpublished study examining leptospirosis in Macropods (kangaroos), found that a significant proportion of the study animals had serological titres that would indicate exposure to L. weilii sv. Topaz. (personal communication, M Roberts and L Smythe).¹¹

Overall, L. weilii sv. Topaz infections represent only 1.9% of the 1,288 reported leptospirosis infections (based upon Queensland data from 1996 to August 2006), however this figure is likely to be an under-estimate since assays for its identification have only recently (since 2000) been available at the WHO Reference Centre. To assess the geographic distribution of the serovar in Australia, it is recommended that all Australian laboratories performing leptospirosis testing include L. weilii sv. Topaz in their MAT panels or alternatively, that the samples with serovar Tarrasovi titres are forwarded to the WHO Reference Centre for confirmatory testing.

The epidemiology of L. weilii sv. Topaz infections is similar to that of other leptospirosis infections in Australia.¹¹ There is a higher likelihood of Leptospira infection in the period from January to May, as this is the peak rainfall period for Far North Queensland. The high rainfall combined with relatively high ambient temperatures provides ideal survival conditions for Leptospira in the environment, translating to a higher risk to humans.

Males of working age (18 to 60) are the most at risk. The median age of 33 years at infection with L. weilli sv. Topaz is consistent with other studies of leptospirosis in Australia.¹¹ The majority of L. weilli sv. Topaz patients report symptoms classically associated with leptospirosis. Banana farming and dairy/beef cattle farming appear to be the most at risk occupation groups. Both these occupations require contact directly and indirectly with animals and at times require close contact with contaminated soil and water. The other major occupation at risk is the tourist operator/tourist sector, accounting for 4 (17%) of the total number of L. weilii sv. Topaz infections (Table 2). A recently study by Slack, et al demonstrated that 17.8% (n=883) of Leptospira infections in Queensland were from recreational exposure.¹¹

The isolation of L. weilii sv. Topaz from a bovine source may require further risk assessments to determine the risk posed to the domestic animal industries or workers in these industries in Australia. Additionally, further studies are required to determine what are the carriage rates and the level of disease caused by this serovar in Australian animal herds and wildlife.

FAFLP has been successfully used to examine the molecular epidemiology of Leptospira isolates.^16,17 Using FAFLP we were able to demonstrate a high level of relatedness amongst 2 animal and 2 human isolates (LT1414 and LT952, LT722 and LT974). These molecular tools allow links to be made between the carriage of this serovar in animals and human disease.

In conclusion, we have described both the descriptive and molecular epidemiology of L. weilii sv. Topaz in Australia. This research provides evidence for the presence of this serovar in native, domesticated animals and humans, however the role and burden of this serovar in animal health and disease needs to be further defined by additional research. Further research is needed to establish the prevalence of this serovar in Australian native fauna and animal industries.

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Acknowledgements

The authors would like to thank the staff of the Queensland Health Population Health Units and other submitting laboratories for their assistance in the completion of surveillance questionnaires. The authors also wish to thank Queensland Health and the Queensland Department of Primary Industries and Fisheries for their past and present support of leptospirosis research in Australia.

Author details

Andrew T Slack¹

Meegan L Symonds¹

Michael F Dohnt¹

Bruce G Corney²

Lee D Smythe¹

1. WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis, Western Pacific Region, Centre for Public Health Sciences, Queensland Health Scientific Services, Brisbane, Queensland

2. Animal Research Institute, Queensland Department of Primary Industries and Fisheries, Yeerongpilly, Queensland

Corresponding author: Mr Lee Smythe, Principal Scientist, WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis, 39 Kessels Road, Coopers Plains QLD 4108. Telephone: +61 7 3274 9061. Facsimile: +61 7 3274 9175. Email: Lee_Smythe@health.qld.gov.au

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References

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This report was published in Communicable Diseases Intelligence Vol 31 No 2, June 2007.