Field rats form a major infection source of leptospirosis in and around Madurai, IndiaCG Priya1, KT Hoogendijk2, MVD Berg2, SR Rathinam3, A Ahmed4, VR Muthukkaruppan1, RA Hartskeerl4
1 Department of Immunology, Aravind Medical Research Foundation, No.1, Anna Nagar, Madurai - 625 020, Tamil Nadu, India
2 Department of Public Health and Food Safety, University of Utrecht, Utrecht, Netherlands
3 Uvea Clinic, Aravind Eye Hospital and PG. Institute of Ophthalmology, No.1, Anna Nagar, Madurai - 625 020, Tamil Nadu, India
4 KIT Biomedical Research, WHO/FAO/OIE Reference Centre for Leptospirosis, Meibergdreef 39, 1105 AZ Amsterdam, Netherlands
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0022-3859.37511
Source of Support: None, Conflict of Interest: None
Aims: To determine the seroprevalence of leptospires and to isolate Leptospira spp. from field rats and bandicoots in and around Madurai.
Keywords: DNA typing, isolation of leptospires, leptospiral serology, leptospirosis, rats, serotyping
Leptospirosis is a widespread zoonosis caused by pathogenic spirochaetes of the genus Leptospira. It assumes zoonotic importance since rodents and certain species of domestic animals such as dogs, pigs and cattle are known to be natural carriers of various serovars thus forming infection reservoirs in the transmission of infection to other animals and man, especially to professionally vulnerable groups such as sewage workers, farmers, butchers and veterinarians.  In humans, the severity ranges from a mild flu-like illness to a severe infection with renal and hepatic failure, pulmonary disease, myocarditis, hemorrhages and death.  Ocular manifestation (uveitis) of systemic leptospirosis occurs at a later stage and has been reported even after one year of systemic illness.  This form of uveitis is referred to as leptospiral uveitis. 
There are several reports indicating more than 30% seropositivity for leptospirosis in Southern India including Chennai and Madurai in Tamil Nadu. ,,, In 1993, after a heavy rainfall and unexpected flooding, many residents in and around Madurai developed an acute febrile illness. After the subsidence of fever, many of these patients developed uveitis. Following this, extensive studies on the seroprevalence of leptospirosis among humans were done at Aravind Eye Hospital, Madurai and Australis, Autumnalis, Icterohaemorrhagiae and Louisiana were identified as the most common serogroups in this region. Till date, around 200 new cases (10% of total uveitis cases) are reported every year at the Uvea clinic, Aravind Eye Hospital.  Madurai, situated on the banks of river Vaigai, has many rice fields on its outskirts. Leptospiral uveitis has often been encountered in connection with rice harvesting and a major proportion of the uveitis patients were farmers. ,,, Since the epidemiology of leptospirosis of any region is best reflected by the serovars carried by the rodents in that region, the present study was undertaken to determine the seroprevalence of leptospirosis and to isolate Leptospira spp. from rats and bandicoots in and around Madurai. Serotyping and DNA typing of the isolates were carried out to identify the infecting serovar and the results were compared with the most prevalent infecting serovar among leptospirosis patients in this region.
Samples: Thirteen rats (presumably Rattus rattus ) and five bandicoots were trapped alive from fields in and around Madurai. All animals had a healthy appearance. The animals were anaesthetized by means of chloroform inhalation. Blood samples for serology were collected by cardiac puncture. The abdomen was then opened and urine was collected from the bladder under sterile condition using a 1 ml syringe and finally one kidney was removed. The urine and kidney were used for isolation of leptospires. The study was carried out with the permission from the Government Multispeciality Veterinary Hospital, Madurai.
Serum: Blood (5 ml) was collected in vacutainer tubes and allowed to clot at room temperature. After centrifuging for 20 min at 2000 rpm, serum was separated, aliquoted and stored at -20 o C. It was not possible to collect serum from rat number 3.
Microscopic agglutination test (MAT): A panel of 18 serovars was used for MAT [Table - 1]. MAT was performed as per the standard procedure starting with a serum dilution of 1:20 up to 1:20480.  The highest dilution of serum showing 50% reduction in free-moving leptospires under dark field microscope was considered the end-titer.
Culture medium: For isolation, medium of Ellinghausen-McCullough as modified by Johnson and Harris (EMJH) supplemented with 200 µg/ml 5-fluorouracil (EMJH-FU) was used. ,,
BSA-Tween 40/80 medium containing 1% rabbit serum and 100 µg/ml 5-FU, 10 µg/ml rifampicin and 2 µg/ml amphotericin B was a kind gift from Dr. Brem, Germany and Dr. Ellis, United Kingdom.
Culture: The urine and macerated kidney samples were inoculated in EMJH-FU medium within two hours after removal. The pH of the samples was alkalinized by adding PBS of pH 8.
Kidney: Kidneys were rinsed with sterile distilled water and put in a sterile Petri dish More Details. In the laminar hood, a piece of 0.5 cm 3 , containing mostly the cortex was cut and macerated using mortar and pestle. It was then suspended in 0.5 ml EMJH medium and 0.5 ml PBS and sodium hydroxide (pH 8.0). The suspension was then centrifuged for 5 min at 800 rpm to remove the tissue material. 0.5 ml of the supernatant was inoculated in 5 ml EMJH-FU medium and further two 10-fold dilutions were made from the initial inoculum to dilute potential growth inhibitor.
Urine: Urine was centrifuged for 15 min at 4000 rpm. Strongly acid urines were made alkaline by adding PBS, pH 8.0 . 0.5 ml of the sediment was inoculated in 5 ml EMJH-FU medium and two 10-fold serial dilutions from the initial inoculum were made in the same culture medium.
The samples were incubated at 28-30 o C up to four months and checked regularly for the presence of Dinger's disc and for live leptospires under dark field microscope. The cultures were checked once a week in the first month, once in two weeks in the second and once in a month in the third and fourth months.
Contaminated positive cultures were subcultured in BSA-Tween 40/80 medium.
Microscopy and staining
The morphological characters of the isolates were studied using dark-field microscopy and modified silver staining technique . 
13° C test: To determine the pathogenic status of Leptospira isolates, growth rates in EMJH medium at 13 o C and 30 o C were determined according to Johnson and Harris (1967). 
Briefly, 0.1 ml cultures were inoculated into 10 ml EMJH medium in four tubes and duplicates incubated at 13 o C and at 30 o C to determine the growth rates at a low and high temperature respectively. Leptospira growth was determined by dark field microscopy up to 12 days. Pathogenic strain Wijnberg and saprophytic strain Patoc I were included in the test for comparison of growth responses.
Serological typing: MAT with group sera and monoclonal antibodies: To identify the isolate up to serogroup status, MAT was performed following standard procedure using a panel of 41 anti- Leptospira rabbit antibodies.  Isolates were further typed at the serovar level by performing MAT with a panel of monoclonal antibodies (mAbs) that characteristically agglutinate serovars from the serogroups Autumnalis (F64C2, F64C6, F64C7, F64C8, F64C10, F65C3, F69C2, F69C8, F69C9, F69C10, F69C11, F69C12, F69C14, F69C15) and Javanica (F12C3, F20C3, F20C4, F70C20, F98C4, F98C5, F98C8, F98C12, F98C17, F98C19 and F98C20).  Genetic characterization: Strains and isolates were grown at 30 o C in EMJH medium and harvested by centrifugation during the late logarithmic phase. The DNA was isolated as described by Boom et al .  PCR was performed on the DNA extracts using the following sets of primers: (i) PCR with primer pairs Lepat1/2 and Sapro1/2 according to the procedure described by Murgia et al .  Lepat1/2 is specific for pathogenic leptospires while sapro1/2 specifically amplifies DNA from saprophytic leptospires. And (ii) G1/G2 and B64-I/B64-II.  Primer set G1/G2 amplifies DNA from all pathogenic species except L. kirschneri and primer set B64-I/B64-II specifically amplifies DNA from species L. kirschneri . The PCR conditions and controls were done as previously described. , The PCR products were further analyzed by agarose gel electrophoresis on gels containing 1.5% agarose, stained with ethidium bromide using standard procedures and subsequently judged by eye under UV illumination.
Southern blot analysis of G1/G2-generated PCR products was performed with DIG-labelled probe G195-28 according to Bal et al . , with a modification that hybridization of the probe was done at 38 o C. 
For sequencing, DNA concentration of PCR products were adjusted in the range of 10-20 ng per reaction and applied to the sequence reaction using the BigDye® Terminator V1.1 Cycle Sequencing Kit (Applied Biosystems, United Kingdom) and subsequently analyzed on an ABI PRISM TM 310 Genetic Analyzer (Applied Biosystems, United Kingdom). The DNA sequence clustal alignments were done using the LaserGene software package (DNASTAR). Species determination was done on basis of a higher sequence identity of PCR products from strains within a single species compared to sequence identities from strains belonging to different species. ,
In the MAT, none of the bandicoots were positive for leptospirosis.
Seven out of 12 (58%) field rats were seropositive by MAT. According to highest titers, one serum sample had a titer of 1:20 against the Australis group, two sera had a titer of 1:40 against the Hebdomadis and Tarassovi groups and four sera had titers ranging from 1:80 to 1:1280 against serogroups Australis (1x), Autumnalis (2x) and Hebdomadis (1x). Cross-reacting serogroups included Javanica, Hebdomadis, Australis and Tarassovi. No reactions were detected against serogroups Canicola, Grippotyphosa, Icterohaemorrhagiae, Mini and Sejroe.
Leptospira were isolated from two urine samples (rats numbered 11 and 13) and six kidney samples of rats (rats numbered 4, 5, 8, 10, 11 and 13). No isolates were obtained from urine and kidney samples of bandicoots.
Morphology and staining
All isolates showed typical morphology and characteristic motility of the genus Leptospira under dark-field microscopic examination. The cells were stained well by silver impregnation technique [Figure - 1].
All isolates from the kidney or urine of rats numbered 4, 5, 8, 10, 11 and 13, together with the control strains Wijnberg (pathogenic) and Patoc I (saprophytic) did grow well at 30°C. However, at 13°C only Isolate 5 and Strain Patoc I did grow while growth of Isolates 4, 8, 10, 11 and 13 together with Wijnberg was severely hampered. This result is suggestive of a pathogenic status for Isolates 4, 8, 10, 11 and 13 and a saprophytic status for Isolate 5.
In the PCR and subsequent agarose gel electrophoresis analysis, the isolate from the kidney of rat number 5 (KR 5) gave a strong product with sapro1/2 primer and only a very weak band with lepat1/2 primer, supporting its saprophytic status. All other isolates gave a strong signal with lepat1/2 primer and no signal with sapro1/2 primer (results not shown). The DNA of all isolates amplified a 285 bp product in PCR using primer pairs G1/G2. The DNA from Isolate KR-5 gave a non-specific signal with B64-I/B64-II primer while all other isolates were negative. Thus none of the isolates belonged to L. kirschneri .
When applying Southern blotting on the G1/G2 generated products, all products except from KR-5 gave a positive signal with DIG-labeled probe G195-28, indicating that the G1/G2-generated amplicon from KR-5 represents an anomalous product. This finding confirms the results of the conventional 13 o C test and pathogenic-specific PCR that all isolates except KR-5 are pathogenic leptospires.
MAT with 41 serogroup-specific sera followed by panels with mAbs characteristically agglutinating serovars of the serogroups Autumnalis and Javanica revealed that six isolates (isolates from urine of rats numbered 11 and 13 and from kidneys of rats numbered 4, 10, 11 and 13) belonged to serovar Autumnalis, strain Akiyami A and one isolate (Rat 8) belonged to Javanica, strain Veldrat Batavia 46 (results not shown). The isolate from Rat 5 was non-reactive with the polyclonal antibodies tested which further implicates that this is a saprophytic isolate.
Isolates and MAT
To determine homologous titers, rat sera were subjected to MAT using the isolates as antigen. Only the sera from Rats 10 and 11 produced titers. Agglutination titers against their own isolates did not differ significantly from those obtained with isolates from Rats 4, 8 and 13 [Table - 2]. For Isolates 4 and 13 this was not unexpected as all four isolates, 4, 10, 11 and 13 were typed as the same serovar Autumnalis, strain Akiyami A. Isolate 8, typed as serovar Javanica, is not related to serovar Autumnalis. Therefore, the titers produced with Isolate 8 are rather due to exposure to serovar Javanica than to cross-agglutination of the sera. No agglutination titers were found with the sera from Rats 1, 2, 4 - 9, 12 and 13 using the isolates as antigen in the MAT. Moreover, field rats numbered 4, 8 and 13, did not show any serological proof of leptospirosis, including in the MAT with reference strains, though leptospires were isolated from their urinary tract or kidneys. None of the sera from the rats agglutinated with the Leptospira isolate from Rat 5, which is in accordance with the saprophytic status of this isolate.
Serogroup Autumnalis, serovar Autumnalis, strain Akiyami A belongs to species Leptospira interrogans sensu stricto while serogroup Javanica, serovar Javanica, strain Veldrat Batavia 46 belongs to L. borgpetersenii .
Sequencing of G1/G2-generated products and subsequent homology analysis were in concordance with this classification. The DNA sequences of the isolates from Rats 4, 10, 11 and 13 showed highest homology with sequences of reference strains belonging to L. interrogans s.s. and the product of Rat 8 shared the highest sequence identity with sequences from reference strains of L. borgpetersenii .
Rodents have been shown to be the main carriers of pathogenic leptospires and their local abundance can be an indicator of the potential transmission of Leptospira spp. to humans and livestock. , In India, the natural leptospiral infection in rats was first documented in Kolkata, subsequently in Mumbai and in Kerala with the isolation of Autumnalis, Hebdomadis and Javanica in rats and bandicoots. ,, Javanica was isolated in Karnataka and in Tamil Nadu, isolation of Autumnalis and Javanica was possible from rats, bandicoots and goats (unpublished observations). , A 46% positivity of viable leptospires belonging to the serogroups Autumnalis and Javanica from the field rats in our study is consistent with these previous findings and indicates that these rats might represent the natural carriers of serovars from these serogroups.
Paragoankar (1957) demonstrated 11% seropositivity against Leptospira among rats in Andhra Pradesh but the seropositivity was 52% in Tamil Nadu mostly to serogroup Autumnalis. , Earlier studies in Tamil Nadu have revealed high titers to Autumnalis followed by Icterohaemorrhagiae, Panama and Sejroe in patients with systemic leptospirosis. , In patients with leptospiral uveitis, Australis, Autumnalis, Icterohaemorrhagiae and Louisiana have been identified as the most common serogroups. ,,, A 58% seropositivity with the highest titer to Autumnalis in rats in our study is in accordance with Autumnalis being a dominant infecting serogroup. The high seroprevalence and the presence of viable leptospires in field rats signify that these rats could be a major source of leptospiral infection to humans in Madurai and its surroundings.
The results of MAT and culturing revealed that Rats 4, 8, 10, 11 and 13 were asymptomatic carriers of pathogenic leptospires. Three of these five rats (60%) had a negative serology in the MAT. This phenomenon of isolation positive, serologically negative rodents has been observed repeatedly by different authors independently on the infecting serovars, indicating the limitations of serology to determine the carrier status of natural hosts. 
Even though no isolation or seropositivity to leptospires was observed in bandicoots in this study, reports are available from India that they too are carriers of serogroups Autumnalis and Javanica. , In a concomitant random serosurvey on cows and dogs in and around Madurai, we found 90% of the cows and 70% of the dogs to be seropositive. In cows there was a wide variety of reacting serovars. Notably serovars of the serogroups Mini, Sejroe, Hebdomadis, Grippotyphosa, Iceterohaemorrhagiae and Pyrogenes agglutinated till high titers (reciprocal titers 1280 to 5120). In dogs, significant titers (≥160) were found against serogroups Autumnalis, Icterohaemorrhagiae, Sejroe and Canicola (unpublished observations). Agglutination and cross-agglutination with a variety of serovars was also observed in the sera of 12 rats. Apparently, there is a high Leptospira infection rate between domestic and wild animals. We therefore hypothesize that humans in the region of Madurai are also at high risk for acquiring leptospirosis and subsequently leptospiral uveitis from various infection sources.
Isolation of leptospires belonging to serogroup Autumnalis from a high proportion of rats identifies these rodents as a major natural carrier and shedder of leptospires and hence as an important source of infection to humans and other animals. Future research will include isolation and typing of leptospires from human and animal patients to confirm Autumnalis as a common serogroup with rats a major source in the region.
[Figure - 1]
[Table - 1], [Table - 2]