Leptospires in field Rats in and around the laboratory animal facilities of Banglore , India

The present study was undertaken to determine the prevalence of leptospires in field rats in and around laboratory animal facilities in Bangalore. 34 rats were trapped alive in and around the laboratory animal facilities in Bangalore. Urine and serum samples from theses field rats were collected. Serum samples were tested for anti-leptospiral antibodies by microscopic agglutination test, while urine samples were subjected for dark field microscopy and polymerase chain reaction to detect the presence of leptospiral antigens. Serology revealed the presence of antileptospiral antibodies in 19 (61.29 percent) field rats and dark field microscopy revealed the presence of leptospiral antigens in 3 (8.82 percent) and 6 (17.65 percent) of urine samples of these field rats. Among the serovars, Icterohaemorrhagiae was predominant followed by Autumnalis and Pyrogens. Serology dark field microscopy and polymerase chain reaction reveals that field rats are major natural carriers and shedders of leptospires.


Introduction
species of rodents that are distributed worldwide and are commonly associated with leptospiral infection: Leptospirosis is wide spread zoonosis caused by Mus musculus (house mouse), Rattus norvegicus pathogenic spirochetes of the genus Leptospira.It (brown rat) and Rattus rattus (black rat).All three assumes zoonotic importance since rodents and species belong to the order Rodentia, family Muridae, certain species of domestic animals such as dogs, pigs subfamily Murinae.In rodents, especially the and cattle are known to be natural carriers of various common rat (Rattus norvegicus) and other reservoir serovars thus forming infection reservoirs in species, the organisms persist indefinitely in the transmission of infection to other animals and man, convoluted tubules of the kidney without causing especially professionally vulnerable group such as apparent disease, and are shed into the urine in massive veterinarians, animal care takers.Certain vertebrate numbers ( Dutta and Christopher et al., 2005).Rattus animal species have a commensal relationship with rattus wroughtoni hinton, Rattuis rattus rufesens, leptospires in which they are the natural hosts for Bandicota indica and Bandicota bengalenses are the pathogenic leptospires that live in their kidneys.Such rodent species involved in spreading leptospirosis to leptospires do little or no detectable harm to these human and live-stock in India (Gangadhar et al., hosts but they maintain the infection and are therefore 2005).With this in view the present study has been known as natural maintenance hosts.If other animals undertaken with the objectives to record the that are not natural maintenance hosts (including prevalence of leptospirosis in field rats in and around humans) are infected by the same pathogenic laboratory animal facilities in Bangalore.leptospires, they often become ill.In addition, if a maintenance host for a particular leptospire is infected

Materials and Methods
with another serovar it may develop symptoms and Present study was undertaken at veterinary signs of Leptospirosis (WHO 2003).
college Bangalore.34 urine and 31 serum samples Rodents are carriers of leptospires throughout from field rats trapped alive in and around five the world and are important reservoirs of infection for different laboratory animal colonies in Bangalore man and domestic animals (Twigg et al., 1969; were collected.Field rats were anesthetized by means Hathaway and Blackmore, 1981).There are three of Diethyl Ether inhalation.Blood samples for denaturation for 5 min at 94°C.The number of cycles 33 were used.The last elongation step was extended to serology were collected by cardiac puncture.The 6 min.After amplification a 10 µl portion of each abdomen was then opened and urine was collected sample was subjected to electrophoresis on a 1.3% from bladder under sterile conditions using 2 ml agarose gel at 100 V for 20 to 30 minutes until the syringe.Collected urine was then transferred to 5 ml tracking dye migrated more than two third of the test tube filled with 2 ml of Phosphate Buffer Saline (PBS).Cold chain was maintained while transporting length of the gel tray in the buffer and the results were these samples.The tubes containing blood samples documented in a gel documentation system (Biorad).were kept in a slanting position for 1 to 2 hours, serum

Results
was separated from the clotted blood by centrifuging Seroprevalence of leptospiral antibodies by at 2000 rpm for 2 min and transferred to 2 ml screw-0 capped plastic vials and were stored at -20 C until MAT among field rodents trapped from within and/or tested.
around the premises of different laboratory animal Microscopic agglutination test: A panel of four facilities revealed 19 (61.29 per cent) samples were positive by agglutinating with one or more of the four serovars was used for the MAT (Table 1).MAT was leptospira antigens tested, out of 31 sera tested.The conducted as per the OIE ( 2004) in 96 well U bottom serovar distribution among laboratory rats in the titration plates with initial dilution of 1:100 upto descending order was Icterohaemorrhagiae (11; 57.89 1:1280.the highest dilution of serum showing 50 per per cent), Autumnalis (5; 26.32 per cent), Pyrogens (3; cent reduction in number of free leptospires was considered as the end-titer. 15.78 per cent).The positive titres in field rats were Dark field microscopy: Urine samples collected in 1:100 (5.26 per cent), 1:200 (5.26 per cent), 1:400 phosphate buffer saline were entrifuged at 12000 rpm (15.79 per cent), 1:800 (5.26 per cent), 1:1600 (31.58 and the sediment suspended in small volume of PBS to per cent), 1:3200 (26.32 per cent), 1:6400 (5.26 per form wet mount preparation and was examined under cent) and 1:12800 (5.26 per cent).dark field microscope.
Dark Field Microscopy (DFM) detected Polymerase chain reaction: PCR assay was carried leptospires in 3 (8.82 per cent) field rat urine samples out as per the method described by Gravekap et al., of the total 34 samples.Where as PCR detected (1993) with slight modifications.The urine samples leptospiral DNA in 6 (17.65 per cent) field rat urine collected in phosphate buffer saline were centrifuged samples of the total 31 samples.at 10,000 rpm for 30 minutes.The pellets were Discussion resuspended in 0.1 ml distilled water and washed two more times with distilled water.The final pellets were The overall seopositivity of field rats in this resuspended in 20 µl of distilled water.The pellet study was 61.29 per cent.Earlier reports from India 0 have shown seopositivity at different levels of 88 per samples were denatured at 96 C for10 minutes and cent (Saadi, 1976), 7•1 per cent (Sharma et al., 2003), snap cooled in icebox.This was used as a DNA 58 per cent (Priya et al., 2008).The magnitude of the template.Amplification of DNA was performed in a seropositivity alters from region to region largely total volume of 25µl.The reaction mixture contained attributed to climatic and environmental conditions 12.5 µl of master mix, 9.5 µl of distilled water, 1 µl of (WHO 2003).Serovar Icterohaemorrhagiae was each primer G1 and G2 (Primer G1: 5'-CTG AAT predominant in these field rats.But in earlier reports CGC TGT ATA AAA GT-3', Primer G 2:5'-GGA AAA the predominant serovar was autumnalis in and around CAA ATG CTC GGA AG-3') and 2 µl of DNA Madurai (Priya et al., 2008) and also in and around template.The samples were placed in automatic PCR Bangalore (Syed, 1978).57.9 per cent were having processor (M J Research minicycler).One amplifititre of 1600 to 3200, where as above 3200 was 10.52 cation cycle consisted of annealing of primers for 1 and lower than 1600 was 31.57.Titre above 800 min at 55°C, elongation for 2 min at 72°C, and Leptospires in field Rats in and around the laboratory animal facilities of Banglore, India