A new serovar of a new serogroup of leptospires

While investigating outbreaks of leptospirosis in Andaman islands, four isolates were recovered from patients. Three of them were identified as belonging to serogroup Grippotyphosa. However, serological tests done on the fourth isolate did not yield conclusive evidence to place it in any of the known serogrops.

The isolate coded as DS-2, and control strains (Jez bratislava,  Wijnberg, Patoc 1and Veldrat Semarang) did not grow well at 13,  30⁰C or in the  presence of  8-azaguanine at the concentration of 225 mg/ml.  As a first step to identify the isolates up to serogroup status, MAT was performed on the isolates using 23 group specific rabbit anti sera representing 23 serogroups.  Since serogroup status of the isolate could not be made by using 23 group specific sera, the isolates was screened against remainig 236 reference rabbit anti sera of serovars belonging to all sero-groups. Antisera was raised against the isolate in rabbits and tested against all the reference strains of serogroups Batavia, Grippotyphosa Pyrogenes, Sarmin, Ranarum, canicola, Hebdomadis, Icterohaemorrhagiae Javanica, Mini, Pyrogenes, and Shermani. as one or two reference rabbit antiserum belonging to these serogroups gave  a titre of 80 or above against isolate.  The percentage of relation was calculated using the standard methodology.

Fig shows Isolate DS-2 under light microscope

(Fontana stained smear)

The Agglutination Absorption Test (AAT) was performed on the serovars which showed more than 10% relation in Cross Agglutination Test using standard procedure to access the serovar status of the isolate. The test was performed repeatedly to confirm the findings. MAT was performed using a panel of mouse monoclonal antibodies that generate characteristic agglutination profiles, were used to further confirm our findings obtained by polyclonal rabbit antisera. These MCAs belonged to serogroup Grippotyphosa, Icterohaemorrhagiae, Canicola, Batavia, Hebdomadis, Pyrogenes, and Shermani.

The isolate showed typical morphology and characteristic motility of the genus Leptospira under dark field microscopic examination.  DS2 as well as the pathogenic reference strains Wijnberg, Jez Bratislava did not show growth at 13⁰C and in the presence of 8-azaguanine even at the 21 days of incubation indicating the pathogenic nature of the isolate.

Fig Shows Electron microscopic view of isolate DS-2

Growth curve of DS2 and reference strains at 13oC

Agglutination of the isolate with the serogroup-representative panel of anti-Leptospira rabbit antisera did not give a conclusive titre and thus did not reveal candidate serogroups for the isolate. Therefore, agglutination was performed with rabbit antisera against all known reference strains. Again no conclusive results were obtained . However several rabit atisera belonging to nine different serogroups gave titres ranging from 160  to 1280. Serovas belonging to  these nine serogroups were selected for CAAT. Cross agglutinatin test showed more than 10% of relation in the case of serovars fluminesis, vanderhoedeni,  manzhuang and szwajizak.

Absorption test was performed on these serovars

The titre obtained after absorption was always ranging form 50 to 100% of the titre before absorption in the case of all serovars. Thus CAAT excluded the identification of the isolate as belonged to these serovars.  Opposite side of the CAAT  using rabbit antisera raised against the isolate was omitted as one-sided CAAT gave conclusive results. The isolate was also screened against a panel of monoclonal antibodies against several serogroups. However the isolate did not react against any of the monoclonal in MAT indicating that the isolate did not belongs to any of these serogroups.

Growth curve of DS2 and reference strains in presence of 8-AG

The results obtained on the isolate by using polyclonal rabbit antisera and monoclonal antibodies were consistent on repeated experiments. The results of the serological experiments on the isolate did not fulfill the criteria laid down by the International Committee on Systematic Bacteriology, Sub-committee on the taxonomy of leptospira (1986) to place an isolate into any serovars/serogroup. Therefore we propose that the isolate is a new serovar of a new serogroup.