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Canine leptospirosis--a seroprevalence study of 253 dogs.


Leptospirosis, a spirochaetal zoonosis has emerged as a serious global Veterinary and public health problem. The disease has gained much importance as it is often undiagnosed (Iqball et al., 2011). Many places in South India are known to be endemic for leptospirosis (WHO, 2007). The pathogenic leptospires are divided into more than 250 different serovars and most common serovars affecting dogs include canicola, icterohaemorrhagiae, pomona, bratislava, grippotyphosa and australis (Koteeswaran, 2006). Despite routine vaccinations, there are increasing reports of canine leptospirosis in Chennai. The commercial vaccines are becoming less effective because as immunity is serovar specific and new serovars are emerging (Lau et al., 2012). Therefore for successful immunization, it is essential to identify the most predominant serovar in specific region. Hence, present study was undertaken to identify prevalence of various serogroups involved in canine leptospirosis in Chennai city.

Materials and Methods

A total of 253 canine serum samples were collected during the period of October' 2013-April' 2014. The samples included those collected from 148 vaccinated and 105 unvaccinated dogs. All samples were subjected to Microscopic Agglutination Test (MAT). A battery of six pathogenic serogroups viz. Leptospira interrogans serogroup Autumnalis, canicola, grippotyphosa, icterohaemorrhagiae, pomona and pyrogens are used in this study. The antigens used in the test were procured from Zoonoses Research Laboratory, TANUVAS, Madhavaram, Chennai. These cultures were maintained in Leptospira liquid culture medium (Difco) with bovine serum albumin supplement. The test was carried out in 96 well U- bottom microtitre plates. Test sera were first diluted to 1:50 and then serially diluted two fold in phosphate buffered saline, to obtain dilutions of 1:100 to 1:1600. To 20 [micro]l each of serum dilution, 20 pl of six day old live antigen was added. Approximate antigen controls were set with 20[micro]l and 20[micro]l of antigen and plates were incubated at 37[degrees]C for two hours. After incubation, the result was read by examining a drop of serumantigen mixture from each well under dark field microscope. The antibody titre was highest dilution of serum showing agglutination of 50 percent or more leptospiral organisms (OIE, 2004) (Fig. 1). Reciprocal agglutination of greater than or equal to 100 was considered as positive reactions. The serogroup reacting at the highest titre was considered to be infecting serogroup. Sera samples showing same agglutination titres to more than one serogroup were considered as mixed equals.




Of the total 253 sera samples, 148 were vaccinated and 70 (47.29%) were found positive. Unvaccinated dogs comprised of 105 and 50 (47.61%) were detected positive (Fig. 1 and 2).

Serogroup distribution

Serogroup status with corresponding MAT titre in vaccinated canine was assessed in 148 dog sera samples. The highest predominant serogroup in vaccinated canine for 1:100 titre was grippotyphosa and icterohaemorrhagiae followed by canicola, pomona, autumnalis and pyrogenes. For 1:200 titres highest prevalent serogroup was canicola followed by pomona, autumnalis, icterohaemorrhagiae and pyrogens. For 1:400 titres most prevalent serogroup was icterohaemorrhagiae followed by autumnalis and grippotyphosa. For 1:800 titres high prevalence was shown for autumnalis followed by canicola, icterohaemorrhagiae, pomona and pyrogens. For 1:1600 titres most prevalent serogroup was autumnalis followed by pyrogens, canicola and grippotyphosa (Table 1 and Fig. 3).



Serogroup status with corresponding MAT titre was detected in 1 05 in unvaccinated canine sera samples. The highest predominant serogroup for 1:100 titres was autumnalis followed by pomona, canicola, grippotyphosa, icterohae-morrhagiae and pyrogens. For 1:200 titres most common serogroup was autumnalis followed by canicola, icterohaemorrhagiae, pomona and pyrogens. For 1:400 titres autumnalis was prevalent followed by grippotyphosa, canicola, icterohaemorrhagiae sero groups. For 1:800 titres autumnalis was more predominant serogroup followed by canicola and autumnalis was prevalent serogroup for 1:1600 MAT titre followed by pyrogens (Table 1 and Fig 4).


Domestic animals such as dogs although vaccinated against Leptospira can shed the organisms in their urine and resulting in domestic transmission in humans (Feigin et al., 1973). Most cases of leptospirosis are diagnosed by serology. Antibodies are detectable in the blood approximately 5 -7 days after onset of symptoms in animals (Levett, 2001).

The overall seropositivity among vaccinated and unvaccinated dogs in study was 47.29 percent and 47.61 percent respectively. A titre of 1:100 and above indicates positive (OIE, 2008). Earlier reports by Lai et al. (2005) had shown sero-prevalence of 45.6 percent in dogs, whereas Ambily et al. (2013) reported higher seropositivity of 71.12 percent. In present study a higher seropositivity was seen in unvaccinated dogs which may be because of constant exposure to leptospiral organisms. MAT titres become positive one week after infection and remain positive for months after natural infection (Greene et al, 2006).

In the study, serogroup status with corresponding MAT titres in vaccinated and unvaccinated canines showed distribution of several serogroups. Prevalence of antibodies was highest to serogroup autumnalis, icterohaemorrhagiae and canicola in vaccinated dogs, while there was significant predominance of serogroup autumnalis, c anicola and pomona in unvaccinated dogs. Cross reactivity between various serogroups may be reason for elevated titres as observed in the study in vaccinated dogs. Ambily et al. (2013) found autumnalis as the most prevalent serogroup followed by australis, pomona, canicola, pyrogens, icterohae-morrhagiae. Weekes et al. (1997) in surveillance, reported serogroup autumnalis was the most common reactor. In vaccinated canines, antibody titre found was low and it may be because titres due to vaccination are usually low and even if higher vaccination titres of 1:800 to serogroups autumnalis and canicola develop, it generally does not persist for more than three months (Greene et al., 2006).


Ambily, R., Mini, M., Joseph, S., Krishna, S.V. and Abinay, G. (2013). Canine Leptospirosis- A seroprevalence study from Kerala, India. Vet. World. 6: 42-44.

Feigin, R.D., Lobes, L.A., Anderson, D. and Pickering, L. (1973). Human leptospirosis from immunized dogs.Ann. Intern. Med. 79: 777-85.

Greene, C.E, Sykes, J.E., Brownie, C.A. and Hartman, K. (2006). Leptospirosis in: Green CE, ed. Infectious diseases of Dog and Cat, 3rd Edn St. Louis, MO: Sunder Elsevier., p.402-17.

Iqbal, A., Singh, R., Wazir, V.S. and Ahmad, I. (2011) Current Status of Leptospirosis in Dogs--An Overview. Indian Pet J. Online. 3: 13-23 http//www. indianpet journal. com.

Koteeswaran, A. (2006). Seroprevalence of leptospirosis in man and animals in Tamilnadu Indian J. Med. Microbiol. 24: 329-31.

Lai, C.J., Liu,C.C., Debble, H.O. and Pan, M.J. (2005). Seroprevalence of Leptospira Infection Amomg Stray Dogs at Northern Taiwan. Taiwan.Vet. J. 31: 1-8.

Levett, P.N. (2001). Leptospirosis. Clin. Microbiol. Rev. 14 : 296-26.

Lau, C.L., Skelly, C., Smythe, L.D., Craig, S.B. and Weinstein, P. (2012). Emergence of new leptospiral serovarsin American Samoa--ascertainment or ecological change? BMC Infectious Diseases 12:19.

Office Internationale des Epizootis (2004). Leptospirosis. In: Manual of Diagnostic Tests and Vaccines for Terrestrial, Animals. Volume 1.5th edition. OIE Biological Standards Commission, Paris, France, p. 316-27.

Office International-des-Epizootics (2008). Terrestial Manual. Leptospirosis.

Weekes, C.C., Everard, C.O.R. and Levett, P.N. (1997). Seroepidemiology of canine leptospirosis on the island of Barbados.Vet. Microbiol. 57: 215-22.

World Health Organization (2007). Leptospirosis Laboratory Manual. p. 69.

(1.) Post Graduate Scholar and Corresponding author. E-mail:

(2.) Professor

(3.) Dean, VCRI, Namakkal

(4.) Professor and Head, Zoonoses Research Laboratory

Netra B. Aswar (1), M. Sekar (2), L. Gunaseelan (3) and G. Ravikumar (4)

Department of Veterinary Public Health and Epidemiology

Madras Veterinary College

Tamil Nadu Veterinary and Animal Sciences University (TANUVAS)

Chennai--600007 (Tamil Nadu)
Table 1: Leptospira Serogroup status with corresponding antibody
titre in vaccinated and unvaccinated canines.

Reacting                         Vaccinated (n=148)
Serogroups            100   200   400   800   1600

Autumnalis            3      2     2     5     7
Canicola              4      4    --     3     1
Grippotyphosa         7     --     1    --     1
Icterohaemorrhagiae   7      2     3     2     --
Pomona                4      3    --     2     --
Pyrogens              1      1    --     2     3
Total                 26    12     5    14     12

Reacting                        Un--vaccinated (n=105Total
Serogroups            100   200   400   800   1600

Autumnalis             6     6     4     7     3      45
Canicola               2     4     1     1     --     20
Grippotyphosa          1    --     2    --     --     12
Icterohaemorrhagiae    1     2     1    --     --     18
Pomona                 3     1     2    --     --     15
Pyrogens               1     1    --    --     1      10
Total                 14    15     8     8     4      120
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Title Annotation:Research Article
Author:Aswar, Netra B.; Sekar, M.; Gunaseelan, L.; Ravikumar, G.
Publication:Intas Polivet
Article Type:Report
Date:Jul 1, 2015
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