SERUM ANTIBODY TITRES FOR LEPTOSPIRA AND ITS ASSOCIATION TO THE IMMUNIZATION AND REARING PATTERN IN A SAMPLE OF DOGS FROM SUBURBS OF COLOMBO : A PRELIMINARY STUDY

INTRODUCTION but, the real disease burden in animal population is not evaluated in the country. The l are normally carried in the kidney Leptospirosis refers to a range of disease conditions in tissues of rodents and other small mammals and are man and animals caused by the spirochetes belonging to excreted in their urine (Greenwood et al., 1992). The genus Leptospira (Evangelista and Coburn, 2010). It is species in which infection is endemic and transferring an important zoonotic disease occurring worldwide from one animal to another animal by direct contact are (Radostits, 1989). There are saprophytic and pathogenic considered as maintenance host. The disease is leptospira species. Pathogenic leptospira can be maintained as a chronic infection in the renal tubules of classified into Leptospira interrogans with more than the maintenance host. Humans as well as animals can be 250 serovars and Leptospira borgpetersenii (Adler et accidental (incidental) host of leptospirosis. Animals al., 2011; Bulach et al., 2006). Strains or serovars are may be maintenance hosts of some serovars but identified based on the characteristics of their surface incidental hosts of others. If a maintenance host for a proteins. particular leptospiral serovar is infected with another Leptospirosis is considered as an emerging public serovar it may develop symptoms (WHO, 2003). Dogs health problem worldwide due to increasing incident are the maintenance host for the serovar, rate in (Evangelista and (WHO, 2003). The maintenance host ensures the Coburn, 2010; Vijayachari et al., 2008). It is primarily continuous circulation of a particular leptospiral serovar considered as an occupational hazard to man. Increasing in a geographical area even without incidental hosts number of leptospirosis cases are reported every year in (WHO, 2003). Sri Lanka among human beings (Victoriano et al., 2009) eptospires


INTRODUCTION
but, the real disease burden in animal population is not evaluated in the country.The l are normally carried in the kidney Leptospirosis refers to a range of disease conditions in tissues of rodents and other small mammals and are man and animals caused by the spirochetes belonging to excreted in their urine (Greenwood et al., 1992).The genus Leptospira (Evangelista and Coburn, 2010).It is species in which infection is endemic and transferring an important zoonotic disease occurring worldwide from one animal to another animal by direct contact are (Radostits, 1989).There are saprophytic and pathogenic considered as maintenance host.The disease is leptospira species.Pathogenic leptospira can be maintained as a chronic infection in the renal tubules of classified into Leptospira interrogans with more than the maintenance host.Humans as well as animals can be 250 serovars and Leptospira borgpetersenii (Adler et accidental (incidental) host of leptospirosis.Animals al., 2011;Bulach et al., 2006).Strains or serovars are may be maintenance hosts of some serovars but identified based on the characteristics of their surface incidental hosts of others.If a maintenance host for a proteins.
particular leptospiral serovar is infected with another Leptospirosis is considered as an emerging public serovar it may develop symptoms (WHO, 2003).Dogs health problem worldwide due to increasing incident are the maintenance host for the serovar, rate in (Evangelista and (WHO, 2003).The maintenance host ensures the Coburn, 2010;Vijayachari et al., 2008).It is primarily continuous circulation of a particular leptospiral serovar considered as an occupational hazard to man.Increasing in a geographical area even without incidental hosts number of leptospirosis cases are reported every year in (WHO, 2003).Sri Lanka among human beings (Victoriano et al., 2009) eptospires

SUMMARY: Leptospirosis is an important zoonotic disease in man and animals. Increasing numbers of human Leptospirosis
reported each year in Sri Lanka.Rodents are most often the natural maintenance host of leptospira, but the potential of transmitting the disease from ruminants and dogs to human should not be overlooked.Aim of this study was to determine the serum antibody titres for leptospira in 139 dogs in selected suburbs of Colombo, Sri Lanka and evaluate its association with dog rearing pattern and immunization history.A questionnaire based survey was conducted to collect data on dog rearing pattern and vaccination history of dogs.In addition, the potential transmission of leptospira from dog to human and public awareness on canine leptospirosis was also assessed respective strains.Strains 62% antibody titres ranging from 1:100 -1:3200, indicating an acute or convalescent stage of infection.Moreover, of primarily vaccinated dogs, 17.9% of regularly vaccinated dogs and of randomly vaccinated dogs failed to seroconvert.Serum samples subjected to sero grouping were all found to be within pathogenic genomic species Leptospira interrogans.Single sero groups L. canicola, L. australis, L. Icterohaemorragica and L. djsiman and mixed sero-groups L. australis and L. canicola were predominantly observed.No clinically proven Leptospirosis was reported among the owners of the dogs, their family members or other household pets even if they had close contact with their dogs.Among the surveyed population, only one person was aware of (0.7%) possible transmission of leptospirosis from dogs.Statistically significant number of primarily vaccinated dogs was in indoor habitats and randomly vaccinated dogs were in outdoor habitats (p< 0.05).That trend was not observed in regularly vaccinated and unvaccinated dog population (p> 0.05).Leptospirosis being a highly infectious zoonotic disease, outdoor living unvaccinated and randomly vaccinated domestic canine populations may contribute to increasing incidence of Leptospirosis in man as well as in other domestic mammals.
are .(Radostits, 1994).When there Questionnaire based survey was conducted at six favourable climatic conditions for , animals randomly selected as well as human beings can get the infection.As in humans, animals that are incidental hosts may become ill with severe disease.Mild leptospiral infection in domestic animals may remain unnoticed.
Convenient In all domestic animals, clinical manifestations of sampling method was used for data collection.A total of leptospirosis vary from acute to sub-acute and chronic 139 dogs aged between three months to 15 years were infection.Chronic infection is localized in the kidneys recruited for the study.The veterinary (sometimes also in the genital tract) usually without fi ll ed th e qu es ti on na ir es by detectable clinical evidence of illness.Animal carriers interviewing the owners of the dogs enrolled in the study.may shed leptospires for months or years in their urine, Information on the age of the dog, sex, and vaccination sometimes in very large numbers, contaminating moist history, any past medical history of laboratory confirmed soil or sources of drinking water (Radostits, 1994).The leptospirosis in selected dogs were also collected.In pollution of surface waters poses a risk of transmission of addition, pet keeping habits of the owners with particular infection to other animals and humans, resulting in water emphasis on closeness of the association with the dogs borne outbreaks of infection in humans.
(keeping dogs inside or outside the dwellings Leptospira interrogans has multiple serovars that could transmission of leptospirosis from dogs and infect dogs.Infected dogs may suffer from a chronic any incidence of leptospirosis in dog owners or their disease leading to kidney damage, or may develop an families were evaluated.Parallel data collection was acute Weil's-like disease syndrome after infection with done in all the selected veterinary clinics.certain serovars (Nelson and Couto, 2004).
Statistical significance of dog rearing and vaccination patterns were calculated by Chi square test using the SPSS statistical package.
Collection of Blood samples from dogs (Nelson and Couto, 2004).Clinical Venous blood samples (2 ml) were drawn from each manifestations of leptospirosis in dogs are variable, as in animal aseptically, after obtaining informed consent from other species of animals, can range from a total lack of the owner.Blood samples were allowed to clot and symptoms (renal carrier state), which is most common, to centrifuged at 3500 rpm for 15 minutes at room severe icterohaemorrhagic disease with acute interstitial temperature to separate serum.nephritis ( et al., 2011).Unvaccinated puppies (aged up to 1 year) suffer from the most severe forms of Microscopic Agglutination Test the disease, sometimes with fatal outcome.
Microscopic Agglutination Test (MAT) detects both Annual vaccination in dogs can prevent the infection IgM and IgG antibodies and was conducted using the caused by specific serovars indicated in the vaccine and standard method practiced at the Medical Research 0 can be recommended for dogs at risk.In Sri Lanka dogs Institute, Colombo.Serum samples were incubated at 56 are generally vaccinated against L. Canicola and L. C for 30 minutes to inactivate any non-specific antibodies icterohaemorragica specific his present.A series of diluted serum was mixed with a vaccine is given as a primary vaccine when the dog is less suspension of live non-pathogenic Leptospira biflexa 0 than three months old and subsequently as a regular Patoc strain in microtitre plates and incubated at C for annual booster.
2 hours.The results were read under dark-field Dogs infected with may directly transmit the microscopy to identify the last dilution with > 50 % disease to humans or do indirect transmission through agglutinated leptospires.Titres reported > 1: 100 were environmental contamination (Barragan et al., 2017).considered as positive.After performing MAT, only 43 There are no published data available on sero prevalence serum samples were adequate enough to carry out the of leptospirosis in domestic and stray dog populations in strain specific MAT test.Strain specific MAT was Sri Lanka.This is a preliminary study to assess the performed against 12 serovars of pathogenic strains of antibody titres for leptospira in vaccinated and Leptospira interrogans including L. pyrogens, L. gem, L. unvaccinated dogs in suburban settings in Colombo.A ceylonica, L. australis, L. tarrasovi, L. pomona, L. hardjo, questionnaire based study was conducted to identify the L. canicola, L. ratnapura, L. weerasighe, L. dog keeping pattern and immunization history.Final icterohaemorragiea and L. Djisimea at the Veterinary objective is to determine the predominant strains of Research Institute, Gannoruwa, Peradeniya in the selected canine population.

Sykes
) and the awareness on quantitative) selected serovar of leptospira described in whilst some serum samples were positive for mixed manual of WHO/FAO reference laboratory on sero-groups.Six samples were positive for L. australis leptospirosis (Smythe, 1993).and L. canicola and two samples were positive for L. Ethical clearance for this study was obtained from the australis and L. Djsimea.L. canicola was the most Ethics Review Committee of the Medical Research predominant single strain observed in the study nd population.Amongst the mixed infections L. australis Institute, Colombo on 2 August 2011 and ethics review and L. canicola were predominantly observed.Among number is 15-2011.
these strains, L. Interrogans serovar L. canicola is the most important one due to its high infection rate in dogs.
Out of the surveyed population (139) 58.3% had received at least a single dose of a combined vaccine (DHL) containing leptospira, while remaining (41.7%) dogs have never been vaccinated against the disease .Vaccinated dogs were categorized in to four main groups for the purpose of data analysis, i.e. (i) dogs less than one year and given primary vaccination and subsequent booster, (ii) regularly vaccinated dogs (iii) randomly vaccinated and (iv) unvaccinated.Results of the percentage of dog population in different subgroups with their antibody (Ab) titres are summarized.Out of the dogs that received primary immunization, (25 %) had no positive Ab titres by the time of testing.Majority of the primarily vaccinated dogs (60%) had antibody titre in the range of 1:100 -1:200 whilst very small fraction (15%) had antibody titres as high as 1:1600.Among the dogs that have been regularly immunized against leptospirosis, (17.94 %) did not have any immune response.Of them 72.1% of the dogs had antibody titres in the range of 1:100 -1:200, 7% had antibody titres in the range of 1:400 -1:800 and 2.3% of the population has antibody titres as high as 1:1600 -1:3200.Few dogs that were over 10 years and regularly vaccinated showed very high antibody titres (1:800 & 1:3200).In the surveyed population, (15.7%) were randomly vaccinated and of them 36.8 % have not had positive titres against leptospira when tested.Only one dog (5.2%) had antibody titres as high as 1:400 whilst all the other dogs (58%) had titres in the range of 1:100 -1:200.Out of the survey population (41.7%) were never been vaccinated against leptospira but (62%) of those dogs had antibodies against leptospira.Titres were in the range of 1:100 -1:3200.Some dogs had antibody titres > 1:400 indicating either acute infection or recovering from infection.
probability of domestic dogs to intermingle with stray dog populations and other peridomestic animals such as mongoose, bandicoots which are commonly observed in the area.
According to the questionnaire based study, 48.9 % of dogs were kept inside Serum samples were subjected to sero grouping and were all found to be within pathogenic genomospecies Leptospira interrogans.Some samples were of single serogroup namely L.canicola (n=12), L. australis(n=2), L. Icterohaemorragica (n=1), and L. djsimea (n=1) According to the study, statistically significant number of primarily vaccinated dogs was in indoor habitats and randomly vaccinated dogs were in outdoor habitats (p< 0.05).That trend was not observed in regularly vaccinated and unvaccinated dog population (p> 0.05).There was no significance difference observed in unvaccinated and regularly vaccinated dogs and they were equally distributed in indoor as well as in outdoor habitats (Table 1).

DISCUSSION
High antibody titres for leptospira were observed in domestic unvaccinated dogs indicating an acute, past or convalescent stage of infection.Leptospirosis is a highly infectious zoonotic disease and unvaccinated domestic canine population is likely to get infected in their own environments and contribute to the increasing incidence of leptospirosis in man as well as in other domestic mammals.In suburban settings in Sri Lanka there may be very high None of the dog owners or their family members had clinically proven ptospirosis and there was no proven evidence of leptospirosis among their pet dogs.Of the In suburban surveyed population only one person knew that the settings in Sri Lanka there is a requirement to encourage domestic dogs are possible source of human dog owners to immunize their dogs against leptospirosis.
leptiospirosis since people have very close contact with their pet animals (Thammitiyagodage, 2011).A study the houses, whilst 51.1% were in conducted in Michigen, USA has indicated high the garden or outside (Table 1).
prevalence of leptospirosis in dogs.Out of 1241 dogs enrolled in their study, all the dogs were positive for at least one strain of leptospira antibody revealing previous exposure to the organism (Stokes et al., 2007).
According to the current study, predominantly observed strains in vaccinated and unvaccinated dog le population in Sri Lanka are L. canicola, L. australis, L. antibody titres than regularly vaccinated dogs.As such Icterohaemorragica and L. djsiman.A study conducted in regular vaccination is essential to have better antibody Thamilnadu, India have shown that 57% vaccinated and titres among the dog population and protect them from unvaccinated dogs were positive for one or more serovars getting the infection.As there is no distinct correlation of leptospira and predominantly observed strains were L. observed in vaccination pattern and dog habitats more icterohaemorrhagiae, L. canicola, L. Pomona and L. systematic in-depth analysis of dog population with their Gripotyphosa (Senthil, 2013).Another study conducted antibody titres is required.in India has shown that the most common serova identified in clinically suspected cases of leptospira was L.
ACKNOWLEDGEMENT pyrogen (Patil, 2014).Though dogs in Thamilnadu are vaccinated using inactivated Leptospira interogans Authors thank MRI financial grant and veterinary serova Icterohaemorrhagiae and Canicola, antibodies for surgeons from See pet Animal Clinic, Pellawatta, Fauna different strains using MAT test were identified in Animal Clinic, Battaramulla, Pet Vet Clinic, Longdon vaccinated dog population.It is reported that multiple Place, Colombo, Rajagiriya Animal Clinic, Animal ser ova s bei ng cir cul ated amo ng vac cin ated and Clinic, Koswatta for their support extended to complete unvaccinated dogs worldwide (Senthil, 2013).According the questionnaire based study and blood to a sero-survey performed in Washington, USA, 22 out samples.Special thank goes to Dr Renuka Jayatissa, of 115 healthy dogs had antibody titres > 1:100 to any Consultant Medical Nutritionist from the MRI for the serovar suggesting that the potential for exposure in assistance given in statistical analysis.Washington state is not uncommon (Davis, 2008).These studies give some insight to the preliminary study carried out in Sri Lanka.Study conducted in New Zealand with 466 apparently healthy dogs has shown that the serovar Copenhageni was more common (O'Keefe et al., 2002).These results clearly show that there is a geographical variation in the pattern of strains available in different regions.
Significant number of primarily vaccinated dogs was living in indoor habitats which mean that dog owners protect them when they are young and significantly higher number of randomly vaccinated dogs living in outdoor habitats may be due to dog owners neglecting their dogs when they grow up.This indicates that dog owners think when dogs are regularly vaccinated they could be let free as they are adequately protected after vaccination and unvaccinated dogs needs to be kept indoors as they are not protected from the diseases.
In Sri Lanka, most important reservoirs of infection with large herbivores as additional significant sources of infection (Haake and Levett, 2015).

CONCLUSION
Unvaccinated dogs had high antibody titres indicating that they are exposed to the disease.These dogs may excrete leptospira spirochetes in their urine and can be potential carriers of the disease.According to this preliminary study randomly vaccinated dogs had less for collection of REFERENCES usually dog owners take their dogs to private veterinarians to immunize them as there is no government programme to vaccinate them regularly.Among the vaccinated population 17.64% of the regularly vaccinated dogs and 36.8% of randomly vaccinated dogs were negative for leptospirosis antibodies using MAT.This could be due to a lack of systematic vaccination programme in the study population or unable to sero convert the animals in the selected dog population.It is necessary to carry out more systematic studies among vaccinated populations to identify the causative reasons.Small mammals are considered as the

Table 1 :
Categorization of dogs according to type of vaccination and their habitats