Leptospira spp. in Cats in Estonia: Seroprevalence and Risk Factors for Seropositivity

Abstract

Leptospirosis is a zoonotic bacterial disease that affects humans and animals worldwide. Recently, more attention has been paid to Leptospira spp. infections in domestic cats. In this seroepidemiological study, we estimated the prevalence of anti-Leptospira spp. antibodies in domestic cats in Estonia and evaluated risk factors for the seropositivity. A total of 546 surplus feline plasma and serum samples, collected in collaboration with small animal clinics and an animal shelter in 2013 and 2015, were included in this study. The samples were tested for antibodies against Leptospira spp. using a microscopic agglutination test. The panel included Leptospira interrogans serovars Pomona, Icterohaemorrhagiae, Bratislava, Canicola, and Hardjo, and Leptospira kirschneri serovar Grippotyphosa. Titers ≥100 (positive reactions at dilutions ≥1/100) were considered positive. Anti-Leptospira spp. antibodies were detected in 12.8% of the cats. The percentage of cats that tested positive for antibodies against L. interrogans serovars Pomona, Icterohaemorrhagiae, Bratislava, Canicola, and Hardjo were 9.3%, 3.5%, 2.4%, 0.4%, and 0.2%, respectively, and the percentage of cats that tested positive for antibodies against L. kirschneri serovar Grippotyphosa was 7.3%. Of the seropositive cats, 46.5%, 35.2%, 12.7%, 4.2%, and 1.4% tested positive for 1, 2, 3, 4, and 5 serovars, respectively. The prevalence of anti-Leptospira spp. antibodies was 11.2% in pet cats and 16.3% in shelter cats. Among pet cats, the seroprevalence was over four times higher in cats that had access to the outdoors (17.2%) than in indoor cats (3.9%). Multivariable models, one based on data on pet cats only and another including also data on shelter cats, identified having access to the outdoors, being a shelter cat, and being from Western Estonia as the risk factors for seropositivity. Cats could be better protected from exposure to Leptospira spp. by not allowing them to roam freely outdoors.

Introduction

Leptospirosis is a zoonotic infectious disease occurring worldwide. The causative bacteria Leptospira spp. can infect more than 150 different mammalian species and are then excreted in urine that can contaminate the environment (Bharti et al. 2003, Hartmann et al. 2013, Schuller et al. 2015). Recently, more attention has been paid to Leptospira spp. in domestic cats (Lapointe et al. 2013, Betance et al. 2017). While studies on cats have indicated exposure, shedding, and clinical disease (Arbour et al. 2012, Weis et al. 2017), the overall number of published studies on Leptospira spp. in cats is limited, particularly from Europe (e.g., Agunloye and Nash 1996, Mylonakis et al. 2005, Markovich et al. 2012, Lapointe et al. 2013, Shropshire et al. 2016, Pratt et al. 2017, Weis et al. 2017, Palerme et al. 2019, Sprißler et al. 2019).

Estonia is a country located in northeastern Europe. In 2006–2018, 0–7 cases of leptospirosis were registered in humans annually (Health Board 2019). Antibodies against Leptospira spp. have been detected in several domestic animal species (Estonian Veterinary and Food Laboratory 2019a) and the clinical disease has been diagnosed in dogs (Estonian Veterinary and Food Laboratory 2019b). There is little information and no publications about leptospirosis in cats in the country. Leptospirosis is a zoonosis that may become more common in the future due to effects of climate change (Lau et al. 2010), and baseline data are therefore needed.

Cats may acquire Leptospira spp. by ingestion of infected rodents or from contaminated environment (Shophet and Marshall 1980, Arbour et al. 2012). A large proportion of cats in Estonia have access to the outdoors and are allowed to hunt prey (Must et al. 2015). Cats in Estonia could thus be exposed to Leptospira spp. and might have a role in spreading it to other animals and humans. In this seroepidemiological study, we estimated Leptospira spp. seroprevalence and evaluated risk factors for seropositivity in cats in Estonia.

Materials and Methods

Ethics statement

We used surplus of feline sera and plasma samples that had been collected for unrelated diagnostic or clinical purposes. No samples were taken for the purpose of this study. Participation was voluntary. The owners of the pet cats gave written informed consent to use the surplus of samples for research on infectious diseases, and a formal agreement was made with the veterinarians at a cat shelter for using surplus of samples collected from shelter cats. Anonymous questionnaires were used to collect background information of the cats. No personal information on owners was handled. All data were treated confidentially.

Study design and setting

The study was a cross-sectional seroepidemiological study set in Estonia. It has been estimated that there are ∼280,000 cats in the country (FEDIAF 2017).

The sample was a convenience sample. The samples were primarily collected for unrelated diagnostic and clinical purposes. There were two sampling periods, the first in 2013 and the second in 2015. Multicenter design (several small animal clinics and one animal shelter) was used to obtain samples from different geographical regions.

Sample size calculation

The sample size calculation was done using EpiTools epidemiological calculators (Sergeant 2019). On the basis of expected seroprevalence of 15% (Agunloye and Nash 1996, Weis et al. 2017), precision of 5%, test sensitivity of 97% and test specificity of 95% (Vijayachari et al. 2001), and 95% confidence level, the minimum sample size was 278 cats. The population size (using the estimated 280,000 [FEDIAF 2017] or infinite population) did not affect the minimum sample size needed.

Collection of blood samples

The samples used in this study were collected from two subgroups of the Estonian domestic cat population: pet cats and shelter cats. We included samples that were accompanied with background information about the cat, which indicated the cat was from Estonia and allowed categorizing it to be either from Eastern or Western Estonia. Counties Tartumaa, Võrumaa, Põlvamaa, Valgamaa, Jõgevamaa, and Ida-Virumaa were considered Eastern Estonia, and counties Harjumaa, Järvamaa, Pärnumaa, Viljandimaa, Läänemaa, Raplamaa, Lääne-Virumaa, Hiiumaa, and Saaremaa were considered Western Estonia.

Samples from two sampling periods were included in this study. First, 485 samples collected previously for a seroepidemiological study on Toxoplasma gondii in cats were included to make further use of already collected samples. The informed consent for the original study included permission for further use of samples in other studies (Must et al. 2015). These samples had been obtained from four small animal clinics and one animal shelter, all located in Tartu (Eastern Estonia, 58°23′N 26°43′E), and they were collected from January 2013 to December 2013 (Must et al. 2015). The second sampling was organized from January 2015 to July 2015 with the aim to include cats from other geographical regions in Estonia in this study. From the second sampling, 61 feline blood samples were included. These samples were from cats presented at small animal clinics located in the capital Tallinn (Western Estonia, 59°26′N 24°44′E), the city Pärnu (Western Estonia, 58°23′N 24°30′E), and the town Kuressaare, which is located on island Saaremaa (Western Estonia, 58°15′N 22°29′E). The total sample size was thus 546 samples.

The reasons why the cats were presented to the small animal clinics and had a blood sample taken ranged from a routine health check to a variety of clinical signs (details were not collected). Shelter cats had a blood sample taken as part of a routine health check performed by shelter veterinarians. The sera and plasmas were separated and stored at −20°C until the analyses.

Serology

The sera and plasma samples of the cats were tested for antibodies against Leptospira spp. using a microscopic agglutination test, performed according to the World Organisation for Animal Health (OIE) guidelines (OIE 2019), at the Estonian Veterinary and Food Laboratory, Tartu, Estonia. The serological analyses were performed in 2014–2015. The live bacterial cultures and positive control sera were obtained from Leptospirosis Reference Centre, Amsterdam, The Netherlands. The microscopic agglutination test panel covered Leptospira interrogans serovars Pomona, Icterohaemorrhagiae, Bratislava, Canicola, and Hardjo, and Leptospira kirschneri serovar Grippotyphosa. Titers ≥100 (positive reaction with dilutions ≥1/100) were considered positive. A cat that tested positive for antibodies against at least one of the serovars was considered Leptospira spp. seropositive.

Questionnaires for the cat owners and the shelter veterinarians

Two anonymous questionnaires were used to collect background information of the cats for the risk factor analyses—one for the cat owners and one for the shelter veterinarians. The questionnaire for the cat owners included questions about the cat's signalment (age, sex, and breed) and lifestyle, and location where the cat lived. The questionnaire for the shelter veterinarians included questions about the cat's age group estimate, sex, and breed, and place where the cat had been found.

Statistical analyses

EpiTools was used to estimate true prevalence from survey testing using Blaker's confidence intervals (CIs) (Sergeant 2019). Two-by-two tables were used for univariable analysis of differences in seroprevalence between groups (Dean et al. 2019). Stata 14.0 (StataCorp) software was used for further analyses of the data. Multivariable logistic regression models for predicting seropositivity were built, including all variables, followed by backward elimination, until only variables with p-values ≤0.05 and confounders were retained in the model. The county the cat originated from was used as random factor in the models. Two final models were developed. One included all cats and the other only pet cats with complete background information. The models were based on data from those cats from which we had data for all the variables that were included in the model.

Results

The final study comprised samples from 546 cats, of which 66% (n = 362) were pet cats and 34% (n = 184) were shelter cats. Most (75%, n = 411) of the cats were from Eastern Estonia. The samples originated from 11 of the 15 counties: Tartumaa, Võrumaa, Põlvamaa, Valgamaa, Jõgevamaa, and Ida-Virumaa (Eastern Estonia), and Harjumaa, Pärnumaa, Viljandimaa, Läänemaa, and Saaremaa (Western Estonia). The majority (92%, n = 501) of the cats were older than 1 year. There were both males (54%, n = 284) and females (46%, n = 246). A large proportion had access to the outdoors (48%, n = 174), and a large proportion had reportedly been hunting (46%, n = 166).

The overall apparent prevalence of anti-Leptospira spp. antibodies was 12.8%. The true seroprevalence (adjusted for test sensitivity and specificity) was estimated to be 8.5% (95% CI 5.8–11.9). Table 1 shows the number and proportion of cats that tested seropositive against Leptospira spp. and each of the serovars. Of the seropositive cats, 47.7%, 35.7%, 12.9%, 4.3%, and 1.4% tested positive for 1, 2, 3, 4, and 5 serovars, respectively. The highest titers observed were against serovars Pomona (titer 3200; dilution 1/3200) and Grippotyphosa (titer 800; dilution 1/800).

Table 1.

Proportion of Domestic Cats from Estonia That Tested Positive for Antibodies Against Leptospira spp. Using a Microscopic Agglutination Test Panel

	N tested	n positive	%	95% CI
Leptospira interrogans serovar Bratislava	546	13	2.4	1.3–4.0
L. interrogans serovar Canicola	546	2	0.4	0.0–1.3
L. interrogans serovar Hardjo	546	1	0.2	0.0–1.0
L. interrogans serovar Icterohaemorrhagiae	546	19	3.5	2.1–5.4
L. interrogans serovar Pomona	546	51	9.3	7.0–12.1
Leptospira kirschneri serovar Grippotyphosa	546	40	7.3	5.3–9.8
All Leptospira spp.	546	70	12.8	10.1–16.0

Exact CI (Clopper-Pearson) method was used for CI calculations.

CI, confidence interval.

The apparent seroprevalence by the investigated categories is shown in Table 2. The multivariable model developed using data from all cats was based on data from 490 cats and identified being a shelter cat or having access to the outdoors, in comparison to being an indoor cat, and being from Western Estonia as risk factors for seropositivity (Table 3). The model using data from pet cats only was based on data from 314 of the cats and identified having access to the outdoors and being from Western Estonia as risk factors for seropositivity. Other factors became removed from the models as nonsignificant.

Table 2.

Leptospira spp. Seroprevalence in Domestic Cats in Estonia, by Categories

	N total	n positive	%	95% CI
Young (<1 year)	35	2	5.7	0.7–19.1
Adult (≥1 year)	501	66	13.2	10.3–16.5
Female	246	28	11.4	7.7–16.0
Male	284	39	13.7	10.0–18.3
Purebred	80	0	0.0	0.0–4.5
Not purebred	433	66	15.2	12.0–19.0
From a town or city	382	40	10.5	7.6–14.0
From countryside	106	18	17.0	10.4–25.5
From Eastern Estonia	411	47	11.4	8.5–14.9
From Western Estonia	84	13	15.5	8.5–25.0
Shelter cat	184	30	16.3	11.3–22.5
Pet cat	362	40	11.0	8.0–14.7
Pet cat that received no raw meat	187	18	9.6	5.8–14.8
Pet cat that received raw meat	125	16	12.8	7.5–20.0
Pet cat kept indoors	155	6	3.9	1.4–8.2
Pet cat with access to the outdoors	174	30	17.2	11.9–23.7
Pet cat that had reportedly never been hunting	156	8	5.1	2.2–9.9
Pet cat that had reportedly been hunting	166	28	16.9	11.5–23.4
Sampled in 2013	485	62	12.8	9.9–16.1
Sampled in 2015	61	8	13.1	5.8–24.2
Total	546	70	12.8	10.1–15.9

Exact CI (Clopper-Pearson) method was used for CI calculations. Age group was unknown for 10 cats, sex for 16 cats, breed for 33 cats, being from town or countryside for 58 cats, receiving raw meat or not for 234 cats, having access to the outdoors or not for 217 cats, and hunting or not for 224 cats.

Table 3.

Results of Logistic Regression Model Where Presence of Anti-Leptospira Antibodies in Domestic Cats, Including Pet Cats and Shelter Cats, Was The Outcome Variable and “County” Was Included as a Random Factor

Variable	N	OR	95% CI	p
Pet cat kept indoors	145	1
Pet cat with access to the outdoors	169	7.45	2.51–22.12	<0.001
Shelter cat	176	9.98	3.15–31.57	<0.001
Western Estonia	81	1
Eastern Estonia	409	0.36	0.16–0.81	0.013

The model was based on data from 490 cats for which we had data for all the variables that were included in the model.

OR, odds ratio.

Discussion

Our study is the first to estimate Leptospira spp. seroprevalence in cats in Estonia. The seroprevalence estimate indicates that exposure to the zoonotic pathogen was common in the investigated cat population. It needs, however, to be emphasized that the sample was a convenience sample that may not represent the local cat population well—older cats and cats from Eastern Estonia were overrepresented in the sample, which comprised samples from cats that were taken to a veterinarian or animal shelter. Nevertheless, the sample size was sufficiently large for a good first estimate of Leptospira spp. seroprevalence in cats in the country.

The epidemiological situation may have changed since the samples were collected, and the sampling was carried out in two different time periods. Having two sampling periods did not appear to have an effect on the results—the seroprevalence was similar for both periods and “sampling period” was not a significant variable in the models. Analyzing the data together was thus justified.

Our estimate of the apparent seroprevalence is similar to those reported from other countries. Outside Europe, Leptospira spp. seroprevalence estimates in cats range from 5% to 25% (e.g., Markovich et al. 2012, Lapointe et al. 2013, Palerme et al. 2019, Sprißler et al. 2019). In Europe, most estimates have been mainly within similar range as well, for example, 9% in the United Kingdom (Agunloye and Nash 1995), 18% in Germany (Weis et al. 2017), and 33% in Greece (Mylonakis et al. 2005).

The results of the serology panel (Table 1) indicated diversity in the serovars circulating in Estonia. This is in line with results from other studies in Europe (e.g., Mylonakis et al. 2005, Weis et al. 2017). It is especially noteworthy that we detected a high prevalence and high titers of antibodies against serovar Pomona and serovar Grippotyphosa in the investigated cats. In Estonia, high titers of antibodies against these serovars have been detected in dogs (Estonian Veterinary and Food Laboratory 2019b, Mik 2019). Moreover, the same serovars are among those reported from human cases (Health Board 2016).

Indoor cats are considered to have a very low risk to acquire Leptospira spp. infection (Hartmann et al. 2013). In our study, however, 6 (3.9%) of the 155 reportedly indoor cats tested seropositive, and these 6 cats reportedly did not hunt. It should be emphasized that whether the cat had access to the outdoors and whether the cat had been hunting were reported by the cat owners, and these data were not confirmed by the authors. Recall bias and not knowing are possible. The main result from the risk factor analyses was that having access to the outdoors was a major risk factor. This was expected, because roaming outdoors allows opportunities to encounter Leptospira spp., if Leptospira spp. is present in rodents or in the environment. Indeed, the variable “outdoor access” was highly collinear with the variable “hunting” in our analysis. Moreover, on the basis of temperature data, conditions for survival of Leptospira spp. in the environment are suitable in Estonia from April to November (Andre-Fontaine et al. 2015, Estonian Weather Service 2019). There is a minor difference (1°C) in annual mean temperature between Eastern and Western Estonia (Estonian Weather Service 2019), and further studies could investigate whether this explains that being from Western Estonia appeared as a risk factor for seropositivity in this study (Table 3).

Feline Leptospira spp. infections are of veterinary and potentially of public health importance. The clinical disease in cats should be suspected, for example, in cats with acute or chronic renal insufficiency, especially in the presence of known risk factors for the infection (Arbour et al. 2012). Currently, while there is no vaccine available for cats, avoiding risk factors for the infection is the available prevention option. Not allowing cats roam freely outdoors would protect domestic cats substantially from infections with Leptospira spp. Having access to the outdoors was also a risk factor for T. gondii seropositivity in our earlier study (Must et al. 2015), and together, these two results from Estonia and the results of several other studies from other countries (e.g., Näreaho et al. 2012, Chalkowski et al. 2019) illustrate that having access to the outdoors is a relevant risk factor for exposure of domestic cats to various zoonotic pathogens.

Conclusions

The results of our study add to the limited knowledge on extent of exposure to Leptospira spp. in domestic cats. Based on the results of the risk factor analysis, to protect cats from exposure to Leptospira spp., cat owners should be advised against allowing cats to roam outdoors.

Footnotes

Acknowledgments

The cat owners and shelter veterinarians are warmly thanked for their collaboration, Mare Viigipuu for performing the serological analyses, and Karen A. Krogfelt for commenting on the article during its preparation.

Author Disclosure Statement

A.L. has received lecture fee from Estonian Veterinary Association. The other authors declare no conflicts of interest.

Funding Information

Financial support was provided by the health promotion research program TerVe 3.2.1002.11-0002 EKZE_SS from the Estonian Research Council.

References

Agunloye

, Nash

. Investigation of possible leptospiral infection in cats in Scotland. J Small Anim Pract, 1996; 37:126–129.

Andre-Fontaine

, Florence

, Chantal

. Waterborne leptospirosis: Survival leptospirosis and preservation of the virulence of pathogenic Leptospira spp. in fresh water. Curr Microbiol, 2015; 71:136–142.

Arbour

, Blais

, Carioto

, Sylvestre

. Clinical leptospirosis in three cats (2001–2009). J Am Anim Hosp Assoc, 2012; 48:256–260.

Betance

, Peda

, Conan

, Ribeiro

. Seroprevalence of leptospirosis in the feral cat population of St. Kitts. J Anim Res Technol, 2017; 2017:38–42.

Bharti

, Nally

, Ricaldi

, Matthias

, et al. Leptospirosis: A zoonotic disease of global importance. Lancet Infect Dis, 2003; 3:757–771.

Chalkowski

, Wilson

, Lepczyk

, Zohdy

. Who let the cats out? A global meta-analysis on risk of parasitic infection in indoor versus outdoor domestic cats (Felis catus). Biol Lett, 2019; 15:20180840.

Dean

, Sullivan

, Soe

. OpenEpi: Open source epidemiologic statistics for public health. 2019. Version 3.1, updated April 6, 2013; cited August 1. Available at www.OpenEpi.com

Estonian Veterinary and Food Laboratory. Aastaaruanded [Annual reports]. 2019a. Cited August 1, 2019. Available at www.vetlab.ee/?a=page&page=42f088c48f3e323aa1bbc

Estonian Veterinary and Food Laboratory. Leptospiroos koertel ja kassidel [Leptospirosis in dogs and cats]. 2019b. Cited January 5, 2020. Available at www.vetlab.ee/?a=mypage&path_id=42f068951029381274db1&story_id=533e7182e33c904d370b7

10.

Estonian Weather Service. Observation data. 2019. 2019. Cited August 1, 2019. Available at www.ilmateenistus.ee/?lang=en

11.

FEDIAF (The European Pet Food Industry). European facts & figures. 2017. Cited August 1, 2019. Available at www.fediaf.org/component/attachments/attachments.html?task=attachment&id=2019

12.

Hartmann

, Egberink

, Pennisi

, Lloret

, et al. Leptospira species infection in cats: ABCD guidelines on prevention and management. J Feline Med Surg, 2013; 15:576–581.

13.

Health

Board

. Communicable disease statistics in

Estonia

, part 16. 2016. Cited August 1, 2016. Available at www.terviseamet.ee/sites/default/files/content-editor/Nakkushaigused/Statistika/Statistikakogumikud/nakkus-ja-parasiithaigused_eestis-16.pdf

14.

Health Board. Nakkushaigustesse haigestumine. [Incidence of infectious diseases]. 2019. Cited August 1, 2019. Available at www.terviseamet.ee/et/nakkushaigused-menuu/tervishoiutootajale/nakkushaigustesse-haigestumine

15.

Lapointe

, Plamondon

, Dunn

. Feline leptospirosis serosurvey from a Quebec referral hospital. Can Vet J, 2013; 54:497–499.

16.

Lau

, Smythe

, Craig

, Weinstein

. Climate change, flooding, urbanisation and leptospirosis: Fuelling the fire?. Trans R Soc Trop Med Hyg, 2010; 104:631–638.

17.

Markovich

, Ross

, McCobb

. The prevalence of leptospiral antibodies in free roaming cats in Worcester County, Massachusetts. J Vet Intern Med, 2012; 26:688–689.

18.

Mik

L-L.

Leptospiroos koertel Eestis [Leptospirosis in dogs in Estonia]. Graduation Thesis. Tartu: Estonian University of Life Sciences, 2019. Available at https://dspace.emu.ee/xmlui/handle/10492/4854

19.

Must

, Lassen

, Jokelainen

. Seroprevalence of and risk factors for Toxoplasma gondii infection in cats in Estonia. Vector Borne Zoonotic Dis, 2015; 15:597–601.

20.

Mylonakis

, Bourtzi-Hatzopoulou

, Koutinas

, Petridou

, et al. Leptospiral seroepidemiology in a feline hospital population in Greece. Vet Rec, 2005; 156:615–616.

21.

Näreaho

, Puomio

, Saarinen

, Jokelainen

, et al. Feline intestinal parasites in Finland: Prevalence, risk factors and anthelmintic treatment practices. J Feline Med Surg, 2012; 14:378–383.

22.

OIE (The World Organisation for Animal Health). Leptospirosis. OIE terrestrial manual. 2018. Cited August 1, 2019. Available at www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.01.12_LEPTO.pdf

23.

Palerme

, Lamperelli

, Gagne

, Cazlan

, et al. Seroprevalence of Leptospira spp., Toxoplasma gondii, and Dirofilaria immitis in free-roaming cats in Iowa. Vector Borne Zoonotic Dis, 2019; 19:193–198.

24.

Pratt

, Conan

, Rajeev

. Leptospira seroprevalence in domestic dogs and cats on the Caribbean island of Saint Kitts. Vet Med Int, 2017; 2017:5904757.

25.

Schuller

, Francey

, Hartmann

, Hugonnard

, et al. European consensus statement on leptospirosis in dogs and cats. J Small Anim Pract, 2015; 56:159–179.

26.

Sergeant

ESG

. Epitools epidemiological calculators. Ausvet Pty

Ltd.

, 2019. Cited August 1, 2019. Available at http://epitools.ausvet.com.au

27.

Shophet

, Marshall

. An experimentally induced predator chain transmission of Leptospira ballum from mice to cats. Br Vet J, 1980; 36:265–270.

28.

Shropshire

, Veir

, Morris

, Lappin

. Evaluation of the Leptospira species microscopic agglutination test in experimentally vaccinated cats and Leptospira species seropositivity in aged azotemic client-owned cats. J Feline Med Surg, 2016; 18:768–772.

29.

Sprißler

, Jongwattanapisan

, Luengyosluechakul

, Pusoonthornthum

, et al. Leptospira infection and shedding in cats in Thailand. Transbound Emerg Dis, 2019; 66:948–956.

30.

Vijayachari

, Sugunan

, Sehgal

. Evaluation of microscopic agglutination test as a diagnostic tool during acute stage of leptospirosis in high & low endemic areas. Indian J Med Res, 2001; 114:99–106.

31.

Weis

, Rettinger

, Bergmann

, Llewellyn

, et al. Detection of Leptospira DNA in urine and presence of specific antibodies in outdoor cats in Germany. J Feline Med Surg, 2017; 19:470–476.