A SEROLOGICAL SURVEY OF CANINE LEPTOSPIROSIS IN THE CITY OF BELGRADE, SERBIA

Canine leptospirosis is a zoonosis caused by bacteria belonging to the genus Leptospira . Dogs are one of the animal species involved in the cycle of preservation and transmission of leptospirosis in urban areas. Serological testing for the presence of specific antibodies against Leptospira spp. in dogs was continuously performed between 2010 and 2020 in the city of Belgrade. At the request of the owners themselves, other veterinary laboratories or laboratory clinics, 179 blood sera from 179 dogs were examined in the Laboratory for Immunology, Scientific Institute of Veterinary Medicine of Serbia. Blood sera samples from dogs were examined using the standard microscopic agglutination test (MAT) for the presence of specific antibodies against seven different serovars of Leptospira : Pomona , Icterohaemorrhagiae , Grippotyphosa , Sejroe , Canicola , Bataviae , and Australis . The number of seropositive dogs was 17/179 (9.5%). Among all examined sera, the highest titre of seropositive samples was to serovar Icterohaemorrhagiae (10/17, 58.8%), followed by Pomona (4/17, 23.5%), and serovar Canicola (3/17, 17.6%). Specific antibodies for serovars Grippotyphosa , Sejroe , Bataviae and Australis were not detected in any of the dog sera. Cross-reaction (the presence of two or three titres with different values where one of them was higher than others) between different serovars was diagnosed in a low number of sera (n=4), with the following serovars: Icterohaemorrhagiae and Pomona (n=3) and Pomona and Canicola (n=1). The confirmed specific antibody titres for Leptospira spp. were between 1:100 to 1:3000 (5 sera had titres of 1:100, 7 had titres of 1:300, 4 had titres of 1:1000, and 1 serum had a titre 1:3000). Monitoring canine leptospirosis is a useful tool in preventing leptospirosis in Belgrade.


INTRODUCTION
On a global scale, leptospirosis is one of the most widespread zoonotic diseases (Adler and de la Peña Moctezuma, 2010;Langston and Heuter, 2003). The causative agent of leptospirosis is a pathogenic, Gram negative, spirochete bacterium from the Leptospiraceae family, genus Leptospira. Pathogenic Leptospira are divided into 25 serogroups, with more than 300 serovars (Picardeau, 2013). Leptospira survives in moist soil and standing waters, and the cycle of transmission involves susceptible species, natural reservoirs, and the environment. Risk factors for the occurrence of leptospirosis are deficient hygiene, water spills due to increased rainfalls, floods, high temperatures, increase in animal population density including reservoir species, inadequate waste disposal, poor drainage, and sewers (Ebi and Schmier, 2005;Levett, 2001). The presence of Leptospira reservoirs, i.e. the field mouse (Apodemus agrarius) and the common vole (Microtus arvalis) in high densities in fields, as well as the grey rat (Rattus norvegicus), the black rat (Rattus rattus), and the house mouse (Mus musculus) from the immediate farm and human environment present a constant threat for leptospiral infection of domestic animals and humans (Stanojević et al., 2003).
From an epizootiological aspect, dogs in urban areas are significant animal species included in the natural leptospirosis cycle and transmission of the pathogen to humans. Certain Leptospira serovars are host specific. In dogs, serovars Canicola and Icterohaemorrhagiae are commonly present, although recently, there was a report of increasing detections of uncharacteristic Leptospira spp., for which dogs are not recognized as the primary hosts (Samokovlija et al., 2010).
The course of canine leptospirosis can be peracute, acute (most common), subacute, or chronic. The clinical symptoms include fever, dehydration, and inflammatory processes in the muscles, meninges, or kidneys. Moreover, a loss of appetite, abdominal pain, icterus, nosebleeds, fatigue, and/or hind leg paralysis is commonly seen in the diseased dogs (Valčić et al., 2014). Dogs that recover occasionally excrete Leptospira in their urine for a very long time, several months to two years after the infection, and they can also be lifelong carriers (Dmitrović, 2002;Faine et al., 1999).
Differing Leptospira serovars can be present depending on the geographical region. In Europe, the most common serovars in dogs are Icterohaemorrhagiae, Grippotyphosa, Bratislava, Pomona, and Canicola (Ellis, 2010). The first research on leptospirosis in Serbia began after World War II. In Serbia, dog leptospirosis was first described as caused by serovar Sejröe (Trbić and Paunović 1955), and positive serological tests for antibodies against leptospirosis were found in 43.2% of dogs in Belgrade and Pančevo (Trbić and Vučković, 1959). Between 1975 and 1980, serological tests were performed on 844 blood sera from dogs in the territory of Belgrade in five urban districts, revealing 67 (7.94) sera from dogs were positive for Leptospira antibodies, and Icterohaemorrhagiae was the most common serovar (Trifunović et al., 1977). A more recent study of 317 blood sera from street dogs confirmed the following serovars: Icterohaemorrhagiae (17, O n L i n e F i r s t 5.36%), Pomona (6, 1.89%), Canicola (2, 0.63%), Grippotyphosa (1, 0.31%) and Bataviae (1, 0.31%) (Elezović et al., 2011).
Considering the recorded occurrence of leptospirosis in dogs in Serbia, the aim of this paper was to present data on seropositivity and the presence of examined serovars of Leptospira spp. in dogs living in the city of Belgrade in the period from 2010 to 2020.

MATERIALS AND METHODS
Whole blood samples from dogs living in Belgrade were submitted to the Laboratory of Immunology of the Scientific Institute of Veterinary Medicine of Serbia, Belgrade in the period from 2010 to 2020. Samples were examined at the request of owners or were submitted by other veterinary laboratories or small animal veterinary clinics. Only one sample per dog was submitted for examination upon the reasonable suspicion of canine leptospirosis (dogs presenting sudden fever, stiffness in muscles and legs, weakness, depression, lack of appetite, vomiting, diarrhoea, petechiae, yellow skin, mild swelling of the lymph nodes, difficulties in breathing). Therefore, there was no possibility for follow-up or repetitive diagnostics. Furthermore, there are no available data on vaccination against canine leptospirosis. Blood sera were extracted from the whole blood samples and analysed.
The cut-off titre for positive samples was 1:100, and this was also used as a preliminary blood serum test. After the preliminary test, those sera with specific Leptospira spp. antibodies were examined, and the titre values were determined for sera dilutions 1:300, 1:1000, 1:3000. The reaction was visualized on an Axiostar plus dark field microscope, Carl Zeiss, Jena, Germany. O n L i n e F i r s t
Cross-reaction (the presence of two or three serovar reacting titres with different values, where one of them was higher than others) between different serovars was diagnosed in a low number of sera (n=4), with the following serovars: Icterohaemorrhagiae and Pomona (n=3) and Pomona and Canicola (n=1).

DISCUSSION
In our study, we found 17/179 (9.5%) of dog sera from Belgrade, Serbia, were seropositive to at least one of the Leptospira serovars examined. This rate of seropositivity was lower than those in Bulgaria (57.9%) (Sabev et al. 2015), Spain (25.8%) (Lόpez et al., 2019), Croatia (23.9%) (Slavica et al., 2016) and Bosnia and Herzegovina (22.3%) (Lindtner Knific et al., 2019), even though the examined population types of dogs differed. This might be explained by the number of sera/dogs that were analysed as well as by the fact that most of the dogs included in this study were living in flats and so were presumably without constant exposure to Leptospira, even though they demonstrated clinical symptoms possibly attributed to canine leptospirosis. Furthermore, we lack data about previous vaccination against canine leptospirosis in the examined dogs. Therefore, some of the positive results might also be due to the presence of vaccinal antibodies.
Examination of dog sera for specific antibodies against Leptospira spp. in this study proved the long-lasting presence of serovar Icterohaemorrhagiae in Belgrade dogs, followed by Pomona and Canicola. The serovar Icterohaemorrhagiae was previously recorded as the most abundant (Vojinović et al., 2015;Elezović, et al. 2011;Trifunović, et al., 1977). Dogs in urban areas, and presumably in Belgrade too, can have frequent contact with rodents and their excreta. Since rodents are reservoirs for serovar Icterohaemorrhagiae (Vukićević et al., 1999), this might offer an explanation as to why this serovar predominated in our study. However, vaccination against canine leptospirosis, for which we have no data, might also be a possible explanation. The results shown in this research regarding the prevalence of serovar Icterohaemorrhagiae are in compliance with results by Lόpez et al. (2019), Vojinović et al. (2015) and Sabev et al. (2015). Historically, the dominant serovar in Belgrade's dog population was Icterohaemorrhagiae. The same is true for the swine population in the territory of Belgrade, in which this serovar was noted as one of the most frequent serovars (Vojinović et al., 2014). This speaks in favour of the spread and importance of Icterohaemorrhagiae as a causative agent of leptospirosis in the territory of Belgrade.
Similar results of serovar composition and prevalence were described in other European cities. For example, in Sofia (Bulgaria), authors found serovars Icterohaemorrhagiae (52.3%) and Canicola (43.1%) in blood sera from stray dogs to be the most common O n L i n e F i r s t (Sabev et al. 2015). In Spain between 2015 and 2017, the most common serovar in owned dogs was Icterohaemorrhagiae at 19.4% among 1310 blood sera from dogs examined by MAT with a panel of eight Leptospira serovars (Lόpez et al., 2019).
Regarding our neighbouring countries, in Bosnia and Herzegovina and Croatia, serovar Pomona was the most frequently found. A greater number of dogs that tested positive for antibodies against Pomona serovar was observed also in our current study compared with previous studies in Belgrade. In Bosnia and Herzegovina, authors analysed 300 blood sera from dogs in three categories from 12 cities. MAT with a panel of 12 Leptospira serovars was used, and for eight of them, seropositive antibodies were confirmed. Altogether 22.3% (67/300) dogs were seropositive, and peak seropositivity was confirmed for serovar Pomona (38, 42.7%) (Lindtner Knific et al., 2019). In Croatia between 2006 to 2015, 535 sera from domestic carnivores (dogs and cats) were examined for 12 different Leptospira serovars by MAT (Slavica et al., 2016). They established a seroprevalence of 23.9%, and the most common serovars in dog populations were Pomona (30.8%), Icterohaemorrhagiae (20.1%), and Grippotyphosa (20.1%) (Slavica et al., 2016).
Cross-reaction between serovars, otherwise commonly present in leptospirosis diagnostics by MAT test (Adler and Faine 1978), was established in a small number of our dog sera (n=4): serovar Icterohaemorrhagiae and serovar Pomona (n=3) and serovars Pomona and Canicola (n=1). Interpretation of the results can be difficult due to crossreactivity between serovars, especially in the acute phase of the disease (Levett, 2001). In order to be sure of the diagnosis of acute infection in positive sera, it is necessary to examine paired sera and determine a fourfold and higher increase in antibody titre. This was, unfortunately, not possible in this study. Nonetheless, in the case of serum from a dog with the clinical symptoms testing positive for the presence of antibodies against Leptospira spp., it is necessary to examine paired sera and monitor the level of antibody titre to determine if the obtained results actually indicate acute canine leptospirosis or not.

CONCLUSION
In the Belgrade area in the period from 2010 to 2020, 17/179 (9.5%) of submitted dog sera (each from an individual dog) were seropositive for Leptospira spp. by MAT. As the number of dogs in Belgrade increases, we recommend this population (especially stray and hunting dogs) be examined in a larger scale epizootiological monitoring programme, considering the potential threat to human health. Furthermore, suitable collaboration with veterinarians in the field needs to be established to ensure adequate sample collection when leptospirosis is suspected. Ustanovljeni titar specifičnih antitela na Leptospira spp. kretao se od 1:100 do 1:3000 (5 uzoraka titra 1:100, 7 titra 1:300, 4 titra 1:1000 i 1 uzorak titra 1:3000). Praćenje leptospiroze pasa je koristan postupak u prevenciji pojave leptospiroze u Beogradu.

Acknowledgements
Ključne reči: Beograd, psi, Leptospira spp., MAT O n L i n e F i r s t