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Research in the field of vector borne diseases and zoonozes became a topic of interest in Serbia, during the last decade. Climate changes as well as higher frequency of human and animal movement and travel, especially of dogs, is rising a threat of dirofilariosis and leismaniasis. The presence of native mosquito and sandfly vectors has already been confirmed in the country and some invasive/previously not detected were found. Dirofilariosis and leishmaniasis, which are found more or less often in dogs, cause clinical symptoms which are not obvious and therefore they represent a danger for public health with dogs acting as reservoirs of the infection.
Scientific Veterinary Institute “Novi Sad,” Novi Sad, Serbia
Branka Vidic
Scientific Veterinary Institute “Novi Sad,” Novi Sad, Serbia
Zivoslav Grgic
Scientific Veterinary Institute “Novi Sad,” Novi Sad, Serbia
Tamas Petrovic
Scientific Veterinary Institute “Novi Sad,” Novi Sad, Serbia
Alekasandar Potkonjak
Department for Veterinary Medicine, Faculty of Agriculture, University of Novi Sad, Serbia
Aleksandra Ignjatovic Cupina
Laboratory for Medical and Veterinary Entomology, Faculty of Agriculture, University of Novi Sad, Serbia
Slavica Vaselek
Laboratory for Medical and Veterinary Entomology, Faculty of Agriculture, University of Novi Sad, Serbia
Dusan Petric
Laboratory for Medical and Veterinary Entomology, Faculty of Agriculture, University of Novi Sad, Serbia
*Address all correspondence to: sara@niv.ns.ac.rs
1. Introduction
Research in the field of vector-borne diseases and zoonozes became a topic of interest in Serbia during the last decade. Impact of climate changes in the country is evident, compared to the weather conditions from 10 or more years ago, in terms of higher temperatures during the summer, higher humidity in summer, shorter spring and autumn periods, and shorter period of low temperature during winter. The influence of climate change has already been highlighted [1]. Also, the frequency of human and animal movement and travel, especially of dogs, is significantly higher, not only in European countries but also in overseas countries. The importation of dogs is done on a pretty flexible basis with health status analysis only for rabies. The presence of vectors of Dirofilaria (mosquitoes) and Leishmania (sandfly) parasites has already been confirmed in the country. Current research in the domain of infectious diseases in dogs mostly includes diseases that drastically endanger health and population of dogs. Some of those infectious diseases, like dirofilariosis and leishmaniasis, which are found more or less often in dogs, cause clinical symptoms that are not so characteristic and expressed. These diseases are zoonozes, and therefore they represent a danger for public health with dogs acting as reservoir of the infection. For a transmission of vector-borne diseases among dogs and from dogs to humans, vectors are essential because a part of the pathogen’s life cycle takes place in vectors.
Vectors of dirofilariosis are mosquitoes. Female mosquitoes which feed on mammals can transfer microfilaria from one infected organism to another non infected one. Dirofilaria immitis is a nematode, intravascular parasite that lives in bloodstream of host, usually pulmonary vessels. Prepatent period is at least 6-7 months in definitive hosts. Maturation of organisms in mosquitoes is temperature dependant and over 14°C is needed.
Dirofilariosis and leishmaniasis were earlier recognized as Mediterranean vector-borne diseases. They both have a zoonotic potential. Vectors necessary for the transmission of dirofilariosis are mosquitoes and for leishmaniasis are sandflies. Today there is evidence of dirofilariosis in different countries around the world and also evidence of presence of vectors for dirofilariosis and leishmaniasis in countries other than Mediterranean [2–5].
Dirofilariosis is a vector-borne zoonosis mostly caused by Dirofilaria immitis and Dirofilariarepens. Even though dirofilariosis was primary known as a disease found in Mediterranean countries only, it has spread out to the North and West of Europe through the years, so now clinical cases of dirofilariosis can be found in middle Europe, including Serbia [6–12].
The first published research on dirofilariosis in Serbia (previously known as part of Yugoslavia) was done during the 1990s, when the first cases were discovered in humans and dogs [13– 16]. Since that time, there is a follow-up on dirofilariosis in several regions of Serbia. Diagnostics of dirofilariosis in Serbia has started approximately 10 years ago. Since 2004 until nowadays, veterinary services have started a regular, routine check up in dogs for dirofilariosis. Cases of dirofilariosis (D. immitis and D. repens) in Serbia have been found so far both in humans and dogs. Several cases of dirofilariosis in humans have been represented, in different studies [17–22].
The first cases of dirofilariosis in Serbia, in dogs, were discovered as a side finding during dissections [43]. The actual first case of canine dirofilariosis in Serbia was considered to be in a dog imported from USA. A number of studies were done on the outbreaks of dirofilariosis in dogs and seroprevalence in different regions [23–27]. In the northern part of Serbia, Vojvodina province, several studies have been done during the previous period on seroprevalence and diagnostic methods [28–32]. Some research was also done on seroprevalence to dirofilariosis in working and military dogs and in pet dogs [33] after the first findings of Dirofilara as a side finding during dissections (Figure 1).
Figure 1.
Dirofilaria immitis found in heart at dissection of a dog.
Vectors of dirofilariosis are mosquitoes. Female mosquitoes that feed on mammals can transfer microfilaria from infected to not infected organism. Over 70 mosquito species can be vectors of dirofilariosis out of 3500 mosquito species worldwide [34]. Female Culex pipiens mosquitoes are most important vectors that can be found in high numbers in Serbia during the warm period of the year, from May to October.
Dirofilariosis can appear with different severity, from asymptomatic to mild, or it can also progress to fatal. Definitive hosts of the parasite can be domestic dogs and wild candies, such as wolfs, coyotes, and foxes. Reservoirs of dirofilariosis in wildlife are raccoons, wolverines, coyotes, dears, and bears. Dirofilariosis has a zoonotic potential. Humans are not definitive hosts for Dirofilaria, but occasionally the disease can occur, most usually under the skin or in the eye.
Dirofilaria immitis is a nematode, intravascular parasite that lives in bloodstream of host, usually pulmonary vessels. Prepatent period is at least 6–7 months in definitive hosts. The maturation of organisms in mosquitoes is temperature dependant, and over 14°C is needed. When mosquitoes feed on the blood of an infected dog, they ingest first-stage (L1) larvae (microfilariae), which are produced over many years by the mature heartworm in the dog. Within the mosquito, larvae mature from stage 1 to stage 3. Most of their development takes place in the Malphigian tubes of the mosquito. Once developed to the infective (L3) larval stage, they migrate through the body to the head cavities of the mosquito, where they wait to infect another host by leaving the mosquito during the blood meal. The prepatent period between initial infection of the dog and the maturation of the worms into adults living in the heart takes 6 to 7 months in dogs. The (L3) larvae of heartworms deposited by the mosquito into dog’s skin grow for a week or two and then molt to the next larval stage (L4) under the skin at the site of the mosquito bite. Then they migrate to the muscles of the chest and abdomen, and 45 to 60 days after infection, they molt to the next larval stage (L5). Between 75 and 120 days after infection, these immature heartworms then enter the bloodstream and are carried through the heart to reside in the pulmonary artery. Over the next 3 to 4 months, they increase in size. Seven months after infection, the adult worms have mated, which has a consequence of the appearance of microfilariae in the blood stream of the host. Microfilariae may circulate in the bloodstream for up to 2 years, and be taken by a bloodsucking mosquito. The extrinsic incubation period required to reach the stage when microfilariae become transmittable to another host can vary from 2 to 6 weeks, depending on the temperature. It is possible that there are no evident clinical symptoms in a host for even a year after infection. In humans, Dirofilariaimmitis never reaches the adult stage, and they can never be found in the heart of humans because humans are accidental hosts [34] (Figure 2).
Dirofilariosis in dogs is most frequently located in the right side of the heart, pulmonal arteries, and rarely in the lungs. Clinical symptoms in dogs are unspecific: lethargy, weakness, fatigue, exercise intolerance, dyspnea, cough, anorexia, weight loss, vomiting, diarrhea, collapse, seizures, and sudden death.
Figure 2.
Adults of Dirofilaria immitis taken out from the heart of a dog.
Diagnostic methods for dirofilariosis are many, but several are used most frequently: Enzyme Linked Immunosorbent Assay (ELISA) and immunoenzyme “fast” or “snap” test for detection of Dirofilaria spp. antigens; then modified Knott test for direct microscopic detection of parasite in blood stream, as well as molecular methods like Polymerase Chain Reaction (PCR) for detection and confirmation of the presence of Dirofilaria spp. genome. ELISA is a highly sensitive and specific serological diagnostic test (Figure 3). It is easy to perform diagnostic assay, but it has to be done in a laboratory. Besides ELISA high specificity and sensitivity, false-positive reaction can happened as a consequence of a cross reaction with antigens of another filarial species. Also, there can be a false-negative finding, if the analysis is performed too early after the infection and the dog still does not have a detectable level of antibodies.
Figure 3.
ELISA method for diagnostics—positive and negative control and positive and negative samples.
Antigens of Dirofilria sp. can also be detected by an immunoenzyme (immunochromatographic) test, usually called “fast” or “snap” tests. The “snap” test is an enzyme immunoassay for in vitro diagnostic for the semiquantitative detection of Dirofilaria immitis (D. immitis) antigen in canine and feline whole blood, serum or plasma.
It is a user-friendly one- or two-step test that can be performed anywhere. No laboratory conditions are needed for the performance of the test, so it can be done at veterinary practice or even in the field. The results of the tests are ready to be read within 10–15 minutes, and the sensitivity and specificity of fast tests is good compared to the other available tests (Figure 4).
Figure 4.
Immunoenzyme fast test—positive (two dots) and negative (one dot) findings.
The most “popular” and most used diagnostic test for dirofilariosis in veterinary labs is the modified Knott test. With this test, circulating microfilaria from the blood stream can be found, colored, and seen with a microscope. The procedure is not complex but requires some laboratory equipment; time and skills are also needed, with a good knowledge of microfilarial morphology. This method is highly specific and sensitive in dogs, and microfilariae belonging to different species can be determined [35]. The modified Knott test is the preferred method for observing morphology and measuring body dimensions to differentiate D. immitis from non-pathogenic filarial species, such as Acanthocheilonema (formerly Dipetalonema) reconditum. Although screening may be based entirely on antigen testing, antigen-positive dogs should also be tested for microfilariae, because a microfilaremia validates the serologic results and identifies the patient as a reservoir of infection [47].
PCR is a molecular method the can be used for highly sensitive and specific detection and characterization of Dirofilaria sp. genomic DNA. This method can be used to discriminate microfilariae from other different filarial worms in dogs. It is a good conformation test and a research tool. If dirofilariosis is detected by snap tests, ELISA, or modified Knott test, the presence of the DNA of pathogen can be confirmed by PCR method [36].
Leishmaniasis are vector-borne zoonotic diseases caused by a protozoan parasite belonging to genus Leishmania. Diseases are transmitted by the bite of infected female sand fly (Psychodidae, Phlebotominae) and most common hosts are mammals, including humans. Human and canine laishmaniasis caused by Leishmania infantum present a threat to re-emerge in Europe. Since 2009, human cases of leishmaniasis were reported in Europe countries (Greece, Cyprus, France, Italy, Malta, Portugal, Spain, Former Yugoslavia Republic of Macedonia, Albania [5] and Bulgaria [37]. Canine leishmaniasis is present worldwide, but recently clinical cases were reported in non endemic countries like Romania [38] and Hungary [39].
Throughout the history, there is evidence of leishmaniasis in humans and in dogs in Serbia and first human cases were reported in 1945. During the ten year period (1945-1955) visceral leishmaniasis was spreading through south-east Serbia in epidemic waves recording over 930 cases [49]. The first autochthonous cases of visceral leishmaniasis were found in the southern part of Serbia (region around city of Nis) back in 1945. During the period of 1946–1949, there were 350 registered cases of human visceral leishmaniasis in Serbia, and some cases were even registered around city of Belgrade [45]. At that same time, about 2% of dogs in the region around city of Nis were found to have asymptomatic leishmaniasis, and dogs were identified as main reservoir of infection [45]. After this period sudden fail in number of cases was detected and leishmaniasis was soon considered eradicated. During the period from 1968 to 1969, rare cases of autochthonous visceral leishmaniasis were reported in the southern part of Serbia. After Second World War conditions for sandfly development in South-East Serbia were more favorable and sandfly fauna was rich in diversity. Abundance of vector species was high which affected rapid spread of the disease during the mentioned ten year period after the war [48]. After application of control measures rapid decline in numbers of collected species and samples was recorded. Cases of leishmaniasis became rare and sporadic and finally in 1969 the last case was found. Soon after that all entomological research was neglected. As province of Vojvodina is the most northern part of Serbia, it is not a suitable region for sandfly development. During a short period of research (1948-1951) in this area only three species were detected (Phlebotomus papatasi, Phlebotomus major and Phlebotonus perfiliewi) [50]. With low diversity and abundance of the species, Vojvodina was never considered as potential ground for leishmaniasis spreading. First infections were the ones confirmed as introduced infections in humans (Montenegro, Former Yugoslavia Republic of Macedonia and Greece mostly during holiday season) [40] and dogs [41, 42] and then recently, cases that could be described as autochthonous cases of canine leishmaniasis appeared (Figures 5 and 6). Within the last 3 years, positive findings were identified in dogs that have never left their homes in Serbia. Three separate cases of dogs were found with clinical symptoms that could indicate leishmaniasis (epistaxis, cachexia, pale mucosa, skin problems, blindness, and lethargy), they were found seropositive for leishmaniasis and after therapy, their condition has improved [41, 42].
Domestic and wild canines are the main host species for leishmaniasis, but the domestic dog is the only epidemiologically important reservoir. Causative organisms are protozoa Leishmania donovani (in Asia, Middle East, and Africa) and Leishmania infantum (in Asia, Middle East, Europe, and South America). The transmission of the disease occurs via sandfly bites, and dogs are the reservoir hosts. Humans are accidental hosts. Transmission of the disease between dogs and humans directly is not possible.Presence of human autochthonous leishmaniasis in Vojvodina hasn’t been confirmed, but new cases of canine leishmaniasis are appearing. Clinical symptoms of leishmaniasis in dogs are nonspecific. They can be as fever, weakness, lethargy, weight loss, muscle wasting, lymphadenopathy, pallor, anemia, thrombocytopenia, conjunctivitis and eye problems, skin lesions and alopecia, etc.
Newly gathered entomological data indicate disease circulation since presence of L. infantum was detected in sandflies. This finding stressed out the need for further more detail research on potential hosts and reservoirs of the disease [46].
Diagnostic procedures for leishmaniasis are many. The most certain method is the demonstration of the parasite from bone marrow, splenic, or lymph node aspirates. Less invasive methods are serologic tests that are used for detection of anti-leishmania antibodies, like immunofluorescent test (IFAT) and enzyme-linked immunosorbent assay (ELISA) [44]. Immunochromatography-based assays are easy to use and provide rapid qualitative results on the spot, but their performance is still not optimal [51, 52]. Detection of Leishmania DNA in tissues by PCR allows sensitive and specific diagnosis of infection. PCR can be performed on DNA extracted from tissues, blood, body fluids or even from histopathologic specimens [53]. Cytological or histological identification of Leishmania amastigotes, free or contained within macrophages, in stained specimens from lymph nodes, spleen, skin, bone marrow or other organs can provide a potentially quick diagnosis of infection. However, these are neither sensitive nor specific assays as dogs with overt clinical disease may have few or no demonstrable tissue parasites and because other objects viewed by light microscopy can be erroneously considered amastigotes [54].
Figure 5.
Bitch with skin lesions, positive serological finding for leishmaniasis.
Figure 6.
Dog with skin lesions, positive serological finding for leishmaniasis.
Materials for the research were samples from dogs and samples of vectors. The research was planned as serological examination of dog blood samples for dirofilariosis and leishmaniasis. The vectors (mosquitoes) were collected, identified, and analyzed for the presence of causative agents of dirofilariosis in the northern part of Serbia.
During spring and summer of 2014 (May–September), 292 samples of mosquitoes were collected and identified. Collecting was done with NS2 type preps baited with dry ice and without light. The morphological identification of mosquitoes was done at Faculty of Agriculture, University of Novi Sad, according to the illustrated key [55]. Only specimens of the main vector species, Cx.pipiens were submitted to further analysis. The analysis of vectors for the presence of Dirofilaria sp. was done by conventional PCR. PCR analysis was performed at the Scientific Veterinary Institute of “Novi Sad.” Female specimens were randomly taken from samples collected at different locations in Vojvodina. The collected mosquitoes were tested in pools (20 mosquitoes were pooled as one sample). DNA extraction was done with commercial kit QIAmp DNA Mini Kit (Qiagen, Hilden, Germany), and PCR was performed by using HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and with primers described by Rishniw et al. 2006 [36]. Briefly, For the amplification of 302 bp fragment of part of 5.8S, internal transcribed spacer region 2 (ITS2) and 28S ribosomal DNA sequences regions of D. immitis, previously described as pan filarial oligonucleotide primers: forward DIDR-F1 5′-AGTGCGAATTGCAGACGCATTGAG-3′ and reverse DIDR-R1 5′-AGCGGGTAATCACGACTGAGTTGA-3′ were used [36].]. The amplification reaction was carried out in a volume of 25 μl containing 5 μl of DNA sample, 12.5 μl of master mix from "HotStar Taq Master Mix Kit" (Qiagen, Hilden, Germany), and 25 µM of each primer. Amplification conditions were as follows: 950C 15 min, 40 cycles of 950C 30s, 600C 30s, 720C 30s, and a final extension at 720C for 10 minutes. PCR fragments were separated by electrophoresis on 1.5% agarose gel and were visualized on a transilluminator.
In total, 170 of blood samples obtained by venous punctuation of dogs were examined for dirofilariosis and leishmaniasis. Samples were obtained by centrifugation and kept on –20°C until ELISA was performed (2-3 weeks at the most).The blood samples were divided into three groups, according to the way of life of the dogs:
Group of hunting and military dogs (79 samples)—in this group, samples were analyzed from dogs that are actively used for hunting. They had their owners, and they mostly did not receive any preventive treatment against parasites. Not one of these dogs has ever left Serbia.
Group of dogs from asylum for homeless dogs (64 samples)—in this group were dogs kept in the asylum, but for a long time, and they have all received preventive treatment against parasites annually. Not one of these dogs has ever left Serbia since they were in asylum, but for many of them, the history of their previous life and origin is unknown.
Group of pet dogs (27 samples)—in this group were dogs that came to veterinary practice for numerous reasons, with nonspecific clinical symptoms, or no clinical symptoms at all. Not one of the owners thought that their dog has dirofilariosis or leishmaniasis. Some of the dogs have received antiparasitic prevention and some did not. Even the ones which did receive preventive treatment did not receive it annually, only during spring and summer. Not one of these dogs has ever left Serbia.
Methods used in the study were the following: modified Knott test and PCR for dirofilariosis and ELISA test for leishmaniasis.
Analysis for dirofilariosis was done with the modified Knott test for the detection of microfilaria in circulation. Analysis for all the samples was done on the same day of sampling or the next day. Samples were taken with anticoagulant. The procedure of the modified Knott test was performed according to the instructions of Genchi et al. [35]. The modified Knott test was done in all the samples—from dogs with clinical symptoms as well as from dogs without any clinical symptoms for dirofilariosis. Positive samples found by the modified Knott test were then selected for molecular analysis to be done by PCR. DNA extraction was done with commercial kit QIAmp Blood DNA Mini Kit (Qiagen, Hilden, Germany), and PCR was performed by using HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and with same primers and protocol described for detection of D. immitis in vectors [36].
Detection for antibodies against Leishmania (IgG) was performed in same samples, by ELISA method using a commercial kit by Ingenasa and following the manufacturer’s instructions.
Analysis of vectors for dirofilariosis: In total, 292 samples of Cx.pipiens mosquito females were collected and tested with conventional PCR method. In 292 mosquitoes randomly collected, the presence of DNA of causative agent for dirofilariosis (Dirofilaria immitis) was not found.
The presence of DNA of D.immitis was not found in any sample of this vector that feeds on dogs during the night. Prevalence of Culex pipiens mosquitoes infested with D. immitis in an endemic area of north-eastern Italy ranged from 0.21 to 1.11%, according to date and site [57]. In Turkey, an infestation rate of 0.12% was recorded in Cx. pipiens [58]. These rather low vector infestation rates might explain our negative results. Another vector of D. immitis, Ae. albopictus [59, 60] is active throughout the whole day, especially in urban habitats. Aedes albopictus has been intercepted in the western and southwestern part of the Serbia. It has been present for the past five years (2011-2015) on the Croatian border, and on the Montenegro border since 2013 [61], Petrić unpublished data]. If Ae.albopictus became established, the sympatric occurrence of both vectors, having diurnal and nocturnal biting activities, may enhance the risk of infestation to dogs and humans, thus increasing the vector-host contact and, eventually, the number of vectors which may carry filarioids in endemic areas throughout the day [62]. The factors enhancing the exposure of the host to the vector (i.e., the dog’s size, the age and the outside habitation) may further increase the risk of D. immitis infestation [63,64] as well as dog’s activities i.e., guard, hunting, stray dogs vs. pet dogs [62]. The number of dogs with no travel records infected with D. immitis in Vojvodina is rising. This will, in the future, allow us to sample at the residence of positive dogs and not from randomly chosen locations as we did.
The results of microscopic analysis of blood samples originating from three groups of dogs (170 samples in total) for the presence of microfilaria by modified Knott test are shown in Table 1.
Group of dogs
Total number of examined dogs
Number of positive dogs
Percentage of positive dogs
Hunting and military dogs
79
18
22.78
Dogs from asylum for homeless dogs
64
2
3.12
Pet dogs
27
6
22
Total
170
26
15.29
Table 1.
Results of the analysis of blood samples (modified Knott test) originating from three groups of dogs for presence of microfilaria
In the group of hunting and military dogs, prevalence for dirofilariosis was found to be 22.78%. In the group of dogs from asylum, a lower prevalence for dirofilariosis was found —3.12% and in the group of pet dogs, prevalence for dirofilariosis was found to be 22%. The prevalence of the infection in different groups was different. It depended on the received prevention treatment against parasites and the lifestyle of dogs. Prevalence was the lowest (3.12%) in dogs living in asylum with regular prevention care. These dogs received preventive treatment monthly during the whole period when mosquitoes can be found (March/April–October). It is important to highlight that even two positive dogs found in asylum were new dogs that came from another place, less than 1 month previously to the sampling. The highest prevalence (22.78%) was found in hunting dogs with no prevention treatment in most of the cases. Prevalence found in pet dogs was not much different than the one found in hunting dogs. This would refer to the fact that not many pet dogs are under preventive treatment, or even if they are, it is not being repeated enough times. Most of the pet owners are not aware enough of the existence of dirofilariosis as a disease in dogs, and so they believe that it is enough if they give the preventive treatment to their pet dogs once or rarely twice during the whole period of the year when mosquitoes are present (March/April–September). Also, the fact that there are no clinical symptoms in dogs usually for a long time after infection makes the owners believe that their dog is healthy.
During a 2-year period, 170 dog blood samples were analyzed. Most of the dogs did not have any clinical symptoms. Only several dogs had clinical symptoms such as cough, lethargy, tiredness, and heart failure symptoms. Modified Knott test gives us a direct overview into the existence of larvae of Dirofilaria (microfilaria) in dogs’ circulation. A total average prevalence for the whole three groups of dogs was found to be 15.29%, but the highest prevalence was found to be in hunting and military dogs, followed very closely by pet dogs. Hunting and military dogs live in most cases outside in backyards and are in constant contact with vectors. They have a long time of outside activities in the regions where vectors can be found. Also, quite a lot of dogs from this group is not protected constantly with preventive antiectoparasitic treatment.
Modified Knott test is a fast and reliable diagnostic tool recognized in the world as a method for the detection of microfilaria in circulation of dogs. In veterinary practices, fast tests can be used for routine checkup of patients. However, in the case of positive finding or if there are recognizable clinical symptoms in a dog, a confirmation of diagnosis has to be done with the modified Knott test. With this test, an identification of Dirofilaria can be done with distinction between Dirofilaria immitis and Dirofilaria repens [35] (Figure 7).
Figure 7.
Diagnostics of dirofilariosis by the modified Knott test.
Figure 8.
PCR reaction for Dirofilaria immitis in blood samples of dogs positive to dirofilariosis, determination based on 302 bp – Samples 1-13; negative control (depc-treato water) – 14; DNA ladder 100bp – 15; positive control (dog blood positive for D.immitis) - 16
After positive samples were found by the modified Knott test, a PCR analysis was done for the conformation of Dirofilaria immitis. PCR analysis was done from blood samples of dogs in which microfilariae were found (26 samples). PCR is a very sensitive, specific, and accurate method that could be used for the determination of Dirofilaria species. It is more a research tool than a diagnostic tool because it is a demanding procedure in equipment and skills. From 26 blood samples from dogs in which Dirofilaria was detected by the modified Knott test, a positive result was confirmed by the PCR method in 24 samples (92.3%) (Figure 8).
These data can be compared to the data collected during the last several years in the same region by the same authors, shown in Table 2. The first official acknowledgment of dirofilariosis in Serbia was published by Dimitrijevic in 1999 [43]. After that time, several authors have been following the development of this disease in dogs in different regions of Serbia. For the northern part of Serbia, data have been collected for more than 10 years now.
Year
Percentage of positive dog samples
2003–2004
5.9–7
2006–2007, dogs with no clinical symptoms
10–11
2006–2007, dogs with clinical symptoms
80
2010
14
2010, only pet dogs
11
2011–2013
5 human cases
2013–2014 hunting and military dogs, dogs from asylum and pet dogs
15.29
Table 2.
An overview of data collection during a 10-year period on the prevalence of dirofilariosis in dogs in northern Serbia
By comparing the data during the last decade, it can be stated that there is a constant increase of prevalence for dirofilariosis in dogs in Serbia over the years. After these findings were published, diagnostic methods for dirofilariosis were introduced into the routine checkup of dogs in veterinary practices—modified Knott test, fast test for detection of microfilariae, and ELISA test for antigen detection. Also, fast tests became available to the practitioners and became the mostly used diagnostic tool in veterinary practices. Preventive treatment is present and offered to the owners, but the awareness of the owners is not quite high enough. Further research on the presence of causative pathogen (Dirofilaria immitis) must be done in vectors so that a risk estimation can be made.
Definitely, dirofilariosis is present in the northern part of Serbia in the percentage that justifies the fact that this disease should always be considered during clinical examination of dogs in veterinary practice. Also, there are already human cases of dirofilariosis in the region, so attention should be paid to this disease in the meaning of “One Health” point of view.
Clinical cases of canine dirofilariosis in Serbia are still often found after dissections, and still mostly as a side finding (Figure 9).
Figure 9.
Dirofilariosis in one of the military dogs from the survey.
It appears that dirofilariosis is a disease more and more frequent in dogs, so lately there are more dog owner demands for testing dirofilaria presence in a routine health status checkup in dogs. The owners are not enough aware that disease can occur without any clinical symptoms for a certain period of time. During the period of our study, positive findings for dirofilariosis were present all the time in dogs, which makes therapy and prevention necessary in the region.
The awareness of the fact that dirofilariosis is a zoonotic disease is higher over the time. Cases of human dirofilariosis are also present in the northern part of Serbia but are still neglected within diagnostic procedure. Medical doctors are still not completely aware of the diagnostics of dirofilariosis in humans, and there are still no reliable, noninvasive diagnostic methods on the market [21].
Apart from the modified Knott test done from the blood samples of dogs, an identification of the pathogen has been confirmed by PCR method too. Positive finding were gained by PCR method, at the matching rate of 92.3% with the modified Knott test.
3.2. Leishmaniasis
During the same period of study, 170 blood samples were examined for antibodies against Leishmania. Three of the examined dogs had skin lesions that would not heal and bad skin condition in general and all of the other dogs did not have any visible clinical symptoms. All blood samples derived from the dogs that never left their dwelling place.
From total number of dog’s sera, 18 dogs (10.59%), tested positive for the presence of specific antibodies against Leishmania infantum.
Seroprevalence of antibodies against Leishmania infantum from three groups of dogs (170 samples in total) for leishmaniasis with ELISA test is shown in Table 3.
Group of dogs
Total number of examined dogs
Number of positive dogs
Percentage of positive dogs
Hunting and military dogs
79
8
10.12
Dogs from asylum for homeless dogs
64
7
10.33
Pet dogs
27
3
11.11
Total
170
18
10.59
Table 3.
Seroprevalence of antibodies against L.infantum from three groups of dogs
In the group of hunting and military dogs, seroprevalence for leishmaniasis was found to be 10.12%. In the group of dogs from asylum, seroprevalence was found to be 10.33%, and in the group of pet dogs, seroprevalence for leishmaniasis was 11.11%. Total seroprevalence for leishmaniasis among whole tested dog population (170 animals) was 10.59%. There seems to be no difference in seroprevalence for leishmaniasis between different groups of dogs, unlike the prevalence to dirofilariosis. There is a constant presence of ethiological agent among dog population in the northern part of Serbia. In the near past, Vojvodina was considered as leishmaniasis free region of Serbia, since there were no reported cases of disease and fauna and abundance of sandflies was fairly low. Along with the first reported cases of canine leishmaniasis in Vojvodina, interest in entomological research of vectors has rised. After more than 60 years, in 2013 entomological research of sandflies in Vojvodina was resumed. Phlebotomus tobbi, a proven vector of L. infantum was recorded for the first time, and presence of the L. infantum in their non specific vectors Phlebotomus papatasi was found [46]. There is a reasonable doubt that canine leishmaniasis appears as a disease in the northern part of Serbia, in Vojvodina.
Nowdays, as the presence of vectors has been identified, as well as the existing seroprevalence in dogs with and without clinical symptoms, it can be suggested that there is an existence of the reservoirs of infection and a possibility of disease reemergence in Vojvodina. Leishmaniasis in humans has been identified so far only in people who have traveled to Mediterranean countries and not as an autochthonous infection. Diversity and abundance of sandflies in Vojvodina is still relatively low, but due to climate changes and increased summer temperatures, conditions that favor sandfly development are more and more pronounced. With potential risk of the disease development, more research has to be done, especially on vectors and reservoirs of the infection, with a precise identification of the pathogen. High prevalence of asymptomatic human carriers of L. infantum in Europe has been previously confirmed [56]. Findings of a L. infantum in sandflies, presence of a proven vector species, along with the findings of asymptomatic/symptomatic autochthonous cases of seropositivity in dogs as reservoirs, suggest that leishmaniasis may become a question of veterinary and public health in Serbia in the close future.
Today, Serbia is surrounded with several countries that have leishmaniasis for sure (Croatia, Montenegro, and FYROM), countries where vectors are identified so far (Hungary), and countries in which there is also a reasonable doubt that leishmaniasis exists in dogs (Romania). More research has to be done, especially on vectors and reservoirs of the infection, with a precise identification of the pathogen.
This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant TR31084) and also was done under the fram of EurNegVec COST Action TD1303.
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Written By
Sara Savic, Branka Vidic, Zivoslav Grgic, Tamas Petrovic, Alekasandar
Potkonjak, Aleksandra Cupina, Slavica Vaselek and Dusan Petric
Submitted: 26 January 2015Reviewed: 16 October 2015Published: 02 December 2015
Open access peer-reviewed chapter
