**Dirofilariosis and Leishmaniasis in the Northern Region of Serbia**

Sara Savic, Branka Vidic, Zivoslav Grgic, Tamas Petrovic, Alekasandar Potkonjak, Aleksandra Cupina, Slavica Vaselek and Dusan Petric

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61761

#### **Abstract**

Research in the field of vector borne diseases and zoonozes became a topic of interest in Serbia, during the last decade. Climate changes in the country (and the region) are evident. Also, significantly is higher frequency of human and animal movement and travel, especially of dogs, within the European countries, but with overseas countries as well. The presence of vectors has already been confirmed in the country and all the surrounding countries. Current research in the domain of infectious diseases in dogs mostly includes diseases which 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 which are not obvious and therefore they represent a danger for public health with dogs acting as reservoirs of the infection.

Vectors necessary for the transmission of dirofilariosis are mosquitoes and for leishmaniasis are sand flies. Vectors of dirofilariosis are mosquitoes. Female mosqui‐ toes 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 14oC is needed. Diagnostic methods for dirofilariosis are many, but several serological methods can be used: ELISA modified Knott test, immunoem‐ zyme fast test and then molecular method (PCR), etc.

Leishmaniasis is a vector borne zoonotic disease caused by a pathogen of *Leishmania* species. For the transmission of the disease, sand flies are needed as vectors from

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Lutzomyia spp. Female sand flies are bloodsucking organisms which can transfer the pathogen from one host to another during their feeding time. The presence of *Phlebotominae* (commonly known as "sand flies") has been identified in Serbia. The most certain method for diagnostic is demonstration of the parasite from bone marrow, splenic or lymph node aspirates, but there are other less invasive methods, like IFAT (immunofluorescent test) and ELISA (enzyme-linked immunosorbent assay).

Material for the research were samples from dogs and samples of vectors. In total, 292 samples of mosquitoes were collected and identified and 170 of blood samples from dogs were examined for dirofilariosis and leishmaniasis. Methods used in the study were modified Knott test and PCR for dirofilariosis and ELISA test for leismaniasis. For dirofialriosis a total prevalence of the disease in dogs was found to be 15,29%, (with different values from 3-22%) for different groups of dogs (hunting and military dogs, dogs from asylum and pet dogs). Total seroprevalence for all 170 blood samples was 10,59% for leishmaniasis. Overall, there is acually no difference in seroprevalnce for leishmaniosis, between different groups of dogs (hunting and military dogs, dogs from asylum and pet dogs). There is a reasonable doubt that leismaniasis appears as a disease in the Northern part of Serbia, in region of Vojvodina. The presence of vectors has been identified (*Phlebotomus papatasi*, *Laroussius tobbi*) as well as existing seropre‐ valence in dogs with and without clinical symptoms. All of this suggests that there is an existence of the reservoirs of infection.

**Keywords:** dirofilariosis, leishmaniasis, diagnostics, dogs

### **1. Introduction**

Research in the field of vector-borne diseases and zoonozes became a topic of interest in Serbia during the last decade. Climate changes in the country (and the region) are 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 has already been confirmed in the country and all the surrounding countries. 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 reservoirs 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.

Lutzomyia spp. Female sand flies are bloodsucking organisms which can transfer the pathogen from one host to another during their feeding time. The presence of *Phlebotominae* (commonly known as "sand flies") has been identified in Serbia. The most certain method for diagnostic is demonstration of the parasite from bone marrow, splenic or lymph node aspirates, but there are other less invasive methods, like IFAT (immunofluorescent test) and ELISA (enzyme-linked immunosorbent

Material for the research were samples from dogs and samples of vectors. In total, 292 samples of mosquitoes were collected and identified and 170 of blood samples from dogs were examined for dirofilariosis and leishmaniasis. Methods used in the study were modified Knott test and PCR for dirofilariosis and ELISA test for leismaniasis. For dirofialriosis a total prevalence of the disease in dogs was found to be 15,29%, (with different values from 3-22%) for different groups of dogs (hunting and military dogs, dogs from asylum and pet dogs). Total seroprevalence for all 170 blood samples was 10,59% for leishmaniasis. Overall, there is acually no difference in seroprevalnce for leishmaniosis, between different groups of dogs (hunting and military dogs, dogs from asylum and pet dogs). There is a reasonable doubt that leismaniasis appears as a disease in the Northern part of Serbia, in region of Vojvodina. The presence of vectors has been identified (*Phlebotomus papatasi*, *Laroussius tobbi*) as well as existing seropre‐ valence in dogs with and without clinical symptoms. All of this suggests that there is

Research in the field of vector-borne diseases and zoonozes became a topic of interest in Serbia during the last decade. Climate changes in the country (and the region) are 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 has already been confirmed in the country and all the surrounding countries. 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 reservoirs of the

assay).

108 An Overview of Tropical Diseases

**1. Introduction**

an existence of the reservoirs of infection.

**Keywords:** dirofilariosis, leishmaniasis, diagnostics, dogs

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 sand flies. 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 *Dirofilaria repens*. 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 (ex, like previously known as Yugosla‐ via) 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. Diagnos‐ tics of dirofilariosis in Serbia has started approximately 10 years ago. Since 2004 until nowa‐ days, veterinary services have started a regular, routine check up in dogs for dirofilariosis. Cases of dirofilariosis (*Dirofilaria immitis* and *Dirofilaria repens*) in Serbia have been found so far both in humans and dogs. Several cases of dirofilariosis in humans have been represented, and few studies have been done [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 seropre‐ valence and diagnostic methods [28–32]. Some research was also done on seroprevalence to dirofilariosis in working and military dogs and in pet dogs [33] (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 one infected organism to another noninfected one. Female mosquitoes are vectors that can be found in high numbers in Serbia during the warm period of the year, from May to October. Over 70 mosquito species can be vectors of dirofilario‐ sis out of 3000 mosquito species worldwide. Three of those species can be found in Serbia —*Aedes*, *Anopheles*, and *Culex* [34].

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, waiting for 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, *Dirofilaria immitis* 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.** *Dirofilaria immitis* taken out from the heart of a dog.

Vectors of dirofilariosis are mosquitoes. Female mosquitoes that feed on mammals can transfer microfilaria from one infected organism to another noninfected one. Female mosquitoes are vectors that can be found in high numbers in Serbia during the warm period of the year, from May to October. Over 70 mosquito species can be vectors of dirofilario‐ sis out of 3000 mosquito species worldwide. Three of those species can be found in Serbia

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

*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, waiting for 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, *Dirofilaria immitis* never reaches the adult stage, and they can never be found in the heart of humans

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,

—*Aedes*, *Anopheles*, and *Culex* [34].

110 An Overview of Tropical Diseases

because humans are accidental hosts [34] (Figure 2).

seizures, and sudden death.

the eye.

Diagnostic methods for dirofilariosis are many, but several serological methods can be used: ELISA modified Knott test, immunoenzyme fast test, and then molecular method (PCR). Antibodies formed against the antigens of *Dirofilaria* sp. can be detected by ELISA method.

ELISA is a very sensitive and specific test, easy to perform, but it has to be done in a laboratory. There can be a false-positive reaction if there is a cross reaction with another antigen. 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 level of antibodies high enough (Figure 3).

**Figure 3.** ELISA method for diagnostics—positive and negative control and positive and negative samples.

Antibodies formed against *Dirofilria* sp. can also be detected by an immunoenzyme test, usually called "fast" or "snap" tests. 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 among veterinarians 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].

PCR is a molecular method with which DNA of *Dirofilaria immitis* is detected. This is a sensitive and accurate method 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 is a vector-borne zoonotic disease caused by a pathogen of *Leishmania* species. For the transmission of the disease, sand flies are needed as vectors from *Lutzomyia* spp. Female sand flies are bloodsucking organisms that can transfer the pathogen from one host to another during their feeding time. In the overview of human leishmaniasis from 2009, in Europe there are cases described in Greece, Cyprus, France, Italy, Malta, Portugal, Spain, FYROM, and Albania [5]. Later on, there are data published on cases of leishmaniasis in humans in Bulgaria [37], in dogs in Romania [38], and in dogs in Hungary [39].

In Serbia, leishmaniasis is considered, so far, an imported disease, and there is no official data that the disease exists as an autochthonous infection in humans or in animals. There are cases of humans with leishmaniasis in Serbia, but all of them were imported from Montenegro, FYOM (Former Yugoslavia Republic of Macedonia), or Greece during holiday season [40]. There were some notifications about leishmaniasis in dogs during the last several years. Three separate cases of dogs were found with clinical symptoms that could indicate leishmaniasis, and they were found seropositive to leishmaniasis. After therapy, their condition has improved [41, 42].

Antibodies formed against *Dirofilria* sp. can also be detected by an immunoenzyme test, usually called "fast" or "snap" tests. 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

The most "popular" and most used diagnostic test for dirofilariosis among veterinarians 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

PCR is a molecular method with which DNA of *Dirofilaria immitis* is detected. This is a sensitive and accurate method 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

Leishmaniasis is a vector-borne zoonotic disease caused by a pathogen of *Leishmania* species. For the transmission of the disease, sand flies are needed as vectors from *Lutzomyia* spp. Female sand flies are bloodsucking organisms that can transfer the pathogen from one host to another during their feeding time. In the overview of human leishmaniasis from 2009, in Europe there are cases described in Greece, Cyprus, France, Italy, Malta, Portugal, Spain, FYROM, and Albania [5]. Later on, there are data published on cases of leishmaniasis in humans in Bulgaria

In Serbia, leishmaniasis is considered, so far, an imported disease, and there is no official data that the disease exists as an autochthonous infection in humans or in animals. There are cases of humans with leishmaniasis in Serbia, but all of them were imported from Montenegro, FYOM (Former Yugoslavia Republic of Macedonia), or Greece during holiday season [40].

**Figure 4.** Immunoenzyme fast test—positive (two dots) and negative (one dot) findings.

to the other available tests (Figure 4).

112 An Overview of Tropical Diseases

to different species can be determined [35].

[37], in dogs in Romania [38], and in dogs in Hungary [39].

method [36].

The presence of *Phlebotominae* (commonly known as "sand flies") has been identified in the southern part of Serbia a long time ago, and these vectors are known in Mediterranean countries as vectors of leishmaniasis. During the late 1950s and early 1960s, studies were done on the presence of *Phlebotominae* in the southern part of Serbia, but after that period, nothing else was published [43]. During a previous period, several dogs were found in the region, as clinical cases suspicious to leishmaniasis (epistaxis, cachexia, pale mucosa, skin problems, blindness, and lethargy), with seropositive findings for this disease [42].

**Figure 5.** Bitch with skin lesions, positive serological finding for leishmaniasis.

**Figure 6.** Dog with skin lesions, positive serological finding for leishmaniasis.

The first clinical cases of leishmaniasis in humans and dogs in Serbia were infections coming from abroad (Montenegro, Greece, Former Yugoslavia Republic of Macedonia, and Croatia), mostly after summer holidays. Within the last 3 years, positive findings were identified in dogs that have never left their homes in Serbia (Figures 5 and 6) [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 *Leishma‐ nia 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 sand fly bites, and dogs are the reservoir hosts. Humans are accidental hosts. Transmission of the disease between dogs and humans directly is not possible.

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.

Diagnostic procedures for leishmaniasis are several. The most certain method is the demon‐ stration of the parasite from bone marrow, splenic, or lymph node aspirates, but there are other less invasive methods too. Serologic tests are most commonly immunofluorescent test (IFAT) and enzyme-linked immunosorbent assay (ELISA) [44].
