**3. Results and discussion**

#### **3.1. Dirofilariosis**

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

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

Clinical symptoms of leishmaniasis in dogs are nonspecific. They can be as fever, weakness, lethargy, weight loss, muscle wasting, lymphadenopathy, pallor, anemia, thrombocytopenia,

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)

Materials for the research were samples from dogs and samples of vectors. The research was planed as serological examination of dog blood samples for dirofilariosis and leishmaniasis. The vectors (mosquitoes) were collected, identified, and analyzed for the presence of causative

During spring and summer of 2014 (May–September), 292 samples of mosquitoes were collected and identified. Collecting was done with lamps with dry ice. The identification of mosquitoes (for gender and species) was done at Faculty of Agriculture, University of Novi Sad. Vector identification was done with microscopic observation. The analysis of vectors for the presence of causative agent for dirofilariosis was done by a molecular method (PCR). PCR analysis were performed at the Scientific Veterinary Institute of "Novi Sad." The samples were pooled as 20 mosquitoes into one pool. In collected mosquitoes, a molecular method of PCR was performed. DNA extraction was done with commercial kits from Quiagen (QIAmp), during a 2-day protocol. PCR was done according to the prescription of Rishniw et al. [36]. Primers used for PCR analysis were primers 5′–3′, forward: DIDR-F1\_for AGTGCGAATTG‐ CAGACGCATTGAG and reverse: DIDR-R1\_rev AGCGGGTAATCACGACTGAGTTGA.

In total, 170 of blood samples from dogs were examined for dirofilariosis and leishmaniasis. Serological analysis for dirofilariosis and leishmaniasis were done from blood samples of dogs obtained by venous punctation. The blood samples were divided into three groups, according

**•** 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

that have never left their homes in Serbia (Figures 5 and 6) [41, 42].

conjunctivitis and eye problems, skin lesions and alopecia, etc.

and enzyme-linked immunosorbent assay (ELISA) [44].

agents of dirofilariosis in the northern part of Serbia.

Determination was done based on 542 bp for *D. immitis*.

dogs and humans directly is not possible.

114 An Overview of Tropical Diseases

**2. Materials and methods**

to the way of life of the dogs:

Analysis of vectors for dirofilariosis: In total, 292 samples of mosquitoes were collected. After determination, it was found that they belong to *Culex* (*Culex pipiens* and *Culex culex*) and *Aedes* species. The samples were collected as random samples from different locations of the northern part of Serbia. In 292 mosquitoes randomly collected, the presence of DNA of causative agent for dirofilariosis (*Dirofilaria immitis*) was not found. Mosquitoes were collected randomly, and weather conditions during the collection of samples were not favorable. Weather conditions were bad for the lamps and the collection process because there was a lot of wind and often rain during the time of collection. Also, the outside temperature was lower than usual for that the time of the year. The results found after the analysis of mosquito samples indicate that sampling was perhaps not done in the best way. The samples should have been collected at the residence of positive dogs and not from several randomly chosen locations. Also, the weather conditions may have influenced the development of L3 larval stage of microfilariae in the mosquito because the temperature for many days was not too much above 14°C. All of these may have influenced the absence of *Dirofilaria* sp. DNA in mosquito samples.

The results of analysis of blood samples from three groups of dogs (170 samples in total) to dirofilariosis by the modified Knott test are shown in Table 1.


**Table 1.** Results of the analysis of blood samples from three groups of dogs to dirofilariosis

In the group of hunting and military dogs, a seroprevalence for dirofilariosis was found to be 22.78%. In the group of dogs from asylum, a lower seroprevalence for dirofilariosis was found —3.12% and in the group of pet dogs, and seroprevalence for dirofilariosis was found to be 22%. In the case of dirofilariosis, the seroprevalence of the disease in different groups was different. It depended on the received prevention treatment against parasites and the lifestyle of dogs. Seroprevalence was the lowest (3.12%) in dogs living in asylum with regular preven‐ tion 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 seroprevalence (22.78%) was found in hunting dogs with no prevention treatment in most of the cases. Seroprevalence 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. The modified Knott test gives us a direct overview into the existence of larvae of *Dirofilaria* (microfilaria) in dogs' circulation. A total average sero‐ prevalence for the whole three groups of dogs was found to be 15.29%, but the highest seroprevalence 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 ectoantiparasitic treatment.

The 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.

part of Serbia. In 292 mosquitoes randomly collected, the presence of DNA of causative agent for dirofilariosis (*Dirofilaria immitis*) was not found. Mosquitoes were collected randomly, and weather conditions during the collection of samples were not favorable. Weather conditions were bad for the lamps and the collection process because there was a lot of wind and often rain during the time of collection. Also, the outside temperature was lower than usual for that the time of the year. The results found after the analysis of mosquito samples indicate that sampling was perhaps not done in the best way. The samples should have been collected at the residence of positive dogs and not from several randomly chosen locations. Also, the weather conditions may have influenced the development of L3 larval stage of microfilariae in the mosquito because the temperature for many days was not too much above 14°C. All of

these may have influenced the absence of *Dirofilaria* sp. DNA in mosquito samples.

Hunting and military dogs 79 18 22.78

Pet dogs 27 6 22 Total 170 26 15.29

In the group of hunting and military dogs, a seroprevalence for dirofilariosis was found to be 22.78%. In the group of dogs from asylum, a lower seroprevalence for dirofilariosis was found —3.12% and in the group of pet dogs, and seroprevalence for dirofilariosis was found to be 22%. In the case of dirofilariosis, the seroprevalence of the disease in different groups was different. It depended on the received prevention treatment against parasites and the lifestyle of dogs. Seroprevalence was the lowest (3.12%) in dogs living in asylum with regular preven‐ tion 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 seroprevalence (22.78%) was found in hunting dogs with no prevention treatment in most of the cases. Seroprevalence 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

dirofilariosis by the modified Knott test are shown in Table 1.

**Total number of examined**

**Table 1.** Results of the analysis of blood samples from three groups of dogs to dirofilariosis

for a long time after infection makes the owners believe that their dog is healthy.

**Group of dogs**

116 An Overview of Tropical Diseases

Dogs from asylum for homeless dogs

The results of analysis of blood samples from three groups of dogs (170 samples in total) to

**dogs Number of positive dogs Percentage of positive**

64 2 3.12

**dogs**

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). The isolation of *Dirofilaria*'s DNA from 200 μl of blood samples was done with QIAmp set kit (Quiagen). PCR procedure was done as described by Rishniw et al. [36]. PCR is a very sensitive, specific, and accurate method with which determination of *Dirofilaria* species is possible. 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 found by the modified Knott test, a positive result was found by the PCR method in 24 samples (92.3%) (Figure 8).

**Figure 8.** PCR reaction for *Dirofilaria immitis* in blood samples of dogs positive to dirofilariosis by the modified Knott test, determination based on 542 bp.

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 dirofilar‐ iosis 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.


**Table 2.** An overview of data collection during a 10-year period on the seroprevalence 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 seroprevalence 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, and ELISA test. 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 when looking at a patient in veterinary practice. Also, there are already human cases 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 there is more demand for control of health status for dirofilariosis within a routine 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, seropositive 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, and this makes the disease a danger for public health. 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**

**Figure 8.** PCR reaction for *Dirofilaria immitis* in blood samples of dogs positive to dirofilariosis by the modified Knott

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 dirofilar‐ iosis 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

2003–2004 5.9–7

2010 14 2010, only pet dogs 11

**Table 2.** An overview of data collection during a 10-year period on the seroprevalence of dirofilariosis in dogs in

By comparing the data during the last decade, it can be stated that there is a constant increase of seroprevalence 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, and ELISA test. 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,

2011–2013 5 human cases

**Year Percentage of positive dog samples**

15.29

northern part of Serbia, data have been collected for more than 10 years now.

2006–2007, dogs with no clinical symptoms 10–11 2006–2007, dogs with clinical symptoms 80

test, determination based on 542 bp.

118 An Overview of Tropical Diseases

2013–2014 hunting and military dogs, dogs from asylum and pet dogs

northern Serbia

During the same period of study, 170 blood samples were examined for leishmaniasis from dogs that did or did not have clinical symptoms of the disease. After serological testing of the samples, positive findings for leishmaniasis were gained. Blood samples were analyzed for

the presence of specific antibodies against *Leishmania* sp. with the ELISA method (Ingezim Leishmania, Ingenasa, 1.5.LSH.K.1). From total number of samples, in 10.59% of samples, the presence of specific antibodies against *Leishmania infantum* was found*.* It is important to highlight that not one of the examined dogs has ever left their dwelling place. In 18 dogs, positive serological findings for leishmaniasis were obtained. Three of the examined dogs had skin lesions that would not heal and bad skin condition in general.

The findings after the analysis of blood samples from three groups of dogs (170 samples in total) for leishmaniasis with ELISA test are shown in Table 3.


**Table 3.** Results of the analysis of blood samples from three groups of dogs for leishmaniasis

In the group of hunting and military dogs, a 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%. The total seroprevalence for all 170 blood samples was 10.59% for leishmaniasis. Overall, there is actually no difference in seroprevalence for leishmaniasis between different groups of dogs. There is a similar seropre‐ valence in all three groups of dogs, unlike the seroprevalence to dirofilariosis. There is a constant presence of causative pathogen among the dog population in the northern part of Serbia. There is a reasonable doubt that leishmaniasis appears as a disease in the northern part of Serbia, in Vojvodina. The presence of vectors has been identified (*Phlebotomus papatasi* and *Laroussius tobbi*) (Vaselek, unpublished data), as well as the existing seroprevalence in dogs with and without clinical symptoms. All of this suggests that there is an existence of the reservoirs of infection. Leishmaniasis in humans has been identified so far only in people who have traveled to Mediterranean countries and not as an autochthonous infection. Due to climate changes, summer temperatures and conditions in the northern part of Serbia are more and more in favor of the life cycle of vectors—sand flies.

In our history, there is evidence of leishmaniasis in humans and in dogs in Serbia, but over 60 years ago. 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]. During the period from 1968 to 1969, rare cases of autoch‐ thonous visceral leishmaniasis were reported in the southern part of Serbia. At that time, the vectors of leishmaniasis were detected: *P. major*, *P. simici*, and *P. perfiliewi* [46]. In the northern part of Serbia, the disease or vectors have never been identified before. After this period of studies and interest of public into leishmaniasis, no more data were found, and no research has been done until now. No vectors have been identified any more, or they were just not looked for until 50 years later. There is a question on the existence of leishmaniasis in Serbia, but at the moment, there is evidence of the existence of vectors and clinical disease in dogs, with serological conformation of the disease and successful therapy. Today, Serbia is sur‐ rounded 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.
