Results of the analysis of blood samples (modified Knott test) originating from three groups of dogs for presence of microfilaria
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.
- sandfly vectors
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 . 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
Vectors of dirofilariosis are mosquitoes. Female mosquitoes which feed on mammals can transfer microfilaria from one infected organism to another non infected one.
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
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 (
The first cases of dirofilariosis in Serbia, in dogs, were discovered as a side finding during dissections . 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  after the first findings of
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 . Female
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
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.
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
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).
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 . The modified Knott test is the preferred method for observing morphology and measuring body dimensions to differentiate
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 .
Leishmaniasis are vector-borne zoonotic diseases caused by a protozoan parasite belonging to genus
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 . 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 . 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 . 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 . 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
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
Newly gathered entomological data indicate disease circulation since presence of
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) . 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 . 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 .
2. Materials and methods
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 . Only specimens of the main vector species,
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. . 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
Detection for antibodies against
3. Results and discussion
Analysis of vectors for dirofilariosis: In total, 292 samples of
The presence of DNA of
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.
|Hunting and military dogs||79||18||22.78|
|Dogs from asylum for homeless dogs||64||2||3.12|
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
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
After positive samples were found by the modified Knott test, a PCR analysis was done for the conformation of
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 . 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.
|2006–2007, dogs with no clinical symptoms||10–11|
|2006–2007, dogs with clinical symptoms||80|
|2010, only pet dogs||11|
|2011–2013||5 human cases|
|2013–2014 hunting and military dogs, dogs from asylum and pet dogs||15.29|
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 (
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).
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 .
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.
During the same period of study, 170 blood samples were examined for antibodies against
From total number of dog’s sera, 18 dogs (10.59%), tested positive for the presence of specific antibodies against
Seroprevalence of antibodies against
|Hunting and military dogs||79||8||10.12|
|Dogs from asylum for homeless dogs||64||7||10.33|
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.
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
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.
Ready PD (2008) Leishmaniasis emergence and climate change. In: S de la Roque, ed‐ itor. Climate change: the impact on the epidemiology and control of animal diseases. Rev Sci Tech Off Int Epiz. 27(2):399–412.
Jacsó O, Kiss G, Krassóvári D, Kiss HJ, Gyurkovszky M, Fok E. (2014) Epidemiologi‐ cal view about dirofilarioses in dogs of Hungary. Fourth European Dirofilaria and Angiostrongylus Days (FEDAD), 2–4 July, 2014, Budapest, Hungary. Edited by Eva Fok, Istvan Kucsera, Final Program and Abstracts, Budapest, European Society of Dirofilaria and Angiostrongylus.
Miterpáková M, Iglódyová A, Antolová D, Hurníková Z. (2014) Canine dirofilariosis in Slovakia—the results of 8-year epidemiological research. Fourth European Dirofi‐ laria and Angiostrongylus Days (FEDAD), 2–4 July, 2014, Budapest, Hungary. Edited by Eva Fok, Istvan Kucsera, Final Program and Abstracts, Budapest, European Soci‐ ety of Dirofilaria and Angiostrongylus.
Farkas R1, Tánczos B, Bongiorno G, Maroli M, Dereure J, Ready PD. (2011) First sur‐ veys to investigate the presence of canine leishmaniasis and its phlebotomine vectors in Hungary. Vector Borne Zoonotic Dis. 11(7):823–34. doi: 10.1089/vbz.2010.0186. Jan 22.
Ready PD. (2010) Leishmaniasis emergence in Europe. Euro Surveill. 15(10):pii=19505. Available online: http://www.eurosurveillance.org
Draghici S, Jarca A. (2012) Dirofilariasis-case file. European Multicolloquium of Para‐ sitology (EMOP XI). Program and Abstract book, Romania, p. 386.
Hamel D, Silaghi C, Mihalkov A, Maurer U, Pfister K. (2012) Vector-borne infections in imported dogs—a serological and molecular survey. European Multicolloquium of Parasitology (EMOP XI). Program and Abstract book, Romania, p. 300.
Simon F, Gonzles-Miguel J, Morchon R, Mellado I, Del Mar Siles-Luca M. (2012) Pro‐ teomic analyses of Dirofilariaspecies. European Multicolloquium of Parasitology (EMOP XI). Program and Abstract book, Romania, p. 234.
Otasevic S, Tasic A, Gabrielli S, Trenkic Bozinovic M, Cancrini G. (2014) Canine and human Dirofilaria infections: what is new in the Balkan Peninsula. Fourth European Dirofilaria and Angiostrongylus Days (FEDAD), 2–4 July, 2014, Budapest, Hungary. Edited by Eva Fok, Istvan Kucsera. Final Program and Abstracts, Budapest, Europe‐ an Society of Dirofilaria and Angiostrongylus.
Diakou A, Kapantaidakis E. (2014) Epidemiology of dirofilariosis in dogs in Greece: previous and last information. Fourth European Dirofilaria and Angiostrongylus Days (FEDAD), 2–4 July, 2014, Budapest, Hungary. Edited by Eva Fok, Istvan Kuc‐ sera. Final Program and Abstracts, Budapest, European Society of Dirofilaria and Angiostrongylus.
Margarida Alho A, Gomes L, Pereira da Fonseca I, Meireles J, Madeira de Carvalho L. (2014) Canine dirofilariosis in Lisbon, Portugal, over a 14-year period: prevalence, diagnosis, and routine laboratory approaches. Fourth European Dirofilaria and An‐ giostrongylus Days (FEDAD), 2–4 July, 2014, Budapest, Hungary. Edited by Eva Fok, Istvan Kucsera. Final Program and Abstracts, Budapest, European Society of Dirofi‐ laria and Angiostrongylus.
Fuehrer H-P, Auer H, Silbermayr K, Duscher GG. (2014) Dirofilaria repens and Diro‐ filaria immitis in Austria. Fourth European Dirofilaria and Angiostrongylus Days (FEDAD), 2–4 July, 2014, Budapest, Hungary. Edited by Eva Fok, Istvan Kucsera. Fi‐ nal Program and Abstracts, Budapest, European Society of Dirofilaria and Angios‐ trongylus.
Kulišić Z, Kranjčić-Zec I, Mitrović S, Radojičić B. (1989) New case of human dirofilar‐ iosis in Jugoslavia (serb), 6. Kongres mikrobiologa Jugoslavije Maribor, Proceedings.
Kulišić Z, Milosavljević P. (1994) Modern techniques in diagnostic of dirofilariosis in dogs (serb). Veterinarski Glasnik. 48, 9: 745–9.
Kulišić Z, Mišić Z, Milosavljević P, Popović N. (1995) Dirofilariosis in dogs in Yugo‐ slavia (serb), 8. Symposium of Veterinarians in Serbia, Zlatibor, Proceedings.
Dimitrijević S. (1999) Dirofilarioza ante portas, Clinica Veterinaria 1, Proceedings, p. 58.
Lalošević D. (2004) Dirofilariaspp parasite like in mesenterium removed by surgery from mesenterium (serb). Medicinski Pregled, 5–6: 307–8.
Tasić A, Tasić S, Miladinović-Tasić N, Zdravković D, Đorđević. (2007) Dirofilaria re‐ pens—potential risk for public health. Acta Medica Medianae; 46(3): 53–6.
Džamić AM, Čolović IV, Arsić-Arsenijević VS, Stepanović S, Boričić I, Džamić Z, Mi‐ trović SM, Rašić DM, Stefanović I, Latković Z, Kranjčić-Zec IF. (2009) Human Dirofi‐ laria repens infection in Serbia. J Helminthol; 83: 129–37.
Tasić S, Stojiljković N, Miladinović-Tasić N, Tasić A, Mihailović D, Đorđević J. (2009) Human subcutaneous dirofilariosis in South Serbia—case report. u: Dirofilaria days (II), Salamanca, Spain, P-12.
Đorđević J, Tasić S, Miladinović-Tasić N, Tasić A. (2010) Diagnosis and clinical value of human dirofilariosis (serb). Acta Facultatis Medicae Naissensis; 27(2): 81–4.
Tasić-Otašević S, Gabrielli S, Tasić A, Miladinović Tasić N, Kostić J, Ignjatović A, Po‐ pović Dragonjić L, Milošević Z, Cancrini G. (2014). Seroreactivity to Dirofilaria anti‐ gens in people from different areas of Serbia. BMC Infect. Dis; 14: 68.
Tasić A, Katić-Radivojević S, Klun I, Mišić Z, Ilić T, Dimitrijević S. (2003) Prevalence of canine filariasis in some parts of Vojvodina. Veterinary Symposium of Serbia (15), Zlatibor, 09–13. 09., Proceedings.
Tasić A, Tasić S, Miladinović-Tasić N, Zdravković D, Đorđević J. (2007) Prevalence of Dirofilariae repensas causative agent of zoonozes in dogs. Acta Facultatis Medicae Naissensis; 24(2): 71–4.
Tasic A, Rossi L, Tasic SA, Miladinovic-Tasic N, Ilic TD, Dimitrijevic SM. (2008) Sur‐ vey of canine dirofilariasis in Vojvodina, Serbia.
Spasojević-Kosić L, Lalošević V, Lalošević D, Naglić A. (2011) Heart worm disease— a case study. Veterinarski Glasnik; 65(3–4): 257–67.
Spasojević-Kosić L, Lalošević V, Lalošević D, Simin S, Vasić I, Kuruca L. (2012) Prev‐ alence of dirofilariosis in pet dogs in Novi Sad. Saremena Poljoprivreda; 61(3–4): 247–54.
Savić-Jevđenić S, Vidić B, Grgić Ž, Milovanović A. (2004) Fast diagnostic of dirofilar‐ iosis in Novi Sad region (serb). Veterinarski Glasnik; 58(5–6): 693–698. ISSN 0350-2457.
Savić-Jevđenić S, Milovanović A, Grgić Ž, Kojić S. (2006) Outspread of dirofilariosis in Novi Sad region-six years later (serb), Proceedings, VIII epizootiological days, Banja Vrdnik, p. 60.
Savić-Jevđenić S, Vidić B, Grgić Ž, Lolić Z. (2007) The appearances of dirofilariosis in Serbia - Vojvodina. First European Dirofilaria Days, Zagreb, Croatia, Proceedings, p. 202.
Savić S, Grgić Z, Vujkov B, Fenjac I, Pajković D, Žekić M. (2009) Determination of ca‐ nine dirofilariasis by ELISA method and modified Knott’s test. Arhiv Veterinarske Medicine; 2(2): 71-77. ISSN 1820-9955.
Savić S, Vidić B, Pajkovič D, Spasojević-Kosić L, Medić S, Potkonjak A, Otašević S. (2014) Overview of dirofilariosis in Serbia during the last ten years 2004–2014 and current status of the disease. Fourth European Dirofilaria and Angiostrongylus Days (FEDAD), 2–4 July, 2014, Budapest, Hungary. Edited by Eva Fok, Istvan Kucsera, Fi‐ nal Program and Abstracts, str. 46, Budapest, European Society of Dirofilaria and Angiostrongylus.
Pajković D, Savić S, Veljković P, Grgić Z. (2010) Study on dirofilariosis in working dogs servicing the Army of Serbia. Arhiv Veterinarske Medicine; 3(2): 53–58.
Newcomb EC, Moorhead A. (2013) Dirofilaria immitis, American Association of Zoo Veterinarians Infectious Disease Committee Manual, 2013 Infectious Disease Manual 2013, 2nd Edition.
Genchi C, Venco L, Genchi M. (2007) Guideline for the laboratory diagnosis of canine and feline Dirofilariainfections, Dirofilaria immitis, and D. repensin dog and cat and human infections. Edited by Genchi C, Rinaldi L, Cringoli Giuseppe. Mappe parasi‐ tologiche 8, ISBN 88-89132-14-0, pp. 139–144.
Rishniw M, Barr SC, Simpsom KW, Frongillo MF, Franz M, Dominguez-Alpizar JL. (2006) Discrimination between six species of canine microfilariae by single polymer‐ ase chain reaction. Veterinary Parasitology 135, pp. 303–314.
Harizanov R, Rainova I, Tzvetkova N, Kaftandjiev I, Bikov I, Mikov O. (2013) Geo‐ graphical distribution and epidemiological characteristics of visceral leishmaniasis in Bulgaria, 1988 to 2012, Eurosurveillance, Volume 18, Issue 29, 18 July 2013, Surveil‐ lance and outbreak reports.
Mircean V, Dumitrache MO, Mircean M, Bolfa P, Györke A, Mihalca AD. (2014) Au‐ tochthonous canine leishmaniasis in Romania: neglected or (re)emerging? Parasit Vectors. 7: 135. http://www.parasitesandvectors.com/content/7/1/135
Tánczos B1, Balogh N, Király L, Biksi I, Szeredi L, Gyurkovsky M, Scalone A, Fioren‐ tino E, Gramiccia M, Farkas R. (2012 July) First record of autochthonous canine leish‐ maniasis in Hungary. Vector Borne Zoonotic Dis. 12(7): 588–94. doi: 10.1089/vbz. 2011.0906. Epub 2012 May 18.
Dakic ZD1, Pelemis MR, Stevanovic GD, Poluga JL, Lavadinovic LS, Milosevic IS, In‐ djic NK, Ofori-Belic IV, Pavlovic MD. (2009) Epidemiology and diagnostics of viscer‐ al leishmaniasis in Serbia. Clin Microbiol Infect. 15(12):1173–6.
Savić-Jevđenić S, Grgić Ž, Vidić B, Vujkov B. (2007) Leishmaniasis in dog—clinical case, IX regional meeting in clinical pathology and therapy in animals Palic, Proceed‐ ings, p. 2, Belgrade, Faculty of Veterinary Medicine.
Savić S, Vidić B, Fenjac I, Bekvalac R, Radičević M, Lolić Z. (2010) Leishmaniasis in dogs in Serbia—existence or not? Symposium of Small Animal Practitioners SIVE‐ MAP 2010, Belgrade, Proceedings, pp. 241–243.
Dimitrijević S, (1999) Dirofilarioza ante portas. Meeting on Clinical Pathology and Therapy of Animals, Proceedings, Budva, pp. 58–61.
Fiorello C. (2013) Visceral leishmaniasis. American Association of Zoo Veterinarians Infectious Disease Committee Manual, 2013 Infectious Disease Manual 2013, 2nd Ed‐ ition.
Simić C. (1957) Protozoe paraziti čoveka i domaćih životinja. Naučna knjiga, Beo‐ grad.
Vaselek S., Savić S., Di Muccio T., Erisoz Kasap O., Gradoni L., Alten B., Petrić D. (2015) Possible re-emergence of leishmaniasis in Serbia – oral presentation;
2nd Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec); Abstract book.
Nelson, C. T. (2005) "2005 Guidelines for the diagnosis, prevention and management of heartworm (Dirofilaria immitis) infection in dogs." Veterinary parasitology 133.2-3 (2005): 255.
Zivkovic V. (1967) Phlebotominae (Diptera, Psychodidae) south-east and east Serbia Glas Srpske akademije nauka i umetnosti, Odeljenje medicinskih nauka, knjiga 20.
Milovanović, M., Popović, D., (1960): Contribution to the study of kala-azar epidemic in PR Serbia. Glasnik higijenskog instituta 23-27.
Simić, Č., Kostić, D., Nežić, E. i Zivković, V., (1951): Prilog poznavanju flebolomina Jugoslavije.VI deo. Flebotomine Vojvodine, Bosne, Hercegovine, Dalmacije i Istre. Glas Srpske akademije nauka, Odeljenje medicinskih nauka, 81-86.
Mettler M, Grimm F, Capelli G, Camp H, Deplazes P (2005): Evaluation of enzymelinked immunosorbent assays, an immunofluorescent-antibody test, and two rapid tests (immunochromatographic-dipstick and gel tests) for serological diagnosis of symptomatic and asymptomatic Leishmania infections in dogs. J Clin Microbiol 2005, 43(11):5515-5519. 45.
Mohebali M, Taran M, Zarei Z (2004): Rapid detection of Leishmania infantum infection in dogs: comparative study using an immunochromatographic dipstick rk39 test and direct agglutination. Vet Parasitol 2004, 121(3- 4):239-245. 46.
Solano-Gallego, Laia, Guadalupe Miró, Alek Koutinas, Luis Cardoso, Maria Grazia Pennisi, Luis Ferrer, Patrick Bourdeau, Gaetano Oliva, and Gad Baneth. (2011) "LeishVet guidelines for the practical management of canine leishmaniosis." Parasit Vectors 4, no. 1: 86.
Baneth, Gad, and Itamar Aroch. (2008) "Canine leishmaniasis: a diagnostic and clinical challenge." The Veterinary Journal 175.1 : 14-15.
Becker N, Petrić D, Zgomba M, Boase C, Madon M, Dahl C, Kaiser A (2010): Mosquitoes and their control. Berlin Heidelberg: Springer – Verlag.
Moral L, Rubio EM, Moya M. (2002), A leishmanin skin test survey in the human population of l'Alacantí region (Spain): implications for the epidemiology of Leishmania infantum infection in southern Europe. Trans R Soc Trop Med Hyg. Mar-Apr;96(2):129-32.
Latrofa MS, Montarsi F, Ciocchetta S, Annoscia G, Dantas-Torres F, Ravagnan S, Capelli G, Otranto D (2012): Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis. Parasit Vectors, 5:76.
Yildirim A, Inci A, Duzlu O, Biskin Z, Ica A, Sahin I (2011): Aedes vexans and Culex pipiens as the potential vectors of Dirofilaria immitis in Central Turkey. Vet Parasitol, 178:143–147.
Cancrini G, Ricci I, Tessarin C, Gabrielli S, Pietrobelli M (2003): Aedes albopictus is a natural vector of Dirofilaria immitis in Italy. Vet Parasitol, 118:195–202.
Cancrini G, Scaramozzino P, Gabrielli S, Di Paolo M, Toma L, Romi R (2007): Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in central Italy. J Med Entomol, 44:1064–1066.
Petrić D, Zgomba M, Bellini R, Becker N (2012): Surveillance of Mosquito Populations: A Key Element to Understanding the Spread of Invasive Vector Species and Vector-Borne Diseases in Europe.: Essays on Fundamental and Applied Environmental Topics.
Otranto D, Dantas-Torres F, Brianti E, Traversa D, Petric D, Genchi C, Capelli G (2013): Vector-borne helminths of dogs and humans in Europe. Parasit vectors, 6:16.
McCall JW, Genchi C, Kramer LH, Guerrero J, Venco L (2008): Heartworm disease in animals and humans. Adv Parasitol, 66:193–285.
Yuasa Y, Hsu TH, Chou CC, Huang CC, Huang WC, Chang CC (2012): The comparison of spatial variation and risk factors between mosquito-borne and tick-borne diseases: Seroepidemiology of Ehrlichia canis, Anaplasma species, and Dirofilaria immitis in dogs. Comp Immunol Microbiol Infect Dis 2012 Dec;35(6):599-606.