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Objective To provide an updated overview of the leishmaniasis epidemiology in the Balkans, as this region presents "blank space" on the map of leishmaniasis knowledge in Europe. Methods Following the PRISMA guideline, literature on the presence and circulation of human leishmaniasis in Balkan countries was screened in PubMed, Google Scholar, Science Direct, Scopus, Web of Science, Matica Srpska Library and the National Library of Serbia. Results Sixty nine publications from 1919 until September 2020 were reviewed. Historical records indicate that leishmaniasis was previously present mainly in the southern parts of the Balkans, while newer data demonstrate its northward spread. Conclusions Human leishmaniasis is re-emerging in the Balkans and in some areas is already widespread.

Leishmania major is the main causative agent of cutaneous leishmaniasis in the Old World. In Leishmania parasites, the lack of transcriptional control is mostly compensated by post-transcriptional mechanisms. Methylation of arginine is a conserved post-translational modification executed by Protein Arginine Methyltransferase (PRMTs). The genome from L. major encodes five PRMT homologs, including the cytosolic protein associated with several RNA-binding proteins, LmjPRMT7. It has been previously reported that LmjPRMT7 could impact parasite infectivity. In addition, a more recent work has clearly shown the importance of LmjPRMT7 in RNA-binding capacity and protein stability of methylation targets, demonstrating the role of this enzyme as an important epigenetic regulator of mRNA metabolism. In this study, we unveil the impact of PRMT7-mediated methylation on parasite development and virulence. Our data reveals that higher levels of LmjPRMT7 can impair parasite pathogenicity, and that deletion of this enzyme rescues the pathogenic phenotype of an attenuated strain of L. major. Interestingly, lesion formation caused by LmjPRMT7 knockout parasites is associated with an exacerbated inflammatory reaction in the tissue correlated with an excessive neutrophil recruitment. Moreover, the absence of LmjPRMT7 also impairs parasite development within the sand fly vector Phlebotomus duboscqi. Finally, a transcriptome analysis shed light onto possible genes affected by depletion of this enzyme. Taken together, this study highlights how post-transcriptional regulation can affect different aspects of the parasite biology., Author summary Understanding the genetics of Leishmania, a protozoan parasite causing leishmaniasis, is relevant for understanding fundamental questions on the pathogen’s biology and its interaction with hosts. We explore mechanisms used by Leishmania to promptly adapt to different hosts investigating the control of gene expression occurring at the post-transcriptional level in the parasite. Methylation of arginine performed by Protein Arginine Methyltransferase (PRMTs), among other post-translational modifications, may alter the function and interactions of target proteins, some of them are RNA binding proteins, known regulators of gene expression. In this study, we unveil the impact of PRMT7 on parasite development and pathogenicity. In addition to a negative correlation between the levels of LmjPRMT7 and parasite pathogenicity, we observed an impairment of the parasite development in the sand fly vector. Remarkably, despite a severe lesion development in mice, we observed no differences in parasite burden between infections with the pathogenic LmjPRMT7 knockout parasite or the attenuated parental line. Instead, the severe pathology observed is associated with an exacerbated inflammatory response correlated with excessive neutrophil recruitment.

Leishmania infantum causes visceral leishmaniasis, a deadly vector-borne disease introduced to the Americas during the colonial era. This non-native trypanosomatid parasite has since established widespread transmission cycles using alternative vectors, and human infection has become a significant concern to public health, especially in Brazil. A multi-kilobase deletion was recently detected in Brazilian L. infantum genomes and is suggested to reduce susceptibility to the anti-leishmanial drug miltefosine. We show that deletion-carrying strains occur in at least 15 Brazilian states and describe diversity patterns suggesting that these derive from common ancestral mutants rather than from recurrent independent mutation events. We also show that the deleted locus and associated enzymatic activity is restored by hybridization with non-deletion type strains. Genetic exchange appears common in areas of secondary contact but also among closely related parasites. We examine demographic and ecological scenarios underlying this complex L. infantum population structure and discuss implications for disease control., Philipp Schwabl, Mariana Boité, and colleagues analyze 126 Leishmania infantum genomes to determine how demographic and selective consequences of the parasite’s invasive history have contributed to intricate population genetic heterogeneity across Brazil. Their data suggest a complex interplay of population expansion, secondary contact and genetic exchange events underlying diversity patterns at short and long-distance scales. These processes also appear pivotal to the proliferation of a drug resistance-associated multi-gene deletion on chromosome 31.

Canine leishmaniasis is a parasitic zoonotic infection of dogs caused by the Leishmania spp. parasites. In Europe, canine leishmaniasis is among the most important vector-borne parasitic diseases of dogs, with majority of data originating from countries of southwestern Europe. With its scarce and outdated records, the Balkan region presents the "blank space" in the European map of canine leishmaniasis knowledge. Numerous records obtained during the past decade and a half indicate the emergence of autochthonous canine leishmaniasis in new settings all across Balkan, a significant increase in the numbers of infected dogs in previously endemic areas, and northward spread of the disease. As the majority of canine leishmaniasis data from Balkan countries are several decades old and generally written in native Balkan languages, hence not available to the broader area of scientists, comprehensive search of scientific literature published from 1934 to January 2021 was conducted in order to provide a better perspective to the past and current canine leishmaniasis situation in Balkan. This review provides exhaustive information on all leishmaniasis records in dogs, wild animals and cats, available from 10 countries of the Balkan region. It identifies dogs as the main disease reservoirs and highlights the importance of wild animals, and potentially cats, as disease reservoirs, which is of great public and veterinary health concern. Additionally, certain shortcomings in canine leishmaniasis research, management, surveillance and control were identified.

In the past decade, leishmaniasis seems to be re-emerging in Balkan countries. There are serious implications that Kosovo is a visceral leishmaniasis endemic region with autochthonous transmission; nevertheless, surveillance of vectors, reservoirs or the disease is not yet established. Gaining knowledge about sandfly vector species is a prerequisite for the development of a monitoring and control plan in the future. After a long gap in research of over 70 years, sandfly studies in Kosovo were resumed in 2014. During this presence/absence study, nine sandfly species were detected: Phlebotomus papatasi, Ph. perfiliewi, Ph. tobbi, Ph. neglectus, Ph. simici, Ph. balcanicus, Ph. alexandri, Ph. mascittii and Sergentomyia minuta. Three species are new with regard to the fauna of Kosovo - Ph. alexandri, Ph. balcanicus and Ph. mascittii. Besides increased diversity, changes in the number of collected specimens and distribution range of species were recorded, with Ph. neglectus being the most dominant species with the widest distribution. Testing of randomly chosen females for Leishmania spp. DNA resulted the in detection of L. tropica in a specimen of Ph. neglectus. the presence of numerous vector species in the sandfly fauna of Kosovo pose a threat for the re-emergence of vector-borne diseases. Therefore, continuous surveillance is recommended with regular updates on vector distribution and abundance., EurNegVec COST ActionEuropean Cooperation in Science and Technology (COST) [TD1303]; VectorNet project - European Food Safety Authority (EFSA); European Centre for Disease Prevention and Control (ECDC) contract [OC/FSA/AHAW/2013/02-FWC1]; European Virus Archive goes Global project (EVAg) - European Union's Horizon 2020 research and innovation programme [653316]; Ministry of Education and Science of the Republic of Serbia [III43007, TR31084]; European Commission, Horizon 2020 Infrastructure Infravec2 projectEuropean Commission, The authors express their gratitude to Vid Srdic who designed the map for this manuscript. the work of Slavica Vaselek, Yusuf Ozbel, Bulen Alten and Dusan Petric was carried out under the frame of EurNegVec COST Action TD1303. the work was supported by VectorNet project funded by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) contract OC/FSA/AHAW/2013/02-FWC1; European Virus Archive goes Global project (EVAg) funded by the European Union's Horizon 2020 research and innovation programme (grant agreement No 653316); and projects III43007 and TR31084 financed by the Ministry of Education and Science of the Republic of Serbia and European Commission, Horizon 2020 Infrastructure Infravec2 project (https://infravec2.eu).

Protein phosphorylation/dephosphorylation is an important regulatory mechanism that controls many key physiological processes. Numerous pathogens successfully use kinases and phosphatases to internalize, replicate, and survive, modifying the host&prime, s phosphorylation profile or signal transduction pathways. Multiple phosphatases and kinases from diverse bacterial pathogens have been implicated in human infections before. In this work, we have identified and characterized the dual specificity protein/lipid phosphatase LmDUSP1 as a novel virulence factor governing Leishmania mexicana infection. The LmDUSP1-encoding gene (LmxM.22.0250 in L. mexicana) has been acquired from bacteria via horizontal gene transfer. Importantly, its orthologues have been associated with virulence in several bacterial species, such as Mycobacterium tuberculosis and Listeria monocytogenes. Leishmania mexicana with ablated LmxM.22.0250 demonstrated severely attenuated virulence in the experimental infection of primary mouse macrophages, suggesting that this gene facilitates Leishmania pathogenicity in vertebrates. Despite significant upregulation of LmxM.22.0250 expression in metacyclic promastigotes, its ablation did not affect the ability of mutant cells to differentiate into virulent stages in insects. It remains to be further investigated which specific biochemical pathways involve LmDUSP1 and how this facilitates the parasite&prime, s survival in the host. One of the interesting possibilities is that LmDUSP1 may target host&prime, s substrate(s), thereby affecting its signal transduction pathways.

Halada, Petr/0000-0002-7229-3450; Vaselek, Slavica/0000-0002-9105-7138; Dvorak, Vit/0000-0001-5636-7483; Ivovic, Vladimir/0000-0002-1562-9528; OZBEL, YUSUF/0000-0001-8335-1997, WOS: 000500182400034, PubMed: 31207207, Serbia was a country endemic for leishmaniasis with rich and abundant sand fly fauna during the middle of the 20th century. After 1968, the disease was considered as eradicated, and as a consequence, all research on vectors and pathogens was neglected. the recent detection of Leishmania infantum in sand flies and confirmed cases of leishmaniasis in humans and dogs indicated the risk of re-emergence in Serbia. Also, for millions of refugees/immigrants, Serbia is a transit route between East-Mediterranean and Middle-East countries, and Central/North Europe, and it is under constant risk of vector and disease introduction. Primary objectives of this research were to determine which sand fly species are present around transit routes, whether they include vectors of the Leishmania spp. moreover, is/are Leishmania spp. present in the vectors. Surveys were conducted at 55 locations, organised in four clusters, which covered main immigrant routes and shelters/camps as well as tourist/trade transit routes. in total, 367 sand fly specimens were collected. Nine species were identified: Phlebotomus papatasi, P. perfiliewi, P. tobbi, P. neglectus, P. sergenti, P. alexandri, P. simici, P. balcanicus and P. mascittii. Detection of P. alexandri represents the first record in Serbia. the diversity of sand fly species increased while the number of collected specimens per night decreased during the period of research neglection. Phlebotomus neglectus, a proven vector of L. infantum, was a predominant species in all surveyed clusters, and in 56,52% of locations, it was the only species present. Although all detected species are regarded either as proven or suspected vectors of Leishmania spp., screening of females for Leishmania presence resulted negative. Our study provides insight into the significant changes of sand fly fauna in Serbia during the end of XX and beginning of XXI century. Diverse sand fly fauna in Serbia suggests that the establishment of new leishmaniasis foci is possible., European Food Safety Authority (EFSA) [OC/EFSA/AHAW/2013/02-FWC1]; European Centre for Disease Prevention and Control (ECDC); Czech Science FoundationGrant Agency of the Czech Republic [15-04329S]; Institute of Microbiology [RVO61388971]; European Virus Archive goes Global goes Global project (EVAg) - European Union's Horizon 2020 research and innovation program [653316]; EDENext FP7 EU project [261504], The work was carried out under the VectorNet project, a European network for sharing data on the geographic distribution of arthropod vectors, transmitting human and animal disease agents (Contract OC/EFSA/AHAW/2013/02-FWC1) funded by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC). the study was partially supported by the Czech Science Foundation (15-04329S) and the Institute of Microbiology (RVO61388971), European Virus Archive goes Global goes Global project (EVAg) funded by the European Union's Horizon 2020 research and innovation program (grant agreement No 653316), and the EDENext FP7- no261504 EU project (http://www.edenext.eu).

Efforts to detect West Nile virus (WNV) in the Vojvodina province, northern Serbia, commenced with human and mosquito surveillance in 2005, followed by horse (2009) and wild bird (2012) surveillance. The knowledge obtained regarding WNV circulation, combined with the need for timely detection of virus activity and risk assessment resulted in the implementation of a national surveillance programme integrating mosquito, horse and bird surveillance in 2014. From 2013, the system showed highly satisfactory results in terms of area specificity (the capacity to indicate the spatial distribution of the risk for human cases of West Nile neuroinvasive disease - WNND) and sensitivity to detect virus circulation even at the enzootic level. A small number (n = 50) of Culex pipiens (pipiens and molestus biotypes, and their hybrids) females analysed per trap/night, combined with a high number of specimens in the sample, provided variable results in the early detection capacity at different administrative levels (NUTS2 versus NUTS3). The clustering of infected mosquitoes, horses, birds and human cases of WNND in 2014-2015 was highly significant, following the south-west to north-east direction in Vojvodina (NUTS2 administrative level). Human WNND cases grouped closest with infected mosquitoes in 2014, and with wild birds/mosquitoes in 2015. In 2014, sentinel horses showed better spatial correspondence with human WNND cases than sentinel chickens. Strong correlations were observed between the vector index values and the incidence of human WNND cases recorded at the NUTS2 and NUTS3 levels. From 2010, West Nile virus was detected in mosquitoes sampled at 43 different trap stations across Vojvodina. At 14 stations (32.56%), WNV was detected in two different (consecutive or alternate) years, at 2 stations in 3 different years, and in 1 station during 5 different years. Based on these results, integrated surveillance will be progressively improved to allow evidence-based adoption of preventive public health and mosquito control measures.

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.

Cutaneous leishmaniasis due to Leishmania tropica is increasingly documented in Europe and the Middle East. Besides its specific vector, Phlebotomus sergenti, permissive Phlebotomus sand flies are suspected as potential vectors of L. tropica. We investigated the susceptibility of two widely distributed species, Phlebotomus perniciosus and Phlebotomus tobbi. Laboratory-reared sand flies were infected experimentally with L. tropica strains differing in lipophosphoglycan epitopes, geographical distribution and epidemiology. High infection rates, heavy parasite loads and fully developed late-stage infections including colonization of the stomodeal valve were observed in all parasite-vector combinations. Our findings demonstrate that P. perniciosus and P. tobbi are susceptible to different L. tropica strains and may play a role in their circulation in endemic foci of Europe, the Middle East and North Africa.