Phlebotomine sand-fly-borne infections are an emerging threat to human and animal health in Mediterranean countries, highlighting the need for improved surveillance and control strategies. Dogs are ideal sentinel hosts owing to their central role in the transmission of zoonotic Leishmania infantum, frequent exposure to sand fly bites, capacity to develop antibodies to phleboviruses, and close contact with humans. This study reports cross-sectional surveys of antibodies to Leishmania, Toscana virus (TOSV), and Sicilian sand fly virus (SFSV) in dogs from Portugal, Spain, Italy, Croatian Istria, Turkey, and Israel, as well as antibodies to salivary proteins of Phlebotomus perniciosus (Portugal, Spain, and Italy) and Phlebotomus papatasi (Spain and Italy), conducted within the Climate Monitoring and Decision Support Framework for the Detection and Mitigation of Sand fly Diseases with Cost–Benefit and Climate Policy Measures (CLIMOS) project. Blood samples and epidemiological data were collected from 2500 dogs. Antibodies to Leishmania were detected by indirect immunofluorescence, phlebovirus antibodies by seroneutralization assays, and antibodies to P. perniciosus and P. papatasi salivary antigens by enzyme-linked immunosorbent assay (ELISA) using recombinant proteins rSP03B and rSP36, respectively. Sources of antibody variability were evaluated using mixed-effects logistic regression models. Antibodies to L. infantum, phleboviruses, and sand fly saliva were widely detected, although seroprevalence varied markedly by region. No Leishmania-seropositive dogs were found in Istria, parts of northern Spain, or several districts in Israel, whereas seroprevalence exceeded 30

Circoviruses cause diseases in a wide range of hosts, including avian species. One of the most studied circoviruses is beak and feather disease virus (BFDV), which has spread worldwide due to global trade of birds and characteristics of the virus that allow it to persist in the environment. The Eurasian griffon vulture (Gyps fulvus) is one of the largest birds in the world and is among several vulture species that are considered endangered today, and currently the only remaining vulture species in Croatia. Several juvenile Eurasian griffon vultures undergoing rehabilitation at the Beli Visitor Centre and Rescue Centre for Griffon Vultures on the island of Cres, Croatia, had feather lesions similar to those caused by BFDV in psittacine birds. Signs included partially retained feather sheaths, discoloration and circumferential constrictions of the feather shafts, gnawed feather tips, keratin and egg deposits near the feather shafts, as well as feather breakage and spontaneous feather loss. Feather samples were collected for molecular detection of viral pathogens and macroscopic detection of ectoparasites; blood samples were taken for complete blood count (CBC), biochemical analysis and oxidative stress analysis. The molecular analysis confirmed a circovirus infection, which was supported by the blood analyses indicating a viral infection. The collected ectoparasites were identified as chewing lice - Colpocephalum turbinatum and Falcolipeurus quadripustulatus, which have previously been detected in Eurasian griffon vultures. To our knowledge, this is the first report of circovirus infection in Eurasian griffon vultures. Considering that circoviruses are highly contagious, very resistant and easily transmissible, it is extremely important to perform continuous monitoring in order to improve the breeding and survival conditions of Eurasian griffon vulture populations worldwide.

Introduction Proper identification and management of reservoir post-kala-azar dermal leishmaniasis (PKDL) and canine visceral leishmaniasis (CVL) cases are prerequisites to the effective control of visceral leishmaniasis (VL) worldwide. Unlike PKDL, CVL still awaits effective improvement because of its cryptic nature, absence of Leishmania parasite in lesions or lymph nodes, and insensitivity of tools in current use. Because of the need for certain skills and equipment, both the liquid direct agglutination test (LQ-DAT) and freeze-dried direct agglutination test (FD-DAT) versions, present in comparison with the indirect immunofluorescence (IFAT) or enzyme-linked immunosorbent assay (ELISA), practical and feasible diagnostic alternatives. Aim Validate the performance of an improved FD-DAT to suit routine and large-scale applications in CVL endemic areas. Methodology Introducing of citrate-saline formaldehyde (CSF) as an anti-clumping agent to replace normal saline for antigen reconstitution and drastically however eligibly lowering the concentration of promastigotes (1.4X107) in comparison with the original reference FD-DAT (> 5X107/ml), To ensure optimal safety, β-mercaptoethanol (β-ME) was replaced by urea or sodium dodecyl sulphate (SDS) as a serum reducing agent. Results Through improving the procedure for reconstitution of FD-DAT antigen with CSF a 150% reduction in the test application cost was achieved. Expired test batches (+ 4 years earlier), were successfully revitalized to full validity. As compared to 48-hour shelf-life time for the original, an FD-DAT batch re-constituted here with CSF maintained validity for + 12 months. Conclusions The highly concordant results with IFAT and ELISA (One Way ANOVA Test P = 0.142, Homogeneity of Variances P = 0.009) as routine CVL diagnostics further motivate the application of the improved FD-DAT for detection of the disease in endemic areas.

Control of canine infections with Leishmania infantum (L. infantum), a major zoonotic disease in Brazil and southern Europe, is becoming increasingly important due to its close proximity to humans, the increasing import of dogs from endemic regions and the impact of climate change on vector spreading. Simple, rapid and reliable diagnostic tests are therefore needed to detect infected dogs. Here, we re-evaluated different serological methods for the diagnosis of canine leishmaniosis (CanL) in Croatia and Brazil. The diagnostic performance of the indirect fluorescent antibody test (IFAT) and the VetLine® Leishmania ELISA (GSD Frankfurt, Germany) was compared with three rKLi8.3-based diagnostic test systems, the rKLi8.3 ELISA (GSD Frankfurt, Germany), the INgezim® Leishma CROM (GSD Madrid, Spain) lateral flow test (LFT) and the VetBlot® Leishmania LineBlot (GSD Frankfurt, Germany). CanL symptomatic dogs were efficiently diagnosed by all tests, except the VetLine® Leishmania ELISA, which is based on whole Leishmania antigens. The advantage of rKLi8.3 was also observed in oligo- and asymptomatic dogs from Brazil and Croatia, although with reduced diagnostic efficiency compared to symptomatic dogs. Similar to IFAT and rKLi8.3 ELISA, the LFT did not cross-react with other common canine pathogens; it showed very high specificity for healthy dogs from endemic regions in both countries and did not react with healthy, vaccinated dogs in Brazil. In conclusion, serodiagnostic tests based on the rKLi8.3 antigens are superior to whole parasite antigens, and the LFT has the advantage of providing a laboratory-independent, rapid and specific diagnosis of CanL.

Comprehensive profiling of serum proteome provides valuable clues of health status and pathophysiological processes, making it the main strategy in biomarker discovery. However, the high dynamic range significantly decreases the number of detectable proteins, obstructing the insights into the underlying biological processes. To circumvent various serum enrichment methods, obtain high-quality proteome wide information using the next-generation proteomic, and study host response in canine leishmaniosis, we applied data-independent acquisition mass spectrometry (DIA-MS) for deep proteomic profiling of clinical samples. The non-depleted serum samples of healthy and naturally Leishmania-infected dogs were analyzed using the label-free 60-min gradient sequential window acquisition of all theoretical mass spectra (SWATH-MS) method. As a result, we identified 554 proteins, 140 of which differed significantly in abundance. Those were included in lipid metabolism, hematological abnormalities, immune response, and oxidative stress, providing valuable information about the complex molecular basis of the clinical and pathological landscape in canine leishmaniosis. Our results show that DIA-MS is a method of choice for understanding complex pathophysiological processes in serum and serum biomarker development.

Invazija parazitima roda Leishmania rezultira razvojem lišmanioze, sve raširenije skupine bolesti koja zahvaća veliki broj ljudi diljem svijeta. Lišmanioza može imati različite ishode u rasponu od kožnih lezija i lezija sluznice do visceralizacije, ovisno o vrsti parazita i imunosnom odgovoru nositelja. Kao obligatni intracelularni parazit koji živi unutar makrofaga, lišmanija je evoluirala u strogom kontaktu s imunosnim sustavom nositelja razvijajući različite mehanizme za izbjegavanje ili moduliranje imunosnog odgovora. Tijekom invazija različitih vrsta lišmanija opaženi su i različiti odgovori imunosnog sustava nositelja koji su rezultirali uklanjanjem parazita, ali i doprinosom patogenezi čime se povećala složenost tijeka bolesti. Kao zaštitno sučelje između utrobnih organa i okoliša, koža se susreće s mnoštvom toksina, patogenih organizama i fizičkih stresora. Za suzbijanje navedenih napada na njeno mikrookruženje, koža ima veću funkciju od one fizičke barijere - predstavlja i aktivan organ imunosnog sustava. Imunosni odgovori u koži uključuju opremljenost imunokompetentnim stanicama i topljivim modifikatorima biološkog odgovora uključujući citokine. Glavni zadatak stanica imunosnog sustava kože je zadržavanje invadirajućeg patogenog uzročnika radi spriječavanja njegovog širenja na unutarnje organe i/ili, ako je moguće, njegovo uklanjanje.

Large American liver fluke (Fascioloides magna) was introduced to Europe at least twice, via invaded North American deer species. These imports resulted in formation of three permanent foci of fascioloidosis - Italian, Czech and Danube floodplain forests. Molecular analysis revealed that flukes in Italian foci represent separate genetic group, and that flukes from Czech and Danube foci are genetically identical. Since previous studies of genetic structure of F. magna from Croatia were based on samples collected from red deer in one limited area (Baranja), the aim of this study was to analyse genetic diversity of 111 fluke samples collected from various hosts (red, roe and fallow deer, wild boar) and geographic locations in Croatia. On 355 base pair long cytochrome oxidase subunit 1 sequence, three haplotypes were identified - previously published CO1-Ha3 and CO1-Ha4, and one new haplotype named CO1-Ha36. Results obtained in our study confirmed as expected, that F. magna in Croatia has a Danube origin and that haplotypes are shared among different final host species.

Lišmanioza je invazijska bolest uzrokovana obligatnim intracelularnim parazitima roda Leishmania. Prenose ih ubodominvadirane ženke papatača (insekti potporodice Phlebotominae). Pripadnici roda Phlebotomus su biološki vektori na području Starog, a roda Lutzomyia na području Novog svijeta. Žarišta lišmanioze ljudi se nalaze u otprilike 90 zemalja tropskih i subtropskih regija te južne Evrope. Procijenjena je godišnja pojava od 50 000 do 90 000 visceralne te 600 000 do 1 miliona novih slučajeva kutane lišmanioze širom svijeta. Na području Sirijske Arapske Republike, Islamskog Emirata Afganistana, Republike Irak te Islamske Republike Iran najčešći izolirani uzročnici kožne lišmanioze su Leishmania tropica i L. major, dok je uzročnik visceralne lišmanioze L. infantum, osim u Republici Irak gdje je izolirana L. donovani. U Evropi, na području mediteranske regije visceralni i kožni oblik uzrokuje L. infantum, dok je L. tropica uzročnik kožnog oblika bolesti u Grčkoj. Posljednjih godina je zabilježeno povećanje broja oboljelih od lišmanioze uz širenje endemskih žarišta i pojavu bolesti u ne-endemskim područjima. Faktorom rizika se smatra prisutnost bioloških vektora roda Phlebotomus, stoga je potreban razvoj strategija za smanjenje broja vektora tokom sezone njihove visoke aktivnosti. Za određivanje potencijalnih promjena u pojavnosti lišmanioze na području Evrope potrebne su informacije o prisutnosti bioloških vektora i njihovom kretanju. U mobilnim populacijama poput migranata i izbjeglica, pojava bolesti prenosivih biološkim vektorima nije neočekivana, zato su osiguranje prava na zdravstvenu zaštitu i univerzalni pristup javnim zdravstvenim uslugama važni u prevenciji sekundarnih slučajeva i izbijanja bolesti.

Prilikom stimulacije, različite ćelije imunološkog sistema brzo su regrutirane u kožu iz krvi ili limfatičnih tkiva što dovodi do rezultirajućih imunoloških odgovora. Ovisno o prirodi podražaja, različite imunološke ćelije mogu biti regrutirane kao odgovor na različite patogene uzročnike. Osim toga, slijed događaja je često kritičan za ishod bolesti ili kontrolu patogenog uzročnika. Nakon inokulacije parazita roda Leishmania papatačima, za manifestaciju bolesti su potrebni mehanizmi koji parazitima barem u početku omogućuju umnožavanje u nositelju i pružanje otpora urođenom i stečenom imunološkom odgovoru. Isto tako, doživotna perzistencija parazita, kakva se javlja i u slučajevima kliničkog izlječenja invazije, ukazuje na postojanje strategija koje omogućuju parazitu djelomično zaobilaženje zaštitnog stečenog imunološkog odgovora nositelja. Promjene u signalnim putevima i staničnom fenotipu, kao i povećana proizvodnja niza proupalnih i protuupalnih medijatora mogu na temeljan način pridonijeti negativnoj modulaciji imunološkog odgovora u početnim trenucima invazije. Navedeni su mehanizmi nastali kao posljedica dugog procesa koevolucije između parazita i nositelja. Nedavni napredak u tehnologijama snimanja, kao i istraživanja koja kombiniraju imunološke i genetske manipulacije, omogućila su bolje razumijevanje međudjelovanja lišmanija i njihovih nositelja sisara. Daljnje istraživanje stanica sistema urođene imunosti i lišmanija može dovesti do boljeg razumijevanja procesa invazije i razvoja kvalitetnijih vakcina protiv lišmanioze.

Zbog izloženosti riziku razvoja različitih bolesti tokom putovanja (migracije) te prilikom boravka u zemlji domaćina, potrebno je brzo i primjereno rješavanje situacije vezane za zdravstveno zbrinjavanje izbjeglica i migranata s obzirom na njihovu veliku brojnost u svijetu. Unatoč porijeklu navedenih skupina iz područja u kojima su prisutne određene endemske bolesti, nije nužno da su one i njihovi nosioci te da će ih širiti na domaću ili populaciju u tranziciji. Šistosomoza je endemska bolest Sirijske Arapske Republike i Republike Irak. Autohtona je parazitska zoonoza akutnog i hroničnog oblika uzrokovana metiljima roda Schistosoma te pripada skupini zanemarenih tropskih bolesti od kojih obično obolijeva najsiromašnije stanovništvo. Loši sanitarni uslovi u izbjegličkim kampovima predstavljaju rizik ponovnog širenja bolesti. S obzirom na veliki broj izbjeglica iz navedenih republika smještenih na području zemalja regije Srednjeg istoka i Sjeverne Afrike (engl. Middle East and North Africa – MENA) u kojima se slučajevi šistosomoze još uvijek pojavljuju, mogućnost održavanja i širenja bolesti postoji ako se ponovno stvore odgovarajući uslovi, odnosno područja u kojima će u isto vrijeme opstajati nosioci (invadirane osobe), posrednici (slatkovodni puževi određenih rodova) te rezervoari (različite vrste životinja invadirane uzročnikom). Na području Evrope, najveći broj izbjeglica je prisutan u Njemačkoj i Švedskoj. U Evropi su žarišta šistosomoze utvrđena na južnoj Korzici u Francuskoj. Istraživanja su ukazala da se uzročnici mogu širiti i zadržavati u umjerenim klimatskim područjima Francuske, Italije, Portugala, Španije i Grčke te da je širenje šistosomoze prema sjeveru Evrope trenutno ograničeno nesposobnošću prilagodbe posrednika hladnijim temperaturama, ali ipak moguće putem invadiranih turista, migranata i izbjeglica, što je već bio slučaj u Rumuniji i Mađarskoj.

A wild male mouflon (Ovis musimon) was shot due to the observed weakness. Necropsy revealed consolidated lungs and traces of black pigment and fibrin on the liver. On the cut surface, a juvenile fl uke was found in the lungs, while traces of destroyed fl ukes' migratory channels were found in the liver. F. magna infection in both, wild and domestic ruminants, causes three types of species-specific host-parasite interactions; definitive, dead-end and aberrant. mouflon are classifi ed as aberrant hosts and here we report unsuccessful migration of a juvenile fl uke that led to a severe pneumonia.

Sandfly-borne phleboviruses are distributed widely throughout the Mediterranean Basin, presenting a threat to public health in areas where they circulate. However, the true diversity and distribution of pathogenic and apathogenic sandfly-borne phleboviruses remains a key issue to be studied. In the Balkans, most published data rely on serology-based studies although virus isolation has occasionally been reported. Here, we report the discovery of two novel sandfly-borne phleboviruses, provisionally named Zaba virus (ZABAV) and Bregalaka virus (BREV), which were isolated in Croatia and North Macedonia, respectively. This constitutes the first isolation of phleboviruses in both countries. Genetic analysis based on complete coding sequences indicated that ZABAV and BREV are distinct from each other and belong to the genus Phlebovirus, family Phenuiviridae. Phylogenetic and amino acid modelling of viral polymerase shows that ZABAV and BREV are new members of the Salehabad phlebovirus species and the Adana phlebovirus species, respectively. Moreover, sequence-based vector identification suggests that ZABAV is mainly transmitted by Phlebotomus neglectus and BREV is mainly transmitted by Phlebotomus perfiliewi. BREV neutralizing antibodies were detected in 3.3% of human sera with rates up to 16.7% in certain districts, demonstrating that BREV frequently infects humans in North Macedonia. In vitro viral growth kinetics experiments demonstrated viral replication of both viruses in mammalian and mosquito cells. In vivo experimental studies in mice suggest that ZABAV and BREV exhibit characteristics making them possible human pathogens.

Tapeworms of the genus Mesocestoides (Cestoda: Cyclophyllidea: Mesocestoididae) are still enigmatic to scientists, due to their high morphological variability, low host specificity, and unknown details of their life cycle. They are found worldwide, with carnivorous mammals as the main definitive hosts, and the disease is potentially zoonotic. After ingestion by a definitive host, the tetrathyridium can occasionally migrate through the intestinal wall and reach the peritoneal cavity or abdominal organs causing peritoneal metacestodosis. Here, we report on a case of metacestodosis of a European wild cat (Felis silvestris silvestris) found dead in Croatia. At necropsy, a large number of white, rice-like structures were found free in the abdominal and thoracic cavities, as well as along the serous surfaces and in the lungs. DNA isolated from the nodules was genotyped and based on a 320-base pair long 12S fragment classified as Mesocestoides vogae. Although post-mortem changes were advanced, severe emaciation due to the severe parasitic infection and gastrointestinal bleeding was diagnosed as the likely cause of death. Intestinal cestodosis was previously reported in wild cats, but according to our knowledge, this is the first description of peritoneal and pleural metacestodosis caused by M. vogae tetrathyridia (metacestodes) in any wild carnivore species.

Ehinokokozu ljudi uzrokuju larvalni stadiji četiri vrste trakavica roda Echinococcus: E. granulosus (sensu lato), E. multilocularis, E. vogeli i E. oligarthrus. Od navedenih vrsta, uzročnik cistične ehinokokoze E. granulosus je najčešći. Veliki broj područja u kojima je cistična ehinokokoza najraširenija, siromašne su i udaljene zajednice sa ograničenim pristupom adekvatnom medicinskom tretmanu. Bolest je proširena po cijelom svijetu no najveća je prevalencija prisutna u zajednicama u kojima prevladava pašno držanje životinja što uključuje područja svih zemalja na granici sa Sredozemljem te veliki broj regija i zemalja središnje Azije, dijelova Kine, Australije i Južne Amerike. Alveolarna ehinokokoza uzrokovana je larvalnim stadijem trakavice E. multilocularis. Iako je bolest rijetka, opaža se na sjevernoj hemisferi, u geografskim područjima gdje je moguće nesmetano odvijanje silvatičkog razvojnog ciklusa uzročnika. Najveći broj slučajeva alveolarne ehinokokoze u ljudi zabilježen je u određenim djelovima zapadne Kine. U Europi je u posljednja dva desetljeća uočeno širenje endemskih područja od kojih se glavno nalazi u Litvi. Obje su bolesti autohtone parazitske zoonoze Sirijske Arapske Republike, Islamske Republike Afganistan, Republike Irak te Islamske Republike Iran, zemalja iz kojih dolazi veliki broj izbjeglica i migranata. Mnogi potječu iz regija u kojima javni zdravstveni sistemi gotovo ne postoje. Brojni nedostaci u praćenju broja oboljelog autohtonog stanovništva te neadekvatan zdravstveni pregled pristigle populacije izbjeglica i migranata, onemogućuju utvrđivanje točnog uzroka porasta navedenih bolesti u zemljama domaćina. U isto vrijeme postoji velika opasnost od širenja već postojećih žarišta zbog formiranja velikih aglomeracija ljudi koji žive u izrazito nepovoljnim uvjetima, a koji su idealni za održavanje razvojnog ciklusa uzročnika bolesti.