Use of wild bird surveillance, human case data and GIS spatial analysis for predicting spatial distributions of West Nile Virus in Greece

West Nile Virus (WNV) is the causative agent of a vector-borne, zoonotic disease with a worldwide distribution. Recent expansion and introduction of WNV into new areas, including southern Europe, has been associated with severe disease in humans and equids, and has increased concerns regarding the need to prevent and control future WNV outbreaks. Since 2010, 524 confirmed human cases of the disease have been reported in Greece with greater than 10% mortality. Infected mosquitoes, wild birds, equids, and chickens have been detected and associated with human disease. The aim of our study was to establish a monitoring system with wild birds and reported human cases data using Geographical Information System (GIS). Potential distribution of WNV was modelled by combining wild bird serological surveillance data with environmental factors (e.g. elevation, slope, land use, vegetation density, temperature, precipitation indices, and population density). Local factors including areas of low altitude and proximity to water were important predictors of appearance of both human and wild bird cases (Odds Ratio = 1,001 95%CI = 0,723–1,386). Using GIS analysis, the identified risk factors were applied across Greece identifying the northern part of Greece (Macedonia, Thrace) western Greece and a number of Greek islands as being at highest risk of future outbreaks. The results of the analysis were evaluated and confirmed using the 161 reported human cases of the 2012 outbreak predicting correctly (Odds = 130/31 = 4,194 95%CI = 2,841–6,189) and more areas were identified for potential dispersion in the following years. Our approach verified that WNV risk can be modelled in a fast cost-effective way indicating high risk areas where prevention measures should be implemented in order to reduce the disease incidence

Ο ρόλος του καφέ ευρωπαϊκού λαγού (Lepus europaeus) ως πηγή αναδυόμενων και επαναδυόμενων παθογόνων παραγόντων

This thesis aimed to investigate the role of European brown hare (Lepus europaeus) as a source of important emerging and re-emerging pathogens. The serological and molecular surveys herein conducted investigated for the first time the occurrence of infection and/or exposure of brown hare to Toxoplasma gondii, Neospora caninum, Leishmania infantum, Bartonella spp, Brucella spp, Coxiella burnetii, and Francisella tularensis in Greece. Moreover, the Ecological Niche of Toxoplasma gondii and Leishmania infantum in seropositive hares has been defined. The role of this species in the epidemiology of Leishmaniasis was further investigated through GIS analysis for identification of the environmental parameters related to Leishmania infection in hares as well as phylogenetic analysis of the Leishmania parasites detected in hares. The phylogenetic position of the Leishmania spp detected in humans, dogs, cats and hares in Thessaly, Central Greece and the first indication for the circulation of L.donovani in Greece are also part of this thesis. In the first chapter of this thesis the tools that have been developed for molecular diagnosis of Leishmaniasis, species identification and phylogenetic analysis presenting different sensitivity and discriminatory power were summarized.The first evidence of Leishmania infection in European Brown Hare in Greece was reported in the second chapter of this thesis. The Leishmania DNA prevalence in hares was found to be 23.49% in Northern Greece (prefectures of Thessaloniki and Chalkidiki) from 2007-2011. The phylogenetic analysis showed that the hare Leishmania sequences belong in the Leishmania donovani complex. The homology of the nucleotide sequences with canine Leishmania sequences of the same area was 98.9% suggesting a possible overlapping of wild and domestic transmission cycles of Leishmania spp in the region. There was no indication that there is an association between the human leishmaniasis cases and the infected hares in the prefectures studied while the hypothesis made was that leishmaniasis is still in the sylvatic transmission cycle in Chalkidiki whereas it seems that an overlapping of sylvatic and domestic transmission cycles occurs in Thessaloniki. Precipitation was recognized as the environmental parameter influencing the presence of Leishmania DNA in hares with an increase of one unit in the precipitation seasonality to be expected to increase the odds of hares to be Leishmania positive by 6.1%.The third chapter concerns the serological and molecular survey for parasitic pathogens in European brown hares from Northern and central Greece during a two year hunting period which showed that the examined hares were N.caninum (3.8%) and L.infantum (9.6%) infected while, 5.7%, 0.95% and 12.4% of the hares reacted positively for the presence of antibodies against T.gondii, N.caninum and L.infantum respectively. Geographical information system (GIS) analysis together with the Ecological Niche Model (ENM) showed that rainfall indices and land uses are significantly influential for hare exposure to T.gondii and L.infantum. The high-risk areas for hare exposure to T.gondii and L.infantum were identified and a risk map for the regions of northern and central Greece was created. The phylogenetic position of Leishmania spp detected in humans, dogs, cats and hares in Thessaly, Central Greece was defined through phylogenetic and analysis of ITS1 for microsatellite repeat numbers and it is described in the fourth chapter of this thesis. The Leishmania sequences derived from different hosts belonged in the L. donovani complex. L. infantum was identified in all the Leishmania sequences included in the study supporting the predominance of L.infantum circulation in different hosts in Thessaly, the existence of the zoonotic transmission cycle of L.infantum in the study area and the possible overlapping between domestic and sylvatic transmission cycles. Unexpectedly, one Leishmania sequence was identified as L. donovani in one feline sample indicating the possible L. donovani infection in a cat from the study area giving the impetus for further investigation.The last chapter concerns the occurrence of infection and/or exposure of European Brown hares to Bartonella spp, Brucella spp, Coxiella burnetii, and Francisella tularensis which are investigated during two-year hunting periods in northern and central Greece. Antibodies against Bartonella spp were detected in 11.4% of the examined hares while none of them was seropositive for Brucella spp, C. burnetii, and F. tularensis. The presence of Bartonella, Brucella spp, C. burnetii, and F. tularensis DNA was not detected in the samples examined. Ιt seems that the role of European Brown hare in the epidemiology of Brucella spp, C. burnetii, and F. tularensis is of minor importance in Greece while this species is exposed to Bartonella spp which gives the impetus for further investigation of its role as another host of this bacterium.This thesis gives insight into the role of an important game species in Greece, European brown hare, in the epidemiology of parasitic and bacterial pathogens of zoonotic potential. This species has a limited home range, being thus prone to many infectious diseases of natural nidality while sharing the same living space with other wild and free-ranging animals including canids, felids and large birds of prey, which comprise its natural predators and hunting grounds with domestic dogs. Its importance is further enhanced by cross border and long-distance movements possibly as part of live animal translocations, as the host of vectors and the prey of carnivores and omnivores that travel in great distances and get into contact with domestic animals and humans. Besides, its short life span makes this species an excellent indicator for recent pathogen transmission in the area and its intensive contact with vectors such as ticks, mosquitoes and sandflies has implicated hare in the epidemiology of important vector borne diseases such as Leishmaniasis. The continuous surveillance of hare populations could provide information on the population health status and the pathogens circulating in the area posing risk for wildlife, domestic animals and humans.Η παρούσα διατριβή, είχε στόχο τη διερεύνηση του ρόλου του Καφέ Ευρωπαϊκού λαγού (Lepus europaeus), ως πηγή σημαντικών αναδυόμενων και επαναδυόμενων παθογόνων παραγόντων. Οι ορολογικές και μοριακές εξετάσεις που διεξήχθησαν, αποτέλεσαν την πρώτη διερεύνηση της παρουσίας μόλυνσης ή / και της έκθεσης του Καφέ Ευρωπαϊκού λαγού σε παρασιτικούς και βακτηριακούς παθογόνους παράγοντες, συμπεριλαμβανομένων των Toxoplasma gondii, Neospora caninum, Leishmania infantum, Bartonella spp, Brucella spp, Coxiella burnetii και Francisella tularensis στην Ελλάδα. Επιπλέον, καθορίστηκε η oικολογική θέση των οροθετικών λαγών στα πρωτόζωα T. gondii και L. infantum. Ο ρόλος αυτού του είδους στην επιδημιολογία της λεϊσμανίασης, διερευνήθηκε περαιτέρω με τη χρήση Συστημάτων Γεωγραφικών Πληροφοριών (ΣΓΠ) για την αναγνώριση των περιβαλλοντικών παραμέτρων, οι οποίες σχετίζονται με τη μόλυνση των λαγών από τα είδη του πρωτόζωου Leishmania καθώς και μέσω της φυλογενετικής ανάλυσης των αλληλουχιών των πρωτόζωων Leishmania που ανιχνεύθηκαν σε λαγούς. Η φυλογενετική θέση των πρωτόζωων Leishmania spp που ανιχνεύθηκαν σε ανθρώπους, σκύλους, γάτες και λαγούς στην περιοχή της Θεσσαλίας, καθώς και η πρώτη ένδειξη για την κυκλοφορία της L.donovani στην Ελλάδα αποτελούν επίσης μέρος αυτής της διατριβής.Στο πρώτο κεφάλαιο, έγινε μια ανασκόπηση των εργαλείων που έχουν αναπτυχθεί για τη μοριακή διάγνωση της Λεϊσμανίασης, την ταυτοποίηση των ειδών και τη φυλογενετική τους ανάλυση, εργαλεία τα οποία παρουσιάζουν διαφορετική ευαισθησία και διακριτική ισχύ.Η πρώτη αναφορά της παρουσίας μόλυνσης από είδη του πρωτόζωου Leishmania στην Ελλάδα, γίνεται στο δεύτερο κεφάλαιο αυτής της διατριβής. Ο επιπολασμός μόλυνσης από πρωτόζωα Leishmania στους λαγούς βρέθηκε να είναι 23.49% στη Βόρεια Ελλάδα (νομοί Θεσσαλονίκης και Χαλκιδικής) από το 2007-2011. Η φυλογενετική ανάλυση, έδειξε ότι οι αλληλουχίες των πρωτόζωων Leishmania που ανιχνεύθηκαν σε λαγούς, ανήκουν στο σύμπλεγμα Leishmania donovani. Η ομολογία των αλληλουχιών των νουκλεοτιδίων μεταξύ των πρωτόζωων Leishmania που ανιχνεύθηκαν σε σκύλους της ίδιας περιοχής και αυτών που ανιχνεύθηκαν σε λαγούς ήταν 98,9%, υποδηλώνοντας την πιθανή αλληλεπικάλυψη του άγριου και οικόσιτου κύκλου μετάδοσης του πρωτόζωου Leishmania spp στην περιοχή. Δεν υπήρξαν ενδείξεις σύνδεσης των περιστατικών Λεϊσμανίασης σε ανθρώπους και των μολυσμένων λαγών στους νομούς που μελετήθηκαν. Η υπόθεση που διατυπώθηκε ήταν ότι στην περιοχή της Χαλκιδικής επικρατεί ο άγριος κύκλος μετάδοσης της Λεϊσμανίασης, ενώ στην περιοχή της Θεσσαλονίκης φαίνεται να υπάρχει αλληλεπικάλυψη του άγριου και οικόσιτου κύκλου μετάδοσης του πρωτόζωου Leishmania. Η βροχόπτωση αναγνωρίστηκε ως η περιβαλλοντική παράμετρος που επηρεάζει την παρουσία του DNA του πρωτόζωου Leishmania στους λαγούς, καθώς η αύξηση της βροχόπτωσης κατά μια μονάδα, αναμένεται να αυξήσει την πιθανότητα παρουσίας λαγών μολυσμένων με το πρωτόζωο Leishmania κατά 6,1%.Το τρίτο κεφάλαιο, αφορά στην μοριακή και ορολογική διερεύνηση για την παρουσία μόλυνσης ή / και έκθεσης των Καφέ Ευρωπαϊκών λαγών από τη Βόρεια και Κεντρική Ελλάδα, σε παρασιτικούς παθογόνους παράγοντες κατά τη διάρκεια θηρευτικών περιόδων δύο ετών, η οποία έδειξε ότι το 5.7%, 0.95% και 12.4% των λαγών αντέδρασαν θετικά στην παρουσία αντισωμάτων έναντι των T.gondii, N.caninum και L.infantum. Αντίστοιχα, οι εξεταζόμενοι λαγοί ήταν μολυσμένοι με τα πρωτόζωα N.caninum (3,8%) και L.infantum (9,6%). Η ανάλυση με ΣΓΠ σε συνδυασμό με το Οικολογικό Μοντέλο Θέσης έδειξαν ότι η βροχόπτωση και οι χρήσεις γης επηρεάζουν σημαντικά την έκθεση του λαγού στα πρωτόζωα T.gondii και L.infantum. Επιπλέον, εντοπίστηκαν oι περιοχές υψηλού κινδύνου για την έκθεση του λαγού σε T.gondii και L.infantum και δημιουργήθηκε ένας χάρτης πρόβλεψης του κινδύνου έκθεσής τους στα εν λόγω πρωτόζωα για τις περιοχές της βόρειας και κεντρικής Ελλάδας.Η φυλογενετική θέση των πρωτόζωων Leishmania spp που ανιχνεύθηκαν σε ανθρώπους, σκύλους, γάτες και λαγούς στη Θεσσαλία, προσδιορίστηκε μέσω της φυλογενετικής ανάλυσης και της ανάλυσης της περιοχής ITS1 του γενώματος του πρωτόζωου Leishmania και περιγράφεται στο τέταρτο κεφάλαιο αυτής της διατριβής. Οι αλληλουχίες Leishmania που προήλθαν από διαφορετικούς ξενιστές βρέθηκε ότι ανήκουν στο σύμπλεγμα Leishmania donovani. Το είδος L. infantum εντοπίστηκε σε όλες τις αλληλουχίες Leishmania που συμπεριλήφθηκαν στη μελέτη, υποδεικνύοντας ότι αποτελεί το κυρίαρχο είδος που κυκλοφορεί σε διαφορετικούς ξενιστές στη Θεσσαλία. Επίσης, την ύπαρξη του ζωονοτικού κύκλου μετάδοσης της L.infantum στην περιοχή μελέτης και την πιθανή αλληλεπικάλυψη μεταξύ του οικόσιτου και του άγριου κύκλου μετάδοσης του πρωτόζωου. Απροσδόκητα, μία αλληλουχία Leishmania ταυτοποιήθηκε ως L. donovani σε ένα δείγμα γάτας, υποδεικνύοντας την πιθανή παρουσία μόλυνσης από L. donovani, σε μια γάτα στην περιοχή της μελέτης, δίνοντας το έναυσμα για περαιτέρω διερεύνηση.Το τελευταίο κεφάλαιο ,αφορά στην εμφάνιση μόλυνσης ή / και έκθεσης του Καφέ Ευρωπαϊκού λαγού, σε βακτηριακούς παθογόνους παράγοντες συμπεριλαμβανομένων των Bartonella spp, Brucella spp, Coxiella burnetii και Francisella tularensis. Η διερεύνηση που πραγματοποιήθηκε κατά τη διάρκεια θηρευτικών περιόδων δύο ετών, στη βόρεια και κεντρική Ελλάδα. Αντισώματα κατά της Bartonella spp ανιχνεύθηκαν στο 11,4% των εξεταζόμενων λαγών, ενώ κανένα δείγμα δεν βρέθηκε οροθετικό για τα βακτήρια Brucella spp, C. burnetii και F. tularensis. Η παρουσία του DNA των βακτηρίων Bartonella spp, Brucella spp, C. burnetii και F. tularensis δεν ανιχνεύθηκε στα δείγματα που εξετάστηκαν. Φαίνεται ότι ο ρόλος του Καφέ Ευρωπαϊκού λαγού ,στην επιδημιολογία των παθογόνων μικροοργανισμών Brucella spp, C. burnetii και F. tularensis είναι ελάχιστης σημασίας στην Ελλάδα ενώ τογεγονός ότι το είδος αυτό, βρέθηκε να έχει εκτεθεί στη Bartonella spp αξίζει περαιτέρω διερεύνηση για την αναγνώριση του ρόλου του ως εναλλακτικού ξενιστή αυτού του βακτηρίου.Η παρούσα διατριβή, παρέχει μια εικόνα για το ρόλο ενός σημαντικού είδους θηραμάτων στην Ελλάδα, του Καφέ ευρωπαϊκού λαγού, στην επιδημιολογία παρασιτικών και βακτηριακών ζωονοτικών παθογόνων παραγόντων. Το είδος αυτό έχει περιορισμένη περιοχή ενδημίας, είναι επομένως επιρρεπές σε παθογόνους παράγοντες που υπάρχουν στο φυσικό του περιβάλλον. Ωστόσο, μοιράζεται τον ίδιο χώρο διαβίωσης με άλλα άγρια και ελεύθερης εκτροφής ζώα, συμπεριλαμβανομένων των κυνοειδών, των αιλουροειδών και των μεγάλων αρπακτικών πτηνών, που αποτελούν τους φυσικούς του θηρευτές καθώς επίσης και με τους κυνηγετικούς σκύλους. Η σπουδαιότητά του, ενισχύεται περαιτέρω, λόγω των μετακινήσεων ζώντων ζώων τόσο σε διασυνοριακό επίπεδο όσο και σε μεγάλες αποστάσεις, καθώς επίσης και λόγω του ότι αποτελεί ξενιστή διαβιβαστών και θήραμα πολλών σαρκοφάγων και παμφάγων, τα οποία μετακινούνται καλύπτοντας εκτεταμένες αποστάσεις και έρχονται σε επαφή με τα κατοικίδια ζώα και τον άνθρωπο. Εξάλλου, η σύντομη διάρκεια ζωής του, καθιστά αυτό το είδος, έναν εξαιρετικό δείκτη για την πρόσφατη μετάδοση παθογόνων παραγόντων στην περιοχή. Η εντατική επαφή του, με διαβιβαστές όπως κρότωνες, κουνούπια και σκνίπες, έχει εμπλέξει τους λαγούς στην επιδημιολογία σημαντικών νοσημάτων που προκαλούνται από διαβιβαστές όπως η Λεϊσμανίαση. Η συνεχής επιτήρηση των πληθυσμών των λαγών, θα μπορούσε να παρέχει πληροφορίες σχετικά με την κατάσταση της υγείας του πληθυσμού του λαγού, καθώς και τους παθογόνους παράγοντες που κυκλοφορούν στην περιοχή διαβίωσής του. Παθογόνοι παράγοντες, οι οποίοι συνιστούν κίνδυνο για την άγρια πανίδα, τα κατοικίδια ζώα και φυσικά τον άνθρωπο

Current Data on Rickettsia felis Occurrence in Vectors, Human and Animal Hosts in Europe: A Scoping Review

Rickettsia felis is an emerging pathogen with increasing reports of human cases and detection in arthropod and animal host species worldwide. In this scoping review we record the newest data reported for R. felis in Europe: the vector and host species found to be infected, and the geographical distribution and prevalence of R. felis infection in vectors and hosts. A total of 15 European countries reported the occurrence of R. felis in hosts and vectors during 2017–2022. The vectors found to be infected by R. felis were flea, tick and mite species; Ctenocephalides felis and Ixodes ricinus were the dominant ones. The hosts found to be infected and/or exposed to R. felis were humans, cats and small mammals. Physicians should be aware of the epidemiology and include illness caused by R. felis in the differential diagnosis of febrile disease. Veterinarians should keep training pet owners on the need for effective year-round arthropod control on their pets, especially for fleas

Current Data on <i>Rickettsia felis</i> Occurrence in Vectors, Human and Animal Hosts in Europe: A Scoping Review

Rickettsia felis is an emerging pathogen with increasing reports of human cases and detection in arthropod and animal host species worldwide. In this scoping review we record the newest data reported for R. felis in Europe: the vector and host species found to be infected, and the geographical distribution and prevalence of R. felis infection in vectors and hosts. A total of 15 European countries reported the occurrence of R. felis in hosts and vectors during 2017–2022. The vectors found to be infected by R. felis were flea, tick and mite species; Ctenocephalides felis and Ixodes ricinus were the dominant ones. The hosts found to be infected and/or exposed to R. felis were humans, cats and small mammals. Physicians should be aware of the epidemiology and include illness caused by R. felis in the differential diagnosis of febrile disease. Veterinarians should keep training pet owners on the need for effective year-round arthropod control on their pets, especially for fleas

Wildlife and parasitic infections: A One Health perspective in Greece

While research on the parasitic fauna of wildlife in Greece is currently limited, conducted studies have provided valuable insights into the prevalence of parasitic infections in wild carnivores, omnivores, and herbivores. This review consolidates the existing data on the endoparasites detected in wild animals in Greece, specifically focusing on those that pose established or potential zoonotic risks. Over the last 60 years, various parasite species such as Leishmania infantum, Cryptosporidium spp., Toxoplasma gondii, Sarcocystis spp., Toxocara canis, Ancylostoma caninum, Capillaria spp., Baylisascaris spp., Trichinella spp., Thelazia callipaeda, Dirofilaria immitis, Echinococcus granulosus, Mesocestoides sp., Taenia spp., Alaria alata, and Dicrocoelium dendriticum have been identified in wildlife in Greece. These findings have become increasingly relevant due to the growing interaction between humans and wild animals, which now extends to urban environments, as well as the increased contact between wild and domestic animals. This is further complicated by the geographical expansion of vector-borne diseases due to global warming and the increased movements of humans and animals. Surveillance and monitoring of parasitic infections in Greek wildlife is warranted, and it should be based on interdisciplinary investigations considering the interconnectedness of human, wild, and domestic animals, as well as environmental health, in line with the One Health approach

Current Applications of Digital PCR in Veterinary Parasitology: An Overview

Digital PCR (dPCR) is an emerging technology that enables the absolute quantification of the targeted nucleic acids. The body of research on the potential applications of this novel tool is growing in human and veterinary medicine. Most of the research on dPCR applications in veterinary parasitology is concentrated on developing and validating new assays to detect and quantify parasites of great financial impact in the food-producing animal industry. Several studies describe the utility of dPCR for individualized medicine in companion animals. Most frequently, dPCR performance is assessed compared to quantitative PCR or Next Generation Sequencing platforms, while others also compare the accuracy of dPCR with traditional parasitological techniques considered gold standard methods. Other researchers describe dPCR assays for surveillance purposes, species identification, and quantification in mixed parasitic infections, the detection of mutations indicative of anthelmintic resistance, and the identification of new targets for drug development. This review provides an overview of the studies that employed dPCR in investigating animal parasites and parasitic diseases from a veterinary perspective and discusses how this novel technology could advance and facilitate diagnosis, surveillance, and the monitoring of response to treatment, or shed light on current gaps in our knowledge of the epidemiology of significant veterinary parasitic diseases

Leishmaniosis in Greece: The Veterinary Perspective

Leishmaniosis caused by the protozoon Leishmania infantum that is transmitted through the bites of infected phlebotomine sandflies is of major veterinary concern in Greece. The country is endemic with particularly favourable environmental conditions for the spread of this infection. Moreover, Greece remains a popular touristic destination, and the continuous travel of pets raises concern regarding the possible dissemination of infection from endemic to non-endemic areas. Dogs are the main reservoir host, although other animal species, including humans, may also be infected. Canine leishmaniosis manifests as a visceral disease that can result in death if left untreated. Serological and molecular epizootiological studies have confirmed circulation of the parasite in Greek canine and feline populations as well as in other mammals. As a result, constant surveillance and identification of high-risk localities are necessary to establish chemoprophylactic protocols for travelling animals to safeguard animal and public health

Natural Co-Exposure to <i>Borrelia burgdorferi</i> s.l. and <i>Anaplasma phagocytophilum</i>: Unraveling the Hematological Profile in Sheep

The occurrence of co-infected hosts and questing ticks with more than one tick-borne pathogen—as in the case of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato—is expected in endemic regions. Their synergy—in terms of pathogenesis and disease severity—has been suggested previously in humans. Limited data exist on the clinicopathological alterations in co-infected sheep. In this study, we investigated the impact of A. phagocytophilum and B. burgdorferi s.l. seropositivity, alone and in combination, on the hematological parameters of naturally infected sheep. A complete blood count was performed, and indirect immunofluorescence assays were used to detect IgG antibodies against A. phagocytophilum and IgG and IgM antibodies against B. burgdorferi s.l. Single natural exposure to B. burgdorferi s.l. was characterized by low Packed Cell Volume (PCV) values and platelet (PLT) counts, while single exposure to A. phagocytophilum was characterized by low PCV values, low white blood cell (WBC) counts, and an increased risk for leukopenia and neutropenia. Co-exposure resulted in the most severe blood abnormalities; all the blood parameters decreased, and the sheep presented an increased risk for anemia. Our study showed that natural co-exposure to A. phagocytophilum and B. burgdorferi s.l. in sheep leads to more severe blood abnormalities and enhances the pathogenic processes. More studies are needed to clarify the possible background mechanisms

Wild Rabbit Exposure to <i>Leishmania infantum</i>, <i>Toxoplasma gondii</i>, <i>Anaplasma phagocytophilum</i> and <i>Babesia caballi</i> Evidenced by Serum and Aqueous Humor Antibody Detection

Wild rabbits (Oryctolagus cuniculus) can be important sentinel species for the presence of zoonotic pathogens. Therefore, we collected blood samples from wild rabbits harvested by hunters during the hunting season 2019–2020 on the island of Lemnos, to determine exposure of wild rabbits to the zoonotic pathogens Leishmania infantum, Toxoplasma gondii, Anaplasma phagocytophilum and Babesia caballi, as well as aqueous humor to assess its diagnostic performance in terms of sensitivity, specificity, positive and negative likelihood ratios. Antibodies against these pathogens were detected by Indirect Immunofluorescence Antibody (IFA) assay. Out of the 72 wild rabbits included in the study, 4.2%, 5.5%, 18% and 9.7% were seropositive to L. infantum, T. gondii, A. phagocytophilum and B. caballi, respectively. Although less frequently, antibodies were also detected in aqueous humor of wild rabbits. The antibody detection in aqueous humor presented 100% specificity but decreased sensitivity compared to serum suggesting that aqueous humor could be successfully used in epidemiological studies to confirm exposure at the population level but has little diagnostic value at the individual level. This is the first report on the seropositivity of wild rabbits to A. phagocytophilum and B. caballi and the detection of antibodies against A. phagocytopylum, L. infantum, T. gondii and B. caballi in the aqueous humor