Phlebotomine sand flies (Diptera: Psychodidae) of Bosnia and Herzegovina: distribution, ecology and environmental preferences.

Sand flies (Diptera: Phlebotominae) are the principal vectors for the protozoan parasites Leishmania spp. and for phleboviruses. The sand fly fauna on the Balkan Peninsula, including Bosnia and Herzegovina (BIH), is diverse and the circulation of Leishmania infantum as well as phleboviruses has been proven. However, recent data on the sand fly fauna in BIH are scarce. In this study, we surveyed understudied regions in central and northeastern BIH to update the sand fly distribution and gain insights into the ecological and environmental factors shaping their appearance. CDC light trapping was conducted in 2022 and 2023 and a combination of morphological and molecular methods (cytochrome oxidase I barcoding) was performed for species identifications. We mapped the currently known distribution, modelled climatic suitability patterns and performed environmental analyses by applying machine learning methods. In addition, we analyzed blood meals by host gene sequencing and MALDI-TOF peptide mass mapping and screened for Leishmania spp. DNA and Phlebovirus RNA. Altogether, 591 sand flies of four species were trapped, predominantly Phlebotomus neglectus (97%), but also Ph. balcanicus, Ph. mascittii, and Ph. papatasi. Records of seven sand fly species known to be endemic were plotted onto distribution maps based on 101 datapoints, identifying Ph. neglectus as the overall predominant species. The environmental analyses of sand fly species indicated variation in altitudinal, thermal, and precipitation conditions across the sand fly-positive sites. Phlebotomus simici, Phlebotomus tobbi, and Sergentomyia minuta are typically found exclusively in Mediterranean and subtropical climate zones, whereas other species typically inhabit continental regions. The Inverse Distance Weighted (IDW) interpolation of sand fly species numbers and Shannon entropy values suggest the southeastern coastal region of BIH as a primary focus for sand fly occurrence. This finding was corroborated by modeled average climatic suitability patterns for sand flies, depicting four distinct meso-regions for sand fly occurrence. The results of the ensemble method highlight the importance of annual precipitation to distinguish between positive and negative sand fly trapping sites in BIH. In total, 55 blood meals of two sand fly species, Ph. neglectus and Ph. balcanicus, were analyzed and five host species identified. Our comprehensive assessment of ecological and environmental preferences of sand flies in BIH may support further entomological surveys and help to better understand and evaluate potential hot spots of disease transmission in the country.

First case of feline cryptococcosis in Bosnia and Herzegovina.

Case summary: A 2-year-old domestic longhair crossbred female cat was referred for a second opinion on a non-healing surgical wound after left eye enucleation. In addition to the left orbital lesion, ulcerative granular masses protruding from the left nostril and on the base of the left ear were noted. A diagnosis of cryptococcosis was established using histopathological examination and a latex cryptococcal antigen agglutination test. The cat was successfully treated with itraconazole. Relevance and novel information: Cryptococcosis, commonly reported in Australia, western Canada and the western USA, is rarely reported in companion animals in Europe. This marks the first report of cryptococcosis in cats in Bosnia and Herzegovina, emphasising the need to raise awareness within the veterinary community, both local and regional, about this disease.

Metagenomic Sequencing of Lloviu Virus from Dead Schreiber's Bats in Bosnia and Herzegovina.

Bats are a natural host for a number of viruses, many of which are zoonotic and thus present a threat to human health. RNA viruses of the family Filoviridae, many of which cause disease in humans, have been associated with specific bat hosts. Lloviu virus is a Filovirus which has been connected to mass mortality events in Miniopterus schreibersii colonies in Spain and Hungary, and some studies have indicated its immense zoonotic potential. A die-off has been recorded among Miniopterus schreibersii in eastern Bosnia and Herzegovina for the first time, prompting the investigation to determine the causative agent. Bat carcasses were collected and subjected to pathological examination, after which the lung samples with notable histopathological changes, lung samples with no changes and guano were analyzed using metagenomic sequencing and RT-PCR. A partial Lloviu virus genome was sequenced from lung samples with histopathological changes and found to be closely related to Hungarian and Italian virus sequences. Further accumulation of mutations on the GP gene, coding the glycoprotein responsible for cell tropism and host preference, enhances the need for further characterization and monitoring of this virus to prevent spillover events and protect human health.

First detection of Echinococcus multilocularis in Bosnia and Herzegovina.

Echinococcus multilocularis has been spreading through Central Eastern Europe but has not yet been reported in Bosnia and Herzegovina (B&H). Recently, this parasite is confirmed in Croatia suggesting the movement of the parasite's distribution limit further south. Given that there is no surveillance or monitoring system for echinococcosis in B&H, our study was designed as a pilot study of E. multilocularis. A total of 57 red foxes originating from 24 localities all over the country were collected during the routine rabies monitoring, autopsied and examined for the presence of echinococcosis. Based on intestinal scraping technique and microscopy, E. multilocularis adult worms have been detected in one (1/57, 1.75%) red fox. To verify this finding and to differentiate Echinococcus spp., DNA extracted from adult worms was subjected to species-specific PCR targeting part of the mitochondrial 12S ribosomal RNA gene. E. multilocularis PCR-positive samples were further confirmed by NGS sequencing of a 203 bp amplified fragment of 12S rRNA, which has been deposited in GenBank (Accession no.: OP047920). This finding represents the first detection of E. multilocularis in B&H, strongly suggesting its presence in the country. The confirmation of the parasite in the same locality where migrants/refugees temporarily stay on their route to Western Europe highlights the need for a One Health approach in addressing all future questions. Moreover, the first detection of E. multilocularis in B&H warrants the need for the implementation of an appropriate state surveillance program.

A Unique Case of Fatal Coinfection Caused by Leptospira spp. and Hepatozoon canis in a Red Fox Cub (Vulpes vulpes).

Red foxes are the most abundant wild carnivore species in Europe commonly exposed to pathogenic Leptospira and Hepatozoon canis. Despite high seroprevalence, the clinical disease caused by these pathogens in red foxes has never been reported. Herein, we report the first-ever case of a fatal Leptospira spp. and H. canis coinfection in a two-month-old red fox cub with acute haemolytic anaemia, mild bronchopneumonia, intraalveolar haemorrhage, and tubulonephrosis. The presence of pathogenic Leptospira spp. DNA was detected in the kidney and lung tissues of the infected animal. In contrast to our previous knowledge, we believe that such fatal cases due to concomitant infection by Leptospira spp. and H. canis, especially in young animals, may commonly occur in nature. However, further studies are required to identify other factors that possibly contribute to the severity and the pathogenic effect of Leptospira spp. and H. canis infections in red foxes.

Echinococcus ortleppi, the cattle strain in a crested porcupine (Hystrix cristata): A new host record.

Echinococcus granulosus sensu lato, a dog tapeworm is a species complex causing cystic echinococcosis or hydatid disease in a great variety of mammalian intermediate hosts, including humans. This complex comprises five species including Echinococcus ortleppi (G5 genotype or cattle strain). In the present paper, we report the first case of infection with the larval stage of latter cestode in a captive crested porcupine (Hystrix cristata), molecularly confirmed by PCR and sequencing of the cox1 and nad1 genes. The food contaminated with the parasite's eggs is the most likely source of the infection. Our data broaden the knowledge on the host range and geographical distribution of this rarely reported species of Echinococcus in Europe.

Eulimdana clava (Nematoda: Filarioidea) infection in domestic pigeons (Columba livia domestica): Molecular characterization and pathological changes.

Filarial nematodes of the Eulimdana genus inhabit subcutaneous tissue of various avian species, mostly Charadriiforme birds. In domestic pigeons, E. clava is the only species recorded in the subcutaneous tissue in a number of isolated cases. In the present study, we discuss the morphology and histopathology of filarial nematodes recovered from subcutaneous tissue of domestic pigeons in Bosnia and Herzegovina. In total 110 pigeons were submitted to necropsy at the Department of Pathology of the Faculty of Veterinary Medicine in Sarajevo. At necropsy, in four pigeons (3.6%) numerous thread-like 0.9-2.1 cm long nematode parasites were observed in the subcutaneous tissue, peritracheal and periesophageal connective tissue. In one pigeon, the parasites were also found free in the body cavity around the heart and lungs. In addition, several 80-90 µm long microfilariae were noted in the tissue cross-sections. No significant lesions were observed associated with adult parasites or microfilariae. Based on morphology, host species and localization detected parasites were identified as E. clava. Molecular analyses of the cox1 and 12S rRNA nucleotide sequences herein generated revealed the close genetic relationship to other filarioid nematodes. The importance of the nematodes in pigeons and the lack of sequences in genetic databases for comparison of avian filarial parasites are emphasized.