High seroprevalence for Crimean-Congo haemorrhagic fever virus in ruminants in the absence of reported human cases in many regions of Bulgaria.

Crimean-Congo haemorrhagic fever (CCHF) is a severe tick-borne zoonotic disease in humans caused by CCHF virus. It has been observed in Bulgaria since 1952 and over the years more than 1600 cases have been reported in the country. Close contact with viraemic livestock was shown as one of the main causes of the infection. Detection of CCHF virus specific antibodies in livestock can be used as an indicator for virus circulation and risk assessment. CCHF seroprevalence was investigated in 843 cattle, 88 goats and 130 sheep, originating from all 28 districts of Bulgaria. CCHF virus-specific IgG antibodies were observed in 165 cattle (19.6, 95% CI 17.0-22.4%), in 20 goats (22.7, 95% CI 15.2-32.5%) and in 10 sheep (7.7, 95% CI 4.2-13.6%). The highest seroprevalence was detected in the district of Kardzhali, South Bulgaria (86.7, 95% CI 73.8-93.7%), a well-known focus of CCHF in humans. The other two districts with human CCHF cases, Blagoevgrad and Burgas, located in southwest and southeast Bulgaria, showed overall seroprevalences in livestock of 41.9% (95% CI 28.4-56.7%) and 31.3% (95% CI 22.2-42.1%), respectively. Seroprevalences in districts with no history of human CCHF cases varied between 55% (95% CI 39.8-69.3%) and 22.5% (95% CI 12.3-37.5%). These results suggest frequent CCHF virus infections even in regions without human CCHF cases and an enhanced risk of infection for humans in close contact with the infected livestock.

A nationwide seroprevalence screening for West Nile virus and Tick-borne encephalitis virus in the population of Bulgaria.

To assess local circulation and risk for human infections with West Nile virus (WNV) and Tick-borne encephalitis virus (TBEV) in Bulgaria, a nationwide seroprevalence study was conducted. In total, 1451 residents of all 28 districts in Bulgaria were tested for WNV-specific and TBEV-specific IgG antibodies. The survey found overall seroprevalence of 1.5% and 0.6%, respectively. The highest WNV seroprevalence was found in Sofia Province and districts near the river Danube. TBEV circulation was detected among residents of six districts. The results showed that the two virus infections seem to be more wide-spread in the country as has been described.

Clinical manifestations and diagnostic approaches in cases of canine leishmaniasis in Bulgaria.

Leishmaniasis, a parasitic disease found in many parts of southern Europe, is transmitted in both humans and canines through the bite of phlebotomine sandflies, and can present in a variety of ways, such as cutaneous, mucocutaneous, diffuse, and visceral. In Bulgaria there are endemic areas of canine leishmaniasis, with sporadic cases in humans. However, no detailed studies of the animal population and vectors have been performed. Here we describe a few clinical cases of canine visceral leishmaniasis in two districts in western Bulgaria: one endemic and one without previously detected cases in humans or dogs. Diagnosis was confirmed serologically and molecularly using both real time and conventional PCR. Specific anti-leishmanial antibodies were confirmed in three of the cases via ELISA, with 50% of them returning extremely high values. In the majority of the cases DNA fragments were detected in the skin or lymph node aspirate but not in the blood. This paper highlights the need for further studies updating the current knowledge on the epidemiology, diagnosis, and control of visceral leishmaniasis in the reservoir host population.

Detection of Leishmania infantum Antibodies in Stray Dogs from Nonendemic Areas in Bulgaria.

Background: Leishmaniasis is a zoonotic protozoan infection which is endemic in parts of Europe. Dogs are commonly affected by Leishmania infantum and are regarded as the main reservoir for humans' infection. Sporadic human cases are reported annually, mainly from the endemic south part of Bulgaria. However, no recent data on canine leishmaniasis geographical distribution in Bulgaria have been published. The aim of this study was to investigate the current seroprevalence rate of L. infantum in stray dogs from areas where this infection was not previously reported. Results: The estimated overall seroprevalence rate was 8.3% (95% confidence interval [CI]: 2.6-14.0) with no significant difference between the two examined districts [χ2 (1, N = 90) = 0.32, p = 0.69]. In Sofia, the seropositivity was 7.2% (95% CI: 0.9-13.5) and in Sofia (stolitsa) 11.0% (95% CI: 0-23.0). No association between sex and serological status [χ2 (1, N = 90) = 1.83, p = 0.27] and age and seropositivity [χ2 (2, N = 90) = 1.98, p = 0.52] was detected. High levels of antibodies (fourfold increase from the assay's cut-off) were observed in half of the positive dogs.

Development of anti-Crimean-Congo hemorrhagic fever virus Gc and NP-specific ELISA for detection of antibodies in domestic animal sera.

Crimean-Congo hemorrhagic fever (CCHF) is a priority emerging disease. CCHF, caused by the CCHF virus (CCHFV), can lead to hemorrhagic fever in humans with severe cases often having fatal outcomes. CCHFV is maintained within a tick-vertebrate-tick cycle, which includes domestic animals. Domestic animals infected with CCHFV do not show clinical signs of the disease and the presence of antibodies in the serum can provide evidence of their exposure to the virus. Current serological tests are specific to either one CCHFV antigen or the whole virus antigen. Here, we present the development of two in-house ELISAs for the detection of serum IgG that is specific for two different CCHFV antigens: glycoprotein Gc (CCHFV Gc) and nucleoprotein (CCHFV NP). We demonstrate that these two assays were able to detect anti-CCHFV Gc-specific and anti-CCHFV NP-specific IgG in sheep from endemic CCHFV areas with high specificity, providing new insight into the heterogeneity of the immune response induced by natural infection with CCHFV in domestic animals.

Sand flies (Diptera: Psychodidae) in eight Balkan countries: historical review and region-wide entomological survey.

BACKGROUND: Sand flies (Diptera: Psychodidae) are medically important vectors of human and veterinary disease-causing agents. Among these, the genus Leishmania (Kinetoplastida: Trypanosomatidae), and phleboviruses are of utmost importance. Despite such significance, updated information about sand fly fauna is missing for Balkan countries where both sand flies and autochtonous leishmaniases are historically present and recently re-emerging. Therefore, a review of historical data on sand fly species composition and distribution in the region was followed by a large-scale entomological survey in eight Balkan countries to provide a recent update on local sand fly fauna. METHODS: The literature search involved the period 1910-2019. The entomological survey was conducted at 1189 sampling stations in eight countries (Bulgaria, Bosnia and Herzegovina, Croatia, Kosovo, Montenegro, North Macedonia, Serbia and Slovenia), covering 49 settlements and 358 sampling sites between June and October in the years 2014 and 2016, accumulating 130 sampling days. We performed a total of 1189 trapping nights at these stations using two types of traps (light and CO2 attraction traps) in each location. Sampling was performed with a minimal duration of 6 (Montenegro) and a maximal of 47 days (Serbia) between 0-1000 m.a.s.l. Collected sand flies were morphologically identified. RESULTS: In total, 8490 sand fly specimens were collected. Morphological identification showed presence of 14 species belonging to genera Phlebotomus and Sergentomyia. Historical data were critically reviewed and updated with our recent findings. Six species were identified in Bosnia and Herzegovina (2 new records), 5 in Montenegro (2 new records), 5 in Croatia (2 new records), 9 in Bulgaria (5 new records), 11 in North Macedonia (1 new record), 10 in Serbia (no new records), 9 in Kosovo (3 new records) and 4 in Slovenia (no new records). CONCLUSIONS: This study presents results of the first integrated sand fly fauna survey of such scale for the Balkan region, providing first data on sand fly populations for four countries in the study area and presenting new species records for six countries and updated species lists for all surveyed countries. Our findings demonstrate presence of proven and suspected vectors of several Leishmania species.

Correction to: The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus complex (Diptera: Ceratopogonidae) at the European level.

An amendment to this paper has been published and can be accessed via the original article.

The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus Complex (Diptera: Ceratopogonidae) at the European level.

BACKGROUND: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. METHODS: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. RESULTS: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. CONCLUSIONS: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.