Ilias Chaligiannis

Anisakidae infection in fish of the Aegean Sea

Nematode worms of the family Anisakidae are the causative agents of infections in humans when fish is consumed raw and of serious allergies up to the death, when fish is consumed raw or cooked by previously sensitized people. From April until November 2009, 462 fish belonging to 26 species, fished in three areas of the Aegean Sea were tested for Anisakidae larvae. Anisakidae larvae were detected in 87 (18.83%) fish of 13 species. These larvae were identified by morphology as the third-stage larvae of the genera Hysterothylacium sp. or Anisakis. Larvae of the genus Anisakis were identified by PCR-RFLP as belonging to A. simplex s.str., A. pegreffii, or as hybrids between A. simplex s.str and A. pegreffii.

A novel AP92-like Crimean-Congo hemorrhagic fever virus strain, Greece.

Ticks were collected from various regions of northern Greece and tested for the presence of Crimean-Congo hemorrhagic fever virus (CCHFV) RNA. Human and animal sera were collected in the regions where CCHFV-positive ticks were detected, and they were tested for the presence of IgG antibodies against the virus. A CCHFV strain was detected in Rhipicephalus bursa ticks collected from sheep in Kastoria regional unit, differing by 9.7% at the nucleotide level from the AP92 strain, which was isolated in 1975 in another region of Greece. Up to date, CCHF cases have not been reported in these regions. The human seroprevalence in the area was estimated at 6%, while IgG-positive sheep was detected in two of the four neighboring farms tested. The circulation of this specific CCHFV lineage in Greece, especially in a region where the seroprevalence is high, together with the lack of human CCHF cases, suggests a probable antigenic, but non- or low-pathogenic character of this lineage. Further studies on these strains will increase our knowledge about the role of AP92-like strains in the CCHF epidemiology, which might be useful for drug and vaccine design.

Ticks Parasitizing Humans in Greece

In summer 2008, two fatal cases were observed in Northeastern Greece: a Crimean-Congo hemorrhagic fever (CCHF) case (first report in Greece) and a Mediterranean spotted fever case. In total, 537 ticks removed from humans who referred for this reason to the two hospitals of the region during June-September 2008 were identified. The vast majority of them (81.5%) were Rhipicephalus sanguineus, which is the main vector of Rickettsia conorii, while Hyalomma marginatum, the main vector of CCHF virus, accounted for 5.2%. The increased aggressiveness of R. sanguineus might be related to the weather conditions occurred during 2007-2008, while a variety of factors, including climate, might play a role in CCHF emergence.

Echinococcus granulosus infection dynamics in livestock of Greece.

An epidemiological and molecular survey on the occurrence of Echinococcus hydatid cysts in livestock was conducted in Greece. In total 898 sheep, 483 goats, 38 buffaloes, 273 wild boars and 15 deer were examined and 30.2% (6.45% cyst fertility), 7.86% (3.2% cyst fertility), 42% (7.9% cyst fertility), 1.1% (0% cyst fertility), 0% of them were found infected, respectively. Infection rate in different geographical regions varied between 26.1 and 53.8% (cyst fertility 2.04 and 34.6%) in sheep, 7.33 and 13.3% (cyst fertility 0 and 3.2%) in goats. Genotyping, based on cox1 and nad1 analyses, demonstrated the predominance of E. granulosus s.s. (G1 genotype). The presence of one single genotype-complex within a relatively large spectrum of intermediate host species in Greece indicates the presence of a dominant transmission dog-sheep cycle involving additional host species which may act as disease reservoir for human infections.

Novel phleboviruses detected in ticks, Greece.

Since 2009, when severe fever with thrombocytopenia syndrome virus and Heartland virus have been identified and associated with disease in humans, the interest on tick-borne phleboviruses is increasing rapidly. The aim of the present study was to investigate the presence of tick-borne phleboviruses in Greece and compare them with respective ones detected worldwide. Ticks collected from goats and sheep in 60 sites of 13 regional units of Greece were grouped in pools (1-3 ticks per pool) and tested for the presence of phleboviral RNA. Six of 210 pools were positive; they consisted of Rhipicephalus sanguineus ticks collected from sheep in 3 regional units of Greece: Pella (2/30, 6.7%), Imathia (2/21, 9.5%), and Ioannina (2/28, 7.1%). The overall tick minimum infection rate was 2.1%. The sequences of the Greek phlebovirus (provisionally named Antigone virus) form a distinct clade in the tick-borne phleboviruses, differing by >40% from the currently known phleboviruses. Any probable implication of these viruses to public health remains to be elucidated.

Rickettsia species in human-parasitizing ticks in Greece.

BACKGROUND Ticks serve as vectors and reservoirs for a variety of bacterial, viral and protozoan pathogens affecting humans and animals. Unusual increased tick aggressiveness was observed in 2008-2009 in northeastern Greece. The aim of the study was to check ticks removed from persons during 2009 for infection with Rickettsia species. METHODS A total of 159 ticks were removed from 147 persons who sought medical advice in a hospital. Tick identification was performed morphologically using taxonomic keys. DNA was extracted from each individual tick and a PCR assay targeting the rickettsial outer membrane protein A gene of Rickettsia spp. was applied. RESULTS Most of the adult ticks (132/153, 86.3%) were Rhipicephalus sanguineus. Rickettsiae were detected in 23 of the 153 (15.0%) adult ticks. Five Rickettsiae species were identified: R. aeschlimannii, R. africae (n=6), R. massilae (4), R. monacensis (1), and Candidatus R. barbariae (1). To our knowledge, this is the first report of R. africae, R. monacensis, and Candidatus R. barbariae in Greece. CONCLUSIONS Several Rickettsia species were identified in ticks removed from humans in Greece, including those that are prevalent in northern and southern latitudes.

Ticks feeding on ruminants and humans in Greece

Ticks are important vectors of disease and transmit an extensive range of viral, bacterial and protozoan pathogens to livestock in a wide variety of habitats. In recent years, diseases such as babesiosis, ehrlichiosis and anaplasmosis have all shown evidence of increased prevalence and distribution in various parts of Europe. However data concerning the prevalence of ticks and tick borne diseases present in livestock and humans in Greece are limited. In order to fill this gap we performed the current study to define the existence and prevalence of different tick species found in farm animals and humans. As regards livestock, we focused on ruminants (mainly sheep and goats and, in a lesser extent, cattle) since they are the only ones spend time on pastures. A sufficient number of farms all over the country were visited during 2 tick seasons (from March to October), taking different habitats and animal density around Greece into account. Ticks collected from humans originated from infected individuals who visited hospitals. In total, 2676 ticks were collected from 26 different prefectures (mainland and islands) all over Greece. From those, 1,883 were coming from sheep (1201) and goats (681) and identified as: Rhipicephalus sanguineus 1,216 (64.65%); R. bursa 495 (26.3%); R. camicasi 12 (0.6%); R. turanicus 70 (3.7%); Ixodes ricinus 1 (0.05%); Dermacentor marginatus 47 (2.5%); Hyalomma marginatum 5 (0.3%); H. excavatum 2 (0.1%), H. dromedarii 31 (1.6%) H. rufipes 2 (0.1%); H. impeltatum 1 (0.05%); and Rhipicephalus nymph 1 (0.05%). More than half (54.3%) of the above were found in an altitude of 0-300 meters, 37.1% in an altitude of 301-800m and 3.2% in an altitude of >800m. 142 ticks originated from cattle and were identified as: R. sanguineus 15 (10.5%); R. bursa 6 (4.2%); R. camicasi 5 (3.5%); R. turanicus 6 (4.2%); H. marginatum 24 17%); H. excavatum 8 (5.6%); H. dromedarii 67 (47.2%); H. rufipes 4 (2.8%); H. impeltatum 1 (0.7%); H. anatolicum 3 (2.1%) and H. turanicum 3 (2.1%). Finally, 701 ticks were coming from humans and identified as: R. sanguineus 562 (80.17%); R. bursa 23 (3.28%); R. turanicus 34 (4.85%); R. annulatus 5 (0.71%); H. marginatum 30 (4.28%); H. excavatum 2 (0.28%); H. rufipes 11 (1.57%); Dermacentor marginatus 2 (0.28%); Ixodes ricinus 6 (0.85%); I. gibosus 6 (0.85%) and Rhipicephalus nymphs 20 (2.85%). In conclusion, the majority of ticks found in both animal species and humans examined belonged in the Rhipicephalus sanguineus group which is the main vector of Rickettsia conorii, while Hyalomma marginatum, the vectors of CCHF virus, were also present. The above results were more or less anticipated given the climatic conditions of the area, fact that also explains the low prevalence of Ixodes spp.

Molecular characterization of Echinococcus granulosus isolates from Bulgarian human cystic echinococcosis patients

Although cystic echinococcosis (CE) is highly endemic in Bulgaria, there is still scarce information about species and/or genotypes of the Echinococcus granulosus complex that infect humans. Our study tackled the genetic diversity of E. granulosus complex in a cohort of 30 Bulgarian CE patients. Ten animal E. granulosus isolates from neighboring Greece were additionally included. Specimens were comparatively analyzed for partial sequences of five mitochondrial (mt) (cox I, nad I, rrnS, rrnL, and atp6) and three nuclear (nc) genes (act II, hbx 2, and ef-1α) using a PCR-sequencing approach. All 30 Bulgarian isolates were identified as E. granulosus sensu stricto (s.s.) and were showing identical sequences for each of the three examined partial nc gene markers. Based upon concatenated sequences from partial mtDNA markers, we detected 10 haplotypes: 6 haplotypes (H1-H6) clustering with E. granulosus s.s. (G1) and 4 haplotypes (H9-H13) grouping with E. granulosus s.s. (G3), with H1 and H10 being the most frequent in Bulgarian patients. The haplotypes H1, H4, and H11 were also present in Greek hydatid cyst samples of animal origin. In conclusion, E. granulosus s.s. (G1 and G3 genotypes) is the only causative agent found so far to cause human CE in Bulgaria. However, further studies including larger sample sizes and other additional geographic regions in Bulgaria will have to be performed to confirm our results.

Combination of RT-PCR and proteomics for the identification of Crimean-Congo hemorrhagic fever virus in ticks

Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne zoonotic disease caused by the CCHF virus (CCHFV). In this study, an experimental approach combining RT-PCR and proteomics was used for the identification and characterization of CCHFV in 106 ticks from 7 species that were collected from small ruminants in Greece. The methodological approach included an initial screening for CCHFV by RT-PCR followed by proteomics analysis of positive and control negative tick samples. This novel approach allowed the identification of CCHFV-positive ticks and provided additional information to corroborate the RT-PCR findings using a different approach. Two ticks, Dermacentor marginatus and Haemaphysalis parva collected from a goat and a sheep, respectively were positive for CCHFV. The sequences for CCHFV RNA segments S and L were characterized by RT-PCR and proteomics analysis of tick samples, respectively. These results showed the possibility of combining analyses at the RNA and protein levels using RT-PCR and proteomics for the characterization of CCHFV in ticks. The results supported that the CCHFV identified in ticks are genetic variants of the AP92 strain. Although the AP92-like strains probably do not represent a high risk of CCHF to the population, the circulation of genetically diverse CCHFV strains could potentially result in the appearance of novel viral genotypes with increased pathogenicity and fitness.

Molecular detection of Crimean-Congo hemorrhagic fever virus in ticks, Greece, 2012–2014

Crimean-Congo hemorrhagic fever virus (CCHFV) is transmitted to humans mainly through the bite of infected ticks. In Greece, only one clinical case has been observed, in 2008, but the seroprevalence in humans is relatively high (4.2%). To have a first insight into the circulation of CCHFV in Greece, 2000 ticks collected from livestock during 2012–2014 were tested. CCHFV was detected in 36 of the 1290 (2.8%) tick pools (1–5 ticks per pool). Two genetic lineages were identified: Europe 1 and Europe 2. Most Europe 1 sequences were obtained from Rhipicephalus sanguineus sensu lato ticks, while most Europe 2 sequences were recovered from Rhipicephalus bursa ticks. The number of collected Hyalomma marginatum ticks (the principal vector of CCHFV) was low (0.5% of ticks) and all were CCHFV negative. Since it is not known how efficient ticks of the Rhipicephalus genus are as vectors of the virus, laboratory studies will be required to explore the role of Rhipicephalus spp. ticks in CCHFV maintenance and transmission.