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

Coxiella burnetii Shedding in Milk and Molecular Typing of Strains Infecting Dairy Cows in Greece

Ruminants are considered the commonest animal reservoir for human infection of Coxiella burnetii, the Q fever causative agent. Considering the recently described importance of human Q fever in Greece, we aimed at providing the first comprehensive direct evidence of C. burnetii in dairy cows in Greece, including the genetic characterization of strains. The 462 examined dairy farms represented all geographical areas of Greece. One bulk tank milk sample was collected from every farm and tested for the presence of C. burnetii. Molecular genotyping of strains, performed directly on samples, revealed the existence of two separate clades characterized by single nucleotide polymorphism (SNP) genotypes of type 1 and type 2. The two clades were clearly distinguished in multiple locus variable-number tandem repeat analysis (MLVA) by two discriminative loci: MS30 and MS28. Whereas MLVA profiles of SNP-type 2 clade were closely related to strains described in other European cattle populations, the MLVA profile observed within the SNP type 1 clade highlighted a peculiar genetic signature for Greece, related to genotypes found in sheep and goats in Europe. The shedding of C. burnetii bearing this genotype might have yet undefined human epidemiological consequences. Surveillance of the genetic distribution of C. burnetii from different sources is needed to fully understand the epidemiology of Q fever in Greece

Lack of Effect of Sleep Apnea on Oxidative Stress in Obstructive Sleep Apnea Syndrome (OSAS) Patients

PURPOSE: The aim of this study was to evaluate markers of systemic oxidative stress and antioxidant capacity in subjects with and without OSAS in order to investigate the most important factors that determine the oxidant-antioxidant status. METHODS: A total of 66 subjects referred to our Sleep laboratory were examined by full polysomnography. Oxidative stress and antioxidant activity were assessed by measurement of the derivatives of reactive oxygen metabolites (d-ROMs) and the biological antioxidant capacity (BAP) in blood samples taken in the morning after the sleep study. Known risk factors for oxidative stress, such as age, sex, obesity, smoking, hypelipidemia, and hypertension, were investigated as possible confounding factors. RESULTS: 42 patients with OSAS (Apnea-Hypopnea index >15 events/hour) were compared with 24 controls (AHI<5). The levels of d-ROMS were significantly higher (p = 0.005) in the control group but the levels of antioxidant capacity were significantly lower (p = 0.004) in OSAS patients. The most important factors predicting the variance of oxidative stress were obesity, smoking habit, and sex. Parameters of sleep apnea severity were not associated with oxidative stress. Minimal oxygen desaturation and smoking habit were the most important predicting factors of BAP levels. CONCLUSION: Obesity, smoking, and sex are the most important determinants of oxidative stress in OSAS subjects. Sleep apnea might enhance oxidative stress by the reduction of antioxidant capacity of blood due to nocturnal hypoxia

Overview of cattle diseases listed under category C, D or E in the animal health law for wich control programmes are in place within Europe

13 páginas, 5 figuras, 3 tablas.The COST action “Standardising output-based surveillance to control non-regulated diseases of cattle in the European Union (SOUND control),” aims to harmonise the results of surveillance and control programmes (CPs) for non-EU regulated cattle diseases to facilitate safe trade and improve overall control of cattle infectious diseases. In this paper we aimed to provide an overview on the diversity of control for these diseases in Europe. A non-EU regulated cattle disease was defined as an infectious disease of cattle with no or limited control at EU level, which is not included in the European Union Animal health law Categories A or B under Commission Implementing Regulation (EU) 2020/2002. A CP was defined as surveillance and/or intervention strategies designed to lower the incidence, prevalence, mortality or prove freedom from a specific disease in a region or country. Passive surveillance, and active surveillance of breeding bulls under Council Directive 88/407/EEC were not considered as CPs. A questionnaire was designed to obtain country-specific information about CPs for each disease. Animal health experts from 33 European countries completed the questionnaire. Overall, there are 23 diseases for which a CP exists in one or more of the countries studied. The diseases for which CPs exist in the highest number of countries are enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis, bovine viral diarrhoea and anthrax (CPs reported by between 16 and 31 countries). Every participating country has on average, 6 CPs (min–max: 1–13) in place. Most programmes are implemented at a national level (86%) and are applied to both dairy and non-dairy cattle (75%). Approximately one-third of the CPs are voluntary, and the funding structure is divided between government and private resources. Countries that have eradicated diseases like enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis and bovine viral diarrhoea have implemented CPs for other diseases to further improve the health status of cattle in their country. The control of non-EU regulated cattle diseases is very heterogenous in Europe. Therefore, the standardising of the outputs of these programmes to enable comparison represents a challenge.Peer reviewe

Cystic echinococcosis in Greece. Past and present

Cystic echinococcosis is a zoonotic disease with a wide geographical distribution, Greece included, and is considered to be a serious problem for the public health and the livestock economy. Although the disease was widespread in Greece since ancient times, cystic echinococcosis was identified as a serious problem around 1970, and since then national surveillance programmes are running, based on meat inspection and stray dogs management. Ever since, there are official records of the parasite’s prevalence in humans and livestock which show a continuous decline. More precisely, human hydatidosis, according to the official records, declines from an annual incidence of 14.8 per 100,000 inhabitants during 1967-1971 to 0.3 in 2008. Late surveys reveal that in Greece the prevalence of echinococcosis was 23-39.2 % for sheep, 7.6-14.7 % for goats, 0 % in cattle and 0.6 % in pigs, while further molecular analyses in Southern Greece showed the existence of the genotypes G1 and G3 in sheep and G7 in goats in that area. All data presented demonstrate that the parasite is still present in Greece. Surveillance is nowadays being performed under EU regulations but it is highly important to improve and adopt corrective and preventive measures to avoid animal and human infection

The geographical distribution of hard ticks (Acari: Ixodidae) in Greece and their role in the epidemiology of Crimean-Congo hemorrhagic fever

Crimean-Congo Hemorrhagic Fever (CCHF) is a severe acute febrile illness often accompanied by hemorrhagic manifestations; the fatality rate ranges between 5% - 30%. The causative agent is the Crimean-Congo hemorrhagic fever virus (CCHFV, genus Orthonairovirus, family Nairoaviridae). It is transmitted to humans by bites of infected ticks, mainly of the Hyalomma genus, or by direct contact with blood or tissues of patients or livestock at viremic phase. CCHFV is the most widespread tick-borne viral disease and has been reported in more than 30 countries in Africa, Asia, Eastern and Southern Europe and the Middle East. The disease has a wide distribution that correlates with the global distribution of Hyalomma ticks (50o north latitude limit). Nine genetic clades are currently recognized (four from Africa, three from Europe and two from Asia), which present a geographic correlation: “Africa 1”, “Africa 2”, “Africa 3” and “Africa 4” with strains from Central and South Africa; “Asia 1” from Middle East; “Asia 2” from China, Tajikistan, Kazakhstan and Uzbekistan; “Europe 1” from Balkan peninsula, Russia and Turkey; “Europe 2” consisting of the AP92 strain from Greece and “Europe 3” with strains from Russia. The strains that are circulating in Greece belong to the lineages “Europe 1” and “Europe 2” which differ genetically and in virulence level. The only human (fatal) case was reported in June 2008 and the strain belonged to lineage “Europe 1”, while the AP92 strain which belongs to lineage “Europe 2”, was isolated in 1975 from R. bursa ticks collected from goats in Vergina village in Central Macedonia. The purpose of the present study was to provide an overview on the prevalence and distribution of tick species in various regions of Greece (especially in areas where seroprevalence studies in humans have been previously performed), to identify the geographical parameters of the tick collection sites through the use of geographic and spatial analysis systems (GIS-SA), to estimate the CCHFV infection rate in ticks and to identify the genetic lineage of the circulating CCHFV strains. Therefore, a cross sectional field study on the distribution of Ixodidae ticks was carried out over three consecutive tick seasons (2012 - 2014); ticks were collected from small ruminants in 309 farms located in the mainland and five islands of Greece. In total, 2108 ticks were collected from sheep and goats. The identification of ticks to species level was carried out using taxonomic keys; two species of Rhipicephalus, two of Ixodes, five of Hyalomma, three of Haemaphysalis, and one of Dermacentor were identified. The species with the widest distribution were Rhipicephalus sanguineus sensu lato (s.l.) (64.8 %), R. bursa (25.9 %), and Dermacentor marginatus (4.1 %), whereas the least frequently collected species were Ixodes ricinus, I. gibbosus, Haemaphysalis parva, H. sulcata, H. punctata, Hyalomma marginatum, H. excavatum, H. dromedarii, H. rufipes, and H. impeltatum representing together less than 5.3 % of the collection. Grouping of ticks by collection date, location, animal host, tick species, and sex resulted in 1290 pools. Following tick homogenization and RNA extraction, an RT-nested PCR was applied for the detection of CCHFV. CCHFV was detected in 2.8% (36 of 1290) of the tick pools. Specifically, CCHFV was detected in 3.2% (27/843) of the R. sanguineus s.l. tick pools and in 2.4% (9 of 377) of the R. bursa tick pools. The regions with the highest CCHFV detection rate were Central and Western Macedonia (8.8%), Crete (6.2%) and Peloponnese (5.7%), while in Thessaly, Thrace and Epirus the percentage of positive pools were 3.1%, 1.4% and 1.2%, respectively. All H. marginatum ticks (the principal vector of CCHFV) were CCHFV negative. Phylogenetic analyses of the virus showed that sequences clustered into two genetic lineages, “Europe 1” (19/27) and “Europe 2” (8/27). Sequences of CCHFV lineage “Europe 1” were obtained mainly from R. sanguineus s.l. ticks (16/19), while sequences of lineage “Europe 2” were taken from R. bursa ticks (6/8). The sequences of the CCHFV lineage “Europe 2” of the present study differed from the prototype AP92 strain by 9.7% at nucleotide level. The present study gave a first insight into the tick fauna from mainland and islands in Greece. To date, there are no reports of CCHF cases in Greece (except the one in 2008 which was caused by a CCHFV strain belonging to the “Europe 1” lineage). The circulation of CCHFV strain(s) of lineage “Europe 2” in Greece, especially in regions where the seroprevalence is relatively high, together with the lack of human CCHF cases, suggests these strains are antigenic, with non- or low-pathogenic to humans. 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. Regarding lineage “Europe 1”, it is probable that ticks of the Rhipicephalus genus are not competent virus vectors as are the ticks of Hyalomma genus. Further laboratory studies are needed to explore the role of Rhipicephalus spp. ticks in CCHFV maintenance and transmission.Ο αιμορραγικός πυρετός Κριμαίας-Κογκό (Crimean - Congo Hemorrhagic Fever– CCHF), είναι σοβαρή οξεία εμπύρετη νόσος που συνοδεύεται συχνά από αιμορραγικές εκδηλώσεις και παρουσιάζει θνητότητα που κυμαίνεται από 5%-30%. Αιτιολογικός παράγοντας της νόσου είναι ο ιός του αιμορραγικού πυρετού Κριμαίας-Κογκό (CCHFV, genus Orthonairovirus, family Nairoaviridae). Η μετάδοση της νόσου στον άνθρωπο γίνεται με νύγμα κροτώνων, κυρίως του γένους Hyalomma, ή μετά από άμεση επαφή των βλεννογόνων ή του δέρματος με αίμα, εκκρίματα ή ιστούς μολυσμένων ζώων ή ανθρώπων. Η γεωγραφική διασπορά του CCHFV είναι η πιο εκτεταμένη από τους ιούς που μεταδίδονται με κρότωνες, καθ΄ όσον η παρουσία του αναφέρθηκε σε περισσότερες από 30 χώρες στην Αφρική, Ασία, Ν.Α Ευρώπη και στη Μέση Ανατολή. Η κατανομή του ιού ακολουθεί τη διασπορά των κροτώνων του γένους Hyalomma, οι οποίοι εξαπλώνονται έως το 50° βόρειο γεωγραφικό πλάτος. Τα στελέχη του CCHF διαχωρίζονται σε 9 γονότυπους: οι γονότυποι “Africa 1”, “Africa 2” , “Africa 3” και “Africa 4” περιλαμβάνουν στελέχη διαφόρων χωρών της Αφρικής, ο γονότυπος “Asia 1” με στελέχη της Μέσης Ανατολής, ο “Asia 2” με στελέχη από την Κίνα, το Τατζικιστάν, το Καζακστάν, το Ουζμπεκιστάν, ο “Europe 1” με στελέχη από τα Βαλκάνια, τη Ρωσία και την Τουρκία, ο “Europe 2” που περιλαμβάνει ένα μόνο στέλεχος, το ελληνικό στέλεχος AP92 και ο γονότυπος “Europe 3” με στελέχη από τη Ρωσία. Στην Ελλάδα ανιχνεύθηκαν στελέχη που ανήκουν στους γονοτύπους “Europe 1” και “Europe 2”, τα οποία διαφέρουν σημαντικά στο γονιδίωμα και στη λοιμογόνο δύναμή τους. Η μόνη (και θανατηφόρα) περίπτωση CCHF στην Ελλάδα παρατηρήθηκε τον Ιούνιο του 2008 και το στέλεχος ανήκε στον γονότυπο “Europe 1” ενώ το στέλεχος AP92 που ανήκει στον γονότυπο “Europe 2” απομονώθηκε το 1975 από κρότωνες Rhipicephalus bursa στην Βεργίνα της Κεντρικής Μακεδονίας. Σκοπός της παρούσας μελέτης ήταν η καταγραφή της διασποράς και συχνότητας των κροτώνων σε διάφορες περιοχές της Ελλάδας (ιδιαίτερα σε αυτές όπου είχαν πραγματοποιηθεί προηγουμένως οροεπιδημιολογικές μελέτες στον γενικό πληθυσμό), η χαρτογράφησή τους μέσω χρήσης συστημάτων γεωγραφικής και χωρικής ανάλυσης (GIS- SA), ο έλεγχος των κροτώνων για μόλυνση με τον CCHFV, καθώς και η γενετική και η φυλογενετική ανάλυση των στελεχών της μελέτης. Συγκεκριμένα, διεξήχθη μια εκτεταμένη μελέτη του πληθυσμού και της διασποράς των σκληρών κρότωνων (Ixodidae) σε τρεις διαδοχικές περιόδους (2012 - 2014) σε 309 κτηνοτροφικές μονάδες αιγών και προβάτων που βρίσκονται στην ηπειρωτική χώρα και σε πέντε νησιά της Ελλάδας. Συνολικά συλλέχθηκαν 2108 κρότωνες. Η ταυτοποίησή τους με ειδικές για αρθρόποδα διχοτομικές κλείδες ανέδειξε δύο είδη Rhipicephalus, δύο Ixodes, πέντε Hyalomma, τρία Haemaphysalis και ένα Dermacentor. Τα είδη με την ευρύτερη κατανομή ήταν τα Rhipicephalus sanguineus sensu lato (s.l.) (64,8%), R. bursa (25,9%) και Dermacentor marginatus (4,1%), και ακολουθούσαν τα Ixodes ricinus, Ι. gibbosus, Haemaphysalis parva, Η. sulcata, Η. punctata, Hyalomma marginatum, Η. excavatum, Η. dromedarii, Η. rufipes και Η. impeltatum που όλα μαζί αντιπροσωπεύουν περίπου το 5,3% της συλλογής. Οι κρότωνες διαχωρίστηκαν σε 1290 ομάδες ανάλογα με το είδος, το φύλο, το ξενιστή, την ημερομηνία και την περιοχή συλλογής τους. Μετά από ομογενοποίηση, απομονώθηκε το ιικό RNA και εφαρμόστηκε αλυσιδωτή αντίδραση πολυμεράσης (RT-nested PCR) για την ανίχνευση του CCHFV. Ο ιός ανιχνεύθηκε σε 36 από τις 1290 (2,8%) ομάδες κροτώνων. Ειδικότερα, ανιχνεύθηκε σε 27 από τις 843 (3,2%) ομάδες κροτώνων του είδους R. sanguineus s.l. και σε 9 από τις 377 (2,4%) ομάδες του είδους R. bursa. Οι περιφέρειες με τα υψηλότερα ποσοστά ανίχνευσης του CCHFV ήταν η Κεντρική και Δυτική Μακεδονία (8,8%), η Κρήτη (6,2%) και η Πελοπόννησος (5,7%), ενώ στις Θεσσαλία, Θράκη και Ήπειρο τα ποσοστά ήταν 3,1%, 1,4% και 1,2% αντίστοιχα. Όλοι οι κρότωνες του είδους H. marginatum (που αποτελεί τον κύριο φορέα του CCHFV) ήταν αρνητικοί. Από την φυλογενετική ανάλυση των αλληλουχιών του ιού που ανιχνεύθηκαν σε 27 ομάδες κροτώνων φάνηκε η παρουσία των γονοτύπων “Europe 1” (19/27) και “Europe 2” (8/27). Ο γονότυπος “Europe 1” βρέθηκε κυρίως σε κρότωνες του είδους R. sanguineus s.l. (16/19), ενώ ο “Europe 2” σε κρότωνες του είδους R. bursa (6/8). Το γονιδίωμα του στελέχους CCHFV γονοτύπου “Europe 2” που ανιχνεύθηκε σε κρότωνες R. bursa διέφερε κατά 9,7% σε επίπεδο νουκλεοτιδίων από το πρότυπο στέλεχος AP92, το οποίο είχε απομονωθεί το 1975.Συμπερασματικά, η παρούσα μελέτη έδωσε μία πρώτη αποτύπωση σχετικά με τον πληθυσμό των κροτώνων στην ηπειρωτική χώρα και τα νησιά της Ελλάδας. Μέχρι σήμερα, δεν έχουν αναφερθεί κλινικές περιπτώσεις CCHF σε ανθρώπους (εκτός από μία περίπτωση το 2008 που οφειλόταν σε στέλεχος του γονοτύπου “Europe 1”). Η κυκλοφορία του CCHFV του γονοτύπου “Europe 2” σε περιοχές όπου ο οροεπιπολασμός είναι σχετικά υψηλός, σε συνδυασμό με την απουσία κλινικών περιστατικών, υποδηλώνει ότι αυτά τα στελέχη παρουσιάζουν αντιγονικότητα, αλλά έχουν μικρή η μηδαμινή λοιμογόνο δύναμη. Περαιτέρω μελέτες σε στελέχη του γονοτύπου “Europe 2” θα αυξήσουν τις γνώσεις μας σχετικά με το ρόλο αυτών των στελεχών τύπου ΑΡ92 στην επιδημιολογία του CCHF, οι οποίες θα μπορούσαν να είναι χρήσιμες για το σχεδιασμό φαρμάκων και εμβολίων. Όσον αφορά τον γονότυπο “Europe 1”, είναι πιθανό, οι κρότωνες του γένους Rhipicephalus να μην είναι ικανοί φορείς του ιού όπως είναι οι κρότωνες του γένους Hyalomma, οπότε θα απαιτηθούν περαιτέρω εργαστηριακές μελέτες για να διερευνηθεί ο ακριβής ρόλος τους στη συντήρηση και μετάδοση του CCHFV

Cystic echinococcosis in Greece. Past and present

Cystic echinococcosis is a zoonotic disease with a wide geographical distribution, Greece included, and is considered to be a serious problem for the public health and the livestock economy. Although the disease was widespread in Greece since ancient times, cystic echinococcosis was identified as a serious problem around 1970, and since then national surveillance programmes are running, based on meat inspection and stray dogs management. Ever since, there are official records of the parasite’s prevalence in humans and livestock which show a continuous decline. More precisely, human hydatidosis, according to the official records, declines from an annual incidence of 14.8 per 100,000 inhabitants during 1967-1971 to 0.3 in 2008. Late surveys reveal that in Greece the prevalence of echinococcosis was 23-39.2 % for sheep, 7.6-14.7 % for goats, 0 % in cattle and 0.6 % in pigs, while further molecular analyses in Southern Greece showed the existence of the genotypes G1 and G3 in sheep and G7 in goats in that area. All data presented demonstrate that the parasite is still present in Greece. Surveillance is nowadays being performed under EU regulations but it is highly important to improve and adopt corrective and preventive measures to avoid animal and human infection

Species diversity and spatial distribution of ixodid ticks on small ruminants in Greece

A cross sectional field study on the distribution of Ixodidae ticks was carried out over two consecutive tick seasons (2012 and 2013) in small ruminants (309 farms) located in mainland and five islands of Greece. A total of 2108 ticks (1199 females, 908 males, and 1 nymph) were collected from sheep and goats. Two species of Rhipicephalus, two of Ixodes, five of Hyalomma, three of Haemaphysalis, and one of Dermacentor were detected. The species with the widest distribution were Rhipicephalus sanguineus s.l. (64.8 %), R. bursa (25.9 %), and Dermacentor marginatus (4.1 %), whereas the least frequently collected species were Ixodes ricinus, I. gibbosus, Haemaphysalis parva, H. sulcata, H. punctata, Hyalomma marginatum, H. excavatum, H. dromedarii, H. rufipes, and H. impeltatum representing together less than 5.3 % of the collections. R. sanguineus s.l. was the predominant species, while D. marginatus and I. gibbosus were restricted to the northeastern Aegean Sea islands. This is the first survey on tick fauna from mainland and islands in Greece.The work is part of the FP7 project “ANTIcipating the Global Onset of Novel Epidemics” (Grant agreement no. 278976) supported by the European Commission.Peer Reviewe