Abstract. Between July and October 1996, a West Nile (WN) fever epidemic occurred in the southern plain and Danube Valley of Romania and in the capital city of Bucharest, resulting in hundreds of neurologic cases and 17 fatalities. In early October 1996, entomologic and avian investigations of the epidemic were conducted in the city of Bucharest and nearby rural areas. Thirty (41%) of 73 domestic fowl sampled had neutralizing antibody to WN virus, including 5 of 13 ducks (38%), 1 of 1 goose, 19 of 52 chickens (37%), 1 of 1 peahen, and 4 of 6 turkeys (67%). Seroprevalence in domestic fowl (27%, or 7 of 26) from the urban Bucharest site was not significantly different (P ϭ 0.08, by Fisher's exact test) than rates at three rural sites (50%, or 23 of 46). Serum collected from one of 12 Passeriformes, an Erithacus rubecula, was positive for neutralizing antibody to WN virus. A total of 5,577 mosquitoes representing seven taxa were collected. Culex pipiens pipiens accounted for 96% of the mosquitoes collected. A single virus isolate, RO97-50, was obtained from a pool of 30 Cx. p. pipiens females aspirated from the walls and ceiling of a blockhouse located near the center of Bucharest, resulting in a minimum infection rate of 0.19 per 1,000. Antisera prepared against RO97-50 failed to distinguish among RO97-50, WN virus strain Eg101, and Kunjin (KUN) virus strain MRM16. A 2,323-basepair DNA fragment of the envelope (E) glycoprotein gene from RO97-50 and a Romanian WN virus strain obtained from a human cerebrospinal fluid sample, RO96-1030, were sequenced. Phylogenetic analyses of 23 WN virus strains and one KUN virus strain using the amino acid and nucleotide sequences for a small portion of the E gene suggest the existence of two large lineages of viruses. Bootstrap analysis of the nucleotide alignment indicated strong support (95%) for a lineage composed of WN virus strains from northern Africa, including isolates from Egypt and Algeria, and west, central, and east Africa, all of the European isolates, those from France and Romania, an Israeli isolate, and an isolate of KUN virus from Australia. The nucleotide sequence of RO97-50 was identical to the sequence of a WN virus isolate obtained from Cx. neavei mosquitoes from Senegal and Cx.

Mosquitoes of the Anopheles maculipennis group were collected in five districts of Romania (Constant,a, Giurgiu, Ilfov, Mehedint,i and Suceava) between March 2000 and June 2003. Two hundred and ninety-seven specimens were identified by molecular methods. Nuclear rDNA ITS2 sequences of 178 specimens were compared with GenBank sequences for nine known Palaearctic species of the group, and 119 specimens were identified using an ITS2 PCR-RFLP assay developed during the study. Five genetically distinct species of the group were identified: A. atroparvus van Thiel, A. maculipennis Meigen, A. melanoon Hackett and A. messeae Falleroni and a previously unrecognized species. The new species, herein formally described and named A. daciae sp. n., was collected in the Black Sea coastal region and plains adjacent to the Danube River in southern Romania. Anopheles daciae is most similar to and sympatric with A. messeae. It is contrasted with A. messeae and characterized on the basis of unique nuclear ITS2 and mitochondrial COI DNA sequences and morphological characters of the eggs. The larval, pupal and adults stages of the two species were also compared, but no reliable characters were found to distinguish them. It seems likely that A. daciae is more widespread in eastern Europe and the Balkan States, and could be responsible for malaria transmission in these regions that is currently attributed to A. messeae. Anopheles melanoon is reported from Romania for the first time.

In response to the 1996 West Nile (WN) fever epidemic that occurred in Bucharest and southeastern Romania, a surveillance program was established. The surveillance system detected 39 clinical human WN fever cases during the period 1997-2000: 14 cases in 1997, 5 cases in 1998, 7 cases in 1999, and 13 cases in 2000. Thirty-eight of the 39 case-patients lived in the greater Danube Valley of southern Romania, and 1 case-patient resided in the district of Vaslui, located on the Moldavian plateau. The estimated annual case incidence rate for the surveillance area during the period 1997-2000 was 0.95 cases per million residents. Thirty-four cases were serologically confirmed, and 5 cases were classified as probable. Twenty-four case-patients presented with clinical symptoms of meningitis (62%), 12 with meningoencephalitis (31%), 1 with encephalitis (3%), and 2 with febrile exanthema (5%). Five of the 39 cases were fatal (13%). Fourteen case-patients resided in rural areas, and 25 in urban and suburban areas, including 7 case-patients who resided in Bucharest. The ages of case-patients ranged from 8 to 76 years with a median age of 45 years. Twenty-four case-patients were males and 15 were females. Dates of onset of illness occurred from May 24 through September 25, with 82% of onset dates occurring in August and September. Limited entomological surveillance failed to detect WN virus. Retrospective sampling of domestic fowl in the vicinity of case-patient residences during the years 1997-2000 demonstrated seroprevalence rates of 7.8%-29%. Limited wild bird surveillance demonstrated seroprevalence rates of 5%-8%. The surveillance data suggest that WN virus persists focally for several years in poorly understood transmission cycles after sporadic introductions or that WN virus is introduced into Romania at relatively high rates, and persists seasonally in small foci.

In this study 1,868 questing Ixodes ricinus ticks (nymphs and adults), collected in six sites from three counties--Giurgiu, Sibiu, and Tulcea--in Romania, were examined by polymerase chain reaction (PCR) followed by reverse line blot (RLB) for detection of Borrelia burgdorferi sensu lato presence. The bacteria were found in 18% of the investigated ticks. The prevalence of infection did not differ significantly between nymphs (19.1%) and adults (15.4%). Three B. burgdorferi s.l. genospecies were detected: B. afzelii (61.1%), B. garinii (31.2%), and B. valaisiana (7.7%). No mixed infections were detected. The highest infection prevalence in nymphs was detected at Cristian (Sibiu County)--22.0%, whereas in adults it was at Comana (Giurgiu County)--19.8%. This preliminary study provides evidence that Lyme disease spirochetes are present in various regions of Romania, and at a relatively high prevalence in their vectors, thus posing a risk of infection to human subjects in the areas infested by ticks.

By integrating the literature data with those derived from personal investigations, the authors present the distribution of the 27 tick species (25 ixodids and 2 argasids) identified up to now in Romania, as well as some aspects regarding their abundance, seasonality and host associations. Altogether, 1439 tick records (of which 256, covering 25 counties, belong to the authors themselves) were georeferenced using EpiMap (an ArcView®-compatible GIS) from CDC's EpiInfo TM software package (v. 3.5.1), on a level 2 LAU (Local Administrative Units) shapefile. The resultant distribution maps and the data on seasonality and host associations may prove to be a useful reference system for subsequent studies on different tick species' distribution, ecology and biology, as well as a predictive tool for human and veterinary medicine, bearing in mind the vectorial role that ticks play in some dangerous diseases for man and livestock.Résumé. En intégrant les données de la littérature avec les résultats d'enquêtes personnelles, les auteurs présentent la distribution des 27 espèces de tiques (25 ixodides et deux argasides) identifiées en Roumanie à ce jour, et quelques données sur l'abondance, la saisonnalité et l'association avec l'hôte. Tous les 1439 enregistrements (dont 256 de 25 comtés, appartiennent aux auteurs) ont été cartographiés, à l'aide de EpiMap (un système d'information géographique compatible avec ArcView®) du logiciel EpiInfo TM (v. 3.5.1) lancée par CDC, sur un fichier vectoriel niveau LAU2 (Unités Administratives Locales). Les cartes de distribution résultées, les données de saisonnalité et celles de l'associaton avec l'hôte, peuvent alors être utiles en tant que système de référence pour des futures études sur la répartition, l'écologie et biologie des différentes espèces de tiques, ainsi que comme outils de prévision pour la médecine humaine et vétérinaire, compte tenu du rôle des tiques dans la transmission des maladies dangereuses pour l'homme et les animaux d'intérêt économique.

We examined 200 questing Ixodes ricinus ticks (nymphs and adults) collected in three different sites in Sibiu County, Romania by polymerase chain reaction (PCR) followed by reverse line blot (RLB) for Borrelia burgdorferi s.l.. We detected the bacteria in 19% of the investigated ticks. The prevalence of infection was higher in nymphs (27%) than in adults (12%). Two B. burgdorferi sensu lato genospecies were detected: B. garinii (20%) and B. afzelii (80%). We did not detect any mixed infections in the investigated ticks. In two of the investigated sites B. burgdorferi prevalence values were comparable (25%), while in the third site the prevalence was lower ( approximately 7%). Our preliminary study provides evidence that Lyme disease spirochetes are present in various areas and at a relatively high prevalence in their vectors, thus posing a risk of infection to human subjects that undergo work or leisure activities in those areas.

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