First evidence of simultaneous occurrence of West Nile virus and Usutu virus neuroinvasive disease in humans in Croatia during the 2013 outbreak.

PURPOSE: We report on first evidence of simultaneous occurrence of West Nile virus (WNV) and Usutu virus (USUV) neuroinvasive infection in humans in Croatia during the transmission season 2013. METHODS: From June to December 2013, a total of 95 patients with clinically suspected WNV infection (WNV fever and neuroinvasive disease) were tested for WNV IgM/IgG antibodies using enzyme-linked immunosorbent assay. Twenty-six reactive samples were further tested by virus neutralization test for confirmation. RESULTS: WNV neuroinvasive infection was confirmed in 20 patients, while in three patients USUV neutralizing antibodies were detected. Cases occurred during the 11-week interval (from 24 July to 07 October 2013). Both WNV and USUV cases were distributed in three north-western Croatian counties. In addition to human cases, recent asymptomatic WNV infection (detection of IgM antibodies) was recorded in 9/3,460 (0.3 %) tested sentinel horses. Infected animals were recorded in two eastern and one north-western county. CONCLUSIONS: Our results indicate co-circulation of WNV and USUV in Croatia. WNV infection could be misdiagnosed with other emerging infectious diseases presenting with neurological symptoms such as USUV infection.

First evidence of bluetongue virus serotype 16 in Croatia.

This study described the first report of BTV-16 in Croatia. Serological evidence occurred in cattle at the end of September and continued during October and November 2004. All positive animals were in the Dubrovnik-Neretva County, a region located in the southernmost part of Croatia. BTV-16 infection was also detected in goats and sheep. Apart from few cases reported in Greece between 1999 and 2000, BTV-16 has never been reported in the Balkanic peninsula before. The BTV strain was isolated from cattle blood samples and typed as BTV-16. When the S5 was sequenced, it showed 100% homology with the BTV-16 vaccine isolate produced by Ondersterpoort Biological Product (SA) and used in Italy during the 2004 BT vaccination campaign. On the other hand no complete homology was found when the same RNA segment sequence was compared with that of the homologous Italian field isolate. As no evidence of livestock movements from Italy was demonstrated, an eolic transmission of the infection through infected Culicoides was hypothesised. According to the local meteostations, in several occasions, during the 2004 summer months, the west-east breeze blew with a speed above 50 km/h from Italy towards the Dubrovnik County. It is concluded that the BTV-16 which infected Croatian livestock was similar to the homologous OBP vaccine isolate and it is likely that it was introduced from Italy into the Southern regions of Croatia through infected Culicoides carried by the wind.

An outbreak of Salmonella Abortusovis abortions in sheep in south Croatia.

The aim of the study was to give an account of the epidemic of abortions in sheep caused by Salmonella enterica ssp. enterica serovar Abortusovis, which occurred in Dalmatia, south Croatia, in winter 2003-2004. Five sheep flocks with rate of abortion ranging from 22% to 38% during the last-third of gestation were examined. Salmonella Abortusovis was isolated from 13 vaginal smears and two fetuses. Direct inoculation was found to be superior to pre-enrichment and enrichment in selective broth for Salmonella Abortusovis isolation. The isolates were biochemically identified, and characterized by serotyping and polymerase chain reaction based on the amplification of the IS200 sequence specific for Salmonella Abortusovis. A fragment of 900 bp was detected in all Salmonella Abortusovis isolates. The sensitivity testing of the isolates, carried out by the disk diffusion method and the determination of the minimal inhibitory concentrations, resulted in a high sensitivity to almost all antimicrobials used. Only two isolates were moderately sensitive to oxytetracycline, whereas one isolate showed resistance to streptomycin. Campylobacter fetus ssp. fetus and Listeria monocytogenes were excluded as causative agents of abortion in sheep by culture testing, and brucellosis, leptospirosis, Q fever and chlamydiosis by serological testing.

Serological evidence of bluetongue and a preliminary entomological study in southern Croatia.

In December 2001, bluetongue (BT) was confirmed serologically by the Croatian Veterinary Institute using the competitive enzyme-linked immunosorbent assay (c-ELISA). Results of the serological testing of blood samples from ruminants in the Dubrovacko-Neretvanska County are presented (3,318 sera of ruminants from 53 herds were examined). In total, 357 bovine sera (178 or 49.9% positive), 1,268 ovine sera (174 or 13.7% positive) and 1,693 caprine sera (270 or 15.9% positive) were tested. Antibodies to BT virus serotype 9 were detected in 212 of the positive sera by serum neutralisation. A preliminary light-trap survey for midges of the Culicoides genus was also performed in the Dubrovacko-Neretvanska County. Fourteen light-trap collections from seven locations were examined and yielded a total of 4,872 Culicoides of which 4,492 (92%) belonged to the Obsoletus Complex (including C. obsoletus and C. scoticus).

Spatial distribution of bluetongue in cattle in southern Croatia in the last quarter of 2002.

The domestic ruminant population of southern Croatia was affected by bluetongue (BT) in late 2001. A sentinel cattle scheme was developed to detect the presence of bluetongue virus (BTV) activity in the domestic cattle population in the protection zone (based on the distribution of BT in 2001: Dubrovacko-Neretvanska County and the southern area of the Splitsko-Dalmatinska County) as well as in the surveillance zone (the northern area of the Splitsko-Dalmatinska County). Twenty-five villages were selected to serve as sentinel locations during the observation period which lasted from 15 September to 15 December 2002. Seroconversion was not detected in cattle in sentinel locations in the surveillance zone. However, in the protection zone, serum antibodies to BTV serotype 9 were detected in eight cattle in five of the ten sentinel locations. Although no clinical case of BT disease was detected in sheep on mainland Croatia in late 2002, BTV activity was present in sentinel cattle in the protection zone. When compared with 2001, spatial distribution of the locations in which cattle seroconverted to BTV-9 in the last quarter of the 2002 suggests a northward trend to the spread of BTV in the cattle of southern Croatia.