Progressive Invasion of Aedes albopictus in Northern Spain in The Period 2013-2018 and A Possible Association with the Increase in Insect Bites.

1) Background: Aedes albopictus has rapidly expanded throughout Europe, becoming a public health concern in the Mediterranean Basin. 2) Methods: Following the detection of Ae. albopictus in the southwestern French region of Aquitaine in 2012, an entomological surveillance programme was implemented in the Basque Country (Northern Spain) in 2013. 3) Results: Ae. albopictus eggs were first detected in 2014 in a transited parking area in the northeastern sampling point, 22 km away from the nearest French site with recorded presence of tiger mosquito. At this site, eggs were found throughout the study (2014-2018). Other western and southern municipalities became positive in 2017 and 2018. Ae. albopictus adults were first captured in 2018 by aspiration of the vegetation in an area where eggs had been detected since 2015, suggesting a progressive establishment of a self-sustained population. Incidence of insect bites in humans was roughly constant over the study period except for a significant increase in 2018 in the Health County where eggs had been detected since 2014. Densities of Ae. albopictus eggs in positive areas remained at similar levels over the years. 4) Conclusion: Multiple approaches and standardized methods are necessary to successfully control this vector.

A Q fever outbreak associated to courier transport of pets.

On August 3rd, 2017, a Q fever outbreak alert was issued at a courier company that in addition to urgent freight transport offered pet delivery services. The epidemiological investigation set the exposition period between June 1 and August 8. In this period, 180 workers from two operational platforms for parcel distribution located in two provinces of the Basque Country (Bizkaia and Araba) were exposed; 64 filled a questionnaire and provided blood samples for serological testing, resulting in 10 confirmed cases (15.6%) and six (9.4%) probable cases. Nine workers (8 confirmed and 1 probable) showed Q fever symptoms, including pneumonia (five cases), and required medical care services, including one hospital admission. The attack rate was 25% (16/64), being higher among workers that visited the Bizkaia platform. This suggested that the origin of the outbreak was in the Bizkaia platform, where animals in transit waited at a pet holding site until being moved to their destination. Environmental samples consisting on 19 surface dust and two aerosol samples were collected at the Bizkaia platform to investigate the presence of C. burnetti DNA. All dust samples were positive by real time PCR, the lowest Ct values being found in dust collected at the pet holding facilities, and therefore suggesting that contamination originated at the pet holding site. The genotype identified in dust was SNP1/MST13, one of the most commonly identified genotypes in goats and sheep in the Basque Country. During the exposure period, two deliveries of miniature goats were made, of which only one could be investigated and tested negative. Although the contamination source could not be unequivocally identified, transport of ruminants was banned at the company, and Q fever was included among the occupational-associated health risks.

Stable levels of Coxiella burnetii prevalence in dairy sheep flocks but changes in genotype distribution after a 10-year period in northern Spain.

Bulk tank milk (BTM) samples were collected from 81 sheep flocks in the Basque Country, Spain, in 2015 and were analysed for antibodies against Coxiella burnetii by ELISA and for C. burnetii DNA by real-time PCR. Thirty-two percent of the flocks had BTM antibodies against C. burnetii. Presence of C. burnetii DNA in BTM was detected in 23% of the flocks, suggesting recent C. burnetii infections. Retrospective data of BTM samples obtained from 154 sheep flocks investigated in 2005 in the same geographic area were compiled to assess temporal changes in C. burnetii infection. The overall percentage of infected sheep flocks did not significantly change after the 10-year period. Among the 46 flocks sampled in both periods, 11 flocks that were negative in 2005 were positive in 2015, 18 maintained their initial status (positive or negative), and 17 positive flocks were negative in 2015. These findings indicate that C. burnetii infection is a dynamic process in dairy sheep in northern Spain. Single nucleotide polymorphism (SNP) genotyping of positive samples identified three genotypes, SNP1 being the most prevalent in 2015 and SNP8 in 2005; SNP4 was only detected once in 2005. These results suggest possible changes in the pattern of genotype infection over time.

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain.

BACKGROUND: Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. RESULTS: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. CONCLUSIONS: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.

Independent lineage of lymphocytic choriomeningitis virus in wood mice (Apodemus sylvaticus), Spain.

To clarify the presence of lymphocytic choriomeningitis virus (LCMV) in Spain, we examined blood and tissue specimens from 866 small mammals. LCMV RNA was detected in 3 of 694 wood mice (Apodemus sylvaticus). Phylogenetic analyses suggest that the strains constitute a new evolutionary lineage. LCMV antibodies were detected in 4 of 10 rodent species tested.