Outbreaks In Europe Research Articles

Knowledge, Attitude and Practices towards the Tiger Mosquito Aedes Albopictus. A Questionnaire Based Survey in Lazio Region (Italy) before the 2017 Chikungunya Outbreak.

The invasion of Aedes albopictus has played a major role in the resurgence of mosquito-borne diseases in Italy, generating the two largest chikungunya outbreaks in Europe (2007, 2017). Knowledge, attitude and practice (KAP) are important in order to prevent Aedes-borne disease transmission, yet so far they have not been assessed. To this scope we used multivariate logistic regression to investigate KAP of citizen-to-Aedes ecology and transmitted diseases. Data were collated by a structured questionnaire (18 questions) in 2016. Participants were selected in the Lazio region from members of native populations and two resident communities (RC) originating from the Indian subcontinent where Aedes-transmitted diseases are endemic. Results showed that compared to Italians, RC respondents had a higher knowledge and concern of Aedes-transmitted diseases (Odds Ratio = 2.61 (95%CI: 1.03–6.05); OR = 3.13 (2.15–4.65)) as well as their life cycles (OR = 2.49 (1.75–3.56); OR = 9.04 (6.22–13.66)). In contrast, they perceived a lower nuisance due to the presence of Ae. albopictus (OR = 0.2 (0.13–0.32); OR = 0.55 (0.38–0.78). These findings suggest that citizens in the Lazio region are not prepared to face a potential outbreak of arboviruses and further efforts should be made to increase knowledge, awareness and best practices.

Origin of HAV strains responsible for 2016-2017 outbreak among MSM: Viral phylodynamics in Lazio region.

In Europe HAV infection occurs mainly among specific risk groups, such as consumers of specific food. Sexual transmission of HAV has been demonstrated, particularly among Men-Who-Have-Sex-With-Men (MSM), causing MSM-specific outbreaksin Europe. Here we report a molecular epidemiologic and phylodynamic analysis on HAV sequences in Lazio (central Italy)to identify genetic background and the phylogenetic relations, and test the HAV infection dynamics during a large outbreak through phylodynamic model.Among all HAV sequences found during 2013–2018 in Lazio, low genetic diversity was observed in HAV population in 2016 and 2017, along with high frequenciesVRD_521_2016and RIVM-HAV16-090, suggesting a large expansion event of viral population. The initial expansion of both VRD_521_2016 and RIVM-HAV16-090 clusters dated back to 2012 (95% HPD:2006–2015). During the2016-2017outbreak in Lazio region, the Re peaked around mid-2016, with a value of 1.73 (95% HPD: 1.03–2.37), consistent with incidence trend of AHA cases in Lazio between 2016 and mid-2017. This study showed the magnitude of HAV outbreak in Lazio during 2016–2017, demonstrating the epidemic continuity to MSM-specific outbreak in Europe. The HAV dataset is available on interactive phylodynamic platform https://nextstrain.org to real-time update of future outbreaks.

Estimating the size of undetected cases of the COVID-19 outbreak in Europe: an upper bound estimator

Abstract Background While the number of detected COVID-19 infections are widely available, an understanding of the extent of undetected cases is urgently needed for an effective tackling of the pandemic. The aim of this work is to estimate the true number of COVID-19 (detected and undetected) infections in several European countries. The question being asked is: How many cases have actually occurred? Methods We propose an upper bound estimator under cumulative data distributions, in an open population, based on a day-wise estimator that allows for heterogeneity. The estimator is data-driven and can be easily computed from the distributions of daily cases and deaths. Uncertainty surrounding the estimates is obtained using bootstrap methods. Results We focus on the ratio of the total estimated cases to the observed cases at April 17th. Differences arise at the country level, and we get estimates ranging from the 3.93 times of Norway to the 7.94 times of France. Accurate estimates are obtained, as bootstrap-based intervals are rather narrow. Conclusions Many parametric or semi-parametric models have been developed to estimate the population size from aggregated counts leading to an approximation of the missed population and/or to the estimate of the threshold under which the number of missed people cannot fall (i.e. a lower bound). Here, we provide a methodological contribution introducing an upper bound estimator and provide reliable estimates on the dark number, i.e. how many undetected cases are going around for several European countries, where the epidemic spreads differently.

West Nile virus in Europe: after action reviews of preparedness and response to the 2018 transmission season in Italy, Slovenia, Serbia and Greece

BackgroundAfter Action Reviews (AAR) with a One Health perspective were performed in Slovenia, Italy, Serbia and Greece following a severe West Nile virus (WNV) transmission season in 2018. A protocol combining traditional techniques and organizational process analysis was developed and then implemented in each country.ResultsIn 2018, response to the unusually intense transmission season of WNV in Slovenia, Italy, Serbia and Greece took place through routine response mechanisms. None of the four countries declared a national or subnational emergency. We found a very strong consensus on the strengths identified in responding to this event. All countries indicated the availability of One Health Plans for surveillance and response; very high laboratory diagnostic capacity in the human, veterinary and entomology sectors and strong inter-sectoral collaboration with strong commitment of engaged institutions as critical in the management of the event. Finally, countries implementing One Health surveillance for WNV (in terms of early warning and early activation of prevention measures) consistently reported a positive impact on their activities, in particular when combining mosquito and bird surveillance with surveillance of cases in humans and equids. Recurring priority areas for improvement included: increasing knowledge on vector-control measures, ensuring the sustainability of vector monitoring and surveillance, and improving capacity to manage media pressure.ConclusionsThe AARs presented here demonstrate the benefit of cross-sectoral and cross-disciplinary approaches to preparedness for West Nile virus outbreaks in Europe. In the coming years, priorities include fostering and strengthening arrangements that: enable coordinated One Health surveillance and response during WNV transmission seasons; ensure adequate laboratory capacities; strengthen risk communication; and fund longer-term research to address the knowledge gaps identified in this study.

Equine influenza vaccination as reported by horse owners and factors influencing their decision to vaccinate or not

BackgroundEquine influenza virus is a highly contagious respiratory pathogen that causes pyrexia, anorexia, lethargy and coughing in immunologically naïve horses. Vaccines against equine influenza are available and vaccination is mandatory for horses that participate in affiliated competitions, but this group forms a small proportion of the total horse population. The aims of this study were to: i) identify the equine influenza vaccination rate as reported in 2016 by horse owners in the United Kingdom (UK); ii) examine the demographics of owners and horses which were associated with significantly lower influenza vaccination rates and iii) explore factors that influence horse owners’ decisions around influenza vaccine uptake. ResultsResponses from 4837 UK horse owners who were responsible for 10,501 horses were analysed. An overall equine influenza vaccination rate of 80% (8385/10501) was reported. Several owner demographic characteristics were associated with significantly lower (p<0.05) reported equine influenza vaccination rates including: some geographical locations, increasing horse owner age, annual household income of less that £15,000 and owning more than one horse. Horse-related features which were associated with significantly lower reported equine influenza vaccination rates included age ranges of <4 years and > 20 years, use as a companion or breeding animal or leaving their home premises either never or at most once a year. The most common reasons cited for failing to vaccinate horses was no competition activity, lack of exposure to influenza and expense of vaccines. In contrast, the most common underlying reasons given by horse owners who vaccinated their horse were protection of the individual horse against disease, veterinary advice and to protect the national herd. Owners of vaccinated horses had less previous experience of an influenza outbreak or adverse reaction to vaccination compared with owners of unvaccinated horses. ConclusionsThis study documented a high rate of equine influenza vaccination as reported by owners in a substantial number of horses in the UK, but this does not reflect the level of protection. Sub-populations of horses which were less likely to be vaccinated and the factors that influence each owner’s decision around vaccination of their horses against equine influenza were identified, but may alter following the 2019 European influenza outbreak. This information may nevertheless help veterinary surgeons identify “at-risk” patients and communicate more personalised advice to their horse-owning clients. It may also influence educational campaigns about equine influenza directed to horse owners, which aim to improve uptake of vaccination against this pathogen.

Time Course of COVID-19 Cases in Austria.

COVID-19 is an infectious disease caused by a novel coronavirus, which first appeared in China in late 2019, and reached pandemic distribution in early 2020. The first major outbreak in Europe occurred in Northern Italy where it spread to neighboring countries, notably to Austria, where skiing resorts served as a main transmission hub. Soon, the Austrian government introduced strict measures to curb the spread of the virus. Using publicly available data, we assessed the efficiency of the governmental measures. We assumed an average incubation period of one week and an average duration of infectivity of 10 days. One week after the introduction of strict measures, the increase in daily new cases was reversed, and the reproduction number dropped. The crude estimates tended to overestimate the reproduction rate in the early phase. Publicly available data provide a first estimate about the effectiveness of public health measures. However, more data are needed for an unbiased assessment.

Cooking chicken at home: Common or recommended approaches to judge doneness may not assure sufficient inactivation of pathogens.

About one third of foodborne illness outbreaks in Europe are acquired in the home and eating undercooked poultry is among consumption practices associated with illness. The aim of this study was to investigate whether actual and recommended practices for monitoring chicken doneness are safe. Seventy-five European households from five European countries were interviewed and videoed while cooking chicken in their private kitchens, including young single men, families with infants/in pregnancy and elderly over seventy years. A cross-national web-survey collected cooking practices for chicken from 3969 households. In a laboratory kitchen, chicken breast fillets were injected with cocktails of Salmonella and Campylobacter and cooked to core temperatures between 55 and 70°C. Microbial survival in the core and surface of the meat were determined. In a parallel experiment, core colour, colour of juice and texture were recorded. Finally, a range of cooking thermometers from the consumer market were evaluated. The field study identified nine practical approaches for deciding if the chicken was properly cooked. Among these, checking the colour of the meat was commonly used and perceived as a way of mitigating risks among the consumers. Meanwhile, chicken was perceived as hedonically vulnerable to long cooking time. The quantitative survey revealed that households prevalently check cooking status from the inside colour (49.6%) and/or inside texture (39.2%) of the meat. Young men rely more often on the outside colour of the meat (34.7%) and less often on the juices (16.5%) than the elderly (>65 years old; 25.8% and 24.6%, respectively). The lab study showed that colour change of chicken meat happened below 60°C, corresponding to less than 3 log reduction of Salmonella and Campylobacter. At a core temperature of 70°C, pathogens survived on the fillet surface not in contact with the frying pan. No correlation between meat texture and microbial inactivation was found. A minority of respondents used a food thermometer, and a challenge with cooking thermometers for home use was long response time. In conclusion, the recommendations from the authorities on monitoring doneness of chicken and current consumer practices do not ensure reduction of pathogens to safe levels. For the domestic cook, determining doneness is both a question of avoiding potential harm and achieving a pleasurable meal. It is discussed how lack of an easy “rule-of-thumb” or tools to check safe cooking at consumer level, as well as national differences in contamination levels, food culture and economy make it difficult to develop international recommendations that are both safe and easily implemented.

Clinical characteristics of coronavirus disease (COVID-19) early findings from a teaching hospital in Pavia, North Italy, 21 to 28 February 2020.

We describe clinical characteristics, treatments and outcomes of 44 Caucasian patients with coronavirus disease (COVID-19) at a single hospital in Pavia, Italy, from 21–28 February 2020, at the beginning of the outbreak in Europe. Seventeen patients developed severe disease, two died. After a median of 6 days, 14 patients were discharged from hospital. Predictors of lower odds of discharge were age > 65 years, antiviral treatment and for severe disease, lactate dehydrogenase > 300 mg/dL.

Human West Nile Virus Lineage 2 Infection: Epidemiological, Clinical, and Virological Findings.

West Nile virus (WNV) lineage 2 is expanding and causing large outbreaks in Europe. In this study, we analyzed the epidemiological, clinical, and virological features of WNV lineage 2 infection during the large outbreak that occurred in northern Italy in 2018. The study population included 86 patients with neuroinvasive disease (WNND), 307 with fever (WNF), and 34 blood donors. Phylogenetic analysis of WNV full genome sequences from patients’ samples showed that the virus belonged to the widespread central/southern European clade of WNV lineage 2 and was circulating in the area at least since 2014. The incidence of WNND and WNF progressively increased with age and was higher in males than in females. Among WNND patients, the case fatality rate was 22%. About 70% of blood donors reported symptoms during follow-up. Within the first week after symptom onset, WNV RNA was detectable in the blood or urine of 80% of patients, while 20% and 40% of WNND and WNF patients, respectively, were WNV IgM-seronegative. In CSF samples of WNND patients, WNV RNA was typically detectable when WNV IgM antibodies were absent. Blunted or no WNV IgM response and high WNV IgG levels were observed in seven patients with previous flavivirus immunity.

Examining the Direct and Indirect Effects of Climatic Variables on Plague Dynamics

Climate change can influence infectious disease dynamics both directly, by affecting the disease ecology, and indirectly, through altering economic systems. However, despite that climate-driven human plague dynamics have been extensively studied in recent years, little is known about the relative importance of the direct and indirect effects of climate change on plague outbreak. By using Structural Equation Modeling, we estimated the direct influence of climate change on human plague dynamics and the impact of climate-driven economic change on human plague outbreak. After studying human plague outbreak in Europe during AD1347–1760, we detected no direct climatic effect on plague dynamics; instead, all of the climatic impacts on plague dynamics were indirect, and were operationalized via economic changes. Through a series of sensitivity checks, we further proved that temperature-induced economic changes drove plague dynamics during cold and wet periods, while precipitation-induced economic changes drove plague dynamics during the cold periods. Our results suggest that we should not dismiss the role of economic systems when examining how climate change altered plague dynamics in human history.

Comparing South Korea and Italy's healthcare systems and initiatives to combat COVID-19.

ABSTRACTItaly and South Korea have two distinctly different healthcare systems, causing them to respond to public health crises such as the COVID-19 pandemic in markedly different ways. Differences exist in medical education for both countries, allowing South Korean medical graduates to have a more holistic education in comparison to their Italian counterparts, who specialize in medical education earlier on. Additionally, there are fewer South Korean physicians per 1000 people in South Korea compared to Italian physicians per 1000 people in Italy. However, both countries have a national healthcare system with universal healthcare coverage. Despite this underlying similarity, the two countries addressed COVID-19 in nearly opposite manners. South Korea employed technology and the holistic education of its physician community, despite having a smaller proportion of physicians in society, to its advantage by implementing efficacious drive-through centers that test suspected individuals rapidly and with little to no contact with healthcare staff, decreasing the possibility of transmission of COVID-19. Conversely, Italy is presently considered the epicenter of the outbreak in Europe and has recorded the highest death toll of any country outside of mainland China. This is partially due to the reactionary nature of Italy’s public health measures compared to South Korea’s proactive response. The different healthcare responses of South Korea and Italy can inform decisions made by public health bodies in other countries, especially in countries across the Americas, which can selectively adopt policies that have worked in curtailing the spread of COVID-19 and learn from mistakes made by both countries.

Estimating the Case Fatality Rate for COVID-19: A Markov Model Application

Europe has became the new epicentre of the COVID-19 pandemic, according to the WHO on 13th March 2020. Sums and ratios of death and confirmed cases were reported daily, however, such statistics vary significantly by country and it is therefore challenging to understand and measure the risk and severity of the novel disease. Prior to the European outbreak, the COVID-19 virus infected more than 80,000 people in China since late 2019 and took the life of several thousands during the past few months. In this paper, a 3-state model Markov model is applied on the data from China to study the dynamics of the disease and the impact of containment strategies. The long-run stable transition probability obtained from the Markov model provides a convenient approach to estimate the case fatality rate of the COVID-19. Also, the estimated life expectancy give a reasonable estimate of time between first symptom and death. Considering the containment strategy implemented in China, the analysis is done for Hubei province and the rest of China respectively. Comparison of daily estimated results over the whole observation period highlight the impact of the strategy while supporting the measures and controls in place. The proposed Markov model produce reasonable and intuitive estimates that help to measure the virulence of the disease and understand the prevalence overtime. While uncertainty persists as the pandemic goes on, our results show that the Markov approaches provide a useful tool for prognosis and epidemic control.

CORONAVIRUS EPIDEMIC OUTBREAK IN EUROPE. COMPARISON WITH THE DYNAMICS IN MAINLAND CHINA

Unfortunately, we can already talk about the coronavirus COVID-19 epidemic outbreak in Europe. This is evidenced by the large number of confirmed cases in Italy, registered in recent days. Prospects for epidemic development can be estimated from the accumulated information on the cases growth rate in mainland China. Comparison with the data on China will allow us to evaluate the effectiveness of quarantine measures and the level of individual protection, since we are dealing with the same pathogen. This short message provides a simple method of data comparison that can be useful for both governmental organizations and anyone. The first data points for the epidemic in Italy corresponded to the epidemic dynamics in mainland China. Fortunately, the growth rate of coronavirus patients in Europe has not become as threatening since February 29, 2020.

Hindcasting the First Tornado Forecast in Europe: 25 June 1967

Abstract The tornado outbreak of 24–25 June 1967 was the most damaging in the history of western Europe, producing 7 F2–F5 tornadoes, 232 injuries, and 15 fatalities across France, Belgium, and the Netherlands. Following tornadoes in France on 24 June, the Royal Netherlands Meteorological Institute (KNMI) issued a tornado forecast for 25 June, which became the first ever—and first verified—tornado forecast in Europe. Fifty-two years later, tornadoes are still not usually forecast by most European national meteorological services, and a pan-European counterpart to the NOAA/NWS/Storm Prediction Center (SPC) does not exist to provide convective outlook guidance; yet, tornadoes remain an extant threat. This article asks, “What would a modern-day forecast of the 24–25 June 1967 outbreak look like?” To answer this question, a model simulation of the event is used in three ways: 20-km grid-spacing output to produce a SPC-style convective outlook provided by the European Storm Forecast Experiment (ESTOFEX), 800-m grid-spacing output to analyze simulated reflectivity and surface winds in a nowcasting analog, and 800-m grid-spacing output to produce storm-total footprints of updraft helicity maxima to compare to observed tornado tracks. The model simulates a large supercell on 24 June and weaker embedded mesocyclones on 25 June forming along a stationary front, allowing the ESTOFEX outlooks to correctly identify the threat. Updraft helicity footprints indicate multiple mesocyclones on both days within 40–50 km and 3–4 h of observed tornado tracks, demonstrating the ability to hindcast a large European tornado outbreak.

Molecular detection and phylogenetic analysis of lumpy skin disease virus from outbreaks in Uganda 2017\u20132018

BackgroundLumpy skin disease (LSD) is an infectious viral disease of cattle caused by a Capripoxvirus. LSD has substantial economic implications, with infection resulting in permanent damage to the skin of affected animals which lowers their commercial value. In Uganda, LSD is endemic and cases of the disease are frequently reported to government authorities. This study was undertaken to molecularly characterize lumpy skin disease virus (LSDV) strains that have been circulating in Uganda between 2017 and 2018. Secondly, the study aimed to determine the phylogenetic relatedness of Ugandan LSDV sequences with published sequences, available in GenBank.ResultsA total of 7 blood samples and 16 skin nodule biopsies were screened for LSDV using PCR to confirm presence of LSDV nucleic acids. PCR positive samples were then characterised by amplifying the GPCR gene. These amplified genes were sequenced and phylogenetic trees were constructed. Out of the 23 samples analysed, 15 were positive for LSDV by PCR (65.2%). The LSDV GPCR sequences analysed contained the unique signatures of LSDV (A11, T12, T34, S99, and P199) which further confirmed their identity. Sequence comparison with vaccine strains revealed a 12 bp deletion unique to Ugandan outbreak strains. Phylogenetic analysis indicated that the LSDV sequences from this study clustered closely with sequences from neighboring East African countries and with LSDV strains from recent outbreaks in Europe. It was noted that the sequence diversity amongst LSDV strains from Africa was higher than diversity from Eurasia.ConclusionThe LSDV strains circulating in Uganda were closely related with sequences from neighboring African countries and from Eurasia. Comparison of the GPCR gene showed that outbreak strains differed from vaccine strains. This information is necessary to understand LSDV molecular epidemiology and to contribute knowledge towards the development of control strategies by the Government of Uganda.

Coxsackievirus A10 causing hand-foot-and-mouth disease in Uruguay

We report for the first time in South America an HFMD case associated with Coxsackievirus A10. The viral strain belongs to a lineage involved in important European outbreaks and probably entered Uruguay after 2017 with a Greek origin. These findings call for strengthening the regional surveillance of HFMD.

Emergence of a Plant Pathogen in Europe Associated with Multiple Intercontinental Introductions.

Pathogen introductions have led to numerous disease outbreaks in naive regions of the globe. The plant pathogen Xylella fastidiosa has been associated with various recent epidemics in Europe affecting agricultural crops, such as almond, grapevine, and olive, but also endemic species occurring in natural forest landscapes and ornamental plants. We compared whole-genome sequences of X. fastidiosa subspecies multiplex from America and strains associated with recent outbreaks in southern Europe to infer their likely origins and paths of introduction within and between the two continents. Phylogenetic analyses indicated multiple introductions of X. fastidiosa subspecies multiplex into Italy, Spain, and France, most of which emerged from a clade with limited genetic diversity with a likely origin in California, USA. The limited genetic diversity observed in X. fastidiosa subspecies multiplex strains originating from California is likely due to the clade itself being an introduction from X. fastidiosa subspecies multiplex populations in the southeastern United States, where this subspecies is most likely endemic. Despite the genetic diversity found in some areas in Europe, there was no clear evidence of recombination occurring among introduced X. fastidiosa strains in Europe. Sequence type taxonomy, based on multilocus sequence typing (MLST), was shown, at least in one case, to not lead to monophyletic clades of this pathogen; whole-genome sequence data were more informative in resolving the history of introductions than MLST data. Although additional data are necessary to carefully tease out the paths of these recent dispersal events, our results indicate that whole-genome sequence data should be considered when developing management strategies for X. fastidiosa outbreaks.IMPORTANCEXylella fastidiosa is an economically important plant-pathogenic bacterium that has emerged as a pathogen of global importance associated with a devastating epidemic in olive trees in Italy associated with X. fastidiosa subspecies pauca and other outbreaks in Europe, such as X. fastidiosa subspecies fastidiosa and X. fastidiosa subspecies multiplex in Spain and X. fastidiosa subspecies multiplex in France. We present evidence of multiple introductions of X. fastidiosa subspecies multiplex, likely from the United States, into Spain, Italy, and France. These introductions illustrate the risks associated with the commercial trade of plant material at global scales and the need to develop effective policy to limit the likelihood of pathogen pollution into naive regions. Our study demonstrates the need to utilize whole-genome sequence data to study X. fastidiosa introductions at outbreak stages, since a limited number of genetic markers does not provide sufficient phylogenetic resolution to determine dispersal paths or relationships among strains that are of biological and quarantine relevance.

Leishmania sp. detection and blood-feeding behaviour of Sergentomyia minuta collected in the human leishmaniasis focus of southwestern Madrid, Spain (2012-2017).

Phlebotomine sand flies are the only known vectors of Leishmania spp. protozoan which causes leishmaniasis in 98 countries. In Spain, 11 sand fly species are described, but only Phlebotomus perniciosus and Phlebotomus ariasi are proven vectors of the disease. On the other hand, Sergentomyia minuta is one of the most abundant and ubiquitous sand flies in this territory, although scarce information is available about this species. Sand flies from this genus are known for their preference to feed on cold-blooded animals and are traditionally involved in the transmission of reptile Leishmania. However, studies have suggested that Sergentomyia spp. could be implicated in the transmission of human pathogenic Leishmania. This study analyses blood meal preferences and Leishmania sp. infection of S.minuta sand flies from the largest human leishmaniasis outbreak in Europe. Sand flies were collected during entomological surveillance carries out from 2012 to 2017 in the active season of these dipterans, from May to October. Molecular detection of Leishmania spp. showed 68 positive specimens of S.minuta out of 377 (18%). The analysis of blood meal preferences by amplification of 359bp fragment of cytochrome b gene revealed that blood preference of S.minuta is not only limited to reptiles, but they also feed on mammals, including humans. Results suggest the presence of a Leishmania sp., related to Leishmania tarentolae, cycle in S.minuta from the studied area. Although there is no evidence about its incrimination in the L.infantum transmission more investigation is needed to elucidate the intravectorial cycle of Leishmania spp. in S.minuta sand flies, their feeding behaviour and their potential contribution in Leishmania spp. epidemiology in the country.

Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues.

Xylella fastidiosa (Xf) is an insect-borne bacterium confined to the xylem vessels of plants. This plant pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with different but overlapping host ranges have been described, but only three subspecies are widely accepted, namely subspecies fastidiosa, multiplex, and pauca. Initially limited to the Americas, Xf has been detected in Europe since 2013. As management of X. fastidiosa outbreaks in Europe depends on the identification of the subspecies, accurate determination of the subspecies in infected plants as early as possible is of major interest. Thus, we developed various tetraplex and triplex quantitative PCR (qPCR) assays for X. fastidiosa detection and subspecies identification in planta in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on k-mers, to detect specific signatures of the species and subspecies from a data set of 58 genome sequences representative of X. fastidiosa diversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different plant species spiked with strains of the different subspecies of X. fastidiosa, and on samples from various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity than the reference protocol and allowed X. fastidiosa detection in all spiked matrices up to 103 cells.ml−1. Moreover, mix infections of two to three subspecies could be detected in the same sample with tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies identification when the current method based on multilocus sequence typing failed. The qPCR assays described here are robust and modular tools that are efficient for differentiating X. fastidiosa subspecies directly in plant samples.

Major Outbreaks in the Nineteenth Century Shaped Grape Phylloxera Contemporary Genetic Structure in Europe

Grape phylloxera is native to North America, where Vitis spp. acquired different mechanisms of resistance to leaf and root attack. Its appearance in European vineyards at the beginning of the 1860s, where the phylloxera-susceptible grapevine species V. vinifera L. is majorly cultivated, caused the devastation of a great number of vineyards, generating a deep crisis in the European wine production and trade industries. However, the origin and genetic structure of this pest across European vineyards still remain controversial and uncertain. Herein, we analysed the genetic structure of 1173 grape phylloxera individuals collected from 100 locations across eight European countries. Structure and phylogenetic analyses show that contemporary grape phylloxera populations in Europe are the result of at least two independent introductions from the native range that mirrors the historical records that also suggest two major outbreaks in Europe. The comparative analysis with samples from the native range trace back one of these two genetic groups to plants imported from the North East coast of North America, where the American species V. riparia and V. labrusca dominate. This study clarifies the level of genetic diversity of grape phylloxera in Europe and provides relevant information to resolve previous controversy about its origin.