Genetic diversity and population structure of Culex modestus across Europe: does recent appearance in the United Kingdom reveal a tendency for geographical spread?

In mainland Europe, the mosquito species Culex modestus Ficalbi (1890) is a bridge vector for West Nile virus (WNV) from its natural bird-mosquito cycle to mammals. The present study assessed the genetic diversity of Cx. modestus, as well as related Culex species, using the mitochondrial COI DNA barcoding region and compared this with the population structure across Europe. A haplotype network was mapped to determine genealogical relationships among specimens. The intraspecific genetic diversity within individual Culex species was below 2%, whereas the interspecific genetic divergence varied from 2.99% to 13.74%. In total, 76 haplotypes were identified among 198 sequences. A median-joining network determined from 198 COI sequences identified two major lineages that were separated by at least four mutation steps. A high level of intraspecific genetic diversity was not detected in Cx. modestus in samples submitted from different European populations, which indicates that morphologically identified specimens represent a single species and not a species complex. Therefore, it is deduced that different populations of Cx. modestus will show a similar potential to transmit WNV, lending support to concerns that the population present in southeast England represents a risk of transmission to humans.

Comparative Risk Analysis of Two Culicoides-Borne Diseases in Horses: Equine Encephalosis More Likely to Enter France than African Horse Sickness.

African horse sickness (AHS) and equine encephalosis (EE) are Culicoides-borne viral diseases that could have the potential to spread across Europe if introduced, thus being potential threats for the European equine industry. Both share similar epidemiology, transmission patterns and geographical distribution. Using stochastic spatiotemporal models of virus entry, we assessed and compared the probabilities of both viruses entering France via two pathways: importation of live-infected animals or importation of infected vectors. Analyses were performed for three consecutive years (2010-2012). Seasonal and regional differences in virus entry probabilities were the same for both diseases. However, the probability of EE entry was much higher than the probability of AHS entry. Interestingly, the most likely entry route differed between AHS and EE: AHS has a higher probability to enter through an infected vector and EE has a higher probability to enter through an infectious host. Consequently, different effective protective measures were identified by 'what-if' scenarios for the two diseases. The implementation of vector protection on all animals (equine and bovine) coming from low-risk regions before their importation was the most effective in reducing the probability of AHS entry. On the other hand, the most significant reduction in the probability of EE entry was obtained by the implementation of quarantine before import for horses coming from both EU and non-EU countries. The developed models can be useful to implement risk-based surveillance.

Rapid spread of Schmallenberg virus-infected biting midges (Culicoides spp.) across Denmark in 2012.

Detection of Schmallenberg virus RNA, using real-time RT-PCR, in biting midges (Culicoides spp.) caught at 48 locations in 2011 and four well-separated farms during 2012 in Denmark, revealed a remarkably rapid spread of virus-infected midges across the country. During 2012, some 213 pools of obsoletus group midges (10 specimens per pool) were examined, and of these, 35 of the 174 parous pools were Schmallenberg virus RNA positive and 11 of them were positive in the heads. Culicoides species-specific PCRs identified both C. obsoletus and C. dewulfi as vectors of Schmallenberg virus.

Dynamic changes in antibody levels as an early warning of Salmonella Dublin in bovine dairy herds.

Salmonella Dublin is a bacterium that causes disease and production losses in cattle herds. In Denmark, a surveillance and control program was initiated in 2002 to monitor and reduce the prevalence of Salmonella Dublin. In dairy herds, the surveillance includes herd classification based on bulk tank milk measurements of antibodies directed against Salmonella Dublin at 3-mo intervals. In this study, an "alarm herd" concept, based on the dynamic progression of these repeated measurements, was formulated such that it contains predictive power for Salmonella Dublin herd classification change from "likely free of infection" to "likely infected" in the following quarter of the year, thus warning the farmer 3 mo earlier than the present system. The alarm herd concept was defined through aberrations from a stable development over time of antibody levels. For suitable parameter choices, alarm herd status was a positive predictor for Salmonella Dublin status change in dairy herds, in that alarm herds had a higher risk of changing status in the following quarter compared with nonalarm herds. This was despite the fact that both alarm and nonalarm herds had antibody levels that did not indicate the herds being "likely infected" according to the existing classification system in the present quarter. The alarm herd concept can be used as a new early warning element in the existing surveillance program. Additionally, to improve accuracy of herd classification, the alarm herd concept could be incorporated into a model including other known risk factors for change in herd classification. Furthermore, the model could be extended to other diseases monitored in similar ways.

First detection of tick-borne "Candidatus Neoehrlichia mikurensis" in Denmark 2011.

This is the first reporting of the tick-borne zoonotic bacterium "Candidatus Neoehrlichia mikurensis" in Denmark. A total of 2,625 Ixodes ricinus ticks from 58 locations in Denmark were collected and analysed for "Ca. Neoehrlichia mikurensis". A nested PCR revealed the presence of the bacterium at three geographically separate locations, which indicates that it is widely established in ticks.

First identification of tick-borne encephalitis in Denmark outside of Bornholm, August 2009.

The incidence of tick-borne encephalitis (TBE) in Scandinavia is increasing and spreading geographically. Following two clinical cases of TBE hospitalised after tick bites in northern Zealand, Denmark, specific IgM and IgG antibodies against tick-borne encephalitis virus (TBEV) were demonstrated in acute serum samples of these patients. TBEV was identified by RT-PCR in ticks collected from the same location. This is the first report of TBEV in Ixodes ricinus leading to clinical cases in Denmark outside of Bornholm island.

Spatial scan statistics to assess sampling strategy of antimicrobial resistance monitoring program.

The collection and analysis of data on antimicrobial resistance in human and animal populations are important for establishing a baseline of the occurrence of resistance and for determining trends over time. In animals, targeted monitoring with a stratified sampling plan is normally used. However, to our knowledge it has not previously been analyzed whether animals have a random chance of being sampled by these programs, regardless of their spatial distribution. In this study, we used spatial scan statistics, based on a Poisson model, as a tool to evaluate the geographical distribution of animals sampled by the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP), by identifying spatial clusters of samples and detecting areas with significantly high or low sampling rates. These analyses were performed for each year and for the total 5-year study period for all collected and susceptibility tested pig samples in Denmark between 2002 and 2006. For the yearly analysis, both high and low sampling rates areas were significant, with two clusters in 2002 (relative risk [RR]: 2.91, p < 0.01 and RR: 0.06, p < 0.01) and one in 2005 (RR: < 0.01, p < 0.01). For the 5-year analysis, one high sampling rate cluster was detected (RR: 2.56, p = 0.01). These findings allowed subsequent investigation to clarify the source of the sampling clusters. Overall, the detected clusters presented different spatial locations over the years and we can conclude that they were more associated to temporary sampling problems than to a failure in the sampling strategy adopted by the monitoring program. Spatial scan statistics proved to be a useful tool for assessment of the randomness of the sampling distribution, which is important when evaluating the validity of the results obtained by an antimicrobial monitoring program.

Growth inhibitory factors in bovine faeces impairs detection of Salmonella Dublin by conventional culture procedure.

AIMS: To analyse the relative importance of different biological and technical factors on the analytical sensitivity of conventional culture methods for detection of Salmonella Dublin in cattle faeces. METHODS AND RESULTS: Faeces samples collected from six adult bovines from different salmonella-negative herds were split into subpools and spiked with three strains of S. Dublin at a concentration level of c. 10 CFU g(-1) faeces. Each of the 18 strain-pools was divided into two sets of triplicates of four volumes of faecal matter (1, 5, 10 and 25 g). The two sets were pre-enriched with and without novobiocin, followed by combinations of culture media (three types) and selective media (two types). The sensitivity of each combination and sources of variation in detection were determined by a generalized linear mixed model using a split-plot design. CONCLUSIONS: Biological factors, such as faecal origin and S. Dublin strain influenced the sensitivity more than technical factors. Overall, the modified semi-solid Rappaport Vassiliadis (MSRV)-culture medium had the most reliable detection capability, whereas detection with selenite cystine broth and Mueller Kauffman tetrathionate broth combinations varied more in sensitivity and rarely reached the same level of detection as MSRV in this experiment. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that for MSRV-culture medium and xylose lysine decarboxylase agar as the indicative medium, the sensitivity of the faecal culture method may be improved by focusing on the strain variations and the ecology of the faecal sample. Detailed investigation of the faecal flora (pathogens and normal flora) and the interaction with chemical factors may result in developing an improved method for detection of S. Dublin.

Effect of topography on the risk of malaria infection in the Usambara Mountains, Tanzania.

We investigated whether the risk of infection with malaria parasites was related to topography in the Usambara Mountains, Tanzania. Clinical surveys were carried out in seven villages, situated at altitudes from 300 m to 1650 m. Each village was mapped and incorporated into a Digital Terrain Model. Univariate analysis showed that the risk of splenomegaly declined with increasing altitude and with decreasing potential for water to accumulate. Logistic regression showed that altitude alone could correctly predict 73% of households where an occupant had an enlarged spleen or not. The inclusion of land where water is likely to accumulate within 400 m of each household increased the accuracy of the overall model slightly to 76%, but significantly improved predictions between 1000 m and 1200 m, where malaria is unstable, and likely to be epidemic. This novel approach illustrates how topography could help identify local areas prone to epidemics in the African highlands.

Relationship between altitude and intensity of malaria transmission in the Usambara Mountains, Tanzania.

There is a consensus that malaria is a growing problem in African highlands. This is surprising because many parts of the highlands were considered too cold to support transmission. In this report, we examined how transmission of Plasmodium falciparum in six villages changed along an altitude transect in the Usambara Mountains, Tanzania, from 300 m to 1700 m. Routine entomological collections were made using spray catches and light traps for 15 mo. Direct estimates of entomological inoculation rates and indirect estimates of vectorial capacity suggested a >1000-fold reduction in transmission intensity between the holoendemic lowland and the hypoendemic highland plateau. Lowland transmission was perennial with a significant peak in the cool season after the long rains in May, when vectors densities were high. In the highlands, low temperatures prevented parasite development in mosquitoes during the cool season rains, and highland transmission was therefore limited to the warm dry season when vector densities were low. The primary effect of increasing altitude was a log-linear reduction in vector abundance and, to a lesser extent, a reduction in the proportion of infective mosquitoes. Highland malaria transmission was maintained at extraordinarily low vector densities. We discuss herein the implications of these findings for modeling malaria and suggest that process-based models of malaria transmission risk should be improved by considering the direct effect of temperature on vector densities. Our findings suggest that variation in the short rains in November and changes in agricultural practices are likely to be important generators of epidemics in the Usambaras.

Addition of Novobiocin in pre-enrichment step can improve Salmonella culture protocol of modified semisolid Rappaport-Vassiliadis.

The aim was to investigate the effect of addition of Novobiocin to the non-selective buffered peptone water (BPW) for pre-enrichment of Salmonella in connection with plating on modified semisolid Rappaport-Vassiliadis (MSRV). In a semi-quantitative study, the level of Salmonella following pre-enrichment of 32 presumably naturally contaminated swine fecal samples were assessed for BPW with and without addition of Novobiocin (22 microg/ml). In another experiment, a total of 400 swine fecal samples were screened for the presence of Salmonella spp., in order to compare the performance of the non-selective pre-enrichment broth with BPW made semi-selective by addition of Novobiocin. The semi-quantitative assessment of the Salmonella level showed that addition of Novobiocin in the pre-enrichment step on average increased the level of Salmonella 1.2 log dilution steps. When growth was scored at five levels, 90 samples opposed to 50 yielded a strong positive reading (+++) when Novobiocin was applied. Growth was on average 0.3 scores higher when pre-enriched with Novobiocin. The difference in growth score medians of the two methods was highly significant (Sign test; p<0.001). Despite the increased sensitivity, 13 culture-positive samples were missed when using the Novobiocin-containing BPW. In conclusion, a simple addition of Novobiocin in the BPW pre-enrichment step of fecal samples may facilitate reading and thereby detection of Salmonella on MSRV. The increase of Salmonella in the semi-quantitative study may be caused by a reduction in the number of competitive microorganisms.

Diversity of Plasmodium falciparum populations and complexity of infections in relation to transmission intensity and host age: a study from the Usambara Mountains, Tanzania.

Diversity and complexity of infections with Plasmodium falciparum were described from cross-sectional surveys in November-December 1996 in 6 villages in the Usambara Mountains, Tanzania, where transmission ranged markedly from 0.03 to 91 infective bites per individual per year. Forty-eight samples, stratified for age and parasite densities, were examined from each village (n = 288). Genotyping was performed by a nested PCR method using primers specific for allele families of genes for the merozoite surface protein 1 (msp-1) and merozoite surface protein 2 (msp-2). A high degree of genetic diversity was found within each village but there were no differences found among the 6 villages. Poisson regressions showed significant effects of host age, village and interaction between host age and village on the complexity of infection. There was a positive, non-linear relationship between complexity of infection and transmission intensity with a maximal number of genotypes found per individual even at high transmission intensities. Furthermore there was a significantly lower complexity found in adults (> 15 years) as compared to children (< 15 years) in the lowland village. This difference was not found as transmission intensity decreased. By comparing data from the same geographical area, using the same methods, and taking into account confounding factors, the present study provides evidence for an effect of both age and transmission intensity on complexity of infection with P. falciparum.

Effect of 1997-98 El Niño on highland malaria in Tanzania.

We compared the level of malaria infection in children from 22 communities in an area of unstable transmission in the Usambara Mountains, Tanzania, immediately before and after one of the strongest recorded El Niño southern oscillation events. Although this event resulted in 2.4 times more rainfall than normal, we found strikingly less malaria than in the preceding year.

Aminopropeptide of human procollagen type I: a marker for the identification of blood from children in the mosquito blood meal.

A capture enzyme-linked immunosorbent assay (ELISA) to distinguish between blood from children and adults in the mosquito blood meal was examined using the alpha 1 chain of the aminopropeptide of human procollagen type I (PINP) as antigenic marker. Rabbit anti-human PINP (alpha 1) antibody was used as catching antibody, and either normal serum from 288 African and 58 Caucasian children and adults, or blood meals from 93 fed Aedes aegypti, were examined. PINP in excess of 40 optical density units (ODU) was detected in all children aged 0-13 years, whereas adults aged 21-77 years had PINP levels less than 25 ODU. In the transitional age group (14-20 years), the PINP levels ranged from 1 to 166 ODU. The PINP levels in 95% of the mosquito blood meals obtained from children exceeded the control levels, using 13 ODU as a cut-off value, whereas none of the blood meals from adults exceeded 13 ODU. The PINP levels in the mosquito blood meals were constant 1 and 8 h after ingestion, but they had decreased significantly after 16-19 h. Our data suggest that the test can be used to identify host preferences in studies of mosquitoes collected within 16 h after the blood meal. A field evaluation is necessary to determine the potential of the antigenic marker PINP as a tool in the identification of mosquito host preference.