Metabolisation of thiamethoxam (a neonicotinoid pesticide) and interaction with the Chronic bee paralysis virus in honeybees.

Pathogens and pesticides are likely to co-occur in honeybee hives, but much remains to be investigated regarding their potential interactions. Here, we first investigated the metabolisation kinetics of thiamethoxam in chronically fed honeybees. We show that thiamethoxam, at a dose of 0.25ng/bee/day, is quickly and effectively metabolised into clothianidin, throughout a 20day exposure period. Using a similar chronic exposure to pesticide, we then studied, in a separate experiment, the impact of thiamethoxam and Chronic bee paralysis virus (CBPV) co-exposure in honeybees. The honeybees were exposed to the virus by contact, mimicking the natural transmission route in the hive. We demonstrate that a high dose of thiamethoxam (5.0ng/bee/day) can cause a synergistic increase in mortality in co-exposed honeybees after 8 to 10days of exposure, with no increase in viral loads. At a lower dose (2.5ng/bee/day), there was no synergistic increase of mortality, but viral loads were significantly higher in naturally dead honeybees, compared with sacrificed honeybees exposed to the same conditions. These results show that the interactions between pathogens and pesticides in honeybees can be complex: increasing pesticide doses may not necessarily be linked to a rise in viral loads, suggesting that honeybee tolerance to the viral infection might change with pesticide exposure.

Q fever in humans and farm animals in four European countries, 1982 to 2010.

Q fever is a disease of humans, caused by Coxiella burnetii, and a large range of animals can be infected. This paper presents a review of the epidemiology of Q fever in humans and farm animals between 1982 and 2010, using case studies from four European countries (Bulgaria, France, Germany and the Netherlands). The Netherlands had a large outbreak between 2007 and 2010, and the other countries a history of Q fever and Q fever research. Within all four countries, the serological prevalence of C. burnetii infection and reported incidence of Q fever varies broadly in both farm animals and humans. Proximity to farm animals and contact with infected animals or their birth products have been identified as the most important risk factors for human disease. Intrinsic farm factors, such as production systems and management, influence the number of outbreaks in an area. A number of disease control options have been used in these four countries, including measures to increase diagnostic accuracy and general awareness, and actions to reduce spillover (of infection from farm animals to humans) and human exposure. This study highlights gaps in knowledge, and future research needs.

Comparative analysis of both genomic segments of betanodaviruses isolated from epizootic outbreaks in farmed fish species provides evidence for genetic reassortment.

Sequencing of the full coding region of both genomic segments of seven betanodavirus strains isolated from different farmed species in Spain and Portugal revealed that six were reassortants, exhibiting a red-spotted grouper nervous necrosis virus (RGNNV)-type RNA1 and a striped jack nervous necrosis virus (SJNNV)-type RNA2. Analysis of sequences of reassortant strains at both the genomic and protein levels revealed the existence of differences compared with type strains of both genotypes. These differences were greater in the polymerase sequence, which is remarkable because viral structural proteins generally diverge more rapidly than non-structural proteins. Changes in two amino acids observed in the SJNNV capsid protein might be involved in the colonization of new host species by these reassortant strains. In addition, a more extensive phylogenetic analysis, including partial sequences of both RNA segments of 16 other Iberian nodaviruses, confirmed the existence of reassortment between RGNNV and SJNNV.

Genetic differences among Staphylococcus aureus isolates from dairy ruminant species: a single-dye DNA microarray approach.

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating sheep, goats and cows. The present study was carried out to compare 65 S. aureus isolates mainly obtained from nasal carriage and subclinical mastitis in dairy sheep and 43 isolates obtained from subclinical mastitis from 22 goats and 21 cows. A DNA microarray, containing probes against 190 true or putative virulence factors, was used to detect the presence of the virulence genes. Their presence/absence was independently assessed by PCR for the genes of interest. Sheep isolates obtained from the nostrils or the udders did not show any significant tissue specific virulence factor. The dominant pulse-field electrophoresis profile (OV/OV'), associated with spa clonal complex spa-CC 1773, matched mainly with the agr group III and was only found in ovine and caprine isolates. This clone was more specifically characterized by the prevalence of the following virulence genes: lpl4, ssl6, bsaA1, bsaB, bsaP, SAV0812. Moreover, seven virulence-associated genes (lpl1, sel, sec, tst, lukF-PV-like component, lukM, SAV0876) were associated with isolates from small ruminants, while the egc cluster, fhuD1, abiF and SAV2496 with bovine isolates. This genomic study suggests the existence of lineage- and host-specific genes leading to the development of host-specific pathogenic traits of S. aureus isolates.

[Influence of acute stroke care organization on the implementation of clinical practice guidelines]. / Influence de la structure unité neurovasculaire sur la mise en pratique des recommandations professionnelles.

INTRODUCTION: Patients who receive care in a dedicated stroke unit are more likely to survive and become independent. Specific guidelines describe evidence-based care practices. We examined the results of a French audit validation campaign to determine whether the presence of a stroke unit had an influence on the implementation of these recommendations. METHODS: Eleven hospital centers volunteered for self-evaluation. Care delivered to patients in the emergency room and in the hospital unit (dedicated stroke unit or not) was assessed with the clinical audit method. RESULTS: Compared with non-dedicated units, care delivered in stroke units was significantly more compliant with published recommendations. All aspects of acute stroke care were concerned: initial evaluation, acute phase treatment, screening for complications and their treatment, multidisciplinary team coordination, discharge preparation. Care delivered in dedicated stroke units was more reproducible, protocols were more widely used, acute phase risks were better prevented, and acute and postacute care was better coordinated between professionals. A second audit one year later showed increased quality of care in both dedicated and non-dedicated units, with more items improved in the latter. CONCLUSION: Although statistical bias cannot be excluded, this study suggests that recommendations are applied better in dedicated stroke units. A second audit showed better compliance with recommendations, especially in non-dedicated units.

Evaluation of the presence of the bap gene in Staphylococcus aureus isolates recovered from human and animals species.

The implication of biofilm in chronic bacterial infection in many species has triggered an increasing interest in the characterization of genes involved in biofilm formation. The bap gene is a newly identified gene that encodes the biofilm-associated protein, BAP, which is involved in biofilm formation in Staphylococcus aureus. So far the bap gene has only been found in a small proportion of S. aureus strains from bovine mastitis in Spain. In order to study the presence of the bap gene in S. aureus isolates obtained from other species and various locations, a collection of 262 isolates was tested by PCR, using published primers and dot-blot. The results indicated that none of the isolates carried the bap gene suggesting that the prevalence of this gene among S. aureus isolates should be very low.

Characterization of Coxiella burnetii strains from ruminants in a Galleria mellonella host-based model.

Coxiella burnetii is a small Gram-negative intracellular bacterium and is the causative agent of Q fever, which is a zoonotic disease with a worldwide distribution. Domesticated ruminants are the main reservoir of the disease, but the bacterium is able to infect a wide range of hosts, including humans, arthropods and invertebrates. Virulence studies of Coxiella strains usually require a suitable animal model. However, mammalian models are costly and are associated with many ethical constraints. An alternative infection model using Galleria mellonella has been used to study the virulence of several bacterial as well as fungal pathogens. Moreover, the G. mellonella larvae model has been used to identify virulence genes using phase II C. burnetii strain Nine Mile mutants. In our study we describe its use for the characterization of C. burnetii strains isolated from ruminants.

Serological and molecular evidence of Q fever among small ruminant flocks in Algeria.

Q fever, a commonly reported zoonosis worldwide, is caused by infection with Coxiella burnetii, an obligate intracellular bacterium. The infection is often asymptomatic in ruminants, but it can lead to reproductive disorders with bacterial shedding into the environment. Between 2011 and 2013, a study was undertaken in small ruminant flocks in different regions of Algeria. A total of 35 flocks were visited and 227 sera and 267 genital swabs were collected from females after abortions or the lambing period to investigate Q fever infection. Indirect ELISA was used to detect specific antibodies against C. burnetii and real-time PCR for detecting bacterial DNA. Our survey indicated that 58% (95% CI=40-76%) of flocks had at least one positive animal (17 seropositive flocks) and individual seroprevalence was estimated at 14.1% (95% CI=11.8-16.4%) (32 seropositive animals). Bacterial excretion was observed in 21 flocks (60%), and 57 females showed evidence of C. burnetii shedding (21.3%). These results suggest that C. burnetii distribution is high at the flock level and that seropositive and infected (shedder) animals can be found all over the country. Further studies are needed in other regions and on different animal species to better understand the distribution and incidence of Q fever, as well as human exposure, and to develop an adequate prophylaxis program.

Phorbol 12-myristate 13-acetate modulates the cAMP-induced light-scattering response of a Dictyostelium discoïdeum cell population.

The effect of phorbol 12-myristate 13-acetate upon the light-scattering response to cAMP of a D. discoïdeum cell suspension was investigated. It was found that the first spike of the cAMP-mediated light-scattering change (peaking at about 15-20 s after stimulation) was inhibited by the phorbol ester. This effect was concentration dependent with an half-maximum value for the inhibition of 4 nM. The inhibition was found to be maximal after a 10-20 min incubation time. The phorbol ester was shown to affect the dose-response relationship between the cAMP concentration and the relative amplitude of the light-scattering change, more by decreasing the number of cAMP receptors than by decreasing their apparent affinity for cAMP.

Natural outbreak of viral encephalopathy and retinopathy in juvenile sea bass, Dicentrarchus labrax: study by nested reverse transcriptase-polymerase chain reaction.

In order to improve the sensitivity of the diagnosis of viral encephalopathy and retinopathy (VER) in sea bass, a nested reverse transcriptase-polymerase chain reaction (RT-PCR) detection method was developed. The reverse transcription step and the first stage PCR were performed using outer primers specific for the coat protein gene, whereas a new primer set was used as inner primers for the second stage PCR. Fish were collected just before, during and after a VER outbreak occurring in a mediterranean fish farm. For each time point, ten different fish were analysed individually by nested RT-PCR, single step PCR and virus cultivation. The results showed that the frequency of positive samples was always higher using the nested RT-PCR assay. In particular, it was possible to detect nodavirus specific signals 1 month before the appearance of the first mortalities, but only by nested RT-PCR. Altogether these results showed that the sensitivity of nodavirus detection is greatly improved using a nested RT-PCR method. In particular, it was possible to monitor the presence of viral genome in asymptomatic carrier fish using this method.

A fluorescence-based quantitative PCR method for investigation of pseudorabies virus latency.

A quantitative PCR method was developed in order to quantitate the number of copies of Pseudorabies virus (PRV) genome present in tissues from infected pigs. The method is based on the use of an internal standard that differs from the target DNA by a deletion of ten base pairs, and that is co-amplified with the target DNA. The resulting PCR products are labelled with a fluorescent primer and are then separated and detected by means of an automated sequencer. The assay was found to be specific and sensitive, allowing the detection of five copies of viral DNA among 10(6) host cells. The method was used successfully to quantitate the number of PRV DNA copies in trigeminal ganglia samples from infected pigs during the acute and the latent stages of the infection. Between 12 and 3.10(5) copies of viral genome per 10(6) neuronal cells were detected in these tissues which is consistent with data published previously.

Phylogenetic analysis of viral haemorrhagic septicaemia virus (VHSV) isolates from France (1971-1999).

The nucleotide sequences of a specific region of the glycoprotein gene were compared among 63 strains of viral haemorrhagic septicaemia virus (VHSV) isolated from fish in France between 1971 and 1999. The analysis was performed on a region corresponding to amino acids 238 to 331 of the glycoprotein gene, also designated the V2 region and previously shown to accumulate most of the mutations. The sequences of many VHSV isolates were found to be identical or very conserved. An isolate, designated L59X, obtained from elver in the Loire estuary, depicted a higher degree of divergence compared to the other French isolates. The deduced amino-acid sequences were analysed together with the results of neutralisation tests performed using monoclonal antibody 168m4 specific to serotype 1. Non-neutralised VHSV strains had mutations in the region corresponding to the previously described 168m4 epitope. Phylogenetic analysis showed that all the VHSV isolates studied, except L59X, belong to genotype I, previously described as containing VHSV strains isolated from continental Europe. Most of the VHSV isolates studied were found to be genetically related to one of the previously described VHSV strains representative of the major serotypes. Isolate L59X, which was the only French marine strain studied, was found to belong to genotype II, previously shown to encompass the VHSV strains isolated from the British Isles coastal waters. Overall there was a good correlation between the geographical origin of the studied isolates and their genetic characteristics.

Comparative study of viral encephalopathy and retinopathy in juvenile sea bass Dicentrarchus labrax infected in different ways.

The transmission of viral encephalopathy and retinopathy (VER) was investigated in juvenile sea bass (3 g) Dicentrarchus labrax by using cell culture supernatant (SSN-1 cell line) containing nodavirus. Five methods of infection were tested: intramuscular injection (IM), intraperitoneal injection (IP), oral infection, bath exposure and cohabitation of healthy fish with infected fish. Some differences were observed in time of disease onset and severity of symptoms depending on the mode of infection used. Clinical symptoms such as whirling swimming and lethargic or hyperactive behaviour were generally reproduced, except for fish infected via oral and IP infection. First mortalities occurred 3 d after IM and IP infection and 6 d after for the other modes of infection. Cumulative mortalities were also variable: 100% after IM infection, 10% after IP infection, 32% for bath exposure, 43% after cohabitation and 24% via oral infection. Histopathologically, vacuolation was observed in the central nervous tissues and in the retina. The observed lesions were more or less severe depending on the mode of infection, the sampling time and the organs: lesions on the surviving fish (42 days post infection, d p.i.) seemed to be generally more conspicuous in the retina than in the brain of the same fish. In most cases, the presence of nodavirus was confirmed in the same samples of brain and retina by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). The virus was not detected in other organs examined. The present results suggest that 2 forms of VER can be induced: IM injection leads to an acute form (severe nervous disorders with high and fast mortality) whereas oral infection, bath exposure and cohabitation induce a subacute form (less severe disorders and weak daily mortality). This experiment demonstrates experimentally induced horizontal transmission of VER in sea bass for the first time.

An RT-PCR-based method for the diagnosis of the sleeping disease virus in experimentally and naturally infected salmonids.

The sleeping disease (SD) of rainbow trout (Oncorhynchus mykiss) is a worldwide disease for which the causative agent, the sleeping disease virus (SDV), has been recently characterized as an atypical alphavirus (Villoing et al. 2000). Up to now, no diagnostic tools were available and thus no epidemiological studies have been undertaken to evaluate the occurrence of this disease on the field. We present in this paper a sensitive and highly specific 1 working day method, which allows the detection of SDV from experimentally and naturally infected fishes. This method, based on a reverse transcriptase/polymerase chain reaction (RT-PCR) assay on total RNA extracted from SDV-infected fish organs, enables the specific DNA amplification of part of the gene encoding the SDV glycoprotein E2, as early as 2 d post-infection (d.p.i.) and as late as 70 d.p.i., at which time clinical signs of infection are no longer apparent. Moreover, we show that this RT-PCR method can be successfully used for the diagnosis of fish infected by a closely related virus, namely salmon pancreas disease virus (SPDV). This report is the first description of a very powerful diagnostic assay which could provide a more accurate replacement for the classical virological, histological and immunochemistry methods.

Sea bream Sparus aurata, an asymptomatic contagious fish host for nodavirus.

During an epidemiological survey of viral encephalopathy and retinopathy (VER) in diseased sea bass Dicentrarchus labrax, a nodavirus isolate was recovered from net pen-reared sea bream Sparus aurata harboured in the same farming premises. After the virus was isolated and identified by immunofluorescence on SSN-1 cells, sequence analysis with a PCR product from the T4 region of the capsid protein gene indicated that the virus shared 100% identity with a pathogenic virus strain isolated from sea bass. Infection trials demonstrated the pathogenicity of the sea bream virus isolate for juvenile sea bass whereas sea bream infected with the same virus isolate remained asymptomatic even following intramuscular injection of virus. Nevertheless, the sea bream appeared to be a potential carrier of nodavirus, as juvenile sea bass became infected when maintained in a tank containing experimentally contaminated sea bream.

Pseudorabies virus latency: a quantitative approach by polymerase chain reaction.

Some data dealing with the establishment of a quantitative PCR assay are presented. The assay is based on the use of an internal standard (mimic) which differs from the target by a deletion of a few base pairs and which is co-amplified with the target DNA. The resulting PCR products are labelled with fluorescent primers and then separated and detected by an automated sequencer. A highly specific and sensitive PCR assay for the envelope glycoprotein gp50 gene has been developed. This assay is highly reproducible with a detection limit of one copy of PRV DNA. Several mimics were then constructed. As a result we can confirm that the strategy that we have chosen is adequate for the quantification of low amounts of virus DNA present in latently infected swine.

Chemiluminescent detection of amplified pseudorabies virus gp50 DNA with immobilized probes on microtiter wells.

A highly specific and sensitive PCR assay for the envelope glycoprotein gp50 has been developed for the detection of PRV-DNA sequences. Primer pairs from PRV gp50 gene were used with the enzyme uracil N-glycosylase and dUTP instead dTTP to prevent contamination due to PCR product carry-over. Biotinylated PCR products were captured in microtiter wells by specific oligonucleotide covalently linked to the polystyrene wells. After recognition of the biotinylated PCR product with a streptavidin phosphatase conjugate, a chemiluminescent detection was realised. Different factors influencing the binding, the hybridization and the detection efficiency have been tested.

Validation of a novel approach for the rapid production of immunogenic virus-like particles for bluetongue virus.

Bluetongue virus causes an emerging disease of ruminants, principally affecting sheep and cattle. Since 1998, there have been multiple separate outbreaks of bluetongue disease in Europe that have highlighted the need for a safe, efficacious, DIVA compliant vaccine. We report here a new baculovirus expression strategy which allowed pre-integration of the genes encoding the BTV inner capsid proteins at one baculovirus locus and those encoding the outer capsid proteins at a different locus. A modified baculovirus with two marker proteins to facilitate the phenotypic selection of recombinant viruses was developed. The utility of this approach is demonstrated by the production of BTV VLPs to a number of serotypes. For a proof of concept, VLPs of one serotype was then tested for protective immune response. VLPs were demonstrated to be safe, highly effective, immunogens in sheep, reducing post-challenge viraemia to levels below the threshold detection limit of quantitative RT-PCR when vaccinated animals were challenged with virulent virus.