F74 plasmids are major vectors of virulence genes in bovine NTEC2.

Necrotoxigenic Escherichia coli 2 (NTEC2) are defined as E. coli producing the toxin known as cytotoxic necrotizing factor 2 (CNF2), a potent toxin primarily found in bovine but also in humans. NTEC2 are mostly associated with bovine, and cnf2 is known to be carried by pVir-like plasmids. In this study, we looked for NTEC2 in a collection of E. coli collected between 2011 and 2018 in French bovine. Thirty-two isolates, collected from both sick (n = 19) and healthy (n = 13) animals, were identified and characterized using whole-genome sequencing. One F74 plasmid of this bacterial collection was long-read sequenced: its size was 138 121 bp and it carried the cnf2, F17cA-eG, cdtB, iutA, iucC and ompP virulence factors (VFs), but no resistance gene. A large variety of genetic backgrounds was observed, but all cnf2-carrying plasmids belonged to the IncF family, and most of them (78·1%) were of the F74 group. Similar F74 plasmids were also reported from bovine in the United Kingdom and the United States, as identified in the publically available databases. Consequently, these F74 plasmids, which are widely disseminated among E. coli from cattle in the French territory, are vectors of virulence determinants that largely went unnoticed until now.

In vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis.

OBJECTIVE: The aim of this study was to assess the efficacy of lytic bacteriophages on Staphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine mastitis models. METHODS: Between May and December 2016, ten S. aureus (five methicillin-resistant and five methicillin-sensitive) isolates were isolated from milk samples of cattle with mastitis in Belgium and Norway. The isolates were assessed in vitro for their susceptibility to four lytic bacteriophages (Romulus, Remus, ISP and DSM105264) and subsequently in vivo in G. mellonella larvae and in murine mastitis model. RESULTS: Romulus, Remus and ISP showed a lytic activity against the S. aureus isolates in vitro. A larvae survival rate below 50% was observed at 4 days post-inoculation (DPI) in the groups infected with a methicillin-sensitive S. aureus isolate and treated with these three phages in vivo. An incomplete recovery of the mouse mastitis was observed at 48h post-inoculation (HPI) in the groups infected and treated with the ISP phage in vivo. CONCLUSIONS: The observations are much more pronounced statistically between the infected- phosphate buffered saline (PBS)-treated and infected-phage-treated groups in G. mellonella and the murine mastitis model demonstrating an effect of the phages against S. aureus associated with bovine mastitis.

Overall changes in the transcriptome of Escherichia coli O26:H11 induced by a subinhibitory concentration of ciprofloxacin.

AIMS: The goal was to explore the effects of subinhibitory concentration (SIC) (0·5 MIC = 20 µg l-1 ) of ciprofloxacin on the transcriptome of enterohaemorrhagic Escherichia coli O26:H11 isolate by 60 minutes of exposure. MATERIALS AND RESULTS: We used a combination of comparative genomic and transcriptomic (RNAseq) analyses. The whole genome of the E. coli O26:H11 #30934 strain of bovine origin was sequenced and assembled. This genome was next used as reference for the differential gene expression analysis. A whole-genome-based analysis of 36 publicly available E. coli O26:H11 genomes was performed to define the core and the accessory transcriptome of E. coli O26:H11. Using RNAseq and RT-qPCR analysis we observed overexpression of the SOS response and of T3SS effectors, together with the inhibition of specific motility-associated genes. Among the large set of transposases present, only three were activated, suggesting moderate transposition of genes with low doses of ciprofloxacin. Our results illustrated that transcriptional repressors, such as the CopG family protein, belonging to the core genome of E. coli O26:H11, are altered in response to fluoroquinolone exposure. The gene ontology enrichment analysis showed SIC of ciprofloxacin induced binding functions and catalytic activities, including mostly transferase and hydrolase proteins. The amino acid pathways involved in metabolic processes were significantly enhanced after the treatment. CONCLUSIONS: Although the core genome of E. coli O26:H11 constituted only 54·5% of the whole genome, we demonstrated that most differentially expressed genes were associated with the core genome of E. coli O26:H11, and that effects on the mobile genetic element, phage, and plasmid-related genes were rare. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time the effect of low dose of ciprofloxacin on the core transcriptome of E. coli O26:H11 was described. The effects on the main biological functions and protein classes including transcriptional regulators were illustrated.

Complete genome sequence of bluetongue virus serotype 4 that emerged on the French island of Corsica in December 2016.

In November 2016, sheep located in the south of Corsica island exhibited clinical signs suggestive of bluetongue virus (BTV) infection. Laboratory analyses allowed to isolate and identify a BTV strain of serotype 4. The analysis of the full viral genome showed that all the 10 genomic segments were closely related to those of the BTV-4 present in Hungary in 2014 and involved in a large BT outbreak in the Balkan Peninsula. These results together with epidemiological data suggest that BTV-4 has been introduced to Corsica from Italy (Sardinia) where BTV-4 outbreaks have been reported in autumn 2016. This is the first report of the introduction in Corsica of a BTV strain previously spreading in eastern Europe.

Preparation for emergence of an Eastern European porcine reproductive and respiratory syndrome virus (PRRSV) strain in Western Europe: Immunization with modified live virus vaccines or a field strain confers partial protection.

The porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic losses for the swine industry worldwide. In the past several years, highly pathogenic strains that lead to even greater losses have emerged. For the Western European swine industry, one threat is the possible introduction of Eastern European PRRSV strains (example Lena genotype 1.3) which were shown to be more virulent than common Western resident strains under experimental conditions. To prepare for the possible emergence of this strain in Western Europe, we immunized piglets with a Western European PRRSV field strain (Finistere: Fini, genotype 1.1), a new genotype 1 commercial modified live virus (MLV) vaccine (MLV1) or a genotype 2 commercial MLV vaccine (MLV2) to evaluate and compare the level of protection that these strains conferred upon challenge with the Lena strain 4 weeks later. Results show that immunization with Fini, MLV1 or MLV2 strains shortened the Lena-induced hyperthermia. In the Fini group, a positive effect was also demonstrated in growth performance. The level of Lena viremia was reduced for all immunized groups (significantly so for Fini and MLV2). This reduction in Lena viremia was correlated with the level of Lena-specific IFNγ-secreting cells. In conclusion, we showed that a commercial MLV vaccine of genotype 1 or 2, as well as a field strain of genotype 1.1 may provide partial clinical and virological protection upon challenge with the Lena strain. The cross-protection induced by these immunizing strains was not related with the level of genetic similarity to the Lena strain. The slightly higher level of protection established with the field strain is attributed to a better cell-mediated immune response.

First complete genome sequence of European turkey coronavirus suggests complex recombination history related with US turkey and guinea fowl coronaviruses.

A full-length genome sequence of 27,739  nt was determined for the only known European turkey coronavirus (TCoV) isolate. In general, the order, number and size of ORFs were consistent with other gammacoronaviruses. Three points of recombination were predicted, one towards the end of 1a, a second in 1b just upstream of S and a third in 3b. Phylogenetic analysis of the four regions defined by these three points supported the previous notion that European and American viruses do indeed have different evolutionary pathways. Very close relationships were revealed between the European TCoV and the European guinea fowl coronavirus in all regions except one, and both were shown to be closely related to the European infectious bronchitis virus (IBV) Italy 2005. None of these regions of sequence grouped European and American TCoVs. The region of sequence containing the S gene was unique in grouping all turkey and guinea fowl coronaviruses together, separating them from IBVs. Interestingly the French guinea fowl virus was more closely related to the North American viruses. These data demonstrate that European turkey and guinea fowl coronaviruses share a common genetic backbone (most likely an ancestor of IBV Italy 2005) and suggest that this recombined in two separate events with different, yet related, unknown avian coronaviruses, acquiring their S-3a genes. The data also showed that the North American viruses do not share a common backbone with European turkey and guinea fowl viruses; however, they do share similar S-3a genes with guinea fowl virus.

Impact of ceftiofur injection on gut microbiota and Escherichia coli resistance in pigs.

Resistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here, we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut microbiota and ESC resistance in Escherichia coli. Pigs were orally inoculated with an ESC-resistant E. coli M63 strain harboring a conjugative plasmid carrying a gene conferring resistance, bla CTX-M-1. On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota were studied using quantitative PCR analysis of the main bacterial groups and quantification of short-chain fatty acids. E. coli and ESC-resistant E. coli were determined by culture methods, and the ESC-resistant E. coli isolates were characterized. The copies of the bla CTX-M-1 gene were quantified. After ceftiofur injection, the main change in gut microbiota was the significant but transitory decrease in the E. coli population. Acetate and butyrate levels were significantly lower in the treated group. In all inoculated groups, E. coli M63 persisted in most pigs, and the bla CTX-M-1 gene was transferred to other E. coli. Culture and PCR results showed that the ceftiofur-treated group shed significantly more resistant strains 1 and 3 days after ESC injection. Thereafter, on most dates, there were no differences between the groups, but notably, one pig in the nontreated group regularly excreted very high numbers of ESC-resistant E. coli, probably leading to a higher contamination level in its pen. In conclusion, the use of ESCs, and also the presence of high-shedding animals, are important features in the spread of ESC resistance.

[Prevention of post-transfusional malaria in France: a descriptive survey]. / Prévention du paludisme post-transfusionnel en France: enquête descriptive.

Due to an expansion in international travel, an increasing number of blood donors are at risk of exposure to malaria. Preventions of transfusional malaria is required by law in France. Acting as National Reference Center for Malarial Immunology we noticed that this regulation was not being respected by all blood banks. We conducted a questionnaire study concerning 181 blood banks to evaluate under which circumstances serological tests were requested, the policy employed in case of seropositivity, the cost of screening, and wishes concerning modification of the law. The response rate was 46.9%. We noticed a great variability in attitude: only 21% of blood banks followed the regulation; 63% added extra criteria to the law; 16% did not follow the regulation. Indirect immunofluorescence was the main method used by 91.5% of blood banks. However the specificity threshold fluctuated. The fate of the blood unit in case of seropositivity was variable (discard of blood bags, plasma fractionation, blood used). Centers who answered to questionnaire performed 2,513,687 blood donations during the survey. A serology was carried out in 2.6% of donations, and was positive in 9.4% of cases i.e. 0.24% of donations. The average cost of screening was 35 FF. The profit loss due to discard of positive blood was estimated at 1.7 million FF. for the duration of the survey and the blood banks studied. 99% of centers answering the questionnaire expressed desire for standardisation of screening method.