Investigation on eggshell apex abnormality (EAA) syndrome in France: isolation of Mycoplasma synoviae is frequently associated with Mycoplasma pullorum.

BACKGROUND: Mycoplasma synoviae (MS) is known to cause Eggshell Apex Abnormality (EAA) syndrome characterized by an altered shell surface with increased translucency on the apex. However, no large-scale studies have been conducted to obtain prevalence data of EAA and MS isolates associated to this syndrome. This manuscript reports the results of two field studies performed in the French poultry industry (2015-2017): focusing mainly on investigation of presence and prevalence of EAA in different types of laying hen flocks (phase 1), and isolation of MS strains from EAA-infected flocks (phase 2). RESULTS: The first survey included 77 farms of commercial layers in three French egg-production regions, hosting 40 flocks in alternative systems (ALT) and 56 in furnished cages (FC). Seven flocks (4 FC and 3 ALT) presented EAA clinical signs, giving a prevalence of 7.3% in this studied sample. A second independent field study was conducted to identify MS by in vitro cultivation and PCR in samples from 28 flocks with clinical signs of EAA. Different types of biological specimens were collected in EAA-affected flocks and submitted to the laboratory. M. synoviae was detected in 25/28 flocks, from both production systems (5/5 ALT and 20/23 FC). Detection of MS was significantly higher in tracheal swabs (59%) than in cloacal (10.5%), albumen (3.6%) and egg yolk (1.1%) swabs. It is worth to mention that attempts to clone MS from positive samples were often hampered by the presence of another Mycoplasma species, which showed fast growing behaviour in the selective media used in this study (Frey Medium 4 and Frey Medium 4 supplemented with erythromycin). The use of MALDI-TOF mass spectrometry in combination with next-generation sequencing (NGS) results allowed the identification of this fast growing mycoplasma as Mycoplasma pullorum, which was detected in 14 of the 25 (56%) MS-positive flocks. CONCLUSIONS: These results confirmed the presence of the EAA syndrome in MS-positive flocks of layers in France, reared in different regions and in different production systems (ALT and FC). Studies need to be conducted to test whether M. pullorum may influence the expression of clinical signs of EAA in MS-infected layer farms.

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.

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.

First detection of Cyprinid Herpesvirus 2 (CyHV-2) in goldfish (Carassius auratus) in France.

Massive mortalities of Carassius auratus (L.) occurred in a farm in France during summer 2014. Fish presented anorexia, loss of scales and large amounts of mucus on the gills. Necrosis of the distal tip of the filament and the lamellae, combined with fusion of the lamellae, was observed, as well as necrosis in the hematopoietic organs and in the digestive tract. The histological examination led to hypothesize the implication of a virus in the mortality. The presence of cyprinid herpesvirus 2 (CyHV-2) in dead fish was demonstrated by amplification and sequencing of portions of the DNA polymerase and helicase genes, both sequences exhibiting 100% identity with CyHV-2 from Japan. In an attempt to find genetic markers of variation, two regions containing tandem repeats in the Japanese genome were amplified from a virus-positive sample from the present outbreak. A first region (mB) was fully identical to the Japanese isolate. However, the second region (mA) exhibited a range of deletions and substitutions compared to CyHV-2 from Japan. This is the first report of CyHV-2 in France in association with mortality of goldfish and the first identification of a molecular marker for its tracing.

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.

First isolation of hirame rhabdovirus from freshwater fish in Europe.

A rhabdovirus was isolated in cell culture inoculated with tissue material from diseased grayling, Thymallus thymallus (L.), originating from a fish farm affected by a mortality episode in Poland. Diagnostics tests showed that the virus was not related to novirhabdoviruses known in Europe, nor to vesiculovirus-like species, except perch rhabdovirus (PRhV) with which it shared moderate serological relations. However, RT-PCR with PRhV probes gave negative results. To identify the virus, a random-priming sequence-independent single primer amplification was adopted. Surprisingly, two of the obtained sequences exhibited a high identity (>99%) with hirame rhabdovirus (HIRRV), a novirhabdovirus usually found in fish in marine Asiatic countries, for instance Japan, China and Korea. The full-length sequence of the phosphoprotein gene (P) demonstrated a higher identity of the present isolate with HIRRV from China compared with the Korean isolate. An identical viral sequence was also found in brown trout, Salmo trutta trutta L., affected by mortalities in a second farm in the same region, after a likely contamination from the grayling farm. To our knowledge, this is the first report of HIRRV in Europe, and in two hosts from fresh water that have not been described before as susceptible species.

Pig anelloviruses are highly prevalent in swine herds in France.

A survey of anelloviruses in swine herds from Britanny, France, is reported. By using PCR targeted to the conserved untranslated region, prevalences of 93 and 73 % were found among 15 herds and 33 animals, respectively. The lung was the organ found to be positive most frequently among the five organs tested from 32 animals. The highest identity levels of our nucleotide sequences were found with pig isolates from Japan and with an isolate from Tupaia belangeri. Interestingly, when aligning all available swine isolates from France and Japan, at least two phylogenetic groups were identified, each one containing clones from France and Japan. Some animals carried clones from both groups, demonstrating intra-individual variability. Despite the putative harmlessness of anelloviruses, the potential inoculum carried by pigs must be further evaluated as a sanitary threat.

[Animal circoviruses and associated diseases]. / Circovirus et pathologies associées chez les animaux.

Animal circoviruses belong to the Circovirus genus of the Circoviridae family. Nowadays, only swine and birds were identified as circovirus hosts. Circoviruses have a single-stranded circular genome of approximately 2000 nucleotide long. DNA of these viruses possesses : (i) a nonanucleotide sequence essential for replication, flanked by inverted repeat sequences, a palindrome that has the potential to form a stem-loop structure and (ii) two major ORFs, located on the viral and complementary strands, which encode respectively the replication-associated protein (Rep) and the capsid protein (Cap). All the circoviruses described at the present time, except porcine circovirus of type 1, are associated with immunosuppressive or immunodepressive diseases. Histopathological lesions such as cytoplasmic inclusions of virus in histiocytic cells and T and B lymphocyte depletion in lymphoid organs, are commonly noticed. No medical prophylaxis of circovirus infections is currently available.

Antigenic and genetic diversity of early European isolates of Infectious bursal disease virus prior to the emergence of the very virulent viruses: early European epidemiology of Infectious bursal disease virus revisited?

Eleven Polish and Hungarian isolates of Infectious bursal disease virus (IBDVs) obtained in the 70/80s (early IBDV) and in the 90s (recent IBDV) were characterized in an Antigen-Capture-ELISA with a panel of neutralizing monoclonal antibodies (Mabs), and by nucleotide sequencing of the VP2 variable domain (vVP2). The viruses were compared with reference IBDV strains, among others with Faragher 52/70 (F52/70, classical, isolated 1970), 89163 (typical very virulent-vvIBDV, isolated 1989) and 91168 (antigenically modified vvIBDV, isolated 1991). Only one of the early isolates (Hungarian strain P1) proved antigenically and genetically similar to F52/70. Other early isolates exhibited no reactivity versus Mabs 3, 4, 5 and/or 8 and had a common previously unrecognized combination of amino acid changes in vVP2. The recent isolates all proved antigenically and genetically related to typical vvIBDV strain 89163, except the Polish isolate 93/35 which proved related to the 91168 strain although no epidemiological relationship had been documented between these viruses in the field. Phylogenetic analysis confirmed that the non-P1 early IBDVs represent a previously unrecognized group among serotype 1 IBDVs. It is discussed whether these early isolates are derivatives of the F52/70-like viruses that might still be present in the field, or whether they represent early IBDV strains that might have been present prior to and progressively replaced by the F52/70-like viruses, as the latter have been replaced by vvIBDVs in the late eighties.

Subgroup C avian metapneumovirus (MPV) and the recently isolated human MPV exhibit a common organization but have extensive sequence divergence in their putative SH and G genes.

The genes encoding the putative small hydrophobic (SH), attachment (G) and polymerase (L) proteins of the Colorado isolate of subgroup C avian pneumovirus (APV) were entirely or partially sequenced. They all included metapneumovirus (MPV)-like gene start and gene end sequences. The deduced Colorado SH protein shared 26.9 and 21.7 % aa identity with its counterpart in human MPV (hMPV) and APV subgroup A, respectively, but its only significant aa similarities were to hMPV. Conserved features included a common hydrophobicity profile with an unique transmembrane domain and the conservation of most extracellular cysteine residues. The Colorado putative G gene encoded several ORFs, the longer of which encoded a 252 aa long type II glycoprotein with aa similarities to hMPV G only (20.6 % overall aa identity with seven conserved N-terminal residues). The putative Colorado G protein shared, at best, 21.0 % aa identity with its counterparts in the other APV subgroups and did not contain the extracellular cysteine residues and short aa stretch highly conserved in other APVs. The N-terminal end of the Colorado L protein exhibited 73.6 and 54.9 % aa identity with hMPV and APV subgroup A, respectively, with four aa blocks highly conserved among Pneumovirus: Phylogenetic analysis performed on the nt sequences confirmed that the L sequences from MPVs were genetically related, whereas analysis of the G sequences revealed that among MPVs, only APV subgroups A, B and D clustered together, independently of both the Colorado isolate and hMPV, which shared weak genetic relatedness at the G gene level.

Phylogenetic analysis of rabbit haemorrhagic disease virus in France between 1993 and 2000, and the characterisation of RHDV antigenic variants.

The first molecular epidemiological study of Rabbit haemorrhagic disease virus undertaken in France between 1988 and 1995, identified three genogroups, two of which (G1, G2) disappeared quickly. We used immunocapture-RT-PCR and sequencing to analyse 104 new RHDV isolates collected between 1993 and 2000. One isolate was obtained in 2000 from a French overseas territory, the Reunion Island. The nucleotide sequences of these isolates were aligned with those of some French RHDV isolates representative of the three genogroups previously identified, of some reference strains and German and American RHDV antigenic variants. Despite the low degree of nucleotide sequence variation, three new genogroups (G4 to G6) were identified with significant bootstrap values. Two of these genogroups (G4 and G5) were related to the year in which the RHDV isolates were collected. Genogroup G4 emerged from genogroup G3, which has now disappeared. Genogroup G5 is a new independent group. The genogroup G6 contained an isolate collected in mainland France in 1999 and the isolate collected from the Reunion Island, as well as German and American RHDV variants. Multiple sequence alignments of the VP60 gene and antigenic analysis with monoclonal antibodies demonstrated that these French isolates are two new isolates of the RHDV variant.

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.