Evidence for the existence of a new genus Chlamydiifrater gen. nov. inside the family Chlamydiaceae with two new species isolated from flamingo (Phoenicopterus roseus): Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov.

The family Chlamydiaceae currently comprises a single genus Chlamydia, with 11 validly published species and seven more taxa. It includes the human pathogens Chlamydia (C.) trachomatis, C. pneumoniae and C. psittaci, a zoonotic agent causing avian chlamydiosis and human psittacosis, as well as other proven or potential pathogens in ruminants, birds, snakes, reptiles and turtles. During routine testing of 15 apparently healthy captive flamingos in a zoo in 2011, an atypical strain of Chlamydiaceae was detected by real-time PCR of cloacal swab samples. Sequence analysis of the 16S rRNA gene revealed high similarity to the uncultured Chlamydiales bacterium clone 122, which previously had been found in gulls. As more samples were collected during annual campaigns of the flamingo ringing program in southern France from 2012 to 2015, Chlamydiaceae-specific DNA was detected by PCR in 30.9% of wild birds. From these samples, three strains were successfully grown in cell culture. Ultrastructural analysis, comparison of 16S and 23S rRNA gene sequences, whole-genome analysis based on de novo hybrid-assembled sequences of the new strains as well as subsequent calculation of taxonomic parameters revealed that the relatedness of the flamingo isolates to established members of the family Chlamydiaceae was sufficiently distant to indicate that the three strains belong to two distinct species within a new genus. Based on these data, we propose the introduction of Chlamydiifrater gen. nov., as a new genus, and Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov., as two new species of the genus.

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.

The small hydrophobic (SH) gene of North American turkey AMPV-C does not attenuate nor modify host tropism in recombinant European duck AMPV-C.

Subgroup C Avian Metapneumoviruses (AMPV-C) has two lineages, one mostly in turkeys and one mostly in ducks. To investigate the molecular basis of AMPV-C host tropism, a reverse genetics system for a duck AMPV-C virus was developed. A recombinant copy and a recombinant virus in which the SH protein had been exchanged for that of a turkey AMPV-C were rescued. No change in cytopathogenic effect or replication profile in vitro were observed for either virus compared to the wild type. In SPF Muscovy ducks the wild type and its recombinant copy were equally pathogenic. Exchanging the SH in the recombinant copy produced the same results. In SPF turkeys, neither recombinant virus was pathogenic, although both showed a low level of replication. Thus, from the current model, it appears that AMPV-C SH proteins derived from the different species are compatible and that turkey SH does not affect duck AMPV-C pathogenicity.

Emergence and multiple reassortments of French 2015-2016 highly pathogenic H5 avian influenza viruses.

From November 2015 to August 2016, 81 outbreaks of highly pathogenic (HP) H5 avian influenza virus were detected in poultry farms from South-Western France. These viruses were mainly detected in farms raising waterfowl, but also in chicken or guinea fowl flocks, and did not induce severe signs in waterfowl although they did meet the HP criteria. Three different types of neuraminidases (N1, N2 and N9) were associated with the HP H5 gene. Full genomes sequences of 24 H5HP and 6 LP viruses that circulated in the same period were obtained by next generation sequencing, from direct field samples or after virus isolation in SPF embryonated eggs. Phylogenetic analyses of the eight viral segments confirmed that they were all related to the avian Eurasian lineage. In addition, analyses of the "Time of the Most Recent Common Ancestor" showed that the common ancestor of the H5HP sequences from South-Western France could date back to early 2014 (±1 year). This pre-dated the first detection of H5 HP in poultry farms and was consistent with a silent circulation of these viruses for several months. Finally, the phylogenetic study of the different segments showed that several phylogenetic groups could be established. Twelve genotypes of H5HP were detected implying that at least eleven reassortment events did occur after the H5HP cleavage site emerged. This indicates that a large number of co-infections with both highly pathogenic H5 and other avian influenza viruses must have occurred, a finding that lends further support to prolonged silent circulation.

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.

Genome areas with high gene density and CpG island neighborhood strongly attract porcine endogenous retrovirus for integration and favor the formation of hot spots.

Porcine endogenous retroviruses (PERV) are members of the gammaretrovirus genus and display integration preferences around transcription start sites, a finding which is similar to the preferences of the murine leukemia virus (MLV). Our new genome-wide analysis of the integration profile of a recombinant PERV (PERV A/C), enabled us to examine more than 1,900 integration sites and identify 224 integration hot spots. Investigation of the possible genome features involved in hot-spot formation revealed that the expression level of the genes did not influence distribution of the integration sites of gammaretroviruses (PERV and MLV) or the formation of integration hot spots. However, PERV integration and the presence of hot spots was found to be greater in areas of the genome with high densities of genes with CpG islands. Surprisingly, this was not true for MLV. Simulation of integration profiles revealed that retrovirus integration studies involving the use of the restriction enzyme MseI (widely used in genome integration studies of MLV and gammaretroviral vector) underestimated integration near CpG islands and in gene-dense areas. These results suggest that the integration of gammaretrovirus or gammaretroviral vectors might occur preferentially in genome areas that are highly enriched in genes under CpG island promoter regulation.

[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.

Interrater reliability of early intervention providers scoring the alberta infant motor scale.

PURPOSE: This study was designed to examine the interrater reliability of early intervention providers scoring of the Alberta Infant Motor Scale (AIMS) and to examine whether training on the AIMS would improve their interrater reliability. METHODS: Eight early intervention providers were randomly assigned to two groups. Participants in Group 1 scored the AIMS on seven videotapes of infants prior to receiving training and after training on another set of seven videotapes of infants. Participants in Group 2 scored the AIMS on all 14 videotapes of the infants after receiving training. RESULTS: Overall interrater reliability before and after training was high with intraclass correlation coefficients ranging from 0.98 to 0.99. Detailed examination of the results showed that training improved the reliability of the supine subscale in a subgroup of infants between the ages of five and seven months. Training also had an effect on the classification of infants as normal or abnormal in their motor development based on their percentile rankings. CONCLUSION: The AIMS manual provides sufficient information to attain high interrater reliability without training, but revisions regarding scoring are strongly recommended.

[The neonatal behavioral observation: a pertinent source of medical informations]. / L'observation du comportement du nouveau-né: une source pertinente d'informations médicales.

The neonatal behavioral observation is an important source of medical information in three domains: 1) assessment of development which can be done with the Assessment of Preterm Infant Behavior, 2) assessment of pain with the analysis of facial expression using validated pain scales such as the Neonatal Facial Coding System, 3) assessment of brain injuries with the Quality Assessment of General Movements. Such a behavioral observation of the newborn using validated tools is a useful complement of the neuro-imaging techniques.

Descriptive epidemiology of non-Hodgkin lymphomas in a southwestern French hematology center: absence of significant relationship with hepatitis C virus infection.

From January 1995 to June 1998, 136 new cases of non-Hodgkin lymphomas (NHLs) were seen in our center which serves all the French Basque Country (300000 inhabitants). The crude and standardized incidence rates were respectively 13.2 and 7.6 cases/100000/year. The distribution of histologic subtypes according to the REAL classification showed a good correlation with the previous published data. Eleven patients (8%) were HIV-positive and two (1.4%) were HCV carriers. Hence, in our region, contrary to Italy, there is no evidence of relationship between HCV and NHLs.

Incidence of cytomegalovirus disease in the Aquitaine cohort of HIV-infected patients: a retrospective survey, 1987-1993. Groupe d'Epidémilogie Clinique du SIDA en Aquitaine (GECSA).

We estimated the incidence of the first episodes of cytomegalovirus (CMV) disease in the Aquitaine cohort of HIV-infected subjects, south-western France. Cases were retrospectively investigated using standardized definition criteria. Retinitis was confirmed by an ophthalmologist. Gastro-intestinal lesions were confirmed histologically. Encephalitis was histologically confirmed; it was considered possible if TDM or magnetic resonance imaging (MRI) and symptomatology suggested this diagnosis. Pneumopathy was probable in case of hypoxemia, interstitial X-Ray images and response to CMV treatment; it was confirmed if intranuclear inclusions were identified on biopsy or brushing specimen. In the cohort (n = 3525) followed for an average of 46 months, 158 patients had a first episode of CMV disease. The cumulative incidence was 4.5% and the incidence rate (IR) 1.2 per 100 person-years. The IR was higher for homosexuals (2.0) than for heterosexuals (1.0) and intravenous drug users (0.5). Retinitis was the most frequent site (90 cases), followed by digestive system (40), lung (three confirmed and 17 probable) and central nervous system (eight confirmed and three possible). Sixty-eight percent of the patients were at the AIDS stage when CMV disease was diagnosed, with a mean CD4 count of 42/mm3. The cumulative probability of developing CMV disease 2 years after falling below 200 CD4 lymphocytes/mm3 was 8.0%. Retinitis is by far the most common site for CMV disease. Homosexual transmission of HIV, clinical AIDS and low CD4 count are associated with the occurrence of the first episode of CMV disease.

In vivo, synergestic inhibition of MAT-LyLu rat prostatic adenocarcinoma growth by polyamine deprivation and low-dose cyclophosphamide.

Polyamine deprivation in vivo produces significant tumor growth inhibition of the hormone-resistant, metastatic Dunning Mat-LyLu murine prostatic carcinoma. In order to produce a cytotoxic effect in addition to the cytostatic effect of polyamine deprivation, various chemotherapy regimens, combined with drug-containing polyamine-deficient chow (DC-PDC), were assessed. Triple chemotherapy combining methotrexate, cyclophosphamide and vindesine; and monochemotherapy with high-dose cyclophosphamide (90 mg. kg-1) and low-dose cyclophosphamide (20 mg.kg-1) were studied alone and in combination with DC-PDC. A variant of DC-PDC excluding the polyamine oxidase inhibitor MDL 72527 was also studied in combination with low-dose cyclophosphamide. The triple-chemotherapy regimen alone or in combination with polyamine deprivation was effective on tumor growth inhibition but was also toxic. High-dose cyclophosphamide alone produced significant tumor growth inhibition and an increase in life span. High-dose cyclophosphamide in combination with DC-PDC was also effective on tumor growth but was also toxic. Low-dose cyclophosphamide alone was moderately effective on tumor growth inhibition with a marginal increase in life span. When combined with polyamine deprivation, results with low-dose cyclophosphamide compared favourably with those of high-dose cyclophosphamide alone and prevented the formation of lung metastases. The polyamine oxidase inhibitor does not appear to be mandatory to achieve this effect if DC-PDC is combined with low-dose cyclophosphamide. Polyamine deprivation appears to be an important tool in anticancer therapy, allowing the use of reduced doses of cytotoxic agents with the same antitumoral efficacy.

Polyamine deprivation: a new tool in cancer treatment.

The fact that tumors require polyamines for growth has been demonstrated in vitro and in vivo and widely reported. This finding led to the use of polyamine biosynthetic enzymes as targets for antitumor drug design. Highly efficient in vitro selective inhibitors of ornithine decarboxylase such as DFMO do not produce important antitumoral effects in vivo, due to the ability of tumor cells to uptake extracellular polyamines. A new strategy was developed, combining a systematic blockade of all endogenous and exogenous sources of polyamines in vivo. Sources of exogenous polyamines were eliminated by administration of a polyamine-free diet to the animals and decontamination of their gastrointestinal tract. Important antitumoral effects were obtained with this polyamine deprivation and are presented with two experimental models of tumors (Lewis lung carcinoma, Mat Lylu prostatic carcinoma). Biological parameters, modified in cases of cancer, were restored to normal values in treated animals: blood counts and NK cytotoxic activity. Number of metastases was significantly reduced. Given that in man cancer treatment remains unsatisfactory due to incomplete cell kill, development of resistance to treatment and secondary effects of chemotherapy, we chose to investigate the potential interest of polyamine deprivation in this field. By combining clinically applied cytotoxic drugs with polyamine deprivation, we observed an improvement of their antitumoral efficiency: a considerable retardation of tumor growth paired with a marked increase in life-span of the treated animals. Our observations confirm that polyamines absorbed from exogenous sources, mainly food and gastrointestinal tract, play an important role in tumor growth control. Furthermore, the study shows that polyamine deprivation represents an important potential therapeutic tool in improved management of cancer treatment.

Ultrastructural and immunocytochemical study of P1 protamine localization in human testis.

Two monoclonal antibodies (MAbs) directed against human protamine P1 were realized. Anti-P1 specificity was assessed by western-blot and confirmed by ELISA. Monoclonal antibody 97-3 was selected. Protamine P1 was specifically demonstrated in human testis by immunoelectron microscopy, using 97-3 MAb and an indirect post-embedding immunogold technique. Our results clearly demonstrated the precise time of appearance of P1 protamine in the nuclei of human spermatids. P1 first appeared in the nucleus of step 5 spermatids and its concentration was increased in steps 6-8 spermatids, cytoplasm was not labelled.

Decondensation of human sperm nuclei and HP1 protamine degradation from normospermia and asthenospermia in Xenopus egg extracts.

The process of human sperm decondensation has been studied in vitro in cytoplasmic extracts prepared from unfertilized Xenopus laevis eggs. The chromatin decondensation-recondensation cycle was divided into four stages according to chromatin appearance. Spermatozoa from normospermia and asthenospermia were evaluated according to their capacity to reach these stages, and their DNA integrity was assessed by acridine orange (AO) staining. We observed a significant difference between normospermia and asthenozoospermia in the ability to achieve the cycle of chromatin decondensation-recondensation. These results correlated with AO staining. The role of human protamine 1 degradation in the decondensation process was evaluated by immunostaining. It was found not to be a prerequisite for the earlier stage of chromatin decondensation and it was not implied in the latest stages of pronuclear development.

Depletion in nuclear spermine during human spermatogenesis, a natural process of cell differentiation.

Polyamines (PA), polycations present in all mammalian cells, are essential for cell proliferation and differentiation. In vitro, PA are known to bind to DNA with a high affinity. In vivo, the intimate association of endogenous PA with highly condensed chromatin has been reported. During spermatogenesis, when processes of cell proliferation and differentiation take place, the potential role of polyamines has not been studied in depth. We report here the PA levels measured in human spermatogenic cell nuclei at different stages of differentiation. Cell populations (spermatocytes and round, elongating, or elongated spermatids) were obtained after submitting human testes to a trypsin-deoxyribonuclease digestion, then to a centrifugal elutriation and Percoll gradient centrifugation. A significant and progressive nuclear spermine level decrease was observed from primary spermatocytes to elongated spermatids. This release of spermine from nuclei was concomitant with three major events in mammalian spermiogenesis: the reduction of DNA transcription activity, the replacement of histone proteins by protamines, and the compaction of chromatin. This is the first report arguing a release of nuclear spermine during an in vivo physiological cell differentiation process.

Preparation of spermatogenic cell populations at specific stages of differentiation in the human.

Studying biochemical events in human spermatogenesis requires separated populations of spermatogenic cells. Dissociation of these cells was performed by a Trypsin-DNAse method adapted from the technique used for rodents. Cell separation was performed by centrifugal elutriation. Seven populations were collected, one further purified by Percoll gradient centrifugation, giving nine different cell populations. The efficiency of the cell separation was evaluated by phase contrast microscopy, flow cytometric DNA analysis, and electron microscopy. Five populations were enriched in spermatids: two in round spermatids (87% and 73%), another in round (52%) and elongating (44%) spermatids, another constituted by 80% elongating spermatids, and the last by 90% elongated spermatids. Two of the four remaining populations were enriched in primary spermatocytes (74% and 54%); another population was the upper part of the Percoll gradient and constituted cytoplasmic lobes and residual bodies (89%); the last population was made up of various cells, with no specific enrichment. Electron microscopic observations revealed good preservation of the separated cells; only the flagella from elongated spermatids were lost. Furthermore, an unusual pattern of nucleoplasm distribution during stages 2-4 of spermatid differentiation was observed and its signification is discussed with regard to the shape of the human spermatozoon.