No evidence of PEDV infection in French artificial insemination centers in 2015.

Pigs infected by porcine epidemic diarrhea virus (PEDV) are affected by severe diarrhea, vomiting and dehydration. The severity of clinical signs depends on the virus strain. Two genetically different PEDV strains are known to infect pigs, the PEDV S-InDel strains which circulate on all continents and the highly virulent PEDV S-non-InDel strains found in Asia and in America. We have previously demonstrated the presence of PEDV RNA in semen from boars experimentally infected with an S-non-InDel PEDV strain. If naturally infected boars may shed PEDV in semen, this would have important consequences for the breeding sector. Thus we sought to determine whether PEDV has been circulating in populations of breeding boars from French artificial insemination (AI) centers. The current study reports on a serological survey conducted on one hundred and twenty boars from six AI centers, representing 18.6% of the total population of breeding boars in French AI centers in 2015. All of them were found negative for PEDV antibodies, showing no evidence of PEDV circulation in French AI centers at that time.

Immunome differences between porcine ileal and jejunal Peyer's patches revealed by global transcriptome sequencing of gut-associated lymphoid tissues.

The epithelium of the intestinal mucosa and the gut-associated lymphoid tissues (GALT) constitute an essential physical and immunological barrier against pathogens. In order to study the specificities of the GALT transcriptome in pigs, we compared the transcriptome profiles of jejunal and ileal Peyer's patches (PPs), mesenteric lymph nodes (MLNs) and peripheral blood (PB) of four male piglets by RNA-Seq. We identified 1,103 differentially expressed (DE) genes between ileal PPs (IPPs) and jejunal PPs (JPPs), and six times more DE genes between PPs and MLNs. The master regulator genes FOXP3, GATA3, STAT4, TBX21 and RORC were less expressed in IPPs compared to JPPs, whereas the transcription factor BCL6 was found more expressed in IPPs. In comparison between IPPs and JPPs, our analyses revealed predominant differential expression related to the differentiation of T cells into Th1, Th2, Th17 and iTreg in JPPs. Our results were consistent with previous reports regarding a higher T/B cells ratio in JPPs compared to IPPs. We found antisense transcription for respectively 24%, 22% and 14% of the transcripts detected in MLNs, PPs and PB, and significant positive correlations between PB and GALT transcriptomes. Allele-specific expression analyses revealed both shared and tissue-specific cis-genetic control of gene expression.

Molecular investigation of Coxiella burnetii infections in aborted sheep in eastern Turkey.

Q fever is a zoonotic disease that occurs worldwide and is caused by the obligate intracellular bacterium Coxiella burnetii. The aim of this study was to investigate the presence of C. burnetii infection in aborted sheep in eastern Turkey using PCR. A total of 200 fetuses were collected from aborted sheep belonging to 200 herds in different locations in the eastern part of Turkey. Foetal organ samples such as liver, spleen, lung and stomach were taken and the DNA was purified from two hundred pooled samples. PCR analysis of C. burnetii presence in infected organs was performed, and 4 samples (2%) were found positive. In addition, the pooled organ suspensions were inoculated to embryonated chicken eggs, and PCR analysis of yolk sacs showed C. burnetii DNA in 5 samples (2.5%). This study shows that C. burnetii infection has an important role in sheep abortions in eastern Anatolia region.

What can we learn from old microdeletion syndromes using array-CGH screening?

Most microdeletion syndromes identified before the implementation of array-comparative genomic hybridization (array-CGH) were presumed to be well-defined clinical entities. However, the introduction of whole-genome screening led not only to the description of new syndromes but also to the recognition of a broader spectrum of features for well-known syndromes. Here, we report on 10 patients presenting with mental retardation associated with atypical features not suggestive of a known microdeletion and a normal standard karyotype. Array-CGH analyses revealed five microdeletions in the DiGeorge region, three microdeletions in the Williams-Beuren region and two microdeletions in the Smith-Magenis region. Reevaluation in these patients confirmed that the diagnosis remained difficult on clinical grounds and emphasized that well-known genomic disorders can have a phenotype that is heterogeneous and more variable than originally thought. The widespread use of array-CGH shows that such patients may be more readily achieved on the basis of genotype rather than phenotype.

Comparison of Coxiella burnetii shedding in milk of dairy bovine, caprine, and ovine herds.

The shedding of Coxiella burnetii in bovine, caprine, and ovine milk was measured using PCR, in 3 herds for each species, the bulk tank milk samples of which were positive at the time of their selection. Milk samples of 95 cows, 120 goats, and 90 ewes were sampled over 16 wk, as was the bulk tank milk. The shedding of C. burnetii in vaginal mucus and feces was checked at the beginning of the experiment and 2 mo later. The clinical signs in the selected herds as well as the duration and the shedding routes differed among the 3 species. The cows were asymptomatic and shed C. burnetii almost exclusively in milk. In one of the caprine herds, abortions due to C. burnetii were reported. The goats excreted the bacteria mainly in milk. In contrast, the ewes, which came from flocks with abortions due to Q fever (C. burnetii infection), shed the bacteria mostly in feces and in vaginal mucus. This could explain why human outbreaks of Q fever are more often related to ovine flocks than to bovine herds. These excretions did not seem more frequent when the samples were taken close to parturition. The samples were taken from 0 to 421 d after parturition in bovine herds and from 5 to 119 d and 11 to 238 d after parturition in the caprine and ovine herds, respectively. The shedding in milk was sometimes intermittent, and several animals shed the bacteria but were negative by ELISA: 80% of the ewes were seronegative, underscoring the lack of sensitivity of the ELISA tests available for veterinary diagnosis. The detection of antibodies in milk seems more sensitive than it is in serum.

Goats may experience reproductive failures and shed Coxiella burnetii at two successive parturitions after a Q fever infection.

Q fever is a zoonosis caused by the obligate intracellular bacterium, Coxiella burnetii. Aborting domestic ruminants are the main source of human infection. In January 2003, an abortion episode occurred in a dairy caprine herd where 18/60 (30%) goats experienced reproductive problems: 4/60 (7%) aborted and 14/60 (23%) had stillbirths. Serological screening for abortion-related infectious diseases suggested Q fever. The diagnosis of C. burnetii infection was confirmed with PCR based on the occurrence of C. burnetii shedding into vaginal mucus, faeces and colostrums taken after kidding from the affected animals. The pregnancy following this episode resulted in one abortion and four stillbirths; three of those goats had already experienced reproductive failure during the previous kidding season. The seroprevalence of C. burnetii infection and the bacteria shedding were investigated using both ELISA and PCR assays, respectively, during the course of the initial and subsequent kidding seasons. Serological testing, performed on the whole herd 6 weeks after the abortion episode, showed 48/60 (80%) of ELISA positive goats. PCR assay performed on both vaginal swab and milk samples showed that the bacterium was shed for almost four months after the outbreak. C. burnetii DNA was also amplified from vaginal swab and milk samples taken from goats after the second kidding season. Furthermore, the bacteria were found into 14 vaginal swabs and 12 milk samples taken from infected females at both kidding seasons.

Spread of Coxiella burnetii infection in a flock of sheep after an episode of Q fever.

In October 1998, two abortions associated with Coxiella burnetii occurred in a group of 34 pregnant ewes in the sheep flock belonging to INRA Tours-Nouzilly. The flock was kept in groups of approximately 40 ewes, which were housed together in the same accommodation. The prevalence of C burnetii infection in the groups was investigated by using ELISA and PCR tests, which revealed a high prevalence of C burnetii. The ewes were treated with oxytetracycline to reduce the shedding of C burnetii and to prevent further abortions. Nevertheless, five abortions attributed to C burnetii occurred in January and March 1999 in three groups of ewes, and 24 of the ewes still shed the bacteria into their vaginal tracts. In addition, a serological study was carried out during the first year of life of the female lambs born in 1999 and 2000; 12 per cent of 113 lambs born in 1999 were seropositive for C burnetii by ELISA, and half of the ELISA-positive lambs were born either to serologically positive ewes or to dams that excreted the pathogen into their vaginal tracts. However, all the 150 lambs born in 2000 were ELISA-negative, suggesting that the preventive measures undertaken had suppressed both the abortions and the shedding of C burnetii, and reduced the transmission of the agent.

Molecular characterisation and ovine live vaccine 1B evaluation toward a Chlamydophila abortus strain isolated from springbok antelope abortion.

Chlamydiosis is a zoonosis with a worldwide distribution. The reservoir of susceptible hosts is large and includes birds and both domestic and wild mammals. Chlamydial infection, determined serologically, seems to be widespread among wild ruminants in the Paris zoo (France). In February 2003, an abortion case was reported within the springbok (Antidorcas marsupialis) herd of the zoo. PCR assay using primers targeting the polymorph membrane protein gene (pmp) family was performed on both vaginal swab and placenta samples revealing the presence of Chlamydophila. The inoculation into chicken embryos of an infected placenta extract led to the successful isolation of a C. abortus strain referred to as ASb1. The omp1 gene coding the major outer membrane protein (momp) and the 16S-23S rRNA spacer region of ASb1 were compared to those of various strains by restriction fragment length polymorphism (RFLP). The RFLP analysis showed that this isolate belonged to Chlamydophila abortus species and is highly related to known domestic ruminant's strains causing abortion. The efficacy of a live vaccine 1B, based on a temperature-sensitive mutant of the ovine abortion reference strain AB7, was tested. Protection-challenge experiments in a mouse model show that the ASb1 strain led to mice abortions and that vaccination with 1B vaccine provided them with effective protection.

Titin-actin interaction in mouse myocardium: passive tension modulation and its regulation by calcium/S100A1.

Passive tension in striated muscles derives primarily from the extension of the giant protein titin. However, several studies have suggested that, in cardiac muscle, interactions between titin and actin might also contribute to passive tension. We expressed recombinant fragments representing the subdomains of the extensible region of cardiac N2B titin (tandem-Ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin. The PEVK fragment bound F-actin, but no binding was detected for the other fragments. Comparison with a skeletal muscle PEVK fragment revealed that only the cardiac PEVK binds actin at physiological ionic strengths. The significance of PEVK-actin interaction was investigated using in vitro motility and single-myocyte mechanics. As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F-actin retarded filament sliding. Myocyte results suggest that a similar interaction makes a significant contribution to the passive tension. We also investigated the effect of calcium on PEVK-actin interaction. Although calcium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK-actin interaction in a calcium-dependent manner. Gel overlay analysis revealed that S100A1 bound the PEVK region in vitro in a calcium-dependent manner, and S100A1 binding was observed at several sites along titin's extensible region in situ, including the PEVK domain. In vitro motility results indicate that S100A1-PEVK interaction reduces the force that arises as F-actin slides relative to the PEVK domain, and we speculate that S100A1 may provide a mechanism to free the thin filament from titin and reduce titin-based tension before active contraction.

Relationships between the shedding of Coxiella burnetii, clinical signs and serological responses of 34 sheep.

Two abortions associated with Coxiella burnetii occurred in a group of 34 pregnant ewes. The seroprevalence of C. burnetii infection was studied by using an ELISA and the immunofluorescence (IF) assay was applied to the contents of vaginal swabs. In addition, a PCR assay, with primers based on a transposon-like repetitive region of the C. burnetii genome (trans-PcR), was used for the highly sensitive and specific detection of C. burnetii in vaginal swabs, milk and faeces. Of the 34 animals tested at parturition, eight (24 per cent) were positive by ELISA, 11 (32 per cent) were positive by IF, and 15 (44 per cent) were positive when the vaginal swab extract was subjected to the trans-PCR assay. C. burnetii was therefore detected by PCR in the vaginal swabs of seven seronegative ewes. However, five weeks after lambing, 16 (47 per cent) of the animals tested were ELISA positive but only two animals (6 per cent) were positive by PCR. Among the ELISA- and PCR-positive animals, eight (25 per cent) shed coxiella in their milk and six (18 per cent) did so in their faeces.

Sequence and mechanical implications of titin's PEVK region.

A widely used titin monoclonal antibody (9D10) was epitope mapped to the PEVK region in the I-band portion of titin. Sequence analysis of the titin PEVK region revealed a large number of 28 amino acid modules (termed "PPAK" repeats) alternating with glutamic acid rich segments. Species differences in cardiac rest tension could not be ascribed to differences in the PEVK length of the N2B titin isoform. The low rest tension generated by dog cardiac muscle also does not appear to be explained by the N2 and PEVK segment lengths in the N2A titin isoform.

The detection of Coxiella burnetii from ovine genital swabs, milk and fecal samples by the use of a single touchdown polymerase chain reaction.

The polymerase chain reaction (PCR), targeting the repetitive transposon-like region of Coxiella burnetii (Trans-PCR), was evaluated for its ability to detect directly C. burnetii in genital swabs, milk and fecal specimens of ewes. By using a combination of centrifugation step, DNA purification using Qiamp Tissue kit followed by Trans-PCR assay, the efficiency for detection of coxiella in ewes milk samples was further improved and one C. burnetii-cell could be detected in 1ml of milk. In addition, an effective, simple and rapid method to remove PCR-inhibitory substances from fecal specimens by simply diluting the DNA template to 1:100 is described, which made the detection of one microorganism per mg of sample then possible. The results obtained from seropositive ewes proved that C. burnetii could also be detected in milk and fecal samples of naturally infected animals.

Purification and characterization of a new potential in vivo inhibitor of cathepsin L from bovine skeletal muscle.

Four papain-inhibiting peaks, labeled F-I, F-II, F-III, and F-IV, were fractionated from a crude bovine muscle extract by gel filtration chromatography on Sephadex G100, and the F-III fraction was analyzed. From F-III, a cysteine proteinase inhibitor was purified by two successive anionic exchange chromatography steps on Q-Sepharose and Mono-Q columns. This inhibitor has a molecular weight of about 30 kDa. Regarding its specificity toward different proteinases, the purified 30 kDa inhibitor was inactive against serine (trypsin and chymotrypsin) and aspartyl (pepsin) families. In contrast, cathepsin L, H, B, and papain, four enzymes of the cysteine class were strongly inhibited suggesting that this inhibitor was specific to the cysteine proteinase group. However, no inhibitory activity was shown against calpains. Kinetic parameters, including inhibition constants (Ki), rate constant for association (kass) and time required for almost complete inhibition of proteinase in vivo were determined. The values are consistent with a possible physiological function for this inhibitor protein in controlling in vivo cathepsin L activity.

Tissue distribution and characterization of a 30-kDa cysteine proteinase inhibitor from bovine skeletal muscle.

Gel chromatography on a Sephadex G100 column of a crude extract obtained from bovine diaphragma muscle separated four fractions (F-I, F-II, F-III and F-IV) in the range of 12-70 kDa that were active against either papain, trypsin or both. From the F-III fraction, a cysteine proteinase inhibitor was purified by two successive anionic exchange chromatographies on Q-Sepharose and Mono Q columns. The pooled active fraction had a Mw of approximately 30 kDa, and isoelectrofocusing revealed one band with a pI of 6.7. The papain-inhibiting activity was unaffected by dithiothreital or 2-mercaptoethanol treatment, and only one band was obtained after SDS-poly acrylamide gel electrophoresis under both reducing and non-reducing conditions. These results suggest that the 30-kDa muscle cysteine proteinase inhibitor did not contain disulphide bonds essential for activity and the protein was a monomer. This proteinase inhibitor is stable between 40 and 80 degrees C and pH 5-12. Furthermore, the 30-kDa inhibitor is stable to papain proteolysis. The tissue distribution of this inhibitor was investigated using double immunodiffusion and Western blot techniques that provided evidence for its presence in bovine heart, spleen, liver and lung and its absence in bovine plasma.

Purification and characterisation of a polymorphic low M(r) bovine muscle cysteine proteinase inhibitor: structural identity with fatty-acid-binding proteins.

Three low molecular mass cysteine proteinase inhibitors were purified from a bovine skeletal muscle crude extract using a three-step procedure. The crude extract was first subjected to gel filtration on a Sephadex G100 column which separated five active fractions (F-I to F-V). Three papain inhibitors, P1, P2 and P3, were fractionated from the F-V fraction by chromatofocalisation on a poly buffer exchanger column. Purification was completed by chromatography on a Mono Q column. After SDS-PAGE, the three inhibitors showed only one band with an M(r) of 14,300. P1, P2 and P3 appeared to be highly resistant to temperature (40-90 degrees C), pH (3-10), reducing agents (5-50 mM) and to be specific for cysteine proteinases since no activity was detected against either serine or aspartyl proteinases. Although to a varying extent, P1, P2 and P3 inhibited papain, cathepsin B and cathepsin L. Analysis of the peptide mixtures of these inhibitors by RP-HPLC after hydrolysis with CNBr or aspartly endoproteinase N together with their amino acid composition revealed that P1, P2 and P3 cysteine proteinase inhibitors are isoforms of the same protein. As their N-terminal ends were blocked, partial sequence of some of these peptides was determined. Computer search in protein identification resources did not reveal any homology of these sequences with proteinase inhibitors of known primary structure. In contrast, they matched well with different parts of the total sequence of a fatty acid binding protein isolated from bovine heart. This homology was supported by the ability of these inhibitors to bind long chain fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)