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.

Attachment of Coxiella burnetii to the zona pellucida of in vitro produced goat embryos.

Previous work demonstrated that after infection of in vivo derived caprine embryos, Coxiella burnetti (C. burnetii) showed a strong tendency to adhere to the zona pellicida (ZP). To investigate the risk of C. burnetii transmission via embryo transfer of in vitro-produced goat embryos the aim of this study was, (i) to evaluate the ability of C. burnetii to adhere to the intact zona pellicida of in vitro-produced goat embryos and to determine by confocal microscopy the location of the bacteria, (ii) to test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii. One hundred ZP-intact caprine embryos, produced in vitro, at the 8 to 16 cell stage, were randomly divided into 11 batches of eight to nine embryos. Nine batches were incubated for 18 h with 109Coxiella/ml of CbB1 strain (IASP, INRA Tours). The embryos then were recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, two batches of embryos were subjected to similar procedures but without exposure to C. burnetii, to serve as the control group. One of the nine batches of infected embryos and one of the two non-infected control batches were separated to perform immunolabeling to locate the bacteria. C. burnetii DNA was detected by C-PCR in all eight batches of infected embryos after 10 successive washings. However, bacterial DNA was not detected in the embryo control batch. The first five washing media of the infected group were consistently found to be positive and Coxiella DNA was detected in the wash bath up to the 10th wash for two batches. After immunolabeling, the observation of embryos under confocal microscopy allowed C. burnetti to be found on the external part of the zona pellucida without deep penetration. This study clearly demonstrates that C. burnetii, after in vitro infection at 109Coxiella/ml, stick strongly to the external part of the zona pellucida of in vitro produced caprine embryos without deap penetration and that the 10 washings protocol recommended by IETS to eliminate the pathogenic agents of bovine embryos is unable to eliminate these bacteria from in vitro-produced goat embryo.

Estimation of the frequency of Q fever in sheep, goat and cattle herds in France: results of a 3-year study of the seroprevalence of Q fever and excretion level of Coxiella burnetii in abortive episodes.

A study was carried out, from 2012 to 2015, in 10 French départements to estimate the serological prevalence of Q fever and the frequency of abortive episodes potentially related to Coxiella burnetii in a large sample of cattle, sheep and goat herds. The serological survey covered 731 cattle, 522 sheep and 349 goat herds, randomly sampled. The frequency of abortive episodes potentially related to C. burnetii was estimated by investigating series of abortions in 2695 cattle, 658 sheep and 105 goat herds using quantitative polymerase chain reaction analyses and complementary serological results when needed. The average between-herd seroprevalence was significantly lower for cattle (36·0%) than for sheep (55·7%) and goats (61·0%) and significantly higher for dairy herds (64·9% for cattle and 75·6% for sheep) than for meat herds (18·9% for cattle and 39·8% for sheep). Within-herd seroprevalence was also significantly higher for goats (41·5%) than for cattle (22·2%) and sheep (25·7%). During the study period, we estimated that 2·7% (n = 90), 6·2% (n = 48) and 16·7% (n = 19) of the abortive episodes investigated could be 'potentially related to C. burnetii'in cattle, sheep and goat herds, respectively. Overall, strong variability was observed between départements and species, suggesting that risk factors such as herd density and farming practices play a role in disease transmission and maintenance.

Coxiella burnetii Circulation in a Naturally Infected Flock of Sheep: Individual Follow-Up of Antibodies in Serum and Milk.

The control of Q fever, a zoonotic disease caused by the Coxiella burnetii bacterium, remains a scientific challenge. Domestic ruminants are considered the main reservoir, shedding C. burnetii essentially through parturition products during abortion or birth. Sheep are particularly frequently associated with human outbreaks, but there are insufficient field data to fully understand disease dynamics and to instigate efficient control measures. A longitudinal follow-up study of a naturally infected sheep flock was performed (i) to investigate relationships between seropositivity and bacterial shedding in the vaginal mucus, (ii) to describe the kinetics of antibodies, including responses to vaccination, (iii) to monitor maternal antibodies in ewe lambs, and (iv) to compare serological results for milk and serum samples. For 8 months, we collected blood samples every 3 weeks from 11 aborting and 26 nonaborting dairy ewes, 20 nonaborting suckler ewes, and 9 ewe lambs. Individual milk samples were also obtained from lactating females. All serum and milk samples were tested by enzyme-linked immunosorbent assay (ELISA), whereas vaginal swabs were tested by quantitative PCR. We found that some dairy females did not seroconvert despite shedding C. burnetii in their vaginal mucus. Overall, antibody levels in adult females were found to remain stable over time, with exceptions during the mating and lambing periods. Maternal antibodies decreased during the first month after birth. Interestingly, antibody levels in milk were correlated with those in serum. This study provides valuable field data that will help improve Q fever surveillance and within-flock management measures.IMPORTANCE Field data are necessary to improve the surveillance, diagnosis, and sanitary management of Q fever in livestock. Here, we provide extensive serological data obtained from serum and milk samples from infected and vaccinated ewes belonging to a naturally infected flock of sheep. We show that antibody levels are stable over time and seropositivity and vaginal shedding are not clearly correlated, whereas antibody levels in milk are strongly correlated with those in serum. Accordingly, we find that antibody levels in bulk tank milk are consistent with the variations observed in the serum of dairy females over time. We report the existence of maternal antibody transmission to ewe lambs and we show that the presence of maternal antibodies at birth does not prevent the development of a serological response to vaccination at the age of 4 months. Finally, we report that adult ewes generally seroconvert after vaccination, including during pregnancy.

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.

Circulation of Coxiella burnetii in a Naturally Infected Flock of Dairy Sheep: Shedding Dynamics, Environmental Contamination, and Genotype Diversity.

Q fever is a worldwide zoonosis caused by Coxiella burnetii. Domestic ruminants are considered to be the main reservoir. Sheep, in particular, may frequently cause outbreaks in humans. Because within-flock circulation data are essential to implementing optimal management strategies, we performed a follow-up study of a naturally infected flock of dairy sheep. We aimed to (i) describe C. burnetii shedding dynamics by sampling vaginal mucus, feces, and milk, (ii) assess circulating strain diversity, and (iii) quantify barn environmental contamination. For 8 months, we sampled vaginal mucus and feces every 3 weeks from aborting and nonaborting ewes (n=11 and n=26, respectively); for lactating females, milk was obtained as well. We also sampled vaginal mucus from nine ewe lambs. Dust and air samples were collected every 3 and 6 weeks, respectively. All samples were screened using real-time PCR, and strongly positive samples were further analyzed using quantitative PCR. Vaginal and fecal samples with sufficient bacterial burdens were then genotyped by multiple-locus variable-number tandem-repeat analysis (MLVA) using 17 markers. C. burnetii burdens were higher in vaginal mucus and feces than in milk, and they peaked in the first 3 weeks postabortion or postpartum. Primiparous females and aborting females tended to shed C. burnetii longer and have higher bacterial burdens than nonaborting and multiparous females. Six genotype clusters were identified; they were independent of abortion status, and within-individual genotype diversity was observed. C. burnetii was also detected in air and dust samples. Further studies should determine whether the within-flock circulation dynamics observed here are generalizable.

Red-light-driven photocatalytic hydrogen evolution using a ruthenium quaterpyridine complex.

A high-temperature, microwave synthesis of [Ru(qpy)3](2+) (qpy = 4,4':2',2'':4'',4'''-quaterpyridine) affords the photosensitiser in quantitative yield. The complex produces H2 photocatalytically in a range extending from the UV region of the spectrum to the red with greater efficiency when compared to [Ru(bpy)3](2+).

Risk of Coxiella burnetii transmission via embryo transfer using in vitro early bovine embryos.

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Domestic ruminants are the main source of infection for humans. The objectives of this study were to determine (1) whether C. burnetii would adhere to the intact zona pellucida (ZP-intact) of early in vitro-produced bovine embryos; (2) whether the bacteria would adhere to or infect the embryos (ZP-free) after in vitro infection; and (3) the efficacy of the International Embryo Transfer Society (IETS) washing protocol. One hundred and sixty, eight- to 16-cell bovine embryos produced in vitro, were randomly divided into 16 batches of 10 embryos. Twelve batches (eight ZP-intact and four ZP-free) were incubated in a medium containing C. burnetii CbB1 (Infectiologie Animale et Santé Publique, Institut National de Recherche Agronomique Tours, France). After 18 hours of incubation at 37 °C and 5% CO2 in air, the embryos were washed in 10 successive baths of a PBS and 5% fetal calf serum solution in accordance with the IETS guidelines. In parallel, four batches (two ZP-intact and two ZP-free) were subjected to similar procedures but without exposure to C. burnetii to act as controls. Ten washing fluids from each batch were collected and centrifuged for 1 hour at 13,000× g. The embryos and wash pellets were tested using conventional polymerase chain reaction. C. burnetii DNA was found in all ZP-intact and ZP-Free embryos after 10 successive washes. It was also detected in the first four washing fluids for ZP-intact embryos and in the 10th wash fluid for two of the four batches of ZP-free embryos. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. burnetii adheres to and/or penetrates the early embryonic cells and the ZP of in vitro bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients and/or their offspring. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of bovine embryos infected by C. burnetii would eliminate the bacteria from the ZP and to verify if similarly results are obtained with in vivo-derived embryos.

Seroprevalence of Q fever (coxiellosis) in sheep from the Southern Marmara Region, Turkey.

Little information is available in Turkey on Q fever, a zoonose caused by Coxiella burnetii and transmitted from domestic ruminants. This study aimed at investigating the seroprevalence in sheep flocks from three provinces (Bursa, Balikesir and Canakkale). Serosurvey was undertaken on 42 flocks, which were categorised by sizes. Sera were collected randomly from specific age groups within the young population. CHEKIT Q-fever ELISA kit was used to identify the infection in sheep. The results showed that 20% (n=151) of sheep were seropositive. A total of 34 flocks (81%) revealed at least one seropositive animal. Higher seroprevalence was observed in Balikesir region. Larger flocks resulted more infected than medium and small flocks. An association was found between seropositivity and age, when the primiparous ewes (1-year old) had higher antibodies rates than newborn sheep (aged less than 10 months) or biparous ewes (2 years old). These results showed that Q fever infection was common and circulating in the studied region, hence encourage efforts to propose measures that could reduce the spread and the zoonotic risk.

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.

[Bacterial enterotoxin receptors]. / Les récepteurs des entérotoxines bactériennes.

Bacterial enterotoxin receptors. Enteric bacterial toxins display a great diversity in their structure, molecular weight and mechanism of action. The interaction of enterotoxins with the intestinal mucosa either leads to a direct effect on the cell membrane or an effect on signal transduction within eukaryotic cells. However, before a toxin can affect a cell, it must after its secretion by a microorganism, recognise and bind to a specific surface molecule, its receptor. Membrane receptors of bacterial enterotoxins have been identified as protein, glycoprotein or glycolipid in nature. The chemical nature of the molecules acting as receptors is crucial and during evolution they have been carefully selected. Some toxins, after their interaction with a receptor molecule, will transduce a signal across the cell membrane while remaining at the cell surface. Other toxins, after this initial binding step with a receptor, will be internalised. Others can form pores leading to leakage of cellular components and cell lysis. Receptors that have been identified often comprise a saccharidic chain that is directly involved in the recognition and binding of the toxin. Today, models explaining toxin-receptor interactions are more complex, including multistep events. This review summarises the knowledge of the interactions between bacterial toxins and membrane receptors present on intestinal mucosa.

Evidence that Escherichia coli STb enterotoxin binds to lipidic components extracted from the pig jejunal mucosa.

Escherichia coli strains producing the heat-stable enterotoxin STb cause diarrhoea in pigs, but little is known on the receptor binding step initiating the diarrhoeal process. In the present study, pig jejunal mucosa extracts were tested for the presence of binding component(s) for STb. Jejunal epithelial cells and the mucus layer were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated material was transferred to a polyvinylidene difluoride (PVDF) membrane and overlayed with STb. The results indicated that a band migrating with the tracking dye was bound by STb. This band was not stained by Coomassie blue and was thus regarded as non proteinic but rather as a lipidic component. Thus, total lipid extracts were obtained from the epithelial cells and the mucus layer. Compared to SDS-PAGE on 12% gels, a better separation of the low molecular mass components contained in these extracts was obtained using high-density Phastgel. Most of the components were detected following silver staining but not using Coomassie blue. Interestingly, commercially available pure glycolipids could also be visualized, after separation, only following silver staining. In the total lipid extracts, a band migrating in the 2.5-6.5 kDa range was observed. Using a monoclonal antisulfatide antibody, this band was recognized indicating that sulfatide was, in effect, present in the extract. When pure sulfatide was run on the same gels, it showed the same electrophoretic mobility. In addition, a dose dependent binding of STb to sulfatide could be observed. Taken together, these data suggested that sulfatide present on the jejunal mucosa, could represent a natural target binding molecule for STb.

Sulfatide from the pig jejunum brush border epithelial cell surface is involved in binding of Escherichia coli enterotoxin b.

Using a quantitative dot blot overlay assay of polyvinylidene difluoride membranes, we investigated the ability of Escherichia coli heat-stable enterotoxin b (STb) to bind to various glycolipids of defined structure. STb bound strongly to acidic glycosphingolipids, including sulfatide (or 3'-sulfogalactosylceramide) and several gangliosides, but not significantly to their derivatives, galactosylceramide and asialogangliosides, respectively. STb exhibited the highest binding affinity for sulfatide. STb bound to pure sulfatide in a dose-dependent and saturable manner, with a detection level of a few nanograms. The binding was not inhibited by tetramethylurea, which is a strong disrupter of hydrophobic interactions, or by the anionic sulfated polymer of glucose, dextran sulfate, indicating that the binding is not due solely to either hydrophobic or ionic interactions via the sulfate group of the sulfatide. The specificity of the binding was confirmed by the finding that a 500-fold molar excess of sulfatide inhibited STb binding by approximately 45%, whereas no competition was obtained with galactosylceramide under the same conditions. Taken together, our data indicated that a galactose residue linked to a sulfate group is required for the binding specificity of STb. Then, total lipids extracted either from the mucous layer or from the epithelial cells of the pig jejunum brush border, the natural target of STb, were analyzed by thin-layer chromatography (TLC). Both extracts contained a lipidic molecule with a relative mobility on a TLC plate similar to that of the sulfatide standard. The migrated lipid extracted directly from a preparative TLC plate was confirmed to be sulfatide, as it was recognized by laminin, a sulfated glycolipid binding protein, and by a monoclonal antibody directed against sulfatide. In an overlay assay on PVDF membranes, STb bound to the sulfatide prepared from porcine jejunum as well as to the sulfatide standard. Thus, these findings suggest that the terminal oligosaccharide sequence Gal(3SO4)beta1- on sulfatide could mediate binding of STb to its target cells and, in support of a recent report (E. Rousset, J. Harel, and J. D. Dubreuil, Microb. Pathog. 24:277-288, 1998), probably terminal sialic acid residue on another glycosphingolipid. Moreover, pretreatment in the ligated intestinal loop assay with laminin or sulfatase altered the biological activity of STb. In summary, we present data indicating that sulfatide represents a functional receptor for the STb toxin.

Binding characteristics of Escherichia coli enterotoxin b (STb) to the pig jejunum and partial characterization of the molecule involved.

Escherichia coli heat-stable enterotoxin b (STb) causes severe diarrhoea in weaning piglets. STb most probably has to bind to intestinal epithelial cells in order to achieve its effect. Using biotinylated biologically active STb, we developed a semi-quantitative binding assay using indirect fluorescence microscopy. We demonstrated the attachment of the biotinylated toxin to microvilli of the pig jejunum. However, binding was abolished when biotinylated STb was either boiled or treated with 2-mercaptoethanol, treatments known to abolish biological activity. Different characteristics of STb attachment to the pig small intestine were determined. The reaction was rapid and reached maximum intensity after approximately 10 min. The binding was pH dependent showing an optimum at pH 5.8. Incubation at either 4 degrees C, 25 degrees C or 37 degrees C did not affect the binding. No competition was observed with non-biotinylated STb. However, preincubation of biotinylated STb with streptavidin conjugated to horseradish peroxidase completely abolished the binding. Pig tissues other than jejunum demonstrated binding towards STb including duodenum, ileum, caecum, colon, liver, lung, spleen and kidney. The molecule involved was then partially characterized. Metaperiodate treatment of the jejunum sections abrogated binding but protease treatment had no effect. Enzymatic treatments of jejunal sections demonstrated that N- and O-glycosidases, and several exoglycosidases did not affect binding, whereas reduced binding was observed with ceramide glycanase and alpha-glucosidase, and was completely abolished following neuraminidase treatment. Overall, our results suggest that in vitro STb binding was rapid, pH dependent, temperature independent, not restricted to jejunum and involves a molecule that seems to be composed of a ceramide moiety, terminal neuraminic acid and/or alpha-linked terminal glucose residue(s).

An Escherichia coli CS31A fibrillum chimera capable of inducing memory antibodies in outbred mice following booster immunization with the entero-pathogenic coronavirus transmissible gastroenteritis virus.

CS31A fibrillae are thin, flexible, heteropolymeric proteinaceous appendages exposed as a capsule-like material around the cell surface of certain Escherichia coli strains. Two antigenic peptides of the S spike glycoprotein (TGEV-S) amino acids (aa) 363-371 and 521-531 of the transmissible gastroenteritis virus (TGEV) were tandemly introduced in the loop-structured, variable region aa 202-218 of the major ClpG subunit protein composing the bulk of CS31A. The resulting hybrid fibrillae with a 25 aa heterologous peptide were produced at the cell surface. Using a monoclonal antibody (Mab) specific for the TGEV epitopes, purified hybrid fibrillae were analysed in Western blotting under native conditions, which showed that the two viral epitopes were recognized immunologically as an integral part of the hybrid fibrillae, and therefore that they were antigenically active. The immunogenicity of the fusion construct was evaluated with live recombinant bacteria, purified hybrid ClpG monomers, and purified chimeric CS31A polymers. Whatever the form of hybrid used as antigen, intraperitoneally immunized outbred mice elicited serum anti-TGEV peptides antibodies (Abs) with significant titres and capable of recognizing native TGEV particles, indicating that the epitopes are exposed in an immunogenic conformation in all cases. However, virus neutralization titres were only obtained after immunization with either purified polymers or monomers. Furthermore, 4 months after an ultimate immunization with 20 micrograms of hybrid fibrillae mice developed a strong anamnestic Ab response against the two TGEV peptides following booster inoculation with virions. We conclude that CS31A fibrillae carrying a combination of TGEV epitopes as insert can induce an immunological memory in outbred animals infected with TGEV, and therefore that hybrid CS31A fibrillae may prove efficient as components of a subunit vaccine.