Listeria phospholipases subvert host autophagic defenses by stalling pre-autophagosomal structures.

Listeria can escape host autophagy defense pathways through mechanisms that remain poorly understood. We show here that in epithelial cells, Listeriolysin (LLO)-dependent cytosolic escape of Listeria triggered a transient amino-acid starvation host response characterized by GCN2 phosphorylation, ATF3 induction and mTOR inhibition, the latter favouring a pro-autophagic cellular environment. Surprisingly, rapid recovery of mTOR signalling was neither sufficient nor necessary for Listeria avoidance of autophagic targeting. Instead, we observed that Listeria phospholipases PlcA and PlcB reduced autophagic flux and phosphatidylinositol 3-phosphate (PI3P) levels, causing pre-autophagosomal structure stalling and preventing efficient targeting of cytosolic bacteria. In co-infection experiments, wild-type Listeria protected PlcA/B-deficient bacteria from autophagy-mediated clearance. Thus, our results uncover a critical role for Listeria phospholipases C in the inhibition of autophagic flux, favouring bacterial escape from host autophagic defense.

Francisella tularensis harvests nutrients derived via ATG5-independent autophagy to support intracellular growth.

Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.

Invasion and persistence of Mycoplasma bovis in embryonic calf turbinate cells.

Mycoplasma bovis is a wall-less bacterium causing bovine mycoplasmosis, a disease showing a broad range of clinical manifestations in cattle. It leads to enormous economic losses to the beef and dairy industries. Antibiotic treatments are not efficacious and currently no efficient vaccine is available. Moreover, mechanisms of pathogenicity of this bacterium are not clear, as few virulence attributes are known. Microscopic observations of necropsy material suggest the possibility of an intracellular stage of M. bovis. We used a combination of a gentamicin protection assay, a variety of chemical treatments to block mycoplasmas entry in eukaryotic cells, and fluorescence and transmission electron microscopy to investigate the intracellular life of M. bovis in calf turbinate cells. Our findings indicate that M. bovis invades and persists in primary embryonic calf turbinate cells. Moreover, M. bovis can multiply within these cells. The intracellular phase of M. bovis may represent a protective niche for this pathogen and contribute to its escape from the host's immune defense as well as avoidance of antimicrobial agents.

IFNγ inhibits the cytosolic replication of Shigella flexneri via the cytoplasmic RNA sensor RIG-I.

The activation of host cells by interferon gamma (IFNγ) is essential for inhibiting the intracellular replication of most microbial pathogens. Although significant advances have been made in identifying IFNγ-dependent host factors that suppress intracellular bacteria, little is known about how IFNγ enables cells to recognize, or restrict, the growth of pathogens that replicate in the host cytoplasm. The replication of the cytosolic bacterial pathogen Shigella flexneri is significantly inhibited in IFNγ-stimulated cells, however the specific mechanisms that mediate this inhibition have remained elusive. We found that S. flexneri efficiently invades IFNγ-activated mouse embryonic fibroblasts (MEFs) and escapes from the vacuole, suggesting that IFNγ acts by blocking S. flexneri replication in the cytosol. This restriction on cytosolic growth was dependent on interferon regulatory factor 1 (IRF1), an IFNγ-inducible transcription factor capable of inducing IFNγ-mediated cell-autonomous immunity. To identify host factors that restrict S. flexneri growth, we used whole genome microarrays to identify mammalian genes whose expression in S. flexneri-infected cells is controlled by IFNγ and IRF1. Among the genes we identified was the pattern recognition receptor (PRR) retanoic acid-inducible gene I (RIG-I), a cytoplasmic sensor of foreign RNA that had not been previously known to play a role in S. flexneri infection. We found that RIG-I and its downstream signaling adaptor mitochondrial antiviral signaling protein (MAVS)--but not cytosolic Nod-like receptors (NLRs)--are critically important for IFNγ-mediated S. flexneri growth restriction. The recently described RNA polymerase III pathway, which transcribes foreign cytosolic DNA into the RIG-I ligand 5'-triphosphate RNA, appeared to be involved in this restriction. The finding that RIG-I responds to S. flexneri infection during the IFNγ response extends the range of PRRs that are capable of recognizing this bacterium. Additionally, these findings expand our understanding of how IFNγ recognizes, and ultimately restricts, bacterial pathogens within host cells.

Structural and functional integrity of spermatozoa is compromised as a consequence of acute uropathogenic E. coli-associated epididymitis.

Uropathogenic Escherichia coli (UPEC)-associated epididymitis is commonly diagnosed in outpatient settings. Although the infection can be successfully cleared using antimicrobial medications, 40% of patients unexplainably show persistent impaired semen parameters even after treatment. Our aim was to investigate whether pathogenic UPEC and its associated virulence factor hemolysin (hlyA) perturb the structural and functional integrity of both the epididymis and sperm, actions that may be responsible for the observed impairment and possibly a reduction of fertilization capabilities. Semen collected from patients diagnosed with E. coli-only related epididymitis showed that sperm counts were low 14 days postantimicrobial treatment regardless of hlyA status. At Day 84 following treatment, hlyA production correlated with approximately 4-fold lower sperm concentrations than in men with hlyA-negative strains. In vivo experiments with the hlyA-producing UPEC CFT073 strain in a murine epididymitis model showed that just 3 days postinfection, structural damage to the epididymis (epithelial damage, leukocyte infiltration, and edema formation) was present. This was more severe in UPEC CFT073 compared to nonpathogenic E. coli (NPEC 470) infection. Moreover, pathogenic UPEC strains prematurely activated the acrosome in vivo and in vitro. Raman microspectroscopy revealed that UPEC CFT073 undermined sperm integrity by inducing nuclear DNA damage. Consistent with these observations, the in vitro fertilization capability of hlyA-treated mouse sperm was completely abolished, although sperm were motile. These findings provide new insights into understanding the possible processes underlying clinical manifestations of acute epididymitis.

Genetic factors influencing C-type RNA virus induction.

Genetic factors were studied which affect the inducibility of C-type RNA viruses from embyro cultures of mouse strains with high and low incidence of spontaneous leukemia. Virus was inducible by chemical treatment from secondary embryo cultures of several inbred strains. Both virus inducibility and the capacity of the virus to persist in cells from which it was activated were found to be inherited as dominant genetic characteristics. The results show that the factors controlling virus activation and persistence in culture also play an important role in spontaneous virus expression in the animal.

Virus-induced diabetes mellitus. VI. Genetically determined host differences in the replicating of encephalomyocarditis virus in pancreatic beta cells.

Beta cells were isolated from strains of mice that were susceptible and resistant to encephalomyocarditis (EMC) viral-induced diabetes mellitus. Beta cells from susceptible mice that were infected in vivo with EMC virus showed higher viral titers, more severe degranulation, and lower concentrations of immunoreactive insulin than beta cells from resistant mice. Immunofluorescence and infectious center assays revealed that pancreas from susceptible mice contained at least 10 times more infected cells than pancreas from resistant mice. Beta cell cultures prepared from susceptible mice and infected in vitro also showed higher viral titers and more severe cytopathologic changes than beta cell cultures from resistant mice. In contrast to beta cell cultures, virus replicated equally well in primary embryo and kidney cell cultures from susceptible and resistant strains of mice. It is concluded that the development of EMC virus-induced diabetes is related to genetically determined host differences in the capacity of the virus to infect beta cells.

Risk of contamination of germplasm during cryopreservation and cryobanking in IVF units.

Cryopreservation of sperm, embryos and, more recently, oocytes plays an important and increasing role in assisted reproduction, due to improvements of old, and introduction of new technologies. In parallel, concerns are increasing about the technical and biological safety of these procedures. However, published data regarding the confirmed or theoretical hazards of these procedures are sparse and sometimes contradictory. The purpose of this review will summarize data and opinions about one of the most disputed risks, the potential hazard of contamination and disease transmission through cryopreservation. Special attention is concentrated on the weak points of the technology including open vitrification systems, sterilization of liquid nitrogen and safety of commonly used storage tanks including straws and cryovials. Suggestions are also made for practical measures to avoid these dangers while preserving the benefits and perspectives of new cryopreservation technologies.

Isolation of a neurotropic type C virus.

A neurogenic paralysis of the lower limb can be induced and serially transmitted in mice by a nontransforming type C virus strain that originated in an embryo of a wild mouse. The virus exerted a neurotropic effect on the anterior horn neurons.

Immunological Role of the Maternal Uterine Microbiome in Pregnancy: Pregnancies Pathologies and Alterated Microbiota.

Understanding what happens at the time of embryo implantation has been the subject of significant research. Investigators from many differing fields including maternal fetal medicine, microbiology, genetics, reproductive endocrinology and immunology have all been studying the moment the embryo interacts with the maternal endometrium. A perfect relationship between the uterus and the embryo, mediated by a tightly controlled interaction between the embryo and the endometrium, is required for successful implantation. Any factors affecting this communication, such as altered microbiome may lead to poor reproductive outcomes. Current theories suggest that altered microbiota may trigger an inflammatory response in the endometrium that affects the success of embryo implantation, as inflammatory mediators are tightly regulated during the adhesion of the blastocyst to the epithelial endometrial wall. In this review, we will highlight the various microbiome found during the periconceptual period, the microbiomes interaction with immunological responses surrounding the time of implantation, its effect on implantation, placentation and ultimately maternal and neonatal outcomes.

Risk of Chlamydia abortus transmission via embryo transfer using in vitro produced early bovine embryos.

The objectives of this study were to determine (i) whether Chlamydia (C.) abortus would adhere to the intact zona pellucida (ZP-intact) of early in vitro produced bovine embryos; (ii) whether the bacteria would adhere to the embryos (ZP-free) after in vitro infection; and (iii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol. The experimentation was made twice. For each replicate 100 (8-16-cell) bovine embryos produced in vitro were randomly divided into 10 batches. Height batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4 × 107Chlamydia/ml of AB7 strain. After incubation for 18 h at 37 °C in an atmosphere of 5% CO2, the embryos were washed in accordance with the IETS guidelines. In parallel, two batches (1 ZP-intact and 1 ZP-free) of 10 embryos were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13,000×g. Each batch of washed embryos and each wash pellets were tested using PCR. C. abortus DNA was found in all ZP-intact and ZP-free batches of 10 embryos after 10 successive washes. For ZP-intact infected embryos, Chlamydia-DNA was also detected in all 10 wash baths for two batches (2/8) of embryos, whereas for ZP-free infected embryos, Chlamydia-DNA was detected in all 10 wash baths for 6/8 batches of embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. The bacterial load for batches of 10 embryos after the 10 wash baths was significantly higher for batches of ZP-free embryos (20.7 ±â€¯9 × 103 bacteria/mL) than for batches of ZP-intact embryos (0.47 ±â€¯0.19 × 103 bacteria/mL). These results demonstrate that C. abortus adheres to the ZP as well as the early embryonic cells of in vitro produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS fails to remove it.

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.

Bartonella infections in three species of Microtus: prevalence and genetic diversity, vertical transmission and the effect of concurrent Babesia microti infection on its success.

BACKGROUND: Bartonella spp. cause persistent bacterial infections in mammals. Although these bacteria are transmitted by blood-feeding arthropods, there is also evidence for vertical transmission in their mammalian hosts. We aimed to determine: (i) the prevalence and diversity of Bartonella spp. in a Microtus spp. community; (ii) whether vertical transmission occurs from infected female voles to their offspring; (iii) the effect of concurrent Babesia microti infection on the success of vertical transmission of Bartonella; and (iv) the impact of congenital infection on pup survival. RESULTS: We sampled 124 Microtus arvalis, 76 Microtus oeconomus and 17 Microtus agrestis. In total, 115 embryos were isolated from 21 pregnant females. In the following year 11 pregnant females were kept until they had given birth and weaned their pups (n = 62). Blood smears and PCR targeting the Bartonella-specific rpoB gene fragment (333bp) were used for the detection of Bartonella. Bartonella DNA was detected in 66.8% (145/217) of the wild-caught voles. Bartonella infection was detected in 81.8% (36/44) of pregnant female voles. Bartonella-positive individuals were identified among the embryos (47.1%; 40/85) and in 54.8% (34/62) of pups. Congenitally acquired Bartonella infections and co-infection with B. microti had no impact on the survival of pups over a 3-week period post partum. Among 113 Bartonella sequences, four species were detected: Bartonella taylorii, Bartonella grahamii, Bartonella doshiae and a Bartonella rochalimae-like genotype. Bartonella taylorii clade B was the dominant species in wild-caught voles (49%), pregnant females (47%), their embryos (85%), dams (75%) and pups (95%). CONCLUSIONS: High prevalence of Bartonella spp. infection maintained in Microtus spp. community is followed by a high rate of vertical transmission of several rodent species of Bartonella in three species of naturally infected voles, M. arvalis, M. oeconomus and M. agrestis. Congenitally acquired Bartonella infection does not affect the survival of pups. Co-infection with B. microti does not affect the effectiveness of the vertical transmission of Bartonella in voles. Bartonella taylorii clade B was found to be the dominant species in wild-caught voles, including pregnant females and dams, and in their offspring, and was also found to be the most successful in vertical transmission.

Relative risks and approaches to biosecurity in the use of embryo technologies in livestock.

Embryo technologies have been integrated into production systems for a variety of livestock species. As relates to transmission of infectious diseases, our working hypothesis has been that use of embryo transfer for distribution of germ plasm within and between herds and flocks is likely safer than the movement of postnatal animals. Indeed, research and experience generally have been supportive of this hypothesis. However, the relative risks of transmitting infectious agents via embryo transfer vary among donor species. Further, different methods of producing embryos appear to present different risks. This paper provides a comparative overview of the risks of transmitting infectious diseases via transfer of both in vivo- and in vitro-derived embryos in common domesticated livestock species. Also discussed are universal approaches to biosecurity in embryo production and transfer.

Disinfection procedures for controlling microorganisms in the semen and embryos of humans and farm animals.

Semen and embryos generated by assisted reproductive techniques (ARTs) may be contaminated with numerous microorganisms. Contamination may arise from systemic or local reproductive tract infections in donors or the inadvertent introduction of microorganisms during ARTs, and may lead to disease transmission. This review describes sanitary procedures which have been investigated to ascertain whether they are effective in rendering semen and embryos free of pathogenic microorganisms, including internationally adopted washing procedures, which can be supplemented by antibiotics and enzymatic treatments. Other methods include treatment with antibodies or ozone, photoinactivation, acidification, and the use of novel antiviral compounds. In conclusion, despite the wide range of antimicrobial procedures available, none can be recommended as a universal disinfection method for rendering semen and embryos free from all potentially pathogenic microorganisms. However, some procedures are unsuitable, as they can compromise the viability of semen or embryos. In humans, washing by the gradient centrifugation method appears to be effective for reducing the microbial population in semen and is harmless to the spermatozoa. A useful procedure for embryos involving multiple washes in sterile medium has much to commend it for the prevention of disease transmission; furthermore, it is recommended by the International Embryo Transfer Society (IETS).

Whole Genome Amplification of Day 3 or Day 5 Human Embryos Biopsies Provides a Suitable DNA Template for PCR-Based Techniques for Genotyping, a Complement of Preimplantation Genetic Testing.

Our objective was to determine if whole genome amplification (WGA) provides suitable DNA for qPCR-based genotyping for human embryos. Single blastomeres (Day 3) or trophoblastic cells (Day 5) were isolated from 342 embryos for WGA. Comparative Genomic Hybridization determined embryo sex as well as Trisomy 18 or Trisomy 21. To determine the embryo's sex, qPCR melting curve analysis for SRY and DYS14 was used. Logistic regression indicated a 4.4%, 57.1%, or 98.8% probability of a male embryo when neither gene, SRY only, or both genes were detected, respectively (accuracy = 94.1%, kappa = 0.882, and p < 0.001). Fluorescent Capillary Electrophoresis for the amelogenin genes (AMEL) was also used to determine sex. AMELY peak's height was higher and this peak's presence was highly predictive of male embryos (AUC = 0.93, accuracy = 81.7%, kappa = 0.974, and p < 0.001). Trisomy 18 and Trisomy 21 were determined using the threshold cycle difference for RPL17 and TTC3, respectively, which were significantly lower in the corresponding embryos. The Ct difference for TTC3 specifically determined Trisomy 21 (AUC = 0.89) and RPL17 for Trisomy 18 (AUC = 0.94). Here, WGA provides adequate DNA for PCR-based techniques for preimplantation genotyping.

No evidence of Mycobacterium avium subsp. paratuberculosis in in vitro produced cryopreserved embryos derived from subclinically infected cows.

The aim of the project was to ascertain if Mycobacterium avium subsp. paratuberculosis (Map) could be cultured from frozen-thawed in vitro produced (IVP) embryos derived from cows with subclinical Johne's disease (JD). Straws of 109 IVP embryos were obtained from 267 cumulus-oocyte-complexes (COCs) collected from 12 clinically normal cows in which antibodies against Map were detected in blood by an enzyme-linked immunosorbent assay (ELISA). These embryos were processed, washed using the standard protocol as described by the International Embryo Transfer Society (IETS) and frozen in a commercial IVP embryo laboratory. Of the 12 donor cows, 11 had histopathological or bacteriological evidence of infection at post-mortem inspection. The frozen embryos were thawed and the contents of the straws were cultured using the radiometric mycobacterial culture method. No Map was detected in any of the 109 embryos or freezing media. This suggests that the use of in vitro produced and cryopreserved embryos derived from cows with subclinical JD poses very low, if any, risk of spreading infection to susceptible animals.

Risk of transmission of Mycobacterium avium ssp. paratuberculosis by embryo transfer of in vivo and in vitro fertilized bovine embryos.

Over a 5-year interval, experiments were conducted to determine if Mycobacterium avium ssp. paratuberculosis (Map) is associated with in vivo and in vitro fertilized (IVF) embryos and whether it can be transmitted by embryo transfer. The present studies included: collection of embryos from five asymptomatic, naturally infected donors and transfer to uninfected recipients; collection of oocytes from two naturally infected donors with overt clinical signs; exposure of in vivo and IVF embryos to Map and transfer to uninfected recipients; and the inoculation (transfer) of "clean" IVF embryos to the uterine lumen of infected cows. The presence of Map was confirmed in the uterine horns of all asymptomatic, infected donors. None of the tested embryos, which were not used for embryo transfer, or unfertilized ova (two per batch), were positive for Map, as determined by culture (n = 19) or by PCR (n = 13). However, all in vivo fertilized embryos exposed to Map in vitro (and subsequently sequentially washed) tested positive for Map, by both culture (12 batches) and PCR (15 batches), whereas IVF embryos treated in the same manner tested positive on culture (51%, 18/35 batches) and by PCR (28%, 20/71 batches). Transferring both in vivo embryos and IVF embryos potentially contaminated with Map into 28 recipients resulted in 13 pregnancies and eight calves born without evidence of disease transmission to either the recipients or the offspring over the following 5-year period. In samples collected from one of the clinically infected animals, two of seven (28%) cumulus oocyte complexes (COC) and follicular fluid tested positive by PCR and 10/10 cumulus oocyte complexes on culture for Map. From the second clinically infected cow, three of five batches of IVF embryos (n = 20) were positive on PCR and two of four batches containing unfertilized oocytes and embryos were positive on culture. Only 10% of embryos reached the morula and blastocyst stage 10 days after fertilization. In conclusion, Map is unlikely to be transmitted by embryo transfer when the embryos have been washed as recommended by the International Embryo Transfer Society.

Expression of virus-like particles in feline preimplantation embryos.

Embryos recovered from random-source domestic cats between 1 and 7 days after ovulation and unfertilized oocytes recovered within 6 hours of ovulation were examined ultrastructurally for the presence of types A and C viruses. Intracisternal type A particles were found consistently within cells of the inner cell mass in blastocysts but not within trophoblast cells or cells from earlier stages of development. Mature type C viruses were not observed. Novel virus-like particles were found within cytoplasmic cisternae in many of the embryos; these particles did not appear to be associated with a particular stage of embryonic development.

Viruslike particles in hamster embryos, fetuses, and tumors.

Intracisternal viruslike particles were present in outbred Syrian golden and inbred ALB/Mey Pfd hamster blastocysts, embryos, and fetuses. Their morphology was identical to that of the "spoked" viruslike particles found in hamster tumors. They were released in great numbers in the embryonal tissues until day 9 of pregnancy and were found until day 13 in the fetal tissues. Thereafter, these viruslike particles were no longer observed in the fetus. They were never recorded in normal adult hamster tissues.