Experimental inoculation of male rats with Coxiella burnetii: successful infection but no transmission to cage mates

Beginning in 2007, the largest human Q fever outbreak ever described occurred in the Netherlands. Dairy goats from intensive farms were identified as the source, amplifying Coxiella burnetii during gestation and shedding large quantities during abortions. It has been postulated that wild rodents are reservoir hosts from which C. burnetii can be transmitted to domestic animals and humans. However, little is known about the infection dynamics of C. burnetii in wild rodents. The aim of this study was to investigate whether brown rats (Rattus norvegicus) can be experimentally infected with C. burnetii and whether transmission to a cage mates occurs. Fourteen male brown rats (wild type) were intratracheally or intranasally inoculated with a Dutch C. burnetii isolate obtained from a goat. At 3 days postinoculation, a contact rat was placed with each inoculated rat. The pairs were monitored using blood samples and rectal and throat swabs for 8 weeks, and after euthanasia the spleens were collected. Rats became infected by both inoculation routes, and detection of C. burnetii DNA in swabs suggests that excretion occurred. However, based on the negative spleens in PCR and the lack of seroconversion, none of the contact animals was considered infected; thus, no transmission was observed. The reproduction ratio R(0) was estimated to be 0 (95% confidence interval = 0 to 0.6), indicating that it is unlikely that rats act as reservoir host of C. burnetii through sustained transmission between male rats. Future research should focus on other transmission routes, such as vertical transmission or bacterial shedding during parturition.     

Haemodynamics using transthoracic echocardiography in healthy pregnant and non-pregnant baboons (Papio hamadryas)

Background To determine systolic and diastolic function using transthoracic echocardiography in the baboon (Papio hamadryas). Methods Transthoracic echocardiography was performed in eight non‐pregnant female and six pregnant baboons according to American Society of Echocardiography recommendations. Results Haemodynamic measurements were obtained from fourteen baboons. Compared to non‐pregnant baboons, pregnant baboons demonstrated: (mean ± SD, pregnant vs. healthy) increased cardiac output (1615 ± 121 ml/minutes vs. 1317 ± 134 ml/minutes P = 0.001) due to an increased heart rate [120 ± 11 beats per minute (BPM) vs. 105 ± 6 BPM P = 0.018]. The inter‐observer and intra‐observer variability (mean difference ± SD) for the left ventricular outflow tract diameter was 0.05 ± 0.07 cm and 0.01 ± 0.03 cm respectively. There was minimal impact to the animal’s daily activities. Conclusions Transthoracic echocardiography was applicable and reproducible for the assessment of haemodynamics in baboons thus enabling translation of animal results to human studies.     

Iron availability increases the pathogenic potential of Salmonella typhimurium and other enteric pathogens at the intestinal epithelial interface

Recent trials have questioned the safety of untargeted oral iron supplementation in developing regions. Excess of luminal iron could select for enteric pathogens at the expense of beneficial commensals in the human gut microflora, thereby increasing the incidence of infectious diseases. The objective of the current study was to determine the effect of high iron availability on virulence traits of prevalent enteric pathogens at the host-microbe interface. A panel of enteric bacteria was cultured under iron-limiting conditions and in the presence of increasing concentrations of ferric citrate to assess the effect on bacterial growth, epithelial adhesion, invasion, translocation and epithelial damage in vitro. Translocation and epithelial integrity experiments were performed using a transwell system in which Caco-2 cells were allowed to differentiate to a tight epithelial monolayer mimicking the intestinal epithelial barrier. Growth of Salmonella typhimurium and other enteric pathogens was increased in response to iron. Adhesion of S. typhimurium to epithelial cells markedly increased when these bacteria were pre-incubated with increasing iron concentration (P = 0.0001), whereas this was not the case for the non-pathogenic Lactobacillus plantarum (P = 0.42). Cellular invasion and epithelial translocation of S. typhimurium followed the trend of increased adhesion. Epithelial damage was increased upon incubation with S. typhimurium or Citrobacter freundii that were pre-incubated under iron-rich conditions. In conclusion, our data fit with the consensus that oral iron supplementation is not without risk as iron could, in addition to inducing pathogenic overgrowth, also increase the virulence of prevalent enteric pathogens.     

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions ¹. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage ^{2, 3}. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle ^4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection ⁷. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization ⁸. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.     

The Co-Chaperone Hch1 Regulates Hsp90 Function Differently than Its Homologue Aha1 and Confers Sensitivity to Yeast to the Hsp90 Inhibitor NVP-AUY922

Hsp90 is a dimeric ATPase responsible for the activation or maturation of a specific set of substrate proteins termed ‘clients’. This molecular chaperone acts in the context of a structurally dynamic and highly regulated cycle involving ATP, co-chaperone proteins and clients. Co-chaperone proteins regulate conformational transitions that may be impaired in mutant forms of Hsp90. We report here that the in vivo impairment of commonly studied Hsp90 variants harbouring the G313S or A587T mutation are exacerbated by the co-chaperone Hch1p. Deletion of HCH1, but not AHA1, mitigates the temperature sensitive phenotype and high sensitivity to Hsp90 inhibitor drugs observed in Saccharomyces cerevisiae that express either of these two Hsp90 variants. Moreover, the deletion of HCH1 results in high resistance to Hsp90 inhibitors in yeast that express wildtype Hsp90. Conversely, the overexpression of Hch1p greatly increases sensitivity to Hsp90 inhibition in yeast expressing wildtype Hsp90. We conclude that despite the similarity between these two co-chaperones, Hch1p and Aha1p regulate Hsp90 function in distinct ways and likely independent of their roles as ATPase stimulators. We further conclude that Hch1p plays a critical role in regulating Hsp90 inhibitor drug sensitivity in yeast.     

Effect of substrate stiffness on early mouse embryo development

It is becoming increasingly clear that cells are remarkably sensitive to the biophysical cues of their microenvironment and that these cues play a significant role in influencing their behaviors. In this study, we investigated whether the early pre-implantation embryo is sensitive to mechanical cues, i.e. the elasticity of the culture environment. To test this, we have developed a new embryo culture system where the mechanical properties of the embryonic environment can be precisely defined. The contemporary standard environment for embryo culture is the polystyrene petri dish (PD), which has a stiffness (1 GPa) that is six orders of magnitude greater than the uterine epithelium (1 kPa). To approximate more closely the mechanical aspects of the in vivo uterine environment we used polydimethyl-siloxane (PDMS) or fabricated 3D type I collagen gels (1 kPa stiffness, Col-1k group). Mouse embryo development on alternate substrates was compared to that seen on the petri dish; percent development, hatching frequency, and cell number were observed. Our results indicated that embryos are sensitive to the mechanical environment on which they are cultured. Embryos cultured on Col-1k showed a significantly greater frequency of development to 2-cell (68±15% vs. 59±18%), blastocyst (64±9.1% vs. 50±18%) and hatching blastocyst stages (54±25% vs. 21±16%) and an increase in the number of trophectodermal cell (TE,65±13 vs. 49±12 cells) compared to control embryos cultured in PD (mean±S.D.; p<.01). Embryos cultured on Col-1k and PD were transferred to recipient females and observed on embryonic day 12.5. Both groups had the same number of fetuses, however the placentas of the Col-1k fetuses were larger than controls, suggesting a continued effect of the preimplantation environment. In summary, characteristics of the preimplantation microenvironment affect pre- and post-implantation growth.     

Gut bacteria in health and disease: a survey on the interface between intestinal microbiology and colorectal cancer

A healthy human body contains at least tenfold more bacterial cells than human cells and the most abundant and diverse microbial community resides in the intestinal tract. Intestinal health is not only maintained by the human intestine itself and by dietary factors, but is also largely supported by this resident microbial community. Conversely, however, a large body of evidence supports a relationship between bacteria, bacterial activities and human colorectal cancer. Symbiosis in this multifaceted organ is thus crucial to maintain a healthy balance within the host-diet-microbiota triangle and accordingly, changes in any of these three factors may drive a healthy situation into a state of disease. In this review, the factors that sustain health or drive this complex intestinal system into dysbiosis are discussed. Emphasis is on the role of the intestinal microbiota and related mechanisms that can drive the initiation and progression of sporadic colorectal cancer (CRC). These mechanisms comprise the induction of pro-inflammatory and pro-carcinogenic pathways in epithelial cells as well as the production of (geno)toxins and the conversion of pro-carcinogenic dietary factors into carcinogens. A thorough understanding of these processes will provide leads for future research and may ultimately aid in development of new strategies for CRC diagnosis and prevention.     

Spontaneous cyclic embryonic movements in humans and guinea pigs

Motility assessment before birth can be used to evaluate the integrity of the nervous system. Sideways bending (SB) of head and/or rump, the earliest embryonic motility in both humans and guinea pigs, can be visualized sonographically. We know from other species that early embryonic motility is cyclic. This study explores the distribution of SB-to-SB intervals in human and guinea pig embryos before the appearance of more complex movements such as general movements. We hypothesized that the activity in both species is cyclic. We made 15-min sonographic recordings of SBs between 5 weeks and 0 days (5wk0d) and 7wk0d conceptional age (CA) in 18 human embryos of uncomplicated IVF pregnancies (term 38 weeks) and in 20 guinea pig embryos between 3wk4d and 4wk0d CA (term 9 weeks). SB-to-SB interval durations were categorized as long (≥10 s) or short (<10 s) intervals. For human embryos, the median values for long and short intervals were 61 s (range, 10-165 s) and 3 s (range, 1-9 s) respectively; for guinea pigs 38 s (range, 10-288 s) and 5 s (range, 1-9 s), respectively. During development, the duration of long intervals decreased while the number of short intervals increased for both species. The earliest embryonic motility in the human and guinea pig is performed cyclically with distinct developmental milestones. The resemblance of their interval development offers promising possibilities to use the guinea pig as a noninvasive animal model of external influences on motor and neural development.     

Mitochondrial nucleotide variability in invasive populations of the root weevil Diaprepes abbreviatus (Coleoptera: Curculionidae) of Florida and preliminary assessment of Diaprepes sp. from Dominica

Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) is a root weevil introduced into the United States from the Caribbean in 1964. It is associated with >300 plants, including citrus, sugarcane, and potatoes. D. abbreviatus is widespread in Florida, and it has recently been detected in limited areas of California and Texas. The purpose of this research is to evaluate the utility of 16S ribosomal (16S rRNA) and cytochrome oxidase I (COI) mitochondrial markers for the delineation of genetic populations of D. abbreviatus in Florida and for the characterization of patterns of dispersion among these populations. We also assessed these markers as genetic tools for the clarification of taxonomic uncertainties in specimens from Dominica (Lesser Antilles). We analyzed 111 weevils from six Florida populations and six specimens from Dominica. In Florida, we found three haplotypes with only one haplotype in each population. Florida haplotypes differed by one to three nucleotide substitutions, possibly the result of a recent divergence from one source population or three different introductions from closely related populations from the Caribbean. In contrast, specimens from Dominica showed a high genetic variability with three 16S haplotypes and six unique COI haplotypes, delineating two mitochondrial clades. We show that these mitochondrial markers are useful for phylogeographic studies of D. abbreviatus.