Investigation of the Causes of Shigatoxigenic Escherichia coli PCR Positive and Culture Negative Samples

peer-reviewedMolecular methods may reveal the presence of pathogens in samples through the detection of specific target gene(s) associated with microorganisms, but often, the subsequent cultural isolation of the pathogen is not possible. This discrepancy may be related to low concentration of the cells, presence of dead cells, competitive microflora, injured cells and cells in a viable but non-culturable state, free DNA and the presence of free bacteriophages which can carry the target gene causing the PCR-positive/culture-negative results. Shiga-toxigenic Escherichia coli (STEC) was used as a model for studying this phenomenon, based on the phage-encoded cytotoxins genes (Stx family) as the detection target in samples through real-time qPCR. Stx phages can be integrated in the STEC chromosome or can be isolated as free particles in the environment. In this study, a combination of PCR with culturing was used for investigating the presence of the stx1 and stx2 genes in 155 ovine recto-anal junction swab samples (method (a)-PCR). Samples which were PCR-positive and culture-negative were subjected to additional analyses including detection of dead STEC cells (method (b)-PCR-PMA dye assay), presence of Stx phages (method (c)-plaque assays) and inducible integrated phages (method (d)-phage induction). Method (a) showed that even though 121 samples gave a PCR-positive result (78%), only 68 samples yielded a culturable isolate (43.9%). Among the 53 (34.2%) PCR-positive/culture-negative samples, 21 (39.6%) samples were shown to have STEC dead cells only, eight (15.1%) had a combination of dead cells and inducible stx phage, while two samples (3.8%) had a combination of dead cells, inducible phage and free stx phage, and a further two samples had Stx1 free phages only (3.8%). It was thus possible to reduce the samples with no explanation to 20 (37.7% of 53 samples), representing a further step towards an improved understanding of the STEC PCR-positive/culture-negative phenomenon

Daphnia predation on the amphibian chytrid fungus and its impacts on disease risk in tadpoles

Direct predation upon parasites has the potential to reduce infection in host populations. For example, the fungal parasite of amphibians, B atrachochytrium dendrobatidis ( B d), is commonly transmitted through a free‐swimming zoospore stage that may be vulnerable to predation. Potential predators of B d include freshwater zooplankton that graze on organisms in the water column. We tested the ability of two species of freshwater crustacean ( D aphnia magna and D . dentifera ) to consume B d and to reduce B d density in water and infection in tadpoles. In a series of laboratory experiments, we allowed D aphnia to graze in water containing B d while manipulating D aphnia densities, D aphnia species identity, grazing periods and concentrations of suspended algae ( A nkistrodesmus falcatus ). We then exposed tadpoles to the grazed water. We found that high densities of D . magna reduced the amount of Bd detected in water, leading to a reduction in the proportion of tadpoles that became infected. Daphnia dentifera , a smaller species of D aphnia , also reduced B d in water samples, but did not have an effect on tadpole infection. We also found that algae affected B d in complex ways. When D aphnia were absent, less B d was detected in water and tadpole samples when concentrations of algae were higher, indicating a direct negative effect of algae on B d. When D aphnia were present, however, the amount of B d detected in water samples showed the opposite trend, with less B d when densities of algae were lower. Our results indicate that D aphnia can reduce B d levels in water and infection in tadpoles, but these effects vary with species, algal concentration, and D aphnia density. Therefore, the ability of predators to consume parasites and reduce infection is likely to vary depending on ecological context. We tested the ability of two species of freshwater crustacean ( Daphnia magna and D. dentifera ) to consume zoospores of the amphibian parasite, Batrachochytrium dendrobatidis (Bd), and to reduce parasite density in water and infection in tadpoles. In a series of laboratory experiments, we allowed Daphnia to graze in water containing Bd, then exposed tadpoles to the grazed water. Our results show that Daphnia can reduce Bd levels in water and infection in tadpoles, but these effects vary with species, algal concentration and Daphnia density.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/100311/1/ece3777.pd

Mammalian oocytes are targets for prostaglandin E2 (PGE2) action

<p>Abstract</p> <p>Background</p> <p>The ovulatory gonadotropin surge increases synthesis of prostaglandin E2 (PGE2) by the periovulatory follicle. PGE2 actions on granulosa cells are essential for successful ovulation. The aim of the present study is to determine if PGE2 also acts directly at the oocyte to regulate periovulatory events.</p> <p>Methods</p> <p>Oocytes were obtained from monkeys and mice after ovarian follicular stimulation and assessed for PGE2 receptor mRNA and proteins. Oocytes were cultured with vehicle or PGE2 and assessed for cAMP generation, resumption of meiosis, and in vitro fertilization.</p> <p>Results</p> <p>Germinal vesicle intact (GV) oocytes from both monkeys and mice expressed mRNA for the PGE2 receptors EP2, EP3, and EP4. EP2 and EP4 proteins were detected by confocal microscopy in oocytes of both species. Monkey and mouse oocytes responded to PGE2 as well as agonists selective for EP2 and EP4 receptors with elevated cAMP, consistent with previous identification of EP2 and EP4 as Gαs/adenylyl cyclase coupled receptors. Incubation of mouse GV stage oocytes with PGE2 delayed oocyte nuclear maturation in vitro, but PGE2 treatment did not alter the percentage of mouse oocytes that fertilized successfully. PGE2 treatment also decreased the percentage of monkey oocytes that resumed meiosis in vitro. In contrast with mouse oocytes, the percentage of monkey oocytes which fertilized in vitro was lower after treatment with PGE2. Monkey oocytes with intact cumulus showed delayed nuclear maturation, but fertilization rate was not affected by PGE2 treatment.</p> <p>Conclusions</p> <p>Monkey and mouse oocytes express functional PGE2 receptors. PGE2 acts directly at mammalian oocytes to delay nuclear maturation. Surrounding cumulus cells modulate the effect of PGE2 to alter subsequent fertilization.</p

Effect of suckler cow vaccination against glycoprotein E (gE)-negative bovine herpesvirus type 1 (BoHV-1) on passive immunity and physiological response to subsequent bovine respiratory disease vaccination of their progeny

peer-reviewedThe study objectives were: 1) to characterise the development of immunocompetence in beef suckler calves from birth to three months of age, and 2) to trace glycoprotein E (gE)-negative bovine herpesvirus type 1 (BoHV-1) antibodies from dam to calf and subsequent vaccination against pneumonia. Thirty multiparous beef suckler, spring-calving cows, consisting of two genotypes were involved; Limousin × Friesian (LF) and Charolais × Limousin (CL). Cows were immunised against the inactivated antigen strain of BoHV-1 (gE- (IBR marker vaccine) at day − 84 and received a booster at day − 56 relative to the expected calving date (d 0). Calves were immunised at 14 and 42 days of age against PI-3 virus, BRSV and Mannheimia (Pasteurella) haemolytica serotype A1 using a commercial vaccine administered subcutaneously. Additionally, calves were immunised against BoHV-1 at 42 days of age, using 1 dose of a live commercial vaccine administered intranasally. Blood samples were collected from all calves (n = 30) via jugular venipuncture at birth, prior to colostrum feeding (0 h), at 12 h (h), 24 h, 72 h and 168 h after the initial feeding of colostrum, and at d 7, 14, 28, 42, 56 and 84 post birth. The mean ratio of gE negative antibodies circulating in the blood of LF and CL dams pre-partum scored negative to gE ab (S/N ≥ 0.70). Antibody levels of BoHV-1 (wild type (wt)) peaked at 12 h post-birth in calves and declined thereafter, as the maternal antibodies decayed. There was no difference in BoHV-1 and BRSV antibody levels in calves post vaccination.This research was funded by the Irish Government under the National Development Plan 2007-2013, Department of Agriculture, Food and the Marine (DAFM) Research Stimulus Fund ((Grant number: 11/S/131) (B. Earley, Principal Investigator)). Katie Tiernan was in receipt of a post-graduate fellowship as part of 11/S/131

The mental health benefits of having dogs on college campuses

College can be a stressful time for many, especially those dealing with problems like stress, anxiety, and depression. While colleges and universities typically offer counseling services, many students who could benefit do not take advantage of them. Research suggests that animals may play an important role in some forms of treatment for conditions such as anxiety, stress, and depression. A recent trend on college campuses is to offer opportunities to interact with dogs and other animals as a way to relieve stress and help individuals deal with other psychological issues. In this paper, we discuss the current research regarding using animals to assist in treatment for problems commonly faced by college students.We also describe our own experiences with a university-sponsored animal-interaction day and offer tips on how others can plan such an event on their campus

A Signal Model for Forensic DNA Mixtures

For forensic purposes, short tandem repeat allele signals are used as DNA fingerprints. The interpretation of signals measured from samples has traditionally been conducted by applying thresholding. More quantitative approaches have recently been developed, but not for the purposes of identifying an appropriate signal model. By analyzing data from 643 single person samples, we develop such a signal model. Three standard classes of two-parameter distributions, one symmetric (normal) and two right-skewed (gamma and log-normal), were investigated for their ability to adequately describe the data. Our analysis suggests that additive noise is well modeled via the log-normal distribution class and that variability in peak heights is well described by the gamma distribution class. This is a crucial step towards the development of principled techniques for mixed sample signal deconvolution