Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation

The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. An alternative mechanism has been proposed by Morris (2006) that cell damage is a result of an osmotic imbalance encountered during thawing. This paper examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. Horse spermatozoa were cooled at a range of cooling rates from 0.3 to 3,000 degrees C/min in the presence of a cryoprotectant. The ultrastructure of the samples was examined by Cryo Scanning Electron Microscopy (CryoSEM) and freeze substitution, to determine whether intracellular ice formed and to examine alternative mechanisms of cell injury during rapid cooling. No intracellular ice formation was detected at any cooling rate. Differential scanning Calorimetry (DSC) was employed to examine the amount of ice formed at different rate of cooling. It is concluded that cell damage to horse spermatozoa, at cooling rates of up to 3,000 degrees C/min, is not caused by intracellular ice formation. Spermatozoa that have been cooled at high rates are subjected to an osmotic shock when they are thawed.     

First Outbreak Response Using an Oral Cholera Vaccine in Africa: Vaccine Coverage,Acceptability and Surveillance of Adverse Events,Guinea, 2012

Background

Despite World Health Organization (WHO) prequalification of two safe and effective oral cholera vaccines (OCV), concerns about the acceptability, potential diversion of resources, cost and feasibility of implementing timely campaigns has discouraged their use. In 2012, the Ministry of Health of Guinea, with the support of Médecins Sans Frontières organized the first mass vaccination campaign using a two-dose OCV (Shanchol) as an additional control measure to respond to the on-going nationwide epidemic. Overall, 316,250 vaccines were delivered. Here, we present the results of vaccination coverage, acceptability and surveillance of adverse events.

Methodology/Principal Findings

We performed a cross-sectional cluster survey and implemented adverse event surveillance. The study population included individuals older than 12 months, eligible for vaccination, and residing in the areas targeted for vaccination (Forécariah and Boffa, Guinea). Data sources were household interviews with verification by vaccination card and notifications of adverse events from surveillance at vaccination posts and health centres. In total 5,248 people were included in the survey, 3,993 in Boffa and 1,255 in Forécariah. Overall, 89.4% [95%CI:86.4–91.8%] and 87.7% [95%CI:84.2–90.6%] were vaccinated during the first round and 79.8% [95%CI:75.6–83.4%] and 82.9% [95%CI:76.6–87.7%] during the second round in Boffa and Forécariah respectively. The two dose vaccine coverage (including card and oral reporting) was 75.8% [95%CI: 71.2–75.9%] in Boffa and 75.9% [95%CI: 69.8–80.9%] in Forécariah respectively. Vaccination coverage was higher in children. The main reason for non-vaccination was absence. No severe adverse events were notified.

Conclusions/Significance

The well-accepted mass vaccination campaign reached high coverage in a remote area with a mobile population. Although OCV should not be foreseen as the long-term solution for global cholera control, they should be integrated as an additional tool into the response.     

Pregnancy Outcomes after a Mass Vaccination Campaign with an Oral Cholera Vaccine in Guinea: A Retrospective Cohort Study

Introduction

Since 2010, WHO has recommended oral cholera vaccines as an additional strategy for cholera control. During a cholera episode, pregnant women are at high risk of complications, and the risk of fetal death has been reported to be 2–36%. Due to a lack of safety data, pregnant women have been excluded from most cholera vaccination campaigns. In 2012, reactive campaigns using the bivalent killed whole-cell oral cholera vaccine (BivWC), included all people living in the targeted areas aged ≥1 year regardless of pregnancy status, were implemented in Guinea. We aimed to determine whether there was a difference in pregnancy outcomes between vaccinated and non-vaccinated pregnant women.

Methods and Findings

From 11 November to 4 December 2013, we conducted a retrospective cohort study in Boffa prefecture among women who were pregnant in 2012 during or after the vaccination campaign. The primary outcome was pregnancy loss, as reported by the mother, and fetal malformations, after clinical examination. Primary exposure was the intake of the BivWC vaccine (Shanchol) during pregnancy, as determined by a vaccination card or oral history. We compared the risk of pregnancy loss between vaccinated and non-vaccinated women through binomial regression analysis. A total of 2,494 pregnancies were included in the analysis. The crude incidence of pregnancy loss was 3.7% (95%CI 2.7–4.8) for fetuses exposed to BivWC vaccine and 2.6% (0.7–4.5) for non-exposed fetuses. The incidence of malformation was 0.6% (0.1–1.0) and 1.2% (0.0–2.5) in BivWC-exposed and non-exposed fetuses, respectively. In both crude and adjusted analyses, fetal exposure to BivWC was not significantly associated with pregnancy loss (adjusted risk ratio (aRR = 1.09 [95%CI: 0.5–2.25], p = 0.818) or malformations (aRR = 0.50 [95%CI: 0.13–1.91], p = 0.314).

Conclusions

In this large retrospective cohort study, we found no association between fetal exposure to BivWC and risk of pregnancy loss or malformation. Despite the weaknesses of a retrospective design, we can conclude that if a risk exists, it is very low. Additional prospective studies are warranted to add to the evidence base on OCV use during pregnancy. Pregnant women are particularly vulnerable during cholera episodes and should be included in vaccination campaigns when the risk of cholera is high, such as during outbreaks.     

Unexpectedly high susceptibility of micropropagated rhubarb (Rheum rhaponticum L.) to spot disease caused by Ramularia rhei

Rapid, large-scale micropropagation of a Rheum rhaponticum (rhubarb) breeding line, selected for the absence of autumn dormancy, produces changes typical of somaclonal variation. Among these is enhanced susceptibility to foliar infection by the spot disease pathogen Ramularia rhei, with consequential increased spore production on infected leaves. This provides an increased level of inoculum in the late season, leading to commercially unacceptable petiole spotting as spores are washed from the lamina on to the petiole surface. Infection of conventionally propagated plants does not exceed market acceptability. Consequently, to maintain the commercial value of this rhubarb selection micropropagation must be avoided, at least until an acceptably modified protocol is available.     

Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France

The prevalence of Clostridium botulinum types A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France. A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10 C. botulinum spores per 25 g and 100% of samples inoculated with more than 30 C. botulinum spores per 25 g. The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did. The prevalence of C. botulinum in seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2 C. botulinum most-probable-number counts, respectively, and is in the low range of C. botulinum contamination reported elsewhere. The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E. Type F was not detected. The high prevalence of C. botulinum type B in fish samples is relatively unusual compared with the high prevalence of C. botulinum type E reported in many worldwide and northern European surveys. However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism.     

Cellulose microfibril deposition at the plasmalemma surface of regenerating tobacco mesophyll protoplasts: A deep-etch study

Summary The reappearance of cellulose microfibrils at the naked surface of protoplasts enzymatically isolated from tobacco (Nicotiana tabacum L. var. Xanthi) mesophyll tissue has been closely studied using the techniques of thin-sectoining and the deep-etch modification of the freeze fracture procedure.A 16 h lag period was recorded between the time of isolation and the sudden appearance of considerable lengths of cellulose microfibril at the outer protoplast surface. The microfibrils were not associated with any structured particles or apparently differentiated regions of the plasmalemma. Terminal regions of the microfibrils appeared to have tapering ends, or else be sinking into the membrane substance. There was no evidence to suggest transport of intact microfibrils in vesicles through the cytoplasm to the plasmalemma.The reported observations have been discussed with respect to the various working hypotheses which have been proposed for the in vivo construction of cellulose microfibrils.     

Further observations on cell-wall formation around isolated protoplasts of tobacco and tomato

Freeze-etch observations of protoplasts isolated from tobacco (Nicotiana tabacum L.) mesophyll tissue and tomato (Lycopersicum esculentum Mill.) fruit locule tissue are described which clarify earlier observations (Burgess, J., Fleming, E.N., Planta 131, 173–178, 1976; Planta 133, 267–273, 1977), obtained using scanning electron microscopy. of fibres associated with projections from these cell surfaces. It is demonstrated (1) that the fibres consist of bundles of small numbers of microfibrils which have become artifactually thickened by the deposition of coating materials, and (2) that the apparent association between fibres and projections results from microfibrils being lifted preferentially from protoplast surfaces in regions rich in projections (plasmalemmasomes). With the higher resolution available using freeze-etching it can be demonstrated that microfibril deposition does not occur in discontinuous zones on these protoplast surfaces. Globules associated with microfibril termini in radish (Raphanus sativus L.) roots are illustrated and it is proposed that turgor pressure differences between isolated protoplasts and intact tissue may account for the absence of similar globules from isolated protoplast surfaces.     

Developmental rates at constant temperatures of three economically important Ceratitis spp. (Diptera: Tephritidae) from southern Africa

Three species of Ceratitis MacLeay are of economic importance in southern Africa. To learn more about the influence of temperature on the development of these species, the developmental rates of South African populations of Ceratitis (Ceratitis) capitata (Wiedemann), C. (Pterandrus) rosa Karsch, and C. (Ceratalaspis) cosyra (Walker) were compared at constant temperatures of 14, 18, 22, 26, and 30 degrees C. The duration of each life stage and the percentage survival of the immature life stages of each species were determined. One linear and three nonlinear developmental rate models (Briére, Lactin, and Logan-6) were found to fit the data well and were used to generate the minimum, optimum, and maximum developmental thresholds, in addition to the life cycle thermal constants for the three species. These parameter values were 9.6, 28.5, 33.0, and 338 for C. capitata, 9.7, 28.8, 33.2, and 376 for C. cosyra, and 8.6, 27.7, 33.0, and 429 for C. rosa, respectively. The parameters for C. capitata are similar to those found by other researchers for this species in Reunion but the parameters for C. rosa differ substantially from published values for a Reunion population of this species, suggesting that these are different biotypes. The similarities between the developmental parameters for C. capitata and C. cosyra do not support known differences in the distribution of these species so other limiting factors such as relative humidity and the availability of host species may be important. This finding therefore cautions against basing predictions of potential global distributions of species solely on life table or climatic parameter values.