Small Interfering RNA Mediated Messenger RNA Knockdown in the Amphibian Pathogen Batrachochytrium dendrobatidis.

RNA interference (RNAi) has not been tested in the pandemic amphibian pathogen, Batrachochytrium dendrobatidis, but developing this technology could be useful to elucidate virulence mechanisms, identify therapeutic targets, and may present a novel antifungal treatment option for chytridiomycosis. To manipulate and decipher gene function, rationally designed small interfering RNA (siRNA) can initiate the destruction of homologous messenger RNA (mRNA), resulting in the "knockdown" of target gene expression. Here, we investigate whether siRNA can be used to manipulate gene expression in B. dendrobatidis via RNAi using differing siRNA strategies to target genes involved in glutathione and ornithine synthesis. To determine the extent and duration of mRNA knockdown, target mRNA levels were monitored for 24-48 h after delivery of siRNA targeting glutamate-cysteine ligase, with a maximum of ~56% reduction in target transcripts occurring at 36 h. A second siRNA design targeting glutamate-cysteine ligase also resulted in ~53% knockdown at this time point. siRNA directed toward a different gene target, ornithine decarboxylase, achieved 17% reduction in target transcripts. Although no phenotypic effects were observed, these results suggest that RNAi is possible in B. dendrobatidis, and that gene expression can be manipulated in this pathogen. We outline ideas for further optimization steps to increase knockdown efficiency to better harness RNAi techniques for control of B. dendrobatidis.

The impact of antenatal imaging on parent experience and prenatal attachment: a systematic review.

INTRODUCTION: Medical imaging in pregnancy (antenatal imaging) is routine. However, the effect of seeing fetal images on the parent-fetal relationship is not well understood, particularly for fathers or partners, or when using advanced imaging technologies. This review aimed to explore how parent experience and prenatal attachment is impacted by antenatal imaging. METHOD: Database searches were performed between September 2020 and April 2021 Inclusion criteria were English language primary research studies published since 2000, describing or reporting measures of attachment after antenatal imaging in expectant parents. The Pillar Integration Process was used for integrative synthesis. FINDINGS: Twenty-three studies were included. Six pillar themes were developed: 1) the scan experience begins before the scan appointment; 2) the scan as a pregnancy ritual; 3) feeling actively involved in the scan; 4) parents' priorities for knowledge and understanding of the scan change during pregnancy; 5) the importance of the parent-sonographer partnership during scanning; and 6) scans help to create a social identity for the unborn baby. CONCLUSION: Antenatal imaging can enhance prenatal attachment. Parents value working collaboratively with sonographers to be actively involved in the experience. Sonographers can help facilitate attachment by delivering parent-centred care tailored to parents' emotional and knowledge needs.

When birth is not as expected: a systematic review of the impact of a mismatch between expectations and experiences.

BACKGROUND: Pregnancy and childbirth are significant events in women's lives and most women have expectations or plans for how they hope their labour and birth will go. It is possible that strong expectations about labour and birth lead to dissatisfaction or other negative outcomes if these expectations are not met, but it is not clear if this is the case. The aim was therefore to synthesise prospective studies in order to understand whether unmet birth expectations are associated with adverse outcomes for women, their partners and their infants. METHOD: Searches were carried out in Academic Search Complete; CINAHL; Medline; PsycINFO, PsychArticles, PubMed, SCOPUS and Web of Science. Forward and backward searches were also completed. Studies were included if they reported prospective empirical research that examined the association between a mismatch in birth expectations/experience and postnatal outcomes in women, their children and/or their partners. Data were synthesised qualitatively using a narrative approach where study characteristics, context and methodological quality were extracted and summarised and then the differences and similarities among studies were used to draw conclusions. RESULTS: Eleven quantitative studies were identified for inclusion from nine countries. A mismatch between birth expectations and experiences was associated with reduced birth satisfaction. Three studies found a link between a mismatch and the development of postnatal post-traumatic stress disorder (PTSD). The evidence was inconsistent for postnatal depression, and fear of childbirth. Only one study looked at physical outcomes in the form of health-related quality of life. CONCLUSIONS: A mismatch between birth expectations and experiences is associated with birth satisfaction and it may increase the risk of developing postnatal PTSD. However, it is not clear whether a mismatch is associated with other postnatal mental health conditions. Further prospective research is needed to examine gaps in knowledge and provide standardised methods of measuring childbirth expectations-experiences mismatch. To ensure women's expectations are met, and therefore experience a satisfying birth experience, maternity providers should provide sensitive care, which acknowledges women's needs and preferences, is based on open and clear communication, is delivered as early in pregnancy as possible, and enables women to make their own decisions about care. TRIAL REGISTRATION: Protocol registration: PROSPERO CRD42020191081 .

Susceptibility of frogs to chytridiomycosis correlates with increased levels of immunomodulatory serotonin in the skin.

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a skin disease responsible for the global decline of amphibians. Frog species and populations can vary in susceptibility, but this phenomenon remains poorly understood. Here, we investigated serotonin in the skin of infected and uninfected frogs. In more susceptible frog populations, skin serotonin rose with increasing infection intensity, but decreased in later stages of the disease. The more resistant population maintained a basal level of skin serotonin. Serotonin inhibited both Bd sporangial growth and Jurkat lymphocyte proliferation in vitro. However, serotonin accumulates in skin granular glands, and this compartmentalisation may prevent inhibition of Bd growth in vivo. We suggest that skin serotonin increases in susceptible frogs due to pathogen excretion of precursor tryptophan, but that resistant frogs are able to control the levels of serotonin. Overall, the immunosuppressive effects of serotonin may contribute to the susceptibility of frogs to chytridiomycosis.

The efficacy and pharmacokinetics of terbinafine against the frog-killing fungus (Batrachochytrium dendrobatidis).

Captive and wild amphibians are under threat of extinction from the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd). The antifungal drug terbinafine (TBF) is used by pet owners to treat Bd-infected frogs; however, it is not widely used in academic or zoological institutions due to limited veterinary clinical trials. To assess TBF's efficacy, we undertook treatment trials and pharmacokinetic studies to investigate drug absorption and persistence in frog skin; and then we correlated these data to the minimal lethal concentrations (MLC) against Bd. Despite an initial reduction in zoospore load, the recommended treatment (five daily 5 min 0.01% TBF baths) was unable to cure experimentally infected alpine tree frogs and naturally infected common eastern froglets. In vitro and in vivo pharmacokinetics showed that absorbed TBF accumulates in frog skin with increased exposure, indicating its suitability for treating cutaneous pathogens via direct application. The MLC of TBF for zoosporangia was 100 µg/ml for 2 h, while the minimal inhibitory concentration was 2 µg/ml, suggesting that the drug concentration absorbed during 5 min treatments is not sufficient to cure high Bd burdens. With longer treatments of five daily 30 min baths, Bd clearance improved from 12.5% to 50%. A higher dose of 0.02% TBF resulted in 78% of animals cured; however, clearance was not achieved in all individuals due to low TBF skin persistence, as the half-life was less than 2 h. Therefore, the current TBF regime is not recommended as a universal treatment against Bd until protocols are optimized, such as with increased exposure frequency.

Pregnancy related risk perception in pregnant women, midwives & doctors: a cross-sectional survey.

BACKGROUND: Risk perception in relation to pregnancy and birth is a complex process influenced by multiple personal, psychological and societal factors. Traditionally, the risk perception of healthcare professionals has been presented as more objective and authoritative than that of pregnant women. Doctors have been presented as more concerned with biomedical risk than midwives. Such dichotomies oversimplify and obscure the complexity of the process. This study examines pregnancy-related risk perception in women and healthcare professionals, and what women and professionals believe about each other's risk perception. METHODS: A cross sectional survey of set in UK maternity services. Participants were doctors working in obstetrics (N = 53), midwives (N = 59), pregnant women (N = 68). Participants were recruited in person from two hospitals. Doctors were also recruited online. Participants completed a questionnaire measuring the degree of perceived risk in various childbirth-related scenarios; and the extent to which they believed others agreed with them about the degree of risk generally involved in childbirth. Main outcome measures were the degree of risk perceived to the mother in baby in pregnancy scenarios, and beliefs about own perception of risk in comparison to their own group and other groups. RESULTS: There were significant differences in total risk scores between pregnant women, doctors and midwives in perception of risk to the mother in 68/80 scenarios. Doctors most frequently rated risks lowest. Total scores for perceived risk to the baby were not significantly different. There was substantial variation within each group. There was more agreement on the ranking of scenarios according to risk. Each group believed doctors perceived most risk whereas actually doctors most frequently rated risks lowest. Each group incorrectly believed their peers rated risk similarly to themselves. CONCLUSIONS: Individuals cannot assume others share their perception of risk or that they make correct assessments regarding others' risk perception. Further research should consider what factors are taken into account when making risk assessments.

What are women stressed about after birth?

BACKGROUND: Having a baby is associated with a variety of stressors, change, and adjustment. This study aimed to identify what women find stressful during the early postpartum period in contemporary Western society. METHODS: Women (n = 148) 6-12 weeks postpartum wrote anonymously about a situation they found stressful as part of the Health after Birth Trial (HABiT) of expressive writing. Transcripts were analyzed for categories of stressors and cross-cutting themes. RESULTS: Five categories of stressors were identified. Stressors in pregnancy, labor, and the early postpartum period (49.3%) included physical and emotional difficulties, and insensitive treatment by health professionals. Stressors related to adjusting to life with a baby (35.8%) included difficulties coping with a new baby, parenting, juggling responsibilities, changes to physical health, and loneliness. Stressors related to the baby's health (32.4%) included infant digestive problems, acute health problems, long-term impact, and neonatal intensive care unit experiences. Stressors related to breastfeeding (23.7%) included pressure to breastfeed, feeling like a 'bad mum' for not breastfeeding, or wanting to breastfeed and not being able to. Other stressors related to changing relationships (18.2%): with their partner, children, and other family members. Cross-cutting themes that emerged in different stressor categories were women making negative self-appraisals (eg, a bad mum, failure), feeling guilty, and lack of support from others. DISCUSSION: Our findings emphasize the importance of exploring stressors and psychological well-being with women to provide support, help women's adjustment postpartum, and ensure interventions are offered when appropriate.

End of season influenza vaccine effectiveness in adults and children in the United Kingdom in 2017/18.

BackgroundIn the United Kingdom (UK), in recent influenza seasons, children are offered a quadrivalent live attenuated influenza vaccine (LAIV4), and eligible adults mainly trivalent inactivated vaccine (TIV).AimTo estimate the UK end-of-season 2017/18 adjusted vaccine effectiveness (aVE) and the seroprevalence in England of antibodies against influenza viruses cultured in eggs or tissue.MethodsThis observational study employed the test-negative case-control approach to estimate aVE in primary care. The population-based seroprevalence survey used residual age-stratified samples.ResultsInfluenza viruses A(H3N2) (particularly subgroup 3C.2a2) and B (mainly B/Yamagata/16/88-lineage, similar to the quadrivalent vaccine B-virus component but mismatched to TIV) dominated. All-age aVE was 15% (95% confidence interval (CI): -6.3 to 32) against all influenza; -16.4% (95% CI: -59.3 to 14.9) against A(H3N2); 24.7% (95% CI: 1.1 to 42.7) against B and 66.3% (95% CI: 33.4 to 82.9) against A(H1N1)pdm09. For 2-17 year olds, LAIV4 aVE was 26.9% (95% CI: -32.6 to 59.7) against all influenza; -75.5% (95% CI: -289.6 to 21) against A(H3N2); 60.8% (95% CI: 8.2 to 83.3) against B and 90.3% (95% CI: 16.4 to 98.9) against A(H1N1)pdm09. For ≥ 18 year olds, TIV aVE against influenza B was 1.9% (95% CI: -63.6 to 41.2). The 2017 seroprevalence of antibody recognising tissue-grown A(H3N2) virus was significantly lower than that recognising egg-grown virus in all groups except 15-24 year olds.ConclusionsOverall aVE was low driven by no effectiveness against A(H3N2) possibly related to vaccine virus egg-adaption and a new A(H3N2) subgroup emergence. The TIV was not effective against influenza B. LAIV4 against influenza B and A(H1N1)pdm09 was effective.

Influence of length of incision and number of suture lines on the biomechanical properties of incisional gastropexy.

OBJECTIVE: To determine the effect of the length of incision and of the number of suture lines on the load to failure of incisional gastropexy in an ex vivo model. STUDY DESIGN: Ex vivo study. SAMPLE POPULATION: Thirty-six hound-mix fresh canine cadavers. METHODS: Specimens were randomly divided into four groups of incisional gastropexies varying in length of incision (2 or 4 cm) and number of suture lines (one or two). Load to failure was measured. Number of suture bites on each side of the gastropexy and number of inadvertent full thickness gastric suture bites were recorded. RESULTS: Incisional gastropexies performed with one or two suture lines sustained loads to failure of 53.80 ± 12.10 N and 53.30 ± 10.60 N (P = .887), respectively. Loads to failure equal to 49.70 ± 10.80 N and 57.30 ± 10.60 N (P = .048) were measured on incisional gastropexies performed with 2- or 4-cm-suture lines, respectively. There was no interaction between the length of the incision and the number of suture lines (P = .634). CONCLUSION: Length of incision but not number of suture lines influenced the biomechanical properties of gastropexies in this acute cadaveric model. CLINICAL SIGNIFICANCE: According to this acute in vitro experiment, gastropexy can be performed with either one or two suture lines.

Cognitive outcomes following epilepsy in infancy: A longitudinal community-based study.

OBJECTIVE: Onset of epilepsy before 2 years of age is associated with poor cognitive outcome; however, the natural course of the range of epilepsies that occur at this age is unknown. The aim of this prospective community-based study was to investigate the neuropsychological development of infants with newly diagnosed epilepsy longitudinally and to identify the clinical factors that predict long-term impairment. METHODS: Sixty-six infants <24 months of age were enrolled in the baseline phase of this study; 40 were seen again at 1-year follow-up and 40 at 3-year follow-up. Children underwent a neurological and neuropsychological assessment at each time point. RESULTS: More than 55% of children demonstrated impaired cognitive functioning at each assessment, with a similar percentage showing impaired memory and attention at 3-year follow-up. Cognitive scores obtained at each time point were correlated. More than 20 seizures/seizure clusters prior to assessment and an abnormal neurologic examination predicted poor cognitive functioning at baseline, whereas continuing seizures and baseline cognitive score predicted 3-year intelligence quotient (IQ)/cognitive score. SIGNIFICANCE: These findings demonstrate the following: (1) infants who are performing poorly at baseline continue to display impaired development at follow-up, (2) these children are delayed across a range of neuropsychological functions, and (3) a high number of seizures close to initial diagnosis and continuing seizures at follow-up independently predict cognitive impairment. These findings help to identify those infants with new-onset epilepsy who are most at risk for poor developmental outcome and suggest that multimodal interventions should be instituted early in the course of the disorder to improve outcomes.

The City Infant Faces Database: A validated set of infant facial expressions.

Adults need to be able to process infants' emotional expressions accurately to respond appropriately and care for infants. However, research on processing of the emotional expressions of infant faces is hampered by the lack of validated stimuli. Although many sets of photographs of adult faces are available to researchers, there are no corresponding sets of photographs of infant faces. We therefore developed and validated a database of infant faces, which is available via e-mail request. Parents were recruited via social media and asked to send photographs of their infant (0-12 months of age) showing positive, negative, and neutral facial expressions. A total of 195 infant faces were obtained and validated. To validate the images, student midwives and nurses (n = 53) and members of the general public (n = 18) rated each image with respect to its facial expression, intensity of expression, clarity of expression, genuineness of expression, and valence. On the basis of these ratings, a total of 154 images with rating agreements of at least 75% were included in the final database. These comprise 60 photographs of positive infant faces, 54 photographs of negative infant faces, and 40 photographs of neutral infant faces. The images have high criterion validity and good test-retest reliability. This database is therefore a useful and valid tool for researchers.

Microclimates Might Limit Indirect Spillover of the Bat Borne Zoonotic Hendra Virus.

Infectious diseases are transmitted when susceptible hosts are exposed to pathogen particles that can replicate within them. Among factors that limit transmission, the environment is particularly important for indirectly transmitted parasites. To try and assess a pathogens' ability to be transmitted through the environment and mitigate risk, we need to quantify its decay where transmission occurs in space such as the microclimate harbouring the pathogen. Hendra virus, a Henipavirus from Australian Pteropid bats, spills-over to horses and humans, causing high mortality. While a vaccine is available, its limited uptake has reduced opportunities for adequate risk management to humans, hence the need to develop synergistic preventive measures, like disrupting its transmission pathways. Transmission likely occurs shortly after virus excretion in paddocks; however, no survival estimates to date have used real environmental conditions. Here, we recorded microclimate conditions and fitted models that predict temperatures and potential evaporation, which we used to simulate virus survival with a temperature-survival model and modification based on evaporation. Predicted survival was lower than previously estimated and likely to be even lower according to potential evaporation. Our results indicate that transmission should occur shortly after the virus is excreted, in a relatively direct way. When potential evaporation is low, and survival is more similar to temperature dependent estimates, transmission might be indirect because the virus can wait several hours until contact is made. We recommend restricting horses' access to trees during night time and reducing grass under trees to reduce virus survival.

High adult mortality in disease-challenged frog populations increases vulnerability to drought.

Pathogen emergence can drive major changes in host population demography, with implications for population dynamics and sensitivity to environmental fluctuations. The amphibian disease chytridiomycosis, caused by infection with the fungal pathogen Batrachochytrium dendrobatidis (Bd), is implicated in the severe decline of over 200 amphibian species. In species that have declined but not become extinct, Bd persists and can cause substantial ongoing mortality. High rates of mortality associated with Bd may drive major changes in host demography, but this process is poorly understood. Here, we compared population age structure of Bd-infected populations, Bd-free populations and museum specimens collected prior to Bd emergence for the endangered Australian frog, Litoria verreauxii alpina (alpine tree frog). We then used population simulations to investigate how pathogen-associated demographic shifts affect the ability of populations to persist in stochastic environments. We found that Bd-infected populations have a severely truncated age structure associated with very high rates of annual adult mortality. Near-complete annual adult turnover in Bd-infected populations means that individuals breed once, compared with Bd-free populations where adults may breed across multiple years. Our simulations showed that truncated age structure erodes the capacity of populations to withstand periodic recruitment failure; a common challenge for species reproducing in uncertain environments. We document previously undescribed demographic shifts associated with a globally emerging pathogen and demonstrate how these shifts alter host ecology. Truncation of age structure associated with Bd effectively reduces host niche width and can help explain the contraction of L. v. alpina to perennial waterbodies where the risk of drought-induced recruitment failure is low. Reduced capacity to tolerate other sources of mortality may explain variation in decline severity among other chytridiomycosis-challenged species and highlights the potential to mitigate disease impacts through minimizing other sources of mortality.

Cognitive biases in processing infant emotion by women with depression, anxiety and post-traumatic stress disorder in pregnancy or after birth: A systematic review.

Perinatal psychological problems such as post-natal depression are associated with poor mother-baby interaction, but the reason for this is not clear. One explanation is that mothers with negative mood have biased processing of infant emotion. This review aimed to synthesise research on processing of infant emotion by pregnant or post-natal women with anxiety, depression or post-traumatic stress disorder (PTSD). Systematic searches were carried out on 11 electronic databases using terms related to negative affect, childbirth and perception of emotion. Fourteen studies were identified which looked at the effect of depression, anxiety and PTSD on interpretation of infant emotional expressions (k = 10), or reaction times when asked to ignore emotional expressions (k = 4). Results suggest mothers with depression and anxiety are more likely to identify negative emotions (i.e., sadness) and less accurate at identifying positive emotions (i.e., happiness) in infant faces. Additionally, women with depression may disengage faster from positive and negative infant emotional expressions. Very few studies examined PTSD (k = 2), but results suggest biases towards specific infant emotions may be influenced by characteristics of the traumatic event. The implications of this research for mother-infant interaction are explored.

Innovations in Practice: The efficacy of nonviolent resistance groups in treating aggressive and controlling children and young people: a preliminary analysis of pilot NVR groups in Kent.

BACKGROUND: Conduct disorders and adolescent violence have been found to be a significant problem in the United Kingdom. METHOD: Nonviolent Resistance (NVR) Parenting Groups were piloted in Kent to address the demand on CAMHS for young people with this issue, and preliminary analysis on outcome measures was conducted. RESULTS: A significant difference in a positive direction was found on all but one of the measurements used. CONCLUSION: Findings suggest that using NVR methods in a group format is an effective intervention for these families. De-escalation and acts of unconditional love were rated by parents as the most useful interventions.

Glutathione-Mediated Metal Tolerance in an Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis).

The spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis, has resulted in amphibian declines and extinctions worldwide. Some susceptible amphibian species can persist in contaminated habitats, prompting the hypothesis that B. dendrobatidis might be sensitive to heavy metals. We tested a panel of 12 metals to rank their toxicity to B. dendrobatidis zoospores and zoosporangia during a 6-h exposure. To better understand the mechanism for metal detoxification, we also evaluated whether glutathione is required for metal tolerance by depleting cellular glutathione before metal exposure. In addition, we investigated whether prior exposure to low metal concentrations impacted tolerance of subsequent exposure, as well as identifying metal combinations that may act synergistically. Silver (Ag), cadmium (Cd), and copper (Cu) were particularly toxic to B. dendrobatidis, with zoospore minimum lethal concentration values of 0.01 mM (Ag), 0.025 mM (Cd), and 0.5 mM (Cu). These three metals along with zinc (Zn) were also inhibitory to zoosporangia, with minimum inhibitory concentration values of 0.005 mM (Ag), 0.04 mM (Cd), 0.075 mM (Cu), and 0.04 mM (Zn). The fungicidal effects of several metals was reduced when assays were conducted in nutrient medium compared with synthetic pond water, highlighting the need for careful in vitro assay design and interpretation. Glutathione depletion strongly influenced tolerance of Cd and Ag (85% and 75% less growth, respectively) and moderately influenced tolerance of Cu, Zn, and lead (37%, 18%, and 14% less growth, respectively), indicating the importance of glutathione for metal detoxification. In general, the minimum metal concentrations that inhibited growth of B. dendrobatidis far exceeded values detected in contaminated amphibian habitats in Australia, suggesting that metal contamination alone may not have a strong protective effect against chytridiomycosis. We discuss future research directions to futher understand the potential for dissolved metals to create chytrid refuges. Environ Toxicol Chem 2024;43:1583-1591. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The Binding, Infection, and Promoted Growth of Batrachochytrium dendrobatidis by the Ranavirus FV3.

Increasing reports suggest the occurrence of co-infection between Ranaviruses such as Frog Virus 3 (FV3) and the chytrid fungus Batrachochytrium dendrobatidis (Bd) in various amphibian species. However, the potential direct interaction of these two pathogens has not been examined to date. In this study, we investigated whether FV3 can interact with Bd in vitro using qPCR, conventional microscopy, and immunofluorescent microscopy. Our results reveal the unexpected ability of FV3 to bind, promote aggregation, productively infect, and significantly increase Bd growth in vitro. To extend these results in vivo, we assessed the impact of FV3 on Xenopus tropicalis frogs previously infected with Bd. Consistent with in vitro results, FV3 exposure to previously Bd-infected X. tropicalis significantly increased Bd loads and decreased the host's survival.

Glutathione is required for growth and cadmium tolerance in the amphibian chytrid fungus, Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd) is a lethal amphibian pathogen, partly due to its ability to evade the immune system of susceptible frog species. In many pathogenic fungi, the antioxidant glutathione is a virulence factor that helps neutralise oxidative stressors generated from host immune cells, as well as other environmental stressors such as heavy metals. The role of glutathione in stress tolerance in Bd has not been investigated. Here, we examine the changes in the glutathione pool after stress exposure and quantify the effect of glutathione depletion on cell growth and stress tolerance. Depletion of glutathione repressed growth and release of zoospores, suggesting that glutathione is essential for life cycle completion in Bd. Supplementation with <2 mM exogenous glutathione accelerated zoospore development, but concentrations >2 mM were strongly inhibitory to Bd cells. While hydrogen peroxide exposure lowered the total cellular glutathione levels by 42 %, glutathione depletion did not increase the sensitivity to hydrogen peroxide. Exposure to cadmium increased total cellular glutathione levels by 93 %. Glutathione-depleted cells were more sensitive to cadmium, and this effect was attenuated by glutathione supplementation, suggesting that glutathione plays an important role in cadmium tolerance. The effects of heat and salt were exacerbated by the addition of exogenous glutathione. The impact of glutathione levels on Bd stress sensitivity may help explain differences in host susceptibility to chytridiomycosis and may provide opportunities for synergistic therapeutics.

Stable in vitro fluorescence for enhanced live imaging of infection models for Batrachochytrium dendrobatidis.

Realistic and modifiable infection models are required to study the pathogenesis of amphibian chytridiomycosis. Understanding the mechanism by which Batrachochytrium dendrobatidis (Bd) can infect and kill diverse amphibians is key to mitigating this pathogen and preventing further loss of biodiversity. In vitro studies of Bd typically rely on a tryptone based growth media, whereas the recent development of a kidney cell-line infection model has provided a more realistic alternative, without the need for live animals. Here we use expression of a fluorescent reporter to enhance the in vitro cell-line based growth assay, and show that transformed Bd cells are able to invade and grow in an amphibian kidney epithelial cell line (A6) as well as in a new system using a lung fibroblast cell line (DWJ). Both Bd and host cells were modified to express reporter fluorescent proteins, enabling immediate and continuous observation of the infection process without the need for destructive sampling for fixation and staining. Plasmid DNA conferring hygromycin resistance and TdTomato (RFP) expression was delivered to Bd zoospores via electroporation, and continuous antibiotic selection after recovery produced stable fluorescent Bd transformants. Host cells (A6 and DWJ) were transfected before each assay using lipofection to deliver plasmid DNA conferring green fluorescent protein (GFP) and containing an empty shRNA expression cassette. Bd RFP expression allowed easy localisation of fungal cells and identification of endobiotic growth was assisted by host GFP expression, by allowing visualization of the space in the host cell occupied by the invading fungal body. In addition to enabling enhanced live imaging, these methods will facilitate future genetic modification and characterisation of specific genes and their effect on Bd virulence.