Is the future female for turtles? Climate change and wetland configuration predict sex ratios of a freshwater species.

Climate change and land-use change are leading drivers of biodiversity decline, affecting demographic parameters that are important for population persistence. For example, scientists have speculated for decades that climate change may skew adult sex ratios in taxa that express temperature-dependent sex determination (TSD), but limited evidence exists that this phenomenon is occurring in natural settings. For species that are vulnerable to anthropogenic land-use practices, differential mortality among sexes may also skew sex ratios. We sampled the spotted turtle (Clemmys guttata), a freshwater species with TSD, across a large portion of its geographic range (Florida to Maine), to assess the environmental factors influencing adult sex ratios. We present evidence that suggests recent climate change has potentially skewed the adult sex ratio of spotted turtles, with samples following a pattern of increased proportions of females concomitant with warming trends, but only within the warmer areas sampled. At intermediate temperatures, there was no relationship with climate, while in the cooler areas we found the opposite pattern, with samples becoming more male biased with increasing temperatures. These patterns might be explained in part by variation in relative adaptive capacity via phenotypic plasticity in nest site selection. Our findings also suggest that spotted turtles have a context-dependent and multi-scale relationship with land use. We observed a negative relationship between male proportion and the amount of crop cover (within 300 m) when wetlands were less spatially aggregated. However, when wetlands were aggregated, sex ratios remained consistent. This pattern may reflect sex-specific patterns in movement that render males more vulnerable to mortality from agricultural machinery and other threats. Our findings highlight the complexity of species' responses to both climate change and land use, and emphasize the role that landscape structure can play in shaping wildlife population demographics.

Accounting for geographic variation in species-habitat associations during habitat suitability modeling.

Range-wide species conservation efforts are facilitated by spatially explicit estimates of habitat suitability. However, species-environment relationships often vary geographically and models assuming geographically constant relationships may result in misleading inferences. We present the first range-wide habitat suitability model (HSM) for the federally threatened eastern indigo snake (Drymarchon couperi) as a case study illustrating an approach to account for known latitudinal variation in habitat associations. Specifically, we modeled habitat suitability using interactive relationships between minimum winter temperature and several a priori environmental covariates and compared our results to those from models assuming geographically constant relationships. We found that multi-scale models including interactive effects with winter temperature outperformed single-scale models and models not including interactive effects with winter temperature. Our top-ranked model had suitable range-wide predictive performance and identified numerous large (i.e., ≥1000 ha) potential habitat patches throughout the indigo snake range. Predictive performance was greatest in southern Georgia and northern Florida likely reflecting more restrictive indigo snake habitat associations in these regions. This study illustrates how modeling interactive effects between temperature and environmental covariates can improve the performance of HSMs across geographically varying environmental gradients.

Reducing complexity and unidentifiability when modelling human atrial cells.

Mathematical models of a cellular action potential (AP) in cardiac modelling have become increasingly complex, particularly in gating kinetics, which control the opening and closing of individual ion channel currents. As cardiac models advance towards use in personalized medicine to inform clinical decision-making, it is critical to understand the uncertainty hidden in parameter estimates from their calibration to experimental data. This study applies approximate Bayesian computation to re-calibrate the gating kinetics of four ion channels in two existing human atrial cell models to their original datasets, providing a measure of uncertainty and indication of potential issues with selecting a single unique value given the available experimental data. Two approaches are investigated to reduce the uncertainty present: re-calibrating the models to a more complete dataset and using a less complex formulation with fewer parameters to constrain. The re-calibrated models are inserted back into the full cell model to study the overall effect on the AP. The use of more complete datasets does not eliminate uncertainty present in parameter estimates. The less complex model, particularly for the fast sodium current, gave a better fit to experimental data alongside lower parameter uncertainty and improved computational speed. This article is part of the theme issue 'Uncertainty quantification in cardiac and cardiovascular modelling and simulation'.

Niche partitioning and the storage effect facilitate coexistence in an amphibian community.

Virtually all natural community assemblages are dominated by a handful of common species. Dominant species can exert negative impacts on biodiversity through competitive exclusion, and thus there is a strong incentive to understand imbalances in community composition, changes in dominance hierarchies through time, and mechanisms of coexistence. Pond-breeding amphibians that utilize ephemeral wetlands provide an excellent opportunity to evaluate theoretical predictions of community composition in stochastic environments. One of the most striking features of pond-breeding amphibians is the marked stochastic fluctuations in abundance across years. Given strong theoretical and empirical links between evenness and biomass, one would expect community evenness to change from year to year. Moreover, if different species exhibit different boom-and-bust reproductive cycles, then a storage effect may help to explain why one species does not outcompete all others. Here, we explore the interplay between biotic and abiotic conditions in shaping amphibian communities at two ephemeral wetlands on Eglin Air Force Base, Florida. We document consistent community composition over 6 years of monitoring, resulting from a lack of species turnover and similar responses of all community members to environmental conditions. The similar dynamics of species argues against a storage effect as the sole mechanism for coexistence and instead points to niche partitioning as a more important factor. In support of this conclusion, we show that the degree of synchrony in breeding migrations only correlates with environmental conditions within species, not between species. The lack of pattern seen between species implies that individuals are somewhat constrained in the timing of breeding migrations, perhaps owing in part to competition with other community members. We hope that our work reinvigorates interest in amphibian communities and highlights ephemeral wetlands as model systems to study community dynamics in stochastic environments.

Cryptosporidium serpentis Surveillance in Free-Ranging Snakes to Inform a Reintroduction Strategy for the Eastern Indigo Snake (Drymarchon couperi).

Understanding risk factors associated with reintroductions is important for making informed decisions within an adaptive framework. Biosecurity measures minimizing the risk of the introduction or spread of transmissible diseases are a priority when considering the release of captive-reared wildlife. Eastern indigo snake (EIS; Drymarchon couperi) reintroductions have been occurring in Alabama since 2010 and in Florida since 2017. During this effort the pathogen Cryptosporidium serpentis was detected, affecting several of the captive breeding snakes. Infected snakes were quarantined and removed from breeding efforts, which reduced snakes available for the reintroduction projects. To make informed management decisions about future reintroduction strategies, 155 free-ranging snakes were sampled at the two release sites and a third site in Georgia to evaluate the natural occurrence of C. serpentis. Additionally, 72 free-ranging EIS and other species incidentally encountered throughout the EIS range were tested opportunistically. All snakes sampled at the three focal sites tested negative, but one opportunistically tested EIS from South Florida tested positive. These results indicate that C. serpentis is present in the environment in at least one location, but at low levels. Our results suggest that, pending additional surveillance, C. serpentispositive snakes should not be included in reintroduction efforts, and that maintaining a high level of biosecurity is important in captive breeding programs.

Forecasting the flooding dynamics of flatwoods salamander breeding wetlands under future climate change scenarios.

Ephemeral wetlands are globally important systems that are regulated by regular cycles of wetting and drying, which are primarily controlled by responses to relatively short-term weather events (e.g., precipitation and evapotranspiration). Climate change is predicted to have significant effects on many ephemeral wetland systems and the organisms that depend on them through altered filling or drying dates that impact hydroperiod. To examine the potential effects of climate change on pine flatwoods wetlands in the southeastern United States, we created statistical models describing wetland hydrologic regime using an approximately 8-year history of water level monitoring and a variety of climate data inputs. We then assessed how hydrology may change in the future by projecting models forward (2025-2100) under six future climate scenarios (three climate models each with two emission scenarios). We used the model results to assess future breeding conditions for the imperiled Reticulated Flatwoods Salamander (Ambystoma bishopi), which breeds in many of the study wetlands. We found that models generally fit the data well and had good predictability across both training and testing data. Across all models and climate scenarios, there was substantial variation in the predicted suitability for flatwoods salamander reproduction. However, wetlands with longer hydroperiods tended to have fewer model iterations that predicted at least five consecutive years of reproductive failure (an important metric for population persistence). Understanding potential future risk to flatwoods salamander populations can be used to guide conservation and management actions for this imperiled species.

Migration path annotation: cross-continental study of migration-flight response to environmental conditions.

Understanding the movements of animals is pivotal for understanding their ecology and predicting their survival in the face of rapid global changes to climate, land use, and habitats, thus facilitating more effective habitat management. Migration by flying animals is an extreme form of movement that may be especially influenced by weather. With satellite telemetry studies, and the growing availability of information about the Earth's weather and land surface conditions, many data are collected that can advance our understanding about the mechanisms that shape migrations. We present the track annotation approach for movement data analysis using information about weather from the North American Reanalysis data set, a publicly available, regional, high-resolution model-observation hybrid product, and about topography, from a publicly available high-resolution digital elevation model (DEM). As a case study, we present the analysis of the response to environmental conditions in three contrasting populations of Turkey Vultures (Cathartes aura) across North America, tracked with a three-dimensional GPS-based sensor. Two populations in the east and west coasts of the United States responded similarly to weather, indicating use of both slope and thermal soaring. Continental-interior, "Plains populations," exhibited a different migratory pattern primarily indicative of thermal soaring. These differences help us understand the constraints and behaviors of soaring migrants. The track annotation approach allowed large-scale comparative study of movement in an important migratory species, and will enable similar studies at local to global scales.

An outbreak of food-borne botulism in Scotland, United Kingdom, November 2011.

An investigation is currently underway to explain an outbreak of food-borne botulism in Scotland. Three children in the same family were confirmed as having botulism following consumption of a meal made with a jar of korma sauce. Residual sauce from the jar, the jar lid and a remnant of the meal, all tested positive for Clostridium botulinum type A toxin. The children are recovering, although two remain ventilated and in intensive care unit.

Does long-term fire suppression impact leaf litter breakdown and aquatic invertebrate colonization in pine flatwoods wetlands?

Ephemeral wetlands are commonly embedded within pine uplands of the southeastern United States. These wetlands support diverse communities but have often been degraded by a lack of growing-season fires that historically maintained the vegetation structure. In the absence of fire, wetlands develop a dense mid-story of woody vegetation that increases canopy cover and decreases the amount of herbaceous vegetation. To understand how reduced fire frequency impacts wetland processes, we measured leaf litter breakdown rates and invertebrate communities using three common plant species (Longleaf Pine (Pinus palustris), Pineland Threeawn Grass (Aristida stricta), and Black Gum (Nyssa sylvatica)) that occur in pine flatwoods wetlands located on Eglin Air Force Base, Florida. We also tested whether or not the overall habitat type within a wetland (fire maintained or fire suppressed) affected these processes. We placed leaf packs containing 15.0 g of dried leaf litter from each species in both fire-maintained and fire-suppressed sections of three wetlands, removing them after 103-104 days submerged in the wetland. The amount of leaf litter remaining at the end of the study varied across species (N. sylvatica = 7.97 ± 0.17 g, A. stricta = 11.84 ± 0.06 g, and P. palustris = 11.37 ± 0.07 g (mean ± SE)) and was greater in fire-maintained habitat (leaf type: F 2,45 = 437.2, P < 0.001; habitat type: F 1,45 = 4.6, P = 0.037). We identified an average of 260 ± 33.5 (SE) invertebrates per leaf pack (range: 19-1,283), and the most abundant taxonomic groups were Cladocera, Isopoda, Acariformes, and Diptera. Invertebrate relative abundance varied significantly among litter species (approximately 39.9 ± 9.4 invertebrates per gram of leaf litter remaining in N. sylvatica leaf packs, 27.2 ± 5.3 invertebrates per gram of A. stricta, and 14.6 ± 3.1 invertebrates per gram of P. palustris (mean ± SE)) but not habitat type. However, both habitat (pseudo-F1,49 = 4.30, P = 0.003) and leaf litter type (pseudo-F2,49 = 3.62, P = 0.001) had a significant effect on invertebrate community composition. Finally, this work was part of ongoing projects focusing on the conservation of the critically imperiled Reticulated Flatwoods Salamander (Ambystoma bishopi), which breeds exclusively in pine flatwoods wetlands, and we examined the results as they relate to potential prey items for larval flatwoods salamanders. Overall, our results suggest that the vegetation changes associated with a lack of growing-season fires can impact both invertebrate communities and leaf litter breakdown.

Publisher Correction: Ophidiomycosis surveillance of snakes in Georgia, USA reveals new host species and taxonomic associations with disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ophidiomycosis surveillance of snakes in Georgia, USA reveals new host species and taxonomic associations with disease.

Ophidiomycosis (snake fungal disease) is caused by the fungus Ophidiomyces ophiodiicola and threatens snake health worldwide. It has been documented throughout the eastern United States and severe cases have recently been reported in Georgia, USA. To evaluate disease distribution and prevalence in this state, 786 free-ranging snakes were examined for skin lesions consistent with ophidiomycosis and swabbed to detect O. ophiodiicola DNA using qPCR. Sampled snakes represented 34 species and 4 families; 27.5% had skin lesions, 13.3% were positive for O. ophiodiicola DNA, and 77.8% of the qPCR positive individuals had skin lesions. This is the first report of O. ophiodiicola in five of the 22 species that were qPCR positive. Multinomial logistic regression modeling indicated that Drymarchon couperi had a higher relative risk of apparent ophidiomycosis (lesions present and qPCR positive), and the best models predicting qPCR result and ophidiomycosis category included individual factors and excluded temporal and spatial factors. Phylogeny-based bipartite network analysis showed that Nerodia erythrogaster, Nerodia taxispilota, and D. couperi had the highest prevalence of apparent ophidiomycosis; this category was more prevalent in the subfamily Colubrinae and less prevalent in Natricinae. These results provide important information about ophidiomycosis epidemiology, which has implications for snake conservation.

Four precursors of Medicare in Saskatchewan.

T. C. Douglas, on assuming power in June 1944 as the first social democratic premier in North America, began working in a step-like pattern as finances permitted, toward his goal of eventual province-wide Medicare. Douglas and his team were able to build on the success of bold initiatives already in place in the Depression-scarred rural municipalities of Pittville, Miry Creek, Webb, and Riverside. These municipalities developed medical and hospital plans that offered residents comprehensive coverage with freedom of choice of doctor. Built on idealism, prairie pragmatism and tenacity, these formative health plans served not only as models, but provided the leadership required during the creation and early years of Swift Current Health Region #1. Key figures such as Bill Burak, Carl Kjorven, Stewart Robertson, and Charles Haydon brought experience, depth, and ambition to the task at hand. Envisioned as simply a demonstration region by the Saskatchewan government, HR #1 achieved more: a seamless integration of preventative medicine with medical care, combined with a sense of local empowerment.

Ophidiomycosis prevalence in Georgia's Eastern Indigo Snake (Drymarchon couperi) populations.

Wildlife diseases have posed a significant challenge to the conservation of many species in recent years. Diseases have been implicated in population declines over large geographic areas, with severe disease outbreaks leading to either local or complete extinctions of wild populations. Ophidiomycosis, commonly known as snake fungal disease, is caused by the fungus Ophidiomyces ophiodiicola, which has been documented in snake populations across the eastern and southern United States. We collected swab samples from the federally threatened Eastern Indigo Snake (Drymarchon couperi) in populations across the species' Georgia range. We used quantitative PCR to determine the presence of O. ophiodiicola DNA and also recorded skin abnormalities characteristic of ophidiomycosis. From 1 September 2016 to 4 August 2018, Eastern Indigo Snakes tested positive for O. ophiodiicola DNA on 47 of 107 occasions (43.9%) and tested negative for fungal DNA but had skin lesions consistent with ophidiomycosis on 42 occasions (39.3%). Symptomatic and qPCR positive individuals were more likely to be encountered during January and February when compared to November and December. We found no effect of sex (p = 0.517), age-class (p = 0.106), or body size (snout-vent length: p = 0.083; mass: p = 0.206; body condition: p = 0.063) on ophidiomycosis status. Over the two-year study, we encountered individuals in which infection was clearly negatively impacting overall health and also documented individuals in which infection apparently cleared from one year to the next. These results demonstrate that O. ophiodiicola and lesions characteristic of ophidiomycosis are widespread in Georgia's Eastern Indigo Snake populations. However, there are many unanswered questions regarding this disease, including the effects of disease on populations and individuals, the presence of infection vectors, and the change in prevalence over time. More research is needed to address ophidiomycosis and understand its impacts on ongoing conservation efforts.

UK surveillance: provision of quality assured information from combined datasets.

Surveillance information is most useful when provided within a risk framework, which is achieved by presenting results against an appropriate denominator. Often the datasets are captured separately and for different purposes, and will have inherent errors and biases that can be further confounded by the act of merging. The United Kingdom Rapid Analysis and Detection of Animal-related Risks (RADAR) system contains data from several sources and provides both data extracts for research purposes and reports for wider stakeholders. Considerable efforts are made to optimise the data in RADAR during the Extraction, Transformation and Loading (ETL) process. Despite efforts to ensure data quality, the final dataset inevitably contains some data errors and biases, most of which cannot be rectified during subsequent analysis. So, in order for users to establish the 'fitness for purpose' of data merged from more than one data source, Quality Statements are produced as defined within the overarching surveillance Quality Framework. These documents detail identified data errors and biases following ETL and report construction as well as relevant aspects of the datasets from which the data originated. This paper illustrates these issues using RADAR datasets, and describes how they can be minimised.

D2R2: an evidence-based decision support tool to aid prioritisation of animal health issues for government funding.

An evidence-based decision support tool, 'D2R2', has been developed by Defra. It contains a wide range of standardised information about exotic and endemic diseases held in 'disease profiles'. Each profile includes 40 criteria used for scoring, enabling D2R2 to provide relative priority rankings for every disease profiled. D2R2 also provides a range of reports for each disease and the functionality to explore the impact of changes in any criterion or weighting on a disease's ranking. These outputs aid the prioritisation and management of animal diseases by government. D2R2 was developed with wide stakeholder engagement and its design was guided by clear specifications. It uses the weighted scores of a limited number of criteria to generate impact and risk scores for each disease, and relies on evidence drawn from published material wherever possible and maintained up to date. It allows efficient use of expertise, as maintained disease profiles reduce the need for on call, reactive, expert input for policy development and enables rapid simultaneous access to the same information by multiple parties, for example during exotic disease outbreaks. The experience in developing D2R2 has been shared internationally to assist others with their development of disease prioritisation and categorisation systems.

Hindcasting Historical Breeding Conditions for an Endangered Salamander in Ephemeral Wetlands of the Southeastern USA: Implications of Climate Change.

The hydroperiod of ephemeral wetlands is often the most important characteristic determining amphibian breeding success, especially for species with long development times. In mesic and wet pine flatwoods of the southeastern United States, ephemeral wetlands were a common landscape feature. Reticulated flatwoods salamanders (Ambystoma bishopi), a federally endangered species, depend exclusively on ephemeral wetlands and require at least 11 weeks to successfully metamorphose into terrestrial adults. We empirically modeled hydroperiod of 17 A. bishopi breeding wetlands by combining downscaled historical climate-model data with a recent 9-year record (2006-2014) of observed water levels. Empirical models were subsequently used to reconstruct wetland hydrologic conditions from 1896-2014 using the downscaled historical climate datasets. Reconstructed hydroperiods for the 17 wetlands were highly variable through time but were frequently unfavorable for A. bishopi reproduction (e.g., only 61% of years, using a conservative estimate of development time [12 weeks], were conducive to larval development and metamorphosis). Using change-point analysis, we identified significant shifts in average hydroperiod over the last century in all 17 wetlands. Mean hydroperiods were shorter in recent years than at any other point since 1896, and thus less suitable for A. bishopi reproduction. We suggest that climate change will continue to impact the reproductive success of flatwoods salamanders and other ephemeral wetland breeders by reducing the number of years these wetlands have suitable hydroperiods. Consequently, we emphasize the importance of conservation and management for mitigating other forms of habitat degradation, especially maintenance of high quality breeding sites where reproduction can occur during appropriate environmental conditions.

Rapid HbA1c testing in a community setting.

OBJECTIVE: To determine whether the DCA 2000 analyzer provides valid and reliable HbA1c results when used under field conditions and operated by nonmedical personnel. This study was part of a community diabetes education program, the Native American Diabetes Project, in which HbA1c was measured as an indicator of average glycemic control. RESEARCH DESIGN AND METHODS: Two study samples were taken, the first in the spring of 1994 and the second in the spring of 1995. Seven community members in 1994 and six new community members in 1995 were trained over 2 days, using standard protocol, to operate the DCA 2000 HbA1c analyzer and to collect two capillary blood samples from participants in the Native American Diabetes Project. Duplicate DCA 2000 HbA1c measurements performed by the community workers were compared with measurements from a high-performance liquid chromatography (HPLC) system. Validity and reliability measures were calculated. RESULTS: Of the participants, 43 were studied in 1994 and 14 in 1995. Comparison of the mean DCA 2000 results with those of HPLC showed high validity, with the absolute relative difference between the mean DCA 2000 and the external reference of HPLC (magnitude of mean DCA 2000-HPLC magnitude of /HPLC) as 4.0 and 2.0% for 1994 and 1995, respectively. The Pearson correlation coefficients (r) between these two measures were 0.968 and 0.996 for 1994 and 1995, respectively. While the 1994 data appeared to have less validity for values > 10%, they included only one value with a 60-min warm-up of the DCA analyzer. The 1995 data, all collected after a 60-min warm-up, had good correlation throughout the range of values. The within-run reliability was excellent, with an intraclass correlation coefficient of reliability of 0.959 and 0.975 for paired samples, for 1994 and 1995 respectively. The mean coefficient of variation for these paired measures was 3.0% in 1994 and 2.8% in 1995. Both validity and reliability were improved by changing the warm-up period of the DCA 2000 analyzer from 5 to 60 min. All correlation coefficients were statistically significant (P < 0.0001). CONCLUSIONS: The DCA 2000 gave valid and reliable HbA1c results when operated in a community setting by nonmedical personnel. Extending the warm-up period of the device to 60 min slightly improved the validity and reliability of the test.

HIV in Canada: 2009 to 2014.

BACKGROUND: Between 1996 and 2008, the number of newly reported HIV cases in Canada fluctuated between approximately 2,100 and 2,700 cases per year. OBJECTIVE: To describe the recent trends in new diagnoses of HIV in Canada between 2009 and 2014 by age group, sex, exposure category, race/ethnicity, and region, as well as the number of perinatally HIV-exposed infants. METHODS: HIV data were compiled from two databases. The National HIV/AIDS Surveillance System is a passive surveillance system that gathers non-nominal data on a voluntary basis from all the provinces and territories of all cases that meet the national case definition, and includes data on age group, sex, race/ethnicity, country of birth, and exposure categories. The Canadian Perinatal HIV Surveillance Program is a sentinel-based surveillance system; non-nominal data is obtained through a national confidential survey completed by participating physicians. RESULTS: Since 2009 the number of new HIV cases has slowly but steadily declined from 2,391 cases in 2009 to 2,044 in 2014, which is the lowest number of annual HIV cases seen in the last two decades. The largest proportion (32%) of new HIV cases continues to be diagnosed among those 30 to 39 years of age, but the proportion of cases diagnosed among those 50 years of age or over has increased from 15% in 2009 to slightly over 20% in 2014. Approximately 75% of newly diagnosed cases are males and 25% females. In males, the most common exposure category (60%) was men who have sex with men. In females, the most common exposure category (66%) was heterosexual contact followed by injection drug use (27%). Race/ethnicity varied by sex. In males, over 50% were White, 14% were Aboriginal and 13% Black. In females, 35% were Black, 35% were Aboriginal and 22% were White. There were regional variations in HIV rates across Canada. Between 2009 and 2014, the number of perinatally HIV-exposed infants varied between 200 and 249 but the percentage of perinatal treatment increased from 87% in 2009 to 97% in 2014. CONCLUSION: The annual number of reported HIV cases in Canada has been declining in recent years. The proportion of HIV-positive mothers receiving treatment has increased and the number of confirmed HIV-infected infants has decreased.

Cloning and expression of putative cytotonic enterotoxin-encoding genes from Aeromonas hydrophila.

A genomic library from a diarrheal isolate, SSU, of Aeromonas hydrophila was constructed in a cosmid vector, pHC79, and in bacteriophage lambda EMBL3. Cell lysates from various Escherichia coli clones containing the recombinant cosmid were examined for their ability to elongate Chinese hamster ovary (CHO) cells, which is a typical enterotoxic response. Based on restriction analysis, a 4.0-kb SalI DNA fragment from one of the clones that exhibited enterotoxic activity was subcloned into a bacteriophage T7 RNA polymerase/promoter hyperexpression system. The cell lysate from this E. coli [pSL24] clone caused CHO cells to elongate and revealed the presence of a major 35-kDa polypeptide by [35S]methionine labeling and sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE). The toxin was biologically heat labile, losing all activity within 20 min at 56 degrees C. In addition, another enterotoxin-producing clone, E. coli[pSBS32], was isolated from cosmid and lambda bacteriophage libraries. We localized this heat-stable (56 degrees C/20 min) enterotoxin to a 4.8-kb SalI-BamHI fragment. Both enterotoxins caused elevation of cyclic adenosine monophosphate (cAMP) in CHO cells. The DNA fragments encoding these enterotoxins did not hybridize with each other. However, a 4.8-kb SalI-BamHI DNA fragment encoding a heat-stable enterotoxin hybridized to a 3.5-kb BamHI DNA fragment of a plasmid, pHPC100, that contained a cytotonic enterotoxin-encoding gene isolated from A. trota. Our data suggest Aeromonas species produce different structural types of cytotonic enterotoxins that are functionally similar.

Amino-acid residues involved in biological functions of the cytolytic enterotoxin from Aeromonas hydrophila.

Some amino acid (aa) residues within the cytolytic enterotoxin (Act) of Aeromonas hydrophila essential for biological activity were identified. Act is a 52-kDa polypeptide, possessing hemolytic, cytotoxic and enterotoxic activities. By deletion analysis, generation of anti-peptide Ab, and site-directed mutagenesis we showed that two regions in Act (aa 245-274 and 361-405) were very important for biological functions. As shown by competitive inhibition assays, peptide 2 (aa 245-274) blocked cytotoxic activity of Act, and aa Tyr256, Trp270 and Gly274 were essential for cytotoxicity. Within peptide 3 (aa 361-405), Trp394 and Trp396 were important for biological activities. Mutations in other regions of the toxin (e.g., Gly169, Asp170, Gly171, Trp172, Asn177,178, Asp179 and His144,209,355) also decreased biological activity. The reactivity of these mutant toxins with Ab in immunoblots was not altered. Data reported in this study suggested the role of some aa residues in biological function(s) of Act.