Cryptosporidium spp. diagnosis and research in the 21st century.

The protozoan parasite Cryptosporidium has emerged as a leading cause of diarrhoeal illness worldwide, posing a significant threat to young children and immunocompromised patients. While endemic in the vast majority of developing countries, Cryptosporidium also has the potential to cause waterborne epidemics and large scale outbreaks in both developing and developed nations. Anthroponontic and zoonotic transmission routes are well defined, with the ingestion of faecally contaminated food and water supplies a common source of infection. Microscopy, the current diagnostic mainstay, is considered by many to be suboptimal. This has prompted a shift towards alternative diagnostic techniques in the advent of the molecular era. Molecular methods, particularly PCR, are gaining traction in a diagnostic capacity over microscopy in the diagnosis of cryptosporidiosis, given the laborious and often tedious nature of the latter. Until now, developments in the field of Cryptosporidium detection and research have been somewhat hampered by the intractable nature of this parasite. However, recent advances in the field have taken the tentative first steps towards bringing Cryptosporidium research into the 21st century. Herein, we provide a review of these advances.

Development of a novel, high resolution melting analysis based genotyping method for Cryptosporidium parvum.

This study employed the post-real-time PCR application, high resolution melting (HRM) analysis, in order to differentiate between characterised clinical and reference Cryptosporidium parvum samples obtained from Cork University Hospital (Cork, Ireland) and the Cryptosporidium Reference Unit (Swansea, Wales). A sample set composed of 18 distinct C. parvum gp60-subtypes of the IIa gp60-subtype family (an allele family accounting for over 80% of all cryptosporidiosis cases in Ireland) was employed. HRM analysis-based interrogation of the gp60, MM5 and MS9-Mallon tandem repeat loci was found to completely differentiate between 10 of the 18 studied gp60-subtypes. The remaining eight gp60-subtypes were differentiated into three distinct groupings, with the designations within these groupings resolved to two to three potential gp60-subtypes. The current study aimed to develop a novel, reproducible, real-time PCR based multi-locus genotyping method to distinguish between C. parvum gp60-subtypes. These preliminary results support the further expansion of the multi-locus panel in order to increase the discriminatory capabilities of this novel method.

A novel genotyping method for Cryptosporidium hominis.

Cryptosporidiosis remains the leading protozoan induced cause of diarrhoea-associated mortality worldwide. Cryptosporidium hominis, the anthroponotically transmitted species within the Cryptosporidium genus, contributes significantly to the global burden of infection, accounting for the majority of clinical cases in many countries. This study applied high resolution melting analysis, a post-real-time PCR application, to the differentiation of six globally prevalent C. hominisgp60-subtypes. This novel method targeted three microsatellite, tandem repeat containing genetic markers, gp60, the genetic marker upon which current Cryptosporidium subtype nomenclature is based, MSB, and MSE, by which to differentiate between C. hominis isolates. This multi-locus approach successfully differentiated between all six C. hominisgp60-subtypes studied, some of which, such as IbA10G2, are known to exhibit global ubiquity. Thus, this method has the potential to be universally employed as a sensitive, cost effective and highly reproducible means to rapidly differentiate between C. hominisgp60-subtypes. Such a method would be of particular utility in epidemiological studies and outbreak scenarios, providing cost effective, clinically accessible alternative to DNA sequencing. The success of this preliminary study also supports further analysis of an expanded C. hominisgp60-subtype range and the potential expansion of the multi-locus panel in order to improve the discriminatory power of this approach.

Development of novel methodology for the molecular differentiation of Cryptosporidium parvum gp60 subtypes via high resolution melting analysis.

Cryptosporidium species subtypes are generally identified via DNA sequencing of the gp60 gene tandem repeat motif region. Due to the immunogenic nature of its glycoprotein products, gp60 is subject to host selective pressures, genetic recombination and evolutionary processes that drive extensive polymorphism at this locus. The elucidation of the polymorphic nature of this gene has led to the current mainstay in Cryptosporidium subtyping nomenclature. This study aimed to develop a real-time polymerase chain reaction based method utilising a post-PCR application, high resolution melting (HRM) analysis, in conjunction with the abovementioned gp60 nomenclature system, in order to differentiate between Cryptosporidium parvum gp60 subtypes. Subtype differentiation is based on the difference between the melting temperatures of individual subtypes conferred by variations in the polymorphic region of gp60. • Nested gp60 primers were designed to amplify a target region of <200 base pairs for effective HRM analysis • This method presents a rapid, sensitive, cost effective alternative to conventional sequencing. • This method is highly flexible and may be applied to other loci in order to facilitate multi-locus analysis and improve the discriminative abilities of the method.

Climate drives the geography of marine consumption by changing predator communities.

The global distribution of primary production and consumption by humans (fisheries) is well-documented, but we have no map linking the central ecological process of consumption within food webs to temperature and other ecological drivers. Using standardized assays that span 105° of latitude on four continents, we show that rates of bait consumption by generalist predators in shallow marine ecosystems are tightly linked to both temperature and the composition of consumer assemblages. Unexpectedly, rates of consumption peaked at midlatitudes (25 to 35°) in both Northern and Southern Hemispheres across both seagrass and unvegetated sediment habitats. This pattern contrasts with terrestrial systems, where biotic interactions reportedly weaken away from the equator, but it parallels an emerging pattern of a subtropical peak in marine biodiversity. The higher consumption at midlatitudes was closely related to the type of consumers present, which explained rates of consumption better than consumer density, biomass, species diversity, or habitat. Indeed, the apparent effect of temperature on consumption was mostly driven by temperature-associated turnover in consumer community composition. Our findings reinforce the key influence of climate warming on altered species composition and highlight its implications for the functioning of Earth's ecosystems.

Increased diversity and novel subtypes among clinical Cryptosporidium parvum and Cryptosporidium hominis isolates in Southern Ireland.

Reported incidence rates of cryptosporidiosis in Ireland are consistently among the highest in Europe. Despite the national prevalence of this enteric parasite and the compulsory nature of incidence surveillance and reporting, in-depth analyses seeking to genotype clinical isolates of Cryptosporidium on an intra-species level are rarely undertaken in Ireland. This molecular epidemiology study of 163 clinical Cryptosporidium isolates was conducted in Southern Ireland, from 2015 to 2018, in order to ascertain population subtype heterogeneity. Analysis was conducted via real-time PCR amplification and gp60 gene sequencing, which successfully determined the subtype designation of 149 of the 163 (91.4%) tested isolates. Overall, 12 C. parvum and five C. hominis subtypes were identified, with the incidence of the regionally predominant C. parvum species found to primarily occur during springtime months, while C. hominis incidence was largely confined to late summer and autumnal months. Additionally, one C. parvum and four C. hominis subtypes were newly reported by this study, having not been previously identified in clinical or livestock infection in Ireland. Overall, these data give insight into the diversification of the Cryptosporidium population and emergent subtypes, while also allowing comparisons to be made with clinical epidemiological profiles reported previously in Ireland and elsewhere.

Cryptosporidium spp surveillance and epidemiology in Ireland: a longitudinal cohort study employing duplex real-time PCR based speciation of clinical cases.

Cryptosporidium is a leading cause of gastroenteritis (cryptosporidiosis), with significant morbidity and mortality worldwide. Irish cryptosporidiosis incidence rates are consistently the highest reported in Europe. A retrospective, longitudinal study of clinical Cryptosporidium isolates was conducted from 2015 to 2018 in Cork, southern Ireland. Overall, 86.5% of cases were attributed to C. parvum, while the remaining 13.5% were caused by C. hominis Despite the widespread implications of this protozoan parasite in sporadic and outbreak-related illness in Ireland, the current dearth of species-level epidemiological surveillance and clinical studies needs to be addressed in order to elucidate the national impact of this enteric pathogen.

Cobble community DNA as a tool to monitor patterns of biodiversity within kelp forest ecosystems.

Kelp forest ecosystems dominate 150,000 km of global temperate coastline, rivalling the coastal occurrence of coral reefs. Despite the astounding biological diversity and productive ecological communities associated with kelp forests, patterns of species richness and composition are difficult to monitor and compare. Crustose coralline algae are a critically important substrate for propagule settlement for a range of kelp forest species. Coralline-covered cobbles are home to hundreds of species of benthic animals and algae and form a replicable unit for ecological assays. Here, we use DNA metabarcoding of bulk DNA extracts sampled from cobbles to explore patterns of species diversity in kelp forests of the central California coast. The data from 97 cobbles within kelp forest ecosystems at three sites in Central California show the presence of 752 molecular operational taxonomic units (MOTUs) and 53 MOTUs assigned up to the species level with >95% similarity to current databases. We are able to detect spatial patterns of important management targets such as abalone recruits, and localized abundance of sea stars in 2012. Comparison of classic ecological surveys of these sites reveals large differences in species targets for these two approaches. In order to make such comparisons more quantitative, we use Presence/Absence Metabarcoding, using the fraction of replicate cobbles showing a species as a measure of its local abundance. This approach provides a fast and repeatable survey method that can be applied for biodiversity assessments across systems to shed light on the impact of different ecological disturbances and the role played by marine protected areas.

Calcifying algae maintain settlement cues to larval abalone following algal exposure to extreme ocean acidification.

Ocean acidification (OA) increasingly threatens marine systems, and is especially harmful to calcifying organisms. One important question is whether OA will alter species interactions. Crustose coralline algae (CCA) provide space and chemical cues for larval settlement. CCA have shown strongly negative responses to OA in previous studies, including disruption of settlement cues to corals. In California, CCA provide cues for seven species of harvested, threatened, and endangered abalone. We exposed four common CCA genera and a crustose calcifying red algae, Peyssonnelia (collectively CCRA) from California to three pCO2 levels ranging from 419-2,013 µatm for four months. We then evaluated abalone (Haliotis rufescens) settlement under ambient conditions among the CCRA and non-algal controls that had been previously exposed to the pCO2 treatments. Abalone settlement and metamorphosis increased from 11% in the absence of CCRA to 45-69% when CCRA were present, with minor variation among CCRA genera. Though all CCRA genera reduced growth during exposure to increased pCO2, abalone settlement was unaffected by prior CCRA exposure to increased pCO2. Thus, we find no impacts of OA exposure history on CCRA provision of settlement cues. Additionally, there appears to be functional redundancy in genera of CCRA providing cues to abalone, which may further buffer OA effects.

Persistence and change in community composition of reef corals through present, past, and future climates.

The reduction in coral cover on many contemporary tropical reefs suggests a different set of coral community assemblages will dominate future reefs. To evaluate the capacity of reef corals to persist over various time scales, we examined coral community dynamics in contemporary, fossil, and simulated future coral reef ecosystems. Based on studies between 1987 and 2012 at two locations in the Caribbean, and between 1981 and 2013 at five locations in the Indo-Pacific, we show that many coral genera declined in abundance, some showed no change in abundance, and a few coral genera increased in abundance. Whether the abundance of a genus declined, increased, or was conserved, was independent of coral family. An analysis of fossil-reef communities in the Caribbean revealed changes in numerical dominance and relative abundances of coral genera, and demonstrated that neither dominance nor taxon was associated with persistence. As coral family was a poor predictor of performance on contemporary reefs, a trait-based, dynamic, multi-patch model was developed to explore the phenotypic basis of ecological performance in a warmer future. Sensitivity analyses revealed that upon exposure to thermal stress, thermal tolerance, growth rate, and longevity were the most important predictors of coral persistence. Together, our results underscore the high variation in the rates and direction of change in coral abundances on contemporary and fossil reefs. Given this variation, it remains possible that coral reefs will be populated by a subset of the present coral fauna in a future that is warmer than the recent past.

Trophic cascades result in large-scale coralline algae loss through differential grazer effects.

Removal of predators can have strong indirect effects on primary producers through trophic cascades. Crustose coralline algae (CCA) are major primary producers worldwide that may be influenced by predator removal through changes in grazer composition and biomass. CCA have been most widely studied in Caribbean and temperate reefs, where cover increases with increasing grazer biomass due to removal of competitive fleshy algae. However, each of these systems has one dominant grazer type, herbivorous fishes or sea urchins, which may not be functionally equivalent. Where fishes and sea urchins co-occur, fishing can result in a phase shift in the grazing community with subsequent effects on CCA and other substrata. Kenyan reefs have herbivorous fishes and sea urchins, providing an opportunity to determine the relative impacts of each grazer type and evaluate potential human-induced trophic cascades. We hypothesized that fish benefit CCA, abundant sea urchins erode CCA, and that fishing indirectly reduces CCA cover by removing sea urchin predators. We used closures and fished reefs as a large-scale, long-term natural experiment to assess how fishing and resultant changes in communities affect CCA abundance. We used a short-term caging experiment to directly test the effects of grazing on CCA. CCA cover declined with increasing fish and sea urchin abundance, but the negative impact of sea urchin grazing was much stronger than that of fishes. Abundant sea urchins reduced the CCA growth rate to almost zero and prevented CCA accumulation. A warming event (El Niño Southern Oscillation, ENSO) occurred during the 18-year study and had a strong but short-term positive effect on CCA cover. However, the effect of the ENSO on CCA was lower in magnitude than the effect of sea urchin grazing. We compare our results with worldwide literature on bioerosion by fishes and sea urchins. Grazer influence depends on whether benefits of fleshy algae removal outweigh costs of grazer-induced bioerosion. However, the cost-benefit ratio for CCA appears to change with grazer type, grazer abundance, and environment. In Kenya, predator removal leads to a trophic cascade that is expected to reduce net calcification of reefs and therefore reduce reef stability, growth, and resilience.