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In April of 2006, we observed southern leopard frog (Rana sphenocephala) tadpoles in a pond in northeast Georgia that were dying from an unknown pathogen. Examination of affected specimens, as well
as PCR characterization, revealed that all were infected with a novel alveolate pathogen closely related to freshwater and
marine eukaryotic organisms a...
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We monitored the development and growth of a cohort of Northern Leopard Frog (Lithobates pipiens) tadpoles, in one North American Waterfowl Management Plan (NAWMP) permanent basin and in one natural environment, a bay of the St. Lawrence River. We wanted to know if this kind of artificial wetland could be considered as suitable habitat for this dec...
Frogs exhibit extreme plasticity and individual variation in growth and behavior during metamorphosis, driven by interactions of intrinsic state factors and extrinsic environmental factors. In northern red-legged frogs (Rana aurora Baird and Girard, 1852), we studied the timing of departure from the natal pond as it relates to date and size of indi...
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... Batrachochytrium dendrobatidis (hereafter Bd) and Ranavirus (hereafter Rv; Garner et al., 2018;Greenberg & Palen, 2019;Scheele, Skerratt, et al., 2017). A third pathogen, an alveolate protist often referred to as amphibian Perkinsea (hereafter Pr), is associated with anuran mass mortality events in North America and infects diverse amphibian taxa worldwide (Chambouvet et al., 2015(Chambouvet et al., , 2020Davis et al., 2007;Isidoro-Ayza et al., 2017;Landsberg et al., 2013). ...
... However, we know little about global or regional impacts of Pr over space and time due to a lack of baseline research on Pr epidemiology in amphibian hosts. Furthermore, Pr remains formally undescribed and can be found in the literature as Dermomycoides sp., Perkinsus-like organism, mesomycetazoan parasite and Alveolate organism (Chambouvet et al., 2015;Cook, 2008;Davis et al., 2007;Green et al., 2002;Isidoro-Ayza et al., 2017). Because Pr has been minimally studied and differs taxonomically and physiologically from other amphibian pathogens, understanding when and where Pr infections occur, and which factors are tightly associated with infection, is critical for assessing the risk this EID poses globally. ...
Amphibians suffer from large‐scale population declines globally, and emerging infectious diseases contribute heavily to these declines. Amphibian Perkinsea (Pr) is a worldwide anuran pathogen associated with mass mortality events, yet little is known about its epidemiological patterns, especially in comparison to the body of literature on amphibian chytridiomycosis and ranavirosis.
Here, we establish Pr infection patterns in natural anuran populations and identify important covariates including climate, host attributes and co‐infection with Ranavirus (Rv).
We used quantitative (q)PCR to determine the presence and intensity of Pr and Rv across 1234 individuals sampled throughout central Florida in 2017–2019. We then implemented random forest ensemble learning models to predict infection with both pathogens based on physiological and environmental characteristics.
Perkinsea infected 32% of all sampled anurans, and Pr prevalence was significantly elevated in Ranidae frogs, cooler months, metamorphosed individuals and frogs co‐infected with Rv, while Pr intensity was significantly higher in ranid frogs and individuals collected dead. Ranavirus prevalence was 17% overall and was significantly higher in Ranidae frogs, metamorphosed individuals, locations with higher average temperatures, and individuals co‐infected with Pr. Perkinsea prevalence was significantly higher than Rv prevalence across months, regions, life stages and species. Among locations, Pr prevalence was negatively associated with crayfish prevalence and positively associated with relative abundance of microhylids, but Rv prevalence did not associate with any tested co‐variates. Co‐infections were significantly more common than single infections for both pathogens, and we propose that Pr infections may propel Rv infections because seasonal Rv infection peaks followed Pr infection peaks and random forest models found Pr intensity was a leading factor explaining Rv infections.
Our study elucidates epidemiological patterns of Pr in Florida and suggests that Pr may be under‐recognized as a cause of anuran declines, especially in the context of pathogen co‐infection.
... Despite the many contributing factors to amphibian declines, such as habitat loss, overexploitation, climate change or contamination, emerging infectious diseases have been described among the top threats to populations (Collins and Storfer 2003, Rachowicz et al. 2006, Skerratt et al. 2007). Several pathogens have been linked to outbreaks and mass mortality events in amphibian assemblages such as Amphibiocystidium (the causative agent for dermocystidiosis; GonzálezHernández et al. 2010), some bacterial agents of the family Chlamydiaceae (Martel et al. 2012a,b) as well as recently discovered parasitic protists belonging to the phylum Perkinsea (linked to mortality events of larval and juvenile ranid frogs; Davis et al. 2007, Landsberg et al. 2013, IsidroAyza et al. 2017). Yet, there is little overall understanding of these pathogens and their dynamics, which heavily contrasts with the advances of the past three decades in chytridiomycosis (Berger et al. 1998) and ranavirosis research (Cunningham et al. 1996, Bollinger et al. 1999. ...
... However, in April 2006, in a pond in northeast Georgia (United States), Davis et al. (2007) observed a massive mortality event of southern leopard tadpoles, Rana sphenocephala (syn. Lithobates sphenocephalus), attributed to an unknown protist affiliated by phylogenetic analysis to the Perkinsea lineage. ...
... Infectious agents of the PPC clade have been identified as responsible for the die-offs of tadpole throughout the United States (Isidoro-Ayza et al., 2017). Infected tadpoles with pathological symptoms, named "severe Perkinsea infection" (SPI), showed lethargic swimming with enlarged and histopathologic lesions of the liver, mesonephros, spleen, pancreas, gills, gastrointestinal tract, skeletal muscle, dermis, and peritoneum (Green et al., 2002;Davis et al., 2007;Jones et al., 2012;Isidoro-Ayza et al., 2017). Histological examination of infected liver tissues revealed a massive number of round infective cells replacing the normal hepatic parenchyma. ...
The last century has witnessed an increasing rate of new disease emergence across the world leading to permanent loss of biodiversity. Perkinsea is a microeukaryotic parasitic phylum composed of four main lineages of parasitic protists with broad host ranges. Some of them represent major ecological and economical threats because of their geographically invasive ability and pathogenicity (leading to mortality events). In marine environments, three lineages are currently described, the Parviluciferaceae, the Perkinsidae, and the Xcellidae, infecting, respectively, dinoflagellates, mollusks, and fish. In contrast, only one lineage is officially described in freshwater environments: the severe Perkinsea infectious agent infecting frog tadpoles. The advent of high-throughput sequencing methods, mainly based on 18S rRNA assays, showed that Perkinsea is far more diverse than the previously four described lineages especially in freshwater environments. Indeed, some lineages could be parasites of green microalgae, but a formal nature of the interaction needs to be explored. Hence, to date, most of the newly described aquatic clusters are only defined by their environmental sequences and are still not (yet) associated with any host. The unveiling of this microbial black box presents a multitude of research challenges to understand their ecological roles and ultimately to prevent their most negative impacts. This review summarizes the biological and ecological traits of Perkinsea—their diversity, life cycle, host preferences, pathogenicity, and highlights their diversity and ubiquity in association with a wide range of hosts.
... While the prevalence and infection intensities we measured were consistently low, in line with the findings of Chambouvet et al. (2015) in French Guiana (prevalence = 3%), suggesting only sub-clinical infections, the fitness impacts of these infections are unknown. Furthermore, Pr tends to cause mortality in larval frogs (Davis et al., 2007), and no data exist on how this may translate to direct-developing Pristimantis embryos, which are rarely observed, but could represent the life stage experiencing Pr mortality. Our results suggest an effect of elevation on Pr prevalence, with an increase in Pr positives at higher elevations. ...
Three infectious pathogens Batrachochytrium dendrobatidis (Bd), Ranavirus (Rv) and Perkinsea (Pr) are associated with widespread and ongoing amphibian population declines. Although their geographic and host ranges vary widely, recent studies have suggested that the occurrence of these pathogens could be more common than previously thought, even in direct-developing terrestrial species traditionally considered less likely to harbor these largely aquatic pathogens. Here, we characterize Bd, Rv, and Pr infections in direct-developing terrestrial amphibians of the Pristimantis genus from the highland Ecuadorean Andes. We confirm the first detection of Pr in terrestrial-breeding amphibians and in the Andean region, present the first report of Rv in Ecuador, and we add to the handful of studies finding Bd infecting Pristimantis. Infection prevalence did not differ significantly among pathogens, but infection intensity was significantly higher for Bd compared to Pr. Neither prevalence nor intensity differed significantly across locality and elevation for Bd and Rv, although low prevalence in our dataset and lack of seasonal sampling could have prevented important epidemiological patterns from emerging. Our study highlights the importance of incorporating pathogen surveillance in biodiversity monitoring in the Andean region and serves as starting point to understand pathogen dynamics, transmission, and impacts in terrestrial-breeding frogs.
... For example, no genetic information on Rastrimonas subtilis (Brugerolle) infecting the freshwater algae cryptophyte is available at present, but observations of its ultrastructure support that this parasite belongs to the phylum Perkinsozoa (Brugerolle, 2002(Brugerolle, , 2003. Sequences of the infectious agent of tadpoles in freshwater environments, which was possibly associated with amphibian mortality events, have also been reported (Davis et al., 2007). These sequences were clustered with numerous environmental sequences obtained from freshwater environments and formed a clade referred to as Novel Alveolate Group 01 (NAG01) (Chambouvet et al., 2015), whereas no detailed information on their morphology and life cycle is available. ...
... Perkinsozoa members parasitize a diverse array of host organisms, ranging from single-celled eukaryotes such as dinoflagellates and cryptophytes to mollusks and fishes (Mackin et al., 1950;Norén et al., 1999;Brugerolle, 2002;Freeman et al., 2017). Recently, tadpoles and, presumably, green algae were shown or suggested to be hosts of Perkinsozoan parasites (Davis et al., 2007;Mangot et al., 2013;Chambouvet et al., 2015;Jobard et al., 2020); however, they were not formally described. Of note, before the current study, all Perkinsozoan parasites infecting dinoflagellates were phylogenetically placed in the family Parviluciferaceae. ...
The phylum Perkinsozoa is known as an exclusively parasitic group within alveolates and is widely distributed in various aquatic environments from marine to freshwater environments. Nonetheless, their morphology, life cycle, the identity of the host, and physiological characteristics remain still poorly understood. During intensive sampling along the west coast of Korea in October and November 2017, a new parasitoid, which shares several characteristics with the extant families Perkinsidae and Parviluciferaceae, was discovered and three strains of the new parasitoid were successfully established in cultures. Cross-infection experiments showed that among the examined planktonic groups, only dinoflagellates were susceptible to the new parasitoid, with infections observed in species belonging to eight genera. Even though the new parasitoid shared many morphological and developmental characteristics with other Perkinsozoan parasites, it differed from them by its densely packed trophocyte structure without a large vacuole or hyaline material during the growth stage. These characteristics are common among Parviluciferaceae members. Furthermore, through palintomic extracellular sporogenesis, it produced characteristic interconnected sporocytes resembling a string of beads. Phylogenetic analyses based on the small subunit and large subunit ribosomal DNA sequences revealed that the new parasitoid was distantly related to the family Parviluciferaceae and was more closely related to the families Perkinsidae and Xcellidae. Morphological, ultrastructural, and molecular data on the new parasitoid raised the need to erect a new family, i.e., Pararosariidae, within the phylum Perkinsozoa with Pararosarium dinoexitiosum gen. et sp. nov. as the type species. The isolation and establishment in culture of the new parasitoid outside the family Parviluciferaceae in the present study would contribute to the better understanding of the diversity of Perkinsozoan parasites and provide useful material for comparisons to other parasite species in the further study.
... The disease pathology has been associated with a specific group of Perkinsea protists (syn. Perkinsids, Perkinsozoa) called Pathogenic Perkinsea Clade (PPC) based on small-subunit ribosomal DNA (SSU rDNA) sequencing [1,2]. This group is part of a wider clade of the freshwater Perkinsea named Novel-Alveolate-Group-01 (NAG01) [3]. ...
... This group is part of a wider clade of the freshwater Perkinsea named Novel-Alveolate-Group-01 (NAG01) [3]. Tadpole associations with specific clades of NAG01-Perkinsea have been detected across multiple continents [3], yet the PPC clade associated with SPI has only been detected in the USA, and mainly from SPI-diseased tadpoles [1,2]. Formal identification of the disease relationship satisfying Koch's postulates is absent [4]. ...
... The first documented MMEs associated with SPI were recorded in New Hampshire in 1999 [7] and, since then, SPI-associated mortality events have been reported in seven other states across a broad geographic range [1,2,7,8]. Most of these SPI-associated MMEs have occurred in Lithobates spp. ...
Severe Perkinsea infection is an emerging disease of amphibians, specifically tadpoles. Disease presentation correlates with liver infections of a subclade of Perkinsea (Alveolata) protists, named Pathogenic Perkinsea Clade (PPC). Tadpole mortality events associated with PPC infections have been reported across North America, from Alaska to Florida. Here, we investigate the geographic and host range of PPC associations in seemingly healthy tadpoles sampled from Panama, a biogeographic provenance critically affected by amphibian decline. To complement this work, we also investigate a mortality event among Hyla arborea tadpoles in captive-bred UK specimens. PPC SSU rDNA was detected in 10 of 81 Panama tadpoles tested, and H. arborea tadpoles from the UK. Phylogenies of the Perkinsea SSU rDNA sequences demonstrate they are highly similar to PPC sequences sampled from mortality events in the USA, and phylogenetic analysis of tadpole mitochondrial SSU rDNA demonstrates, for the first time, PPC associations in diverse hylids. These data provide further understanding of the biogeography and host range of this putative pathogenic group, factors likely to be important for conservation planning.
... Information regarding members of the paraphyletic group of protists involved in co-infections in amphibians is exceptionally scarce. In the last two decades, many mass mortality events were reported in North American Lithobates sphenocephala tadpoles [117,118], where the causative agent was identified to be an intracellular protist parasite of the phylum Perkinsozoa (genus Perkinsus) belonging to the superphylum Alveolata [119,120]. It appears that the parasite exhibits cryptic genetic diversity and is widespread globally [120]. ...
... It appears that the parasite exhibits cryptic genetic diversity and is widespread globally [120]. Infection with Perkinsus causes pathology in the liver of tadpoles [118], an organ which is also known to be targeted by other alveolate parasites like Nematopsis temporariae [121][122][123] or Goussia sp. [124,125]. ...
Parasites, including viruses, bacteria, fungi, protists, helminths, and arthropods, are ubiquitous in the animal kingdom. Consequently, hosts are frequently infected with more than one parasite species simultaneously. The assessment of such co-infections is of fundamental importance for disease ecology, but relevant studies involving non-domesticated animals have remained scarce. Many amphibians are in decline, and they generally have a highly diverse parasitic fauna. Here we review the literature reporting on field surveys, veterinary case studies, and laboratory experiments on co-infections in amphibians, and we summarize what is known about within-host interactions among parasites, which environmental and intrinsic factors influence the outcomes of these interactions, and what effects co-infections have on hosts. The available literature is piecemeal, and patterns are highly diverse, so that identifying general trends that would fit most host–multiparasite systems in amphibians is difficult. Several examples of additive, antagonistic, neutral, and synergistic effects among different parasites are known, but whether members of some higher taxa usually outcompete and override the effects of others remains unclear. The arrival order of different parasites and the time lag between exposures appear in many cases to fundamentally shape competition and disease progression. The first parasite to arrive can gain a marked reproductive advantage or induce cross-reaction immunity, but by disrupting the skin and associated defences (i.e., skin secretions, skin microbiome) and by immunosuppression, it can also pave the way for subsequent infections. Although there are exceptions, detrimental effects to the host are generally aggravated with increasing numbers of co-infecting parasite species. Finally, because amphibians are ectothermic animals, temperature appears to be the most critical environmental factor that affects co-infections, partly via its influence on amphibian immune function, partly due to its direct effect on the survival and growth of parasites. Besides their importance for our understanding of ecological patterns and processes, detailed knowledge about co-infections is also crucial for the design and implementation of effective wildlife disease management, so that studies concentrating on the identified gaps in our understanding represent rewarding research avenues.
Graphical Abstract
... Bd has been documented as a factor in many species extinctions and population declines globally, particularly in North and South America (Burrowes and De la Riva, 2017a;Carvalho et al., 2017;Fisher et al., 2009;Green et al., 2002;Isidoro-Ayza et al., 2017), and particularly in the Ranidae, Hylidae and Bufonidae families (La Marca et al., 2005;Rothermel et al., 2008;Scheele et al., 2019;Van Rooij et al., 2015;Watters et al., 2018). Pr is an emerging protist pathogen that primarily infects larval and juvenile frogs in the family Ranidae (Cook, 2008;Davis et al., 2007). Based on limited genetic data, Pr is an alveolate that is most similar to the bivalve pathogen Perkinsus, but it remains taxonomically undescribed (Chambouvet et al., 2015;Davis et al., 2007;Isidoro-Ayza et al., 2017;Karwacki et al., 2018;Landsberg et al., 2013). ...
... Pr is an emerging protist pathogen that primarily infects larval and juvenile frogs in the family Ranidae (Cook, 2008;Davis et al., 2007). Based on limited genetic data, Pr is an alveolate that is most similar to the bivalve pathogen Perkinsus, but it remains taxonomically undescribed (Chambouvet et al., 2015;Davis et al., 2007;Isidoro-Ayza et al., 2017;Karwacki et al., 2018;Landsberg et al., 2013). Pr has spore and zoospore life stages, the latter of which penetrates anuran embryos, hatchlings, and tadpoles, or is ingested through the water column by pre-and post-metamorphic anurans (Cook, 2008;Davis et al., 2007). ...
... Based on limited genetic data, Pr is an alveolate that is most similar to the bivalve pathogen Perkinsus, but it remains taxonomically undescribed (Chambouvet et al., 2015;Davis et al., 2007;Isidoro-Ayza et al., 2017;Karwacki et al., 2018;Landsberg et al., 2013). Pr has spore and zoospore life stages, the latter of which penetrates anuran embryos, hatchlings, and tadpoles, or is ingested through the water column by pre-and post-metamorphic anurans (Cook, 2008;Davis et al., 2007). The zoospore embeds in the host's liver, spreads throughout the internal organs, and degrades host tissue leading to organ failure (Davis et al., 2007;Green et al., 2003). ...
Batrachochytrium dendrobatidis (Bd), Perkinsea (Pr), and Ranavirus (Rv) are three leading pathogens linked to amphibian declines, but much remains unknown about their origins and spread. Museum specimens are a critical resource for resolving epidemiological patterns because they can elucidate infection dynamics and their relationship with disease outbreaks. Here, we established a timeline of Bd, Pr, and Rv presence in anurans in Florida, USA, a region of high amphibian diversity. We measured pathogen prevalence and pathogen intensity in museum specimens of the genus Rana, a widely infected host lineage. We also sequenced barcoding regions to determine whether genetic lineages varied over time. Before this study, the first documented cases of Bd, Pr, and Rv in Florida were in 2009, 2002, and 2002, respectively. We detected all three pathogens nearly a century earlier, with the earliest Bd and Pr detections in 1928 and the earliest Rv detection in 1922. Prevalence and intensity varied across host species, geographic regions, life stages, and decades, with unique patterns for each pathogen. While we were unable to recover robust sequence data for Rv, the Bd and Pr sequence data showed no genetic variation over time. We found significantly more co-detection of Pr and Rv compared to any other pathogen combination, and to our knowledge, we also detected the first simultaneous tri-detection of Bd, Pr, and Rv. Overall, this study represents considerable extension of the timing and understanding of amphibian pathogens in the southeastern USA.
... This finding is consistent with previous studies documenting that Pr infections are associated with mortality (Green et al. 2002;Isidoro-Ayza et al. 2017, 2019 and that O. septentrionalis tend to harbor higher Pr infection intensities than other species in the same amphibian communities (Karwacki et al. 2018). Pr infection may be amplified in local amphibian communities where O. septentrionalis occurs, and this is particularly concerning in regions such as New Orleans, because many of the Pr die-offs in native species have been reported from the southeastern U.S. (Davis et al. 2007;Landsberg et al. 2013), suggesting high pathogenicity, host susceptibility, or both. The lack of a significant difference in Fulton's Body Condition among Rv infected individuals suggests O. septentrionalis may exhibit some level of tolerance to Rv, which could lead to amplification of this pathogen as has been documented in other species (Brunner et al. 2019). ...
... Given the smaller number of larvae relative to adults in this study, the different tissues analyzed, and the single sampling date when larvae were collected, further data are needed to draw conclusions about life stage differences in Rv infection dynamics. Similarly, we did not find any difference in Pr prevalence or intensity across life stages, which was an unexpected finding as previous studies have identified that Pr primarily infects larvae (Karwacki et al. 2018) or recently metamorphosed frogs (Davis et al. 2007;Landsberg et al. 2013). Again, our results should be interpreted with caution due to limited sampling events, different tissue types, and small sample sizes. ...
Invasive species can negatively impact ecosystems in numerous ways, including vectoring pathogenic organisms. In amphibians, a lineage globally threatened by multiple pathogens, this spread of disease via invasive species could contribute to declines in native populations. The Cuban Treefrog (Osteopilus septentrionalis) is invasive in the southeastern USA. To assess whether O. septentrionalis is a potential reservoir host for the pathogens Batrachochytrium dendrobatidis (Bd; Amphibian Chytrid Fungus), Ranavirus (Rv), and Perkinsea (Pr), we sampled 82 individuals from a recently invaded site in New Orleans, Louisiana, USA. We used quantitative PCR to assess prevalence and intensity of Bd, Rv, and Pr in mouthparts and tail clips from 22 larvae and in toe clips from 60 metamorphosed frogs. We compared infection prevalence and intensity across host characteristics, including Fulton's Body Condition, sex, and life stage. None of the individuals were infected with Bd, 72% were infected with Rv, and 44% were infected with Pr. Twenty-three individuals (28%) were co-infected with Rv and Pr, but co-infection did not significantly predict the prevalence or intensity of either Rv or Pr. Although we did not observe any disease signs, Pr infections were significantly associated with lower body condition, suggesting sub-lethal fitness costs. Our study establishes that invasive O. septentrionalis in New Orleans are infected with two pathogens of global concern for amphibians. Understanding host-pathogen dynamics in O. septentrionalis in Louisiana is a critical step towards understanding how this invasive species could threaten amphibian biodiversity in the region by transmitting infectious pathogens.
... Tadpole mass mortality events (MMEs) associated with an alveolate protist in the phylum Perkinsea have been reported across a broad geographic range within the USA [1][2][3][4][5]. Recent phylogenetic analysis of Perkinsea identified these putative pathogens as a distinctive clade within a broad monophyletic group of alveolates known as novel alveolate group 01 (NAG01; [6]). ...
... NAG01 includes three clades (NAG01a-c) known to infect tadpoles from a broad range of taxa without causing any identifiable gross or tissue-level symptoms of disease [6], and a discrete clade, designated pathogenic Perkinsea clade (PPC), which is associated with severe Perkinsea infection (SPI) [2]. PPC sequences have been detected in all SPI-associated MMEs described in the USA [1,2], but from only a small proportion of phenotypically normal tadpoles [2], indicating that PPC is the likely causative agent of SPI. Nevertheless, the exact relationship between the PPC clade, the wider NAG01 clade, and SPI-associated disease remains undefined and untested. ...
... The primers used in this study (table 1) target an approximately 287-290 bp fragment of the 18S rRNAencoding gene of NAG01 Perkinsea. Specifically, the PPC assay targets this region in a Rana sphenocephala pathogen (accession: EF675616) [1], while the NAG01a-c assay targets this region in a randomly selected NAG01-targeted clone (accession: KP122572) [6] (figure 1). The probes used in this study (table 1) target an approximately 113-116 bp variant region within this fragment that can be used to distinguish NAG01a-c from PPC (figure 1). ...
Alveolate protists within the phylum Perkinsea have been found to infect amphibians across a broad taxonomic and geographic range. Phylogenetic analysis has suggested the existence of two clades of amphibian Perkinsea: a putatively non-pathogenic clade linked to asymptomatic infections of tadpoles in Africa, Europe and South America, and a putatively pathogenic clade linked to disease and mass mortality events of tadpoles in North America. Here, we describe the development of a duplex TaqMan qPCR assay to detect and discriminate between rDNA sequences from both clades of Perkinsea in amphibian tissues. The assay uses a single primer pair to target an 18S small subunit (SSU) ribosomal RNA (rRNA) gene region shared between the two clades, and two dual-labelled probes to target a region within this fragment that is diagnostic for each clade. This assay enables rapid screening for each of the two Perkinsea groups, allowing for detection, primarily of the phylogenetic group associated with disease outbreaks, and secondarily for the phylogenetic group with no current disease relationship identified. Incorporation of our novel qPCR assay into the routine surveillance of amphibian populations will allow for the assessment of the incidence of each protist clade, thereby providing an improved understanding of Perkinsea infection pervasiveness and a method to underpin future conservation planning.
