Amphibian Disease Research Articles

Whether the Weather Drives Patterns of Endemic Amphibian Chytridiomycosis: A Pathogen Proliferation Approach

The pandemic amphibian disease chytridiomycosis often exhibits strong seasonality in both prevalence and disease-associated mortality once it becomes endemic. One hypothesis that could explain this temporal pattern is that simple weather-driven pathogen proliferation (population growth) is a major driver of chytridiomycosis disease dynamics. Despite various elaborations of this hypothesis in the literature for explaining amphibian declines (e.g., the chytrid thermal-optimum hypothesis) it has not been formally tested on infection patterns in the wild. In this study we developed a simple process-based model to simulate the growth of the pathogen Batrachochytrium dendrobatidis (Bd) under varying weather conditions to provide an a priori test of a weather-linked pathogen proliferation hypothesis for endemic chytridiomycosis. We found strong support for several predictions of the proliferation hypothesis when applied to our model species, Litoria pearsoniana, sampled across multiple sites and years: the weather-driven simulations of pathogen growth potential (represented as a growth index in the 30 days prior to sampling; GI30) were positively related to both the prevalence and intensity of Bd infections, which were themselves strongly and positively correlated. In addition, a machine-learning classifier achieved ∼72% success in classifying positive qPCR results when utilising just three informative predictors 1) GI30, 2) frog body size and 3) rain on the day of sampling. Hence, while intrinsic traits of the individuals sampled (species, size, sex) and nuisance sampling variables (rainfall when sampling) influenced infection patterns obtained when sampling via qPCR, our results also strongly suggest that weather-linked pathogen proliferation plays a key role in the infection dynamics of endemic chytridiomycosis in our study system. Predictive applications of the model include surveillance design, outbreak preparedness and response, climate change scenario modelling and the interpretation of historical patterns of amphibian decline.

Mitigating amphibian chytridiomycosis with bioaugmentation: characteristics of effective probiotics and strategies for their selection and use

Probiotic therapy through bioaugmentation is a feasible disease mitigation strategy based on growing evidence that microbes contribute to host defences of plants and animals. Amphibians are currently threatened by the rapid global spread of the pathogen, Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis. Bioaugmentation of locally occurring protective bacteria on amphibians has mitigated this disease effectively in laboratory trials and one recent field trial. Areas still naïve to Bd provide an opportunity for conservationists to proactively implement probiotic strategies to prevent further amphibian declines. In areas where Bd is endemic, bioaugmentation can facilitate repatriation of susceptible amphibians currently maintained in assurance colonies. Here, we synthesise the current research in amphibian microbial ecology and bioaugmentation to identify characteristics of effective probiotics in relation to their interactions with Bd, their host, other resident microbes and the environment. To target at-risk species and amphibian communities, we develop sampling strategies and filtering protocols that result in probiotics that inhibit Bd under ecologically relevant conditions and persist on susceptible amphibians. This filtering tool can be used proactively to guide amphibian disease mitigation and can be extended to other taxa threatened by emerging infectious diseases.

The pandemic pathogen of amphibians, Batrachochytrium dendrobatidis (Phylum Chytridiomycota), in Italy

Worldwide amphibian declines and species losses are global problems and emerging infectious diseases have been identified as one of the major threats. The worst of these is chytridiomycosis, an amphibian disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Here we review what is known of the distribution of Bd and chytridiomycosis in Italy. We critically summarize the evidence in support of the hypothesis that Bd is an invasive pathogen in Italy. Last we provide recommendations for immediate research needs, both for basic science and applied conservation.

Amphibian Chytrid Prevalence in an Amphibian Community in Arid Australia

[Extract] The amphibian disease chytridiomycosis, caused by the pathogen Batrachochytrium dendrobatidis (Bd), has dramatically affected amphibians, causing population declines in over 200 species worldwide (Fisher et al. 2009). The disease is widespread, driving amphibian declines in North America (Muths et al. 2003; Briggs et al. 2005), Australia (Berger et al. 1998), Central America (Lips et al. 2006) and South America (Catenazzi et al. 2011). The variation in susceptibility to disease and mortality seen among host species, populations and locations is at least partially driven by interplay between external environmental and internal host-specific factors (Woodhams et al. 2007; Searle et al. 2011; Blaustein et al. 2012). While at a single location some species may be locally extirpated, others may persist (Lips et al. 2006). Amphibian infection prevalence and mortality rates due to chytridiomycosis are correlated with ambient environmental conditions: being highest during cooler months and at higher elevations (Berger et al. 1998; Woodhams and Alford 2005; Kriger and Hero 2008).

Prior Infection Does Not Improve Survival against the Amphibian Disease Chytridiomycosis

Many amphibians have declined globally due to introduction of the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Hundreds of species, many in well-protected habitats, remain as small populations at risk of extinction. Currently the only proven conservation strategy is to maintain species in captivity to be reintroduced at a later date. However, methods to abate the disease in the wild are urgently needed so that reintroduced and wild animals can survive in the presence of Bd. Vaccination has been widely suggested as a potential strategy to improve survival. We used captive-bred offspring of critically endangered booroolong frogs (Litoria booroolongensis) to test if vaccination in the form of prior infection improves survival following re exposure. We infected frogs with a local Bd isolate, cleared infection after 30 days (d) using itraconazole just prior to the onset of clinical signs, and then re-exposed animals to Bd at 110 d. We found prior exposure had no effect on survival or infection intensities, clearly showing that real infections do not stimulate a protective adaptive immune response in this species. This result supports recent studies suggesting Bd may evade or suppress host immune functions. Our results suggest vaccination is unlikely to be useful in mitigating chytridiomycosis. However, survival of some individuals from all experimental groups indicates existence of protective innate immunity. Understanding and promoting this innate resistance holds potential for enabling species recovery.

Exserohilum Rostratum Fungal Meningitis Associated With Methylprednisolone Injections

<i>Exserohilum Rostratum</i> Fungal Meningitis Associated With Methylprednisolone Injections

Biodiversity decreases disease through predictable changes in host community competence

Accelerating rates of species extinctions and disease emergence underscore the importance of understanding how changes in biodiversity affect disease outcomes. Over the past decade, a growing number of studies have reported negative correlations between host biodiversity and disease risk, prompting suggestions that biodiversity conservation could promote human and wildlife health. Yet the generality of the diversity-disease linkage remains conjectural, in part because empirical evidence of a relationship between host competence (the ability to maintain and transmit infections) and the order in which communities assemble has proven elusive. Here we integrate high-resolution field data with multi-scale experiments to show that host diversity inhibits transmission of the virulent pathogen Ribeiroia ondatrae and reduces amphibian disease as a result of consistent linkages among species richness, host composition and community competence. Surveys of 345 wetlands indicated that community composition changed nonrandomly with species richness, such that highly competent hosts dominated in species-poor assemblages whereas more resistant species became progressively more common in diverse assemblages. As a result, amphibian species richness strongly moderated pathogen transmission and disease pathology among 24,215 examined hosts, with a 78.4% decline in realized transmission in richer assemblages. Laboratory and mesocosm manipulations revealed an approximately 50% decrease in pathogen transmission and host pathology across a realistic diversity gradient while controlling for host density, helping to establish mechanisms underlying the diversity-disease relationship and their consequences for host fitness. By revealing a consistent link between species richness and community competence, these findings highlight the influence of biodiversity on infection risk and emphasize the benefit of a community-based approach to understanding infectious diseases.

Immune Suppression by Neonicotinoid Insecticides at the Root of Global Wildlife Declines

of infectious diseases in honey bees, fish, amphibians, bats and birds in the past two decades have coincided with the increasing use of systemic insecticides, notably the neonicotinoids and fipronil. A link between insecticides and such diseases is hypothesised. Firstly, the disease outbreaks started in countries and regions where systemic insecticides were used for the first time, and later they spread to other countries. Secondly, recent evidence of immune suppression in bees and fish caused by neonicotinoids has provided an important clue to understand the sub-lethal impact of these insecticides not only on these organisms, but probably on other wildlife affected by emerging infectious diseases. While this is occurring, environmental authorities in developed countries ignore the calls of apiarists (who are most affected) and do not target neonicotinoids in their regular monitoring schedules. Equally, scientists looking for answers to the problem are unaware of the new threat that systemic insecticides have introduced in terrestrial and aquatic ecosystems. Journal of Environmental Immunology and Toxicology 2013; 1:3-12

Chytridiomycosis: a global threat to amphibians

Chytridiomycosis, which is caused by Batrachochytrium dendrobatidis, is an emerging infectious disease of amphibians. The disease is one of the main causes of the global decline in amphibians. The aetiological agent is ubiquitous, with worldwide distribution, and affects a large number of amphibian species in several biomes. In the last decade, scientific research has substantially increased knowledge of the aetiological agent and the associated infection. However, important epidemiological aspects of the environment-mediated interactions between the aetiological agent and the host are not yet clear. The objective of the present review is to describe chytridiomycosis with regard to the major features of the aetiological agent, the host and the environment.

Interactive effects of competition and predator cues on immune responses of leopard frogs at metamorphosis

Recent hypotheses suggest that immunosuppression, resulting from altered environmental conditions, may contribute to the increased incidence of amphibian disease around the world. Antimicrobial peptides (AMPs) in amphibian skin are an important innate immune defense against fungal, viral and bacterial pathogens. Their release is tightly coupled with release of the stress hormone noradrenaline (norepinephrine). During metamorphosis, AMPs may constitute the primary immune response in the skin of some species because acquired immune functions are temporarily suppressed in order to prevent autoimmunity against new adult antigens. Suppression of AMPs during this transitional stage may impact disease rates. We exposed leopard frog tadpoles (Lithobates pipiens) to a factorial combination of competitor and caged-predator environments and measured their development, growth and production of hydrophobic skin peptides after metamorphosis. In the absence of predator cues, or if the exposure to predator cues was late in ontogeny, competition caused more than a 250% increase in mass-standardized hydrophobic skin peptides. Predator cues caused a decrease in mass-standardized hydrophobic skin peptides when the exposure was late in ontogeny under low competition, but otherwise had no effect. Liquid chromatography tandem mass spectrometry of the skin peptides showed that they include six AMPs in the brevinin and temporin families and at least three of these peptides are previously uncharacterized. Both of these peptide families have previously been shown to inhibit harmful microbes including Batrachochytrium dendrobatidis, the fungal pathogen associated with global amphibian declines. Our study shows that amphibians may be able to adjust their skin peptide defenses in response to stressors that are experienced early in ontogeny and that these effects extend through an important life-history transition.

Techniques for minimizing the effects of PCR inhibitors in the chytridiomycosis assay

Chytridiomycosis is an amphibian disease of global conservation concern that is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Since the discovery of Bd in 1998, several methods have been used for detection of Bd; among these polymerase chain reaction (PCR) from skin swabs is accepted as the best method due to its noninvasiveness, high sensitivity and ease of use. However, PCR is not without problems - to be successful, this technique is dependent upon the presence of nondegraded DNA template and reaction contents that are free from inhibitors. Here, we report on an investigation of several techniques aimed at improving the reliability of the Bd PCR assay by minimizing the effects of humic acid (HA), a potent PCR inhibitor. We compared the effectiveness of four DNA extraction kits (DNeasy, QIAamp DNA Stool, PowerLyzer Power Soil and PrepMan Ultra) and four PCR methods (Amplitaq Gold, bovine serum albumin, PowerClean DNA Clean-up and inhibitor resistant Taq Polymerase). The results of this and previous studies indicate that chytridiomycosis studies that use PCR methods for disease detection may be significantly underestimating the occurrence of Bd. Our results suggest that to minimize the inhibitory effects of HA, DNeasy should be used for sample DNA extraction and Amplitaq Gold with bovine serum albumin should be used for the Bd PCR assay. We also outline protocols tested, show the results of our methods comparisons and discuss the pros and cons of each method.

Changes in cutaneous microbial abundance with sloughing: possible implications for infection and disease in amphibians

The emergence of disease as a significant global threat to amphibian diversity has generated considerable interest in amphibian defenses against cutaneous microbial infection and disease. To date, however, the influence of sloughing on the susceptibility of amphibians to infection and disease has been largely overlooked. To investigate the potential for sloughing to regulate topical microbial loads, the abundance of cultivable cutaneous bacteria and fungi in the cane toad Rhinella marina were compared before and after sloughing. Toads were also exposed to fluctuating thermal regimes (10-20 and 20-30°C) and variable photoperiods to investigate possible effects of season and climate on sloughing periodicity. Sloughing substantially reduced the abundance of cultivable cutaneous bacteria and fungi by up to 100%. The intermoult interval of toads maintained at 10-20°C was twice that of animals at 20-30°C and did not appear to thermally acclimate. Photoperiod had no discernable influence on sloughing periodicity. Results of this study suggest that normal sloughing cycles could play a significant role in controlling the persistence and build-up of cutaneous microbes, including pathogens. The loss of non-pathogenic commensal and protective skin microbiota after sloughing may also influence host susceptibility to cutaneous pathogens. We suggest that the spatio-temporal dynamics of chytridiomycosis, the widespread and often fatal disease caused by the fungal pathogen Batrachochytrium dendrobatidis, are related to temperature not only because of its effect on the growth of the fungus, but also because of its effect on the frequency of host sloughing.

Hematologic and plasma biochemical reference intervals for health monitoring of wild Australian tree frogs

Few hematologic and biochemical reference intervals for wild amphibians have been established. Reference values would aid in early detection of emerging infectious diseases, which are a significant problem for amphibian conservation efforts. We aimed to establish reference intervals for a wide range of hematologic and plasma biochemistry variables for 2 species of Australian tree frogs, describe morphologic features of leukocytes, and analyze the effects of season, year, and parasite status on blood values. Blood specimens were collected from reference populations of wild adult Australian tree frogs, Litoria caerulea and L infrafrenata, for analysis of hematologic (manual) variables, plasma biochemical (automated) analytes, and plasma and serum proteins using automated methods, refractometry, and electrophoresis. Inter- and intraspecies differences were found in L caerulea (n = 80) and L infrafrenata (n = 66) frogs for hematologic and biochemical variables. Intraspecies differences were largely associated with seasonal variations. In the dry season, both species had higher WBC counts, with higher lymphocyte counts in L caerulea and higher neutrophil counts in L infrafrenata, and uric acid concentrations. In the wet season, both species had higher glucose and potassium concentrations, L caerulea frogs had higher neutrophil counts, and L infrafrenata frogs had higher total protein, phosphorus, and sodium concentrations, AST activity, PCV, hemoglobin concentration, and RBC, thrombocyte, and basophil counts. Hemogregarines were identified in 19% of blood samples from L infrafrenata frogs; multiple hematologic and biochemical variables were altered in infected frogs. Wide interspecies and seasonal variations highlight the need to establish species- and season-specific reference intervals for amphibians. Hematologic and plasma biochemical reference values should be useful in assessing the health status and in detecting emerging diseases in wild amphibians.

Temperature alters reproductive life history patterns in Batrachochytrium dendrobatidis, a lethal pathogen associated with the global loss of amphibians

Understanding how pathogens respond to changing environmental conditions is a central challenge in disease ecology. The environmentally sensitive fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the amphibian disease chytridiomycosis, has spread globally causing amphibian extirpations in a wide variety of climatic regions. To gain an in-depth understanding of Bd's responses to temperature, we used an integrative approach, combining empirical laboratory experiments with mathematical modeling. First, we selected a single Bd isolate and serially propagated two lineages of the isolate for multiple generations in two stable thermal conditions: 4°C (cold-adapted lineage) and 23°C (warm-adapted lineage). We quantified the production of infectious zoospores (fecundity), the timing of zoospore release, and zoospore activity in reciprocal temperature transplant experiments in which both Bd lineages were grown in either high or low temperature conditions. We then developed population growth models for the Bd lineages under each set of temperature conditions. We found that Bd had lower population growth rates, but longer periods of zoospore activity in the low temperature treatment (4°C) compared to the high temperature treatment (23°C). This effect was more pronounced in Bd lineages that were propagated in the low temperature treatment (4°C), suggesting a shift in Bd's response to low temperature conditions. Our results provide novel insights into the mechanisms by which Bd can thrive in a wide variety of temperature conditions, potentially altering the dynamics of chytridiomycosis and thus, the propensity for Bd to cause amphibian population collapse. We also suggest that the adaptive responses of Bd to thermal conditions warrant further investigation, especially in the face of global climate change.

Cytochemical and immunocytochemical characterization of blood cells and immunohistochemical analysis of spleen cells from 2 species of frog, Rana (Aquarana) catesbeiana and Xenopus laevis

Mechanisms of amphibian diseases are not characterized as well as those in domestic mammalian species. Antemortem laboratory testing is limited in frogs, presenting a diagnostic challenge to zoos, laboratories, and exotic veterinarians. This study aimed to characterize blood cells and splenic cells from 2 anuran species based on characteristics identified by Wright staining, cytochemical staining, and immunochemical analysis and on histologic examination of spleens. Blood specimens and spleens were obtained from 2 species of frog, the American bullfrog (Rana [Aquarana] catesbeiana) and the African clawed frog (Xenopus laevis). Blood smears were evaluated after Wright staining and cytochemical staining for α-naphthyl butyrate esterase (NBE), chloroacetate esterase (CAE), myeloperoxidase (PER), Sudan black B (SBB), and leukocyte alkaline phosphatase (LAP) reactions and for immunoreactivity for antibodies against CD3ε, CD79a, and BLA.36 antigens. Histologic sections of spleen were evaluated after staining with H&E and for immunoreactivity for CD3ε, CD79a, and BLA.36 antigens. In bullfrogs, neutrophils, eosinophils, and monocytes were positive for some or all of the following: NBE, CAE, PER, and SBB; lymphocytes occasionally were positive for CAE. In clawed frogs, neutrophils, basophils, and monocytes were positive for some or all of the following: NBE, CAE, PER, and SBB; eosinophils occasionally were positive for CAE and PER, and lymphocytes were negative for all cytochemical stains. LAP was not a useful marker for any leukocyte type. In both species, peripheral blood lymphocytes were strongly immunoreactive for CD3ε, CD79a, and BLA.36. In splenic tissue, histologic patterns varied and there was diffuse immunoreactivity for CD79a and BLA.36 with focal reactivity for CD3ε, but with different distribution patterns in each species. Cytochemical and immunochemical analysis of cells may be helpful in identification and characterization of amphibian blood cells and splenic cells for evaluation of the health of these animals.

Germ Tube Mediated Invasion of Batrachochytrium dendrobatidis in Amphibian Skin Is Host Dependent

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines.We focussed on the early stages of infection by Bd in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of Alytes muletensis, Litoria caerulea and Xenopus leavis were exposed to Bd in an Ussing chamber for 3 to 5 days. Early interactions of Bd with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations in vitro, comparison was made with skin from experimentally infected frogs. Additional in vitro experiments were performed to elucidate the process of intracellular colonization in L. caerulea.Early interactions of Bd with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of A. muletensis and L. caerulea skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by Bd proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus.In X. laevis, interaction of Bd with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The in vitro data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of B. dendrobatidis is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.

Widespread Co-occurrence of Virulent Pathogens Within California Amphibian Communities

The chytrid fungus Batrachochytrium dendrobatidis, ranaviruses, and trematodes (Ribeiroia ondatrae and echinostomes) are highly virulent pathogens known to infect amphibians, yet the extent to which they co-occur within amphibian communities remains poorly understood. Using field surveillance of 85 wetlands in the East Bay region of California, USA, we found that 68% of wetlands had ≥2 pathogens and 36% had ≥3 pathogens. Wetlands with high pathogen species richness also tended to cluster spatially. Our results underscore the need for greater integration of multiple pathogens and their interactions into amphibian disease research and conservation efforts.

The novel ‘CandidatusAmphibiichlamydia ranarum’ is highly prevalent in invasive exotic bullfrogs (Lithobates catesbeianus)

Knowledge concerning microbial infectious diseases in the current amphibian crisis is rudimentary and largely limited to ranavirosis and chytridiomycosis. The family Chlamydiaceae is gaining attention as a common cause of disease in amphibians and may harbour new and emerging amphibian pathogens. We identified a novel species of Chlamydiales (Candidatus Amphibiichlamydia ranarum) with a prevalence of 71% in exotic invasive bullfrog tadpoles (Lithobates catesbeianus) from an introduced population in the Netherlands. The sequence of a 1474 bp 16S rRNA gene fragment showed that the novel taxon forms a well-defined clade with 'Candidatus Amphibiichlamydia salamandrae' within the Chlamydiaceae family. Although none of the tadpoles examined showed signs of clinical disease, urgent evaluation of its pathogenic potential for native amphibian species is required.

NovelChlamydiaceaeDisease in Captive Salamanders

To the Editor: Although 2 major diseases of amphibians, chytridiomycosis and ranavirosis, have been relatively well studied, enigmatic amphibian disease and death not attributable to any of the known amphibian diseases frequently occur (1). We describe an apparently new disease in salamanders that is associated with a novel genus within the family Chlamydiaceae. The salamanders seen in our clinic belonged to 1 of the following species: Salamandra corsica, the Corsican fire salamander (5 animals from 1 collection); Neurergus crocatus, the yellow spotted newt (11 animals from 3 collections); or N. strauchii, Strauch’s spotted newt (6 animals from 2 collections). All salamanders were captive bred; housed in breeding colonies in private collections in Elsloo and Eindhoven, the Netherlands, Munich, Germany, and Brugge, Belgium; and 1–3 years of age. Disease was characterized by anorexia, lethargy, edema, and markedly abnormal gait. Mortality rate was 100%. Animals in these collections had no histories of disease. All animals were in good nutritional condition. Necropsy did not yield any macroscopic lesions. All animals had mild intestinal nematode or protozoan infections. Results of real-time PCRs for iridoviruses in liver and skin (2) or Batrachochytrium dendrobatidis fungus of skin (3) were negative for all animals. We placed liver suspensions from the dead salamanders on Columbia agar with 5% sheep blood and tryptic soy agar and then incubated the samples up to 14 days at 20°C. No consistent bacterial growth was observed. Histologic examination of 2 Corsican fire salamanders and 1 yellow spotted newt revealed hepatitis in 1 of the Corsican fire salamanders and the yellow spotted newt. Hepatitis was characterized by high numbers of melanomacrophages and a marked infiltration of granulocytic leukocytes. Immunohistochemical staining for chlamydia (IMAGEN Chlamydia; Oxoid, Basingstone, UK) showed cell-associated fluorescently stained aggregates in liver tissue, suggestive of Chlamydiales bacteria. Transmission electron microscopic examination of the liver of a yellow spotted newt revealed intracellular inclusions containing particles matching the morphology of reticulate or elementary bodies of Chlamydiaceae (Technical Appendix). A PCR (4) to detect the 16S rRNA of all Chlamydiales bacteria, performed on liver tissue samples from all animals, yielded positive results in all 5 Corsican fire salamanders; in 4/7, 1/3, and 1/1 yellow spotted newts; and in 4/5 and 1/1 Strauch’s spotted newts. For taxon identification, the 16S rRNA gene of the Chlamydiales bacteria was amplified and sequenced from the livers from 2 yellow spotted newts (1 from the collection in Elsloo, the Netherlands and 1 from the collection in Munich, Germany), 1 Strauch’s spotted newt, and 5 Corsican fire salamanders. The sequences shared >90% nt identity with the 16S rRNA gene of C. abortus B577 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:D85709,term_id:2114161,term_text:D85709}}D85709) and therefore can be identified as a member of the family Chlamydiaceae (5). The closest 16S rRNA similarity (92%) was observed with C. psittaci strain CPX0308 ({type:entrez-nucleotide,attrs:{text:AB285329,term_id:148537245,term_text:AB285329}}AB285329). The sequence obtained from all spotted newt species specimens was identical (GenBank accession no. {type:entrez-nucleotide,attrs:{text:JN392920,term_id:358009359,term_text:JN392920}}JN392920) but differed slightly (1%) from that obtained from the fire salamander species specimens (GenBank accession no. {type:entrez-nucleotide,attrs:{text:JN392919,term_id:358009358,term_text:JN392919}}JN392919). These sequence differences point to the existence of multiple strains with possible host adaptation. We determined the phylogenetic position of the novel taxon, named Candidatus Amphibiichlamydia salamandrae (Technical Appendix), identified by using neighbor-joining analysis with Kodon software (Applied Maths, Sint-Martens-Latem, Belgium). The novel Chlamydiales forms a distinct branch in the well-supported monophyletic clade with the genera Chlamydia and Candidatus Clavochlamydia salmonicola (family Chlamydiaceae) (Figure). Maximum parsimony and unweighted pair group with arithmetic mean analyses yielded cladograms with the same topology (results not shown). Previous reports of members of the family Chlamydiaceae in amphibians concerned species occurring in other vertebrate taxa as well: C. psittaci, C. pneumoniae, C. abortus, and C. suis (6–10). To our knowledge, this member of the family Chlamydiaceae has been seen in amphibians, but not in other vertebrate hosts. The 16S rRNA analysis showed this taxon to belong to a clade with Candidatus Clavochlamydia salmonicola, a taxon found in fish. The phylogenetic position of the novel taxon in the family Chlamydiaceae thus roughly reflects the phylogenetic relation between the host species, providing evidence for host–bacterium co-evolution in the family Chlamydiaceae. Figure Topology of the novel amphibian Chlamydiaceae (Candidatus Amphibiichlamydia salamandrae) within the phylogenetic tree obtained by neighbor-joining and based on 16S rRNA gene data from representative species. Numbers show the percentage of times each branch ... Although the results obtained are not conclusive with regard to the pathogenic potential of this novel genus and species of Chlamydiales, we were not able to attribute the clinical signs to any known disease. We therefore suggest that we discovered a novel bacterial taxon with possible considerable impact on amphibian health.

Qualitative risk analysis of introducing Batrachochytrium dendrobatidis to the UK through the importation of live amphibians

The international amphibian trade is implicated in the emergence and spread of the amphibian fungal disease chytridiomycosis, which has resulted in amphibian declines and extinctions globally. The establishment of the causal pathogen, Batrachochytrium dendrobatidis (Bd), in the UK could negatively affect the survival of native amphibian populations. In recognition of the ongoing threat that it poses to amphibians, Bd was recently included in the World Organisation for Animal Health Aquatic Animal Health Code, and therefore is in the list of international notifiable diseases. Using standardised risk analysis guidelines, we investigated the likelihood that Bd would be introduced to and become established in wild amphibians in the UK through the importation of live amphibians. We obtained data on the volume and origin of the amphibian trade entering the UK and detected Bd infection in amphibians being imported for the pet and private collection trade and also in amphibians already held in captive pet, laboratory and zoological collections. We found that current systems for recording amphibian trade into the UK underestimate the volume of non-European Union trade by almost 10-fold. We identified high likelihoods of entry, establishment and spread of Bd in the UK and the resulting major overall impact. Despite uncertainties, we determined that the overall risk estimation for the introduction of Bd to the UK through the importation of live amphibians is high and that risk management measures are required, whilst ensuring that negative effects on legal trade are minimised.