Quantifying the disease transmission function: effects of density on Batrachochytrium dendrobatidis transmission in the mountain yellow-legged frog Rana muscosa

1. Chytridiomycosis is an emerging infectious disease of amphibians, caused by the fungal pathogen Batrachochytrium dendrobatidis, which has been implicated recently in population declines and possible extinctions throughout the world. 2. The transmission rate of this pathogen was quantified in the mountain yellow-legged frog Rana muscosa through laboratory and field experiments, and a maximum likelihood approach was used to determine the form of the transmission function that was best supported by the experimental data. 3. The proportion of R. muscosa tadpole hosts that became infected increased with the number of previously infected R. muscosa tadpoles to which they were exposed, as would be expected in an infectious disease. 4. The laboratory experiment revealed some support for a transmission function in which the transmission rate levels off as the density of infected individuals increases. However, there was not enough power to distinguish between a frequency-dependent form and several other asymptotic forms of the transmission function. 5. The impacts of crowding and temperature on transmission were also investigated; however, neither of these factors significantly affected the transmission rate.     

Pathophysiology in mountain yellow-legged frogs (Rana muscosa) during a chytridiomycosis outbreak

The disease chytridiomycosis is responsible for declines and extirpations of amphibians worldwide. Chytridiomycosis is caused by a fungal pathogen (Batrachochytrium dendrobatidis) that infects amphibian skin. Although we have a basic understanding of the pathophysiology from laboratory experiments, many mechanistic details remain unresolved and it is unknown if disease development is similar in wild amphibian populations. To gain a better understanding of chytridiomycosis pathophysiology in wild amphibian populations, we collected blood biochemistry measurements during an outbreak in mountain yellow-legged frogs (Rana muscosa) in the Sierra Nevada Mountains of California. We found that pathogen load is associated with disruptions in fluid and electrolyte balance, yet is not associated with fluctuations acid-base balance. These findings enhance our knowledge of the pathophysiology of this disease and indicate that disease development is consistent across multiple species and in both laboratory and natural conditions. We recommend integrating an understanding of chytridiomycosis pathophysiology with mitigation practices to improve amphibian conservation.     

Batrachochytrium dendrobatidis infection dynamics in the Columbia spotted frog Rana luteiventris in north Idaho, USA

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) is contributing to amphibian declines worldwide. Temperature plays an important role in both pathogen growth and host immune function, but little is known about seasonal dynamics of Bd infection in north temperate regions. Our objective was to increase understanding of Bd disease ecology by investigating patterns of Bd infection of Columbia spotted frogs Rana luteiventris across seasons, age classes, and sexes in north Idaho, USA. We collected skin swabs from 223 R. luteiventris in spring, summer, and fall 2009 at 7 ponds in the Palouse region and quantified Bd zoospores for each sample using quantitative PCR. Across seasons, Bd prevalence of adults was higher in summer than in spring or fall, suggesting that individuals may be clearing low-level infections over the summer. Among age classes, all but one late stage tadpole (Gosner stage 43-45) tested negative for Bd. Conversely, 100% of metamorphs tested positive for Bd and had the highest Bd loads of all age classes, suggesting they may be the most vulnerable age class. Adult R. luteiventris had high infection prevalence (> 60%) in all seasons, indicating that Bd infection is maintained within populations and that adults likely serve as disease reservoirs across seasons. Among adults, we also found weak evidence for females having higher infection prevalence than males. Further laboratory and field studies are needed to determine whether there are individual and population impacts from Bd on R. luteiventris and other amphibians in north Idaho.     

Widespread Occurrence of the Chytrid Fungus Batrachochytrium dendrobatidis on Oregon Spotted Frogs (Rana pretiosa)

The pathogen Batrachochytrium dendrobatidis (Bd) has been associated with amphibian declines in multiple continents, including western North America. We investigated Bd prevalence in Oregon spotted frog (Rana pretiosa), a species that has declined across its range in the Pacific Northwest. Polymerase chain reaction analysis of skin swabs indicated that Bd was prevalent within populations (420 of 617 juvenile and adults) and widespread among populations (36 of 36 sites) where we sampled R. pretiosa in Oregon and Washington. We rarely detected Bd in R. pretiosa larvae (2 of 72). Prevalence of Bd in postmetamorphic R. pretiosa was inversely related to frog size. We found support for an interactive effect of elevation and sampling date on Bd: prevalence of Bd generally increased with date, but this effect was more pronounced at lower elevations. We also found evidence that the body condition of juvenile R. pretiosa with Bd decreased after their first winter. Our data indicate that some Oregon spotted frog populations are currently persisting with relatively high Bd prevalence, but the risk posed by Bd is unknown.     

Elevated temperature as a treatment for Batrachochytrium dendrobatidis infection in captive frogs

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines worldwide. In vitro laboratory studies and those done on wild populations indicate that Bd grows best at cool temperatures between 17 and 25 degrees C. In the present study, we tested whether moderately elevating the ambient temperature to 30 degrees C could be an effective treatment for frogs infected with Bd. We acquired 35 bullfrogs Rana catesbeiana from breeding facilities and 36 northern cricket frogs Acris crepitans from the wild and acclimated them to either 23 or 26 degrees C for 1 mo. Following the acclimation period, frogs were tested for the presence of Bd using qPCR TaqMan assays. The 12 R. catesbeiana and 16 A. crepitans that tested positive for Bd were subjected to 30 degrees C for 10 consecutive days before returning frogs to their starting temperatures. Post-treatment testing revealed that 27 of the 28 frogs that had tested positive were no longer infected with Bd; only a single A. crepitans remained infected following treatment. This result indicates that elevating ambient temperature to a moderate 30 degrees C can be effective as a treatment for Bd infection in captive amphibians, and suggests that heat may be a superior alternative to antifungal drugs.     

Altitudinal distribution of chytrid (Batrachochytrium dendrobatidis) infection in subtropical Australian frogs

The disappearance of amphibian populations from seemingly pristine upland areas worldwide has become a major focus of conservation efforts in the last two decades, and a parasitic chytrid fungus, Batrachochytrium dendrobatidis, is thought to be the causative agent of the population declines. We examined the altitudinal distribution of chytrid infections in three stream‐dwelling frog species (Litoria wilcoxii, L. pearsoniana and L. chloris) in southeast Queensland, Australia, and hypothesized that if B. dendrobatidis were responsible for the disappearance of high‐altitude frog populations, infection prevalence and intensity would be greatest at higher altitudes. Overall, 37.7% of the 798 adult frogs we sampled were infected with B. dendrobatidis, and infections were found in all the populations we examined. Contrary to our initial hypothesis, we found no consistent evidence that high‐altitude frogs were more likely to be infected than were lowland frogs. Further, the intensity of fungal infections (number of zoospores) on high‐altitude frogs did not differ significantly from that of lowland frogs. Batrachochytrium dendrobatidis appears to be capable of infecting frogs at all altitudes in the subtropics, suggesting that all populations are at risk of decline when conditions favour disease outbreaks. We did find evidence, however, that chytrid infections persist longer into summer in upland as compared with lowland areas, suggesting that montane amphibian populations remain susceptible to disease outbreaks for longer periods than do lowland populations. Further, we found that at high altitudes, temperatures optimal for chytrid growth and reproduction coincide with frog metamorphosis, the life‐stage at which frogs are most susceptible to chytrid infections. While these altitudinal differences may account for the differential population‐level responses to the presence of B. dendrobatidis, the reason why many of southeast Queensland's montane frog populations declined precipitously while lowland populations remained stable has yet to be resolved.     

Batrachochytrium dendrobatidis in the live frog trade of Telmatobius (Anura: Ceratophryidae) in the tropical Andes

Species of frogs in the genus Telmatobius are traded and sold for human consumption in the Andes and in coastal cities of Peru and Bolivia. These frogs are harvested from wild populations. We report high prevalence of infection by the pathogenic fungus Batrachochytrium dendrobatidis in live frogs purchased at the main market in Cusco, Peru, from January 2008 to January 2010. We suggest that the transport of native anurans through the live frog trade could facilitate the spread of this fungus among Andean frogs. The tropical Andes are the most important biodiversity hotspot for amphibians. Because many neotropical taxa are known to be susceptible to chytridiomycosis, the presence of a large reservoir of infection in the frog trade poses a significant threat to amphibian conservation.     

Field and laboratory studies of the susceptibility of the green treefrog (Hyla cinerea) to Batrachochytrium dendrobatidis infection

Amphibians worldwide are experiencing devastating declines, some of which are due to the amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd). Populations in the southeastern United States, however, have not been noticeably affected by the pathogen. The green treefrog (Hyla cinerea) is abundant and widespread in the southeastern United States, but has not been documented to harbor Bd infection. This study examined the susceptibility of H. cinerea to two strains of Bd in the lab and the prevalence of infection in wild populations of this species in southeastern Louisiana. Although we were able to infect H. cinerea with Bd in the lab, we did not observe any clinical signs of chytridiomycosis. Furthermore, infection by Bd does not appear to negatively affect body condition or growth rate of post-metamorphic individuals. We found no evidence of infection in surveys of wild H. cinerea. Our results suggest that H. cinerea is not susceptible to chytridiomycosis post-metamorphosis and probably is not an important carrier of the fungal pathogen Bd in the southeastern United States, although susceptibility at the larval stage remains unknown.     

Experimental infection of self-cured Leiopelma archeyi with the amphibian chytrid Batrachochytrium dendrobatidis

The susceptibility of Archey's frog Leiopelma archeyi to Batrachochytrium dendrobatidis (Bd) is unknown, although one large population is thought to have declined sharply due to chytridiomycosis. As primary infection experiments were not permitted in this endangered New Zealand species, 6 wild-caught L. archeyi that naturally cleared infections with Bd while in captivity were exposed again to Bd to assess their immunity. These frogs were from an infected population at Whareorino, which has no known declines. All 6 L. archeyi became reinfected at low intensities, but rapidly self cured, most by 2 wk. Six Litoria ewingii were used as positive controls and developed heavier infections and clinical signs by 3 wk, demonstrating that the zoospore inoculum was virulent. Six negative controls of each species remained uninfected and healthy. Our results show that L. archeyi that have self cured have resistance to chytridiomycosis when exposed. The pattern is consistent with innate or acquired immunity to Bd, and immunological studies are needed to confirm this.     

Persistence of the emerging pathogen Batrachochytrium dendrobatidis outside the amphibian host greatly increases the probability of host extinction

Pathogens do not normally drive their hosts to extinction; however, Batrachochytrium dendrobatidis, which causes amphibian chytridiomycosis, has been able to do so. Theory predicts that extinction can be caused by long-lived or saprobic free-living stages. The hypothesis that such a stage occurs in B. dendrobatidis is supported by the recent discovery of an apparently encysted form of the pathogen. To investigate the effect of a free-living stage of B. dendrobatidis on host population dynamics, a mathematical model was developed to describe the introduction of chytridiomycosis into a breeding population of Bufo bufo, parametrized from laboratory infection and transmission experiments. The model predicted that the longer that B. dendrobatidis was able to persist in water, either due to an increased zoospore lifespan or saprobic reproduction, the more likely it was that it could cause local B. bufo extinction (defined as decrease below a threshold level). Establishment of endemic B. dendrobatidis infection in B. bufo, with severe host population depression, was also possible, in agreement with field observations. Although this model is able to predict clear trends, more precise predictions will only be possible when the life history of B. dendrobatidis, including free-living stages of the life cycle, is better understood.     

Batrachochytrium dendrobatidis Infection and Lethal Chytridiomycosis in Caecilian Amphibians (Gymnophiona)

Batrachochytrium dendrobatidis (Bd) is commonly termed the ‘amphibian chytrid fungus’ but thus far has been documented to be a pathogen of only batrachian amphibians (anurans and caudatans). It is not proven to infect the limbless, generally poorly known, and mostly soil-dwelling caecilians (Gymnophiona). We conducted the largest qPCR survey of Bd in caecilians to date, for more than 200 field-swabbed specimens from five countries in Africa and South America, representing nearly 20 species, 12 genera, and 8 families. Positive results were recovered for 58 specimens from Tanzania and Cameroon (4 families, 6 genera, 6+ species). Quantities of Bd were not exceptionally high, with genomic equivalent (GE) values of 0.052–17.339. In addition, we report the first evidence of lethal chytridiomycosis in caecilians. Mortality in captive (wild-caught, commercial pet trade) Geotrypetes seraphini was associated with GE scores similar to those we detected for field-swabbed, wild animals.     

High Prevalence of Batrachochytrium dendrobatidis in Wild Populations of Lowland Leopard Frogs Rana yavapaiensis in Arizona

Batrachochytrium dendrobatidis (Bd) is a fungus that can potentially lead to chytridiomycosis, an amphibian disease implicated in die-offs and population declines in many regions of the world. Winter field surveys in the last decade have documented die-offs in populations of the lowland leopard frog Rana yavapaiensis with chytridiomycosis. To test whether the fungus persists in host populations between episodes of observed host mortality, we quantified field-based Bd infection rates during nonwinter months. We used PCR to sample for the presence of Bd in live individuals from nine seemingly healthy populations of the lowland leopard frog as well as four of the American bullfrog R. catesbeiana (a putative vector for Bd) from Arizona. We found Bd in 10 of 13 sampled populations. The overall prevalence of Bd was 43% in lowland leopard frogs and 18% in American bullfrogs. Our results suggest that Bd is widespread in Arizona during nonwinter months and may become virulent only in winter in conjunction with other cofactors, or is now benign in these species. The absence of Bd from two populations associated with thermal springs (water >30°C), despite its presence in nearby ambient waters, suggests that these microhabitats represent refugia from Bd and chytridiomycosis.     

Prevalence of Batrachochytrium dendrobatidis infection is extremely low in direct-developing Australian microhylids

The emerging infectious disease chytridiomycosis has been implicated in declines and disappearances of amphibian populations around the world. However, susceptibility to infection and the extent of pathological effects of infection vary among hosts, and species with life histories that include parental care of direct-developing terrestrial eggs may tend to be less susceptible. We examined samples from a total of 595 individuals of 9 species of direct-developing Australian frogs in the family Microhylidae for the presence of infection by Batrachochytrium dendrobatidis (Bd). Between 1995 and 2004, 336 samples were collected; 102 of these were analysed histologically and 234 were tissues stored in alcohol, which were examined using diagnostic quantitative PCR (qPCR). Swab samples were collected from 259 frogs from 2005 to 2008 and were examined using qPCR. None of the 595 samples showed evidence of infection by Bd. If these data are regarded as a single sample representative of Australian microhylids, the upper 95% binomial confidence limit for the prevalence of infection in frogs of this family is 0.0062 (<1%). Even if only the data from the more powerful diagnostic qPCR tests are used, the upper 95% confidence limit for prevalence is 0.0075 (<1%). Our data suggest that Australian microhylids have a very low prevalence of infection by Bd in nature, and thus are either not susceptible, or are only slightly susceptible, to chytridiomycosis. This could be due solely to, or in combination with, low rates of transmission and to factors that promote resistance to infection, including ecological or behavioural characteristics, innate immune functions such as antimicrobial skin peptides, or antimicrobial symbionts in skin flora.     

Chemotaxis of the amphibian pathogen Batrachochytrium dendrobatidis and its response to a variety of attractants

Batrachochytrium dendrobatidis is a fungal pathogen of amphibians that is increasingly implicated as a major cause of large-scale mortalities of amphibian species worldwide. Previous studies indicate that motile zoospores of B. dendrobatidis colonize the keratinized tissues of susceptible amphibians. Infections spread to adults and cause destruction of epidermal tissue. In an effort to understand how the chytrid cues into its host we developed an assay to study chemotaxis in the fungus. Here we show that zoospores exhibit positive movement toward a variety of attractants including sugars, proteins and amino acids. These observations suggest that the chytrid can respond to nutritional cues, including those of host origin. Implications of these observations to amphibian susceptibility to infection and chytrid virulence are discussed.     

Ratio-dependent functional response of two common Cladocera present in farmland ponds to Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis is responsible for amphibian declines worldwide. Decreasing the aquatic density of this chytrid through consumption of its infectious zoospores by Cladocera (water fleas) may mitigate the impact of chytridiomycosis. Understanding this predator-prey relationship requires insights in the zoospore ingestion rate of an average water flea, but such data are almost non-existent. We investigated the functional response of Simocephalus vetulus and Chydorus sphaericus feeding on B. dendrobatidis zoospores. These Cladocera commonly occur in farmland ponds, which may represent a major habitat for disease control. Both water fleas actively ingested zoospores and their per capita ingestion rate was best modelled in function of zoospore-to-Cladocera ratio, implying mutual interference among water fleas during zoospore feeding. The larger S. vetulus substantially consumed more zoospores, characterised by a maximum ingestion rate of 2.5 × 10⁵ zoospores.Cladocera⁻¹.h⁻¹.mL⁻¹, which is about 12 times higher than for C. sphaericus. These findings are useful to support model-based management of chytridiomycosis.     

Non-detection of mycoviruses in amphibian chytrid fungus (Batrachochytrium dendrobatidis) from Australia

Mycoviruses may influence the pathogenicity of disease-causing fungi. Although mycoviruses have been found in some chytrid fungi, limited testing has not detected them in Batrachochytrium dendrobatidis (Bd), the cause of the devastating amphibian disease, chytridiomycosis. Here we conducted a survey for mycovirus presence in 38 Bd isolates from Australia (n = 31), Brazil (n = 5) and South Korea (n = 2) with a combination of modern high-throughput sequencing and conventional dsRNA cellulose chromatography. Mycoviruses were not detected in any isolates. This result was unexpected, given the long evolutionary history of Bd, as well as the high prevalence of mycoviruses in related fungal species. Given our widespread sampling in Australia and the limited number of Bd introductions, we suggest that mycoviruses are uncommon or absent from Australian Bd. Testing more isolates from regions where Bd originated, as well as regions with high diversity or low fungal virulence may identify mycoviruses that could aid in disease control.     

Season dependent response of the Marsh frog (Rana ridibunda) to cadmium exposure

Cadmium toxicity related to cysteine metabolism and glutathione levels in several tissues of the Marsh frog (Rana ridibunda) collected in late spring were investigated after exposure to 80 mg CdCl2 L(-1) for 168 h. The results were compared to those obtained in a previous experiment carried out in autumn. The most striking changes involved the brain which could not maintain a proper glutathione level and the testes in which neither GSH nor sulfane sulfur levels recovered. Substantial damage is expected in the presence of Cd which decreases the antioxidant status of these tissues. It seems that in spring, frogs had lesser tolerance for Cd in comparison with frogs in autumn. This may be caused by the transition to aerobic respiration after hibernation.     

Presence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in threatened corroboree frog populations in the Australian Alps

Since the early 1980s, the southern corroboree frog Pseudophryne corroboree and northern corroboree frog P. pengilleyi have been in a state of decline from their sub-alpine and high montane bog environments on the southern tablelands of New South Wales, Australia. To date, there has been no adequate explanation as to what is causing the decline of these species. We investigated the possibility that a pathogen associated with other recent frog declines in Australia, the amphibian chytrid fungus Batrachochytrium dendrobatidis, may have been implicated in the decline of the corroboree frogs. We used histology of toe material and real-time PCR of skin swabs to investigate the presence and infection rates with B. dendrobatidis in historic and extant populations of both corroboree frog species. Using histology, we did not detect any B. dendrobatidis infections in corroboree frog populations prior to their decline. However, using the same technique, high rates of infection were observed in populations of both species after the onset of substantial population declines. The real-time PCR screening of skin swabs identified high overall infection rates in extant populations of P. corroboree (between 44 and 59%), while significantly lower rates of infection were observed in low-altitude P. pengilleyi populations (14%). These results suggest that the initial and continued decline of the corroboree frogs may well be attributed to the emergence of B. dendrobatidis in populations of these species.     

Experimental Evidence for American Bullfrog (Lithobates catesbeianus) Susceptibility to Chytrid Fungus (Batrachochytrium dendrobatidis)

The emerging fungal pathogen, Batrachochytrium dendrobatidis (Bd), has been associated with global amphibian population declines and extinctions. American bullfrogs (Lithobates catesbeianus) are widely reported to be a tolerant host and a carrier of Bd that spreads the pathogen to less tolerant hosts. Here, we examined whether bullfrogs raised from eggs to metamorphosis in outdoor mesocosms were susceptible to Bd. We experimentally exposed metamorphic juveniles to Bd in the laboratory and compared mortality rates of pathogen-exposed animals to controls (non-exposed) in two separate experiments; one using a Bd strain isolated from a Western toad and another using a strain isolated from an American bullfrog. We wanted to examine whether metamorphic bullfrogs were susceptible to either of these strains. We show that bullfrogs were susceptible to one strain of Bd and not the other. In both experiments, infection load detected in the skin decreased over time, suggesting that metamorphic bullfrogs from some populations may be inefficient long-term carriers of Bd.     

Seasonality of Batrachochytrium dendrobatidis infection in direct-developing frogs suggests a mechanism for persistence

Batrachochytrium dendrobatidis (Bd), a disease-causing amphibian-specific fungus, is widely distributed in Puerto Rico, but is restricted to elevations above 600 m. The effect of this pathogen in the wild was studied by monitoring Eleutherodactylus coqui and E. portoricensis in 2 upland forests at El Yunque, a site characterized by historic population declines in the presence of chytridiomycosis. We tested a potential synergistic interaction between climate and Bd by measuring prevalence of infection and level of infection per individual sampled (number of zoospores), across the dry and wet seasons for 2 yr (between 2005 and 2007). Infection levels in adult frogs were significantly higher during the dry season in both species studied, suggesting a cyclic pattern of dry/ cool-wet/warm climate-driven synergistic interaction. These results are consistent with ex situ experiments in which E. coqui infected with Bd were more susceptible to chytridiomycosis when subjected to limited water treatments resembling drought. Long-term data on the prevalence of Bd in the populations studied versus intensity of infection in individual frogs provided contradictory information. However, the conflicting nature of these data was essential to understand the status of Bd in the species and geographical area studied. We conclude that in Puerto Rico, Bd is enzootic, and vulnerability of eleutherodactylid frogs to this pathogen is related to seasonal climatic variables. Our data suggest a mechanism by which this disease can persist in tropical frog communities without decimation of its hosts, but that complex interactions during severe droughts may lead to population crashes.