The range of interactions among Bd, skin bacteria, host and environment leads us to propose sampling strategies and filtering protocols that are designed to guide selection of effective probiotics for protecting individual species and amphibian communities. The filtering protocol differs from listing effective probiotic characteristics as presented in other studies (Fuller 1989; Kesarcodi-Watson et al. 2008). Isolates are placed through a series of tests that progressively filter out ineffective ones, leaving the most promising candidates. A species-specific approach focuses on treating at-risk individuals with probiotic baths while a community-based approach targets amphibian assemblages by treating ponds or local areas with a broad-spectrum probiotic. We stress that bioaugmentation approaches must use microbes found in the local environment to improve success and minimise biosafety concerns.
Species-specific probiotics should target individuals being repatriated from survival assurance colonies (Becker et al. 2011) and individuals of critically endangered species in front of an advancing Bd wave (Woodhams et al. 2007b; Vredenburg et al. 2010). Assurance colonies have been implemented to rescue species when they are experiencing rapid declines across their range or when there is an imminent threat to amphibian populations due to the anticipated arrival of Bd. The goal of assurance colonies is to reintroduce threatened species to their native habitats and establish persisting populations; however, releasing susceptible individuals will be unsuccessful because Bd persists in the natural environment on reservoir species (Reeder et al. 2012). In addition, there are situations where susceptible species in front of an advancing Bd wave in the wild are not in assurance colonies (Vredenburg et al. 2010). In both cases, individuals can be treated with a probiotic derived from the appropriate sampling strategy and that successfully passes through the filtering protocol outlined below.
The sampling strategy for obtaining a species-specific probiotic for assurance colony species and endangered wild species will differ for species that have some populations in the wild coexisting with Bd (e.g. Anaxyrus boreas and R. muscosa), and species that are extirpated from the wild (e.g. Atelopus zeteki). If there are populations coexisting with Bd, it is essential to sample and culture microbes from members of these populations (Fig. 3a). For species that have been extirpated from the wild, it will be necessary to focus microbe sampling on related species that have a similar life-history, are found in similar habitats and locations, and are coexisting with Bd (Fig. 3b). Individuals in populations coexisting with Bd are surviving with Bd infection and are more likely to have anti-Bd bacteria.
Figure 3. Sampling strategies and filtering processes for the selection of species-specific and community-based probiotics. Notations in parentheses link the elements of the figure to expanded discussion in the text. SS = species-specific; CB = community based.
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Once microbes are collected from amphibians using standard methods (Harris et al. 2006), they must pass through the filtering criteria, which leaves a progressively smaller number of probiotic candidates. The candidate probiotics must inhibit Bd under ecologically relevant conditions of the intended host (Fig. 3 (SS1), Box 2). For example, it is essential that probiotics inhibit at temperatures at which the amphibian is most vulnerable to Bd infection (Daskin & Alford 2012). Preference should be given to inhibitory isolates that are present on a large proportion of sampled individuals since ubiquity suggests the isolate will persist on the target amphibians. The remaining candidate probiotics must colonise and persist on target amphibians at all life-history stages while not harming the host (Fig. 3 (SS2), Box 2). If bacteria are collected from surviving individuals of the intended host, the likelihood of bacterial persistence is high. If persistence is observed, it indicates that the host's immune system or resident microbes do not inhibit the isolate. It will be important to eliminate isolates that inhibit Bd in vitro, but do not persist or provide continual inhibition of Bd on amphibians (Box 2). The remaining candidates must inhibit Bd in clinical trials with all life-history stages to confirm in vivo effectiveness of the candidate probiotic to prevent disease (Fig. 3 (SS3), Box 2). Successful probiotics will decrease mortality and sub-lethal effects for all stages. Lastly, selected isolates must inhibit Bd in a small-scale field trial to assess effectiveness in the natural environment (Fig. 3(SS4), Box 2). At this point, remaining candidates have a high likelihood of being effective probiotics for the target amphibians.
Two amphibian species currently established in assurance colonies, the boreal toad, An. boreas, and the Panamanian golden frog (At. zeteki), are targets for pre-release probiotic treatment. The toad An. boreas is a species that has experienced population declines (Muths et al. 2003); however, there are wild populations persisting with Bd infection that should be sampled to obtain probiotic candidates (Fig. 3a). The collected microbes should be screened through the four-step filter discussed above. At. zeteki is a species that is likely extirpated from the wild. Becker et al. (2011) tested a probiotic candidate, J. lividum, on At. zeteki, which was isolated from North American salamanders. The probiotic treatment kept infection loads low initially, but the probiotic abundance declined and mortality occurred. Subsequently, 600 isolates were collected from related species coexisting with Bd in the same locations and habitats where At. zeteki was found and are currently being screened using the criteria listed above. Importantly, inhibition trials (step 1) removed 85% of isolates from consideration as a probiotic (Fig. 3b).
The susceptible frog in the Sierra Nevadas, R. muscosa, is an example of a species where populations in front of an advancing Bd wave are in need of protection. Probiotic candidates have been collected from populations that have persisted through the arrival of Bd and are therefore more likely to possess Bd-inhibitory bacteria (Fig. 3a) (Woodhams et al. 2007b; Lam et al. 2010). One R. muscosa population under threat from imminent Bd arrival, and predicted to be decimated, provided an opportunity for probiotic application. Due to the short lead-in time available, it was not possible to apply all elements of the filtering process. However, the probiotic J. lividum was chosen due to its success in previous experiments (Harris et al. 2009a) and its presence on a number of amphibian species across many locations, including the Sierra Nevadas. This trial was successful: greater survival was seen for treated individuals, and Bd loads remained low compared to untreated controls (Vredenburg et al. 2011). Therefore, when immediate treatment is necessary, and little time exists for a full filtering process, priority can be given to probiotics that have been successful in other studies, assuming that a strain of the probiotic can be found on amphibians in the intended application area.
Box 2. Methodologies of filtering protocol
To determine the inhibitory nature of the candidate probiotics, we advocate the following protocols. The bacterial isolate should be co-cultured with Bd, because it will induce the bacteria to produce anti-Bd metabolites. In addition, isolates that are inhibited by Bd will be excluded. The culture filtrate (cell-free supernatant) that includes bacterial metabolites from the co-culture is assayed for Bd inhibition in 96-well microtiter plates (Bell et al. 2013). A negative control (heat-killed Bd), positive control (Bd without culture filtrate but with the equivalent volume of medium) and a control for Bd-produced metabolites (culture filtrate from a Bd culture) should be included. Inhibition assays can also be carried out on agar plates (Harris et al. 2006). In this protocol, Bd is spread evenly across the tryptone-agar plate and bacteria are streaked across the Bd-covered plate. After 72–96 h of incubation, the inhibition zone is measured. Trials should be replicated to accurately estimate inhibition and allow for statistical tests.
Colonisation & persistence trials
To assess colonisation and persistence, candidate probiotics are inoculated onto amphibians of all life-history stages in laboratory trials. For species-specific treatment strategies, amphibians are bathed in probiotic baths; for community treatment strategies, the housing substrate is inoculated with the probiotic. Colonisation and persistence can be assessed using culture-based or molecular methods (Becker et al. 2011). If culture-based techniques are used, artificial selection of bacterial isolates for rifampicin resistance can facilitate tracking during experiments (Muletz et al. 2012). For molecular detection, polymerase chain reaction (PCR) can be used to confirm colonisation and persistence of the probiotic. This technique requires the use of species-specific primers, which have been developed for some species such as J. lividum (Harris et al. 2009a). In all experiments, control groups of untreated amphibians are required. Ideally, during these trials swabbing or bathing should be used to periodically collect amphibian skin secretions, which are a mixture of defensive products of amphibians and their microbial symbionts. This protocol is currently being optimised. These secretions are used in Bd inhibition assays to compare control treatments (no probiotic) to probiotic treatments as a measure of the probiotic's in vivo effectiveness against Bd. Because these bioassays assess in vivo effectiveness of potential probiotics, they reduce the possibility of unsuccessful clinical trials.
Environmental persistence trials
Probiotic persistence in the environment is determined through laboratory trials where an environmental substrate is inoculated with the probiotic candidate (Muletz et al. 2012) and monitored over time. Depending on the habitat of the intended hosts, trials are conducted with water or soil as the substrate. Probiotic transmission can also be assessed if amphibians are housed in the inoculated substrate. Transfer of the probiotic to the host and persistence in the environment can be measured using culture-based or molecular methods (Becker et al. 2009; Muletz et al. 2012). A similar protocol can be used for trials conducted in nature.
Laboratory-based clinical trials for species-specific probiotic treatment involve bathing amphibians in the probiotic and exposing both treated individuals and untreated controls to Bd in randomised, replicated trials (Harris et al. 2009a). Clinical trials for community-based probiotics involve inoculating the laboratory environment (water or soil) with the candidate probiotic and housing the selected host amphibians in these treated environments as well as housing a set of individuals in untreated control environments. Amphibians in both treatments should be exposed to Bd and monitored for survival and sublethal effects (i.e. growth rate, behaviours) (Harris et al. 2009a,b). Estimating Bd loads via qPCR (Hyatt et al. 2007) can be helpful in determining whether the probiotic kept Bd loads below a lethal threshold. These trials need to be replicated and conducted under ecologically relevant conditions. In addition, they should be conducted on all life-history stages, (i.e. larvae, juvenile, adult) to ensure the probiotic is effective across all stages.
Small-scale probiotic field trials should be completed at locations where appropriate regulatory approval has been obtained. For species-specific strategies, field trials involve treatment of individuals with and without a probiotic bath and release at the field location (Vredenburg et al. 2011). Monitoring of Bd infection, the establishment of the probiotic on amphibians and ultimately the survival of released individuals will determine the outcome of the experiment. Field trials for community-based environmental treatment involve inoculation of soil or water with a probiotic and release of amphibians to treated areas. Survival of amphibians at the treated sites, Bd loads and probiotic abundance on the hosts and in the environment should be monitored and compared to control sites to evaluate success.