Estimating Herd Immunity to Amphibian Chytridiomycosis in Madagascar Based on the Defensive Function of Amphibian Skin Bacteria

Abstract

For decades, Amphibians have been globally threatened by the still expanding infectious disease, chytridiomycosis. Madagascar is an amphibian biodiversity hotspot where Batrachochytrium dendrobatidis ( Bd ) has only recently been detected. While no Bd -associated population declines have been reported, the risk of declines is high when invasive virulent lineages become involved. Cutaneous bacteria contribute to host innate immunity by providing defense against pathogens for numerous animals, including amphibians. Little is known, however, about the cutaneous bacterial residents of Malagasy amphibians and the functional capacity they have against Bd . We cultured 3179 skin bacterial isolates from over 90 frog species across Madagascar, identified them via Sanger sequencing of approximately 700 bp of the 16S rRNA gene, and characterized their functional capacity against Bd . A subset of isolates was also tested against multiple Bd genotypes. In addition, we applied the concept of herd immunity to estimate Bd -associated risk for amphibian communities across Madagascar based on bacterial antifungal activity. We found that multiple bacterial isolates (39% of all isolates) cultured from the skin of Malagasy frogs were able to inhibit Bd . Mean inhibition was weakly correlated with bacterial phylogeny, and certain taxonomic groups appear to have a high proportion of inhibitory isolates, such as the Enterobacteriaceae, Pseudomonadaceae, and Xanthamonadaceae (84, 80, and 75% respectively). Functional capacity of bacteria against Bd varied among Bd genotypes; however, there were some bacteria that showed broad spectrum inhibition against all tested Bd genotypes, suggesting that these bacteria would be good candidates for probiotic therapies. We estimated Bd -associated risk for sampled amphibian communities based on the concept of herd immunity. Multiple amphibian communities, including those in the amphibian diversity hotspots, Andasibe and Ranomafana, were estimated to be below the 80% herd immunity threshold, suggesting they may be at higher risk to chytridiomycosis if a lethal Bd genotype emerges in Madagascar. While this predictive approach rests on multiple assumptions, and incorporates only one component of hosts' defense against Bd , their culturable cutaneous bacterial defense, it can serve as a foundation for continued research on Bd -associated risk for the endemic frogs of Madagascar.

Probiotic consortia are not uniformly effective against different amphibian chytrid pathogen isolates

“…In our data, the principal source of variance in inhibition was among bacterial strains, with the number of strains demonstrating broad‐spectrum facilitation of Bd GPL being approximately equal to the number exhibiting broad‐scale inhibition of the pathogen. These data support previous work suggesting B. dendrobatidis inhibition capability is distributed widely across bacterial genera (Antwis et al., ; Becker et al., ; Bletz, Myers, et al, ); several strains demonstrated at least weak inhibition for all 10 Bd GPLs but were spread across multiple genera with no clear pattern. That there is clear functional redundancy among genera in this host‐protective trait suggests it is not prudent to focus on any one genus in the search for beneficial probiotics (Becker et al., ), as highly divergent microbial communities can still possess similar functional traits (e.g., Bletz et al., ; Bletz, Perl, ).…”

“…Laboratory and field studies have shown host‐associated bacterial communities protect amphibians from B. dendrobatidis infection and that it is possible to artificially augment the microbiota with probiotic bacteria to improve survivorship in response to the pathogen (Becker et al., ; Bletz et al., ; Harris, Lauer, Simon, Banning, & Alford, ; Jani & Briggs, ; Kueneman et al., ; Muletz, Myers, Domangue, Herrick, & Harris, ; Walke et al., ). However, inhibitory capabilities of individual bacteria are not uniform across the variation presented by B. dendrobatidis (Antwis, Preziosi, Harrison, & Garner, ; Bletz, Myers, et al, ; Muletz‐Wolz et al., ). In addition, previous work has found either no (Becker et al., ) or weak evidence (Bletz, Myers, et al, ) of a phylogenetic signal in the ability of bacterial genera to inhibit a singular B. dendrobatidis isolate.…”

“…However, inhibitory capabilities of individual bacteria are not uniform across the variation presented by B. dendrobatidis (Antwis, Preziosi, Harrison, & Garner, ; Bletz, Myers, et al, ; Muletz‐Wolz et al., ). In addition, previous work has found either no (Becker et al., ) or weak evidence (Bletz, Myers, et al, ) of a phylogenetic signal in the ability of bacterial genera to inhibit a singular B. dendrobatidis isolate. However, a major gap in our understanding concerns whether some bacterial genera are more likely to show broad‐spectrum inhibition across a range of B. dendrobatidis isolates, allowing a more focused search for effective amphibian probiotics.…”

Probiotics as a tool for disease mitigation in wildlife: insights from food production and medicine

“…Importantly, culturable species can account for a large fraction of the total relative abundance of a microbial community . Assays to determine the ability of a bacterial species to inhibit the pathogen have been conducted on agar plates and in 96‐well microtiter plates . Strains have ranged in effectiveness from highly inhibitory to facilitating pathogen growth in vitro .…”

“…75 Assays to determine the ability of a bacterial species to inhibit the pathogen have been conducted on agar plates and in 96-well microtiter plates. 76 Strains have ranged in effectiveness from highly inhibitory to facilitating pathogen growth in vitro. Clearly, it is necessary to select an inhibitory strain, but it is less clear how inhibitory the strain must be in vitro in order to be effective (e.g., is 90% inhibition in vitro better than 80% inhibition once the strain is inoculated onto the host?).…”