The gut microbiota of Testudines is fundamental to their digestion and overall health, yet remains a poorly investigated area in their biology, particularly in wild freshwater turtle (terrapins) and tortoise populations within South Africa. This study investigated the occurrence, diversity, virulence genes and antibiotic resistance of Escherichia coli isolated from Testudine gut microbiota and sediments at Timbavati Private Nature Reserve, South Africa. Cloacal swab samples were collected from 36 wild Testudines and 20 sediment samples from temporary and permanent water bodies. Presumed E. coli isolates were confirmed by polymerase chain reaction (PCR) targeting the β-D glucuronidase (uidA) gene and further validated through 16 S rRNA gene sequencing. Phenotypic antibiotic resistance was evaluated with the Kirby-Bauer method, whilst resistance and virulence genes were identified using PCR assays. E. coli was detected in 54 (62%) of 87 isolates (23 Testudines and 31 sediments), confirmed by uidA PCR assay. Detected virulence genes included eaeA (42%), virF (22%), stx1 (16%), and stx2 (3%), and isolates exhibited resistance to erythromycin (53%), cephalothin (48%), and spectinomycin (40%). Resistance genes such as mcr-4 (70%), blaSHV (46%), blaTEM (64%), mcr-1 (42%), qnrA (16%), mcr-2 (22%), qnrD (11%), and tetW (2%) were also detected. This study demonstrates that wild Testudines harbour E. coli in their gut and that it also occurs in their surrounding environment, with notable antibiotic resistance and virulence potential. The findings underscore the complexity of host-microbial interactions and the influence of environmental and host factors on microbial diversity, informing potential conservation and health management strategies for these reptilian species.

Invasive and highly virulent parasites are emerging worldwide, transported to new locations and into novel hosts by anthropogenic activities. Multiple introduction events lead to interactions amongst genetically dissimilar genotypes that can result in either competitive exclusion, coexistence, or cycling through a combination of the two. Here, we report how intra-lineage trait variation of the multihost amphibian parasite Batrachochytrium dendrobatidis drives contrasting outcomes of inter-lineage interactions on two continents, Europe and Africa. Through field surveys, competition experiments and mathematical modelling we show that interactions between the same two lineages in the two continents and in different hosts demonstrate both ends of the exclusion/coexistence continuum. Trait variation in one of the two predominating lineages, BdGPL, is responsible for these contrasting outcomes: In Europe, BdGPL is highly competitive and has constrained the distribution of the other, BdCAPE, to two locations. In Africa, BdGPL and BdCAPE can mutually invade host populations when the other lineage is already resident, potentially leading to coinfections and recombination. That these contrasting outcomes are prolonged and contemporaneous for the same two lineages shows that epidemiological models of invasive parasites need to account for trait variation both within and across lineages. ### Competing Interest Statement The authors have declared no competing interest.

Reptiles are carriers of an array of microorganisms, including significant zoonotic bacteria of the genus Salmonella, which cause a disease referred to as salmonellosis that affects both animals and humans. This study investigated the occurrence of Salmonella serovars in wild reptiles at Timbavati Private Game Reserve in Limpopo Province, South Africa, and examined their virulence and antimicrobial resistance gene profiles. A total of 19 wild reptiles were sampled, which resulted in 30 presumptive Salmonella isolates. The isolates were identified using polymerase chain reaction (PCR) by amplifying the invA gene and were further confirmed by 16S rRNA gene sequencing. Salmonella serovars were detected in chameleons (36.8%), lizards (31.6%), snakes (15.8%), and tortoises (15.8%). The use of 16S rRNA gene sequencing revealed that Salmonella enterica subsp. enterica serovar Salamae (30%), S. enterica subsp. enterica (16.7%), S. enterica subsp. enterica serovar Typhimurium (13.3%), and S. enterica subsp. enterica serovar Indiana (13.3%) were the four most common subspecies among the investigated 30 isolates. Detected virulence genes included pagN (100%), hilA (96.7%), ssrB (96.7%), prgH (86.7%), and marT (86.7%). The isolates exhibited resistance to nalidixic acid (43.3%) and kanamycin (43.3%), followed by streptomycin (16.7%) and ciprofoxacin (3.3%). Antibiotic-resistant genes were detected as follows: strA, strB, qnrA, qnrS, parC, aadA, aac(6 ')-Ib, and aac(6 ')-Ib-cr at 33.3%, 6.7%, 16.7, 13.3%, 10%, 23.3%, 6.7%, and 10%, respectively. The findings highlight the necessity of educational initiatives aimed at reducing reptile-related infections. Effective antibiotic treatment appears promising for infection, given the minimal drug resistance observed in reptile Salmonella serovars in the current study.

The amphibian-infecting chytrid fungus, Batrachochytrium dendrobatidis (Bd), is widespread throughout Africa and is linked to declines of populations and species across the continent. While it is well established that the lineage of Bd encodes traits which determine disease severity, knowledge around how lineages are distributed according to environmental envelope is unclear. We here studied the distribution of Bd in South Africa based on the two lineages found, BdGPL and BdCAPE, in terms of their genome and environmental envelope statistically associated with their distribution. We used Bd surveillance data from published studies, as well as data collected during fieldwork from across South Africa, Lesotho, and eSwatini with samples collected along a transect spanning most of South Africa from Lesotho to the west coast. We utilized lineage-typing qPCR to resolve the spatial distribution of BdGPL and BdCAPE across South Africa and used the resulting surveillance data to create a predictive ecological niche model for Bd lineages in South Africa. Phylogenomic analyses were performed on isolates sourced from across the transect. We show that BdGPL demonstrates a strong isolation by distance suggestive of stepping-stone dispersal, while BdCAPE showed two distinct clusters within their genomic structure that appear geographically and temporally clustered, indicating two separate invasions. Our predictive niche model revealed that the two lineages tended to occur in different ecotypes; BdGPL was associated with lower altitude, arid regions while BdCAPE occurred across cooler, higher altitude environs. Niche predictions identified a zone of lineage contact, where genomics identified inter-lineage recombinants. We argue that this zone of recombination should be prioritized for disease surveillance as it is a potential hotspot for the evolution of variants of amphibian chytrid with novel traits that may be epidemiologically relevant.

Outbreaks of emerging infectious diseases are influenced by local biotic and abiotic factors, with host declines occurring when conditions favour the pathogen. Deterioration in the population of the micro-endemic Tanzanian Kihansi spray toad ( Nectophrynoides asperginis ) occurred after the construction of a hydropower dam, implicating habitat modification in this species decline. Population recovery followed habitat augmentation; however, a subsequent outbreak of chytridiomycosis caused by Batrachochytrium dendrobatidis ( Bd ) led to the spray toad's extinction in the wild. We show using spatiotemporal surveillance and mitogenome assembly of Bd from archived toad mortalities that the outbreak was caused by invasion of the Bd CAPE lineage and not the panzootic lineage Bd GPL. Molecular dating reveals an emergence of Bd CAPE across southern Africa overlapping with the timing of the spray toad's extinction. That our post-outbreak surveillance of co-occurring amphibian species in the Udzungwa Mountains shows widespread infection by Bd CAPE yet no signs of ill-health or decline suggests these other species can tolerate Bd when environments are stable. We conclude that, despite transient success in mitigating the impact caused by dams’ construction, invasion by Bd CAPE caused the ultimate die-off that led to the extinction of the Kihansi spray toad.

Nasal drug administration has been identified as a potential alternative to oral drug administration, especially for systemic delivery of large molecular weight compounds. Major advantages of nasal drug delivery include high vascularity and permeability of the epithelial membranes as well as circumvention of first-pass metabolism. RPMI 2650 cell layers (in vitro cell model) and excised sheep nasal mucosal tissues (ex vivo sheep model) were evaluated with regard to epithelial thickness, selected tight junction protein expression (i.e. claudin-1, F-actin chains, zonula occludin-1), extent of p-glycoprotein (P-gp) related efflux of a model compound (Rhodamine-123, R123) and paracellular permeation of a large molecular weight model compound (FITC-dextran 4400, FD4). The cell model grown under liquid cover conditions (LCC) was thinner (24 +/- 4 mu m) than the epithelial layer of the sheep model (53 +/- 4 mu m), whereas the thickness of cell model grown under air liquid interface (ALI) conditions (53 +/- 8 mu m) compared well with that of the sheep model. Although the location and distribution of tight junction proteins and F-actin differed to some extent between the cell model grown under ALI conditions and the sheep model, the extent of paracellular permeation of FD4 was similar (P-app = 0.48 x 10(-6) cm.s(-1) and 0.46 x 10(-6) cm. s(-1), respectively). Furthermore, the bi-directional permeation of R123 yielded the same efflux ratio (ER = 2.33) in both models. The permeation results from this exploratory study indicated similarity in terms of compound permeation between the RPMI 2650 nasal epithelial cell line and the excised sheep nasal epithelial tissue model.

Outbreaks of emerging infectious diseases are trained by local biotic and abiotic factors, with host declines occurring when conditions favour the pathogen. Extinction in the wild of the Tanzanian Kihansi spray toad (Nectophrynoides asperginis) in 2004 was contemporaneous with the construction of a dam, implicating habitat modification in the loss of this species. However, high burdens of a globally emerging infection, Batrachochytrium dendrobatidis (Bd) followed the dams commission indicating infectious disease as the ultimate cause of the toads extinction. Here, we demonstrate that shotgun sequencing skin DNA from archived toad mortalities and assembling pathogen mitogenomes presents a powerful epidemiological tool by proving the outbreak was caused by an invasion of the BdCAPE lineage and not the panzootic lineage BdGPL. Using molecular dating we show an emergence of BdCAPE across Southern Africa overlapping with the timing of the toads extinction. That our post-outbreak surveillance of conspecific amphibian species showed widespread infection yet no signs of ill-health or decline shows these species can tolerate Bd when environments are stable. We conclude that, despite transient success in mitigating the impact caused by dams construction, subsequent invasion of the pathogen caused the catastrophic die-off that led to the loss of the Kihansi spray toad. Similar synergies between infection and environmental change will likely herald wider negative impacts on biodiversity in the Anthropocene.

The ability to detect and monitor infectious disease in a phylogenetically informative manner is critical for their management. Phylogenetically informative diagnostic tests enable patterns of pathogen introduction or changes in the distribution of genotypes to be measured, enabling research into the ecology of the pathogen. Batrachochytrium dendrobatidis ( Bd ), a causative agent of chytridiomycosis in amphibian populations, emerged worldwide in the 21 st century and is composed of six lineages which are display varying levels of virulence in their hosts. Research into the distribution, ecology and pathogenicity of these lineages has been hampered by an inability to type lineage efficiently. Here, we describe a lineage-specific TaqMan qPCR assay that differentiates the two lineages of Bd most commonly associated with chytridiomycosis: Bd GPL and Bd CAPE. We demonstrate how this assay can be used for the surveillance of wild populations of amphibians in Southern Africa using skin swabs, tissue samples and cultured isolates.

Outbreaks of emerging infectious diseases are trained by local biotic and abiotic factors, with host declines occurring when conditions favour the pathogen. Extinction of the Tanzanian Kihansi spray toad ( Nectophrynoides asperginis ) in 2004 was contemporaneous with the construction of a dam, implicating habitat modification in the loss of this species. However, high burdens of a globally emerging infection, Batrachochytrium dendrobatidis ( Bd ) were synchronously observed implicating infectious disease in this toads extinction. Here, by shotgun sequencing skin DNA from archived toad mortalities and assembling chytrid mitogenomes, we prove this outbreak was caused by the Bd CAPE lineage and not the panzootic lineage Bd GPL that is widely associated with global amphibian extinctions. Molecular dating showed an invasion of Bd CAPE across Southern Africa overlapping with the timing of the extinction event. However, post-outbreak surveillance of conspecific species inhabiting this mountainous region showed widespread infection by Bd CAPE yet no signs of amphibian ill-health or species decline. Our findings show that despite efforts to mitigate the environmental impact caused by dams construction, invasion of the pathogen ultimately led to the loss of the Kihansi spray toad; a synergism between emerging infectious disease and environmental change that likely heralds wider negative impacts on biodiversity in the Anthropocene. ### Competing Interest Statement The authors have declared no competing interest.

The Kihansi spray toad, Nectophrynoides asperginis, became extinct in the wild despite population monitoring and conservation management of its habitat in the Kihansi gorge, Tanzania. Anecdotal evidence has indicated human induced habitat modification, predators, pesticides and disease as possible causes of a rapid population decline and the species extirpation. Here, we systematically investigate the role of disease in the extinction event of the wild toad population. The amphibian chytrid fungus, Batrachochytrium dendrobatidis, was detected in spray toads that died during the extinction event and subsequently in other amphibian species in Kihansi Gorge and the adjacent Udagaji Gorge, but not in any toads collected prior to this. Following the population decline, the remnant spray toad population gradually disappeared over a nine-month period. We demonstrate how demographic and behavioral attributes predisposed the spray toads to chytridiomycosis, due to B. dendrobatidis infection, and how epidemic disease could have been exacerbated by altered environmental conditions in the spray wetlands. Our results show that chytridiomycosis was the proximate cause of extinction in the wild of N. asperginis. This represents the first known case of extinction by disease of an amphibian species in Africa. A captive breeding program in the US and Tanzania ensures the survival of the species and a reintroduction program is underway. However, we caution that chytridiomycosis remains an existing threat that requires a comprehensive mitigation strategy before the desired conservation outcome of an established population of repatriated toads can be achieved.

Introduction: The nasal route of drug administration offers an alternative way for oral drug delivery and has the benefit of avoiding first-pass metabolism through drug delivery directly into the systemic circulation. The drug absorption enhancing effects of selected aloe leaf materials have been shown across various delivery routes, but their efficacies in this regard across nasal epithelia have not yet been investigated. The aim of this study was to determine the effects of gel and whole leaf extract materials from three selected aloe species (Aloe vera, Aloe ferox and Aloe muth-muth) on FITC-dextran 4400 permeation across two nasal epithelial models. Methods: Permeation of FITC-dextran 4400 and histological studies were conducted on both RPMI 2650 cell layers and excised sheep nasal mucosa, while toxicity studies were conducted using a neutral red assay on the RPMI 2650 cell model. Results: Significantly increased (P ≤ 0.05) apparent permeability coefficient (Papp) values of FITC-dextran 4400 in the presence of the aloe materials as compared to the control were found with all three aloe species at the highest concentrations (1.5% and 3% w/v) in the RPMI 2650 cell line, while only Aloe muth-muth at the highest concentration exhibited significantly (P ≤ 0.05) higher Papp values across the excised tissue model. Histological and neutral red analysis showed that Aloe vera materials exhibited detrimental effects, Aloe muth-muth only showed slight effects on cell viability and Aloe ferox exhibited no effect on the nasal epithelium. Conclusion: This in vitro study showed for the first time the potential of Aloe ferox and Aloe muth-muth leaf materials to enhance nasal drug delivery without causing damaging effects on the epithelium, while Aloe vera enhanced nasal drug delivery with detrimental effects as determined by means of cytotoxicity assays and histological analysis.

The amphibian chytrid fungus Batrachochytrium dendrobatidis ( Bd ), responsible for numerous amphibian declines and extinctions, was previously thought to originate from the African continent. This was based on infected museum specimens from early 20th century South Africa, Cameroon and Uganda. Further research on archived specimens from other continents eventually revealed early 20th century records also in Brazil and Japan. Recent robust analysis of genomic diversity and phylogeny of Bd has shown origin from Asia to be more plausible. This raises the issue that the threat of Bd for African amphibians as a novel pathogen has been underestimated. There are now cases where dramatic amphibian declines in disparate mountains on the continent could be attributed to Bd , and this necessitates an urgent realigning of hypotheses and the research agenda for amphibian conservation on the continent. Notably, hotspots of amphibian host naivety include West Africa where this pathogen has so far not been detected. We discuss research gaps that amphibian conservationists might focus on, notably more genomic sequencing of the Bd CAPE (the less virulent) lineage to determine its date of emergence, and assessing the susceptibility of different amphibian species to infection, disease and decline to better prioritize conservation actions.

Abstract. Accurate survey methods are required for any wildlife research to yield reliable population data. This constraint finds significance in amphibian research that involves a highly threatened group of animals with a large proportion of cryptic species not easily detected by conventional survey methods. Across a growing spectrum of zoology research, survey outcomes are benefitting from the efficacy of scent detection dogs in assisting with species detection. We investigated the ability of a scent detection dog to locate and identify traces of giant bullfrog, Pyxicephalus adspersus scent and investigate methods of preserving frog scent for use in subsequent conditioning training of dogs. The scent detection dog was able to detect 100,000 times diluted scent with 87% sensitivity and 84% efficacy. High specificity (98,6%) was also achieved while presented with the challenge of detecting P. adspersus scent amid that of other frog species. Detection sensitivity was negatively correlated with scent preservation time but yielded the highest sensitivity for samples that were preserved as skin swabs stored at 4 °C and diluted shortly before use. Conservationists, scientists, and customs officials alike can benefit from scent detection dog detection of amphibians through enhanced sample acquisition rates with reduced collection biases.

The fruit from various pepper plants has been employed for the seasoning of food, as perfuming agents, and also as traditional medicines. Phytochemicals isolated from different pepper species have been found to modulate the pharmacokinetics of orally administered drugs. This study investigated the possibility to apply capsaicin and piperine (extracted alkaloids) as modulators for drug delivery across the nasal epithelium. Both a nasal epithelial cell line (RPMI 2650) and excised sheep nasal tissue were used as models to investigate the effects of the selected pepper compounds on drug permeation. FITC-dextran 4400 (MW 4400 Da) was used as a large molecular weight marker compound for paracellular transport, while rhodamine 123 was used as a marker compound that is a substrate for P-glycoprotein-mediated efflux. From the permeation results, it was clear that capsaicin inhibited P-glycoprotein efflux to a larger extent, while piperine showed drug permeation enhancement via other mechanisms. The cell cytotoxicity studies indicated that capsaicin was noncytotoxic up to a concentration of 200 µM and piperine up to a concentration of 500 µM as indicated by cell viability above 80%. The histological analysis of the excised nasal tissue and cultured RPMI 2650 cell layers indicated that some damage occurred after treatment with 200 µM capsaicin, but no changes were observed for piperine up to a concentration of 50 µM.

Anthropogenic trade and development have broken down dispersal barriers, facilitating the spread of diseases that threaten Earth's biodiversity. We present a global, quantitative assessment of the amphibian chytridiomycosis panzootic, one of the most impactful examples of disease spread, and demonstrate its role in the decline of at least 501 amphibian species over the past half-century, including 90 presumed extinctions. The effects of chytridiomycosis have been greatest in large-bodied, range-restricted anurans in wet climates in the Americas and Australia. Declines peaked in the 1980s, and only 12% of declined species show signs of recovery, whereas 39% are experiencing ongoing decline. There is risk of further chytridiomycosis outbreaks in new areas. The chytridiomycosis panzootic represents the greatest recorded loss of biodiversity attributable to a disease.

Amphibian populations worldwide are at risk of extinction from infectious diseases, including chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Amphibian cutaneous microbiomes interact with Bd and can confer protective benefits to the host. The composition of the microbiome itself is influenced by many environment- and host-related factors. However, little is known about the interacting effects of host population structure, genetic variation and developmental stage on microbiome composition and Bd prevalence across multiple sites. Here we explore these questions in Amietia hymenopus, a disease-affected frog in southern Africa. We use microsatellite genotyping and 16S amplicon sequencing to show that the microbiome associated with tadpole mouthparts is structured spatially, and is influenced by host genotype and developmental stage. We observed strong genetic structure in host populations based on rivers and geographic distances, but this did not correspond to spatial patterns in microbiome composition. These results indicate that demographic and host genetic factors affect microbiome composition within sites, but different factors are responsible for host population structure and microbiome structure at the between-site level. Our results help to elucidate complex within- and among-population drivers of microbiome structure in amphibian populations. That there is a genetic basis to microbiome composition in amphibians could help to inform amphibian conservation efforts against infectious diseases.

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

The Infectious disease chytridiomycosis, which is caused by the fungal pathogen Batrachochytrium dendrobatidis, has been identified as one of the most important drivers of amphibian declines and extinction. In vitro B. dendrobatidis is susceptible to a range of disinfectants, but not all have been tested on animals and some that have been proven effective have harmful side effects on the surrounding environment or the animals being treated. We tested the efficacy of F10SC veterinary disinfectant to treat B. dendrobatidis in experimentally infected tadpole and juvenile Sclerophrys gutturalis and tadpoles of Sclerophrys poweri and Amietia hymenopus. The minimum inhibitory concentration for F10SC on in vitro B. dendrobatidis ranged between 1:7000 for 5-min contact time and 1:10000 for 10-min contact time. Based on the survival data of test animals the no observed effect concentration for 15-min contact time was estimated to be 1:2000 dilution for juveniles, and 1:10000 for tadpoles. In S. gutturalis juveniles an 86% infection clearance rate was achieved after five 15-min doses of 1:3000 dilution. A 100% clearance was achieved in A. hymenopus tadpoles after seven 15-min doses of 1:10000 dilution, and after nine doses of the same treatment in S. poweri tadpoles. F10SC has the benefit of being a concentrated compound that provides a treatment protocol which is nontoxic to tadpoles and post-metamorphic individuals, has a short half-life and is effective against B. dendrobatidis during short contact times, but further testing on different species of amphibians is advised.