Outbreaks of pathogens that threaten both human and nature have increased in recent years. Infectious and transmittable diseases, such as chytridiomycosis, which is caused by the emerging pathogen Batrachochytrium dendrobatidis, has been identified as one of the most important drivers of the current declines in amphibian numbers. This pathogen has spread globally and is not only responsible for the declines in amphibian population numbers, but also for the extinction of species in several countries. As part of the Amphibian Conservation Action Plan, the IUCN recommended ex situ breeding of amphibian species to try and stem the global loss of amphibian species. Due to chytridiomycosis being one of the most eminent threats for amphibians, it poses an additional threat for the ex situ breeding plan. There is thus a need for safe and effective measures to treat chytridiomycosis, especially in breeding programs for endangered species. F10 (Health and Hygiene) is a veterinary antiseptic that has shown to be 100% effective in killing B. Dendrobatidis in vitro. Before any chemical treatment can be applied the efficacy and toxicity of F10 has to be determined to establish if F10 can be effectively applied across different amphibian species and across different life stages. We propose to develop a treatment protocol for F10 for the effective treatment of amphibian chytridiomycosis by challenging juveniles of Amietophrynus gutturalis with B. dendrobatidis and subsequently treating the infection with a proposed concentration of F10. The survival of B. dendrobatidis zoospores was also determined in the presence of F10. The results obtained showed survival of tadpoles at a 1:10,000 concentration of F10 for 30min, and juveniles at a concentration of 1:2000 for 15 min. Furthermore the in vitro tests showed that the B. dendrobatidis zoospores died after 10 min at a 1:10,000 concentration and 30 min at a 1:15,000 concentration. The successful treatment of tadpoles as well as juveniles will increase any species chance for survival, especially when treating tadpoles as the pathogen will then be eradicated before the tadpole metamorphoses and reaches the disease-susceptible life stage. By establishing a partnership between the industry, academic and zoo/wildlife communities we hope to maximise the likelihood of implementing this program in the future and thus ensuring long term sustainability. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Outbreaks of pathogens that threaten both human and nature have increased in recent years. Infectious and transmittable diseases, such as chytridiomycosis, which is caused by the emerging pathogen Batrachochytrium dendrobatidis, has been identified as one of the most important drivers of the current declines in amphibian numbers. This pathogen has spread globally and is not only responsible for the declines in amphibian population numbers, but also for the extinction of species in several countries. As part of the Amphibian Conservation Action Plan, the IUCN recommended ex situ breeding of amphibian species to try and stem the global loss of amphibian species. Due to chytridiomycosis being one of the most eminent threats for amphibians, it poses an additional threat for the ex situ breeding plan. There is thus a need for safe and effective measures to treat chytridiomycosis, especially in breeding programs for endangered species. F10 (Health and Hygiene) is a veterinary antiseptic that has shown to be 100% effective in killing B. Dendrobatidis in vitro. Before any chemical treatment can be applied the efficacy and toxicity of F10 has to be determined to establish if F10 can be effectively applied across different amphibian species and across different life stages. We propose to develop a treatment protocol for F10 for the effective treatment of amphibian chytridiomycosis by challenging juveniles of Amietophrynus gutturalis with B. dendrobatidis and subsequently treating the infection with a proposed concentration of F10. The survival of B. dendrobatidis zoospores was also determined in the presence of F10. The results obtained showed survival of tadpoles at a 1:10,000 concentration of F10 for 30min, and juveniles at a concentration of 1:2000 for 15 min. Furthermore the in vitro tests showed that the B. dendrobatidis zoospores died after 10 min at a 1:10,000 concentration and 30 min at a 1:15,000 concentration. The successful treatment of tadpoles as well as juveniles will increase any species chance for survival, especially when treating tadpoles as the pathogen will then be eradicated before the tadpole metamorphoses and reaches the disease-susceptible life stage. By establishing a partnership between the industry, academic and zoo/wildlife communities we hope to maximise the likelihood of implementing this program in the future and thus ensuring long term sustainability. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines around the world, including the southwestern United States. I studied patterns of Bd infection in Hyla arenicolor, Rana catesbeiana, and R. yavapaiensis in the Rincon Mountains and Tucson Basin in Arizona. Bd prevalence and infection intensity were location dependent in all species and R. yavapaiensis may be a reservoir of Bd for H. arenicolor, where they co-occurred. Treatment of a backyard population of R. yavapaiensis with itraconazole did not reduce winter frog mortality due to Bd. The lethal Bd infection threshold of this population was between 59,847 and 4,237,330 zoospores. Zoospore loads from swabs of freshly dead frogs did not differ significantly from those taken from those same frogs following freezing and thawing. Thus, important information regarding infection intensity and probable cause of death can be gathered from frogs collected by others and frozen until convenient to process.

Amphibians are of great ecological importance and a loss of species will have widespread and dire consequences. Recent population declines and extinctions have resulted in amphibians being labelled the most threatened vertebrate class on a global scale. The unique Okavango ecosystem is well known and documented, yet the amphibians of this region are poorly known. This project aimed at assessing diversity in the Okavango Delta by testing isolation as a possible driver for community composition; determining the effect of hydrology on breeding behaviour; and assessing the status and prevalence of the pathogen Batrachochytrium dendrobatidis (Bd) responsible for the widespread epidemic chytridiomycosis implicated in amphibian decline. Using various monitoring techniques, observations of species occurrence were made at three locations representing different degrees of isolation over a 20 month period. Breeding indicators were observed and frogs were screened for amphibian chytrid fungus. A total of 29 species were recorded, and results indicated that there were no significant differences in community composition between the sampled localities. Species presence, however, was significantly correlated with habitat type. Thus, the availability of suitable habitat appears to be driving amphibian diversity patterns, rather than geographic isolation; and increased habitat diversity near the Delta periphery explains increased amphibian diversity in these areas. Results from breeding indicators suggested that reproduction in continuous breeders was correlated with the annual flood as well as rainfall, whilst that of explosive breeders was correlated with rainfall alone. It is thus proposed that opportunistic breeding behaviour for some amphibian species is driven by the hydrology of the ecosystem; and this may be explained by increased biological production associated with the flood pulse. Outcomes highlight the unique nature of the Okavango Delta system, and emphasises the need for its preservation. A total of 249 swab samples were collected and screened for amphibian chytrid fungus. The geographical distribution of collection samples were evenly spread throughout the localities, and were obtained from at least 25 amphibian species. Analyses proved negative for Bd for the 79.92% swabs analysed thus far and it is concluded that Bd seems absent in the study region, a result which has massive conservation implications for the region. Despite the fact that the Okavango Delta has benefitted from conservation and tourism efforts in the past, the system and its biodiversity remains threatened and effective conservation management strategies must be devised and implemented to ensure its preservation. / Thesis (M.Sc. (Environmental Science))--North-West University, Potchefstroom Campus, 2011.

Amphibians are of great ecological importance and a loss of species will have widespread and dire consequences. Recent population declines and extinctions have resulted in amphibians being labelled the most threatened vertebrate class on a global scale. The unique Okavango ecosystem is well known and documented, yet the amphibians of this region are poorly known. This project aimed at assessing diversity in the Okavango Delta by testing isolation as a possible driver for community composition; determining the effect of hydrology on breeding behaviour; and assessing the status and prevalence of the pathogen Batrachochytrium dendrobatidis (Bd) responsible for the widespread epidemic chytridiomycosis implicated in amphibian decline. Using various monitoring techniques, observations of species occurrence were made at three locations representing different degrees of isolation over a 20 month period. Breeding indicators were observed and frogs were screened for amphibian chytrid fungus. A total of 29 species were recorded, and results indicated that there were no significant differences in community composition between the sampled localities. Species presence, however, was significantly correlated with habitat type. Thus, the availability of suitable habitat appears to be driving amphibian diversity patterns, rather than geographic isolation; and increased habitat diversity near the Delta periphery explains increased amphibian diversity in these areas. Results from breeding indicators suggested that reproduction in continuous breeders was correlated with the annual flood as well as rainfall, whilst that of explosive breeders was correlated with rainfall alone. It is thus proposed that opportunistic breeding behaviour for some amphibian species is driven by the hydrology of the ecosystem; and this may be explained by increased biological production associated with the flood pulse. Outcomes highlight the unique nature of the Okavango Delta system, and emphasises the need for its preservation. A total of 249 swab samples were collected and screened for amphibian chytrid fungus. The geographical distribution of collection samples were evenly spread throughout the localities, and were obtained from at least 25 amphibian species. Analyses proved negative for Bd for the 79.92% swabs analysed thus far and it is concluded that Bd seems absent in the study region, a result which has massive conservation implications for the region. Despite the fact that the Okavango Delta has benefitted from conservation and tourism efforts in the past, the system and its biodiversity remains threatened and effective conservation management strategies must be devised and implemented to ensure its preservation. / Thesis (M.Sc. (Environmental Science))--North-West University, Potchefstroom Campus, 2011.

Batrachochytrium dendrobatidis (Bd) has contributed to population declines and extinction of amphibians worldwide. Disease-caused extinction is rare, but may occur where reservoirs exist or where transmission is independent of host density. Because some species persist after Bd epizootic events, they may act as potential reservoirs of the infection. I studied seasonal and elevational patterns of prevalence and intensity of Bd infection in eight pond-breeding species that persisted after an epizootic event at three low- and three mid-elevation ponds in Central America. I visited each pond three times over the rainy season to sample for Bd and to quantify frog density. I recorded air and water temperature at each pond. I analyzed 1,288 samples and detected Bd in all six ponds and in six of the eight species. Elevation and time affected Bd prevalence (F2,7 = 8.90, p = 0.01) and Bd intensity (F2,8 = 9.09, p = 0.008). Frog density was not correlated with Bd prevalence (F1,7= 4.75, p= 0.06), and temperature did not affect prevalence or intensity of the infection. Bd is enzootic at all 6 sites and six pond-breeding species are reservoirs. The presence of abundant reservoirs at enzootic conditions explains the persistence of Bd in areas where other species declined without showing evidence of recovery. Low intensities of infection explain the survivorship of these reservoir species. I suggest that temperature might not be the principal factor molding Bd dynamics in the humid Neotropical forests, and present evidence that ponds are ecosystems where frogs might clean or reduce their infections

<p>Per-and  polyfluoroalkyl  substances  (PFAS)  are  a  large  group  of  emerging  contaminants  that include astrong carbon-flourine bond that makes the compounds resistant to physical, chemical and  biological  degradation.  They  are  found  in  drinking  water  supplies,  daily  human  products, manufacturing  facilities,  and  in  areas  where  aqueous  film-forming  foam  (AFFF)was  used  to extinguish fires. Toxicity levels of these chemicals can vary depending on the characteristics of the specific chemical; longer carbon chain has shown to be more bioaccumulative and toxic than shorter chain length PFAS. Many studies have recognized perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) to be a substantial concern due to their known toxicity to wildlife. For example, studies show strong evidence that PFOA and PFOS suppress the antibody response from animals. Due to adverse health effects and public concern, the U.S stopped perfluorooctanoic acid (PFOA) manufacturing and switched to the production of an alternative fluorinated compound known  as  hexafluoropropylene  oxide  (HFPO)  dimer  acid  or  GenX,  which  is  thought  to  beless bioaccumulative and therefore, potentially less toxic. These anthropogenic pollutants are one of many stressors acting on aquatic organisms like anurans. Natural stressors such as the devastating fungal  pathogen Batrachocytrium  dendrobatidis(Bd)  is  another  stressor  impacting  amphibian populations.  Despite the co-occurrence of these stressors, no studies have examined interactive effects of the fungal pathogen Bd and PFAS, or whether PFAS effects carry over into the terrestrial environment aftera larvae  exposure. This study  tested the growth and developmental effects of PFOS, PFOA, and GenX, on gray treefrog (Hyla versicolor) tadpoles, followed by a Bd challenge in metamorphs. Our results demonstrate that a PFAS larval exposure interacted witha terrestrial Bdchallenge to influence growth and development. Bdexposed animals were significantly shorter (smaller snout vent length)  and had a significant increase in body condition and mass. This is the first study to report effects on amphibian terrestrial life stages after larval exposure to PFAS and to report an increased sensitivity to Bd. The environmentally relevant concentrations tested in this study  (<10  parts  per  billion)  lend  ecological  significance  to  these  results  however,  additional studies are needed to understand the mechanisms behind these effects.</p>

No description available.

Chytridiomycosis is an emerging cutaneous fungal disease that contributes to recent global declines and extinction of amphibian species, caused by infection of the skin with a fungus known as Batrachochytrium dendrobatidis (Bd). Many species of frogs secrete antimicrobial peptides onto their skin that are capable of killing Bd. This thesis is an investigation of the effect of corticosteroids on cutaneous innate immunity in frogs, in the context of infection with Bd. The general hypothesis was that injections of glucocorticoids would impair the cutaneous synthesis of these antimicrobial peptides, thereby increasing susceptibility to Bd infection. The objective of the first experiment was to measure and compare gene expression levels of cutaneous AMP’s in frogs treated with glucocorticoids with sham-treated controls. Wild-caught Lithobates pipiens were acclimatized and administered either the corticosteroid methylprednisolone or saline every 48 hours. Norepinephrine-elicited cutaneous secretions were collected prior to the first injection of corticosteroid or saline, and then every 8 days for 40 days. Gene expression of the AMP’s brevinin and ranatuerin in the cutaneous secretions was quantified relative to the reference genes EF1-α and RPL8 using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Corticosteroid treatment was associated with a significant (P<0.027) increase in brevinin gene expression, which was most notable at 24-40 days of corticosteroid administration. Ranatuerin expression followed a similar but nonsignificant trend. The second experiment was a pilot study intended to establish a Bd challenge protocol in L. pipiens. Frogs were immersed in water containing 0, 104, 105 or 106 zoospores of Bd strain JEL 423. Cutaneous swabs were collected prior to challenge and tested for Bd by qPCR; unexpectedly, some tested positive, indicating pre-challenge infection. The analysis was complicated by an identified cross-reactivity of the assay with other fungi. The findings of the first experiment refuted the hypothesis, and suggested that corticosteroids promote rather than impair AMP gene expression in the skin of L. pipiens, under these experimental conditions. Further, the second study demonstrated that none of the frogs showed clinical abnormalities or died, despite exposure to Bd zoospores and despite molecular and histologic evidence of cutaneous Bd infection in some frogs. / NSERC Discovery Grant

The waterborne pathogen Batrachochytrium dendrobatidis (Bd), amphibian chytrid, is implicated as being the causative agent for global amphibian declines. The fungus attacks the keratinized skin of adult and postmetamorphic animals and the keratinized mouthparts of tadpoles. Postmetamorphic animals seem to be more susceptible to Bd than tadpoles and adult frogs. Hypotheses exist that the origin of the fungus is in Africa. During the study different aspects of Bd infections in South African frogs were examined including the distribution of Bd, cultivation of Bd, preservation of cultures, the morphology of Bd as an infection as well as in culture and finally differences in host defense. Positive and negative localities for Bd were identified through surveys conducted in South Africa. These data will be contributed to the Bd Mapping Project and the African Bd Database in order to determine whether chytrid has any environmental preferences. Cultures obtained from the positive localities were maintained and cryopreserved for use in numerous experiments. In a future study, DNA extractions from the cultures will be analyzed using multilocus sequence typing in order to determine the sequence type of South African strains in comparison with global strains. This will provide important epidemiological information concerning the origin and control of Bd. The morphology of Bd was also examined using scanning electron microscopy and laser scanning confocal microscopy. Damage due to Bd infections was more severe on the larval mouthparts of Amietia vertebralis than that of Hadromophryne natalensis. The adverse effect of Bd is therefore not limited to postmetamorphic animals. Confocal microscopy uses fluorescent stains and lasers to examine specific structures within organisms. An especially effective stain used during confocal microscopy on Bd is Calcofluor White M2R. Due to its specificity this stain can be used as an effective screening tool for Bd in tissue. The role of antimicrobial skin peptides as a defense against Bd was also examined. A. vertebralis experiences die-offs due to chytrid, while H. natalensis does not experience the same effect in the presence of Bd. H. natalensis possess more antimicrobial skin peptides against Bd with a higher effectiveness than peptides extracted from A. vertebralis. This may explain the observed susceptibility of A. vertebralis to Bd. The relevance of this study is in order to identify areas in South Africa in which the probability of finding Bd is high. This will help in the surveillance of Bd and in the identification of susceptible species to be monitored and protected against the fungus. The effect of Bd on frog species can also be determined by means of exposure experiment using cultures isolated during this study. Through the identification of peptides effective against Bd, predictions can be made with regard to the susceptibility of different frogs to Bd, improving our ability to protect the amphibian biodiversity in South Africa. With the use of confocal microscopy in the examination of Bd, we became the first group to use the method. By the identification of a stain with a high potential as a screening tool, we also contributed to the more efficient identification of Bd in tissue. Keywords: Batrachochytrium dendrobatidis, Bd, amphibian chytrid, distribution, cultivation, antimicrobial skin peptides, laser scanning confocal microscopy, Amietia vertebralis, Hadromophryne natalensis, South Africa / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2009.