Pioneer access of the foam nest bacterial community of Leptodactylidae frogs and its biotechnological potential.

Many anuran amphibians deposit their eggs in foam nests, biostructures that help protect the eggs and tadpoles from predators. Currently, there are no other identification and description studies of the cultivable microbiota role in the nests of the Leptodactylid frogs such as Physalaemus cuvieri, Leptodactylus vastus and Adenomera hylaedactyla. This study aimed to isolate and identify the culturable bacteria from these three anuran species' nests, as well as to prospect enzymes produced by this microbiota. Foam nests samples and environmental samples were diluted and viable cell count was determined. Bacterial morphotypes from foam nest samples were isolated through spread plate technique. Isolates' DNAs were extracted followed by rRNA 16S gene amplification and Sanger sequencing. To evaluate their enzymatic potential, the isolates were cultured in ATGE medium supplemented with starch (0.1% w/v), gelatin (3% w/v) and skimmed milk (1% w/v), to verify amylase and protease activity. A total of 183 bacterial morphotypes were isolated, comprising 33 bacterial genera. Proteobacteria phylum was the most abundant in all the three nests (79%). The genera Pseudomonas and Aeromonas were the most abundant taxon in P. cuvieri and L. vastus. In A. Hylaedactyla, were Enterobacter and Bacillus. Regarding enzymatic activities, 130 isolates displayed protease activity and 45 isolates were positive for amylase activity. Our results provide unprecedented information concerning culturable bacterial microbiota of the foam nests of the Leptodactylid frogs, as well as their potential for biomolecules of biotechnological interest.

High-mortality epizootic Mycobacterium ulcerans ecovar Liflandii in a colony of Zaire Dwarf Clawed Frogs (Hymenochirus boettgeri).

Mycobacterium ulcerans ecovar Liflandii (MuLiflandii) was identified as the causative agent of mycobacteriosis in a research colony of Zaire dwarf clawed frogs (Hymenochirus boettgeri) at the University of Michigan. Clinical presentation included lethargy, generalized septicemia, cutaneous granulomas, coelomic effusion, and acute mortality. Identification of the mycobacterial species was based on molecular, microbiological, and histopathologic characteristics. These findings indicate that MuLiflandii is a primary cause of morbidity and mortality in Zaire dwarf clawed frogs and should be considered in the differential diagnosis of sepsis and coelomic effusion in amphibians. Mycobacterial speciation is important given the variability in pathogenesis within the family Mycobacteriaceae and the implications for both animal and human health as potential zoonoses. The Zaire dwarf clawed frog is a species common in the pet trade, and these findings provide consideration for this pathogen as a potentially important public health concern. This is the first report of MuLiflandii infection in the genus Hymenochirus and illustrates the diagnostic challenges of differentiating among both mycolactone-producing mycobacteria and Mycobacterium marinum. Furthermore, we demonstrate the utility of environmental sampling for this pathogen within the tank system, suggesting this mode of sampling could replace the need for direct frog surveillance.

Antibacterial activity of the skin secretion of Phyllomedusa azurea (Anura: Hylidae) from the Central Brazil Cerrado / Actividad antibacterial de secreciones de la piel de Phyllomedusa azurea (Anura: Hylidae) en Cerrado, Brasil centro

Abstract Phyllomedusa azurea is a frog species well distributed geographically in South America, including Brazilian biomes as Pantanal and Cerrado. Compared with other anurans from the Phyllomedusinae family, there are few reports on the bioactive potential of skin-derived molecules from this species. In this perspective, the aim of the present study was to evaluate the in vitro antibacterial activity of skin secretion of P. azurea by detection of Minimum Inhibitory Concentration (MIC) of the growth of bacterial indicator strains and to determine if occurs a changing in the bacterial cell envelope permeability. The MIC determination was carried out by the microdilution plate method. The absorbance was measured and analyzed statistically using the t-test to compare two groups (0.05 % of significance). The impact of the crude extract on cell envelope permeability of Staphylococcus aureus ATCC 25923 was conducted by the crystal violet assay, and the absorbance was measured spectrophotometry followed by the calculation of the crystal violet uptake percentage. The specific MIC for S. aureus ATCC 25923 and Escherichia coli ATCC 25922 was 31.25 µg/mL, while for Bacillus subtilis ATCC 6633 was 125 µg/mL and Pseudomonas aeruginosa ATCC 27853 was 250 µg/mL. The treatment with crescent concentrations of frog skin secretion increased the crystal violet uptake by S. aureus ATCC 25923 cells, suggesting an action on the cell plasma membrane. The results demonstrated that the skin secretion of P. azurea presents antibacterial activity and merit further investigations to characterize the bioactive molecules.(AU)

Resumen P. azurea es una especie de rana bien distribuida geográficamente en América del Sur, que incluye biomas brasileños como Pantanal y Cerrado. En comparación con otros anuros de Phyllomedusinae, existen pocos informes sobre el potencial bioactivo de las moléculas derivadas de la piel de esta especie. En esta perspectiva, el objetivo del presente estudio fue evaluar la actividad antibacteriana in vitro de la secreción de la piel de P. azurea mediante la detección de la Concentración Inhibitoria Mínima (CIM) del crecimiento de cepas indicadoras bacterianas y determinar si ocurre un cambio en la permeabilidad de la envoltura celular bacteriana. La determinación de MIC se llevó a cabo mediante el método de la placa de microdilución. La absorbancia se midió y se analizó estadísticamente mediante la prueba t para comparar dos grupos (0.05 de significancia). El impacto del extracto crudo sobre la permeabilidad de la envoltura celular de Staphylococcus aureus ATCC 25923 se realizó mediante el ensayo de cristal violeta, y se midió la absorbancia mediante espectrofotometría seguida del cálculo del porcentaje de absorción de violeta cristal. La CIM específica para S. aureus ATCC 25923 y Escherichia coli ATCC 25922 fue 31.25 μg / ml, mientras que para Bacillus subtilis ATCC 6633 de 125 μg / ml y Pseudomonas aeruginosa ATCC 27853 de 250 μg / ml. El tratamiento con concentraciones crecientes de secreción de piel de rana aumentó la absorción de violeta cristal por las células de S. aureus ATCC 25923, sugiriendo una acción sobre la membrana plasmática de la célula. Los resultados demostraron que la secreción de la piel de P. azurea presenta actividad antibacteriana y amerita más investigaciones para caracterizar las moléculas bioactivas.(AU)

Microbiota and skin defense peptides may facilitate coexistence of two sympatric Andean frog species with a lethal pathogen.

Management of hyper-virulent generalist pathogens is an emergent global challenge, yet for most disease systems we lack a basic understanding as to why some host species suffer mass mortalities, while others resist epizootics. We studied two sympatric species of frogs from the Colombian Andes, which coexist with the amphibian pathogen Batrachochytrium dendrobatidis (Bd), to understand why some species did not succumb to the infection. We found high Bd prevalence in juveniles for both species, yet infection intensities remained low. We also found that bacterial community composition and host defense peptides are specific to amphibian life stages. We detected abundant Bd-inhibitory skin bacteria across life stages and Bd-inhibitory defense peptides post-metamorphosis in both species. Bd-inhibitory bacteria were proportionally more abundant in adults of both species than in earlier developmental stages. We tested for activity of peptides against the skin microbiota and found that in general peptides did not negatively affect bacterial growth and in some instances facilitated growth. Our results suggest that symbiotic bacteria and antimicrobial peptides may be co-selected for, and that together they contribute to the ability of Andean amphibian species to coexist with the global pandemic lineage of Bd.

Non-tuberculous Mycobacterium species causing mycobacteriosis in farmed aquatic animals of South Africa.

BACKGROUND: Mycobacteriosis caused by non-tuberculous mycobacteria (NTM), is among the most chronic diseases of aquatic animals. In addition, fish mycobacteriosis has substantial economic consequences especially in the aquaculture and fisheries industry as infections may significantly decrease production and trade. Some fish NTM pathogens are highly virulent and zoonotic; as such, infection of aquaria with these pathogens is a public health concern. In this study, we report isolation of nine different NTM species from sixteen aquatic animals including different fish species, frogs and a crocodile. Given the clinical significance of Mycobacterium marinum and its close relation to Mycobacterium tuberculosis, as well as the significance of ESAT 6 and CFP-10 secretion in mycobacterial virulence, we analysed the esxA and esxB nucleotide sequences of M. marinum isolates identified in this study as well as other mycobacteria in the public databases. RESULTS: Mycobacterium shimoidei, Mycobacterium marinum, Mycobacterium chelonae, Mycobacterium septicum /M. peregrinum and Mycobacterium porcinum were isolated from gold fish, Guppy, exotic fish species in South Africa, koi and undefined fish, Knysna seahorse, as well Natal ghost frogs respectively, presenting tuberculosis like granuloma. Other NTM species were isolated from the studied aquatic animals without any visible lesions, and these include Mycobacterium sp. N845 T, Mycobacterium fortuitum, a member of the Mycobacterium avium complex, and Mycobacterium szulgai. Phylogenetic analysis of mycobacteria, based on esxA and esxB genes, separated slow growing from rapidly growing mycobacteria as well as pathogenic from non-pathogenic mycobacteria in some cases. CONCLUSIONS: Isolation of the different NTM species from samples presenting granuloma suggests the significance of these NTM species in causing mycobacteriosis in these aquatic animals. The study also revealed the potential of esxA and esxB sequences as markers for phylogenetic classification of mycobacteria. Observations regarding use of esxA and esxB sequences for prediction of potential pathogenicity of mycobacteria warrants further investigation of these two genes in a study employing NTM species with well-defined pathogenicity.

Mycobacterial Arthritis and Synovitis in Painted Reed Frogs (Hyperolius marmoratus).

Several species of atypical mycobacteria have been isolated from wild and captive amphibians. In captive anurans, cutaneous and visceral mycobacteriosis are common and can result in significant mortality, particularly when animals are immunocompromised. Mycobacterial arthritis and synovitis are reported rarely in amphibians. We describe 20 cases in painted reed frogs (Hyperolius marmoratus), which presented with cachexia, limb paresis or paralysis or 'spindly leg syndrome'. Histopathology revealed multifocal histiocytic to granulomatous synovitis affecting appendicular, rib or spinal intervertebral joints. Periarticular granulomata, granulomatous cellulitis and skeletal muscle atrophy, necrosis and degeneration were also present. In one case, granulomatous spinal osteomyelitis was recorded. Ziehl-Neelsen stains showed large numbers of acid-fast bacteria in macrophages and histiocytes. The mycobacterial isolates obtained from culture were identified as members of the Mycobacterium chelonae complex (either M. chelonae or Mycobacterium abscessus). This was confirmed by 5'-16S ribosomal ribonucleic acid (rRNA) sequencing. In 17 cases mycobacterial lesions were present only in the joints and skeleton, highlighting the importance of not ruling out mycobacterial infection on the basis of absence of cutaneous or visceral lesions.

A Brucella spp. Isolate from a Pac-Man Frog (Ceratophrys ornata) Reveals Characteristics Departing from Classical Brucellae.

Brucella are highly infectious bacterial pathogens responsible for brucellosis, a frequent worldwide zoonosis. The Brucella genus has recently expanded from 6 to 11 species, all of which were associated with mammals; The natural host range recently expanded to amphibians after some reports of atypical strains from frogs. Here we describe the first in depth phenotypic and genetic characterization of a Brucella strains isolated from a frog. Strain B13-0095 was isolated from a Pac-Man frog (Ceratophyrus ornate) at a veterinary hospital in Texas and was initially misidentified as Ochrobactrum anthropi. We found that B13-0095 belongs to a group of early-diverging brucellae that includes Brucella inopinata strain BO1 and the B. inopinata-like strain BO2, with traits that depart significantly from those of the "classical" Brucella spp. Analysis of B13-0095 genome sequence revealed several specific features that suggest that this isolate represents an intermediate between a soil associated ancestor and the host adapted "classical" species. Like strain BO2, B13-0095 does not possess the genes required to produce the perosamine based LPS found in classical Brucella, but has a set of genes that could encode a rhamnose based O-antigen. Despite this, B13-0095 has a very fast intracellular replication rate in both epithelial cells and macrophages. Finally, another major finding in this study is the bacterial motility observed for strains B13-0095, BO1, and BO2, which is remarkable for this bacterial genus. This study thus highlights several novel characteristics in strains belonging to an emerging group within the Brucella genus. Accurate identification tools for such atypical Brucella isolates and careful evaluation of their zoonotic potential, are urgently required.

Quantitative Proteomics of an Amphibian Pathogen, Batrachochytrium dendrobatidis, following Exposure to Thyroid Hormone.

Batrachochytrium dendrobatidis (Bd), a chytrid fungus, has increasingly been implicated as a major factor in the worldwide decline of amphibian populations. The fungus causes chytridiomycosis in susceptible species leading to massive die-offs of adult amphibians. Although Bd infects the keratinized mouthparts of tadpoles and negatively affects foraging behavior, these infections are non-lethal. An important morphogen controlling amphibian metamorphosis is thyroid hormone (T3). Tadpoles may be infected with Bd and the fungus may be exposed to T3 during metamorphosis. We hypothesize that exposure of Bd to T3 may induce the expression of factors associated with host colonization and pathogenicity. We utilized a proteomics approach to better understand the dynamics of the Bd-T3 interaction. Using liquid chromatography-mass spectrometry (LC-MS), we generated a data set of a large number of cytoplasmic and membrane proteins following exposure of Bd to T3. From these data, we identified a total of 263 proteins whose expression was significantly changed following T3 exposure. We provide evidence for expression of an array of proteins that may play key roles in both genomic and non-genomic actions of T3 in Bd. Additionally, our proteomics study shows an increase in several proteins including proteases and a class of uncommon crinkler and crinkler-like effector proteins suggesting their importance in Bd pathogenicity as well as those involved in metabolism and energy transfer, protein fate, transport and stress responses. This approach provides insights into the mechanistic basis of the Bd-amphibian interaction following T3 exposure.

Host-derived, pore-forming toxin-like protein and trefoil factor complex protects the host against microbial infection.

Aerolysins are virulence factors belonging to the bacterial ß-pore-forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. ßγ-CAT, a complex of an aerolysin-like protein subunit (two ßγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When ßγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1ß release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1ß was potently triggered by ßγ-CAT from the frog peritoneal cells and murine macrophages in vitro. ßγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1ß maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens.

The interactive effects of chytrid fungus, pesticides, and exposure timing on gray treefrog (Hyla versicolor) larvae.

Aquatic organisms are often exposed to a wide variety of perturbations in nature, including pathogens and chemical contaminants. Despite the co-occurrence of these 2 stressors, few studies have examined the effects of chemical contaminants on host-pathogen dynamics. The authors tested the individual and combined effects on gray treefrog (Hyla versicolor) tadpoles of 2 commonly used pesticides (Roundup® and Sevin®) and the pathogenic fungus Batrachochytrium dendrobatidis (Bd). A fully factorial design was used, and tadpoles were exposed to Bd, Roundup, or Sevin alone, or a combination of Bd and either pesticide at 3 points during larval development (early, mid, late). It was predicted that pesticides would mediate the effect of Bd on tadpoles and reduce the likelihood of negative consequences of infection and that timing of exposure would influence these effects. Tadpoles exposed to Bd at the mid point experienced higher survival through metamorphosis than those exposed to Bd at the early or late points, while tadpoles exposed to Sevin at the early point experienced reduced survival compared with those exposed to Roundup or no-pesticide control at the same exposure point. Roundup ameliorated the effects of Bd on survival compared with tadpoles exposed to Bd alone, while there was no interactive effect of Sevin on survival. In addition, Sevin reduced mass of new metamorphs compared with Roundup and reduced snout-vent length compared with all other treatments. The present study supports the hypothesis that pesticides can mitigate the effects of Bd on amphibian hosts and that such effects may depend on the timing of exposure.

Body length of Hylodes cf. ornatus and Lithobates catesbeianus tadpoles, depigmentation of mouthparts, and presence of Batrachochytrium dendrobatidis are related / Tamanho do corpo, despigmentação das partes bucais e presença de Batrachochytrium dendrobatidis estão relacionados em Hylodes cf. ornatus e Lithobates catesbeianus

A fungal pathogen Batrachochytrium dendrobatidis (Bd), which can cause morbidity and death of anurans, has affected amphibian populations on a worldwide basis. Availability of pure cultures of Bd isolates is essential for experimental studies to understand the ecology of this pathogen. We evaluated the relationships of body length of Hylodes cf. ornatus and Lithobates catesbeianus tadpoles to depigmentation of mouthparts and determined if dekeratinization indicated an infection by Batrachochytrium dendrobatidis. A strong association existed for both species, one from South America (Brazil: São Paulo) and one from North America (USA: Maine). We believe it prudent not to kill adult amphibians if avoidable, thus obtaining tissue for isolating Bd from tadpoles is reasonable because infected specimens of some species can be selectively collected based on depigmentation of mouthparts.

O fungo patógeno Batrachochytrium dendrobatidis (Bd) é apontado como o causador de morbidade e morte em anuros, e tem afetado populações de anfíbios em uma base mundial. Avaliar culturas puras de isolados de Bd é essencial para estudos experimentais para o entendimento da ecologia desse patógeno. Avaliou-se a relação entre o comprimento do corpo em girinos de Hylodes cf. ornatus e Lithobates catesbeianus com a despigmentação das peças bucais, para verificar se a desqueratinização indica uma infecção por Batrachochytrium dendrobatidis. Uma forte associação existe para ambas as espécies, uma da América do Sul (Brasil: São Paulo) e uma da América do Norte (USA: Maine). Acredita-se ser prudente este uso, para evitar a morte de anfíbios adultos; dessa forma, obter tecidos para isolar o Bd de girinos é razoável, porque espécimes infectados podem ser coletados seletivamente com base na despigmentação do aparelho bucal.

Post-secretory events alter the peptide content of the skin secretion of Hypsiboas raniceps.

A novel family of antimicrobial peptides, named raniseptins, has been characterized from the skin secretion of the anuran Hypsiboas raniceps. Nine cDNA molecules have been successfully cloned, sequenced, and their respective polypeptides were characterized by mass spectrometry and Edman degradation. The encoded precursors share structural similarities with the dermaseptin prepropeptides from the Phyllomedusinae subfamily and the mature 28-29 residue long peptides undergo further proteolytic cleavage in the crude secretion yielding consistent fragments of 14-15 residues. The biological assays performed demonstrated that the Rsp-1 peptide has antimicrobial activity against different bacterial strains without significant lytic effect against human erythrocytes, whereas the peptide fragments generated by endoproteolysis show limited antibiotic potency. MALDI imaging mass spectrometry in situ studies have demonstrated that the mature raniseptin peptides are in fact secreted as intact molecules within a defined glandular domain of the dorsal skin, challenging the physiological role of the observed raniseptin fragments, identified only as part of the crude secretion. In this sense, stored and secreted antimicrobial peptides may confer distinct protective roles to the frog.

Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis is a fungus belonging to the Phylum Chytridiomycota, Class Chytridiomycetes, Order Chytridiales, and is the highly infectious aetiological agent responsible for a potentially fatal disease, chytridiomycosis, which is currently decimating many of the world's amphibian populations. The fungus infects 2 amphibian orders (Anura and Caudata), 14 families and at least 200 species and is responsible for at least 1 species extinction. Whilst the origin of the agent and routes of transmission are being debated, it has been recognised that successful management of the disease will require effective sampling regimes and detection assays. We have developed a range of unique sampling protocols together with diagnostic assays for the detection of B. dendrobatidis in both living and deceased tadpoles and adults. Here, we formally present our data and discuss them in respect to assay sensitivity, specificity, repeatability and reproducibility. We suggest that compliance with the recommended protocols will avoid the generation of spurious results, thereby providing the international scientific and regulatory community with a set of validated procedures which will assist in the successful management of chytridiomycosis in the future.

Survival of three species of anuran metamorphs exposed to UV-B radiation and the pathogenic fungus Batrachochytrium dendrobatidis.

When exploring the possible factors contributing to population declines, it is necessary to consider multiple, interacting environmental stressors. Here, we investigate the impact of 2 factors, ultraviolet radiation and disease, on the survival of anuran amphibians. Exposure to ultraviolet-B (UV-B) radiation increases mortality and results in various sub-lethal effects for many amphibian species. Infectious diseases can also negatively impact amphibian populations. In this study, we exposed metamorphic individuals (metamorphs) to both UV-B and Batrachochytrium dendrobatidis (BD), a fungal pathogen and cause of the disease chytridiomycosis, and monitored survival for 3 wk. We tested for possible interactions between UV-B and BD in 3 species: the Cascades frog Rana cascadae; the Western toad Bufo boreas; and the Pacific treefrog Hyla regilla. We found strong interspecific differences in susceptibility to BD. For example, R. cascadae suffered a large increase in mortality when exposed to BD; B. boreas also experienced mortality, but this effect was small relative to the R. cascadae response. H. regilla did not show any decrease in survival when exposed to either factor. No synergistic interactions between UV-B and BD were found for any of the test species. A previous study investigating the impact of BD on larval amphibians showed different species responses (Blaustein et al. 2005a). Our results highlight the importance of studying multiple life history stages when determining the impact of environmental stressors. The contrast between these 2 studies emphasizes how vulnerability to a pathogen can vary between life history stages within a single species.

Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals.

Members of most Chryseobacterium species occur in aquatic environments or food products, while strains of some other species are pathogenic to humans and animals. A collection of 52 Chryseobacterium sp. strains isolated from diseased fish, one frog isolate and 22 reference strains were included in a polyphasic taxonomy study. Fourteen clusters of strains were delineated following the comparison of whole-cell protein profiles. Most of these clusters were confirmed when the phenotypic and RAPD profiles and the 16S rRNA gene sequences were compared. Fatty acid composition helped differentiate the Chryseobacterium strains from members of related genera. None of the fish isolates could be allocated to the two species previously reported from fish but two isolates belonged to C. joostei, while the frog isolate was identified as Elizabethkingia meningoseptica, a human pathogen previously included in the genus Chryseobacterium. Three clusters grouping from 3 to 13 isolates will probably constitute the core of new Chryseobacterium species but all other isolates occupied separate or uncertain positions in the genus. This study further demonstrated the overall high similarity displayed by most Chryseobacterium strains whatever the technique used and the resulting difficulty in delineating new species in the genus. Members of this bacterial group should be considered potential emergent pathogens in various fish and frog species, farming conditions and geographical areas.

Possible differences in pathogenicity between cane toad-, frog- and platypus-derived isolates of Mucor amphibiorum, and a platypus-derived isolate of Mucor circinelloides.

Platypuses (Ornithorhynchus anatinus) in the north of the island state of Tasmania, Australia, suffer from a serious disease called ulcerative mycosis, which is responsible for high morbidity and, presumably, mortality rates in areas where it occurs. The disease is caused by the dimorphic fungus Mucor amphibiorum, which is also found in Queensland, New South Wales and Victoria. However, it does not cause disease in platypuses in those states. It has been previously reported that a closely related fungus, Mucor circinelloides, may also be capable of causing this disease. This paper describes pathogenicity trials involving cane toads (Bufo marinus) as the experimental model. The toads were infected with either Tasmanian, platypus-derived M. amphibiorum, West Australian, frog-derived M. amphibiorum, Queensland cane-toad-derived M. amphibiorum or Tasmanian platypus-derived M. circinelloides. The Tasmanian isolates of M. amphibiorum were more likely to cause a serious, long-term infection than were Queensland or West Australian isolates, and (+) mating types caused a more serious infection than the (-) mating type. The isolate of M. circinelloides was incapable of infecting the toads, lending further weight to the theory that it represents an environmental contaminant. The results suggest that an endemic strain of M. amphibiorum has mutated and become pathogenic to platypuses. Alternatively, a pathogenic strain of M. amphibiorum may have been introduced into Tasmania, where it is infecting a naïve population.

Factors associated with the catastrophic decline of a cloudforest frog fauna in Guatemala

Comparison of recent and historical surveys of frog populations in cloudforest habitat in Sierra de las Minas,Guatemala,indicated population declines and local extirpation of several species.Pathological exams of diseased tadpoles indicated infection by amphibian chytridiomycosis. The local habitat has been severely altered by recent establishment of large-scale leatherleaf fern production.Analysis of water chemistry at our study site suggested increased nitrogenation associated with the leatherleaf industry

Una comparación entre un inventario anterior y otro reciente de poblaciones de ranas de bosque nublado en la Sierra de Las Minas de Guatemala demostró disminuciones poblacionales y ausencia localizada de varias especies.El examen patológico de un renacuajo muerto indicó infección por un hongo quítrido propio de los anfibios.El hábitat local ha sido gravemente alterado por el establecimiento reciente de producción a gran escala de helechos ornamentales.El análisis químico del agua en el área de estudio señaló un aumento en nitrogenación asociado al cultivo de helechos

Bacterial pathogens isolated from cultured bullfrogs (Rana castesbeiana).

A commercial bullfrog (Rana castesbeiana) operation in south Georgia had multiple epizootics of systemic bacterial infections over a 3-year period, 1998-2000. A number of potential pathogens (Aeromonas hydrophila, Chryseobacterium (Flavobacterium) meningosepticum, Chryseobacterium (Flavobacterium) indolgenes, Edwardsiella tarda, Citrobacterfreundii, Pseudomonas spp., and (Streptococcus iniae) were isolated from various tissues. Clinically, frogs demonstrated acute onset of torticolis, stupor, and indifference to stimuli. Cutaneous hyperemia, subcutaneous and muscular hemorrhage, and peripheral edema were consistent gross findings. Histologically, clusters of lymphocytes, monocytes, and occasional acidophiles with scattered granulomas occurred in liver, kidney, and spleen. This is the first report of S. inae and C. meningosepticum as potential disease agents in R. castesbeiana. These findings suggest that a variety of bacteria may be associated with redleg and that culture results must be obtained for accurate diagnosis.