Experimental exposure of Canadian toads to Basidiobolus ranarum

Experimental transmission of the fungus Basidiobolus ranarum was induced in two treatment groups of Canadian toads (Bufo hemiophrys) and caused a fatal mycotic dermatitis. Seven of 10 (70%) toads that had their ventral skin mildly abraded and exposed to B. ranarum developed hyperemia, and sloughing of their ventral skin and died. Toads with abraded ventral skin or exposure to infected skin also were affected statistically at a higher rate than those with abraded skin and exposure to pure cultures of B. ranarum inoculated into their water source. Of toads showing clinical disease, B. ranarum was identified by both impression smears and histology in all cases, but not from toads that appeared clinically healthy. The organism was cultured from 5 of 7 (71%) toads with clinical disease but not from any toad that appeared clinically healthy (n = 28). This study documents methods of experimental transmission of B. ranarum, an organism responsible for causing a mycotic dermatitis that is fatal to toads.     

Mortality of captive Canadian toads from Basidiobolus ranarum mycotic dermatitis

Twenty-six adult free-ranging Canadian toads (Bufo hemiophrys) were collected from northeastern North Dakota (USA) during the last week of August 1994 and placed in captivity. During late December and January 1995, 21 Canadian toads died. Clinical signs included increased time sitting in water bowls, darkened dorsal skin, constant arching of their backs, and hyperemia and sloughing of ventral epidermis. The condition progressively worsened until death occurred within 5 to 7 days after onset of clinical disease. Mycotic dermatitis due to Basidiobolus ranarum was diagnosed in all toads and the fungus was isolated from 11 (52%) of 21 toads. Histology of the ventral skin and digits revealed numerous fungal spherules and occasional hyphae without significant inflammatory reaction. This condition clinically resembled red leg associated with Aeromonas hydrophila and many other bacterial organisms, and the diseases could be confused without appropriate diagnostic tests. This also is the first report of B. ranarum causing clinical disease in a toad species.     

Causes of mortality of the Wyoming toad

Wyoming toads (Bufo baxteri) that died from January 1989 to June 1996 were submitted to the Wyoming State Veterinary Laboratory (Laramie, Wyoming, USA) for postmortem evaluation. These consisted of 108 free-ranging toads and 170 animals from six captive populations. Ninety-seven (90%) of 108 free-ranging toad carcasses were submitted during September and October. From 1989 to 1992, 27 (77%) of 35 mortalities in the captive populations occurred in October, November, and December. From 1993 to 1996, mortality in captive toads occurred without a seasonal pattern and coincided with changes in hibernation protocols that no longer mimicked natural cycles. Cause of mortality was determined in 147 (53%) of the 278 cases. Mycotic dermatitis with secondary bacterial septicemia was the most frequent diagnosis in 104 (71%) of 147 toads. Basidiobolus ranarum was found by microscopic examination of skin sections in 100 (96%) of 104 of these mortalities. This fungus was isolated from 30 (56%) of 54 free-ranging and 24 (48%) of 50 captive toads. This research documents the causes of mortality for both free-ranging and captive endangered Wyoming toads over a 7 yr period.     

Mucormycotic dermatitis in captive adult Wyoming toads

During late May 1995, 50 adult captive endangered Wyoming toads (Bufo baxteri) were brought out of hibernation. Approximately 3 to 10 days after hibernation emergence, all toads were hormonally induced to breed, and paired. Each pair was placed in their own breeding tank. Four toads developed clinical signs of disease which included lethargy and multiple (4 to 12) small (2 mm) raised hyperemic nodules with white fuzzy caps on the ventral skin. The condition progressively worsened until death occurred, within 3 to 6 days. Mycotic dermatitis caused by Mucor sp. was diagnosed in the four toads through histology and isolation of the organism. This is the first case report of a Mucor sp. causing a fatal dermatitis in an amphibian without significant inflammatory response and without systemic involvement.     

茄病镰刀菌致ICR小鼠局部皮肤感染的实验研究

目的建立茄病镰刀菌感染皮肤的ICR小鼠模型。方法将茄病镰刀菌三个不同浓度的菌悬液分别接种于正常和免疫抑制ICR小鼠受损及正常未受损的皮肤,于接种后第7、14、21、28天处死动物,取皮疹和各脏器进行真菌镜检、培养和组织病理学检查。结果正常和免疫抑制组皮肤受损的小鼠均出现皮疹,而免疫抑制组皮疹程度重,持续时间长,且真菌镜检、培养及组织病理学大都可见菌丝。皮肤未受损的小鼠不发生皮疹。实验小鼠均未出现系统播散。结论接种109CFU/mL或1010CFU/mL茄病镰刀菌的孢子悬液于免疫抑制ICR小鼠擦伤的皮肤,可有效造成ICR小鼠的茄病镰刀菌皮肤感染。     

Prevalence of infection by Batrachochytrium dendrobatidis and Ranavirus in eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) in eastern Tennessee

Hellbenders (n=97) were collected from the Little and Hiwassee Rivers in eastern Tennessee, USA, during 2009 and 2010. Location and morphometrics for each animal were recorded, and nonlethal tissue samples were collected to estimate the prevalence of infection with Batrachochytrium dendrobatidis (Bd) and Ranavirus in each watershed and year. Real-time polymerase chain reaction was performed on skin swabs for Bd and on tail clips for ranaviruses. Overall prevalences of DNA of Bd, Ranavirus, and coinfections (i.e., detectable DNA of both pathogens in the same individual) were 26%, 19%, and 5%, respectively. Differences in infection prevalence were detected between watersheds and years. Gross lesions were observed in 31 animals (32%), but the types of lesions were not consistent with chytridiomycosis or ranaviral disease. This is the first report of infection of eastern hellbenders with Bd and Ranavirus. Despite infection by both pathogens, it is unclear whether chytridiomycosis or ranaviral disease develops in wild populations of hellbenders. More research is needed to determine the susceptibility of hellbenders to Bd and ranaviruses and their role in the epidemiology of these pathogens.     

Ranavirus infection of free-ranging and captive box turtles and tortoises in the United States

Iridoviruses of the genus Ranavirus are well known for causing mass mortality events of fish and amphibians with sporadic reports of infection in reptiles. This article describes five instances of Ranavirus infection in chelonians between 2003 and 2005 in Georgia, Florida, New York, and Pennsylvania, USA. Affected species included captive Burmese star tortoises (Geochelone platynota), a free-ranging gopher tortoise (Gopherus polyphemus), free-ranging eastern box turtles (Terrapene carolina carolina), and a Florida box turtle (Terrepene carolina bauri). Evidence for Ranavirus infection was also found in archived material from previously unexplained mass mortality events of eastern box turtles from Georgia in 1991 and from Texas in 1998. Consistent lesions in affected animals included necrotizing stomatitis and/or esophagitis, fibrinous and necrotizing splenitis, and multicentric fibrinoid vasculitis. Intracytoplasmic inclusion bodies were rarely observed in affected tissues. A portion of the major capsid protein (MCP) gene was sequenced from each case in 2003-2005 and found to be identical to each other and to Frog virus 3 (FV3) across 420 base pairs. Ranavirus infections were also documented in sympatric species of amphibians at two locations with infected chelonians. The fragment profiles of HindIII-digested whole genomic DNA of Ranavirus, isolated from a dead Burmese star tortoise and a southern leopard frog (Rana utricularia) found nearby, were similar. The box turtle isolate had a low molecular weight fragment that was not seen in the digestion profiles for the other isolates. These results suggest that certain amphibians and chelonians are infected with a similar virus and that different viruses exist among different chelonians. Amphibians may serve as a reservoir host for susceptible chelonians. This report also demonstrated that significant disease associated with Ranavirus infections are likely more widespread in chelonians than previously suspected.     

Fungal root endophytes from natural vegetation in Mediterranean environments with special reference to Fusarium spp

Surveys (in 2002 and 2003) were performed for fungal endophytes in roots of 24 plant species growing at 12 sites (coastal and inland soils, both sandy soils and salt marshes) under either water or salt stress in the Alicante province (Southeast Spain). All plant species examined were colonized by endophytic fungi. A total of 1830 fungal isolates were obtained and identified by morphological and molecular [internal transcribed spacer (ITS) and translation elongation factor-1alpha gene region (TEF-1alpha) sequencing] techniques. One hundred and forty-two fungal species were identified, belonging to 57 genera. Sterile mycelia were assigned to 177 morphospecies. Fusarium and Phoma species were the most frequent genera, followed by Aspergillus, Alternaria and Acremonium. Fungal root endophytic communities were influenced by the soil type where their respective host plants grew, but not by location (coastal or inland sites). Fusarium oxysporum, Aspergillus fumigatus and Alternaria chlamydospora contributed most to the differences found between endophytic communities from sandy and saline soils. Host preference was found for three Fusarium species studied. Fusarium oxysporum and Fusarium solani were especially isolated from plants of the family Leguminosae, while Fusarium equiseti showed a preference for Lygeum spartum (Gramineae). In some cases, specificity could be related to intra-specific variability as shown by sequencing of the TEF-1alpha in the genus Fusarium.     

Context-dependent effects of ranaviral infection on northern leopard frog life history traits

Pathogens have important effects on host life-history traits, but the magnitude of these effects is often strongly context-dependent. The outcome of an interaction between a host and an infectious agent is often associated with the level of stress experienced by the host. Ranavirus causes disease and mortality in amphibian populations in various locations around the world, but most known cases of ranaviral infection have occurred in North America and the United Kingdom. While Ranavirus virulence has been investigated, the outcome of Ranavirus infection has seldom been related to the host environment. In a factorial experiment, we exposed Northern leopard frog (Lithobates pipiens, formerly Rana pipiens) tadpoles to different concentrations of Ranavirus and investigated the effect of host density on certain life-history traits, namely survival, growth rate, developmental stage and number of days from virus exposure to death. Our results suggest a prominent role of density in driving the direction of the interaction between L. pipiens tadpoles and Ranavirus. We showed that increasing animal holding density is detrimental for host fitness as mortality rate is higher, day of death earlier, development longer and growth rate significantly lower in high-density tanks. We observed a linear increase of detrimental effects when Ranavirus doses increased in low-density conditions, with control tadpoles having a significantly higher overall relative fitness. However, this pattern was no longer observed in high-density conditions, where the effects of increasing Ranavirus dose were limited. Infected and control animals fitness were consequently similar. We speculate that the host may eventually diverts the energy required for a metabolic/immune response triggered by the infection (i.e., direct costs of the infection) to better cope with the increase in environmental "stress" associated with high density (i.e., indirect benefits of the infection). Our results illustrate how the net fitness of organisms may be shaped by ecological context and emphasize the necessity of examining the direct/indirect costs and benefits balance to fully understand host-pathogen interactions.     

Variation in the effects of antidiuretic hormone on the isolated skin of the toad, Scaphiopus couchi.

The rate of active sodium transport as measured by short-circuit current across the isolated skin of the toad, Scaphiopus couchi, was elevated following vasopressin (0.2 units/ml) or arginine vasotocin (0.1 units/ml) treatment of skins from active animals at all times of the year tested. Skins from dormant animals showed no such elevation at any time of the year. The rate of active sodium transport was elevated following treatment with dibutyryl cyclic AMP (2.5mM) plus theophylline (10 mM) in all skins tested. The hydraulic conductivity of isolated skins from both active and dormant animals showed no significant change following treatment with vasopressin (0.2 units/ml) or arginine vasotocin (0.1 units/ml except on the first day following emergence from dormancy in the field. A correlation was, therefore, observed between the occurrence of a hydroosmotic response to antidiuretic hormones and the seasonal exposure of S. couchi to standing water. A small but significant elevation of hydraulic conductivity was observed across the skins of dormant toads following treatment with dibutyryl cyclic AMP (2.5 mM) plus theophylline (10 mM) whereas a substantial elevation was observed with the skins of active animals.     

浅部真菌感染和变态反应性皮肤病相关性研究

目的 通过比较合并与未合并浅部真菌感染的变态反应性皮肤病对常用变应原的敏感性,综合从皮肤或指(趾)甲中分离出的菌种情况,评估浅部真菌感染在变态反应性皮肤病的病因学中的作用.方法 受试者包括353例慢性荨麻疹、湿疹及特应性皮炎患者.通过真菌直接镜检法将受试者分为两组.实验组:变态反应性皮肤病合并浅部真菌感染组(n =173);对照组:变态反应性皮肤病无浅部真菌感染组(n=180).对所有实验组及对照组受试者进行9种真菌变应原和9种非真菌变应原皮内试验.实验组患者进一步进行真菌培养以鉴定菌种.结果 慢性荨麻疹患者实验组须发癣菌、新月弯孢霉,特异青霉、烟曲霉变应原阳性率显著高于对照组(P＜0.05),慢性湿疹患者实验组须发癣菌变应原阳性率显著高于对照组(P ＜0.001).慢性湿疹、荨麻疹患者其他真菌变应原及粉尘螨、屋尘螨等非真菌变应原阳性率比较差异均无统计学意义(P＞0.05).134例患者皮肤或指(趾)甲分离鉴定主要为红色毛癣菌(52.86％)、须癣毛癣菌(14.18％)、絮状表皮癣菌(5.22％)、白念珠菌(6.72％),实验组须发癣菌变应原阳性率及皮肤分离皮肤癣菌阳性率比较差异无统计学意义(P＞0.05).结论 实验结果表明,须发癣菌变应原阳性的慢性荨麻疹、湿疹患者往往合并皮肤癣菌感染,皮肤癣菌感染可能在部分慢性荨麻疹、湿疹的病因学中起重要作用.     

Distribution and Pathogenicity of Batrachochytrium dendrobatidis in Boreal Toads from the Grand Teton Area of Western Wyoming

The pathogen Batrachochytrium dendrobatidis (Bd), which causes the skin disease chytridiomycosis, has been linked to amphibian population declines and extinctions worldwide. Bd has been implicated in recent declines of boreal toads, Bufo boreas boreas, in Colorado but populations of boreal toads in western Wyoming have high prevalence of Bd without suffering catastrophic mortality. In a field and laboratory study, we investigated the prevalence of Bd in boreal toads from the Grand Teton ecosystem (GRTE) in Wyoming and tested the pathogenicity of Bd to these toads in several environments. The pathogen was present in breeding adults at all 10 sites sampled, with a mean prevalence of 67%. In an experiment with juvenile toadlets housed individually in wet environments, 10⁶ zoospores of Bd isolated from GRTE caused lethal disease in all Wyoming and Colorado animals within 35 days. Survival time was longer in toadlets from Wyoming than Colorado and in toadlets spending more time in dry sites. In a second trial involving Colorado toadlets exposed to 35% fewer Bd zoospores, infection peaked and subsided over 68 days with no lethal chytridiomycosis in any treatment. However, compared with drier aquaria with dry refuges, Bd infection intensity was 41% higher in more humid aquaria and 81% higher without dry refuges available. Our findings suggest that although widely infected in nature, Wyoming toads may escape chytridiomycosis due to a slight advantage in innate resistance or because their native habitat hinders Bd growth or provides more opportunities to reduce pathogen loads behaviorally than in Colorado.     

RNA silencing of mycotoxin production in Aspergillus and Fusarium species

Mycotoxins are natural fungal products that are defined by their harmful effects on humans and animals. Aflatoxin contamination of maize by Aspergillus species and trichothecene contamination of small grains by Fusarium species are two of the most severe mycotoxin problems in the United States. We are investigating RNA silencing in an effort to identify novel ways to control mycotoxin contamination of crops. Transformation of two Aspergilli (A. flavus and A. parasiticus) and a Fusarium (F. graminearum) with inverted repeat transgenes (IRT) containing sequences of mycotoxin-specific regulatory genes suppressed mycotoxin production in all three plant-pathogenic fungi. This atoxigenic phenotype was stable during infection on corn and wheat, and importantly, F. graminearum IRT strains were less virulent on wheat than were wild type. The IRT did not alter physiological characteristics of the fungi, such as spore production and growth rate on solid media. These results indicate that RNA silencing exists in Aspergillus and Fusarium plant pathogens and suggest that RNA silencing technology may be a useful tool for eliminating mycotoxin contamination of agricultural products.     

Cutaneous blood flow and water absorption by dehydrated toads

Blood cell flux (BCF) in ventral pelvic skin capillaries was measured in toads, Bufo woodhouseii and Bufo punctatus, using a chamber that allowed hydration behavior and water absorption to be observed concurrently in unrestrained animals. Dehydrated B. woodhouseii and B. punctatus placed on a rehydration solution significantly increased BCF relative to that on a dry surface in less than 2 min. Skin contact with a rehydration solution rather than dehydration alone is the primary stimulus for increased seat patch blood flow. In B. woodhouseii, the water absorption response was initiated after the increase in BCF had started but before maximum BCF was reached. BCF and water uptake across the ventral skin of both species placed on deionized water were not different from those of toads placed on 50 mM NaCl. Similarly, no significant correlation between BCF and rate of water uptake could be observed in dehydrated toads of either species. Angiotensin II (AII) injection in hydrated B. punctatus had no effect on BCF, suggesting that factors other than AII are responsible for the increase in blood flow upon water contact in dehydrated toads.     

Tectal responses to potassium loads and subsequent visual stimuli in the toad, Bufo bufo

Calling male toads were tested behaviourally for their prey catching responses to wormlike stimuli and assigned to groups of non-hungry and hungry depending on their prey catching motivation before being prepared for visual unit, massed unit and slow potential shift (SPS) recording from the optic tectum. Control recordings to visual stimuli were made before recording the effects of application of isotonic solutions containing concentrations of 0-41 mM K(+). Application of solution was followed by presentation of the visual stimulus while the solution still bathed the tectum. The best tectal responses were made to large square visual stimuli in the non-hungry toads, perhaps because recordings were made in the breeding season. Responses of the tectum to solution addition were significant in the concentration range of 7-17 mM K(+). Hungry toads showed an earlier, smaller response than non-hungry (sexually motivated) animals. When the visual stimulus was presented, there were unit and massed unit responses at all bathing solution concentrations, which were larger in non-hungry animals. These experiments revealed that toads motivated to feed respond earlier than non-hungry toads to application of artificial CSF to the tectum, though non-hungry toads responded best to the subsequent visual stimulus.     

Massive fungal contamination in animal care facilities traced to bedding supply.

During the course of immunologic studies involving the gastrointestinal colonization of mice with Candida albicans, it became apparent that the animals were being exposed to large numbers of Aspergillus fumigatus spores which interfered with the C. albicans colonization. To determine the source of the A. fumigatus exposure and the extent of fungal contamination of the medical school vivarium and four satellite facilities, fungal analyses of feed, bedding, and air were undertaken. Initial samples from the air were collected with 3-h settle plates; air sampling following cleanup was done with an Anderson air sampler. The source of contamination in the mouse rooms was determined to be Beta Chip bedding, which came from the manufacturer highly contaminated. Beta Chip bedding (1 g) obtained from the manufacturer just prior to testing contained 10(4) CFU of A. fumigatus, 20 CFU of a zygomycete, and 10 CFU of a Penicillium sp. Coarse-grade Beta Chip had approximately one-half those levels of contamination. Pure Cob bedding was highly contaminated also, but with a Fusarium sp. and a Cladosporium sp. Untreated and heat-treated Sani-Chip as well as all other heat-treated preparations obtained from the manufacturer contained no detectable spores. Rodent chow direct from the manufacturer had no A. fumigatus, although it did contain 150 CFU of fungus per g, including 80 CFU of a Rhodotorula sp., 60 CFU of Cryptococcus uniguttulatus, and 1 CFU of a Penicillium sp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the dry-cold season dormancy on the tonic and phasic neural control of heart rate in the toad, Bufo paracnemis

This work examined basal heart rate and autonomic cardiac tone as well as sympathetic cardiac reactivity to hypotension induced by systemic nitroprusside injection in dormant toads (dry-cold season), Bufo paracnemis, comparing the values with those of toads collected during the active months (hot-rainy season). Autonomic tone was calculated according to the method of Lin and Horwath ('72), which allows its evaluation as a percentage of intrinsic heart rate. Specimens were maintained in an outdoors terrarium except for the week preceding surgery, when they were transferred to indoor nonclimatized tanks. The heart rate of dormant toads (27.8 +/- 2.7 beats/min) was lower than that of active toads at rest (38.6 +/- 2. 3). Cholinergic tone was higher than adrenergic tone both in active (26.2% and 7.8%, respectively) and aestivated (19.5% and 4.8%, respectively) animals. Thus, cholinergic tone and adrenergic tone were both lower in dormant animals. The reflex tachycardia elicited by nitroprusside-induced hypotension was lower in aestivated toads (9.3 +/- 0.9 beats/min) when compared to active toads (19.9 +/- 1.0), indicating a reduced sympathetic reactivity. Nitroprusside-induced hypotensions were not different in the two groups. We conclude that at rest Bufo paracnemis heart is under the influence of a double cholinergic and adrenergic tone, and that both influences, as well as the reflex adrenergic reactivity to the unloading produced by nitroprusside-induced hypotension, are reduced in aestivated toads.     

The effect of salt acclimation of the water uptake and osmotic permeability of the skin of the toad (Bufo viridis, L.)

1. Water uptake in vivo, and water fluxes across the isolated skin were studied in salt (NaCl) acclimated toads. 2. Water uptake of acclimated toads maintained in the solution of acclimation, decreased with the environmental salinity. 3. The osmotic water permeability (Pos) of the skin increased upon salt (NaCl) acclimation, both in vivo and in vitro. 4. Pos of the skin of toads acclimated to non-permeant solutes such as sucrose (230 mmol/l) or mannitol (400 nmol/l), was greatly reduced. 5. Oxytocin (syntocinon) increased the Pos both in tap water and salt acclimated toads. In high salt (greater than 200 mmol/l NaCl) acclimated toads however, the increased Pos and water flux at larger osmotic gradients, could not be stimulated further by the hormone. 6. The adaptive nature of the selective changes in the permeability properties of the skin under salt acclimation conditions is discussed.     

PCR detection assays for the trichothecene-producing species Fusarium graminearum, Fusarium culmorum, Fusarium poae, Fusarium equiseti and Fusarium sporotrichioides

Contamination of small-grain cereals with the fungal species Fusarium graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti is an important source of trichothecenes, Zearalenone and other mycotoxins which cause serious diseases in human and animals. Additionally, these species contribute to Fusarium Head Blight, a disease which produces important losses in cereal yield. Early detection and control of these Fusarium species is crucial to prevent toxins entering the food chain and a useful tool in disease management practices. We describe the development of specific PCR assays to F. graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti using DNA from pure fungal cultures as well as from naturally infected wheat seeds, using in this case a rapid and easy protocol for DNA isolation. The specific primers were designed on the basis of IGS sequences (Intergenic Spacer of rDNA), a multicopy region in the genome that permits to enhance the sensitivity of the assay in comparison with PCR assays based on single-copy sequences.