Chytridiomycosis in amphibians--first report in Europe

Declining of amphibian populations is a worldwide phenomenon. A cutaneous mycosis as a cause of death in free-living amphibians as well as in captive ones due to an chytrid fungus (Batrachochytrium dendrobatidis) was reported at first in 1998. This infections were reported hitherto from Australia, North, Central and South America. This is the first report on chytrid infections in captive anurans from Europe. Dendrobates auratus and D. pumilo imported from Costa Rica and P. vittatus imported from French Guayana died with chytridiomycosis within a week after arrival in Europe. Batrachocytrium was also found on captive bred frogs in Germany and Belgium. Clinical signs, diagnosis and conclusions for protecting free-living amphibian populations and captive frogs are discussed.     

Chytridiomycosis in an aquarium collection of frogs: diagnosis, treatment, and control

The introduction of a new group of dendrobatid frogs to an established captive amphibian collection was followed by several acute mortalities in both resident and introduced frog populations. Chytridiomycosis, caused by Batrachochytrium dendrobatidis, was diagnosed by histology in two of the dead frogs. Following the diagnosis, all amphibians were moved to a specially made quarantine room with strict handling protocols and treated with itraconazole. Frogs, being terrestrial amphibians, were treated with itraconazole (Sporanox, 10 mg/ml) at 0.01% in 0.6% saline in a 5-min bath for 11 consecutive days. Axolotls (Ambystoma mexicanum) and Kaup's caecilians (Potymotyphlus kaupii), being aquatic amphibians, were treated with itraconazole administered directly in their primary tank water to achieve a concentration of 0.01% for 30 min every 5 days for four treatments. Itraconazole was removed from the tank water after 30 min by high-rate-of-flow activated charcoal filters. The treatment and quarantine procedures were successful in eradicating the disease. The few amphibian mortalities that occurred in the 18 mo after the start of the treatment have been histologically negative for the presence of chytrid fungi. The collection is now considered free of chytridiomycosis.     

Chloramphenicol with fluid and electrolyte therapy cures terminally ill green tree frogs (Litoria caerulea) with chytridiomycosis

Terminal changes in frogs infected with the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd) include epidermal degeneration leading to inhibited epidermal electrolyte transport, systemic electrolyte disturbances, and asystolic cardiac arrest. There are few reports of successful treatment of chytridiomycosis and none that include curing amphibians with severe disease. Three terminally ill green tree frogs (Litoria caerulea) with heavy Bd infections were cured using a combination of continuous shallow immersion in 20 mg/L chloramphenicol solution for 14 days, parenteral isotonic electrolyte fluid therapy for 6 days, and increased ambient temperature to 28 degrees C for 14 days. All terminally ill frogs recovered rapidly to normal activity levels and appetite within 5 days of commencing treatment. In contrast, five untreated terminally ill L. caerulea with heavy Bd infections died within 24-48 hr of becoming moribund. Subclinical infections in 15 experimentally infected L. caerulea were cured within 28 days by continuous shallow immersion in 20 mg/L chloramphenicol solution without adverse effects. This is the first known report of a clinical treatment protocol for curing terminally ill Bd-infected frogs.     

A survey of Blastocystis infection in anuran and urodele amphibians

Blastocystis infection in amphibians was surveyed in three species of anuran and one species of urodele amphibians captured at two distinct locations in Japan. All three species of frogs were highly infected with Blastocystis, while 69 individual urodele newts, Cynopus pyrrhogaster, were negative for infection. Eleven Blastocystis isolates (47.8%) were recovered from 23 Rana nigromaculata leopard frogs. Twenty-three (92%) of 25 Rana catesbeiana bullfrogs and all (100%) of 24 Bufo japonicus japonicus toads were positive for Blastocystis. Two distinct populations of the toad and bullfrog showed a high prevalence (100 or 84.6%) of Blastocystis infection, while in two populations of the leopard frog only one population was positive for Blastocystis (84.6%). Three Blastocystis isolates from different species of the frogs were established. Since none of the three isolates could survive at 37 degrees C, a temperature tolerance assay was performed to assess the optimal growth temperature and to determine the range of non-lethal temperatures. During the exponential growth phase of 3- or 4-day cultures at 25 degrees C, three isolates were exposed to 4, 28, 31, or 34 degrees C for 3 days and then returned to 25 degrees C to monitor the cell growth. Based on the optimal growth temperatures and different ranges of temperature tolerance among the three new isolates from frogs and two known species, Blastocystis hominis and Blastocystis lapemi, it was established that the three isolates recovered from different species of frogs had different physiological features from B. hominis and B. lapemi.     

蟾蜍壶菌病病原遗传分化研究

壶菌病为近年发现的两栖类动物重要传染病,对野生和养殖种群危害极大,为确定药用经济动物蟾蜍历史上壶菌病感染情况,提高养殖蟾蜍疾病防治水平,选取某博物馆馆藏采集于四川的蟾蜍标本32只,利用Taqman—MGB荧光探针定量PCR技术进行壶菌检测,并对定量PCR产物克隆、测序,通过序列比对和系统发育分析判定其来源。最终得到定量...     

馆藏泽蛙标本壶菌病原实时PCR检测与系统发育分析

为研究、验证我国两栖类壶菌病的历史疫情,从时间和系统进化角度追溯壶菌的来源,该研究选取北京自然博物馆馆藏1982年采集于广东的泽蛙标本39只,利用Taqman-MGB荧光探针定量PCR技术进行壶菌检测,并对定量PCR产物克隆、测序,通过序列比对和系统发育分析判定其来源。最终得到定量PCR标准曲线:Y=-3.1X+32.65;相关系数R2=0.999 8;检测结果为阳性样本12只,检出率30.8%;同时系统发育分析表明,我国的壶菌存在一定程度的分化,一类与北美洲、南美洲、欧洲菌株呈现高度的亲缘关系;另一类则表现出与世界其他地区分布的壶菌有明显的不同,显示为独特类型。该研究把我国壶菌感染的最早记录推进到了20世纪80年代初期。     

Nematode and ciliate nasal infection in captive archey's frogs (Leiopelma archeyi)

Archey's frogs (Leiopelma archeyi) are first on the list of evolutionarily distinct and globally endangered (EDGE) amphibians. Captive breeding is an important strategy for protection of the species, but programs are hampered by a lack of information on diseases present in wild and captive populations. Two novel nematodes (Koerneria sp. and Rhabditis sp.) were found separately in four captive Archey's frogs showing clinical signs of hemorrhagic purulent nasal discharge and weight loss. One of these frogs also had a novel protozoal infection (Tetrahymena) in the nasal cavity. Koerneria, Rhabditis, and Tetrahymena have not previously been reported in amphibians in New Zealand. One frog was treated successfully with oral moxidectin at 0.4 mg/kg for the nematode infection and topical metronidazole at 10 mg/kg for the protozoal infection. The clinical signs abated only after both infections were cleared. The second frog died before treatment could be established. The third and fourth frogs were found dead.     

Effects of temperature and hydric environment on survival of the Panamanian Golden Frog infected with a pathogenic chytrid fungus

Considerable controversy exists concerning whether or not climate changes (particularly global warming) are causing outbreaks of a lethal amphibian pathogen, the chytrid fungus Batrachochytrium dendrobatidis (Longcore, Pessier & D.K. Nichols 1999). In the present study, groups of Panamanian golden frogs (Atelopus zeteki Dunn, 1993), a critically endangered amphibian thought to be nearly extinct in Panama, were exposed to varying dosages of zoospores of Batrachochytrium dendrobatidis, temperatures and hydric environments in order to learn whether this species is susceptible to this pathogen and, if so, how environmental factors affect survival. This pathogen proved to be highly lethal for A. zeteki. Frogs exposed to a dosage of 100 Bd zoospores survived significantly (P < 0.0001) longer than those that had been exposed to 10⁴ or 10⁶ zoospores. Exposed frogs housed at 23 °C survived significantly (P < 0.0001) longer than those that were housed at 17 °C. Exposed frogs held in dry conditions survived significantly longer than those in wet conditions (P < 0.0001). As a laboratory study, these results do not directly test hypotheses about the relation between climate change and the decline of these frogs in the field, but they inform the discussion about how environmental conditions can have an impact on the interaction between a susceptible amphibian and this pathogen. These data do not support the contention that rising global temperatures are necessary to cause the death of amphibians infected with this pathogen because the pathogen was equally lethal at 17 as at 23 °C, and frogs at the warmer temperature lived significantly longer than those at the cooler one.     

Effects of constant environmental temperatures on the performance of laying pullets

1. Two experiments are described in which laying pullets maintained at constant temperatures were fed a range of diets with a view to defining optimum combinations of temperature and nutrient intake. 2. In the first experiment, all combinations of 6 temperatures (15 degrees, 18 degrees, 21 degrees, 24 degrees, 27 degrees and 30 degrees C) 9 diets (three protein concentrations and three energy contents) and two stocks were tested for 34 weeks using 4320 pullets. In experiment 2, all combinations of three rearing temperatures, three laying temperatures (18 degrees, 22.5 degrees and 27 degrees C) three diets (protein concentration) and two stocks were tested for 61 weeks using 2160 pullets. 3. As anticipated, higher dietary protein concentrations were needed to maintain egg output at higher temperatures. If diets suplying adequate amino acid intakes were provided, egg output was unaffected by temperatures in the range 15 degrees to 27 degrees C although, at the highest temperature, egg weight was slightly reduced and rate of lay (particularly in the later part of the laying year) was increased. At 30 degrees C, egg output was depressed whichever diet was fed. 4. Dietary energy content had small but significant effects on egg weight and egg output but did not interact with temperature. It was not possible to maintain egg weight or egg output at 30 degrees C by feeding a high energy, high protein diet. 5. Estimated heat output of the birds increased during the course of the experiment at the lower temperatures but decreased with time at 30 degrees C. Feather loss occurred earlier at the lower temperatures and this is interpreted as an effect of temperature on the timing of the annual moult, which also accounts for the better persistency of lay observed at 27 degrees C.     

Survival of Isospora suis oocysts under controlled environmental conditions

Isospora suis is a coccidian parasite infecting piglets soon after birth. While the gross epidemiology of I. suis is well known, little knowledge exists on the ecology of the oocysts. To study the development and survival of oocysts of I. suis under controlled laboratory conditions, known numbers of oocysts ( approximately 200 in each of 4 replicates) were exposed to all combinations of 4 relative humidities (53-100% RH) and 3 temperatures (20 degrees , 25 degrees , 30 degrees C). Determination of viability was based on morphological and fluorescent properties of the oocyst as well as on the permeability of the oocyst wall characterized by inclusion/exclusion of the fluorescent dye propidium iodide. The viability of the oocysts was studied over time by fluorescence and light microscopy until <5% of the oocysts were considered to be viable. The sporulation rate increased with temperature, however, the infective sporocyst stage was reached within 24h at all temperatures, while RH did not seem to affect sporulation. Results show a rapid reduction in viable oocysts exposed to high temperatures (25 degrees C and 30 degrees C) in combination with low relative humidities (53% RH and 62% RH), at which conditions oocysts died within 24h. Viability was higher when oocysts were exposed to higher relative humidities (75% RH and 100% RH) as well as a lower temperature (20 degrees C). However, even at 75% RH the oocysts died within 24-60 h at 30 degrees C to 20 degrees C, respectively, while the most favourable condition appeared to be 100% RH and 25 degrees C at which condition the percentage of viable oocysts decreased from 100% to 17% in 96 h. The results indicate that it may be possible to reduce the infection pressure of I. suis in modern sow herds by changing the environmental conditions and/or the management within the farrowing pens, and thereby increase animal welfare without relying on the use of routine medication.     

Hematology and serum chemistry values of the European brown hare

Background: Hematology and serum biochemistry values can be useful for evaluating the health of populations of captive and free-living European brown hares ( Lepus europaeus ). However, current information is fragmented and incomplete.
Objective: The objective of this study was to determine normal hematology and serum chemistry values for a population of captive European brown hares and to investigate differences related to sex and age.
Methods: Routine hematology and serum chemistry values were determined for 48 captive European brown hares in Catalonia (northeastern Spain): 21 young (6 males and 15 females) and 27 adult (14 males and 13 females) hares. Blood was obtained by auricular venipuncture while animals were physically restrained. Results were expressed as the mean ± SD, and reference intervals were calculated as the mean ± 2SD.
Results: RBC count, PCV, concentrations of hemoglobin, total protein, albumin, gamma globulin, urea nitrogen, and creatinine, and alanine aminotransferase activity were significantly higher in adults than in young hares, and alkaline phosphatase activity and total bilirubin concentration were lower. Sex differences were not observed.
Conclusions: Hematology and serum chemistry results obtained in this study are considered representative for healthy captive European brown hares. A single reference interval can be used for values from male and female hares, however, age-specific intervals are needed for some analytes.     

A zoospore inhibition technique to evaluate the activity of antifungal compounds against Batrachochytrium dendrobatidis and unsuccessful treatment of experimentally infected green tree frogs (Litoria caerulea) by fluconazole and benzalkonium chloride

Effective and safe treatments of chytridiomycosis in amphibians, caused by Batrachochytrium dendrobatidis, are needed to help prevent mortality in captive programs for threatened species, to reduce the risk of spread, and to better manage the disease in threatened populations. We describe a simple method to determine minimum inhibitory concentrations (MICs) of antifungal agents that involves adding zoospores to various drug concentrations in 96 well plates and microscopic observation after four days. We report results from testing 10 commercially available antifungal compounds: benzalkonium chloride (<0.78 μg/ml), povidone iodine (312.5 μg/ml), amphotericin B (3.125 μg/ml), fluconazole (<1.56 μg/ml), itraconazole (<1.56 μg/ml), enilconazole (<1.56 μg/ml), mercurochrome (6.25 μg/ml), sodium chloride (12.5 mg/ml), methylene blue (<1.56 μg/ml) and Virkon (3.125 μg/ml). For treatment trials of juvenile Litoria caerulea, baths of benzalkonium chloride at 1 mg/L and fluconazole at 25 mg/L were used on 18 experimentally infected frogs per treatment. Although these treatments resulted in longer survival times (mean 43.7 ± 11.3 days) than in the untreated controls (37.9 ± 9.3 days), the mortality rate was still 100%. Higher doses of fluconazole are suggested for further animal trials.     

Pentastomiasis and other parasitic zoonoses from reptiles and amphibians

Reptiles are growing in popularity as pets.The colonization of reptiles and amphibians by parasites and the resulting disease conditions are the most common problems seen in captive animals.This review focuses on pentastomiasis and sparganosis, important parasitic zoonoses of reptiles and amphibians, respectively, and free living-amoebae. Humans are suitable accidental hosts for some pentastomid species (particularly Armillifer and Porocephalus). In geographical areas with special ethnics, such as in West and Central Africa, and East Asia, 8-45% of the human population can be affected. Usually the larvae are coincidentally found during abdominal surgeries. However, fatalities have been described. Extreme caution is necessary when handling infected reptiles. Ocular or cerebral sparganosis is not uncommonly found in humans in East Asia. This disease is caused by spargana, tapeworm larvae (plerocercoids) of Spirometra sp. The infection occurs when uncooked meat from reptiles or amphibians is applied to wounds or eyes and the parasites migrate directly to human tissue, or by consumption of contaminated food or water. As a consequence of the reptile's predatory behaviour, the full spectrum of endo- and ectoparasites from potential prey animals can be found as transiting parasites in the intestinal tract, e. g. Hymenolepis nana, Cryptosporidium (C.) muris, C parvum or Capillaria hepatica. Occasionally, free-living amoebae are also found in reptile faeces (Acanthamoeba, Naegleria, Hartmanella, Vahlkampfia or Echinamoeba sp.).     

Wild‐Caught and Farm‐Reared Amphibians are Important Reservoirs of Salmonella,A Study in North‐East Thailand

The role of amphibians as Salmonella reservoirs has not been as well studied as in reptiles, where the literature is abundant. Recent outbreaks of salmonellosis associated with exotic pet frogs have occurred in United States. Frog farming and wild frog harvesting have increased the international trade in these species. This necessitates a better understanding of the risk of salmonellosis transmission from amphibians to humans. We explored the presence of Salmonella in amphibians (frogs and toads) in Thailand, where farmed and wild frogs as well as toads are present. These live animals are easily found in the local markets and are used as food. Exportation of frog meat from Thailand is common. During March–June 2014, ninety‐seven frogs were collected from several habitats, including frog farms, urban areas and protected natural areas. The collected amphibians were tested for the presence of Salmonella. The overall prevalence of Salmonella was 69.07% (90.00% in farm animals, 0% in urban area animals and 44.83% in protected area animals). Eight serovars of Salmonella were isolated: subsp. diarizonae ser. 50:k:z, Hvittingfoss, Muenchen, Newport, Stanley, Thompson, Panama and Wandsworth. Six of the identified serovars, Hvittingfoss, Newport, Panama, Stanley, Thompson and Wandsworth, have been detected in humans in Thailand. According to our results, amphibians are reservoirs of Salmonella and can be a public health concern when used as a source of protein for humans.     

Corneal lipid deposition in Cuban tree frogs (Osteopilus septentrionalis) and its relationship to serum lipids: an experimental study.

To evaluate the association between corneal lipid infiltration (corneal arcus) and dietary cholesterol in Cuban tree frogs (Osteopilus septentrionalis), 47 wild-caught frogs were fed diets of either regular or high-cholesterol crickets containing 0.7% and 1.7% cholesterol dry matter, respectively. Serum total cholesterol and triglycerides were measured when the frogs were euthanized after 17 mo. In a subsample of frogs, serum lipoproteins were characterized using high-performance liquid chromatography. The first case of corneal lipid deposition occurred in a female frog after 13 mo on the high-cholesterol diet. In the subsequent 4 mo, 5/11 males and 11/35 females developed the disease. Four of these affected frogs were females on the regular diet. Frogs with corneal lipid deposition had elevated serum total cholesterol (27.3 +/- 19.8 mmol/L) and low-density lipoproteins (LDL, 17.8 +/- 18.9 mmol/L) compared with unaffected captive frogs (16.5 +/- 20.4 and 9.0 +/- 7.6 mmol/L, respectively). Corneal lipid deposition was more prevalent in frogs on the high-cholesterol diet, and this group had higher serum total cholesterol (34.1 +/- 15.2 mmol/L in females, 22.8 +/- 14.8 mmol/L in males) than did frogs on the diet of regular crickets (12.3 +/- 8.7 mmol/L in females, 10.4 +/- 3.1 mmol/L in males). Captive frogs on both diets had higher serum total cholesterol than did wild frogs (3.1 +/- 2.1 mmo/L in females, 5.3 +/- 2.6 mmo/L in males). This additional serum cholesterol was primarily carried on very low-density lipoproteins (VLDL) and LDL rather than high-density lipoproteins (HDL), as indicated by the significantly higher ratio of VLDL cholesterol and LDL cholesterol over HDL cholesterol in captive frogs compared with wild frogs. Elevation in this ratio was significantly higher in captive females than in captive males. There was no evidence that increased serum cholesterol in captive females was directly related to the process of vitellogenesis.     

Relative humidity at moderate ambient temperatures: its effect on male broiler chickens and turkeys

1. The effect of relative humidity (RH, 40% to 75%) at moderate ambient temperatures (Ta, 28 degrees and 30 degrees C) on the performance and thermoregulation of male broiler chickens and turkeys was studied at the age of 4 to 8 weeks. 2. Weight gain and food intake of male broiler chickens were significantly higher at 60% to 65% RH but food conversion efficiency was not affected by RH. In male turkeys, no effect of RH on performance was recorded. 3. Both chickens and turkeys controlled body temperature at normothermic levels during exposure to the experimental environmental conditions. 4. The rate of panting estimated from blood pH and pCO2 was lower in chickens exposed to 28 degrees C than to 30 degrees C. In turkeys, the rate was lower than that recorded in chickens at both Ta(s). 5. Plasma T3 was positively and significantly correlated with food intake. 6. It may be concluded that RH plays a role in the performance of chickens exposed to 30 degrees and 28 degrees C. whereas male turkeys must respond to RH at Ta >30 degrees C.     

Plasma pharmacokinetics of selamectin after a single topical administration in the American bullfrog (Rana catesbeiana).

Parasitism is common in wild and captive amphibians; however, pharmacologic data are lacking for anthelmintic drugs. This study was developed to determine the plasma pharmacokinetics of selamectin after topical administration in bullfrogs. Thirty-two adult American bullfrogs (Rana catesbeiana) were randomly assigned into eight groups of four with each group representing a different collection time point. Seven groups received selamectin (6 mg/ kg) topically and the remaining group served as the untreated control group. One group of frogs was euthanized and blood samples immediately collected on days 0 (control), 1, 5, 10, 15, 20, 25, and 30. Plasma was analyzed for selamectin using high performance liquid chromatography with fluorescence detection. Individual samples were analyzed, then data were reported as the mean of the four frogs at each time point. A histologic evaluation of the lung, liver, kidney, and skin tissues was performed and none of the frogs showed histologic evidence of toxicity due to selamectin administration. The mean peak plasma concentration was 162.5 +/- 42.3 ng/ml, area under the curve was 2,856 ng day/ml, mean residence time was 12.2 days, and disappearance half-life was 1.87 days. Based on the plasma pharmacokinetics, bullfrogs appear to absorb selamectin very efficiently, concentrations reach high levels in the plasma, and there were no apparent histologic effects from single dose administration.     

Influence of Temperature during Glycerol Addition and Post-thaw Dilution on the Quality of Canine Frozen Semen

Contents The aim of the present study was to compare the influence of room temperature (27 degrees C) and 4 degrees C during glycerol addition on canine semen cryopreservation and verify the effect of different post-thawing dilutions on canine semen. Ten ejaculates from five stud dogs were collected by digital manipulation. Semen samples were evaluated and further divided into two aliquots. The first aliquot was extended in Tris-egg yolk-glycerol at 27 degrees C and the second one received glycerol at 4 degrees C. Samples were frozen and stored in liquid nitrogen. After 1 week, samples were thawed and submitted to evaluations of progressive sperm motility, morphology, acrosomal integrity, hypo-osmotic swelling (HOST) and thermoresistance tests. For thermoresistance test, aliquots were divided in two portions: one portion was kept undiluted (1 : 0) and the other one was diluted in a 1 : 4 ratio (one part semen to four parts extender). No differences were observed between temperatures for glycerol addition regarding seminal parameters evaluated. Furthermore, post-thawing dilutions demonstrated similar effect on canine semen longevity. Correlations among post-thaw sperm motility and HOST and results from thermoresistance test were observed for both temperatures for glycerol addition. In conclusion, glycerol could be added to canine semen at room temperature (27 degrees C) or at 4 degrees C. Moreover, there is no need to extend canine semen after thawing for the thermoresistance test, but if we need to increase the inseminating volume for artificial inseminations, the addition of extender will not damage the semen.