Effect of season and temperature on mortality in amphibians due to chytridiomycosis

OBJECTIVE: To investigate the distribution and incidence of chytridiomycosis in eastern Australian frogs and to examine the effects of temperature on this disease. DESIGN: A pathological survey and a transmission experiment were conducted. PROCEDURE: Diagnostic pathology examinations were performed on free-living and captive, ill and dead amphibians collected opportunistically from eastern Australia between October 1993 and December 2000. We conducted a transmission experiment in the laboratory to investigate the effects of temperature: eight great barred frogs (Mixophyes fasciolatus) exposed to zoospores of Batrachochytrium dendrobatidis and six unexposed frogs were housed individually in each of three rooms held at 17 degrees C, 23 degrees C and 27 degrees C. RESULTS: Chytridiomycosis was the cause of death or morbidity for 133 (55.2%) of 241 free-living amphibians and for 66 (58.4%) of 113 captive amphibians. This disease occurred in 34 amphibian species, was widespread around the eastern seaboard of Australia and affected amphibians in a variety of habitats at high and low altitudes on or between the Great Dividing Range and the coast. The incidence of chytridiomycosis was higher in winter, with 53% of wild frogs from Queensland and New South Wales dying in July and August. Other diseases were much less common and were detected mostly in spring and summer. In experimental infections, lower temperatures enhanced the pathogenicity of B. dendrobatidis in M. fasciolatus. All 16 frogs exposed to B. dendrobatidis at 17 degrees C and 23 degrees C died, whereas 4 of 8 frogs exposed at 27 degrees C survived. However, the time until death for the frogs that died at 27 degrees C was shorter than at the lower temperatures. Infections in survivors were eliminated by 98 days. CONCLUSION: Chytridiomycosis is a major cause of mortality in free-living and captive amphibians in Australia and mortality rate increases at lower temperatures.     

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.     

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.     

Differences in helminth infections between captive and wild spur-thighed tortoises Testudo graeca in southern Spain: a potential risk of reintroductions of this species

Although the spur-thighed tortoise, Testudo graeca, is one of the most widely distributed species of tortoises, its natural populations are threatened through its whole range. Particularly at south-eastern Spain, the species is mainly threatened by habitat destruction and over-collection, given that this chelonian has been traditionally considered an appreciate pet. As south-eastern Spanish wildlife recovery centers shelter hundreds of captive animals mainly coming from illegal trade or captive-bred, there is a strong debate about what to do with these animals: maintaining them in captivity all along their lives or reintroducing them to wildlife. It is well known that the reintroduction of captive animals supposes a risk for the wild population due to the uncertainty of their genetic origin and to the possible spread of infectious diseases. However, despite the increasing evidence that infectious agents are a potential health hazard for wildlife, little is known about the risk that introduced parasites could suppose for the wild populations of spur-thighed tortoise. The present study investigates for the first time the presence of helminth eggs and worms in faeces from 107 wild and captive individuals collected from mid-March to mid-June 2010, and relates the findings to different environmental and host variables. Sixteen oxyurid species and the ascarid Angusticaecum holopterum were identified. This last nematode and the oxyurid species Tachygonetria palearticus and T. seurati had not been reported in Spanish wild T. graeca previously. The prevalence of oxyurid eggs and worms were 94% and 70%, respectively; while, ascarid eggs and worms were found in 26% and 5% of tortoises, respectively. Ascarid infections affected mostly captive animals and were associated to caparace deformities and symptoms of upper respiratory tract disease (p<0.05). Oxyurid infections were not associated to negative health traits and prevalence increased with age. In free-living tortoises, the distribution of pharingodonid genera also varied according to habitat; moreover, T. longicollis, T. pusilla, T. conica, T. robusta and Mehdiella stylosa where significantly more frequent in wild compared to captive tortoises (p<0.05). Study results highlight important differences in the nematode fauna of captive and free-living tortoises and questions one more time if the reintroductions of captive animals suppose a risk for the wild population since the former ones can harbor and distribute among free populations pathogens like ascarid nematodes.     

A squamous cell carcinoma and an adenocarcinoma in Australian treefrogs

We describe the pathology in a captive adult White lipped treefrog (Litoria infrafrenata) with a squamous cell carcinoma of the skin and a free-living adult Common green treefrog (L. caerulea) with an adenocarcinoma of the skin. Although many amphibian neoplasms have been reported world wide, none had been described in Australian species.     

Systematic assessment of the impact of adenovirus infection on a captive reintroduction project for red squirrels (Sciurus vulgaris)

PCR was used to amplify adenoviral DNA, and transmission electron microscopy (TEM) to detect adenovirus particles in tissue and intestinal content samples from red squirrels (Sciurus vulgaris) associated with a reintroduction study on Anglesey (North Wales), from other populations on the island and from stock held at the Welsh Mountain Zoo, 38 km to the east. Samples were collected during the routine surveillance postmortem examinations of all 60 red squirrels with carcases retrieved in a suitable condition between 2004 and 2010, including 29?captive and 31 free-living animals. Following significant clusters of mortality in captive red squirrels, adenovirus was identified retrospectively in faecal material from 12 of 13 (92 per cent) examined carcases from squirrels captive on Anglesey, and 14 of 16 (88 per cent) from the Welsh Mountain Zoo. Virus was identified in 13 of 31 (42 per cent) free-living wild animals, with evidence of both subclinical and clinically significant enteric adenoviral infections in wild squirrels. Without ancillary PCR and TEM testing, the extent of adenovirus infection in such populations would have been underestimated. Screening protocols that include examinations for adenovirus should, therefore, be part of the routine biosecurity measures protecting reintroduction or captive breeding programmes for red squirrels.     

Ranavirus-associated mass mortality in wild amphibians, the Netherlands, 2010: a first report

In 2010, a mass die-off of over 1000 wild water frogs (Pelophylax spp.) and at least 10 common newts (Lissotriton vulgaris) occurred in a pond in The Netherlands. Haemorrhagic disease with hepatomegaly and splenomegaly was evident. Microscopically, multiple organs presented cells with multifocal intracytoplasmic inclusion bodies, in which ranavirus-like particles were demonstrated ultrastructurally. All specimens examined tested positive for ranavirus by PCR. The sequence obtained showed a 100% identity with the one deposited for common midwife toad virus (CMTV). This is the first report of ranavirus-associated mortality in wild amphibian populations in The Netherlands. It is also the first time CMTV or a CMTV-like virus has been reported in these two species in the adult stage and outside of Spain.     

Concurrent infection with ranavirus, Batrachochytrium dendrobatidis, and Aeromonas in a captive anuran colony

Four species (Dendrobates auratus, Phyllobates terribilis, Pyxicephalus adspersus, and Rhacophorus dennysi) of captive anurans with a clinical history of lethargy and inappetence were found dead and were submitted for necropsy. Gross lesions included irregular patches of sloughed skin and rare dermal ulcerations. Histologic findings included epidermal proliferation that was most pronounced on the digits and that included intracytoplasmic chytrid organisms. Bacteria were often associated with the epidermal lesions. Intracytoplasmic inclusion bodies were observed in hepatocytes. Real-time polymerase chain reaction yielded positive results for both Ranavirus and Batrachochytrium dendrobatidis (Bd). Bacterial culture of internal organs yielded Aeromonas hydrophila. This is the first report of concurrent infections in anurans by Ranavirus and Bd and A. hydrophila.     

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.     

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.     

Amphibian virology.

Amphibians are a diverse group of species; much work remains to be done to elucidate the viruses of amphibians. Viral diseases may play an important role in wild and captive amphibian populations. Iridoviruses are a leading cause of wild amphibian mortality events in the United States and are a common cause of amphibian mortality events worldwide. In addition to frank viral diseases, viral infections may play a role in the establishment of bacterial, fungal, and parasitic diseases and are an underlying cause of neoplasia. It is important for the amphibian clinician to recognize disease syndromes and pathology that are consistent with viral etiology.     

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 New Ranavirus Isolated from Pseudacris clarkii Tadpoles in Playa Wetlands in the Southern High Plains,Texas

Abstract

Mass die-offs of amphibian populations pose a challenging problem for conservation biologists. Ranaviruses often cause systemic infections in amphibians and, in North America, are especially virulent and lethal to larvae and metamorphs. In this paper we describe a novel ranavirus isolate as well as the first recorded occurrence of ranavirus in the southern High Plains of Texas and in associated populations of the spotted chorus frog Pseudacris clarkii. The breeding sites were playas, that is, wetlands that fill via isolated thunderstorms that can occur infrequently; thus, not every playa has water or breeding amphibians annually. We did not detect ranavirus in sympatric anurans, but other reports document ranaviruses in Pseudacris spp. elsewhere. The occurrence of multiple isolates of ranavirus in a number of Pseudacris species suggests that this genus of frogs is highly susceptible to ranaviruses and may experience exceptionally high mortality rates from infection. Thus, the virus may contribute to substantial seasonal population declines and low seasonal recruitment, with negative impacts on populations of breeding adults in successive years.     

Inclusion body myositis in spring peepers (Pseudacris crucifer).

In 2000, 2 adult captive spring peepers (Pseudacris crucifer) from the same zoological park were humanely euthanized. Histologically, both frogs had degeneration, atrophy, and necrosis of striated myofibers of the tongue admixed with chronic lymphohistiocytic inflammation. One frog had similar lesions in the skeletal muscles of the body wall. Several degenerate and necrotic myofibers contained single, eosinophilic, intranuclear inclusion bodies. Ultrastructural examination of the inclusions revealed nonenveloped, icosahedral, virus-like particles averaging 20-24 nm in diameter. This is the first reported case of inclusion body myositis in frogs and is believed to be due to parvoviral infection.     

Ovarian dysgerminomas in two mountain chicken frogs (Leptodactylus fallax).

This report describes the gross, histologic, and immunohistochemical features of ovarian dysgerminomas in two adult female mountain chicken frogs (Leptodactylusfallax) from the same zoological institution. One frog was found dead, and the other frog had been ill for several days with a bloated abdomen and lethargy. On necropsy, large, pale multilobulated masses replaced the left ovary in both frogs, and one frog also had numerous smaller nodules scattered throughout the coelomic viscera. Histologically, these masses were composed of sheets and cords of polyhedral discrete germ cells consistent with the diagnosis of dysgerminoma. Neoplastic cells stained positive with immunohistochemistry for Oct4, which has been reported to detect stem cells including germ cells in a variety of species, including humans. Ovarian tumors are uncommonly encountered in both reptiles and amphibians, and this report is the first report of dysgerminoma in any amphibian species.     

Yolk coelomitis in a white-throated monitor lizard (Varanus albigularis)

Yolk coelomitis as a result of pre-ovulatory follicular stasis is a common disorder in captive reptiles, especially in captive lizards of various genera. The clinical signs are generally fairly non-specific and diagnosis is based on clinical signs together with most of the common diagnostic modalities. The condition is most likely a husbandry and environment-related reproductive disorder. It has not been reported in wild free-living specimens. This report describes the clinical presentation and post mortem lesions in a white-throated monitor lizard that died during treatment for non-specific clinical signs related to a severe yolk coelomitis.     

First report of ophidiomycosis in Asia caused by Ophidiomyces ophiodiicola in captive snakes in Japan

Ophidiomycosis is an emerging infectious disease caused by the fungus Ophidiomyces ophiodiicola, which has been affecting wild and captive snakes in North America, Europe, and Australia. We report 12 cases of suspected ophidiomycosis in captive colubrid snakes in Japan. Pathological and microbiological examinations were performed, and the results confirmed the diagnosis of ophidiomycosis in two snakes, which indicated that the remaining sympatrically raised snakes also had ophidiomycosis since they exhibited similar lesions. This is the first report of ophidiomycosis in Asia caused by O. ophiodiicola. To prevent the expansion of ophidiomycosis in the natural environment in Japan, there is a need to evaluate the ophidiomycosis carrier status of imported snakes, the pathogenicity of the infection in native snakes, and the prevalence and distribution of O. ophiodiicola in wild and captive snakes. Measures also must be taken to prevent endemicity globally.     

Fatal disseminated encephalitozoonosis in a captive, adult Goeldi's monkey (Callimico goeldii) and subsequent serosurvey of the exposed conspecifics

A captive, adult male Goeldi's monkey (Callimico goeldii) (GM) presented in acute respiratory distress 4 yr after importation into the United States from Europe. Radiographs and echocardiogram were consistent with heart failure. The monkey died within 24 hr of presentation. Necropsy findings included multicentric arteritis and aortitis with aneurysm associated with microsporidian organisms morphologically consistent with Encephalitozoon species. Polymerase chain reaction confirmed organisms were Encephalitozoon cuniculi. Sequence analysis of amplicons generated by using primers specific for the polar tube protein of E. cuniculi determined the organism to be genotype II. An E. cuniculi serosurvey of potentially exposed conspecifics that represented approximately 40% of the captive GM population in the United States was conducted. Multiple individuals that had been imported from Europe with the individual of this report were seropositive via an immunofluorescent antibody assay for E. cuniculi. Multiple samples were available from 3 individuals that demonstrated a decrease in titer or reversion to seronegative status within 3 yr of initial positive status. All other GM were negative on serology. This case is unique in that the genotype identified (genotype II) was different than the genotype (genotype III) reported in other New World primate (NWP) species, the patterns of arteritis were different from the typical pattern of microsporidial vasculitis described in other species, and clinical disease was observed in an adult. Most reported cases of clinical disease secondary to E. cuniculi in NWP have been in neonates and juveniles.     

Pestiviruses in wild animals

Pestiviruses are not strictly host-species specific and can infect not only domestic but also wild animals. The most important pestivirus, CSFV, infects domestic pigs and wild boars, which may cause a major problem for successful CSFV eradication programmes. Mainly BVDV specific antibodies have been reported in captive and free-living animals. Virus has been isolated from some of these animal species, but since BVDV can contaminate cell cultures and foetal calf serum, early reports of BVDV isolation have to be considered with caution. Genetic typing of early pestivirus isolates from wild species revealed that the majority were BVDV-1. Of the pestiviruses identified so far three species (CSFV, BVDV-1, giraffe pestivirus) and three genotypes (BDV-2, BDV-4, pronghorn) appear to circulate in wildlife animal populations. The potential for pestiviruses to spread between farm animals and free-living animals is discussed as are epidemiological and technical problems, and the future direction of research.     

Salmonella in free-living exotic and native turtles and in pet exotic turtles from SW Spain

We screened 78 native and 94 exotic turtles from natural ponds and 39 exotic pet turtles for presence of Salmonella, resulting with infection rates of 6.61%, 6.4%, and 5.1%, respectively. Concurrent shedding of multiple serotypes of the bacteria was only detected in one pet turtle. Eleven isolates were obtained in free-living turtles, including serotypes commonly found in reptiles and also the serotype Typhimurium, which is commonly related to human infections. In pet turtles, the five serotypes isolated were different to those isolated in free-living turtles and had been reported to cause reptile-associated salmonellosis in humans. These results confirm the risk of transmission of Salmonella from free-living and pet turtles to humans, demanding the necessity of regulation of pet turtle trade in Europe.