著者

田向 健一

出版者

麻布大学

巻号頁・発行日

2014

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The animal trade has aided in pathogen dispersal and has frequently been the cause of pandemics such as SARS, H5N1, avian influenza, and koi herpes. Its effects may be sufficiently significant to cause declines in wild populations as well as serious economic losses around the world. Batrachochytrium dendrobatidis, a fungus which belongs to the Chytridiomycetes class of the Chytridiales order of fungi and which was first described in 1999, causes chytridiomycosis, a disease which infects amphibians. This fungus is responsible for the decline or extinction of more than 20 families and 200 species of amphibians.Abroad, the International Union for Conservation of Nature (IUCN) has designated B. dendrobatidis a pathogen that requires global monitoring and specific study in order to determine its role as a causative factor of chytridiomycosis, and also to evaluate its effects on ecological systems. B. dendrobatidis is also listed in the obligatory Aquatic Animal Health Code published by the World Organization for Animal Health (OIE). The first cases of chytridiomycosis in Asia were confirmed in captive exotic amphibians in Japan by Une et al. (2007), with some of the cases resulting in death. Japan is home to 63 species of amphibians, 40 species of anurans, and 23 species of urodeles, including endemic species. Of these, 42 species (67%) are listed as endangered and near-threatened on the Red List compiled by the Ministry of the Environment of the Government of Japan.B. dendrobatidis has a broad host range, is highly infectious, and has a high fatality rate. B. dendrobatidis zoospores can spread through water and cause infection rapidly over a wide area. To date, however, no studies have sought to determine the prevalence of B. dendrobatidis in imported exotic amphibians in Japan. In addition, the prevalence of B. dendrobatidis in captive amphibians is unknown, and, moreover, treatment methods and elimination techniques for chytridiomycosis have yet to be established for many of the amphibian species at risk of infection.The aim of this study was to survey imported and captive exotic amphibians in Japan in order to determine the prevalence of B. dendrobatidis, and to establish chytridiomycosis treatment and B. dendrobatidis elimination techniques. An additional goal was to decrease the threat of B. dendrobatidis infection in endemic Japanese amphibians.This study consists of three areas of research, described in Chapters 1 to 3, respectively.Chapter 1. B. dendrobatidis Prevalence and Haplotypes in Domestic and Imported Pet Amphibians in Japan.In order to clarify the infection status of B. dendrobatidis, we surveyed amphibians imported into Japan and those held in captivity for a long period or bred in Japan. Between 2008 and 2011, samples were taken from 820 individuals of 109 amphibian species and were analyzed using nested-PCR assays. A total of 76 samples (9.3%) from these 820 amphibians were identified as B. dendrobatidis-positive. Although B. dendrobatidis prevalence was 6.9% (18/259) in sampled amphibians from private collections and those commercially bred in Japan, it was 10.3% (58/561) in imported amphibians. The high prevalence of B. dendrobatidis in imported animals is possibly a result of the increased opportunity for infection due to the high density of individuals in closed environments in the distribution process, particularly breeding facilities, as well as reduced immunity, resulting from the stress of living in an environment different from their natural habitats.Both captive amphibians and those from the pet trade that were surveyed for this study included a significant number of healthy B. dendrobatidis carriers. We identified the genotypes of this fungus using partial DNA sequences of the internal transcribed spacer (ITS) region. Sequencing the PCR products of all 76 B. dendrobatidis-positive samples revealed 11 haplotypes. The species infected with the greatest variety of haplotypes was the Japanese-bred Xenopus laevis, in which haplotypes A, C, Q, and V were detected. This finding supports the contention that Xenopus laevis is a key B. dendrobatidis host species. Whereas five haplotypes, A, C, Q, V and Bd28, were detected in captive Japanese amphibians, the proportion of B. dendrobatidis haplotypes found in samples from Japan differed from that of those from other countries. Haplotype A (DNA Data Bank of Japan accession number AB435211) was found in 90% (52/58) of imported amphibians. Haplotype A is a hypervirulent strain of the global panzootic lineage (Bd-GPL). In contrast, we respectively detected haplotypes C, A, V, Q, and Bd28 in 44%, 28%, 17%, 5.6%, and 5.6% of the sample collected in Japan. On the basis of these results, we determined the diversity of B. dendrobatidis ITS haplotypes in Japan. The exotic amphibians, which had been kept in captivity for a long period of time, would most likely have come into contact with native amphibians during transport and at rearing facilities, allowing the formation of a variety of haplotype phases.Nine B. dendrobatidis haplotypes (A, C, E, L, Q, V, Bd28, Bd38, and Bd41) were detected in amphibians originating from Asia, whereas only three (A, Bd29, and Bd43) were detected in amphibians from outside of Asia. It is clear that Asian amphibians are infected with a high diversity of B. dendrobatidis haplotypes, a fact that supports the “Chytrid out of Asia” hypothesis described by Goka (2009).Chapter 2. Treatment of Spontaneous Chytridiomycosis in Captive Amphibians Using Itraconazole.In Chapter 1, it was mentioned that exotic amphibians imported for the pet trade had a B. dendrobatidis prevalence of 10.3%, and that we confirmed infection and die-off from chyridiomycosis. B. dendrobatidis infects amphibians not only through direct contact but also through B. dendrobatidis zoospores in water, which can cause rapid widespread infection. One particular set of chyridiomycosis-infected amphibians released a large number of zoospore, necessitating the urgent development of treatment methods and elimination techniques for chytridiomycosis.In Chapter 2, we describe the development of an effective, simple, and safe treatment method that targets clinical cases of chytridiomycosis in various amphibian species. The subjects were 12 amphibians (11 anurans of 4 different species and 1 urodela) diagnosed with chytridiomycosis by clinical signs, microscopic findings of shed skin, and a PCR assay. The treatment protocol consisted of a 10-minute immersion in a 0.01% aqueous solution of itraconazole every other day for a total of 7 treatments. We evaluated the efficacy of the treatment using 3 methods: clinical signs, direct microscopy, and a nested-PCR assay. In addition, re-examination was performed to confirm the elimination of chytridiomycosis after treatment (20–57 days, average 34.4 days). As a result, we succeeded in curing 11 of the amphibians of chytridiomycosis and eliminating B. dendrobatidis. Recurrence of chytridiomycosis has not been observed in the past 12 months. This protocol is the first treatment method to cure a caudata of chytridiomycosis. Using the same protocol, coauthor Dr. Une et al. (2012) succeeded in eradicating B. dendrobatidis in a Japanese giant salamander (Andrias japonicus), an endangered species considered to be a special natural monument of Japan. Therefore, we recommend this as a proven treatment method and elimination technique for chytridiomycosis for use in captive amphibians, including caudata.Chapter 3. Efficacy of Copper Ions (Cu2+) for Eradicating B. dendrobatidis : Assessment of Cu2+ on the African Clawed Frog (Xenopus laevis).In Chapter 2, we described the successful development of a method for treating chytridiomycosis in anuran and caudata. Although the medicinal agent used in that research, Itraconazole, is costly when employed to treat humans, it is very effective for treating chytridiomycosis. In the case of amphibians, test animals were placed in small, individual containers, enabling total immersion in the chemical agent and maximizing the cost performance of the medicine.Although this method is well suited for treating terrestrial amphibians, because of the complexity and high cost of managing breeding water, it is not suitable for treating the African clawed frog (Xenopus laevis) or other aquatic amphibians. The African clawed frog is an important laboratory animal that is widely used in biology, genetics, embryology and other fields around the world. The African clawed frog is also an important natural host and carrier of B. dendrobatidis. Indeed, some studies have proposed that the pervasiveness of the African clawed frog may have facilitated the global spread of B. dendrobatidis. As discussed in Chapter 1, B. dendrobatidis was detected in 26.9% of African clawed frogs surveyed and there is currently no established method for eradicating B. dendrobatidis in this species.In this Chapter, we focus on the use of copper ions (Cu2+) as a safe, simple, inexpensive and effective method for eradicating B. dendrobatidis in the African clawed frog. Cu2+ is already used to control bacterial and fungal diseases in fisheries. A previous study demonstrated the application of a 0.006 ppm Cu2+ solution to control Saprolegnia, a fungus which infects fish eggs. Une et al. (unpubl. data) used a B. dendrobatidis strain to examine the effect of Cu2+ on B. dendrobatidis in vitro, and found that 1 ppm of Cu2+ could inhibit fungal growth, while 5 ppm or more could prevent fungal proliferation. We therefore set out to assess the sensitivity of African clawed frogs to Cu2+ and to investigate the application of Cu2+ as means of eradicating B. dendrobatidis. We prepared a copper standard solution for the control group as well as six additional Cu2+ solutions with ion concentrations ranging from 0.02 to 19.68 ppm. The effects of Cu2+ on the African clawed frog were determined by observing physical changes (including changes in the breeding water), measurement of the 50% lethal time (LT50), blood biochemistry, and histopathological examination. No deaths were observed after exposure to the 0.02 ppm Cu2+ solution, one animal died after 239.5 hours of exposure to the 0.21 ppm solution, and half of the animals died after 287.6 hours of exposure to the 0.31 ppm solution; in other words, LT50 and Cu2+ are inversely proportional, with LT50 decreasing as the Cu2+ concentration increases. The optimum Cu2+ concentration of viable African clawed frog is therefore considered to be approximately 0.2-0.3 ppm However, even in the 0.21 ppm group, the breeding water at the end of the experiment was very cloudy due to excessive mucus secretion and/or the presence of sloughed skin. Histopathological examination of the skin revealed acanthosis, mild hyperkeratosis and detachment of the cornified layer in the low Cu2+ concentration group. There were severe changes in skin structure, such as intercellular dissociation accompanied by single cell necrosis, cleft formation between the epidermal cells, and reticular degeneration in the epidermal layer in the high Cu2+ concentration group. These findings indicate that Cu2+ is capable of harming a specimen being treated, even at 0.21 ppm, and that the severity of any damage could be expected to increase with continued exposure.The results described in this Chapter therefore show that Cu2+ concentrations of 0.2 ppm or higher damage the skin of amphibians. At higher concentrations, Cu2+ may cause chemical burns which result in protein denaturation and corrosion of the skin as well as blood electrolyte abnormalities, which result in the damaged skin being unable to function effectively in osmoregulation. Blood enzyme activities will also increase dramatically as the Cu2+ concentration increases and the solution becomes toxic. Thus, these findings show that Cu2+ is not well suited for eradicating B. dendrobatidis. However, Cu2+ can inhibit the proliferation of Saprolegnia at 0.006 ppm and Vibrio bacteria at 0.1 ppm. Culturing B. dendrobatidis is very difficult and establishing the first strain in Japan took one year. The reason it was so time-consuming was because bacteria from the surface of the frog skin could not be removed from the culture medium. However, it may now be possible to supplement the culture medium with Cu2+ and create a selective medium for B. dendrobatidis.We have clarified the extent of B. dendrobatidis infection in amphibians introduced to Japan from abroad using a molecular biology approach, and have established a method for treatment and eliminating spontaneous chytridiomycosis. The findings from this research now provide us with a method for preventing the proliferation of B. dendrobatidis in captive amphibians and show promise for developing medical treatments for wild amphibians threatened by chytridiomycosis.