- 出版者
- 麻布大学
- 巻号頁・発行日
- 2016
IntroductionChytrid fungs (Batrachochytrium dendrobatidis: Bd) is emerging pathogen that associated with global decline of amphibian species and individuals. We detected chytridiomycosis at first in Japan and in Asia in 2006. The Japanese field survey and pathological examination detected 2 differences with large scale damaged foreign countries; the amphibian mass die-off from Bd epidemic has not detected and diversity of Bd haplotype of Japan were higher than that of damaged foreign countries. These situations were similar in the Asian foreign countries where have not damaged by Bd. Thus, the hypothesis: Bd originated from Asia and Japan (Bd originated Asia hypothesis) has occurred, but this hypothesis has not concluded because Bd plevarence in Asia is very low and it had passed only short term after Bd detected in Asia. On the other hand, pathogenesis of chytridiomycosis has not yet been completely clarified because it is only observed cause of death known that infected frog dead by osmotic imbalance through loss of electrolytes and cardiac arrest after effected skin chytridiomycosis. To clarify pathogenesis of chytridiomycosis, disruption of amphibian unique physiological function by chytridiomycosis must be observed because the integument of frog is a site of regulated transport for water, ions (electrolytes) and respiratory gases. The aim of this study was to identify the origin of Bd by detecting distribution and genetic analysis of Bd in Japan with in mind that Bd originated Asia hypothesis. And to reveal pathogenesis of skin chytridiomycosis focused on the water channel, aquaporin (AQP) that amphibian unique physiological function.Chapter 1. Confirming of oral chytridiomycosis in bullfrog (Lithobates catesbeiana) larvae and establishment of Bd examination method We examined bullfrog (Lithobates catesbeiana) larvae (tadpole) usability as monitor of Bd field survey, since the reasons as follows. Bullfrog distributed over Japan widley, and it is known that this animal harbor Bd highly and its variety of haplotype is widely. We can capture bullfrog tadpole easily, and only examine the larval mouthparts that only distributed keratinized layer that area of Bd infection. Additionally, we established the examination method which can detect Bd infection exactly in this capter. Fifty nine wild-captured bullfrog tadpole mouthparts underwent macroscopic, histopathological examination and nested PCR. Macroscopic lesions were observed in 21 of 59 tadpoles, and its 20 tadpoles were detected Bd infection by nested PCR and/or histopathological examination. Seventeen in 38 tadpoles that were not observed macroscopic lesions, were detected Bd indection. Total prevalence was 62.7% in these tadpoles. The macroscopic lesion characters were depigmentation, thinning of pigmented layer and partial defect of jaw sheaths and whited tooth rows. We concluded that bullfrog tadpole is usable for Bd field survey because the animal is compromised host for Bd infection and its mouthparts legion is easily detected by macroscopic examination. To establish the usable examination method, we compared nested PCR (materials: swab and mouthparts) and histopathological examination of mouthparts. One hundred twenty wild-captured bullfrogs from 2 habitas were divided into 2 groups, each group were 60 individuals, because we unable to conduct all examination methods to same one tadpole. The group 1 was sampled mouthparts swab and dissected mouthparts, and conducted nested PCR. Another group were conducted histopathological examination. Detection rate of each examination was high in order of swab nested PCR (67%), mouthparts nested PCR (65%) and histopathological examination (33%). The mouthparts nested PCR was recommended as Bd survey, however, sensitivity of swab nested PCR was higher than that of mouthparts nested PCR, because the sampling of swab was effected by sampling techniques. We concluded that collecting bullfrog tadpole mouthparts and conduct nested PCR can detect Bd infection situation in Japanese field. Chapter 2. Seasonality of Bd infection of bullfrog tadpole To decide term of Bd field survey, we detected seasonality of Bd infection of bullfrog tadpole. We collected 15 or 30 tadpoles in even months without December 2012, and totally 345 tadpoles were collected in between December 2011 and June 2014. The prevalence rate at each collected month was detected by nested PCR of mouthparts of these tadpoles. The seasonality of Bd prevalence in Japanese bullfrog was revealed: the highest prevalence was April and June that average prevalence was 79%, the lowest prevalence was Augst and October that has not detected Bd infection. February and December detected Bd infection, however, the prevalence of these months were different in collected year that 7 to 63%. Bd infection dynamic cycle amoung the year is seems that Bd grows and diffuses in winter to next spring, and largely decreases in summer that temperature is higher than that of Bd growth optimal temperature, and grows in late autum to winter. We decided that sampling Bd survey conduct between February and June as described above.Chapter 3. Distribution and haplotype of Bd infected in bullfrog in Japanese field We surveyed the Bd situation in Japan such as prevalence and Bd haplotype that infected in wild-captued bullfrog in Japan. 2012 and 2013, we collected 835 tadpoles from 44 areas of 26 counties, between February and June in each year. We conducted nested PCR from these samples, and determined the ITS gene region sequence from Bd positive samples, and conducted phylogenetic analysis with Bd that has been detected in previous studies. Bd detected 266 individuals (32%) from 29 areas of 19 counties. The average prevalence was 51% (3 to 100%) in detected Bd areas. Sixty eight samples were detected sequence from 266 samples and were distinguished 41 haplotypes. Six types were already isolated haplotypes; Bd global pendemic lineage (A type: 19, CW34 Clone N: 1 sample), isolated in China (Bd50: 1, Bd41: 1 sample) and isolated in Japan (P type: 2, Z type: 2 samples). The other 35 types were newly identified haplotypes in this study. Interestingly, A type was detected highly and distributed widely in Japan. We conducted phylogenetic analysis by neighbor-joining method with Bd which isolated in previous study such as China, Africa, Brazil, Australia, Ecuador and Texas. Distinguishable areal difference was not observed in clade of phylogenetic tree, and partial clade was included with Chinese haplotypes. These result likely suggest that Bd is originated from Asia and Japan, and this pathogen seems to coevolved with host amphibians for long history.Chapter 4. The effect on skin water absorption by Bd infection To clarify the pathogenesis of chytridiomycosis, we performed skin water permeability test and histopathological examination of skin and biochemical test with animal experiment. Ten frogs, Litoria caerulea from commercial market were eradiated Bd because some were already infected, and randomly assigned to exposure and control groups (each group were 5 frogs). Infection group frogs were exposed Bd via shallow immersion in bath of diluted water containing 1 ×106, 5,000 or 6,000 zoospores per 1ml. After 24 h frogs were moved to fresh container and keep 6 days. Control group were immersed in diluted water for 24 h, and keep 6 days similarly. Thigh and dorsal skins were sampled and conducted water mermeability test using a modified Ussing chamber. Ventral and dorsal skins were conducted histopathological examination. And collected blood was conducted biochemical test. To obtain Bd infection, we examined skin of all frogs, and conducted nested PCR of 5 frog skins (infection group:3, control group: 2). Three of 5 frogs of infection group showed severe skin sloughing, and 1 frog was assossiated with anorexia and lethargy. Two of 5 frogs of infection group were obtained Bd infection by histopathological examination or nested PCR. Disruption of water regulatory function via thigh skin was observed: water absorption associated arginine vasotocin (AVT) reactive AQPa2s(AQP6vs) significantly decreased, however, water absorption associated with AQP3 that permeable with or without AVT stimulation significantly increased. Dorsal skin water permeability was not observed in all animals. Histologically, thicknning of stratum corneum and skin epidermal hyperplasia of both thigh and dorsal skin were observed in infected frogs, but not detected significant differences. The osomorality, Na, K and Cl level decreased slightly, and Mg and Ca level significantly decreased in biochemical test.