バナメイエビ Litpoenaeus vannameiにおけるVp_PirAB-like毒素抵抗性関連遺伝子の同定

東京海洋大学博士学位論文 平成30年度(2018) 応用生命科学 課程博士 甲第515号指導教員: 廣野育生東京海洋大学201

Report of aquatic animal diseases in Thailand during January – June 2019

Aquaculture is an important industry in Thailand which has been established more than two decades ago. The cultured species are divided into two main groups; shrimp and finfish farming. The major cultured shrimp species are Penaeus vannamei (Pacific white shrimp), P. monodon (black tiger shrimp) and Macrobrachium rosenbergii (giant freshwater prawn), whereas the finfish are Oreochromis sp., Lates calcarifer and Epinephelus sp. Also, ornamental fish such as Cyprinus carpio (Koi carp), Carassius spp. (goldfish), and Betta splendens (fighting fish or betta). Disease outbreaks are the key factor that affect Thailand’s aquaculture production and resulting in economic losses. The infectious diseases in aquaculture are mainly caused by viral and bacterial pathogens. In 2018, the reported shrimp pathogens are white spot syndrome virus (WSSV), yellow head virus (YHV) genotype 1, Taura syndrome virus (TSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VPAHPND), and microsporidian Enterocytozoon hepatopenaei (EHP). On the other hand, the reported pathogens in finfish are Betanodavirus causing viral nervous necrosis (VNN), Tilapia lake virus (TiLV) and Streptococcus sp. etc. In Thailand, the Department of Fisheries (DOF) is the competent authority for various aspects of aquatic animals including aquatic animal health. Strategies to prevent and control diseases in aquatic animals include issuance of legislations/regulations, implementation of biosecurity measures, disease surveillance programs, capacity building, cooperation with international and national organizations. Moreover, DOF has developed contingency plan in dealing with aquatic animal disease emergencies through the provincial fisheries officer. The provincial fisheries officer acts as director of emergency aquatic animal disease control center in each province, while Aquatic Animal Health Research and Development Division (AAHRDD) and Songkhla Aquatic Animal Health Research and Development Center (SAAHRC) serve as disease diagnosis and laboratory testing centers. Because of the above actions, we are capable of preventing and controlling disease outbreaks in the country. But during the occurrence of some diseases, we have no treatment to support and completely solve the problem. Example are viral diseases, unlike bacterial diseases which can be treated by using chemical or drug. Furthermore, there are a few researches that could be applied in farm level. Especially shrimp which has no adaptive immunity, so it is difficult to develop vaccine compare to fish. Therefore, DOF mostly recommended farmers to follow the good management practices on aquatic animal health for promoting sustainable aquaculture

Latest research on acute hepatopancreatic necrosis disease (AHPND) of penaeid shrimps

Acute hepatopancreatic necrosis disease (AHPND) is caused by unique strains of Vibrio parahaemolyticus (VPAHPND) and V. harveyi that have transferrable plasmid carrying the virulent PirAB-like toxin genes. The genomes of VPAHPND strains and V. harveyi from Thailand and Viet Nam, respectively, have been characterized by our group. The genome of VPAHPND strains from Mexico, Viet Nam, and China have also been studied by other groups. We have developed a conventional polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) methods for the detection of AHPND using a primer set that targets the PirAB-like toxin genes of VPAHPND. We have characterized the toxin genes of VPAHPND strains and also constructed a recombinant plasmid (broad host range) carrying PirAB-like toxin genes. Non-VPAHPND strain N7 which does not carry the plasmid and strain FP11 which is carrying a plasmid not coding for the toxin genes were transformed with the plasmid carrying PirAB-like toxin genes. As a result, the transformed N7 and FP11 strains became virulent and killed whiteleg shrimp (Penaeus vannamei) similar to or at par with the virulence of VPAHPND strain. We then fed the whiteleg shrimp with commercial feed containing the formalin-killed VPAHPND strain. After 2 days of feeding, all of the whiteleg shrimp died. These results clearly indicate that the PirAB-like toxin is the virulence factor of VPAHPND. We have been investigating the virulence mechanism of the PirAB-like toxin produced by VPAHPND strains. First, we calculated the copy number of plasmid encoding the PirAB-like toxin genes of several VPAHPND strains. The copy number of the plasmid varied, ranging from 1 to 36 copies. Interestingly, VPAHPND strains carrying low copy number of plasmid were more virulent than VPAHPND strains carrying high copy number of the plasmid. These results imply that the copy number of toxin genes is not an important factor responsible for the degree of virulence of the VPAHPND strains. We are also studying other factors associated with the virulence of PirAB-like toxin. Likewise, we are developing prevention methods against AHPND including the use of formalin-killed cell vaccine, IgY additive in feed, and nano-bubble treatment of rearing water. This paper summarizes the current R&D on the disease