Distribution of Flavobacterium Psychrophilum and Its Gyra Genotypes in a River
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Fisheries Science (2019) 85:913–923 https://doi.org/10.1007/s12562-019-01355-7 ORIGINAL ARTICLE Biology Distribution of Flavobacterium psychrophilum and its gyrA genotypes in a river Erina Fujiwara‑Nagata1 · Yuki Shindoh2,3 · Michitaka Yamamoto4 · Takashi Okamura4,5 · Kentaro Takegami4,5 · Mitsuru Eguchi1 Received: 31 March 2019 / Accepted: 18 August 2019 / Published online: 10 September 2019 © The Author(s) 2019 Abstract Flavobacterium psychrophilum can be divided into three genotypes, G-C type (ayu type), A-C type (multi-fsh type), and A-T type (salmon-trout type), by two single-nucleotide polymorphisms in the DNA gyrase subunit A (gyrA) gene. We isolated F. psychrophilum from various samples collected from the lower basin of a river fowing into Lake Biwa, as a model for bacterial survey, in Shiga Prefecture in June, September, and December from 2010 to 2013 and investigated their gyrA genotypes. All three types of F. psychrophilum were isolated in June when ayu went up the river from the lake. In September, ayu gathered in the lower river basin for spawning and became high in density, almost all of the isolates were of the G-C genotype and the A-T genotype was never isolated. In December, only the A-T type was isolated from the river samples, when Biwa trout Oncorhynchus masou rhodurus came to the river to spawn and were present in the sampling area. In accordance with the seasonal change of the host fsh species in the river, the genotype of F. psychrophilum isolated from the environment seems to have changed as well. Keywords Flavobacterium psychrophilum · Bacterial cold-water disease · Ayu · Plecoglossus altivelis · Lake Biwa basin · gyrA genotyping · Distribution Introduction (Bernardet et al. 1996). The frst report of F. psychrophi- lum infection was from North America in the 1930s. Since Bacterial cold-water disease (BCWD) and rainbow trout then, many countries have been afected, with economic fry syndrome (RTFS) are a problem mainly with salmonid losses reaching millions of dollars (Antaya 2008). The dis- fsh such as coho salmon Oncorhynchus kisutch, rainbow ease caused by F. psychrophilum has been reported as the trout Oncorhynchus mykiss, and ayu Plecoglossus altivelis peduncle disease of rainbow trout (Davis 1946), and the altivelis. The causative agent Flavobacterium psychrophi- bacterium was isolated from juvenile coho salmon (Borg lum is a Gram-negative, aerobic, long, rod-shaped bacterium 1960). In Japan, the ayu afected by F. psychrophilum was * Erina Fujiwara-Nagata 1 Faculty of Agriculture, Kindai University, Nakamachi, [email protected] Nara 631-8505, Japan Yuki Shindoh 2 Graduate School of Agriculture, Kindai University, [email protected] Nakamachi, Nara 631-8505, Japan Michitaka Yamamoto 3 Present Address: FUKAEKASEI CO., LTD., [email protected] Kobe 651-2271, Japan Takashi Okamura 4 Shiga Prefectural Fisheries Experiment Station, Hikone, [email protected] Shiga 522-0057, Japan Kentaro Takegami 5 Present Address: Shiga Prefecture Fisheries Division, Otsu, [email protected] Shiga 520-8577, Japan Mitsuru Eguchi [email protected] Vol.:(0123456789)1 3 914 Fisheries Science (2019) 85:913–923 found in 1987 (Wakabayashi et al. 1994). Since then, almost to accurately understand the distribution of F. psychrophilum all parts of Japan have been afected by F. psychrophilum, and the potential impact on fsheries owing to its occurrence and it has spread not only in aquaculture facilities but also in in important fsh species. natural rivers of Japan. F. psychrophilum has been isolated To contribute to the establishment of efective preven- from various wild fsh including akaza Liobagrus reinii, tion measures for BCWD occurring in rivers, the purpose oikawa Zacco platypus, ookuchibass Micropterus salmoides, of this study was to elucidate the distribution status of each kadayashi Gambusia affinis, kawamutsu Nipponocypris genotype of F. psychrophilum in the natural environment. temminckii, numa-chichibu Tridentiger brevispinis, and We chose a river which fows into Lake Biwa as a model for honmoroko Gnathopogon caerulescens (Arai et al. 2006; bacterial survey, because it was clear and supported vari- Izumi et al. 2007; Fujii et al. 2009), and from environmental ous fsh species including ayu, salmonids, and cyprinids. samples such as river water and algae (Amita et al. 2000). The landlocked lake-produced ayu living in Lake Biwa, the Treatments designed to cure BCWD include chemother- largest freshwater lake in Japan, primarily spawns and dies apy using sulfsozole sodium (Ninomiya and Yamamoto in the downstream area of the infowing rivers from Septem- 2001) and forfenicol, which are the ofcially approved drugs ber to October. The larvae grow in the lake from October to cure BCWD in ayu, and warmed water (28°C) treatment to February of the following year, and from the beginning (Yamamoto et al. 2001; Sugahara et al. 2010a). However, of March they return to the rivers around the lake. In the chemotherapy is restricted and undesirable because of the present study, samples of ayu, other fsh species, river water, occurrence of drug-resistant bacteria (Rangdale et al. 1997; aquatic insects and their nests, algae adhering to the surface Bruun et al. 2000, 2003). In addition, warmed water treat- of stones, sand, and foating mud were collected, separated, ment may cause heat stress, oxygen defciency, water qual- and subjected to bacterial isolation. For the isolates identi- ity degradation, and other diseases. Furthermore, although fed as F. psychrophilum, gyrA genotyping was performed. both methods are efective for closed systems in onshore aquaculture sites, it is impossible to use them in open natu- Materials and methods ral rivers. Thus, prevention of BCWD is an essential and realistic approach to controlling its spread. Many countries Field sampling and companies have been working toward the development of vaccines against BCWD such as injection, oral treatment, Our feld surveys were conducted in the lower basin of the immersion, and live vaccines; however, they have not yet river, which fowed into the northwestern part of Lake Biwa, reached the practical stage. in Shiga Prefecture in June, September, and December of In order to prevent the occurrence of BCWD, it is important 2010–2013 (sampling was missed in December 2010 and to elucidate the infection route of this bacterium. Attempts June 2012). have been made to investigate the distribution of the bacte- Fish including ayu, environmental samples such as river rium, but satisfactory results have yet to be obtained, in part water, aquatic insects and their nests, algae adhering to the because of its host specifcity (Izumi et al. 2003; Yoshiura et al. surface of rocks, and mud from the bottom of the river were 2006; Fujiwara-Nagata et al. 2012). We have reported that F. sampled. With authorization from Shiga Prefecture, fsh psychrophilum can be divided into four genotypes using sin- caught by electrofshing were kept at low temperatures in gle nucleotide polymorphisms of the DNA gyrase subunit A tanks with ice cubes and quickly transported to our labora- (gyrA) gene (Fujiwara-Nagata et al. 2012). The frst is a G-C tory. We caught only fsh bigger than 5 cm in length. type which was isolated from ayu, and the second is the A-T Water samples were collected using sterilized plastic type which was isolated from salmonid fsh. The third, G-T bottles and transferred to the laboratory at a low tempera- type, and the fourth, A-C type, are multi-fsh types which were ture with ice cubes. Water temperature was measured with isolated from various fsh species including ayu, salmonids, a water temperature gauge. Furthermore, from June 2012 to and cyprinids. The G-C type shows virulence in ayu, but the December 2013, the river water temperature was continu- A-T and G-T types show no virulence in the fsh. The A-C ously recorded by a data logger (UA-002-64, Onset Com- type shows both high and low virulence in ayu, that is, the A-C puter Corporation, MA, USA). type isolated from ayu shows high virulence and the A-C type isolated from other than ayu shows low virulence in ayu. The Sample processing G-T type was isolated from rainbow trout, yamame Oncorhyn- chus masou masou, amago Oncorhynchus masou ishikawae, Fish (except lamprey) and ayu from 2002 to 2005, and the isolation areas were also limited. The use of gyrA genotyping makes it possible to iden- Fish were euthanized by severing the spinal cord. Gill sam- tify the host fsh species of F. psychrophilum. As a next step ples were cut into pieces of ~ 25 mm2 and suspended in 1 ml to prevent the occurrence of BCWD in rivers, it is necessary of phosphate-bufered saline (PBS). When gill samples were 1 3 Fisheries Science (2019) 85:913–923 915 collected, up to fve fsh of the same fsh species were pooled inactivation were performed at 55 °C for 30 min and at 95 °C in one microtube to make one specimen. Samples from inter- for 10 min, respectively. nal organs and skin lesions were collected using a sterile loop and streaked directly onto FLP agar plates (Cepada Polymerase chain reaction (PCR) et al. 2004). PCR was performed targeting the 16S rRNA gene with Lamprey nested PCR (Toyama et al. 1994) and DNA topoisomerase IV subunit B (parE) gene (Izumi and Wakabayashi 2000). Using sterilized dissection scissors and tweezers, the head of When the PCR results were positive for both genes, the iso- the lamprey was cut out, minced, and homogenized in 1 ml late was considered to be F. psychrophilum. of PBS. As much as possible, the meat pieces adhering to For the frst round of 16S rDNA nested PCR, a universal the homogenizer pestles were put in PBS, and the samples primer pair, 20F (5′-AGAGTT TGATC(AC)TGG CTC AG-3 ′) were weighed.