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ABSTRACT. The present study describes the in vitro and in vivo survival of Lactococcus garvieae bacteriophages and the potential of the phage for controlling experimental L. garvieae infection in yellowtail. Anti-L. garvieae phages persisted well in various physicochemical (water temperature, salinity, pH) and biological (feed, serum and alimentary tract extracts of yellowtail) conditions, except for low acidity. In the in vivo, the phage PLgY-16 was detected in the spleens of yellowtail until 24 h after intraperitoneal (i.p.) injection, or the phage was recovered from the intestine of yellowtail 3 h after the oral administration of phage-impregnated feed but undetectable 10 h later. Simultaneous administration of Live L. garvieae and phage enhanced recovery of the phage from the spleen or intestine. The survival rate was much higher in yellowtail that received 1.p. injection of the phage after i.p. challenge with L. garvieae, compared with that of control fish without phage injection. When fish were i.p.-injected with phage at different hours after L. garvieae challenge, higher protective effects were demonstrated in fish that received phage treatment at the earlier time. Protection was also obtained in yellowtail receiving phage-impregnated feed, in which fish were challenged by an anal intubation with L. garvieae. Anal-intubated L. garvieae were detected constantly In the spleens of the control fish, while they were detected sporadically and disappeared from the phage-treated fish 48 h later. On the other hand, orally administered phage was detected at high plaque-forming units from the intestines and spleens of the phage-treated fish until 48 h later. These results indicate that intraperitoneally or orally administered anti-L. garvieae phage prevented fish from experimental L, garvieae infection, suggesting potential use of the phage for controlling the disease.

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Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The viruses have been classified into 4 distinct types based on nucleotide sequence similarities in the variable region (the so-called T4 region) of the smaller genomic segment RNA2 (1.4 kb). Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among the viruses. We have previously demonstrated, using reassortants between striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), that RNA2, which encodes the coat protein, strictly controls host specificity. However, because RNA2 is large, we were unable to propose a mechanism underlying this RNA2-based host specificity. To identify the RNA2 region that controls host specificity, we constructed RNA2 chimeric viruses from SJNNV and RGNNV and tested their infectivity in the original host fish, striped jack Pseudocaranx dentex and sevenband grouper Epinephelus septemfasciatus. Among these chimeric viruses, SJNNV mutants containing the variable region of RGNNV RNA2 infected sevenband grouper larvae in a manner similar to RGNNV, while RGNNV mutants containing the variable region of SJNNV RNA2 infected striped jack larvae in a manner similar to SJNNV. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that these chimeric viruses multiplied in the brains, spinal cords and retinas of the infected fish, as in infections by the parental viruses. These results indicate that the variable region of RNA2 is sufficient to control host specificity in SJNNV and RGNNV.

魚病研究 F i s h P a t h o l o g y , 47 ( 4 ) , 1 2 9 -1 3 6 , 2 0 1 2 . 1 2 © 2 0 1 2 T h e J a p a n e s e S o c i e t y o f F i s h P a t h o l o g y S u r v e i l l a n c e o f T y p e 1 Os t r e i d He r p e s v i r u s ( Os HV -1 )V a r i a n t s i n J a p a n Na t i o n a l Re s e a r c h I n s t i t u t e o f A q u a c u l t u r e , F i s h e r i e sRe s e a r c h A g e n c y , Mi e 5 1 9 -0 1 9 3 , J a p a n 2 ABS T RA CT -Os t r e i d h e r p e s v i r u s 1 ( Os HV -1 ) m V a r i s a v a r i a n t o f Os HV -1 a n d s u s p e c t e d o f b e i n g t h e c a u s a t i v e a g e n t o f a c u t e ma s s mo r t a l i t y e v e n t s o f P a c i f i c o y s t e r s d u r i n g s u mme r s i n E u r o p e s i n c e 2 0 0 8 . I n t h i s s t u d y , t h e d i s t r i b u t i o n o f Os HV -1 wa s s u r v e y e d i n t h e s i x ma i n o y s t e r -p r o d u c i n g a r e a s o f J a p a n , u s i n g P CR t a r g e t i n g a C2 / C6 f r a g me n t i n c l u d i n g ORF 4 . P CR p Ho k k a i d o Na t i o n a l F i s h e r i e s Re s e a r c h I n s t i t u t e , F i s h e r i e s Re s e a r c h r o d u c t s we r e a mp l i f i e d f r o m 1 2 3 o u t o f 1 , 7 1 4 o y s t e r s o f t h r e e s p e c i e s o f Cr a s s o s t r e a ( C. g i g a s , C. s i k a me a a n d C. a r i a k e n s i s ), a n d 2 3 d i f f e r e n t n u c l e o t i d e s e q u e n c e s , s h o wi n g 9 6 % t o 9 9 % s i mi l a r i t y t o t h e r e f e r e n c e Os HV -1 , we r e o b t a i n e d . A l t h o u g h 1 8 s e q u e n c e s a mo n g t h e 2 3 o b t a i n e d p o s s e s s e d a mi c r o s a t e l l i t e d e l e t i o n u n i q u e t o Os HV -1 m V a r , a l l P CR p r o d u c t s c o n t a i n e d t wo c o n s e r v e d n u c l e o t i d e s t h a t we r e s h a r e d wi t h t h e r e f e r e n c e Os HV -1 a n d n o t wi t h Os HV -1 m V a r . He r e , we f o u n d v a r i a b l e t y p e s o f Os HV -1 i n o y s t e r s i n J a p a n , b u t t h e i r n u c l e o t i d e s e q u e n c e s we r e n o t i d e n t i c a l t o t h o s e o f Os HV -1 m V a r . Ke y wo r d s : Os t r e i d h e r p e s v i r u s , Os HV -1 , Os HV -1 m V a r , Cr a s s o s t r e a g i g a s , C. s i k a me a , C. a r i a k e n s i s , J a p a n , P a c i f i c o y s t e r* Co r r e s p o n d i n g a u t h o r E -ma i l : o h s e k o @a f f r c . g o . j p * * P r e s e n t a d d r e s s : He a d q u a r t e r s , F i s h e r i e s Re s e a r c h A g e n c y , K a n a g a wa 2 2 0 -6 1 1 5 , J a p a n