Identification of leukocytes on tissue sections is important to elucidate the mechanism of swim bladder lesions. To determine the best fixative solution for a koi carp swim bladder, the swim bladders were fixed in 10% formalin, Bouin's, MFAA and Davidson's solution. The swim bladders fixed in MFAA or Davidson's solution were severely detached and twisted, whereas those fixed in 10% formalin and Bouin's solution kept their external shape. However, the majority of the 10% formalin-fixed specimens showed detachment of the tunica interna from the tunica externa under the light microscope. Therefore, Bouin’s solution was determined to be the most suitable fixing solution for the swim bladder. Imprints (head kidney-touched slides glass were fixed with Bouin's solution) and tissue sections of head kidney fixed in Bouin’s solution were stained with Mayer’s hematoxylin and eosin (HE) and May-Grünwald·Giemsa (MGG), and the best staining method for leukocytes identification was investigated. In the HE-stained specimens, identification of leukocytes by staining was difficult. On the other hand, MGG-stained specimens could be identified by staining. Fixation with Bouin's solution and MGG staining was determined to be the most suitable method for leukocytes identification in the swim bladder.

Viral edema of carp is caused by Carp edema virus and is a major carp disease listed on the WOAH list of emerging infectious diseases. However, an effective experimental infection method that can control virus copy number has not been established. In this study, we compared three experimental infection methods：immersion infection using gill suspensions from diseased fish (immersion method), infection by rearing water from diseased fish (rearing water method) and co-infection method. Cumulative mortality in the immersion groups ranged from 40 to 60%, while that in the rearing water infected and co-infected groups ranged from 80 to 100%. The rearing water method can be used to control the viral DNA copy number and to perform experimental infections when frozen storage is possible. Thus, we performed the same experimental infection using frozen rearing water and found no disease or mortality, indicating that stable experimental infection using the rearing water method is difficult. The immersion method has a lower mortality rate than the other two methods, and it requires a large amount of gill tissue because of the low number of virus DNA copies that can be obtained from the gill tissue. However, it is possible to control the number of virus DNA copies and to preserve the gill tissue suspension by freezing. Therefore, the immersion method was considered the most suitable for stable experimental infection under the same conditions,

Viral edema of carp (VEC) caused by the carp edema virus (CEV) causes economic losses for Japanese koi farms. In this study, we investigated the infectivity and pathogenicity of a domestic CEV isolate (genogroup IIa) in koi carp, common carp and goldfish. The challenge test consisted of 9 groups (n = 15):3 groups each of koi carp, common carp, and goldfish, at 15, 20 and 25℃. These groups were challenged with CEV (3.0×10^3 copies/µL) in duplicate. All koi carp died in the 15 and 20℃ groups, but all survived in the 25℃ group. The surviving koi carp in 25℃ groups showed high PCR positive rates of 66.7 and 73.3%, with VEC histopathological changes observed. For the common carp, 1 and 2 fish died in the 20℃ groups, but no deaths or VEC symptoms were observed in the 15 and 25℃ groups. In all common carp groups, PCR-positive fish were observed along with histopathological changes. For all goldfish groups, no deaths or VEC symptoms were observed. As with the common carp, PCR-positive fish were found in all goldfish groups, yet no VEC histopathological changes were detected. These results demonstrate infectivity of this CEV strain in koi carp, common carp, and goldfish, but low pathogenicity in common carp and goldfish.