Kondo Masakazu

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.

Two types of stratified (two-layered) granules (type 1, NG1a; type 2, NG2) in the neutrophils of inshore hagfish Eptatretus burgei showed eosin-positive (positive site: outer layer (L1) of NG1a and inner layer (L0) of NG2). In contrast, both eosin-positive sites have been reported to be basophilic. From present result, it is clarified that both sites exhibit as follows: L1 of NG1a, eosinophilic and mataazurophilic; L0 of NG2, eosinophilic, orthomethylenophilic and mataazurophilic. The inshore hagfish neutrophils phagocytosed zymosan particles, in vitro. All blood cells showing phagocytosis were identified as neutrophil.

Neutrophil granules (NG) of adult (mature) far eastern brook lamprey Lethenteron reissneri [L. sp. S (souther form)] were classified into two types of stratified [two-layer; inner layer (L0) and outer layer (L1)] granules (type 1, NG1a; type 2, NG2), like as larva (ammocoetes) of this species. The L0 of NG1a and L0 of NG2 in adult neutrophils were chromophobic, and L1 of NG1a and L0 of NG2 showed chromatophilic. The stainability of those chromatophilic layers with May-Grünwald (MG), Giemsa, or MG-Giemsa were varied [chromophobic, eosinophilic or basophilic (orthomethylenophilic or metaazurophilic)] with a slight difference to larva. As seen in larval neutrophils, these layers were positive for alkaline phosphatase,α-naphtyl acetate esterase and Sudan black B, and some enzymes (acid phosphatase, ꞵ-glucuronidase, and naphthol AS-D chloroacetate esterase) were detected in the L0 of NG2. Alpha-naphtyl acetate esterase was positive in L0 of NG2 of adult neutrophils. Both larval and adult neutrophils lacked peroxidase.

Based on the findings of past literature, we speculated the existence of two types of stratified [two-layer; inner layer (L0) and outer layer (L1)] granules in the neutrophils (granulocytes; blood cells) of amphioxus (Cephalocordata), especially Branchiostoma japonicum. Type 1 neutrophil granules (NG1a) consist of chromophobic L0 and chromatophilic L1. Other type (type 2; NG2) have chromatophilic L0 and chromophobic L1. The L1 of NG1a and L0 of NG2 show metaazurophilic (purple to blackish purple) in Giemsa staining preparation. Ultrastructurally, these two granule types will not be separately identified. These granules contain intragranular particles (granulons, g) in the L0 of granules. At least, three types of granulons (g-1, g-2, and g-3) are recognized in a granule and localized in L0. Two of them form an agglomerate with short rod shape and two layers (inner layer consisted by g-2, and outer layer by g-3). The agglomerate have been considered as tubular structure or microtubule (misinterpretation). Neutrophils of cyclostome (hagfish and lamprey) also have NG1 [NG1a or NG1b (chromophobic L0 and L1)] and NG2. Further, neutrophil granules of cyclostome also contain granulons (without forming of agglomerate).