Yasumoto Shinya

The monocytes of lampreys [Lethenteron camtschaticum (adult), L. reissneri (adult), L. hattai (adult, ammocoetes)] were round or oval, low nucleus/cytoplasm ratio, and have striated (two-layered) granules (MoG). The MoG consisted of a basophilic inner layer (L0) and a chromophobic outer layer (L1). The L0 were positive for acid phosphatase, α-naphthyl acetate esterase, naphthol AS-D chloroacetate esterase and Sudan black B. Also, the L0 showed orthochromatic (blue) with toluidine blue. However, ꞵ-glucuronidase, α-naphthyl butyrate esterase and peroxidase were not detected in the monocytes, which were negative for periodic acid Schiff reaction, alcian blue, Sudan III, and oil red O. Kenji Kiyono reported the two types of monocytes (as blood histocytes), real and dubious monocytes, from the blood of hagfish Eptatretus burgeri. He observed both monocyte types in grown-up hagfish, but only real monocytes in undeveloped (details not stated) hagfish. His real monocytes in undeveloped hagfish were speculated as the monocytes of lamprey (mistaking the specimen), and the real monocytes in grown-up hagfish as poorly stained neutrophils. The dubious monocytes of Kiyono were considered as the real monocytes of hagfish.

Artur Pappenheim reported a poor description of the morphology of blood neutrophils from lamprey (Lampetra planeri； adult and ammocoetes) and hagfish (Myxine glutinosa) in Folia Haematologica (volume 8, 1909). Here, we inferred on the morphological characteristics of cyclostome neutrophils observed by Pappenheim based on his descriptions and our previous reports. He recognized neutrophilic leukocytes (mature neutrophils) in lamprey (adult and ammocoetes) and hagfish, and neutrophilic myelocytes (immature neutrophils) in ammocoetes and hagfish. This means the existence of specific granules and azure granules. However, the specific granules were considered as an inner layer (L0) of the striated granule (NG2), and the azure granules outer (L1) layer of the striated granule (NG2). The specific granules (=L0 of NG2) would probably have been stained with triacid (purple), acid dyes, and May-Grünwald·Giemsa (MGG； purple), but not with methylgreen-pyronine (MP). The azure granules (=L1 of NG1a) would also have stained purple with MGG, but negative with triacid, acidic dyes and MP.

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.

Previously, we speculated on the staining principle of triacid staining solution (neutral mixture) containing a dye complex. The principle could be explained solely by the relationship between the stained object and the dye complex. We propose here to extend the staining principles of triacid stain to explain the staining principles of other dye mixtures such as May-Grünwald (MG), Giemsa and MG-Giemsa. In a mixture of acidic and basic dyes, the staining characteristics of the object were presumably determined by the degree of stainability (degree of acidophilicity or basophilicity) of the object, the degree of stainability of both dyes (degree of basophilicity of the acidic dye and degree of acidophilicity of the basic dye), and the ratio and concentration of each dye.

Triacid staining solution (neutral mixture) contains dye complex. Here, we speculate on the structure and characteristics of the dye complex in various triacid staining solutions. It was inferred that the dye complexes in the Ehrlich (methyl green + 2 acid fuchsin, methyl green + 2 orange G, methyl green + acid fuchsin + orange G), Romanowsky (eosin + methylene blue), and Pappenheim (eosin + methylene azure) triacid stains behave as acidic dyes, and that the dye complexes do not dissociate against weakly acidophilic objects, resulting in staining with a complex color (purple). Therefore, it can be said that the objects to which the dye complexes bind without dissociation do not need to be basophilic. When the stained material is highly acidophilic or basic, the dye complex dissociates, and the acidophilic object is stained by the acidic dye and the basiophilic object by the basic dye. The dissociation of dye complexes depends on the degree of acidophilicity and basophilicity of the stained material, and the dissociation depends on the types of dye composing the dye complexes. The dye complex in a triacid stain can be defined as a purple acidic dye in which the bonds between the acidic and basic dyes in the complex can be broken, depending on the acidophilic and basophilic strength of the object.

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,

The term “neutrophilic” and “neutrophilic granule” was first introduced by Paul Ehrlich to identify specific granules of major polymorphonuclear leukocytes (neutrophils). He used an original staining method, triacid stain, for this purpose. The staining solution, called “neutral mixture”, which was mixture of acidic dye solution and basic dye solution, with soluble form in water (not neutral dye). In this paper, we speculate the structure and characteristics of the dye complex in the neutral mixture. The dye complex has free acidic groups and is expected to behave as an acidic dye. The dye complex bound to the neutrophilic granules stains the granules with the color tone of the dye complex (purple) because the acid and basic dyes do not dissociate. On the other hand, in the vicinity of the eosinophilic granules and nucleus, the dye complex dissociates into acid and basic dyes, and the former bind to the acid dye and the latter to the basic dye. It was inferred that this dye complex is not formed during staining with stains containing neutral dyes, such as May-Grünwald (methylene blue eosinate), Wright (polychromtic methylene blue eosinate), and Giamsa (containing azure II eosinate).

Neutrophil granules (NG) of West African lungfish Protopterus annectens were classified into two types of stratified [two-layer; inner layer (L0) and outer layer (L1)] granules (type A, NG-A; type B, NG-B). The L1 of NG-A and both layers of NG-B were chromophobic, and L0 of NG-A showed polychromatophilic [eosinophilic or basophilic (orthomethylenophilic or metaazurophilic)]. The L0 of NG-A showed metachromatic (reddish purple) with toluidine blue. On the other hand, L0 of NG-B were positive for acid phosphatase, α-naphthyl acetate esterase, α-naphthyl butyrate esterase and naphthol AS-D chloroacetate esterase. Both types of granules were negative in periodic acid Schiff reaction, alcian blue, Sudan black B, Sudan III, and oil red O. Alkaline phosphatase, β-glucuronidase, and peroxidase were not detected in either granule.

Structure and development of eosinophilic granules (EG1) in eosinophils from larva (ammocoetes) of far eastern brook lamprey Lethenteron reissneri were speculated. EG1 is stratified granules (two-layer) and consists of inner eosinophilic layer (L0) and chromophobic outer layer (L1). Three subtypes of EG1 are identified based on the optical artificial image (OAI) of inclusion structure (IS) in L0: EG1a, EG1b, and EG1c. The EG1a had no OAI (probably no IS). The EG1b and EG1c contain IS in L0. The OAI of both EG1b and EG1c were larger than IS. The former was round or oval chromophobic area (OAI-1), and the latter was expanded and rugged (three-dimensional) image (OAI-2) surrounded with OAI-1. EG1a was thought to be a prototype of EG1, which would develop into EG1b and then EG1c.

Monocytes were observed in the blood of inshore hagfish Eptatretus burgei. The monocytes were round or oval, high nucleus/cytoplasm ratio, and have several round or oval eosinophilic granules, which show dark red or blackish red color when stained with May-Grünwald (MG), Giemsa, and MG-Giemsa stain. The granules were positive for alkaline phosphatase, acid phosphatase, and α-naphthyl butyrate esterase. Also, the granules show metachromatic (reddish purple) with toluidine blue. However, ꞵ-glucuronidase, α-naphthyl acetate esterase, naphthol AS-D chloroacetate esterase and peroxidase were not detected in the monocytes, and negative for periodic acid Schiff reaction, alcian blue, Sudan black B, Sudan III, and oil red O. The monocytes engulfed many yeast particles (zymosan).

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).

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.

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.

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 granules (two-layer) were observed in the eosinophils (eosinophil granule. EG： type 1, EG1； type 2, EG2) of larva (ammocoetes) of far eastern brook lamprey Lethenteron reissneri collected in a tributary of the Koyagawa River in Yamaguchi Prefecture. The EG1 consisted of inner eosinophilic layer (L0) and chromophobic outer layer (L1). Dark (low light transmittance) inclusion structure (IS), which was various size and morphology (round, oval, rod, or spindle), was observed in the L0 of many EG1 (only one IS in a EG1). The IS was found in the cytochemical staining preparation, but not in the preparation stained with May-Grunwald (MG), Giemsa and MG•Giemsa. Therefore, recognition of IS was affected with the eosin-stained L0 of EG1. The EG1 classified into three subtypes (EG1a, EG1b and EG1c) based on the optical artificial image (OAI) of IS in L0. The EG1a had no OAI (probably no IS). The OAI of both EG1b and EG1c were larger than IS. The former was round or oval chromophobic area (OAI-1), and latter was expanded and rugged (threedimensional) image (OAI-2； chromophobic； round, oval, or rod). The EG1a may be prototype of EG1. The EG1 showed no positive reaction by various cytochemical stains. The EG2 had chromophobic inner layer (L0) and basophilic (orthomethylenophilic) outer layer (L1). Some enzymes (alkaline phosphatase, acid phosphatase, ꞵ-glucuronidase, α-naphtyl acetate esterase, naphthol AS-D chloroacetate esterase) were detected in L0 of EG2. The eosinophils lacked α-naphtyl butyrate esterase and peroxidase.

Two types of stratified [two-layer； inner layer (L0) and outer layer (L1)] granules were observed in the neutrophils (neutrophil granule, NG： type 1, NG1a； type 2, NG2) of larva (ammocoetes) of far eastern brook lamprey Lethenteron reissneri collected in a tributary of the Koyagawa River in Yamaguchi Prefecture. The NG1a consisted of chromophobic L0 and chromatophilic L1. On the other hand, the NG2 had chromatophilic L0 and chromophobic L1. The L1 of NG1a and L0 of NG2 showed a variety of colors [eosinophilic or basophilic (orthomethylenophilic or metaazurophilic)] depending on the staining conditions. These layers were positive for alkaline phosphatase, α-naphtyl acetate esterase and Sudan black B. Some enzymes, such as acid phosphatase, ꞵ-glucuronidase, and naphthol AS-D chloroacetate esterase were detected in the L0 of NG2. The neutrophils lacked α-naphtyl butyrate esterase and peroxidase.