Cytochemistry of leukocytes from the family Macropodidae

The cytochemical staining characteristics of leukocytes from four species of Macropodidae and one species of Potoroidae were studied to investigate the cellular composition of leukocytes within the Macropodidae family and determine markers that may be useful for identifying cell lineage. Blood smears from the tammar wallaby (Macropus eugenii), the western grey kangaroo (Macropus fuliginosis), the red kangaroo (Macropus rufus), quokka (Setonix brachyurus) and a potoroid species, the woylie (Bettongia pencillata) were examined following reaction for Sudan Black B (SBB), peroxidase (PER), chloracetate esterase (CAE), α-naphthyl butyrate esterase (NBE) and alkaline phosphatase (ALP). Similar to domestic animal species, the neutrophils and eosinophils of macropodid species stained for both SBB and PER while monocytes and lymphocytes showed little or no reaction. CAE and NBE, however, were not useful as markers for macropodid neutrophils and monocytes, respectively. Significant variation between species was seen in ALP content. Tammar wallabies and quokkas demonstrated strong ALP activity within their neutrophils and eosinophils. In contrast, western grey kangaroos and red kangaroos contained no ALP activity in any of their leukocytes and staining in woylies was limited to weak reaction within some eosinophils.     

Ultrastructure of nonspecific cytotoxic cells in teleosts. I. Effector-target cell binding in a marine and a freshwater species (Seabream: Sparus aurata L., and Carp: Cyprinus carpio L.)

Background: Fish cytotoxic effectors form a cell population whose ultrastructure and properties of conjugation with target cells have not been completely established. We report the ultrastructure of the non-specific cytotoxic cells in a seawater teleost (Sparus aurata L.) and compare it to a freshwater species (Cyprinus carpio L.). Methods: Blood leucocytes were incubated with HeLa or B16 melanoma cells. Samples were processed for transmission electron microscopic study. Results: Conjugates consisting of leucocytes binding targets were regularly observed after 30 min, 1 hr, or 2 hr of incubation. In both species leucocytes binding to targets showed ultrastructural features of either monocyte-like or lymphocyte-like cells. Monocyte-like cells usually appeared flattened against the targets and seemed to enclose fragments of the target to form cytoplasmic vesicles and the content of their scarce cytoplasmic granules seemed to be delivered into these vesicles. In the seabream lymphocyte-like cells, dense cytoplasmic granules occurred only occasionally, and neither microvilli nor cell processes were present at the contact areas with the targets. In the carp, the contacts were more numerous and formed regularly interdigitating contact areas and the lymphocytes showed granules with characteristic dense and fibrillar contents. Conclusions: We conclude that seabream and carp have a leucocyte cell population with ultrastructural features of either monocytes or lymphocytes showing nonspecific cytotoxic ability. © 1994 Wiley-Liss, Inc.     

Morphology, cytochemistry and immunocytochemistry of the circulating granulocytes of the loggerhead sea turtle Caretta caretta

The eosinophils and heterophils of loggerheads display a similar rounded shape, an eccentric and elongated or slightly lobulated nucleus and a cytoplasm containing acidophilic pleomorphic granules. The characterization of the different types of granulocytes from nine Mediterranean loggerheads (Caretta caretta), based on cytochemical and immunocytochemical reactions and ultrastructural studies, is reported. On the basis of their positivity after myeloperoxidase, alkaline phosphatase and chloroacetate esterase reactions (typical of mammalian neutrophils), the granulocytes were classified as heterophils, while those myeloperoxidase-negative, major basic protein-positive granulocytes (like mammalian eosinophils), displaying a weak expression of interleukin 5 (a growth and differentiation factor, activator and chemoattractant for eosinophils), were classified as eosinophils. The immunocytochemical and TEM studies allowed the identification of these two granulocyte types, both of which show an eosinophilic reaction with May–Grünwald–Giemsa, periodic acid–Schiff and neutral red staining positivity.     

Cell-mediated immune response and T-like cells in thymectomized Oncorhynchus mykiss (Walbaum) infected with or vaccinated against the pathogenic haemoflagellate Cryptobia salmositica Katz, 1951

 T-cell-mediated delayed-type hypersensitivity (T_DTH) reaction was detected in Cryptobia salmositica-infected intact and thymectomized (2 months post-thymectomy) Oncorhynchus mykiss (Walbaum). Both groups of fish showed significant induration at the site of C. salmositica antigen injection at 8, 12 and 16 weeks post-infection. A significant difference was not observed in T_DTH reactions between the thymectomized and intact (control) infected fish. The total numbers of circulating leucocytes detected in infected thymectomized fish were significantly lower than those found in infected sham-thymectomized fish. The numbers of T-like cells determined (using alpha-naphthyl acid esterase assay) in thymectomized fish (9 months post-thymectomy) were similar to those seen in intact fish prior to and at 4 weeks after vaccination with an avirulent strain of C. salmositica. At 2 weeks after challenge with the pathogen the numbers of T-like cells in intact vaccinated fish increased significantly (P < 0.01) and remained high for the duration of the study (15 weeks). However, in vaccinated thymectomized fish the numbers of T-like cells remained low after parasite challenge. These results suggest that thymectomy in adult rainbow trout did not lower T-cell-mediated delayed-type hypersensitivity; however, it reduced the numbers of circulating leucocytes and retarded the proliferation of T-like cells after antigenic stimulation. Received: 18 September 1995 / Accepted: 15 March 1996     

Ultrastructural study of the blood cells of the coelacanth Latimeria chalumnae (Rhipidistia: Coelacanthini)

The blood cells in the renal capillaries of the coelacanth Latimeria chalumnae Smith were studied by transmission electron microscopic methods. On the basis of ultrastructural similarities of cytoplasmic granules of the leukocytes and by comparison with those of the fish and mammalian cells, erythrocytes and three types of granular leukocytes, namely neutrophils, eosinophils and basophils, and three types of agranular leukocytes, i.e., lymphocytes, monocytes and thrombocytes are characterized. The presence of granular and agranular leukocytes in the blood of Latimeria suggests that these cells appeared early in vertebrate evolution. The display of nuclear blebs on the cytoplasmic phase of the nuclear membrane and the presence of nuclear fragments in the cytoplasm of some erythrocytes suggest that these cells undergo apoptosis in order to delete older erythrocytes from the blood stream. The relatively small size of its nucleated erythrocytes and the striking resemblance of the ultrastructural features of its leukocytes to those of higher vertebrate leukocytes support the view that Latimeria is a close living relative of tetrapods.     

The early history of the eosinophil

In 1879 Paul Ehrlich published his technique for staining blood films and his method for differential blood cell counting using coal tar dyes and mentions the eosinophil for the first time. Eosin is a bright red synthetic dye produced by the action of bromine on fluorescein and stains basic proteins due to its acidic nature. It was discovered in 1874 by Heinrich Caro, Director of the German chemical company Badische Anilin‐ und Soda‐Fabrik. Ehrlich introduced the term ‘eosinophil’ to describe cells with granules (which he called alpha‐granules) having an affinity for eosin and other acid dyes. He also observed black‐staining, indulinophilic, beta‐granules in bone marrow‐derived eosinophils, which were probably immature crystalloid granules in eosinophil myelocytes. Ehrlich described the features of the alpha‐granule and the cell's distribution in various species and tissues. He speculated correctly that the alpha‐granule contents were secretory products and described several causes of eosinophilia including asthma, various skin diseases, helminths and reactions to medications. However, the cell was almost certainly observed by others before Ehrlich. In 1846 Thomas Wharton Jones (1808–1891) described ‘granule blood cells’ in the lamprey, frog, fowl, horse, elephant and man. He ‘borrowed’ the term granule cell from Julius Vogel (1814–1880) who had observed similar cells in inflammatory exudates. Vogel in turn was aware of the work of the Gottlieb (Théophile) Gluge (1812–1898) who used the term ‘compound inflammatory globules’ to describe cells in pus and serum. Almost 20 years before Ehrlich developed his staining methods, Max Johann Sigismund Schultze (1825–1874) performed functional experiments on coarse granular cells using a warm stage microscopic technique and showed they had amoeboid movement and phagocytic abilities. Although these early investigators recognised distinct granular cells Ehrlich's use of stains was a landmark contribution, which heralded modern studies on eosinophils and other blood leucocytes.     

A lymphoproliferative disorder of granular lymphocytes with a novel phenotype and suppressor function

In this study we have identified and characterized an expanded granular lymphocyte population in a patient with anemia and granulocytopenia. Granular lymphocytes were identified through the presence of cytoplasmic azurophilic granules, the dispersed granular pattern of cytochemical staining for acid hydrolases, and the ultrastructural localization of acid phosphatase within the granules. The surface phenotype of the granular lymphocytes was E⁺, FcR^–, Leu 4⁺, Leu 2⁺, D 12⁺ (Leu 15⁺), OKM1⁺, and Leu 7⁺. This phenotype has not been reported previously in patients with similar features. Functional studies on FACS-purified populations showed that the patient's granular lymphocytes responded poorly to T-cell mitogens and were inefficient in NK and ADCC assays but exerted a potent suppressor effect on both T-cell proliferation and B-cell differentiation. The phenotype and functions of the expanded granular lymphocyte population correspond to those of a subset of normal E rosette-forming granular lymphocytes.This work was supported by Grants CA 16673 and CA 13148 awarded by the National Cancer Institute, Grant DRR-RR32 from the National Institutes of Health, and Grant AI 18745 from the National Institute of Allergy and Infectious Diseases. Alan Landay is the recipient of NIH Grant T32AI07051.     

Haematology, morphology and blood cells characteristics of male and female Siamese fighting fish (Betta splendens)

The aim of the present study was to obtain baseline data on blood cell size, morphology and haematological parameters in Siamese fighting fish (Betta splendens) since there is limited information in the published literature. Blood samples from the caudal vein of apparently healthy Siamese fighting fish (male: n = 40 and female: n = 36) were collected. Haematological values of the blood samples were determined using standard techniques. The morphological features of blood cells were described according to observations made by light microscopy. The various types of blood cells measurement were carried out with the help of a stage and an ocular micrometre at a magnification of ×1,000. Erythrocytes, thrombocytes and four types of leucocytes: lymphocytes, monocytes, heterophils and eosinophils, were distinguished and characterised. The average size of the erythrocyte cell and nucleus was 97.33 and 16.28 μm², respectively. Results showed a positive correlation between erythrocyte size and nucleus size for Siamese fighting fish (r = 0.470, p < 0.01). We also found sex-dependent differences for total white blood cell count, lymphocytes and heterophils in Siamese fighting fish (p < 0.05). Statistical analysis revealed that differences in other haematological parameters and blood cell morphology, between male and female fish were not statistically significant (p > 0.05).     

Cytochemistry and morphology of granulocytes of the caecilian <Emphasis Type="Italic">Siphonops annulatus</Emphasis> (Amphibia,Gymnophiona)

The caecilians (Amphibia, Gymnophiona) constitute one of the least known groups of terrestrial vertebrates because most species live underground in quite inaccessible environments. Siphonops annulatus is an exclusively fossorial species and is the most extensively distributed caecilian in South America. Little is known of this order concerning circulating granulocytes, including their morphological and cytochemical structure and ultrastructure. This paper is part of a project covering the study of granulocytes in representative species of the order Amphibia. Blood extensions were carried out and submitted to Leishman, Toluidine Blue, Periodic acid Schiff, Sirius Red and hydrogen o-toluidine peroxide methods. Part of the samples was prepared for conventional transmission electron microscopy. Among granular leukocytes, mature and immature neutrophils and eosinophils were identified, plus basophils. The most frequent granulocyte encountered in S. annulatus peripheral blood is the neutrophil. This is a cell with a hyper-segmented nucleus and with a very clear cytoplasm when compared to the eosinophil, which presents large cytoplasmic acidophilic granules. On the other hand, the basophils present basophilic and metachromatic granules. Glycogen was detected in the cytoplasm of the neutrophils and eosinophils, while basic protein rich in amino acids was observed in the eosinophil’s granules. Myeloperoxidase activity was detected in the cytoplasm of the neutrophils and eosinophils. Neutrophils were ultrastructurally detected with three types of small granules: eosinophils with large and small spherical granules and basophils with large spherical granules with lamellate structures.     

Embryonic development of goldfish (Carassius auratus): A model for the study of evolutionary change in developmental mechanisms by artificial selection

Background: Highly divergent morphology among the different goldfish strains (Carassius auratus) may make it a suitable model for investigating how artificial selection has altered developmental mechanisms. Here we describe the embryological development of the common goldfish (the single fin Wakin), which retains the ancestral morphology of this species. Results: We divided goldfish embryonic development into seven periods consisting of 34 stages, using previously reported developmental indices of zebrafish and goldfish. Although several differences were identified in terms of their yolk size, epiboly process, pigmentation patterns, and development rate, our results indicate that the embryonic features of these two teleost species are highly similar in their overall morphology from the zygote to hatching stage. Conclusions: These results provide an opportunity for further study of the evolutionary relationship between domestication and development, through applying well‐established zebrafish molecular biological resources to goldfish embryos. Developmental Dynamics 242:1262–1283, 2013. © 2013 Wiley Periodicals, Inc.     

Antibody response of goldfish to a protein immunogen and to haptenic determinants

The serum antibody response of goldfish (Carassius auratus) to protein immunogen (bovine serum albumin, BSA) and haptenic determinants was studied. The goldfish antibody response was compared with rabbit antibody response to similar antigens. The goldfish received BSA only or BSA with one of three haptens coupled to it. Arsanilic acid, sulphanilic acid and para-amino-benzoic acid were used as haptens. Precipitation and passive haemagglutination were used to demonstrate the presence of antibody. Only one size of antibody could be demonstrated to all antigens (13.2S₂₀). Two populations of antibody could be demonstrated by the use of electrophoresis, and both populations of antibody reacted with BSA in fish receiving only BSA. In fish receiving BSA-hapten, the BSA antibody was found in the slower migrating population, while the haptenic antibody was found in the faster migrating population. The fish antibodies to all immunogens were poor precipitins, but gave titres similar to rabbit antibody when tested by passive haemagglutination. The specificity of fish protein and haptenic antibody is similar to rabbit antibody.     

Molecular and functional characterization of kita and kitla of the goldfish (Carassius auratus L.)

Kit ligand and its type III tyrosine kinase receptor Kit promotes the survival, proliferation and differentiation of progenitor cells involved in mammalian myelopoiesis. In this study we report on the molecular and functional characterization of kit receptor A (kita) and kit ligand A (kitla) from the goldfish. Both kita and kitla were ubiquitously expressed in goldfish tissues, with higher mRNA levels observed in the kidney and spleen, the major hematopoietic organs of fish. Furthermore, both kita and kitla expressions decreased in a time-dependent manner in goldfish primary kidney macrophage (PKM) cultures, as progenitor to macrophage development progressed, and the highest expressions of both the receptor and ligand were observed in sorted progenitor cell populations. Activation of mature macrophage cultures increased both kita and kitla expressions. Kit ligand A induced chemotactic response, proliferation and survival of PKM cells in a dose-dependent manner, but did not induce differentiation of early PKM cells. These results are consistent with the role of kita and kitla during myelopoiesis of higher vertebrates and suggest a conserved mechanism of macrophage development throughout vertebrates.     

The distribution of Neuropeptide Y-immunoreactive neurons and nerve fibers in the forebrain of the carp Cyprinus carpio L.

The present study reports the distribution of Neuropeptide Y (NPY)-immunoreactive neurons and fibers in the forebrain of the adult carp Cyprinus carpio L.. Serial Nissl-stained sections were used for cytoarchitecture and identification of anatomical structures. Immunostaining of NPY-containing neurons and fibers was used as neurochemical marker and tool for comparison with other species, including the goldfish.The general outline of the cytoarchitecture of the carp forebrain is similar to that of other Cypriniformes. However, using NPY immunohistochemistry, we found several specific differences with the goldfish, especially in the diencephalon. In the hypothalamus of the carp NPY-immunoreactive (NPYir) neurons were identified in the n. dorsolateralis thalami, and in the n. ventralis lateralis thalami. In the same location, we observed the n. anterior hypothalami and the n. preglomerulosus pars lateralis, described in the goldfish, as parts of n. prerotundus. However, in the carp we were not able to identify a n. preglomerulosus pars medialis, a n. preglomerulosus pars medialis commissuralis and a n. glomerulosus. We describe a n. rotundus, in which we did not find substructures typical of the goldfish.Further differences with the goldfish, trout and salmon were also noted.     

Kudoa iwatai and two novel Kudoa spp., K. trachuri n. sp. and K. thunni n. sp. (Myxosporea: Multivalvulida), from daily consumed marine fish in western Japan

Infection of marine fish by certain myxosporean species of the genus Kudoa results in unsightly cyst formation in the trunk muscle or post-mortem myoliquefaction, causing a great economic loss to aquaculture industries, capture fisheries, and fish dealers. In addition, consumers encountering unsightly Kudoa cysts in fish fillets believe them to be unknown foreign materials acquired during processing. To identify prevalent Kudoa spp. encountered in daily life by the Japanese population, fresh fish slices (sashimi) or fish fillets with whitish spots were collected during a 7-month period (May to December 2008) at local markets in the city of Yamaguchi, western Japan. Kudoa cysts were found in three Japanese seaperches (Lateolabrax japonicus), two black sea bream (Acanthopagrus schlegelii), two Japanese jack mackerel (Trachurus japonicus), and one albacore (Thunnus alalunga). Kudoa iwatai was identified in all the examined Japanese seaperch and black sea bream from Japan’s Inland Sea, as assessed by morphology and genetic analysis of the 18S and 28S ribosomal RNA gene (rDNA). Kudoa trachuri n. sp. from two Japanese jack mackerel fished in the Japanese Sea off Nagasaki and Kudoa thunni n. sp. from one albacore fished in the Pacific Ocean had a spore, which was semiquadrate in shape in apical views and ovoid in lateral views, with four equal shell valves and drop-like polar capsules. Scanning electron microscopy revealed that these three Kudoa species had different types of small projections at the apex of each valve. The 18S and 28S rDNA sequences of K. trachuri n. sp. and K. thunni n. sp. were found to be closely related to those of Kudoa crumena; however, these sequences were distinct in each of the species, which additionally exhibited different morphological features.     

Ultrastructure of pulmonary alveoli and macrophages in experimental Legionnaires' disease

Guinea pigs, rhesus monkeys and marmosets infected with Legionella pneumophila in small particle aerosols developed an acute fibrinopurulent bronchopneumonia. Changes from 24 hr included exudation into alveoli of protein-rich, often fibrinous fluid and many polymorphonuclear leucocytes (PMN) and macrophages. Damage to alveolar capillary endothelium consisted of widespread cytoplasmic swelling and vesiculation, but necrosis of endothelium and the associated alveolar epithelium was focal and less common. Phagocytosis of L. pneumophila organisms was predominantly by macrophages, but the bacteria were also seen in PMN. Free organisms were present in alveoli and capillary lumina at all stages of the infection but were not observed in lung parenchymal cells. Some infected macrophages and PMN became necrotic and lysed to release intact bacteria. In all species of experimental animal, intracytoplasmic aggregations of granular material, believed to be glycogen, were seen frequently in macrophages and PMN which had phagocytosed L. pneumophila. These deposits of glycogen may reflect either an increased energy demand by the host cell or an interference with its carbohydrate metabolism.     

Morphological and morphometric study of blood leucocytes from Chaetophractus villosus (Mammalia Dasypodidae)

The aim of this work was to study some morphological and morphometric parameters of leucocytes from blood smears of adult armadillosChaetophractus villosus (Mammalia, Dasypodidae). We also analysed the significance of the different sources of variation in these measurements. Blood samples were obtained through heart puncture, using plastic syringes without anticoagulant, in anaesthetised, wild, healthy animals (n=25). Two blood smears of each animal were stained with May Grünwald-Giemsa. Maximum and minimum diameters were measured for each type of leucocyte (n=10 for the most abundant ones: neutrophils and lymphocytes, andn=5 for others: eosinophils, basophils and monocytes). A hierarchical decomposition of the different sources of variation (animals, samples and cells) was carried out using pure model II nest ANOVA. This analysis was applied to both measured parameters. The variation components related to cells were always higher than those related to animals. Estimation of the selected parameters was only marginally changed between samples of the animal. The nuclei of both neutrophils and eosinophils were morphologically similar, generally with four or more lobes. The cytoplasm and neutrophils was only lightly stained. Lymphocytes are the smallest cell type, whereas basophils and lymphocytes are the roundest ones. Eosinophils and basophils are the cell types most variable in size. We suggest that for further studies the effort of sampling be aimed at measuring a larger number of cells from a moderate number of animals. Our results should represent the normal morphological and morphometrical characteristics of leucocytes of wild, healthy animals of this species.     

The in-vitro interaction of eosinophils,neutrophils, macrophages and mast cells with nematode surfaces in the presence of complement or antibodies

The adherence in vitro of leucocytes to the surface of various stages in the life cycle of T. spiralis and N. brasiliensis in the presence of serum was examined. Considerable differences were observed in the behaviour of mast cells, eosinophils, neutrophils and macrophages in this interaction. Mast cells adhered for a short time, did not flatten onto the surface and did not degranulate. Adherence ceased after 4–6 hr. Eosinophils adhered within minutes to the surface of worms, flattened and degranulated; only their cytoplasmic remnants could be seen on the worms' surface after 24 hr in culture. In contrast, only a small area of the cytoplasmic membrane of neutrophils flattened on the surface of the worms and adherence ceased after 2–24 hr. The NBT conversion reaction showed a positive deposit at the interface between neutrophils and parasites during neutrophil adherence. This deposit remained as “foot prints” on the surface of the nematodes following neutrophil detachment. This positive NBT reaction occurred only with neutrophils and not with eosinophils, mast cells or macrcphages. Macrophages adhered permanently to the surface of these worms, they did not flatten and retained their integrity. Under the light microscope the cytoplasmic inclusions appeared to decrease in size during culture. Electron microscopy revealed the presence of fewer granules and an increased number of vacuoles in later cultures of macrophages. These findings are discussed in relationship to the immuno-pathology of nematode infection in vivo.