Haematology, cytochemical and ultrastructural characteristics of blood cells in leopard (Panthera pardus)

The leopard (Panthera pandus) is an endangered Asian big cat found in Thailand and also listed in the CITES, Appendix 1. Blood samples from 17 leopards (six males and 11 females) were collected from the cephalic vein for haematology, cytochemical and ultrastructural studies. Red blood cells (RBCs) were slightly anisocytosis, ranged 5–6.5 μm with 5.6 μm mean diameter. They were easy to form rouleaux, blunt end crenation and some RBCs contained Heinz bodies. The male leopards had a significantly higher packed cell volume (39.3 ± 7.5%) values and absolute eosinophil number (1.658 ± 1.483 × 10⁹/l) than the females (30.7 ± 3.2%; 0.965 ± 0.611 × 10⁹/l). The cytochemical results were: sudanophilia and myeloperoxidase in neutrophils and some monocytes; nonspecific esterase (alpha-naphthyl acetate esterase, ANAE) in the granules of eosinophils, some lymphocytes, some monocytes and platelets; beta-glucuronidase (βG) in granules of basophils, monocytes and some lymphocytes. The ANAE and βG reaction were detected inter-granular of eosinophils. More detailed morphological aspects of all blood cells were observed by means of scanning electron microscope and transmission electron microscope (TEM). The many round granules and homogeneous content under TEM were characteristics of leopard eosinophils. The engulfed RBCs by neutrophils were detected under TEM in one male leopard. This study provides a guide for the haematology, identification of the morphology, cytochemistry and ultrastructure of blood cells in leopards that is useful for zoological veterinarians in leopard conservation.     

Morphological, cytochemical, and ultrastructural observations on the blood cells of the reptile Tupinambis merianae (Squamata)

Tupinambis merianae is a lizard of the Brazilian fauna and belongs to the Squamata order. Blood cell data are scarce. Blood samples from six specimens of adult T. merianae were used to evaluate some hematological parameters. For structural analysis, 2 ml of blood was collected in the presence of EDTA. Part of the blood was used for preparing blood smears which were submitted to the methods of Leishman, Laür, and toluidine blue, and the cytochemical reactions of periodic acid-Schiff, sirius red, sudan black B, and ortho-toluidine-H₂O₂. The remainder was centrifuged and the leukocyte buffy coat was fixed in Karnovsky’s fluid for electron microscopy examination. The following blood cells were identified: mature and immature erythrocytes, spherical and elliptical thrombocytes, heterophilic granulocytes, eosinophils and basophils types I and II, lymphocytes, monocytes, and azurophilic cells. The more significant results obtained were: the presence of glycogen in the cytoplasm of the thrombocytes, heterophils, and basophils; the presence of basic polyaminoacid-rich proteins in the granules of heterophils and eosinophils and myeloperoxidase in the granules of the heterophils, eosinophils, and azurophilic cells; and sudanophilic small granules in the heterophils, eosinophils, and azurophilic cells. More detailed morphological aspects of the cells were observed by means of ultrastructural analysis.     

The effects of vinblastine on the secretory ameloblasts: an ultrastructural, cytochemical, and immunocytochemical study in the rat incisor

Secretory ameloblasts synthesize the organic matrix of enamel and secrete it at two distinct "putative secretory sites" characterized by membrane infoldings (Nanci and Warshawsky, 1984a). The antimicrotubular agent vinblastine sulphate interferes with secretion. We have examined the effect of this drug on the ameloblast secretory sites and re-evaluated the effect on the intracellular organization of the cell by using conditions that optimize fixation, cytochemistry (ZI0), and immunocytochemistry. Associated with the disappearance of secretory granules and Golgi-related structures from Tomes' process was the loss of membrane infoldings at secretory sites. The Golgi apparatus appeared fragmented and numerous granule clusters were found throughout the cell body. These clusters were often seen in relation to extracellular patches of material in which no crystallites were seen. Immunocytochemistry revealed the presence of enamel proteins in the protein synthetic organelles, including various granule types, in lysosomes and in the extracellular patches. These data suggest that ameloblasts under the effect of vinblastine carry on secretory activities, but the product is not routed to the usual sites. It was confirmed that membrane infoldings characterize the sites where enamel proteins are normally secreted.     

Structural and ultrastructural studies of GH, PRL and SMT cells in goat fetus (Capra hircus) using immunocytochemical methods

The first data based on immunolabeling techniques of goat fetus adenohypophysis show that the structure and ultrastructure of growth hormone (GH)-, prolactin (PRL)-, and GH- plus PRL-secreting cells (SMT cells) in fetuses aged 100 days differ from those in the adult. Both cell number and cell size are smaller in the fetus, and the percentage of dark cells decreases with development. The data do not support the hypothesis that SMT cells represent the common origin of GH- and PRL-cells.     

Haematological, cytochemical and ultrastructural characteristics of blood cells and serum chemistry in captive Malayan tapir (Tapirus indicus) in Thailand

The Malayan tapir (Tapirus indicus) is classified as a preserved animal of Thailand. It is listed as an endangered species on the 2008 IUCN red list and also by the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Blood samples from captive Malayan tapirs (n?=?20, 10 males and 10 females) from five zoos under the Zoological Park Organization of Thailand were examined and measured for complete blood counts, serum chemistry and electrolyte analysis. Cytochemical staining including Peroxidase, Sudan Black B, Periodic acid Schiff's reaction (PAS), α-naphthyl acetate esterase (ANAE) and β-glucuronidase (BG) was also analysed. Statistically significant differences were found between sexes including alkaline phosphatase, total protein and red cell distribution widths. Red blood cells were slightly anisocytosis, (range 4.5–8.5 μm) with 6.3-μm mean diameter. Eosinophils and monocytes were positive with all five cytochemical stains. Lymphocytes showed a variable reaction for ANAE and BG. PAS was positive for all type of blood cells except red blood cells. Ultrastructurally, eosinophils had the largest round shape with three-capsule layer granules which were prominent and distinguishable from other species. This investigation provides basic haematology and serum chemistry analyses as well blood cells identification guideline for the Malayan tapir and serves as endangered mammal reference values.     

Morphological and ultrastructural study of the thrombocytes and leukocyte granulocytes of Salminus maxillosus (Characiformes,Characidae)

The thrombocytes and leukocytes of 'dourado' fish Salminus maxilosus peripheral blood were studied by means of light microscopy and transmission electron microscopy techniques. In respect to the morphological and ultrastructural aspects of thrombocytes and granulocytes, the cellular elements that appear with high frequency in the light microscopy preparations stained by the Rosenfeld method, three different types of granulocytes could be differentiated at the electron microscope, based on the ultrastructural aspect of the cytoplasmic elements: type I granulocyte that possibly corresponds to the type I neutrophil described under light microscopy; type II granulocyte is possibly the cell identified as type II neutrophil by the morphological analysis; type III granulocyte is equivalent to the eosinophils described in the samples stained by Rosenfeld methods.     

Fine-needle aspiration biopsy of pleomorphic (giant cell) carcinoma of the pancreas. Cytologic, immunocytochemical, and ultrastructural findings

Four cases of pleomorphic (giant cell) carcinoma (PGC) of the pancreas, diagnosed by fine-needle aspiration biopsy, are presented. Ancillary immunocytochemical studies were also performed on the smears (four cases) and cell blocks (three cases), along with ultrastructural (electron microscopic) examination of the aspirated specimen (two cases). Three of the four patients were elderly (age range: 44-65 years), and all had large pancreatic masses. A spectrum of cytomorphologic features was encountered, including poorly cohesive bizarre mononuclear cells, many multinucleated tumor giant cells, and associated malignant spindle cells. Prominent cellular cannibalism and cytophagocytosis also were seen. Immunoperoxidase studies demonstrated dual staining of the malignant cells for cytokeratin and vimentin in two cases and exclusive staining for vimentin in the remaining two cases. Ultrastructural examination demonstrated epithelial features in both cases studied. One of the cases demonstrated intracytoplasmic whorled bundles of intermediate filaments, corresponding to pale eosinophilic globular material, appreciated both in the Diff-Quik- and Papanicolaou-stained smears and staining with positive results for vimentin. The immunocytochemical studies suggest that the coexpression of vimentin and cytokeratin within the cells is consistent with a pleomorphic malignancy of epithelial origin. It is speculated that the staining of two of the cases exclusively for vimentin can be seen in either epithelial malignancies with sarcomatoid transformation or, less likely, a pure sarcoma of the pancreas.     

Structural and ultrastructural features of the agouti tongue (Dasyprocta aguti Linnaeus, 1766)

The agouti (Dasyprocta aguti Linnaeus, 1766) is a wild rodent belonging to the family Dasyproctidae that is found throughout Brazil and feeds on fruits and seeds. The aim of the present study was to describe the following features of the tongue of agouti: its morphological structures, the three‐dimensional characteristics of the lingual papillae surface, the connective tissue cores (CTCs) and the epithelial cell ultrastructure. Four types of papillae were observed on the dorsal surface of the tongue with a triangular shape: filiform, fungiform, foliate and vallate. Filiform papillae were distributed throughout the tongue surface, and removal of the epithelial surface revealed conical CTCs and multifilaments. Fungiform papillae were observed in the rostral and middle regions, whereas foliate papillae developed in pairs on the lateral margin of the caudal region. Removal of the epithelium in these regions revealed CTCs with parallel laminar conformation. Vallate papillae were arranged in a V‐shape in the caudal region, and their CTCs ranged in shape from elongate to ovoid. The ultrastructural components of the dorsal epithelium were the basal, spinous, granular and keratinised layers. A broad area with cytoplasmic projections was identified in the interface region between the lamina propria and the basal layer. Flattened cells with intermediate filaments were observed in the transitional region between spinous and granular layers. The keratinised layer was composed of superimposed epithelial cells where desmosomes and cell‐surface microridges were observed. These structural features, including the three‐dimensional aspects of the lingual papillae, the CTCs and the epithelial ultrastructure, indicate that when compared with other animals, particularly other rodent species, the morphological features of the tongue of agouti are relatively well developed, especially regarding foliate and vallate papillae.     

Structural and ultrastructural aspects of growth in Oscarella lobularis (Porifera, Demospongiae)

Light and electron microscopy were used to study projections emerging from areas of the sponge Oscarella lobularis. These projections seem to arise from foldings of the exopinacoderm, a process also involving mesohyl cells. Besides folding, exopinacocytes invaginate giving rise to small pockets. These may open directly to the outside and represent the precursors of flagellated chambers. Exopinacocytes lining pockets and spaces between projections transform into endopinacocytelike cells. As pockets become isolated from the exterior, cells further differentiate into choanocytes. Remodeling processes, involving fusion of adjacent protrusions and transformation of the intervening spaces into canals, eventually realize a functional aquiferous system. Allowing the sponge to increase its size, these events represent the steps of a growth mechanism.     

Resident sinusoidal macrophages in the liver of the brown bullhead (Ictalurus nebulosus): an ultrastructural, functional and cytochemical study

Ultrastructural, functional, and cytochemical characteristics of resident sinusoidal macrophages (RSM) in brown bullhead (Ictalurus nebulosus) liver were examined. Following perfusion fixation of the hepatic vascular bed, light micrographs revealed RSM that possessed multiple elongate cytoplasmic processes and frequently contained erythrocytes in various stages of degradation. Following brief perfusion fixation, light microscope examination of vibratome sections of bullhead liver reacted for peroxidase revealed intensely positive RSM. By transmission electron microscopy, peroxidase activity was localized to the nuclear envelope and cytoplasmic granules of RSM and in endothelial and perisinusoidal fat-storing cells. In cryostat sections of fresh-frozen liver, glucose-6-phosphate dehydrogenase (G-6-PDH) was uniformly distributed over hepatocytes, whereas intensely positive punctate staining for G-6-PDH was localized over RSM. To test for phagocytosis by RSM, latex beads (0.81 micron) were injected into a tributary of the hepatic portal vein 2 min prior to perfusion fixation. Latex beads appeared either singly or in dense aggregates within RSM. Ultrastructurally, RSM were characterized by an irregularly shaped, eccentrically located nucleus, electron-dense vacuoles, small patches of granular endoplasmic reticulum, a well-developed Golgi apparatus, elongated mitochondria, desmosomes or desmosome-like densities that served as a source of attachment to endothelial cells, and a centriole with radiating microtubules. Invaginations of the plasma membrane (vermiform processes) characteristic of mammalian Kupffer cells were not observed in bullhead RSM. The results indicated a resident cell population of sinusoidal macrophages in the bullhead liver with properties that partially resembled mammalian Kupffer cells. These results are important for the identification of the normal resident cells in the bullhead liver.     

The ultrastructural characterization of the tegument ofClonorchis sinensis (Cobbold, 1875) cercaria

Summary The tegumental ultrastructure of the cercaria of the liver fluke,Clonorchis sinensis (Cobbold, 1875), was studied by scanning and transmission electron microscopy. The body surface is almost encircled by many rows of regularly arranged spines. The tegumental syncytium of the body contains many rod-shaped dense granules and central electron-lucent bodies, neither of which are present in the tail tegument. There are four rows of hooked teeth and modified spines on the oral cone. These teeth are differentiated morphologically and probably functionally from the other body spines. Disc-shaped papillae with long or short cilia are distributed on the body in a bilaterally symmetrical pattern dorsally and ventrally. There are 30 to 37 papillae with much longer cilia laterally. Four pairs of papillae were found on the tail. From their structure and location these papillae appear to have a mainly tango-or rheoreceptive function. Another type of sheathed papillae is situated around the oral sucker. The cuticular tegument is expanded laterally at the base of the tail, forming a characteristic sac-like structure.This work was supported in part by a Scientific Grant (No. 037036) from the Ministry of Education, Japan     

Morphological, cytochemical, and ultrastructural study of thrombocytes and leukocytes in neotropical fish, Brycon orbignyanus Valenciennes, 1850 (Characidae, Bryconinae)

Morphological, cytochemical and ultrastructural studies are important to demonstrate the function of the blood cells, which is very little understood in teleosts. In peripheral blood of 'piracanjuba' Brycon orbignyanus, thrombocytes, lymphocytes, monocytes, neutrophils and heterophils were studied and characterized. Thrombocytes had a fusiform or oval shape with PAS-positive granules. Lymphocytes presented small size with sparse basophilic cytoplasm. Monocytes were large in size, presented basophilic cytoplasm that may be foamy or vacuolated, with non-specific esterase staining. The neutrophils presented lightly neutrophilic granule cytoplasm, with positivity for PAS and peroxidase. The heterophils were large in size, with eosinophilic and basophilic granules cytoplasm and PAS-positive. Transmission electron microscopy study demonstrated that the thrombocytes, lymphocytes and monocytes features were similar to other teleosts. In ultrastructural study only one type of neutrophils was observed. Cytochemical findings indicated that neutrophils and monocytes of B. orbignyanus may be involved in phagocytosis, and neutrophils play an important microbicidal role.     

Terminal deoxynucleotidyl transferase (TdT) in acute nonlymphocytic leukemia. A clinical, morphologic, cytochemical, immunologic, and ultrastructural study

The classification of acute leukemia is essential for proper therapy and may be based on morphologic, cytochemical, immunologic, or even ultrastructural studies. Terminal deoxynucleotidyl transferase (TdT) is expressed in most patients with acute lymphocytic leukemia (ALL) and a minority of patients with acute nonlymphocytic leukemia (ANLL). Thirteen patients with ANLL and greater than 30% blasts positive for TdT were studied to establish the clinical, light microscopic, cytochemical, immunologic, and ultrastructural correlates of this phenomenon. Most patients demonstrated some morphologic and cytochemical features of monocytic differentiation. On cytochemical stains, nine had greater than 3% Sudan black-positive blasts. Diffuse alpha naphthyl acetate esterase (ANAE) staining of leukemic cells was present in nine cases, though extremely weak in seven. Blasts in ten patients did not express any other markers of lymphoid differentiation except TdT. However, two patient's immature cells bore CD10 common ALL antigen (CALLA) and CD19 (B4). Ultrastructural studies confirmed nonlymphoid differentiation in all ten patients studied, with a prominent monocytic component present in nine. In no case was a second population of lymphoblasts identified to account for TdT positivity. These patients responded poorly to conventional therapy for ANLL, with complete remissions in 3 of 13 (23%). With conventional therapy for ALL, complete remission was achieved in only two of nine (22%) patients. However, four of seven (57%) patients had a complete remission with high-dose cytosine arabinoside regimens. The authors' studies suggest that patients with TdT-positive ANLL represent a distinct subset that usually displays ultrastructural evidence for monocytic differentiation and is clinically significant in that these patients respond poorly to conventional therapy for both ALL and ANLL. Recognition of the monocytic lineage of these cases by light microscopic examination is difficult because they are often poorly differentiated morphologically and express only weak nonspecific esterase positivity.     

Medullary carcinoma of the thyroid: a cytological, immunocytochemical, and ultrastructural study.

The cytomorphological features of 38 cases of medullary carcinoma of the thyroid on FNAC were studied. These cases were retrieved from the files of the cytopathology laboratory. In addition, staining for calcitonin (19 cases), thyroglobulin (5 cases), neurone specific enolase (7 cases), and carcinoembryonic antigen (8 cases) was done. Electron microscopy was done in two cases. On morphology, the presence of amyloid together with cytoplasmic granules was found to be diagnostic. The plasmacytoid cell type and the mixed cell populations were most commonly observed. Pseudononuclear inclusions were seen in 58% of cases. Calcitonin was present in 74% of cases. NSE was noncontributory, whereas CEA was positive in one of the calcitonin negative cases.     

First ultrastructural and cytochemical data on the spermatozoon and its differentiation in progenetic and adult Archigetes sieboldi Leuckart, 1878 (Cestoda,Caryophyllidea, Caryophyllaeidae)

Parasitology Research - Spermiogenesis in progenetic and adult stages of Archigetes sieboldi Leuckart, 1878, a tapeworm parasitic in oligochaetes and fish respectively, has been examined using...     

Morphology and histoenzymology of eosinophilic granulocytes in the circulating blood of the turtle (Chrysemys dorbignih)

The studies on the characterization of eosinophils and neutrophils/heterophils of turtles are contradictory. Some authors have pointed out the existence of two distinct cell types: eosinophils and heterophils. Other authors have proposed that eosinophils and heterophils may be the same cells in different stages of maturation. These interpretations are based only on a morphological analysis. In the blood of the turtle (Chrysemys dorbignih), a South American freshwater species, there are two types of granulocytes with eosinophilic staining pattern: the first with round cytoplasmic granules and the second with ellipsoidal cytoplasmic granules. In the present study by using histoenzymological methods for the analyses of enzymological cellular content, we found that the cells with round cytoplasmic granules were positive for nonspecific esterase and the cells with ellipsoidal granules were positives for acid phosphatase, alkaline phosphatase, nonspecific esterase and peroxidase. The results show that these cells are distinct cells and that the cells with ellipsoidal cytoplasmic granules have the same histoenzymological characteristics as the neutrophils/heterophils of mammalians and other vertebrates.     

Peroxisomal targeting protein 14 (PEX14) from Leishmania donovani. Molecular,biochemical, and immunocytochemical characterization

Pathogens of the Leishmania and Trypanosoma genera compartmentalize glycolytic and other nutritional pathways in glycosomes, unique subcellular organelles related to the peroxisomes of mammals and yeasts. Most glycosomal proteins are targeted to the glycosomes by a COOH-terminal tripeptide signal similar to the peroxisomal targeting signal-1 (PTS-1). It has been proposed that PTS-1 forms a complex with the PEX5 receptor protein which then docks to the glycosomal membrane through interactions with the membrane associated PEX14 protein. To analyze the role of PEX14 in glycosomal protein import, the gene encoding the L. donovani PEX14 (LdPEX14) was isolated and shown to encode a 464 amino acid protein that exhibited very limited sequence homology with peroxisomal PEX14 proteins. In vitro binding experiments with purified recombinant LdPEX14 and LdPEX5 confirmed that LdPEX14-LdPEX5 interacted with a K(d) of 2.75 microM. When LdPEX5 was preloaded with a PTS-1 peptide, the affinity of the LdPEX14-LdPEX5 interaction affinity increased. Furthermore, binding experiments with truncated forms of LdPEX5 and LdPEX14 showed that the interaction domains localized to the amino terminal region of both proteins. Finally, confocal microscopy, subcellular fractionation, and differential extraction experiments indicated that LdPEX14 is a soluble protein that associates tightly with the glycosomal membrane and further support the role of LdPEX14 in forming a docking complex involved in glycosome biogenesis.