Migration of erythroblastic islands toward the sinusoid as erythroid maturation proceeds in rat bone marrow

It has been hitherto considered that mature erythroblasts migrate toward the sinusoid along the cytoplasmic processes of macrophages of erythroblastic islands in bone marrow. Our previous report, however, has demonstrated the morphological features of a mature erythroblastic island passing through the sinusoidal endothelium. In this study, the possibility of migration of erythroblastic islands toward the sinusoid was examined in rat bone marrow by light microscopical histoplanimetry. As a result, the more mature erythroblasts were not regularly arranged in the peripheral direction of the erythroblastic islands. Immature erythroblasts were populated more in the erythroblastic islands away from the sinusoid than in those islands neighboring the sinusoid, whereas mature erythroblasts were more in erythroblastic islands neighboring the sinusoid. These findings suggest that the formation of erythroblastic islands occurs in a region away from the sinusoid, and that erythroblastic islands migrate towards the sinusoids as erythroid maturation proceeds.     

Spontaneous Erythroid Leukemia in a 7-Week-Old Crl:CD (SD) Rat

A young male Crl:CD (SD) rat with erythroid leukemia that presented with emaciation, abdominal distension and a pale visible mucosal membrane was euthanized at 7 weeks of age. At necropsy, enlargement of liver, spleen and pancreatic lymph node was noted. Analysis of blood smear samples revealed many mono- or binucleated erythroblasts that had PAS-positive vacuoles in the cytoplasm. Histopathologically, neoplastic proliferation of atypical cells was observed in the hepatic sinusoids, splenic red pulp, bone marrow, pancreatic lymph node, kidney and lung. Neoplastic cells showed a round to spindle shape, and some neoplastic cells had deeply stained small nuclei and small cytoplasms and resembled erythroblasts. Immunohistochemically, many neoplastic cells were positive for hemoglobin. To our knowledge, this is the first report of erythroid leukemia in a rat of this age. The observed features were similar to those of pure erythroid leukemia in humans.     

Immunohistochemical detection of phosphatidylserine and thrombospondin on denucleating erythroblasts in rat bone marrow

The hypothesis that apoptotic factors play some roles in the denucleation of erythroblasts has been confirmed by the immunohistological detection of both phosphatidylserine and thrombospondin as phagocytosis-inducing factors in general apoptotic events. Both phosphatidylserine and thrombospondin were detected on the surface of cell membrane of mature erythroblasts, while thrombospondin was also detected in more immature erythroblasts. The intensities of their immune reactions increased as the erythroids matured. During denucleation, the positivities of both phosphatidylserine and thrombospondin were restricted on the surface of the cell membrane surrounding the protruding nuclei. Thus, the apoptotic process involves denucleation of erythroblasts and phosphatidylserine, and thrombospondin acts as phagocytosis-inducing factors in the denucleation event.     

Macrophages of the bovine heifer mammary gland: morphological features during initiation and resolution of the inflammatory response

This work characterizes macrophage morphological features during initiation and resolution of an inflammatory response by the bovine mammary gland. The study has been carried out in 20 mammary glands of five virgin heifers by using light microscopy of natural and stained cells and by using transmission electron microscopy (TEM). The inflammatory reaction was induced by an intramammary administration of phosphate-buffered saline (PBS). It has been found that both the initial as well as the resolution phases of the inflammatory reaction are characteristic of the presence of various morphologically different macrophage forms. During the initial phase of the inflammatory response, the major proportion of the macrophage population consisted of monocyte-like macrophages, which represented newly migrated cells. These macrophages were 12-15 mum in size, with spherical or ovoidal shapes, and contained homogenous, fine-granular cytoplasm rich in Golgi complexes, numerous mitochondria, and no lysosomes. The nuclei of the macrophages were kidney-shaped, and surrounded by dark chromatin along the peripheries. Macrophages with phagocytosed apoptotic neutrophils in the cytoplasm were detected already during the initial phase. These macrophages reached the highest proportion 48-72 h after the influx induction and participated in the resolution of the inflammatory reaction. Other cells, also detected during the resolution of the inflammatory reaction, were vacuolized macrophages that formed the largest cells in the lavages of the mammary glands and that were structurally characteristic for the presence of vacuoles in the cytoplasm. In TEM the macrophage vacuoles formed both phagolysosomes with residues of pre-digested material of phagocytosed apoptotic neutrophils and vacuoles that were less electon-dense. Morphologically different forms of macrophages reflected their real-time functions in the inflammation process.     

Primitive neuroectodermal neoplasia in coho salmon, Oncorhynchus kisutch

Twelve coho salmon, approximately 8 weeks old, were each observed to have a single neoplasm involving the dorsolateral axial skeletal musculature. The neoplasm was closely associated with the vertebrae in all cases. The neoplasm was composed of islands containing small cells with round and occasional spindeloid morphology. Neoplastic cells had basophilic cytoplasm and vesicular nuclei. These cells exhibited immuno-positivity only for vimentin and S-100 protein. Ultrastructurally, neoplastic cells had nuclei with a predominance of euchromatin, cytoplasm containing marked amounts of rough endoplasmic reticulum, scant amounts of smooth endoplasmic reticulum, and scattered mitochondria. Rudimentary cell junctions were occasionally observed between adjacent neoplastic cells. Based on the close association of these neoplasms with the vertebrae as well as the histologic, ultrastructural, and immunohistochemical findings, these neoplasms were considered to all be primitive neuroectodermal neoplasms.     

Porcine circovirus induces B lymphocyte depletion in pigs with wasting disease syndrome

To disclose the mechanism of cellular injury following porcine circovirus (PCV) infection, 12 pigs were examined by the terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) method and immunohistochemistry. Histologically, the lymphoid tissues were characterized by marked apoptosis of lymphocytes, lymphocyte depletion, and macrophages and giant cells containing numerous inclusion bodies with or without apoptotic bodies. Immunohistochemically, there were many lysozyme-positive macrophages in the lymphoid follicles, while the number of CD79a-positive B lymphocytes was scanty. Apoptotic cells, which were proved to be TUNEL positive, revealed CD79a positivity. Although detectable mainly in the cytoplasm of macrophages, PCV antigens were found also in the nuclei of macrophages and apoptotic lymphocytes. Ultrastructurally, the presence of PCV virions was confirmed in apoptotic bodies phagocytosed by macrophages. These findings suggested that lymphocyte depletion with apoptotic death of B lymphocytes was caused by PCV, and that some of the inclusion bodies were phagolysosomes derived from the apoptosis. Thus, PCV may trigger the development of wasting disease syndrome by producing an immunocompromised state in pigs.     

Apoptosis induced in vivo by new type gosling viral enteritis virus

In this study, apoptosis was induced by new type gosling viral enteritis virus (NGVEV) in experimentally infected goslings is reported in detail for the first time. After 3-day-old goslings were orally inoculated with a NGVEV-CN strain suspension, the time course of NGVEV effects on apoptotic morphological changes of the internal tissues was evaluated. These changes were observed by histological analysis with light microscopy and ultrastructural analysis with transmission electron microscopy. DNA fragmentation was assessed with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and DNA ladder analysis. A series of characteristic apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, plasma membrane blebbing, and formation of apoptotic bodies were noted. Apoptosis was readily observed in the lymphoid and gastrointestinal organs, and sporadically occurred in other organs after 3 days post-infection (PI). The presence and quantity of TUNEL-positive cells increased with infection time until 9 days PI. DNA extracted from the NGVEV-infected gosling cells displayed characteristic 180~200 bp ladders. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages, monocytes, and epithelial and intestinal cells. Necrosis was subsequently detected during the late NGVEV-infection phase, which was characterized by cell swelling, plasma membrane collapse, and rapidly lysis. Our results suggested that apoptosis may play an important role in the pathogenesis of NGVE disease.     

Developmental changes in cell proliferation and apoptosis in the normal duck thymus

Cell proliferation and apoptosis in the normal duck thymus during embryonic and post-embryonic development were studied. The flow cytometry assay shows that the level of G(0)/G(1) thymic cell population and the proportion of apoptotic cells increased with age, while the levels of S phase, G(2) + M phase and the proliferating index decreased with age. Proliferation cell nuclear antigen (PCNA) was mainly detected in the nuclei of lymphocytes. The number of PCNA-positive cells in the cortex and medulla significantly decreased with age. Transferase-mediated dUTP nick-end labelling (TUNEL) reaction stained apoptotic bodies in the cytoplasm of macrophages and free apoptotic bodies or nuclei with condensed chromatin in lymphocytes. The number of TUNEL-positive cells in the cortex and medulla markedly increased with age. The amount of proliferation and apoptotic cells in the thymic cortex was higher than that in the medulla. The balance between proliferation and apoptosis in the duck thymus may account for the process of thymic development and involution.     

Morphological and biochemical aspects of apoptosis,oncosis and necrosis

Recent investigations have demonstrated the need for a precise differentiation of various forms of cell death such as apoptosis, oncosis, necrosis and programmed cell death. Apoptosis is marked by cellular shrinking, condensation and margination of the chromatin and ruffling of the plasma membrane with eventually breaking up of the cell in apoptotic bodies. Cell death marked by cellular swelling should be called oncosis, whereas the term necrosis refers to the morphological alterations appearing after cell death. Apoptosis and oncosis are therefore pre-mortal processes, while necrosis is a post-mortal condition. The term programmed cell death refers to the 'fixed' pathway followed by dying cells, whether or not with the characteristic morphology of apoptosis. Three mechanisms are actually known to be involved in the apoptotic process: a receptor-ligand mediated mechanism, a mitochondrial pathway and a mechanism in which the endoplasmic reticulum plays a central role. All three mechanisms activate caspases which are responsible for the characteristic morphological changes observed during apoptosis. A review of the different methods used for detecting apoptotic cells demonstrates that most of these techniques are not entirely specific.     

Tissue pool neutrophils of the bovine mammary gland: ultrastructural features during in vitro senescence

The aim of the study was to verify whether the in vitro senescence process of tissue pool neutrophils of the bovine mammary gland is accompanied by similar changes of ultrastructure as typically occurs in in vivo conditions. The experiments were carried out in four clinically healthy, Holstein x Bohemian Red-Pied crossbred heifers aged 14-16 months. With the aid of transmission electron microscopy (TEM), scanning electron microscopy (SEM) and flow cytometry (FCM), neutrophil apoptosis in vivo was detected and during senescence it was monitored in vitro. The neutrophil apoptosis comprised three ultrastructurally different stages: (1) karyopyknosis, (2) zeiosis, and (3) apoptotic bodies. These stages were obvious in the apoptotic neutrophils both in vivo and in vitro. In addition to the common morphological signs, however, ultrastructural differences were also detected in apoptotic neutrophils in vitro. These in vitro ultrastructural differences mostly comprised hyper-vacuolation of the cytoplasm with mega-vacuoles and secondary necrosis of apoptotic neutrophils. Morphological features of apoptosis during in vitro senescence of tissue pool neutrophils of the bovine mammary gland were shown to be in close accordance with these in vivo signs.     

Growth and morphological characteristics of canine venereal tumor cells in vitro

Canine venereal tumor cells were grown in monolayer and tumor tissue fragments were maintained in vitro for 56 days in medium-199. Attachment and cell replication were evident within 17 hours and monolayer was obtained by the tenth day. Growth at first comprised mainly round cells with abundance of cytoplasm and vesicular nuclei, and a few spindle-shaped cells. Later cells became more elongated and like fibroblasts. Cell degeneration started by the 17th day and most were degenerated by the 24th day. Necrosis and depletion of cell population were prominent in tumor explants during the first 2 weeks. Distinct cell multiplication was evident by the 21st day and tumor fragments were repopulated with cells resembling the original tumor by the 56th day. Two distinct morphological cell types were seen: small cells with vesicular, round to oval nuclei and acidophilic cytoplasm; and the large cells with large hyperchromatic nuclei and acidophilic cytoplasm.     

Ultrastructure of phagocytes from mammary glands of non-pregnant heifers

Results of ultramicroscopic investigations of phagocytes isolated from non-secreting and aberrantly secreting juvenile mammary glands of non-pregnant heifers are presented. The two types of phagocytes observed in cell suspensions obtained by lavage of mammary gland cavities were polymorphonuclear leukocytes and macrophages. Polymorphonuclear leukocytes were spherical or irregular in shape and contained segmented nuclei. Azurophilic and specific electron-dense granules, mitochondria, glycogen particles, phagosomes and phagolysosomes in cytoplasma and characteristic pseudopodia on the cell surface were observed. In addition to these normal polymorphonuclear leukocytes, degenerating cells, characterized by spherical nuclei, total absence of pseudopodia, merged nuclear segments and altered granules, other cellular organelles and plasmalemma were present. Two types of macrophages, i.e. vacuolized and non-vacuolized, could be distinguished. Typical of the non-vacuolized type was a kidney-shaped nucleus, a rich Golgi complex and a large amount of lysosomes in the cytoplasm. The vacuolized macrophages contained a large amount of electron-dense vacuoles in the cytoplasm. Unlike non-secreting glands, the cell suspensions collected from aberrantly secreting juvenile mammary glands contained only vacuolized macrophages. The vacuolization results from phagocytosis of corpuscular particles of aberrant milk plasma.     

Haematology of foetal sheep

OBJECTIVE: To describe the ontogeny of blood cells throughout foetal development in sheep. DESIGN: A haematological study on blood and bone marrow from 42 sheep foetuses aged between 19 days gestation and full term. PROCEDURE: Virgin Merino ewes were mated and the developing foetuses removed surgically at different stages of gestation. Blood and bone marrow samples were collected, stained for cytological examination or processed for electron microscopy. Blood samples were also examined haematologically. Foetuses were incubated with 3H-thymidine and autoradiographed. RESULTS: During the first 4 weeks of development primitive erythroblast constituted the majority of the circulating blood cells. Definitive erythroid cells, originating in the liver, first appeared in the blood at around 27 days gestation and entirely replaced the primitive erythroblasts by 50 days gestation. Leukocyte numbers, especially lymphocyte count, increased rapidly after 49 days gestation. Erythropoiesis predominated in the marrow of all foetuses older than 70 days. In more marrow, myelopoiesis was the major activity and lymphopoiesis was not significant. CONCLUSIONS: Red blood cell numbers and haemoglobin content progressively increases during foetal development. Primitive erythroblasts are not the precursors of the definitive erythroblasts. There are no significant differences in morphological features or maturation sequence between hepatic and bone marrow erythroblasts. Myelopoiesis is a major activity of bone marrow rather than of foetal liver.     

Ultrastructure of schizonts in the liver of cats with experimentally induced cytauxzoonosis

Schizonts in the liver of 2 cats with cytauxzoonosis were studied by both light and electron microscopies. By light microscopy, the cytoplasm of macrophages in the sinusoids and small vascular channels contained schizonts with cytomeres or both cytomeres and mature merozoites. By electron microscopy, it was determined that schizogony occurred in 4 stages. The earliest stage was the presence of a multilobed structure containing finely granular protoplasm in the cytoplasm of the macrophage. The 2nd stage was an increase in height and number of the lobulations on the surface of the schizont. The 3rd stage involved the development of cytomeres and the appearance of a polar ring and rhoptries in everted sacculations on the cytomere membrane. Nuclei and mitochondria were incorporated into the sacculations before the release of mature merozoites into the host cell cytoplasm. In the last stage of schizogony, following massive merozoite formation and reduction in size of the schizont, residual nuclei divided by multiple fission. Each nuclear division became incorporated into a developing merozoite having preformed rhoptries, mitochondria, and a polar ring.     

Primary cutaneous extragenital canine transmissible venereal tumour with Leishmania‐laden neoplastic cells: a further suggestion of histiocytic origin?

The clinical signs and histopathological features of a primary extragenital canine transmissible venereal tumour (TVT) are described. Three subcutaneous round alopecic nodules were located on the anterior and caudal dorsal region and in the ventral area of the neck. Cytologically, tumour cells were intermediate in size with a moderate amount of cytoplasm, and the nuclei were immature with finely reticular chromatin. The cytoplasm was lightly to heavily basophilic and contained distinct small vacuoles at the periphery. On the basis of these characteristics, a diagnosis of TVT was made and confirmed by histological and ultrastructural investigations. Leishmania amastigotes were detected in the cytoplasm of macrophages and neoplastic cells of the tumoral mass. The presence of the parasite within neoplastic cells is consistent with a histiocytic origin of TVT.     

Malignant giant cell tumor of soft parts in a mare

Two subcutaneous masses were removed from the elbow of a mare. Histologically they were composed of islands of polygonal to plump spindlelioid cells with large nuclei, coarsely stippled chromatin, and eosinophilic cytoplasm. Findings were diagnostic for a malignant giant cell tumor of soft parts, a rare tumor with a fair prognosis.     

Apoptosis, inflammatory response and parasite load in skin of Leishmania (Leishmania) chagasi naturally infected dogs: A histomorphometric analysis

The skin has an important role in infection by Leishmania chagasi. Apoptosis modulates the inflammatory response acting distinctively either on the progression or regression of the lesions. The parasites interact with multiple regulatory systems inducing apoptosis in host cells, during cell invasion, stabilization and multiplication of pathogens. In this context, the aim of this study was to evaluate cell death within the inflammatory infiltrates, and to correlate these results with parasite load and clinical features of dogs naturally infected with L. chagasi. Fragments of skin pinnas (8 symptomatic+8 asymptomatic+6 negative controls) were used to characterize and measure the inflammatory response, parasite load and apoptosis. Diagnosis of canine leishmaniasis was confirmed by the detection of anti-Leishmania antibodies by IFA and ELISA in serum, direct visualization of the parasite and culture in spleen, liver, pinna, bone marrow and lymph nodes, and PCR (pinna). Histomorphometry was performed with images obtained from 20 representative histological fields in a light microscope. Ultra-thin sections were mounted over a 300 mesh grids, contrasted with 2% uranyl acetate and lead citrate and examined under a Transmission Electronic Microscopy. Amastigotes were only found in the skin of symptomatic animals (31.94±18.81). The number of foci and cellularity of the inflammatory infiltrates in symptomatic dogs were higher than in other groups and in asymptomatics were higher than in controls (p<0.05; Tukey). The average area, perimeter and extreme diameters of the inflammatory infiltrates obtained in symptomatic dogs were higher than in controls (p<0.05; Tukey). The apoptotic index was higher in symptomatic than in other groups and there was no difference between asymptomatics and controls (p<0.05; Tukey). Ultrastructurally, apoptotic cells were shrunken, with condensed nuclear chromatin and cytoplasm. Condensed nuclei were frequently fragmented. Internucleosomal DNA fragmentation occurred only in symptomatic cases. Amastigotes were observed within neutrophils and macrophages. Apoptosis is directly related to parasite load, intensity of inflammatory response and clinical manifestations in L. chagasi naturally infected dogs.     

Filamentous Intranuclear Inclusion Bodies in Psittacine Birds. A Structural and Ultrastructural Study

This paper concerns a disease affecting a group of African grey parrots, which involves intranuclear inclusion bodies composed of filamentous material. The disease was characterized by either sudden death or death within 2–3 days from onset of non-specific symptoms. At necropsy, gross lesions included enlarged liver, mild hepatic congestion and focal necrosis. Samples from five birds were fixed in 10% formol and routinely processed for light and electron microscopy. In four birds, numerous hepatocytes displayed an enlarged nucleus, with peripheral margination of chromatin; the nucleus was partially or wholly filled by a basophilic inclusion body. In the remaining bird, inclusion bodies were acidophilic and completely filled the nucleus; nuclear enlargement was less evident than in the other birds. At ultrastructural examination, and in both types of IIB, nuclei contained looped filaments but no evidence of viral structures. However, virion-like structures were observed in the cytoplasm of some hepatocytes.     

Role of M cells and macrophages in the entrance of Mycobacterium paratuberculosis into domes of ileal Peyer's patches in calves

Ligated ileal loops of calves were inoculated with live and heat-killed Mycobacterium paratuberculosis and were examined by light and electron microscopy. At 5 hours after inoculation, acid-fast bacilli were in subepithelial macrophages, but not in M cells covering domes. At 20 hours, more than 50 acid-fast bacilli per cross section were in subepithelial macrophages in domes. Both living and heat-killed bacilli passed into domes. Addition of anti-M. paratuberculosis bovine serum to the inoculum enhanced entry of bacteria into domes. By electron microscopy, intact bacilli with electron-transparent zones (peribacillary spaces) were in the supranuclear cytoplasm of M cells at 20 hours. M cells also contained vacuoles, including electron-dense material interpreted as degraded bacilli. Subepithelial and intraepithelial macrophages contained bacilli and degraded bacterial material in phagosomes. These results suggest that calf ileal M cells take up bacilli, and that subepithelial and intraepithelial macrophages secondarily accept bacilli or bacterial debris which are expelled from M cells.