Spontaneous micronuclei in peripheral blood erythrocytes from 54 animal species (mammals, reptiles and birds): part two

The normal numbers of micronucleated erythrocytes (MNE) observed in peripheral blood samples differ among species. This depends on the effectiveness of the spleen (or the rest of the reticuloendothelial system) to withdraw them from circulation. In our previous report, we assessed the number of MNE in the peripheral blood of 35 mammalian species. Here we show the results observed in 54 species including mammals, reptiles and birds. We obtained 212 peripheral blood samples from different species. In 14 species, only one individual was studied. Slides were stained with acridine orange. The total number of MNE (normo and polychromatic) in 10,000 erythrocytes per animal are shown. The species that display the higher MNE were: ocelote, lynx, owl, gray squirrel, hedgehog, lion, orange fronted parakeet and common barn owl. For this reason, these species could be tested as monitors for genotoxic events. Another interesting observation was that in the gray squirrel, we found the highest values of MNE in the smaller (younger) animals when compared with the larger (older) of the same species.     

Ultrastructure of experimentally produced subcutaneous haematomas in the rabbit.

Haematomas were produced in rabbits by subcutaneous injection of autologous blood. Clotting and marked lysis of erythrocytes was noted in these haematomas, but there was no evidence of fragmentation of erythrocytes prior to or after ingestion by macrophages as has been reported in other sites such as the peritoneal cavity and the joint cavity. The phagocytosis of intact erythrocytes, lysed erythrocytes and haemoglobin led to the formation of three main types of lysosomal bodies; (1) myelinosomes containing whorled osmiophilic membranes, (2) siderosomes containing haemosiderin, and (3) myelinosiderosomes containing a mixture of osmiophilic membranes and haemosiderin.     

蒙古口蘑子实体凝集素性质初步研究

对蒙古口蘑干燥子实体研磨后,用磷酸盐缓冲液浸提,得到蒙古口蘑子实体的凝集素粗提物。对其性质进行分析表明,蒙古口蘑子实体凝集素对牛血和羊血都能凝集,且对羊血的凝集作用较强;D-果糖、β-葡萄糖、半乳糖和木糖对蒙古口蘑子实体凝集素均具有抑制作用;弱酸或弱碱性浸提液有利于凝集素的提取;蒙古口蘑子实体凝集素具有一定的热稳定性,直到70℃以后凝集红细胞的活力才丧失;凝集素的凝集活性对Ca2+、Mg2+、Mn2+和Fe3+这4种离子有不同程度的依赖。     

Cytamoeba bacterifera Labbé, 1894. I. Morphology and Host Incidence of the Parasite in California.

SVXOPSIS. Parasites belonging to the genus Cytamoeba have been found in amphibians from central and northern California. The infected animals and their numbers were (URODELA) Aneides flavipunctatus (2), A. lugubris (29), Batrachoseps a. atttnuatus (81). Dicamptodon ensatus (2), and (SALIENTIA) Rana b. boyli (1). The rates of infection varied between 0.6% and 29.3%, and the number of parasites per infected cell ranged from one to seven.
Preparations stained with haematoxylin and Romanowsky dyes indicated several types of parasite: some were homogeneous, others possessed several dark staining granules and some possessed large, dark staining central bodies or peripheral dark staining rims. Parasites were seen in the erythrocytes, the plasma and, occasionally, in endothelial cells and monocytes. They were found in the peripheral and heart blood and in the blood of the liver, spleen, kidneys and lungs. Cell division was common in erythrocytes, and yielded daughter cells of identical size. In some cases, chains of 3 to 5 individuals were seen and, rarely, three bodies, in a cloverleaf configuration, were produced as the result of a single division. Following acid hydrolysis, methylene blue-stained parasites showed a peripheral ring of granules, and, in some, a few stained centrally located bodies.     

A possible iridovirus in erythrocytes of Bufo marinus in Costa Rica

Icosahedral viral particles were found in the cytoplasm of erythrocytes and splenic reticular cells of a marine toad (Bufo marinus) collected from Costa Rica. Capsids had a maximum diameter of 312 nm and a spherical core with biphasic electron density. Viruses in erythrocytes were associated with cytoplasmic assembly areas and vacuoles in cytoplasm. Nuclei had finely granular material of decreased electron density located centrally, but contained no viral particles. A group of unenveloped viral particles was seen extracellularly in a splenic vessel. The virus was consistent with an iridovirus. In a blood smear stained with Giemsa round basophilic bodies with average diameters of 1.70 microns and morphologically similar to Pirhemocyton sp. were seen in the cytoplasm of erythrocytes and occasionally in the cytoplasm of monocytes or extracellularly. Erythrocytes containing these bodies had vacuoles and irregular pale-staining areas in the cytoplasm and pale-staining areas in the nucleus. These changes corresponded to the viral particles, assembly areas, vacuoles and nuclear changes at the ultrastructural level.     

Red blood cell phagocytosis and lysis following oxidative damage by phenylhydrazine

Red blood cells exposed in vitro to phenylhydrazine acquired Heinz bodies, bound autologous IgG and were then phagocytized when incubated with autologus mononuclear phagocytes. In vivo, phenylhdyrazine administered to rabbits, caused the appearance of high plasma hemoglobin levels and hemoglobinuria as well as Heinz body formations and IgG binding to erythrocytes. This suggests that while in vitro the main mechanism of red cell removal seems to be phagocytoses, in vivo both intravascular hemolysis and phagocytosis are active processes. Preliminary biochemical studies on phenylhydrazine-exposed erythrocytes showed that together with the well-known appearance of Heinz bodies, methemoglobin and a drop in reduced glutathione, this drug also causes ATP depletion. This is initially concomitant with the appearance of ADP and AMP and subsequently hypoxantine. Thus, irreversible ATP depletion may contribute to the genesis of the hemolytic process observed in vivo.     

Description and ultrastructure of Leishmania zuckermani n. sp. amastigotes detected within the erythrocytes of the South African gecko Pachydactylus turneri Gray, 1864.

In erythrocytes recovered from blood of geckoes of the species Pachydactylus turneri collected in Gauteng Province, Republic of South Africa, leishmania zuckemani n. sp. were detected. Giemsa stained erythrocytes contained amastigotes, either single or numerous, in loose assemblies or in a compact rounded oggregates which may condense to become a round basophilic bodies with a central hollow. This new species of Leishmania differs from all previously described species in being almost exclusively parasitic in circulating erythrocytes. Three to seven amastigotes lodged all within one, or divided between several parasitophorous vacuoles were detected at the EM level. The amastigotes demonstrated essentially all the cytological components characteristic of leishmania species known to parasitize mammals. A point which emphasizes an already suggested close affiliation between mammalian and lizard Leishmania.     

Comparison of the effects of human erythrocyte ghosts and intact erythrocytes on platelet interactions with subendothelium in flowing blood

We investigated whether ghosts behaved similarly to intact erythrocytes to maintain regular primary hemostasis under flow conditions. To this end we performed perfusion experiments with whole blood in which erythrocytes were replaced by pink ghosts, and platelet interaction with the subendothelial surface of a damaged vessel was morphometrically evaluated. The same objective was sought by means of studies with a platelet function analyzer (PFA-100(TM) instrument). Perfusions performed with control blood reconstituted with intact erythrocytes gave rise to 0.4+/-0.2% contact but not spread platelets, 10.8+/-3.4% adhering and spread platelets, 16.3+/-4.6% platelets in thrombi, with 27.5+/-7.4% of the surface covered. Even though the average diameter of the ghosts was smaller than that of intact erythrocytes (5.3 microm vs. 7.7 microm), the values obtained in perfusions performed with ghosts were similar to those of the erythrocyte controls. Studies performed with the PFA-100(TM) analyzer were consistent with those observed in perfusion studies. The viscosity of control blood was compared with that of blood reconstituted with ghosts. At shear rates lower than 450 s(-1), the viscosity of the ghost samples was higher than that of the controls, but the difference progressively decreased as shear rate increased up to 750 s(-1) (3.61+/-0.15 and 3.71+/-0.17 cP, respectively). In conclusion, the results of our study showed that ghosts behaved similarly to intact erythrocytes in maintaining a normal platelet interaction with digested subendothelium, under conditions of moderate shear rate and constant hematocrit (40%). The rheological activity of ghosts, bodies that are metabolically less active, was sufficient for them to satisfactorily act as substitutes for intact erythrocytes in our system.     

Effect of formate on mouse erythrocytes in vivo and in vitro

The effect of formiat was tested in erythrocytes of mice damaged by oxidisation. Decrease of Heinz's bodies, stabilisation of reduced glutathion, and increase of enzyme activities by referring to an example of superoxidismutase could be identified as a positive effect. Possibilities of formiat reactions in the metabolism of red blood cells are discussed.     

Spontaneous cardiomyopathy resembling acute rheumatic heart disease in an owl monkey.

A wild-caught male Aotus vociferans died spontaneously during quarantine. Histologic examination of the heart showed several small myocardial arteries replaced with fibrinoid necrosis and adjacent inflammation that included lymphocytes and large histiocytic cells. Less often, similar inflammatory foci were observed in the myocardium interstitium. The lesions observed in the heart of the owl monkey resembled Aschoff bodies.     

Heinz bodies do not modify the membrane characteristics of common marmoset (Callithrix jacchus) erythrocytes

The possibility was examined that the membrane function of erythrocytes obtained from healthy common marmosets (Callithrix jacchus) was modified by the presence in the cells of Heinz bodies. No significant differences were found in erythrocyte endogenous free malonyl dialdehyde (MDA) or reduced glutathione (GSH) between normal human erythrocytes and marmoset erythrocytes containing Heinz bodies. Membrane fluorescent chromolipids, surface charge and thiol levels were similar in both species but average membrane bulk lipid fluidity was slightly elevated in the marmosets. It was concluded that, in contrast to the situation in human erythrocytes, the presence of Heinz bodies in red cells of marmosets does not adversely affect the properties of the membrane.     

Multiple infection in bovines from the tropics: observation of blood parasites by scanning and transmission electron microscope

Blood samples from a splenectomized bovine, experimentally inoculated with blood from a field cow living in southwestern Venezuela, were processed for transmission and scanning electron microscopy. The blood sample showed multiple infection with hemoparasites of the genera Anaplasma marginale, Eperythrozoon wenyonii and Trypanosoma vivax. Scanning electron microscope showed that the blood from bovines with multiple infection had profound deformation in knob-like protruding structures with reduced cellular volume similar to echinocyte red blood cells. E. wenyonii parasites appear associated with the membrane, grouped in shallow to severe invaginations at the surface of the erythrocytes. The morphology of the parasites is predominantly rod-like; they also appear as coccoid-shaped and bifurcate or triskelion-shaped organisms. The organisms are present in pairs or clusters. T. vivax appeared with double flagella, which indicates active cellular division and infection processes. Transmission electron microscope study showed erythrocytes infected with intracytoplasmic bodies of A. marginale and with E. wenyonii embedded in the external membrane cell, with mature, juvenile and dividing forms present.     

Some observations on the stained blood cellular elements of channel catfish, Ictalurus punctatus

Slides were prepared from blood of channel catfish, Ictalurus punctatus , and evaluated. The cellular blood elements observed were thrombocytes, lymphocytes, neutrophils, monocytes, eosinophils, basophils, granular anucleate bodies, erythrocytes. In channel catfish, thrombocytes, lymphocytes and neutrophils were the predominate leucocytes. Erythrocytes were observed in various stages of development. Smudge cells were observed in all smear preparations. The variations found between the observations in this study and information provided in the literature suggest the need for establishing a standardized terminology.     

Inclusion bodies in loggerhead erythrocytes are associated with unstable hemoglobin and resemble human Heinz bodies

The aim of this study was to clarify the role of the erythrocyte inclusions found during the hematological screening of loggerhead population of the Mediterranean Sea. We studied the erythrocyte inclusions in blood specimens collected from six juvenile and nine adult specimens of the loggerhead turtle, Caretta caretta, from the Adriatic and Tyrrhenian Seas. Our study indicates that the percentage of mature erythrocytes containing inclusions ranged from 3 to 82%. Each erythrocyte contained only one round inclusion body. Inclusion bodies stained with May Grünwald-Giemsa show that their cytochemical and ultrastructure characteristics are identical to those of human Heinz bodies. Because Heinz bodies originate from the precipitation of unstable hemoglobin (Hb) and cause globular osmotic resistance to increase, we analyzed loggerhead Hb using electrophoresis and high-performance liquid chromatography to detect and quantitate Hb fractions. We also tested the resistance of Hb to alkaline pH, heat, isopropanol denaturation, and globular osmosis. Our hemogram results excluded the occurrence of any infection, which could be associated with an inclusion body, in all the specimens. Negative Feulgen staining indicated that the inclusion bodies are not derived from DNA fragmentation. We hypothesize that amino acid substitutions could explain why loggerhead Hb precipitates under normal physiologic conditions, forming Heinz bodies. The identification of inclusion bodies in loggerhead erythrocytes allow us to better understand the haematological characteristics and the physiology of these ancient reptiles, thus aiding efforts to conserve such an endangered species.     

Erythrophagocytosis in the caprine hemal node

Caprine hemal nodes were studied by transmission electron microscopy after glutaraldehyde fixation and epoxy resin embedding. Hemal node macrophages were observed to be engaged in erythrophagocytosis. In the early stages of endocytosis, intact erythrocytes were contained in some of the heterophagic vacuoles of macrophages. Later, granular, electron-dense material appeared on erythrocytes, presumably as a result of lysosomal degradation of their matrices. Subsequently, the matrix fragmented and probably formed 'myelin-like figures' and residual bodies that dominated the macrophage cytoplasm. In addition, images of sinusoidal endothelium, reticular cells, lymphocytes and, rarely, eosinophils were observed that depicted structures resembling various stages of lysosomal digestion of erythrocyte matrix noted in macrophages. Our study provides evidence to support the fact that effete erythrocytes are filtered, besides known organs, also in caprine hemal nodes. The morphology and location of hemal nodes suggest that the organ can be an efficient blood filter.     

Phagocytosis in the spleen of the sunfish Lepomis spp.

A histological investigation of the filtering function of the spleen of the sunfish Lepomis spp. was conducted by light, scanning, and transmission electron microscopy. The parenchyma of the organ is predominantly red pulp, a system of splenic cords and sinuses. The white pulp consists of loose lymphoid tissue which forms a cuff around the pulp arteries. Filtering of particulate matter from the blood occurs in the red pulp by phagocytes of the pulp cords and ellipsoids (periarterial macrophage sheaths). The ellipsoids are pale-staining cuffs of macrophages and reticular cells in a framework of reticular fibres surrounding the arterial capillaries. Destruction of effete blood cells (especially erythrocytes) is confined to the pigment nodules; particulate matter is not taken up by the nodules. These yellow-brown bodies are dispersed throughout the red pulp and are bounded by a reticular capsule. They contain masses of phagocytes and have the appearance of a morula. They are associated with blood vessels and are surrounded by sinusoids. Prussian Blue stain shows the presence of haemosiderin within their phagocytes. The phagocytes of the pigment nodules are filled with inclusions such as residual bodies, siderosomes, and fragments of erythrocytes. The early filtering of particulate matter by the phagocytes of the pulp cords and ellipsoids may allow for a more efficient phagocytosis of erythrocytes by the pigment nodules, followed by storage and reutilization of iron-containing compounds uncontaminated by other phagocytosed material.     

Localization of chitinolytic enzymes in blood of turbot, Scophthalmus maximus, and their possible roles in defence

The intracellular localizations ofchitinase and β-N-acetylglucosaminidase were detected in turbot blood smears, using a novel method employing fluorogenic substrates. The two enzymes showed different distributions, with chitinase being more generally distributed and N-acetylglucosaminidase being strongly associated with distinct intracellular bodies, probably lysosomes. The fluorogenic substrates were used to analyse soluble and membrane fractions of homogenates of red and white blood cells prepared on Percoll gradients. In the leucocytes, the chitinase and N-acetylglucosaminidase activities were mostly in the soluble fraction. In the erythrocytes the activities were lower, at about one-hundredth and one-tenth specific activities, respectively, and were distributed between soluble and membrane-bound fractions at about 2 : 1 and 3 : 1, respectively. In contrast, lysozyme had a soluble distribution in leucocytes and was not detected in erythrocytes. Plasma was rich in chitinase and lysozyme activities but had no detectable N-acetylglucosaminidase. Two possible roles for the chitinolytic enzymes are discussed: defence against pathogens and processing of glycoproteins or glucosaminoglycans. Evidence for a defence role for the chitinase and lysozyme is provided by demonstrating that they had inhibitory activity against the chitinous fungus Mucor mucedo .     

The Effect of Exon 7 Deletion during the Evolution of TRIMCyp Fusion Proteins on Viral Restriction,Cytoplasmic Body Formation and Multimerization

TRIMCyp is a fusion protein consisting of the TRIM5 gene product and retrotransposed Cyclophilin A (CypA). Two primate TRIMCyp fusion proteins with varying anti-HIV-1 activities independently evolved in owl monkeys and Old World monkeys. In addition, Old World monkey TRIMCyps lack exon7, which encodes amino acids in the Linker2 region. Previous studies on TRIM5α indicated that this region affects anti-retroviral activity, cytoplasmic body formation, and multimerization. The effects of exon7 deletion on the functions of the TRIMCyp are unclear. In this study, we found that the cytoplasmic bodies and multimers of owl monkey TRIMCyp (omTRIMCyp) are different from those of northern pig-tailed macaque TRIMCyp (npmTRIMCyp). In addition, we demonstrated that exon7 deletion affected cytoplasmic body formation and multimerization. Moreover, we unexpectedly found two chimeric proteins of omTRIMCyp and npmTRIMCyp that failed to block HIV-1 replication, despite the presence of CypA in omTRIMCyp. Further studies indicated that the cytoplasmic bodies and spontaneous multimerization were not responsible for TRIMCyp anti-HIV-1 activity. Moreover, potent viral restriction is associated with higher amounts of monomeric TRIMCyp when the CypA domain is able to recognize and bind to the HIV-1 capsid. Our results suggested that the deletion of exon7 during the evolution of TRIMCyp affected its function.     

Microsatellite markers characterized in the barn owl (Tyto alba) and of high utility in other owls (Strigiformes: AVES)

We have identified 15 polymorphic microsatellite loci for the barn owl (Tyto alba), five from testing published owl loci and 10 from testing non‐owl loci, including loci known to be of high utility in passerines and shorebirds. All 15 loci were sequenced in barn owl, and new primer sets were designed for eight loci. The 15 polymorphic loci displayed two to 26 alleles in 56–58 barn owls. When tested in 10 other owl species (n = 1–6 individuals), between four and nine loci were polymorphic per species. These loci are suitable for studies of population structure and parentage in owls.     

Characteristics of erythrocyte lipids in blood of tundra voles (Microtus oeconomus Pall.) inhabiting areas with increased natural radioactivity

Interrelations between the lipid characteristics of the blood erythrocytes and 226Ra accumulation in the body of tundra voles (Microtus oeconomus Pall.) inhabiting areas with different levels of the radiation background were investigated. It is shown that the ratio of the phospholipid (PL) fractions which cause the blood erythrocyte structure depends on the phase of the population cycle, as well as on the sex and age of tundra voles. The statistically significant interrelation between lysoforms and the sphingomielin content has been revealed in the blood erythrocyte PL of tundra voles; its scale somewhat differs for the animals from the reference and Ra areas. The peroxide concentration in the blood erythrocyte lipids of tundra voles from the Ra area exceeded the control values in all mature groups of the animals trapped at the depression phase of the population density. The 226Ra content in the bodies of the tundra voles which were trapped in the Ra area at the increased phase of the population cycle is for certain higher than that for the animals from the reference area. Interrelations between the lipid peroxidation parameters in the blood erythrocytes and the body 226Ra content for separate sex-age groups of tundra voles have been revealed.