Crystalline inclusions in erythrocytes parasitized with Babesia equi following treatment of ponies with imidocarb

Four splenectomized Welsh ponies were infected with Babesia equi. Two ponies were treated with imidocarb dipropionate, and two were not treated. By light microscopic examination, 1% to 2% of the parasitized erythrocytes of treated ponies contained crystalline inclusions. The crystals were rectangular, diamond, or burr shaped. They occupied most of the erythrocytic cytoplasm, and, as a result, the remainder of the pale staining cytoplasm was inconspicuous in Wright-Giemsa-stained blood smears. The size and shape of intraerythrocytic inclusions varied when examined by electron microscopy, but in most instances they were either adhered to or were located close to the parasite. The sides of crystals were either smooth or serrated, and corners were either sharp or notched. Fractures or faults were common in large crystals. Parasitized erythrocytes of nontreated ponies and nonparasitized erythrocytes of treated ponies did not contain crystals. Four hemoglobulin types were identified in five noninfected, nontreated Welsh ponies from the same herd.     

Increased generation of superoxide in erythrocytes infected with Babesia gibsoni.

The present study was conducted to clarify the mechanism underlying the oxidative process in erythrocytes infected with Babesia gibsoni. The parasite B. gibsoni was cultured together with erythrocytes from normal dogs for 7 days. When parasitemia reached 12.0-13.4% at Day 7. the production of superoxide in erythrocytes was significantly higher in the parasitized culture than in the control culture (p<0.005). The concentration of thiobarbituric acid reactive substances (TBARS) in erythrocytes in parasitized culture was also significantly increased compared with the control culture (p<0.005), indicating that lipid peroxidation was greater in infected erythrocytes than in non-infected cells. In addition, the rates of superoxide generation in the blood of B. gibsoni-infected dogs were also significantly higher than in non-infected dogs (p<0.001). These results indicate that superoxide anions are increased in erythrocytes parasitized with B. gibsoni. and suggest that oxidative damage, due to lipid peroxidation, might be caused in host erythrocytes by the parasite.     

The development of Plasmodium gallinaceum infections in chickens following single infections with three different dose levels

In the present study, groups of 5-day-old chickens were inoculated intravenously with approximately 10(6), 10(4) or 10(2) P. gallinaceum-infected erythrocytes. The outcome of disease in relation to dose level was evaluated in terms of number of parasitized erythrocytes, change in number of erythrocytes, pathological changes of organs and the course of exo-erythrocytic stages of the parasite in various organs over a period of 8 weeks. Mean weight gain and mortality were also recorded. With regard to differences between the size of the inoculation dose and the severity of disease, relationships could be observed in relation to clinical signs, mortality, prepatent period, exo-erythrocytic parasites and severity of pathological changes in organs, but in relation to weight gain, erythrocyte loss and number of parasitized erythrocytes no relationships were observed.     

Abnormality of osmotic fragility and morphological disorder of bovine erythrocytes infected with Theileria sergenti

The osmotic fragility and the surface structure of erythrocytes obtained from 3 calves infected with Theileria sergenti and from 3 phlebotomized ones were compared. As the parasitemia progressed, the osmotic fragility of the erythrocytes significantly increased in the infected calves. Particularly the hemolysis ratio in the isotonic area (21.5-94.1%) obviously increased. On the other hand, the percentage of parasitized cells in the erythrocytes did not show so much high values (16.1-21.3%). Similar phenomenon was found in each different percentage of erythrocytes suspension which was separated from density gradient centrifugation. No significant difference in the serum osmotic pressure between the infected calves and the phlebotomized calves was found. By scanning microscopy, the erythrocytes of infected calves, which were collected at the crisis period of parasitemia, were almost completely deformed and showed echinocyte form. Moreover, the appearance ratio of echinocyte form in the erythrocytes population was superior to the percentage of parasitized erythrocytes. Similar membranous alterations were also observed in the erythrocytes of grazing cattle in the crisis period of the theileriosis. It was proven that abnormality of osmotic fragility and morphological disorders of erythrocytes occurred not only in parasitized erythrocytes but also in non-parasitized ones in T. sergenti parasitemia.     

Sequestration and phagocytosis of Haemobartonella felis in the spleen.

Spleens of two cats infected with Haemobartonella felis were examined by electron microscopy to determine the means by which the organism was sequestered in this organ. The principal means of sequestration occurred when H felis, located on the erythrocytes was removed by phagocytosis by a cordal macrophage, apparently preceded by the adhesion of extended processes of the macrophage to H felis. The second and least frequent means of removal of H felis was by pitting, a process that did not cause destruction of the host erythrocyte. The H felis was pitted from the parasitized erythrocyte when H felis passed through gaps between reticular cells or when the parasitized erythrocyte passed among the cytoplasmic processes of the reticular cells in the splenic cords. Some H felis were closely associated with the plasmalemma of cordal reticular cells and also were located in intracytoplasmic vacuoles of the cells without being influenced by the phagocytic process.     

Erythrocyte membrane alterations associated with the attachment and replication of Eperythrozoon suis: a light and electron microscopic study

Erythrocytes from pigs with experimental porcine eperythrozoonosis were examined using light microscopy and scanning and transmission electron microscopy. Three distinct forms of Eperythrozoon suis were attached to the plasma surface of erythrocyte membrane. Erythrocytes were initially parasitized by one or several immature forms. Immature forms enlarged and developed into juvenile and mature forms. The parasite replicated by budding of small immature forms from larger immature forms, juvenile forms, and mature forms. Small immature forms attached to adjacent membrane of the same erythrocyte or a nonparasitized erythrocyte. E. suis organisms were intimately associated with, but distinctly separated from erythrocyte membrane by a 30-nm electron lucent zone. Cell membrane in this area was denser than adjacent nonparasitized membrane. Early interaction between erythrocyte membrane and small immature forms resulted in no membrane deformation, but as this form enlarged, it later became embedded in a deep cup-like membrane invagination. As the immature form developed into the juvenile form a shallow, broad-based depression was observed in the membrane. A similar depression that covered a greater surface area was observed in the membrane parasitized by the mature form. The interaction between the parasitic forms and erythrocyte membrane resulted ultimately in severe membrane deformation. Parasite-membrane interactions may play an important role in the development of misdirected immune responses in experimental porcine eperythrozoonosis.     

Trichomonas gallinae: a possible contact-dependent mechanism in the hemolytic activity

The in vitro hemolytic activity of Trichomonas gallinae was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of six adult animals of different species (rabbit, rat, chicken, horse, bovine, and sheep). Results showed that T. gallinae lysed all human erythrocytes groups, as well as rabbit, rat, chicken, horse, bovine and sheep erythrocytes. No hemolysin released by the parasites could be identified. Hemolysis did not occur with trichomonad culture supernatants, with sonicated extracts of T. gallinae, or with killed organisms. The scanning electron microscopy (SEM) showed that the erythrocytes adhered to the parasite surface and were phagocytosed. These observations suggest that the contact between T. gallinae and erythrocytes may be an important mechanism in the injury caused to the erythrocytes. The hemolytic activity of T. gallinae may be an efficient means of obtaining nutrients for the parasite and allow the investigation of the mechanism used by T. gallinae to damage cellular membranes.     

Continuous in vitro cultivation of a recently identified Babesia that infects small ruminants in China

Babesia sp. Xinjiang was isolated from a splenectomised sheep infested by Rhipicephalus sanguineus and Hylomma anatolicum anatolicum, collected from sheep and cattle in Xinjiang province. It was considered to be a novel ovine Babesia species on the basis of its morphology, pathogenicity, vector tick species and alignments of 18S ribosomal RNA (18S rRNA) and internal transcribed spacers (ITS) gene sequences. Continuous in vitro cultures of the ovine parasite were established using infected sheep blood. In RPMI 1640 medium with 7.5% sheep red blood cells (RBCs) maintained in an incubator at 37 °C and 5% CO(2), the percentage of parasitized erythrocytes (PPE) peaked at 10% in 24- and 6-well plates. It increased to 20-50% with the same culture medium but with 2.5% RBC in 75 cm(2) flasks. Two clonal lines of Babesia sp. Xinjiang were screened using the limiting dilution method. Growth characteristics of these lines in vitro were measured by a microtiter-based spectrophotometric method and from the PPE. The generation time in sheep erythrocytes was between 15.20 h and 16.27 h. Furthermore, the host range of parasite was identified with in vitro culture and in vivo infection. Erythrocytes of sheep, cattle, sika deer and humans could be invaded into by lines in vitro, but the parasites could not propagate in human erythrocytes. The parasites could not enter erythrocytes from goats in vitro. However, in vivo, only sheep could be infected by lines. Finally, a Babesia sp. Xinjiang-like parasite (which shared 99.5% identity with the original strain of Babesia sp. Xinjiang) was isolated using this in vitro culture system from 1 of 19 sheep blood samples collected from western Gansu province, China.     

The parotid, mandibular and lateral retropharyngeal lymph nodes of the camel (Camelus dromedarius)

The parotid, mandibular and lateral retropharyngeal lymph nodes of the dromedary camel were examined using both light and electron microscopy. All three lymph nodes were lobulated. They did not show the characteristic medulla, cortex and paracortex of typical lymph nodes. Instead, they contained lymphatic nodules, dense anodular lymphoid tissue and diffuse lymphoid tissue dispersed throughout the lymph node. Networks of sinuses were present in the diffuse lymphoid tissue. The diffuse lymphoid tissue in the periphery of all lymph nodes examined was characterized by numerous erythrocytes within and around its network of sinuses. The nodal sinuses were contiguous with the septal vessels, which are considered the possible source of erythrocytes seen in this study. The lymph nodes that were seen in this study resembled the haemolymph nodes of other mammals with regard to their content of erythrocytes but were unique in being located in sites that were typical of ordinary lymph nodes. Morphometric analysis has shown that the percentage volume densities of the stroma and the various parenchymal components were similar in the three lymph nodes.     

Ultrastructure of Babesai bovis (Babes, 1888)

An electron microscope study of thin sections of bovine erythrocytes parasitized with Babesia bovis (Texas isolate) revealed that the body of the parasite is covered with a pellicular complex consisting of three membranes, two located on the interior and one on the outer part of the pellicle. Parasites observed possessed one nucleus and one nucleolus containing aggregated nuclear material. Each of these aggregations was bounded by two nuclear membranes. Organelles such as polar rings, rhoptries, micronemes, vesicular structures, cisternae of the nuclear membrane, spherical bodies, mitochondria-like structures and Maurer's clefts were indentified and described. The basic similarities and differences in the intra-erythrocytic stages of Babesia bovis, in comparison with other Babesia spp. parasites, are discussed.     

Neuropathology and host-parasite relationship of acute experimental toxoplasmosis of the blue fox (Alopex lagopus)

The neuropathology and host-parasite relationship of experimental infection with the RH-strain of Toxoplasma gondii were studied in 27 blue foxes (Alopex lagopus) aged 0 to 23 days, using light microscopy, including immunohistochemical staining, and transmission and scanning electron microscopy. All cases displayed multifocal necrotic lesions with numerous parasitic tachyzoites in the brain and spinal cord. The gray matter and the meninges were most seriously affected. Although a wide variety of cell types were parasitized, neurons and astrocytes seemed to be the main target cells. Individual parasitophorous vacuoles usually contained only a few tachyzoites, with rosette formations as a prominent feature. The present ultrastructural study supports the theory that the parasites actively invade the host cells by mechanisms that are different from those of phagocytosis. This is apparently the first report indicating that the formation of the network of tubular structures within the parasitophorous vacuole of T. gondii is associated with a transient, sack-like formation in the posterior end of the tachyzoites.     

Experimental Babesia bovis infection in Holstein calves

One intact and two splenectomized Holstein calves were infected intravenously with a Mexican strain of Babesia bovis and killed following the onset of severe clinical disease. A light and electron microscopic study was conducted on selected tissues to examine the relationship between parasitized erythrocytes and microvascular endothelial cells. The pattern and degree of specific organ sequestration of parasitized erythrocytes was assessed and correlated to lesions. Red blood cells infected with Babesia bovis exhibited stellate membrane protrusions. This morphological change appeared to mediate erythrocyte sequestration in the microvascular and capillary beds of the brain, kidney, and adrenal gland by an as yet unknown mechanism(s).     

Scanning electron microscope study of Sendai virus fusion between HeLa cells and chick erythrocytes.

Chick erythrocytes were fused with HeLa cells by Sendai virus and preparations examined by scanning electron microscopy at different times after fusion. Heterokaryons were usually formed by fusion of erythrocyte ghosts with HeLa cells. Occasionally whole erythrocytes were engulfed but there was no evidence that free nuclei fused. Initial inter-cell attachments were usually, and possibly always, made at the site of an attached virus particle. This study helps to correlate topographical findings with previous two-dimensional studies with the transmission electron microscope and may also provide a model system for the fusion of parasitised erythrocytes with eukaryotic cells.     

Sheep as a new experimental host for Babesia divergens

Babesia divergens was cultivated in sheep erythrocytes in RPMI 1640 supplemented with 10% Fetal Calf Serum (FCS) or sheep serum. In vitro cultures in sheep red blood cells were initiated with human erythrocytes infected in vitro with B. divergens Rouen 1987 or with gerbil blood infected with several isolates from bovine origin. After the first subcultures on sheep erythrocytes, a ten-fold multiplication of the parasites was obtained within 48 h. Erythrocytes from three splenectomized sheep were infected in vitro with B. divergens; when parasitaemia reached 10%, the animals were inoculated with homologous parasitized erythrocytes. All sheep expressed hyperthermia with a peak between the 6th and the 9th day post-infection (p-i) and a transitory parasitaemia 10 days p-i. In vitro primary cultures were performed on two of these sheep, demonstrating the parasite persistence at very low parasitaemia in the infected animals. Splenectomized sheep can be used as a new model for B. divergens chronic infection.     

Flow cytometry to evaluate the level of Babesia gibsoni parasitemia in vivo and in vitro by using the fluorescent nucleic acid stain SYTO16

In the present study, we employed flow cytometry to evaluate the level of parasitemia of Babesia gibsoni infecting canine erythrocytes in vivo and in vitro by using fluorescent nucleic acid staining. Peripheral blood samples from a B. gibsoni-infected dog and cultured B. gibsoni parasitizing in canine erythrocytes were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16. In this study, we utilized normal canine erythrocytes (LK erythrocytes) and canine erythrocytes containing high concentrations of potassium, reduced glutathione, and some free amino acids (HK erythrocytes) as host cells for culture. Parasitized cells in vive were discriminated completely from unparasitized cells and a correlation (r = 0.998) between the percentage of SYTO16-positive cells and parasitemia in vivo was observed. On the other hand, erythrocytes in vitro could not be divided clearly into parasitized and unparasitized cells. However, when LK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost the same as, and was well correlated (r = 0.932) with, the level of parasitemia. When HK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost half of, but was correlated (r = 0.982) with, the level of parasitemia. Therefore, we attempted to observe the changes in the percentage of parasitized cells after treatment with antiprotozoal drug or mitochondria inhibitors by using flow cytometry. The changes in the percentage of SYTO16-positive cells corresponded well with the changes of the level of parasitemia when the parasites in HK erythrocytes were cultured with each compound. The present results suggest that flow cytometric detection using SYTO16 is a rapid and reliable method for monitoring parasitemia both in vive and in vitro.     

Ultrastructural localization of anaplasmal antigens (Pawhuska isolate) with ferritin-conjugated antibody.

Antibodies from a cow with an experimentally induced infection of the Pawhuska isolate of bovine anaplasmosis were conjugated with ferritin and used to label antigenic sites in preparations of parasitized erythrocytes. Intact erythrocytes did not label on the extracellular surface. Ferritin-conjugated antibody did not pass through the intact erythrocyte to label the parasite, probably due to the large molecular size of the antibody. Damage to erythrocytic plasmalemma and inclusion body in the hemolyzed erythrocytes and complement-fixation antigen allowed labeling of anaplasmal inclusion structures. The positively labeled structures were outer surface of the pellicle, chromatin of the initial body, and inclusion appendage. Unlabeled structures included inner organismic membrane of the initial body, inclusion membrane, fibrillar protoplasmic network of the initial body, and small electron-dense bodies derived from the initial body.     

Tritrichomonas foetus damages bovine oocytes in vitro

Tritrichomonas foetus is an extracellular parasite of the urogenital tract in cattle. It causes infertility and abortion, but there is no documented information on the susceptibility of bovine oocytes to the parasite, except by one article that claimed no effects of T. foetus on oocytes or embryos. The aim of the present study was to study the effects provoked by T. foetus when in interaction with bovine oocytes. Oocytes were obtained from cow ovaries and divided into two groups: (1) one group contained cumulus cells, whereas (2) a second group was denuded from these cells. Light microscopy, video microscopy and scanning electron microscopy (SEM) revealed that exposure of oocytes to T. foetus caused rapid adhesion of the trichomonads to cumulus cells and to the zona pellucida (ZP). Motile parasites were observed for 12 h. The ZP was completely damaged, and the parasites were able to infiltrate beneath the ZP and reached the oocytes directly when the oocytes were denuded of the cumulus cells. Both the oocytes and the cumulus cells exhibited morphological characteristics compatible with apoptosis after interaction with T. foetus, such as chromatin condensation, the presence of several cytoplasmic vacuoles, with intact cellular membranes and organelles. The results from this study demonstrate that when a large number of T. foetus interacts with oocytes in vitro damage and apoptosis are provoked in the cow's reproductive cells. The behavior of this parasite as one of the causes of cattle infertility is discussed.     

Tritrichomonas foetus: a scanning electron microscopy study of erythrocyte adhesion associated with hemolytic activity

The in vitro hemolytic activity of Tritrichomonas foetus was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of nine adult animals of different species (the rabbit, rat, chicken, cat, dog, swine, horse, bovine, and sheep). The results showed that T. foetus strains (ATCC KV1, K, PAL, 5022, RJ, 90) did not present any hemolytic activity against any human erythrocyte group nor against rabbit, rat, chicken, cat, dog and swine erythrocytes. T. foetus strains, however, lysed horse, bovine, and sheep erythrocytes. No hemolysin released by the parasites could be identified. Hemolysis did not occur with trichomonad culture supernatants, with sonicated extracts of T. foetus, nor with killed organisms. Scanning electron microscopy (SEM) showed that human erythrocytes did not adhere to the trophozoites, in contrast horse erythrocytes adhered to the surface of the parasites and were phagocytosed for up to 90 min. The parasites are able to exert their cytopathic effects through: (a) physical contact established between the two cell surfaces, (b) toxins released from parasites into the interaction media, or (c) the association of both mechanisms. Further studies are necessary to clarify the importance of the hemolytic activity in the biology of T. foetus.     

吉氏巴贝斯虫实验动物模型的研究

用吉氏巴贝斯虫感染置换了犬红细胞的SCID鼠,吉氏巴贝斯虫在SCID鼠体内得到高水平的生长和增殖.虫体大小比在犬体内略增大,而且繁殖型虫体增多,常在一个红细胞内寄生有2、4、8、16和32个虫体.在感染的第10天前后,末梢血液中红细胞的染虫率高达12%左右.从SCID鼠末稍血液能检出虫体的期限为15～18天左右.从而成功地建立了吉氏巴贝斯虫的实验动物模型.     

Efficacy of enrofloxacin against severe experimental Anaplasma marginale infections in splenectomized calves

Four Anaplasma marginale-infected splenectomized calves with greater than 25% parasitized erythrocytes received enrofloxacin at 12.5 mg/kg SC twice, 48 hours apart. Two infected splenectomized calves were designated as untreated controls. A precipitous decline in percent parasitized erythrocytes from 39.13% to less than 1% was observed over 12 days following treatment. However, a self-limiting recrudescence of A. marginale parasites was observed within 30 days after treatment. Untreated control calves became moribund and were euthanized. These data indicate that the regimen of enrofloxacin tested herein ameliorates, but does not eliminate, A. marginale infections in splenectomized calves.