Low prevalence of Babesia hongkongensis infection in community and privately-owned cats in Hong Kong.

Domestic cats are susceptible to infection with at least 11 species of Babesia. In Hong Kong, where dogs are commonly infected with B. gibsoni, a single infection in a cat by a novel species, B. hongkongensis, was reported previously. The aim of this study was to investigate the frequency of Babesia spp. detection in cats in Hong Kong. Residual blood-derived DNA from healthy free-roaming community cats (n = 239), and privately-owned cats with and without anaemia undergoing diagnostic investigations (n = 125) was tested for Babesia spp. DNA using a pan-Babesia PCR targeting mitochondrial Cytochrome B, and a B. hongkongensis specific PCR targeting 18S rRNA. Positive samples were confirmed by sequencing and comparative sequence analysis against the GenBank nucleotide database. Babesia hongkongensis was detected in 4/239 (1.7 %) community cats, and 0/125 (0.0 %) privately-owned cats. Babesia gibsoni was detected in 0/239 community cats and 1/125 (0.8 %) privately-owned cats. Cats infected with B. hongkongensis were clinically healthy at the time of sampling. The B. gibsoni-infected cat was anaemic and thrombocytopenic. Cats in Hong Kong can be infected with B. hongkongensis and B. gibsoni, albeit at low frequency. The tick vector for B. hongkongensis is yet to be identified.

Physico-chemical characterization of pine cone shell and its use as biosorbent and fuel.

Physico-chemical properties of pine cone shell have been determined. Results of characterization study showed that pine cone shell could be used as biosorbent of nickel from aqueous solutions in a fixed-bed column and later as input material in thermochemical processes. To study the behavior of Ni-loaded pine cone shell as fuel, non-isothermal thermogravimetric tests were performed. These tests showed that, in nitrogen atmosphere, the main decomposition occurs from 200°C to 500°C and, in oxidant atmosphere, the behavior is of type "combustion+pyrolysis" (at higher temperatures there is a clear decomposition of residue formed during the initial steps). Finally, the effect of the presence of Ni was analyzed. Thermogravimetric curves did not change their profile and the total amount of nickel was detected in char-ash fraction and not in flue gases. These results suggest that nickel does not form volatile compounds at considered operational conditions.

First appearance, distribution, and origin of macrophages in the early development of the avian central nervous system.

A phagocytic cell system of hemopoietic origin exists in the early avian embryo (Cuadros, Coltey, Nieto, and Martin: Development 115:157-168, '92). In this study we investigated the presence of cells belonging to this system in the central nervous system (CNS) of chick and quail embryos by using both histochemical staining for acid phosphatase and immunolabelling with antibodies recognizing cells of quail hemangioblastic lineage. The origin of these cells was traced in interspecific chick-quail yolk sac chimeras. Hemopoietic cells were detected within the CNS from developmental stage HH15 on, and steadily increased in number at subsequent stages. Analysis of yolk sac chimeras revealed that most of these cells were of yolk sac origin, although some hemopoietic cells of intramebryonic origin were also found in the CNS. Immunocytochemical, histochemical, and ultrastructural characterization allowed us to identify hemopoietic cells in the CNS as macrophages. These cells were consistently found in the brain vesicles and spinal cord, appearing (1) between undifferentiated neuroepithelial cells at dorsal levels of the CNS; (2) in areas of cell death; (3) in the marginal layer in close relationship with developing axons; (4) in large extracellular spaces in the subventricular layer; (5) on vascular buds growing through the marginal and subventricular layers; and (6) in the ventricular lumen. Macrophages in different locations varied in morphology and ultrastructure, suggesting that in addition to their involvement in phagocytosis, they play a role in other processes in the developing CNS, such as axonal growth and vascular development. The first macrophages migrate to the CNS independently of its vascularization, apparently traversing the pial basal lamina to reach the nervous parenchyma. Other macrophages may enter the CNS together with vascular buds at subsequent stages during CNS vascularization.

Origin of microglia in the quail retina: central-to-peripheral and vitreal-to-scleral migration of microglial precursors during development.

The origin, migration, and differentiation of microglial precursors in the avascular quail retina during embryonic and posthatching development were examined in this study. Microglial precursors and developing microglia were immunocytochemically labeled with QH1 antibody in retinal whole mounts and sections. The retina was free of QH1+ macrophages at embryonic day 5 (E5). Ameboid QH1+ macrophages from the pecten entered the retina from E7 on. These macrophages spread from central to peripheral areas in the retina by migrating on the endfeet of the Müller cells and reached the periphery of the retina at E12. While earlier macrophages were migrating along the inner limiting membrane, other macrophages continued to enter the retina from the pecten until hatching (E16). From E9 on, macrophages were seen to colonize progressively more scleral retinal layers as development advanced. Macrophages first appeared in the ganglion cell layer at E9, in the inner plexiform layer at E12, and in the outer plexiform layer at E14. Therefore, it seems that macrophages first migrated tangentially along the inner retinal surface and then migrated from vitreal to scleral levels to gain access to the plexiform layers, where they differentiated into ramified microglia. Macrophages appeared to differentiate shortly after arrival in the plexiform layers, as poorly ramified QH1+ cells were seen as early as E12 in the inner plexiform layer and at E14 in the outer plexiform layer. Radial migration of macrophages toward the outer plexiform layer continued until posthatching day 3, after which retinal microglia showed an adult distribution pattern. We also observed numerous vitreal macrophages intimately adhered to the surface of the pecten during embryonic development, when macrophages migrated into the retina. These vitreal macrophages were not seen from hatching onwards, when no further macrophages entered the retina.

Microglia development in the quail cerebellum.

We used the QH1 antibody to study changes in the morphological features and distribution of microglial cells throughout development in the quail cerebellum. Few microglial precursors were present in the cerebellar anlage before the ninth incubation day (E9), whereas many precursors apparently entered the cerebellum from the meninges in the basal region of the cerebellar peduncles between E9 and E16. From this point of entry into the nervous parenchyma, they spread through the cerebellar white matter, forming a 'stream' of labeled cells that could be seen until hatching (E16). The number of microglial cells in the cerebellar cortex increased during the last days of embryonic life and first posthatching week, whereas microglial density within the white matter decreased after hatching. As a consequence, the differences in microglial cell density observed in the cerebellar cortex and the white matter during embryonic life diminished after hatching, and microglia showed a nearly homogeneous pattern of distribution in adult cerebella. Ameboid and poorly ramified microglial cells were found in developing stages, whereas only mature microglia appeared in adult cerebella. Our observations suggest that microglial precursors enter the cerebellar anlage mainly by traversing the pial surface at the basal region of the peduncles, then migrate along the white matter, and finally move radially to the different cortical layers. Differentiation occurs after the microglial cells have reached their final position. In other brain regions the development of microglia follows similar stages, suggesting that these steps are general rules of microglial development in the central nervous system.

Naturally occurring cell death and migration of microglial precursors in the quail retina during normal development.

We compared chronotopographical patterns of distribution of naturally occurring neuronal death in the ganglion cell layer (GCL) and the inner nuclear layer (INL) with patterns of tangential and radial migration of microglial precursors during quail retinal development. Apoptotic cells were identified by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling technique, and microglial precursors were identified by immunocytochemistry with an antibody recognizing quail microglial cells (QH1 antibody). Apoptotic cells were first detectable in the GCL at the seventh day of incubation (E7), were most abundant at E10, and were absent after E13. In the INL, apoptotic cells first appeared at E7, were most abundant at E12, and disappeared entirely after the third posthatching day (P3). In both retinal layers, cell death first appeared in a small central area of the retina and subsequently spread along three gradients: central-to-peripheral, temporal-to-nasal, and dorsal-to-ventral. The chronology of tangential (between E7 and E16) and radial migration (between E8 and P3) of microglial precursors was highly coincident with that of cell death in the GCL and INL. Comparison of the chronotopographical pattern of distribution of apoptotic nuclei in the GCL with the patterns of tangential and radial migration of microglial precursors neither supported nor refuted the hypothesis that ganglion cell death is the stimulus that triggers the entry and migration of microglial precursors in the developing retina. However, microglial cells in most of the retina traversed the INL only after cell death had ceased in this layer, suggesting that cell death in the INL does not attract microglial precursors migrating radially. Dead cell debris in this layer was phagocytosed by Müller cells, whereas migrating microglial cells were seen phagocytosing apoptotic bodies in the nerve fiber layer and GCL but not in the INL.

Proliferation of actively migrating ameboid microglia in the developing quail retina.

Sheets containing the inner limiting membrane covered by a carpet of Müller cell endfeet were used to show that ameboid microglial cells migrating tangentially in the vitreal part of the developing retina of quail embryos underwent mitosis. Double labeling with anti-beta-tubulin/QH1 or Hoechst 33342/QH1 revealed that some migroglial cells with morphological features typical of active migration were in early prophase. By anaphase and early telophase, microglial cells had retracted their lamellipodia and were ovoid in shape. Later in telophase, but well before completion of cytokinesis, both daughter cells again emitted lamellipodia, thus regaining the typical morphology of migrating cells. We concluded that ameboid microglial cells go through cycles in which migration and mitosis alternate, and that both mechanisms contribute to the spread of microglia throughout the developing retina. The mitotic spindle of dividing microglial cells showed different orientations, which probably influenced the course of subsequent migration. The expression of the proliferating cell nuclear antigen in the nucleus of most tangentially migrating ameboid microglial cells at E9-E10 confirmed their proliferative capability. However, the rate of proliferation of these cells decreased during embryonic development, and was nearly zero at E14.

Localization and distribution of alkaline phosphatase activity in the hepatopancreas of the snail.

Histocytochemical methods were used to investigate alkaline phosphatase activity in the digestive gland (hepatopancreas) of the common garden snail Helix aspersa. Histochemical findings and light microscopic observations showed that enzymatic activity was confined mainly to the basal connective tissue that enveloped the adenomeres. Transmission electron microscopy showed that enzymatic activity was localized in the plasma membrane, and showed an intercellular distribution along the lateral surfaces and the basal portions of the cells in different adenomeres. Alkaline phosphatase activity was also found in the plasma membrane of fibrocytes of the basal connective tissue enveloping the adenomeres. Enzymatic activity was seen around the fat droplets of glandular cells. The possible involvement of alkaline phosphatase in processes or remodelling of the basal connective tissue that envelopes the gland is discussed.

Phosphatase activity in the hepatopancreas of Helix aspersa.

1. Phosphatase acid (PhA) activity in the digestive gland (hepatopancreas) of the common garden snail Helix aspersa has been investigated using cytochemical methods. 2. All the cells composing this gland show PhA activity, the distribution pattern differing according to the cell type. 3. The digestive cells show the most widely distributed reaction product (brush border, phagolysosomes, multivesicular bodies and autophagic vacuoles). 4. In the excretory cells this activity appears in large sacs, while in the calcium cells the reaction product is abundant in the calcium granules. 5. Cellular digestion processes performed by each of these cell types is discussed together with their role in the detoxification of heavy elements derived from the environment.

An electron microscopic study on the muscle cells and nerve fibres in the digestive gland of the snail Cryptomphallus aspersa.

This work is an electron microscopic study of the muscle cells and the nerve fibres which can be found between the connective tissue of the digestive gland of the gastropod mollusc Cryptomphallus aspersa. The muscle cells are smooth and are located immediately below the basal lamina of the secretory epithelium or in more central zones of the connective tissue of the gland. Two types of myofilaments can be found in the sarcoplasm: thin and thick. The thin ones have an approximate diameter of 5 nm, whereas the thick ones vary in diameter between 15 and 65 nm. The possibility exists that the myofilaments of 15 nm might belong to a third type. Nerve fibres can be found in central zones of the connective tissue of the digestive gland, at the base of the glandular tubules and within the secretory epithelium. In these fibres it is characteristic to find a large number of granules, of a diameter between 80 and 250 nm, surrounded by a membrane, the content of which varies in density. Small clear vesicles of 40 . . . 70 nm in diameter can also be observed. Occasionally granules, which fuse their membranes thus mixing their content, can be detected.

Tangential migration of ameboid microglia in the developing quail retina: mechanism of migration and migratory behavior.

Long distance migration of microglial precursors within the central nervous system is essential for microglial colonization of the nervous parenchyma. We studied morphological features of ameboid microglial cells migrating tangentially in the developing quail retina to shed light on the mechanism of migration and migratory behavior of microglial precursors. Many microglial precursors remained attached on retinal sheets containing the inner limiting membrane covered by a carpet of Müller cell endfeet. This demonstrates that most ameboid microglial cells migrate tangentially on Müller cell endfeet. Many of these cells showed a central-to-peripheral polarized morphology, with extensive lamellipodia spreading through grooves flanked by Müller cell radial processes, to which they were frequently anchored. Low protuberances from the vitreal face of microglial precursors were firmly attached to the subjacent basal lamina, which was accessible through gaps in the carpet of Müller cell endfeet. These results suggest a mechanism of migration involving polarized extension of lamellipodia at the leading edge of the cell, strong cell-to-substrate attachment, translocation of the cell body forward, and retraction of the rear of the cell. Other ameboid cells were multipolar, with lamellipodial projections radiating in all directions from the cell body, suggesting that microglial precursors explore the surrounding environment to orient their movement. Central-to-peripheral migration of microglial precursors in the retina does not follow a straight path; instead, these cells perform forward, backward, and sideways movements, as suggested by the occurrence of (a) V-shaped bipolar ameboid cells with their vertex pointing toward either the center or the periphery of the retina, and (b) threadlike processes projecting from either the periphery-facing edge or the center-facing edge of ameboid microglial cells.

Circumferential migration of ameboid microglia in the margin of the developing quail retina.

Central-to-peripheral migration of QH1-positive microglial precursors occurs in the vitrealmost part of the developing quail retina. This study shows that some QH1-positive ameboid cells with morphological features of migrating cells are already present in the margin of the retina before microglial precursors migrating centrally to peripherally arrive in this zone. Because the earlier cells are oriented parallel to the ora serrata, we deduce that some microglial cells migrate circumferentially in the margin of the retina, whereas other microglial precursors migrate from central to peripheral zones. Microglial cells that migrate circumferentially are first seen on embryonic day 6 (E6) and advance in a temporal-to-dorsal-to-nasal direction from the temporoventral quadrant of the retina. When cells migrating centrally to peripherally reach the retinal margin, they meet those migrating circumferentially. From E6 on, some QH1-positive dendritic cells in the ciliary body bear processes that penetrate the retina, where they are oriented circumferentially. These observations suggest that microglial cells that migrate circumferentially in the retinal margin share a common origin with dendritic cells of the ciliary body. Therefore, microglial cells of the quail retina appear to make up a heterogeneous population, with some cells originating from the pecten/optic nerve head area and others from the ciliary body.

Effect of dietary nucleotides on small intestinal repair after diarrhoea. Histological and ultrastructural changes.

The effects of specific nutrients on intestinal maturation and repair after injury are practically unknown. The purpose of this work was to study the effects of dietary nucleotides on the repair of the intestinal mucosa after chronic diarrhoea induced by a lactose enriched diet in the weanling rat. One group of weanling rats was fed with a standard semipurified diet (control group), and another group was fed with the same diet containing lactose as the only soluble carbohydrate (lactose group). After 14 days the lactose group was allowed to recover for four weeks with the control diet (lactose-control group) or with the control diet supplemented with AMP, GMP, IMP, CMP, and UMP 50 mg/100 g each (lactose-nucleotide group). The control group was divided into two subgroups, which were fed with the control diet and the nucleotide supplemented diet for the same period (control-control group and control-nucleotide group). The lactose diet induced diarrhoea after 24 hours of feeding. Two weeks later there were changes in intestinal structure with loss of enterocyte microvillar surface, significant lymphocyte infiltration, supranuclear cytoplasmic vesiculation, decreased number of goblet cells, and enlarged mitochondria with low density and few cristae. After recovery from diarrhoea, animals fed the nucleotide enriched diet showed an intestinal histology and ultrastructure closer to that of the normal control group. Mitochondrial ultrastructure was closer to normal in comparison with the lactose-control diet group. In this second group the number of goblet cells as well as the villous height/crypt depth ratio was reduced and the number of intraepithelial lymphocytes increased compared with the nucleotide supplemented group. These results suggest that dietary nucleotides may be important nutrients for intestinal repair.

Dietary restriction induces biochemical and morphometric changes in the small intestine of nursing piglets.

The purpose of this study was to evaluate the biochemical and morphometric changes in the small intestine of nursing piglets caused by 60% dietary restriction, and to ascertain whether this model reproduces the intestinal alterations caused by malnutrition in human infants. Piglets subjected to dietary restriction had significantly lower levels of mucosal DNA and protein, and significantly reduced segmental disaccharidase and leucine aminopeptidase activities compared with age-matched, freely fed controls. However, greater disaccharidase-specific activities were observed in duodenum and jejunum of diet restricted piglets compared with controls. Other findings included significantly lower thickness of the mucose, villous height and width, and villous surface area, a significantly lower number of goblet cells, and significantly greater mucosal crypt depth, intraepithelial leucocyte number, and infiltrated cells per area of lamina propria. The model reproduces most of the biochemical and morphometric changes observed in the small intestine of young human infants with chronic diarrhea and malnutrition, and may be useful in further investigations of the biochemical and molecular mechanisms of intestinal alterations caused by primary malnutrition in early infancy.