Biomedical subjects

J H Rombout

Publications and source records attributed to J H Rombout.

At least 19 recordsLinked to original sources

PubMed2001

The gill is a major organ for antibody secreting cell production following direct immersion of sea bass (Dicentrarchus labrax, L.) in a Photobacterium damselae ssp. piscicida bacterin: an ontogenetic study.

Extremely high numbers of antibody secreting cells (ASC) were observed in the gills of sea bass fry immunised at three different age/sizes (initial weight of 0.1, 2 and 5 g) by direct immersion in a Photobacterium damselae spp. piscicida bacterin. The relatively low ASC production in the head kidney and spleen suggests that the systemic compartment was only slightly stimulated upon immersion vaccination. There was no response of corresponding magnitude in the gut as the one observed in the gills. A clear age effect was observed in the ASC response of the different groups, especially visible in the gills. Significantly higher numbers of specific ASC were observed in the gills of the two oldest groups (initial weight of 2 and 5 g) compared with the youngest fish (initial weight of 0.1 g), but the oldest groups were not significantly different from each other. Additionally, a more rapid response was observed with the ageing of the fish, with peak responses in all the organs at day 18, 16 and 8 post-immunisation in the smallest to largest fish, respectively. There was no evidence that direct immersion exposure to P. damselae ssp. piscicida at the earliest stages used in the present study (0.1 g) was tolerogenic. In the context of present knowledge, this study strongly supports the importance of the route of immunisation to locally stimulate ASC and the importance that the gills might have in specific responses.

Age Factors↗

PubMed2001

Kinetics of juvenile sea bass (Dicentrarchus labrax, L.) systemic and mucosal antibody secreting cell response to different antigens (Photobacterium damselae spp. piscicida, Vibrio anguillarum and DNP).

The ELISPOT assay was used to measure the number of specific antibody secreting cells (ASC) induced during the primary and secondary immune responses in the spleen, head kidney and gut of juvenile (5 g) sea bass (Dicentrarchus labrax) to bacterial (Vibrio anguillarum and Photobacterium damselae ssp. piscicida) and hapten dinitrophenyl-conjugated to keyhole limpet haemocyanin (DNP-KLH) antigens administered intraperitoneally. High variability among individuals was observed at each sampling day. All fish were bath vaccinated to V. anguillarum at an earlier stage (2 g) in the farm of origin prior to the development of the experiments, and therefore only secondary and tertiary responses were measured in the group immunised with this bacterium. Significant differences to the controls were observed in the primary responses of the head kidney and the spleen to P. damselae ssp. piscicida and DNP, respectively. Frequency analysis of the production of ASC suggests that significant responses in the gut might be masked by the high error variance. The peak of the primary response was observed 4 days earlier to DNP (18-20 days post-immunisation) and it was significantly higher than the response to P. damselae ssp. piscicida. Higher numbers of ASC were observed in the secondary responses of the head kidney and spleen, although they were not statistically significantly different from the primary levels, probably due to the high error variance as supported by the frequency analysis. Nevertheless, together with a faster response (peak at 7 days post-immunisation), the data suggest that memory formation had occurred. Additionally, the data suggest that some suppression of the secondary immune response in the gut might have occurred. The head kidney appears to produce the highest number of specific ASC of the organs tested. It appears that sea bass show a relatively fast but short duration antibody response.

Animals↗

PubMed2001

Monoclonal antibodies against haemocyte molecules of Penaeus monodon shrimp react with haemolymph components of other crustaceans and disparate taxa.

In a previous study, monoclonal antibodies (mAbs) against different haemolymph molecules of the marine shrimp Penaeus monodon were produced and characterised. It was suggested that these mAbs could be used in studying haemocyte differentiation, behaviour and function in P. monodon. In the present study, the reaction of these mAbs on P. monodon was compared with other crustaceans and disparate taxa. The mAbs also reacted with haemolymph components of three freshwater crustaceans, a terrestrial isopod crustacean and with coelomic fluid of an annelid. No reactions were observed with haemolymph of an insect and a mollusc, nor with blood cells of two vertebrates. This comparative study shows reactivity of the mAbs with a wide range of crustaceans and related animals and suggests that well conserved molecules are recognised, which may indicate functional importance. Well-described mAbs can be used in studies of the crustacean defence system and may finally result in a better insight into this system.

Animals↗

PubMed2000

Characterisation of different morphological features of black tiger shrimp (Penaeus monodon) haemocytes using monoclonal antibodies.

Monoclonal antibodies (mabs) specific for Penaeus monodon haemocytes were produced by immunising mice with membrane lysates of shrimp haemocytes. Four mabs (WSH 6, WSH 7, WSH 8 and WSH 16) were characterised using flow cytometry, light microscopy, laser scanning microscopy, electron microscopy and immunoprecipitation. WSH 6 recognised a carbohydrate determinant on an 85 kDa molecule. WSH 7, WSH 8 and WSH 16 recognised 50, 35 and 115 kDa molecules, respectively. For all mabs, differences in amount and intensity of the labelling were found when haemocytes were fixed immediately in 2% formaldehyde in Alsever's Solution (AS), compared with non-fixed haemocytes that were kept in AS (which reduced activation of the haemocytes) or in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of >80% of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mabs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS and in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different size and electron density. Immunoreactive extracellular thread-like material could be observed in cells in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and the lowest for WSH 16. Double labelling revealed that all mabs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis is put forward that immunoreactive molecules recognised by these mabs, are related to haemocyte activation factors.

Animals↗

PubMed2000

Ontogeny of B and T cells in sea bass (Dicentrarchus labrax, L.).

Monoclonal antibodies specific to sea bass Ig heavy (WDI 1) and light (WDI 3) chains and T cells (DLT15) were used in an ontogenetic study of sea bass by flow cytometry and immunocytochemistry. The influence of weight and age, as well as season, on B cell development was studied in the fastest and slowest growing offspring from the same spawn (5-305 days post hatch: dph). Additionally, B and T cell development was followed in samples of different offspring (5-137 dph). The results suggest that DLT15 recognises very early (pre-?) T cells as well as mature T cells and that these very early T cells might have their origin in a different compartment and subsequently mature in the thymus. They also appeared much earlier in ontogeny (between 5-12 dph onwards) than pre-B cells having cytoplasmic Ig (from 52 dph onwards). With the monoclonal antibodies used, adult levels of T and B cells were both reached between 137-145 dph, suggesting that sea bass is immunologically mature from at least that age onwards. As in other teleosts, the thymus appears to be the primary organ for T lymphocytes and head kidney the primary organ for B lymphocytes. For sea bass, age seems to be more important in determining B cell maturation than body weight.

Aging↗

PubMed1998

Distribution of macrophages during fish development: an immunohistochemical study in carp (Cyprinus carpio, L.).

A monoclonal antibody against carp macrophages (WCL15) has been utilised in flow cytometry, immuno-histochemistry and immuno-electron microscopy to assess the distribution of monocytes/macrophages in developing carp lymphoid tissues. In suspensions of living cells WCL15 reacted strongly with cytoplasm and plasmic membrane of macrophages. It also cross-reacted with a subpopulation of thrombocytes, but this reaction could be neglected by double immunostaining in combination with a thrombocyte-specific marker. In Bouin-fixed tissues the antibody distinctly recognised macrophages. Macrophages were found from day 2 post-fertilisation in head kidney and in the dorsal portion of the yolk sac epithelium. From 1 week onwards macrophages were found scattered in thymus and gut and during the second week in spleen. Macrophages increased in number in all lymphoid tissues until the 6-8th week post-fertilisation, but they decreased except in thymus, where they became localised mainly in the cortical-medullary boundary, and in white pulp areas of head kidney. The role of macrophages in allowing an early non-specific defence in young fish and in co-operating during the differentiation processes of T-cells and B-cells is discussed.

Animals↗

PubMed1998

Expression and temperature-dependent regulation of the beta2-microglobulin (Cyca-B2m) gene in a cold-blooded vertebrate, the common carp (Cyprinus carpio L.).

Expression of beta2-microglobulin (beta2m) in the common carp was studied using a polyclonal antibody raised against a recombinant protein obtained from eukaryotic expression of the Cyca-B2m gene. Beta2m is expressed on peripheral blood Ig+ and Ig lymphocytes, but not on erythrocytes and thrombocytes. In spleen and pronephros, dull- and bright-positive populations could be identified correlating with the presence of erythrocytes, thrombocytes, and mature leucocytes or immature and mature cells from the lympho-myeloid lineage, respectively. Thymocytes were shown to be comprised of a single bright-positive population. The Cyca-B2m polyclonal antiserum was used in conjunction with a similarly produced polyclonal antiserum to an MHC class I (Cyca-UA) alpha chain to investigate the expression of class I molecules on peripheral blood leucocytes (PBL) at different permissive temperatures. At 12 degrees C, a temporary downregulation of class I molecules was demonstrated, which recovered to normal levels within 3 days. However, at 6 degrees C, a lasting absence of class I cell-surface expression was observed, which could be restored slowly by transfer to 12 degrees C. The expression of immunoglobulin molecules on B cells was unaffected by temperature changes. The absence of the class I cell-surface expression was shown to be the result of a lack of sufficient Cyca-B2m gene transcription, although Cyca-UA mRNA was present at comparable levels at all temperatures. This suggests that class I expression is regulated by a temperature-sensitive transcription of the Cyca-B2m gene.

Animals↗

PubMed1997

Induction of oral tolerance in carp (Cyprinus carpio L.) after feeding protein antigens.

Induction of oral tolerance against ferritin, recombinant surface glycoprotein of viral haemorrhagic septicemia virus (KLG18) and ovalbumin (OVA) was studied in carp. Feeding of ferritin or KLG18 resulted in lower Ab titres compared to unprimed controls when animals were intramuscularly (i.m.) injected with protein 10 weeks later and sampled 21 days after this injection. After administration of OVA by different routes (oral, anal, i.m.) and i.m. injection with OVA + Freund's incomplete adjuvant 2 months later, only a few fish responded to OVA as measured by serum Ab titres. Responsiveness to OVA appeared to be carp strain dependent. When an isogenic carp strain was selected for an optimal response to i.m. injection with OVA, this carp strain did not develop oral tolerance after feeding. In contrast, 6 x feeding high doses of OVA on subsequent days, resulted in immunological memory formation. Oral tolerance can be induced in carp, but differences in tolerance induction may depend on the protein used. A possible role of genetic factors in the induction of oral tolerance in fish is discussed.

Administration, Oral↗

PubMed1996

Influence of flumequine and oxytetracycline on the resistance of the European eel against the parasitic swimbladder nematode Anguillicola crassus.

The effect of the antibacterial drugs flumequine (FQ) and oxytetracycline (OTC) on the defence system of the European eel (Anguilla anguilla L., 1758) was investigated using an experimentally induced infection with the parasitic swimbladder nematode Anguillicola crassus. Eight weeks after oral administration of infective larvae, the mean recovery of the parasites in FQ-treated eels was lower than in non-medicated control animals, and significantly lower than in OTC-treated eels. Mean numbers of peripheral blood granulocytes and B-lymphocytes, as well as the total number of circulating lymphoid cells, showed a significant increase as a result of the infection, while drug treatment merely affected the quantity of the lymphoid cells. The difference in protection against the parasite after FQ or OTC administration points to a modulation of the fish resistance as a result of the drug treatment. The results favour a modulation of the cellular rather than the humoral response, as no specific antibodies were found.

Air Sacs↗

PubMed1995

Production, characterisation and applicability of monoclonal antibodies to European eel (Anguilla anguilla L., 1758) immunoglobulin.

Monoclonal antibodies (mAbs) to European eel (Anguilla anguilla L., 1758) immunoglobulin (Ig) were produced, characterised and tested for applicability in a number of immuno(cyto)chemical assays. The selected mAbs, WEI 1 and WEI 2, were specifically reactive with Ig heavy and light chain, respectively. WEI 1 appeared to react with all or nearly all Ig molecules, B cells and plasma cells. WEI 2 was reactive with a subpopulation of those cells, indicating that European eel possesses at least two antigenically different light chain types. Both mAbs could be used for detection of antigen-specific antibodies in plasma by means of an enzyme-linked immunosorbent assay (ELISA).

Anguilla↗

PubMed1995

Immunocytochemical analysis of a monoclonal antibody specific for rainbow trout (Oncorhynchus mykiss) granulocytes and thrombocytes.

A monoclonal antibody against rainbow trout peripheral blood leucocytes was selected for its lack of reactivity with rainbow trout immunoglobulin. Its reactivity with leucocytes from peripheral blood, head kidney and spleen was analysed by flow cytometry and electron microscopy, and compared with that of monoclonal antibodies directed against rainbow trout immunoglobulin, which reacted with B cells, B lymphoblasts and plasma cells. The antibody reacted with 5-20% of the peripheral blood leucocytes, 8-9% of head kidney leucocytes and 5-7% of spleen leucocytes. Electron microscopical immunocytochemistry revealed that the antibody reacted strongly with granulocytes and weakly with thrombocytes, and not with erythrocytes, lymphocytes, monocytes or macrophages. The antibody has possible applications in the identification and isolation of rainbow trout leucocytes, either alone or in combination with other monoclonal antibodies.

Animals↗

PubMed1995

Influence of flumequine on in vivo mitogen responses of European eel (Anguilla anguilla L., 1758) lymphoid cells.

The influence of flumequine on mitogen induced lymphoid cell proliferation in European eels (Anguilla anguilla L., 1758) was studied. For this purpose an in vivo test, using peroral drug administration followed by successive intraperitoneal injections with concanavalin A (ConA) or bacterial lipopolysaccharides (LPS) and 5-bromo-2'-deoxyuridine, was applied. Direct counting of proliferated cells in blood smears revealed that flumequine possesses mitogenic properties. A synergistic and an antagonistic effect of the drug was observed after LPS and ConA stimulation, respectively. Flow cytometric analysis of peripheral blood lymphoid cells showed a significant reduction of the mean proportion surface immunoglobulin positive cells in the flumequine-treated animals. It is concluded that flumequine enhances proliferation of lymphoid cells (probably surface immunoglobulin negative cells) in eel under the present experimental conditions.

Animals↗

PubMed1994

Immunocytochemical analysis of mitogen responses of carp (Cyprinus carpio L.) peripheral blood leucocytes.

Phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) responses of surface immunoglobulin-positive (sIg+) and surface immunoglobulin-negative (sIg-) carp peripheral blood leucocytes (PBL) were studied. sIg+ cell-enriched and depleted carp PBL populations (sIg+ and sIg- cell fractions, respectively) were obtained by magnetic cell sorting (MACS) and mitogenic stimulation in vitro was measured by 3H-thymidine incorporation. The mitogen responses of sIg+ and sIg- cells in non-separated carp PBL cultures were analysed by simultaneous detection of incorporated 5-bromo-2'-deoxyuridine (BrdU) and sIg with the fluorescence microscope and flow cytometer. Flow cytometric determination of the percentage of sIg+ cells in combination with absolute cell counting, revealed an increase of sIg+ cells but not of sIg- cells after LPS stimulation while the number of sIg- cells and not of sIg+ cells was enhanced after PHA stimulation. LPS stimulation showed an increased 3H-thymidine incorporation in the sIg- cell fraction compared with non-separated cells and BrdU incorporation was observed in sIg- cells from LPS-stimulated cultures by fluorescence microscopy. However, flow cytometric analysis showed that mainly dull sIg+ cells and not sIg- cells are stimulated by LPS. These dull sIg+ cells were not sorted from sIg- cells with MACS and could apparently not be distinguished from sIg- cells by light microscopy. PHA stimulates sIg- cells and not sIg+ cells as was estimated by all techniques used.

Animals↗

PubMed1994

Response of Ig-positive cells to Goussia carpelli (Protozoa: Apicomplexa) infections in carp (Cyprinus carpio L.).

The kinetics of Ig-positive cell populations in carp tissues was followed during an infection with the gut dwelling coccidian Goussia carpelli Léger et Stankovich, 1921. In cell suspensions of the anterior and posterior sections of the intestine, the proportion of Ig-positive cells increased with the development of the coccidia and peaked during oocyst formation at day 15 post exposure. These results suggest a reaction of the local mucosal immune system. In cell suspensions of pronephros the proportion of Ig-positive cells increased as well, indicating that a systemic immune response was also induced against this intestinal coccidian parasite of carp.

Animals↗

PubMed1992

Effects of vitamin A deficiency and Newcastle disease virus infection on lymphocyte subpopulations in chicken blood.

The effect of vitamin A deficiency and Newcastle disease virus (NDV)-infection on peripheral blood lymphocytes (PBL) was studied by differential cell counting and flow cytometry. Day-old chickens were fed purified diets containing either marginal or adequate levels of vitamin A and at 26 days of age half of the chickens in each group were infected with NDV. The absolute numbers of PBL and their subpopulations were studied until 10 days after infection. Vitamin A deficiency resulted in significantly lower numbers of PBL throughout the experiment. NDV-infection produced lymphopenia during the first 3 days, followed by a strong increase in PBL numbers after 6 days. Both changes in PBL were less pronounced in vitamin A-deficient birds. For flow cytometric analysis monoclonal antibodies reacting specifically with B-cells or a subpopulation of T-cells were used. Vitamin A-induced lymphopenia could be attributed to a decreased number of PBL, negative for both antibodies, and to the absence of an increase in B-cells which normally occurs at this age. The negative cells are suggested to represent, at least partially, cytotoxic T-cells, which may explain the impaired cytotoxic T-cell-activity found in earlier studies. NDV-induced lymphopenia and subsequent increase of PBL could be attributed to all cell types investigated. However, in vitamin A-deficient birds negative cells did not show these reactions. Therefore, it can be concluded that vitamin A deficiency has a detrimental effect on PBL, negative for both antibodies used, and on the normal growth of the number of B-cells at this age.

Animals↗

PubMed1992

Production of chickens with marginal vitamin A deficiency.

Marginally vitamin A-deficient 1-d-old chickens capable of remaining healthy for at least 6 weeks were produced using a two-generation model. In this model, hens fed on diets with a limited vitamin A content were used to obtain 1-d-old chickens which were marginally deficient in vitamin A. Only hens with a narrow range of plasma retinol values (0.60-0.85 mumol/l) were satisfactory for this purpose. Above this range the 1-d-old chickens were not marginally vitamin A deficient. Below this range egg production and hatchability were affected to some extent depending on the degree of vitamin A deficiency. Even when egg production and hatchability remained at a high level in such birds, the 1-d-old chickens produced were not sufficiently strong to survive the first weeks of life. The advantages of the two-generation model for producing marginally vitamin A-deficient chickens are the increased uniformity and predictability of the chickens with respect to body-weight, general health and vitamin A status. However, it does take about 3 months to produce such chickens.

Animals↗

PubMed1992

Effect of vitamin A deficiency and Newcastle disease virus infection on IgA and IgM secretion in chickens.

The effect of vitamin A deficiency or the lentogenic La Sota strain of Newcastle disease virus (NDV) infection, or both, on immunoglobulin (IgA and IgM) levels in bile and plasma were investigated. In addition, tissue distribution of IgA-, IgG- and IgM-containing cells was studied to establish the source of these Ig. Chickens (1-d-old) with limited vitamin A reserves were fed ad lib. on diets containing either marginal or adequate levels of vitamin A. At 4 weeks of age, half the chickens in each group were infected with NDV. The number of IgA- and IgM-containing cells was not significantly affected by vitamin A deficiency, demonstrating that neither class-switching nor homing of Ig-containing cells is influenced by vitamin A deficiency. Although bile IgM levels were not significantly different in vitamin A-deficient chickens compared with normal chickens, IgA levels were significantly lower. This decrease was even more pronounced in deficient NDV-infected chickens, despite the higher number of IgA-containing cells found in these birds. These results, together with the slightly increased levels of IgA in plasma of vitamin A-deficient chickens, suggest that the hepatobiliary transport of IgA is impaired by vitamin A deficiency and possibly also by NDV infection, although disturbed secretion by IgA-containing cells cannot be excluded.

Animals↗

PubMed1992

Epithelia-damaging virus infections affect vitamin A status in chickens.

The effect of infection with infectious bronchitis virus (IBV) and reovirus (RV) on vitamin A status was investigated in chickens with a normal or marginal intake of vitamin A. At the age of 4 wk, chickens were infected with either IBV or RV, primarily affecting the respiratory or intestinal tract, respectively. Both viruses lowered plasma retinol levels significantly. The effect was more pronounced in chickens fed a diet marginally deficient in vitamin A than in those fed a diet adequate in vitamin A. Concentrations of retinol-binding protein, transthyretin and albumin in RV-infected chickens were also significantly lower than in noninfected chickens fed the same diets; in chickens infected with IBV, there was no effect. These results suggest that the reduced vitamin A status of IBV-infected chickens could be attributed to increased rate of utilization by tissues. In RV infection, this mechanism could be involved but impaired absorption of nutrients (including vitamin A) and direct loss of nutrients via the intestinal tract could also be important.

Animals↗