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1.
Little is known about the role of macrophage products in blood-borne parasitic infections of fish. This study determined the effects of soluble products of goldfish macrophages on the growth of the haemoflagellate, Trypanosoma danilewskyi Laveran & Mesnil. Cell-free supernatants were collected from macrophage cultures that were derived from infected or uninfected goldfish. Trypanosomes were seeded into wells containing conditioned or control medium, incubated for seven days, and enumerated using flow cytometry. Supernatants collected from macrophages isolated from infected or uninfected fish, supported parasite growth significantly better ( P < 0.05) compared to control medium. Supernatants, collected on day 12, 15, and 18 from macrophage cultures, induced a greater than 25% increase in the number of parasites. Moreover, the growth of T. danilewskyi was related to the number of macrophages seeded into cultures. This growth-enhancing activity was not specific to species or cell-type, as medium conditioned by mammalian macrophages or fibroblasts (fish or mammalian) significantly enhanced ( P < 0.05) parasite growth. While compound(s) that are necessary for proliferation remain unidentified, our results suggest that over the prolonged evolutionary relationship with teleosts, T. danilewskyi has evolved to utilize soluble products of macrophages and fibroblasts as growth-enhancing factors.  相似文献   

2.
The objective of this study was to determine the growth of Trypanosoma danilewskyi (Laveran & Mesnil, 1904) from goldfish, Carassius auratus (L.), in medium containing no serum, or in medium supplemented with either 10% fish serum (goldfish, carp, or tin foil barb) or 10% mammalian serum (horse or foetal bovine). After 10 days, the number of trypanosomes in flasks containing tin foil barb serum increased by nearly 700% over the initial inoculum. Similarly, a substantial increase in the number of parasites was seen after 10 days in the cultures containing carp and goldfish serum. After 6 weeks, there was more than a 15-fold increase of T. danilewskyi in cultures containing goldfish serum. Medium containing no serum or mammalian serum failed to support the growth of parasites. Therefore, serum-related factors within fish blood are required for the propagation of T. danilewskyi isolated from infected goldfish. Since T. danilewskyi can be propagated in vivo and in vitro in the presence of homologous proteins, cultured and wild-type forms can be compared to determine if cultured parasites can be used as analogues of natural life-cycle stages.  相似文献   

3.
Abstract. Purification and in vitro cultivation techniques were developed for the fish haemoflagellate, Trypanosoma danilewskyi. The parasites were isolated and purified from the peripheral blood of experimentally infected goldfish, using a combination of Ficolt-paque gradient centrifugation to remove fish red blood cells and in vitro incubation to remove the remaining fish leucocytes. A serum-free culture medium for T. danilewskyi supported both short- and long-term cultivation of the haemoflagellates. The serum-free medium is a mixture of reagents available commercially: Leibovitz's L-15 medium, Dulbecco's Modified Eagle Medium and Hank's balanced salt solution. The doubling time was calculated to be 44.4 × 7.8h. Typically, a two- to five-fold increase in the number of cultured parasites was observed on day 7 after subculture with 1 × 106 and 5 × 105 trypanosomes ml−1, respectively. When administered to fish, the in vitro -derived parasites caused an infection and pathology whose characteristics were similar to those observed following infection with trypanosomes obtained from infected goldfish. The freshly isolated and in vitro -grown parasites were successfully cryoprescrvcd in the culture medium containing 10% glycerine at −80°C for at least 3 months. Although the viability of the parasite decreased by 40–50% after thawing, cryoprcserved parasites retained the ability to infect goldfish.
Correspondence: Dr M. Belosevic, Associate Professor, Department of Zoology, CW-312 Biological Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2E9.  相似文献   

4.
This study examined whether in vitro-cultured Trypanosoma danilewskyi were susceptible to lysis in the presence or absence of anti-parasite antibodies and complement. Cultured trypanosomes were resistant to lysis by either immune or non-immune goldfish serum. However, trypanosomes treated with the proteolytic enzyme trypsin, which destroys surface proteins of the parasites, became susceptible to lysis when exposed to either immune or non-immune goldfish serum. The lysis by goldfish serum was dependent on the presence of heat-labile factors and occurred at 4 and 20 degrees C. The lysis was also dependent on the presence of Mg(2+) ions but not Ca(2+) ions. Furthermore, treatment of the parasites with different sialidases did not enhance their susceptibility to lysis by goldfish serum. Trypsinized parasites regained resistance to lysis after at least 6-h cultivation in the absence of trypsin and the restoration of full resistance was observed after 24-h cultivation. The resistance to lysis was abrogated when the protein synthesis inhibitor, puromycin, was added to the cultures. These results suggest that trypsinized trypanosomes were susceptible to lysis by goldfish complement (alternative pathway) and that protective surface proteins of the parasite were required for the resistance of normal trypanosomes to lysis.  相似文献   

5.
Abstract. Growth of the eel trypanosome, Trypanosoma granulosum , was attempted in five semi-defined and three defined media. Growth was poor in four semi-defined and two defined media, but one semi-defined medium, a modified version of SDM-79 without MEM F-14, supported good growth of the trypanosome. In this medium, doubling time was between 1 and 2 days, over 90% of organisms seen in culture after 6 days were trypomastigotes, and 1.8 × 0.1 × 107 trypanosomes ml−1 was achieved in 7 days. When foetal calf serum from the modified SDM-79 was substituted with insulin to create a fully defined medium, a modest but significant increase in cell numbers occurred compared with controls. In vitro experiments with D, L-alpha-difluoromethylomithine (DFMO) showed morphological changes leading to destruction of the trypanosome with increasing concentrations of the drug. A 50-mM concentration of DFMO inhibited growth by more than 90%, and the IC50 was found to be 16 × 2 mM.  相似文献   

6.
Abstract. Common carp, Cyprinus carpio L., were experimentally infected with cloned Trypanoplasma borreli and some haematological data were determined. The haematocrit and total erythrocyte counts were reduced and were lowest at day 29 post-inoculation during the peak of infection. Differential erythrocyte number showed a reduction in mature erythrocytes and an increase in immature erythrocytes. The total leucocyte number was markedly increased and peaked at day 44 post-inoculation. Differential blood cell counts showed an increased abundance of leucocytes, especially granuloblasts and granulocytes.  相似文献   

7.
Abstract. During two summers in Portugal, intertidal fishes were captured with anaesthetic at four sites along the Portuguese Atlantic West Coast. They were examined for the blood parasite Haemogregarina bigemina , for other haematozoa, and for haematophagous eetoparasitcs. Haemogregarina bigemina was found only in biennies, Lipophrys pholis (L.) and Coryphoblennius galerita (L.), and infections in these fish were similar. No other haematozoa were detected. Ectoparasites found attached to biennies included haematophagous praniza larvae of the isopod Gnathia maxillaris and a single leech. Gametocytes like those of fish haemogregarines were found in the anterior hindgut of 25% of pranizae and one contained gametoeytes and stages resembling early oocysts. The leech showed none of these stages. It is concluded that gnathiid pranizae should be considered as possible vectors of H. bigemina in Portugal.  相似文献   

8.
Polyamines are important in the growth, division and differentiation of many cell types, including trypanosomes. The ultrastructure of culture forms of Trypanosoma granulosum was examined following growth in a modified semidefined medium (controls) and in the same medium with added polyamine biosynthesis inhibitors (DFMO, MGBG and Berenil). Untreated trypanosomes had ultrastructural features in common with other cultured flagellates. Those treated with DFMO were generally more rounded in contour. Cytoplasmic vacuoles and swollen mitochondrial membranes suggested osmotic imbalance, perhaps resulting from compromised membrane integrity. An anticlockwise arm dividing subtubule B of the peripheral doublets of the flagellum was noted in some trypanosomes treated with 20 mM DFMO. Vacuolation was also induced by MGBG and flagellar division occurred more frequently at 0.2 mM than in controls. With 1 mM MGBG, mitochondria were difficult to discern and kinetoplasts were disrupted. With 0.2 mM Berenil, vacuoles, swollen mitochondria, membranous whorls, additional microtubules underlying subpellicular tubules and disaggregated kinetoplasts were noted. With 1 mM Berenil, much of the cell structure was destroyed, although the pellicle, flagellum and paraxial rod remained intact. The present study illustrates the importance of hnctional polyamine synthetic pathways for the integrity of membranes, mitochondria, kinetoplasts and possibly microtubules in cultured T. granulosum.  相似文献   

9.
Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus‐2 (CyHV‐2), has affected the commercial production of the goldfish Carassius auratus and gibelio carp Carassius auratus gibelio. High water temperature treatments are reported to reduce the mortality rate of infected goldfish and elicit immunity in the survivors. To define the mechanism by which this intervention induces resistance, clonal ginbuna Carassius auratus langsdorfii, which is closely related to both species and has been used in fish immunology, may represent a promising model species. In this study, we investigated the susceptibility of clonal ginbuna strains to CyHV‐2 and the effect of high water temperature treatment on infected ginbuna and goldfish. Experimental intraperitoneal infection with CyHV‐2 at 25 °C caused 100% mortality in ginbuna strains, which was accompanied by histopathological changes typical of HVHN. Both infected ginbuna S3n strain and goldfish, exposed to high temperature for 6 days [shifting from 25 °C (permissive) to 34 °C (non‐permissive)], showed reduced mortalities after the 1st inoculation, and subsequent 2nd virus challenge to 0%, indicating induction of immunity. It was concluded that ginbuna showed a similar susceptibility and disease development in CyHV‐2 infection compared to goldfish, suggesting that ginbuna can be a useful fish model for the study of CyHV‐2 infection and immunity.  相似文献   

10.
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11.
Goldfish, Carassius auratus (L.), mobilized an acute, non-specific cellular inflammatory response against experimental infection with metacercariae of the digenean Ribeiroia marini in lateral line scale canals. The host cellular response to primary infection consisted of a rapid infiltration of neutrophilic granulocytes and macrophages that adhered to the metacercial cyst wall. Granulocytes released cytoplasmic granules, forming a necrotic zone around the cyst. Outside the necrotic zone, a fibrocytic zone and epithelioid cells formed a matrix upon which reactive leucocytes were attached in a progressive granulomatous encapsulation. Interdigitating epitheliod cells formed a syncytial border separating the necrotic zone from the outer fibrocytic region of the granuloma. Acute inflammatory response preceded the expulsion of most metacercariae from the scale canals. Few cysts remained encapsulated in epidermal tissue. Cellular response to challenge infections was more intense, but no major differences in leucocyte composition between primary and challenge infections suggested a continuum of inflammation and cell-mediated responses. In late primary and challenge infections, the predominant eosinophilic granular cell released cytoplasmic granules. We propose that the eosinophilic granular cell of the goldfish has an antiparasitic function, releasing bioactive granules that alter the environment of the scale canal and cause the expulsion of parasites.  相似文献   

12.
Myxobolus wulii (= Myxosoma magna ) was first described from the gills of goldfish, Carassius auratus auratus, in China. Subsequently, a myxosporean infecting the hepatopancreas of allogynogenetic gibel carp, C. auratus gibelio , was designated as a different species, Myxobolus guanqiaoensis , although the morphological features were almost identical to those of M. wulii . In Japan, an unidentified Myxobolus sp. was found in the gills and hepatopancreas of goldfish. Morphological and molecular analyses in the present study identified these myxosporeans as M. wulii , which was thus shown to use different habitats in the host fish. Phylogenetic analyses of small subunit ribosomal RNA gene sequences showed that M. wulii is closely related to two gill-infecting Myxobolus species, M. ampullicapsulatus and M. longisporus . Fish infected with M. wulii in the hepatopancreas exhibit swollen abdomens and chronic mortality. Hepatopancreas tissues are virtually destroyed and replaced with plasmodia of M. wulii . A remarkable difference in susceptibility to M. wulii between two clones of allogynogenetic gibel carp was observed, suggesting that resistance to the myxosporean infection was established in a clone of fish bred by allogynogenesis.  相似文献   

13.
This study was conducted to identify the possible effect of recombinant vertebrate ancient long (VAL) opsin as a non-visual “photoreceptor” in the deep brain of goldfish, Carassius auratus. In addition, we investigated the effects of green-wavelength light on the predictable reproductive function of VAL-opsin as a green-sensitive pigment in the deep brain. To determine this, we quantified changes in gonadotropin hormone (GTH) [GTHα, follicle stimulating hormone (FSH) and luteinizing hormone (LH)] and estrogen receptor (ER; ERα and ERβ) mRNA expression levels associated with goldfish reproduction as well as changes in plasma FSH, LH, and 17β-estradiol (E2) activities after injection of recombinant VAL-opsin protein in two concentrations (0.1 or 0.5 μg/g body mass) for 4 weeks (injection once weekly) and examined the possible impact of green-wavelength light (500, 520, and 540 nm) on the function of VAL-opsin. As a result, all parameters associated with reproduction significantly increased with time and light-emitting diode (LED) exposure. Based on these results, we suggested that VAL-opsin in the deep brain is involved in goldfish maturation, and it is possible that green-wavelength light improves the ability of VAL-opsin to promote maturation by increasing VAL-opsin expression.  相似文献   

14.
Abstract. The humoral immune response of turbot, Scophthalmus maximus (L.), to antigens from the microsporean parasite Tetramicra brevifilum Matthews & Matthews, 1980, was studied. Thirty days after intraperitoneal immunization with whole T. brevifilum spores in Freund's complete adjuvant, double indirect ELISA indicated that initial production of antibodies to parasite surface antigens was considerably higher than production of antibodies to the antigens contained in a crude extract (CE) of spores. Following re-immunization without adjuvant on day 30, levels of antibodies to surface antigens gradually declined, whilst levels of antibodies to CE antigens increased. The antibody response of intraperitoneally immunized fish was characterized by Western blotting of total soluble antigens obtained by heating and reduction of T. brevifilum spores at 95–100°C in Tris-HCl buffer containing SDS and dithiothreitol: a series of bands with molecular weights between 20 and 53 kDa was recognized by immunized turbot sera. Four additional bands (with molecular weights between 15 and 18kdA) were recognized by serum from re-immunized fish. ELISA studies of sera from naturally infected fish revealed a surprisingly low incidence of strong T. brevifilum seropositivity (61% individuals); antibodies to surface antigens predominated in seropositive individuals. The low background response levels and high sensitivity of the ELISA used in this study indicate that the assay is of value for the monitoring of serum antibody levels in turbot. However, given the relatively low seropositivities observed in naturally infected turbot, particularly to CE antigens, the use of anti- T. brevifilum serum antibody levels for the diagnosis of infection by this parasite may lead to false negative results.  相似文献   

15.
Abstract. Intraperitoneal implants of cortisol (cortisol suspended in hydrogenated coconut oil) were used to induce a graded hypercortisolism in rainbow trout, Salmo gairdneri Richardson. There was no obvious reduction in circulating lymphocytes in cortisol-implanted rainbow trout (70, 140 or 210μg/g body weight). Cortisol-implanted fish infected with Cryptobia salmositica had significantly higher parasitaemia and lower antibody litres compared with controls infected with haemonagellate but given coconut oil implants. These confirm the immunodepressive effects of the steroid. The parasite was also more readily detected at the early stage of the infection (shorter prepatent period, more infected fish and higher parasitaemia) in cortisol-implanted fish (140 and 210 μg/g body weight) than in controls. The mortality of the infected cortisol-implanted fish was higher than that of the infected fish implanted with only coconut oil, or the cortisol-implanted but non-infected fish. This in vivo study suggests that protective immunity against C. salmositica is, in part, due to a humoral response.  相似文献   

16.
Abstract. Electrophoretic analysis of six types of tissue (serum, erythrocyte, liver, kidney, muscle and eye) and seven different isoenzymes (LDH, MDH, Est, ADH, IDH, α-GPDH and SOD) from both healthy grass carp, Ctenopharyngodon idellus Cuvier & Valenciennes, and carp affected by haemorrhagic disease was performed. It was found that: (1) Enzymes from diseased grass carp showed the most drastic changes within each tissue. The absolute activity of LDH in serum from the diseased fish increased to approximately twice that of the healthy fish; whereas the relative activity of A4 and A3B1, the main components of A sub-base in both serum and erythrocytes, was accelerated. (2) The isoenzymic changes in the control groups, diseased fish, artificially infected fish and healthy fish from diseased ponds indicated that biochemical changes occurred in the tissues before pathological change.  相似文献   

17.
研究从患病蝶尾金鱼(Butterfly)肝脏中分离获得的1株病菌,为金鱼疾病防治提供参考。分离纯化获得1株细菌,编号为DW-1,通过细菌形态观察、理化特性、16S rDNA序列分析等方法进行鉴定。结果表明DW-1菌株为革兰氏阴性短杆菌,可发酵葡萄糖产气,赖氨酸脱羧酶、硝酸盐还原等为阳性;进一步通过PCR方法扩增16S rDNA序列,测得长度为1 385 bp,与维氏气单胞菌(Aeromonas veronii)相似性为99%~100%;构建系统发育树与维氏气单胞菌温和生物变种(A.veronii biovar sobria)模式菌株ATCC9071聚为一支。最终鉴定该菌株为维氏气单胞菌温和生物变种。人工感染试验结果表明,对斑马鱼半数致死量(LD_(50))为1.17×10~6CFU/m L。药敏试验结果显示该菌株对头孢克肟、头孢哌酮、链霉素、恩诺沙星、诺氟沙星、左氟沙星、复方新诺明等23种药物敏感。  相似文献   

18.
Vinclozolin (VZ) is a pesticide that acts as an anti-androgen to impair reproduction in mammals. However, VZ-induced disruption of reproduction is largely unknown in fish. In the present study, we have established a combination exposure in which adult goldfish were exposed to VZ (30 and 100 μg/L), anti-androgen flutamide (Flu, 300 μg/L), and androgen testosterone (T, 1 μg/L) to better understand effects of VZ on reproductive endocrine system. mRNA levels of kisspeptin (kiss-1 and kiss-2) and its receptor (gpr54), salmon gonadotropin-releasing hormone (gnrh3) and androgen receptor (ar) in the mid-brain, and luteinizing hormone receptor (lhr) in the testis were analyzed and compared with those of control following 10 days of exposure. kiss-1 mRNA level was increased in goldfish exposed to 100 µg/L VZ and to Flu, while kiss-2 mRNA level was increased following exposure to Flu and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. gpr54 mRNA level was increased in goldfish exposed to Flu and to combination of 30 µg/L VZ with Flu and 100 µg/L VZ with T. gnrh3 mRNA level was increased in goldfish exposed to 100 µg/L VZ, to Flu, and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. The mid-brain ar mRNA level was increased in goldfish exposed to Flu and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. Testicular lhr mRNA level was increased in goldfish exposed to Flu and to combination of 30 µg/L VZ with Flu. These results suggest that VZ and Flu are capable of interfering with kisspeptin and GnRH systems to alter pituitary and testicular horonal functions in adult goldfish and the brain ar mediates VZ-induced disruption of androgen production.  相似文献   

19.
Abstract. Laboratory-raised Cryptobia -susceptible brook charr, Salvelinus fontinalis (Mitchill), and rainbow trout, Oncorhynchus mykiss (Walbaum), were vaccinated intraperitoneally with a live Cryptobia salmositica vaccine (250000 parasites per fish), and 4 weeks later were challenged with the pathogen (250000 parasites per fish). Unvaccinated and infected brook charr had high parasitaemias but no clinical signs of disease, while unvaccinated and infected rainbow trout had anaemia and general oedema. Vaccinated and challenged fish had very low parasitaemias compared to unvaccinated and infected brook charr and rainbow trout. Complement fixing antibodies were detected in vaccinated and challenged fish 2 weeks after challenge. Unvaccinated and infected brook charr had consistently higher litres of complement fixing antibody than unvaccinated and infected rainbow trout. Parasitaemias were lower in all fish in which titres of complement fixing antibody were high. In a second experiment, brook charr inoculated intraperitoneally or intramuscularly with 100000 C. salmositica per fish had high parasitaemias but no anaemia or other clinical signs. The results show that susceptible brook charr do not suffer from cryptobiosis and may serve as reservoir hosts for C. salmositica in areas where the disease is prevalent. Vaccination to reduce the parasitaemia when fish become infected may be a control strategy in these areas.  相似文献   

20.
Abstract. Histopathological changes in bullminnows, Fundulus grandis Baird and Girard, experimentally infected with a non-haemolytic group B Streptococcus sp. were studied. Following experimental exposure to the Streptococcus sp. approximately 40% of the fish developed exophthalmia, dropsy or petechial haemorrhage along the abdomen. The exophthalmic eye showed haemorrhage with neutrophil infiltration. The liver was oedematous and hepatocytes were atrophied, accompanied by a decrease in glycogen and increases in vacuolization, hyaline droplet degeneration and necrosis. The spleen was congested and cells of the sheathed artery were necrotized.  相似文献   

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