博士 / 國立中興大學 / 獸醫學系 / 94 / Abstract
Massive muscle necrosis caused by yeast pathogen (Debaryomyces hansenii) is among the most important diseases affecting giant freshwater prawn (Macrobrachium rosenbergii) during the wintertime in Taiwan. The objectives of this study were to investigate the influence of temperature and β-glucan on phagocytic activity of hemocytes, and to compare the effect of phagocytosed pathogens or non-pathogenic latex beads on hemocytes of giant freshwater prawn. The FITC-labeled yeast pathogen and latex beads were injected into ventral muscle of grouped prawns and maintained at different water temperature (17, 27 and 35C). The hemolymph was withdrawn directly from the heart of prawns at 1, 2, 3, 6, 12, and 24 hours after inoculation. The results showed that phagocytic activity of hemocytes at 27C and 35C groups were higher than 17C group. Moreover, the FITC-labeled latex beads and yeast pathogen were injected into experimental prawns which had been fed with β-glucan (250 µg/prawn/day) and maintained at 27C. The hemolymph was withdrawn from the hearts of the experimental prawns 3 hours post inoculation. The phagocytic activities of a total of 104 hemocytes were assayed according to the ingested cellular fluorescence using flow cytometry. The results revealed that both phagocytic activity against FITC-labeled yeast pathogen or latex beads were significantly enhanced after feeding with β-glucan for 7 and 14 days. Furthermore, after oral administration of β-glucan for one week, experimental prawns were challenged intramuscularly with yeast pathogen and then maintained in water for the survival trials at 17C, 27C and 35C. In the 17C group, mortality reached 100% on the ninth day post inoculation but was 100% on the sixth day in control group. However, the mortality was only 50% even after 10 days post inoculation at 27C, while it reached 100% on the tenth day in control group. In the 35C group, the mortality was 80% but that was 100% in control group on the ninth day.
Hemocytes were also investigated for the induction of apoptosis after phagocytosis of pathogenic yeasts, bacteria and non-pathogenic latex beads in vitro. Isolated hemocytes of M. rosenbergii were cultured at a ratio of 1:50 hemocytes to pathogen with yeasts, with bacteria Aeromonas hydrophila or Enterococcus faecium, or with latex beads at 25C for 2 h, followed by washing to remove free particles. At least two hundred hemocytes were counted to determine the phagocytosis rate, and the results showed that hemocytes engulfed latex beads at a higher rate than the aquatic pathogens. By transmission electron microscopy, the yeast- or bacterium-engulfing hemocytes displayed morphological alterations, characteristics of apoptosis, including formation of cytoplasmic vacuoles, chromatin condensation, and fragmentation of nuclei. The DNA laddering and TUNEL assays further confirmed this pathogen-induced apoptosis. Neither hemocytes treated with latex beads nor un-infected hemocytes (the control group) showed signs of apoptosis up to 48 hours in culture. In summary, the results of the studies indicated that phagocytosis of hemocytes were temperature-dependent in vivo, and both phagocytic activity and survival rate of experimental prawns against yeast pathogen were significantly improved after feeding a diet containing β-glucan for one week. Moreover, phagocytosis of pathogens, but not non-pathogenic particles, could induce apoptosis in hemocytes of the giant freshwater prawn.
| Identifer | oai:union.ndltd.org:TW/094NCHU0541002 |
| Date | January 2006 |
| Creators | Jung-Pin Hsu, 徐榮彬 |
| Contributors | Maw-Sheng Chien, 簡茂盛 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 110 |
Page generated in 0.0087 seconds