Two forms of vitellogenin were isolated by DEAE agarose ion-exchange chromatography from plasma of the tilapia, Oreochromis mossambicus. The monomers have apparent molecular masses of 200 and 130 kDa, as indicated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE),
and a total amount of phosphorus of 1.7 and 0.1%, respectively. Antibodies specific to the two forms, designated tVTG-200
and tVTG-130, were generated in rabbits and used to develop enzyme-linked immunosorbent assays (ELISAs) and in Western blot
analyses of plasma and oocyte extract. SDS-PAGE of the oocyte extract showed a major protein band at 106.6, minor bands at
26.6, 24.2, and 23.7 kDa, and very faint bands at 83.4 and 17.5 kDa. Western blots of the oocyte extract revealed that the
antiserum to tVTG-200 recognized strongly the protein bands at 24.2 and 23.7 kDa, and less strongly the bands at 25.1 and
22.6 kDa, whereas the antiserum to tVTG-130 recognized mainly the protein band at 106.6 kDa. The presence of both VTGs in
untreated male tilapia was detected with the ELISAs using relatively high plasma volumes. Their presence in males was confirmed
by VTG-like immunoreactive materials eluting from the ion-exchange column at the same positions as tVTG-200 and tVTG-130.
The concentrations of the VTGs in males were several orders of magnitude lower than in vitellogenic females. Treatment of
male tilapia with estradiol-17β (E2) induced both VTGs within 24h. After 7 days, tVTG-130 reached a maximum concentration in plasma, whereas tVTG-200 continued
to increase. Our findings demonstrate that the two vitellogenins are biochemically distinct, possibly differentially regulated,
and made by both sexes. 相似文献
The present study aimed to determine whether protection is conferred by immunization of grouper, Epinephelus coioides, against a protozoan parasite, Cryptocaryon irritans. The immunization of E. coioides was carried out by a low level exposure of fish to live C. irritans theronts from predetermined number of tomonts and by an intraperitoneal injection of a vaccine consisting of formalin-killed C. irritans theronts.
Mucus titers detected by ELISA were significantly higher in fingerling and adult grouper subjected to the low level of exposure to C. irritans theronts at 3-week post-exposure compared to fish that had no previous exposure. In addition, significantly smaller tomonts were produced from adult grouper after three successive exposures than the tomonts produced after a single exposure to the parasite.
In the vaccine-immunization experiment, no mortality was monitored in fish that received high dose vaccine (100 μg/fish), while 40% cumulative mortality and 100% cumulative mortality were recorded in low dose group (10 μg/fish) and control group (PBS-injected), respectively. In the succeeding replicate, the vaccine-immunized group (high dose) had 37.5% cumulative mortality and 100% cumulative mortality for the control. In addition, a total of 1830 tomonts were collected at 5-day post-challenge from the control group while none from the vaccine-immunized group. Significantly fewer trophonts and tomonts were enumerated at 5-day and 7-day post-challenge, respectively, in the vaccine-immunized group than the control.
Results suggest that a protective immunity has been conferred on the immunized grouper as indicated by high antibody titers in the mucus of C. irritans-exposed fish and higher survival and fewer parasites in vaccine-immunized fish than the control groups. The conferred immunity played a major role in preventing or limiting the adhesion, invasion, and development of C. irritans theronts on the skin of the immunized grouper. 相似文献
It is hypothesized that the frequency of bacterial coldwater disease outbreaks can be reduced through the detection of the aetiologic agent, Flavobacterium psychrophilum, in broodstock followed by culling of eggs from heavily infected broodstock. Before a culling programme can be instituted, however, it is necessary to determine the sensitivity and specificity of existing assays for the detection of F. psychrophilum. In this study, tissue and ovarian fluid samples were collected from 224 fish at five hatcheries and screened using an enzyme‐linked immunosorbent assay (ELISA), a membrane‐filtration fluorescent antibody test (MF‐FAT), bacteriological culture and nested PCR. Latent class analysis was used to estimate sensitivity and specificity of kidney culture, kidney ELISA, nested PCR and MF‐FAT. Analytical sensitivity of the ELISA varied but was greatest when bacteria were cultured under iron‐limiting conditions. Diagnostic sensitivity estimates ranged from 0.02 (kidney culture) to 0.97 (kidney ELISA). Specificity estimates ranged from 0.02 (MF‐FAT) to 0.98 (kidney ELISA). In a separate challenge experiment, the ELISA confirmed the presence of F. psychrophilum in sub‐clinically infected fish. Results from this study demonstrate that the ELISA is an appropriate tool to screen broodstock and provides an indication of infection severity, which is crucial for implementation of a screening/culling programme. 相似文献
Measurement of cortisol response is an important tool to asses stress in fisheries research. Radioimmunoassay (RIA) is a common
method for the measure of cortisol in fish. Use of enzyme-linked immunosorbent assays (ELISA) to detect cortisol would eliminate
health hazards, costs of handling radioisotopes, and the short stability time associated with RIA. Enzyme-linked immunosorbent
assays have been used for the determination of cortisol in several fish species. However, the ELISA method of cortisol determination
in fish lacks proper validation testing. We conducted validation procedures for multiple commercial cortisol ELISA kits and
compared the results to RIA. The assays were tested for four species: (1) channel catfish Ictalurus punctatus, (2) largemouth bass Micropterus salmoides, (3) red pacu Piaractus brachypomus, and (4) golden shiners Notemigonus crysoleucas. We evaluated the ELISA methods against RIA, and determined that at least one kit is suitable (accuracy: mean recovery of
spiked samples, 102.8%; reproducibility: interassay coefficient of variation < 10.5% for all species; precision: intra-assay
coefficient of variation < 16.8% for all species; linearity: R2 > 0.96 for all species) for the measurement of cortisol response in fish and comparative determination of stress. All of
the ELISA assay results varied by more than 10% from the cortisol concentrations detected by the RIA. The high variability
of the kit results indicates that commercial ELISA kits could be utilized for qualitative determination of cortisol in fish,
but should be fully validated in each laboratory for each species before being used for research. 相似文献
Abscisic acid (ABA), arginine and sucrose were evaluated for their effects on the morphology, germination rates and protein content of date palm somatic embryos (SE). Different concentrations of these supplements in the culture medium were used. The comparative study of SE length and thickness between treated and untreated SE revealed no differences, except for ABA (20 μM), which increased thickness. A decrease of water content (WC) in favor of an increase in dry weight (DW) was observed in all treated SE, especially with sucrose (90 g l−1) and ABA (20 μM). Only ABA (20 and 4 μM) caused a proliferation rate of the cultures higher than those in the control. Although all the tested compounds increased protein content, ABA (20 μM) was more effective in protein enrichment than arginine and sucrose treatments. The SDS-PAGE protein profiles showed a significant difference between treated and untreated SE. A protein band of 22 kDa, identified as glutelin in a previous work, was accumulated after treatment with 20 μM ABA or 3 mM arginine. These findings may contribute to further understanding of the mechanisms involved in the accumulation of specific storage proteins in several plants. 相似文献