Characterization of Aphanomyces invadans by electrophoretic and Western blot analysis

Lilley, J.H.; Thompson, Kim D.; Adams, Alexandra

doi:10.3354/dao030187

Cited by 29 publications

(17 citation statements)

References 14 publications

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“…Purification of SE, SDS-PAGE and sample transfer to nitrocellulose membranes were made as described by Lilley et al (1997), except that Proteinase K solution was not used and protein concentration was determined by Branford's method and adjusted to 1 mg ml -1 . Following transfer, the nitrocellulose membrane was washed with distilled water for 5 min and then blocked with 5% dry skimmed milk in tris-buffered saline (TBS).…”

Section: Methodsmentioning

confidence: 99%

Antibody response of brown trout Salmo trutta injected with pathogenic Saprolegnia parasitica antigenic extracts

et al. 2007

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Brown trout Salmo trutta injected with antigenic extracts from a pathogenic isolate of Saprolegnia parasitica developed specific antibodies that were detected by enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) and Western blotting (WB), but not by immunodiffusion (ID). Three groups of five 2 yr old brown trout were injected intraperitoneally with 3 different antigenic extracts: small hyphal fragments (HF) and soluble extracts from sonicated mycelia grown in medium with or without β-sytosterol (SEB and SE, respectively). In the 2 groups injected with SE and SEB, antibodies were found in 66.7% of the serum samples by ELISA, 54.5% by IF and 48.5% by WB. In the group injected with HF, only 1 trout survived the experiment, and in this fish only 1 sample was positive by ELISA. The results obtained by ELISA and IF were similar and show that there is cross-reaction between the antigens used. By WB, the proteins most frequently recognised were 2 proteins of 25 and 29 kDa. No significant differences were found in the groups injected with SE or SEB. KEY WORDS: Saprolegnia parasitica · Brown trout · Antibody response · Immunological techniques Resale or republication not permitted without written consent of the publisherDis Aquat Org 74: [107][108][109][110][111] 2007 Sohnle & Chusid 1983, Bly et al. 1993. Hodkinson & Hunter (1974) investigated the influence of culture conditions on the antigenic products of Saprolegnia and found that precipitating reactions only occurred between salmon serum and antigens from Saprolegnia grown in medium containing β-sytosterol.The aim of the present research was to study the capacity of brown trout injected with antigenic extracts of Saprolegnia parasitica pathogenic to salmonids to produce specific serum antibodies. MATERIALS AND METHODSFish and antigenic extracts. Three different groups of five 2 yr old brown trout (225 ± 70 g weight and 26.5 ± 2.8 cm length) were injected intraperitoneally with 0.2 ml of antigenic extracts in phosphatebuffered saline (PBS) (approximately 200 µg of total protein) twice at 2 wk intervals. The extracts were mixed with Freund's complete adjuvant at a ratio of 3:1 in the first injection and with Freund's incomplete adjuvant (1:1) in the second injection. A fourth group was injected only with PBS and adjuvant as a negative control group. Three different antigenic extracts were prepared using the pathogenic isolate TRU 8 of Saprolegnia parasitica (formerly Saprolegnia sp.; Fregeneda-Grandes et al. 2000, 2003: (1) small hyphal fragments (HF) obtained after sonication of formalised-mycelial mats growing on glucose-yeast broth (GY: 1% glucose, 0.25% yeast extract); (2) soluble extract (SE) from sonicated formalised-mycelia growing on GY; and (3) SE from sonicated formalisedmycelia growing on GY supplemented with 20 mg l -1 of β-sytosterol (SEB). The experiment was carried out between April and June 2004 at a fish farm in small raceways situated in the open and with a continuous supply of well water. The water temperature varied fro...

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Section: Methodsmentioning

confidence: 99%

Antibody response of brown trout Salmo trutta injected with pathogenic Saprolegnia parasitica antigenic extracts

et al. 2007

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“…Both fungi were isolated from snakehead fish (Channa striata) during outbreaks of EUS in Thailand (Lilley & Roberts, 1997). Zoospore preparations, protein-rich fungal extracts and extracellular products (ECP) were produced according to Lilley et al, 1997b). Briefly, zoospore suspensions were produced by washing mycelium mats five times with sterile distilled water and incubating overnight at 20 C in autoclaved filtered pond water, diluted 1:2 with distilled water (APW).…”

Section: Fungal Preparationmentioning

confidence: 99%

“…The resulting zoospores were enumerated using a haemocytometer. Fungal extracts were prepared by adding double strength glucose peptone yeast medium (Willoughby & Roberts, 1994) to the zoospore suspensions, culturing them for 2 days at 20 C, then extracting the mycelial mats using extraction bu#er as given in Lilley et al, 1997b). Fungal extracts were stored frozen at 70 C until used.…”

Section: Fungal Preparationmentioning

confidence: 99%

The immune response of rainbow trout (Oncorhynchus mykiss) againstAphanomyces invadans

Thompson

Lilley

Chen

et al. 1999

Fish & Shellfish Immunology

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“…12 h of incubation, the secondary zoospores were harvested by centrifugation at 1000 g at 20°C. Fungal extract was prepared as described by Lilley et al (1997), briefly, the zoospore suspensions were added to an equal volume of 2X GPY broth. The germlings were grown for 3 days at 20°C.…”

mentioning

confidence: 99%

Immunisation of Labeo rohita with Antigenic preparation of Aphanomyces invadans

Yadav¹,

Pradhan²,

Sood³

et al. 2017

Indian J. Fish.

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Aphanomyces invadans is an important fungal pathogen infecting freshwater and brackishwater fishes. In the present study, an attempt has been made to determine the effect of immunisation in Labeo rohita advanced fingerlings against A. invadans infection. The efficacy of the immunisation was evaluated by challenge with A. invadans as well as quantification of antibody level by ELISA. Following an initial immunisation in conjunction with adjuvant, the fish were given a booster dose after 35 days. After 14 and 28 days of the booster injection, blood was collected from the immunised rohu for monitoring the antibody level. The fish were also challenged with A. invadans zoospores to determine the relative percent survival. The immunised fish had significantly higher antibody level after 14 days of booster injection as compared to the control fish. However, the antibody level after 28 days of booster injection was not significantly different from the control fish. More importantly, similar to control fish, 100% mortality was observed in the immunised fish challenged after 14 and 28 days of booster immunisation. Therefore, it can be concluded that it may not be suitable to induce protective immunity following immunisation with conventional antigenic preparations.

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Characterization of Aphanomyces invadans by electrophoretic and Western blot analysis

Cited by 29 publications

References 14 publications

Antibody response of brown trout Salmo trutta injected with pathogenic Saprolegnia parasitica antigenic extracts

Antibody response of brown trout Salmo trutta injected with pathogenic Saprolegnia parasitica antigenic extracts

The immune response of rainbow trout (Oncorhynchus mykiss) againstAphanomyces invadans

Immunisation of Labeo rohita with Antigenic preparation of Aphanomyces invadans

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