A Fluorescent Antibody Test for Detection of the Rickettsia Causing Disease in Chilean Salmonids

Lannan, C. N.; Ewing, S. A.; Fryer, J. L.

doi:10.1577/1548-8667(1991)003<0229:afatfd>2.3.co;2

Cited by 58 publications

(41 citation statements)

References 6 publications

(6 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Blood was collected 10 d following the last injection and the serum was separated and stored at -20°C. The anti WSPSLO serum was used in indirect fluorescent antibody tests (IFAT) as described for Piscirickettsia salmonis by Lannan et al (1991). Anti-P. salmonis rabbit serum was provided by J. L. Fryer (Oregon State University, Corvallis).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A Piscirickettsia salmonis-like bacterium associated with mortality of white seabass Atractoscion nobilis

Chen¹,

Yun²,

Marty³

et al. 2000

Dis. Aquat. Org.

View full text Add to dashboard Cite

Mortality among hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was associated with infections by a Piscirickettsia salmonis-like organism (WSPSLO). Infected fish had no consistent external signs other than pale gills, lethargy and impaired swimming behavior. Internally, the kidney and spleen were enlarged, and some fish had livers with multiple pale foci. Smears from infected kidney, liver, and spleen stained with Wright-Giemsa had intracytoplasmic coccoid organisms, often in pairs, that ranged in size from 0.5 to 1.0 µm. Microscopic lesions included multifocal hepatic, renal, and splenic necrosis, and intralesional macrophages often contained the WSPSLO. The bacterium was isolated from infected fish on cell lines of salmonid (CHSE-214) and white seabass (WSBK) origin. The WSPSLO induced plaque formation and destroyed the cell monolayers within 10 to 14 d incubation at temperatures of 15 and 20°C. The bacterium retained infectivity for cell lines up to 14 d at 4 and 13°C, up to 7 d at 20°C, but it was inactivated at 37 and 56°C within 24 and 1 h, respectively. Freezing at -20°C reduced infectivity by 100-fold. Dehydration and resuspension in distilled water completely inactivated the bacterium. In contrast, the WSPSLO retained nearly all of its infectivity for CHSE-214 cells following a 72 h period in seawater at 20°C. Polyclonal rabbit antibodies made to the WSPSLO reacted specifically in indirect fluorescent antibody tests (IFAT) with the bacterium in cell cultures and smears from infected fish tissues. Tissue smears from infected salmon or CHSE-214 cells with P. salmonis reacted weakly with the anti-WSPSLO serum. Conversely, polyclonal anti-P. salmonis serum produced a weakly positive reaction with the WSPSLO from infected CHSE-214 cells. The WSPSLO as propagated in CHSE-214 cells was highly virulent for juvenile coho salmon Oncorhynchus kisutch, inducing 80% mortality within 10 d of intraperitoneal injection of 10 2.5 50% tissue culture infectious doses per fish. We conclude that the bacterium from white seabass possesses antigenic differences from P. salmonis yet possesses virulence for salmon equal to known strains of P. salmonis.

show abstract

Section: Methodsmentioning

confidence: 99%

“…Anti-P. salmonis rabbit serum was provided by J. L. Fryer (Oregon State University, Corvallis). Slides containing fixed P. salmonis from infected coho salmon or infected CHSE-214 cells and WSPSLO from infected CHSE-214 cells were compared in reciprocal IFAT with the 2 rabbit sera according to Lannan et al (1991).…”

Section: Methodsmentioning

confidence: 99%

A Piscirickettsia salmonis-like bacterium associated with mortality of white seabass Atractoscion nobilis

Chen¹,

Yun²,

Marty³

et al. 2000

Dis. Aquat. Org.

View full text Add to dashboard Cite

show abstract

“…13 The tilapia disease agent (0.57 ϫ 0.8 m) is smaller than the reported size of P. salmonis (0.5 ϫ 1.5 m). 9 The combination of characteristics-different host species, smaller cell size, no response to P. salmonis-specific PCR and P. salmonis-specific fluorescent antibody assays-indicates that the bacterium causing epizootics in tilapia in these cases is not P. salmonis.…”

mentioning

confidence: 91%

“…5,15,18 Their cysts have even been demonstrated in dust during dust storms. 13 The habitat of Balamuthia is not known but is believed to be similar to that of Naegleria and Acanthamoeba. 8 Primary amoebic meningoencephalitis (PAM) is the term for the human disease caused by N. fowleri.…”

mentioning

confidence: 99%

Occurrence of Piscirickettsiosis-Like Syndrome in Tilapia in the Continental United States

et al. 2005

View full text Add to dashboard Cite

Abstract. From 2001to 2003 farms in Florida, California, and South Carolina experienced epizootics of a systemic disease causing mortality. The fish exhibited lethargy, occasional exophthalmia, and skin petechia. The gills were often necrotic, with a patchy white and red appearance. Grossly, the spleen and kidneys were granular with whitish irregular nodules throughout. Granulomatous infiltrates were observed in kidney, spleen, testes, and ovary tissues, but not in the liver. The granulomas contained pleomorphic coccoid bacteria, measuring 0.57 Ϯ 0.1 ϫ 0.8 Ϯ 0.2 m, that were Giemsa-positive, acid-fast-negative, and Gram-negative. The bacteria had a double cell wall, variable electron-dense and -lucent areas, and were present in the cytoplasm and within phagolysosomes. The syndrome was associated with cold stress and poor water conditions. These findings are consistent with an infectious process caused by a Piscirickettsia-like bacterium described previously in tilapia in Taiwan and Hawaii. This report involves the first identified cases of a piscirickettsiosis-like syndrome affecting tilapia in the continental United States.Key words: Piscirickettsiosis; Piscirickettsia-like organisms; piscirickettsiosis-like syndrome; Tilapia.Since the identification of Piscirickettsia salmonis as the first rickettsia-like organism recognized as a fish pathogen, 9 a number of Piscirickettsia-like organisms (PLOs) have been observed microscopically or isolated in cell culture from a variety of marine and fresh-water piscine species from locations worldwide. 5,11,12,16 Piscirickettsiosis or piscirickettsiosis-like syndromes can be severe, resulting in high mortality and significant economic losses.The number of reports of piscirickettsiosis, with significant associated economic losses, have been increasing for the last 5-10 years in British Columbia, 2,8 Ireland, 16 and Scotland. 10 PLOs have also been observed in and isolated from seabass in Europe 1,7 and California 3 during epizootics characterized by high mortality and severe economic losses. Tilapia in Taiwan 4,6 and Hawaii 15 have also been severely affected by PLO epizootics. Here we report on the first identified cases of PLO epizootics in tilapia in the continental United States.Between 2001 and 2003, 3 facilities in the continental United States (California, South Carolina, and Florida) reported epizootics with signs consistent with a piscirickettsiosis-like syndrome. All 3 facilities had increased mortalities during times of fish stress due to lowered temperature or water quality. The fish appeared lethargic, with darkened color, and would crowd toward the middle of the pond or tank. Occasionally, petechia were observed on the sides of the fish. Fins had light fraying, some exophthalmia was observed, and gills were necrotic, with a patchy white and red appearance. Internally, the spleen and kidneys were extremely granular with whitish irregular nodules throughout. The liver was a pale reddish tan with patchy lighter areas.In hematoxylin and eosin (HE) sections, the spl...

show abstract

“…The tissue sections were incubated with neat hybridoma supernatant overnight at 4°C in a humidified chamber and then with FITC-labelled anti-mouse Ig for 1 h. The reaction was enhanced with a second cycle (Linsenmayer et al 1988), by repeating the application of the goat serum, MAb supernatant and secondary antibody, and all wash steps described by . After a final wash step, 5% v/v methyl green in PBS was added for 5 min to reduce background staining (Lannan et al 1991). The slides were washed in running water for 5 min, mounted with a cover slip and stored in the dark at 4°C for not more than 7 d before examining.…”

Section: Staining Of Fixed Germlings By Immunofluorescence Antibody Tmentioning

confidence: 99%

Immunofluorescence of the epizootic ulcerative syndrome pathogen, Aphanomyces invadans, using a monoclonal antibody

Miles¹,

Thompson²,

Lilley³

et al. 2003

Dis. Aquat. Org.

View full text Add to dashboard Cite

A monoclonal antibody (MAb), designated 3gJC9, was raised against a protein antigen of Aphanomyces invadans, the oomycete pathogen that causes epizootic ulcerative syndrome (EUS). The antigen was expressed on the surface of hyphae and secreted extracellularly. MAb 3gJC9 did not cross-react with other oomycete or fungal pathogens of fish, although it did react to the crayfish plague pathogen A. astaci. The MAb was used for immunofluorescent staining on histological sections of fish infected with EUS, and was found to be more sensitive than conventional staining methods for detecting A. invadans. It thus has utility in confirming the case definition of EUS. It also revealed very small filamentous structures, the significance of which is unclear, but they may represent an early stage of infection, thus allowing earlier detection of the disease, since they are not detected using conventional staining methods. produced by sporulation, and diluting the pond water containing them in an equal volume of glucosepeptone broth media containing 100 mM calcium chloride to stimulate germination (Deacon & Saxena 1998). They were incubated overnight at 24°C and collected by centrifuging at 3000 × g for 20 min. The germlings were prepared for experimental use by fixing in formaldehyde as described by Burr (1991), and after a final wash with phosphate buffered saline at pH 7.4 (PBS), the germlings were resuspended in PBS and the concentration of the suspension determined by counting the germlings in a haemocytometer.Soluble antigens were collected after disruption of the germlings by shaking with 0.5 mm silicon beads for 160 s in a bead shaker (Biospec Products). The concentration of the soluble antigen was assessed spectrophotometrically.Preparation of monoclonal antibodies: The MAbs were prepared after Adams et al. (1995). Three BALB/c mice received 3 × 100 µl intraperitoneal injections of soluble antigen prepared from germlings of Aphanomyces invadans isolate PA8 over a 60 d period. . The last immunisation was given intravenously 28 d later, when 100 µl of soluble antigen from 2.5 × 10 5 germlings ml -1 prepared in PBS was injected into the caudal vein of the mice. Three days later, the B-cells were harvested from the spleen of the mice and fused with SP2 myeloma cells to make hybridomas.Hybridomas producing antibodies against Aphanomyces invadans were identified by dot-blot and ELISA (Adams et al. 1995), and positive wells were expanded and recloned 3 times until monoclonal cell lines were obtained. The isotypes of the MAbs were determined by ELISA, using isotype-specific secondary antibodies (Sigma Chemical).Characterisation of MAbs by Western blot: To determine the molecular weights of the antigens recognised by the MAbs, 2 samples of soluble PA8 germling extract, and 1 of extracellular products (ECP) from culture, were prepared. All of the protein was digested from 1 sample of soluble extract by digesting it with 1 mg ml -1 proteinase K (Sigma) for 60 min at 60°C.The ECP was prepared by filtering a broth culture ...

show abstract

A Fluorescent Antibody Test for Detection of the Rickettsia Causing Disease in Chilean Salmonids

Cited by 58 publications

References 6 publications

A Piscirickettsia salmonis-like bacterium associated with mortality of white seabass Atractoscion nobilis

A Piscirickettsia salmonis-like bacterium associated with mortality of white seabass Atractoscion nobilis

Occurrence of Piscirickettsiosis-Like Syndrome in Tilapia in the Continental United States

Immunofluorescence of the epizootic ulcerative syndrome pathogen, Aphanomyces invadans, using a monoclonal antibody

Contact Info

Product

Resources

About