Immunological detection techniques

Immunological detection techniques offer many advantages over culture-based techniques, particularly when attempting to detect organisms, such as A. salmonicida, whose growth on laboratory media can be inhibited by the presence of other bacteria. These techniques have been revolutionized in recent years by

Table 10.8. Non-culture-based techniques developed for, or applied to, the detection of typical Aeromonas salmonicida since 1990.

Target sample

Table 10.8. Non-culture-based techniques developed for, or applied to, the detection of typical Aeromonas salmonicida since 1990.

Target sample

Detection principle Assay type

Clinical

Environmental

Reference

Immunological ELISA

Kidney

Adams and Thompson (1990)

ELISA

Kidney

Bernoth (1990b)

IFAT, ELISA

Kidney, liver

Lallier eta/. (1990)

I mmu nofluorescence

Water, sediment

Enger and Thorsen (1992)

ELISA

Blood

Yoshimizu eta/. (1992)

ELISA

Kidney, intestine

Hiney eta/. (1994)

Immunodot-blot

Various tissues

Banneke and Bernoth (1994)

ELISA

Kidney

Sediment

Gilroy and Smith (1995)

ELISA

Kidney, intestine

Sediment

Hiney eta/. (1997a)

ELISA

Kidney, mucus, gills, spleen

Bullock etal. (1998)

Genetic 16SrDNA-PCR

Pure culture

Barry et a/. (1990)

DNA-PCR

Spleen, kidney

Fish faeces, tank effluent

Gustafson etal. (1992)

DNA-PCR

Pure culture

Hiney etal. (1992)

DNA-PCR

Freshwater microcosm

Morgan etal. (1993)

DNA-PCR

Marine sediment, water

Hiney (1994)

DNA-PCR

Hatchery effluent

O'Brien etal. (1994)

DNA-PCR

Blood

Mooney eta/. (1995)

DNA-PCR

Freshwater microcosm

Pickup etal. (1996)

16S rDNA-PCR

Kidney, spleen

Hoie etal. (1996)

DNA-PCR

Kidney

Miyata etal. (1996)

DNA-PCR

Kidney, intestine

Freshwater sediment

Hiney etal. (1997a)

DNA-PCR

Kidney, gill swabs

Hoie etal. (1997)

DNA-PCR

Kidney

Jen$i$and Piano (1997)

DNA-PCR

Kidney, intestine

Freshwater sediment

Pad ley etal. (1997)

DNA-PCR

Kidney

Serum etal. (1998)

IFAT, indirect fluorescent antibody test; rDNA, ribosomal DNA.

IFAT, indirect fluorescent antibody test; rDNA, ribosomal DNA.

the introduction of monoclonal antibodies and ELISAs, which allow for more specific assays that can be semiautomated. As a result, a number of ELISAs have been developed for screening of clinical samples for signs of A. salmonicida (Bernoth, 1997b; Table 10.8). In a comparison of ELISA and an indirect fluorescent antibody test (IFAT), similar to the fluorescent antibody microscopy (FAM) technique, Lallier et al. (1990) found that ELISA was more sensitive than IFAT when tested on pure cultures of A. salmonicida and both methods were found to be more sensitive than bacteriological culture. However, it has been argued that the use of IFAT coupled with experience overcomes the problems of lesser specificity of this technique, making it comparable to ELISA (E.-M. Bernoth, CSIRO, 1995, personal communication). Perhaps the most useful application of immunological assays would be in the detection of covert A. salmonicida infections, and a number of ELISA tests have been applied for this purpose. Good correlation was found between detection of covert A. salmonicida infection by stress testing and ELISA of kidney material from non-stress-tested fish (Scallan, 1983; Rose et al., 1989). These findings were supported by Hiney et al. (1994), who found that ELISA examination of the kidney, mucus and intestine of covertly infected fish detected A. salmonicida antigens in 45% of fish as compared with culture of the organism from the kidney of 24% of a parallel group of stress tested fish. On the other hand, Bullock et al. 1997 found that culture of mucus, gills, kidney and spleen was more sensitive than ELISA for identification of A. salmonicida in fish that had been stress-tested. Enger and Thorsen (1992) have reported on an IFAT which was applied to detection of A. salmonicida antigens in the environment of a fish farm. None of these tests have, however, been applied successfully under true field conditions, nor are they recommended with any conviction in diagnostic manuals (Crane and Bernoth, 1996).

There are a number of problems to resolve when attempting to detect bacterial antigens in situ in tissue or environmental samples. Many of the immunological assays developed do not appear to offer any greater sensitivity or reliability than conventional bacteriological methods (Inglis et al., 1993). The lower limit of detection has been found to be 103 cells ml-1 or greater in both clinical and environmental samples (Sakai et al., 1986; Adams and Thompson, 1990; Bernoth, 1997b). A major problem with cross-reactivity of antisera or antibodies to epitopes expressed by ubiquitous motile Aeromonas species or other aquatic microorganisms has been reported, which requires that confirmatory bacteriological isolation of the pathogen be performed (Bernoth, 1990b). Importantly, immunological assays do not differentiate between live and dead cells (Rose et al., 1989) and A. salmonicida-targeted ELISA has been shown to generate positive results from the spleen and intestine of fish which were immunized with a killed whole-cell furunculosis vaccine (Gilroy and Smith, 1997). Another problem with immunological assays is that the antiserum raised against bacteria grown in vitro must detect the organism as it occurs in situ in a clinical or environmental sample. For A. salmonicida, at least, cells grown in vivo have been found to express novel antigens, including an antigenically new form of lipopolysaccharide (LPS), which were not induced under in vitro growth conditions (Garduño et al., 1993; Thornton et al, 1993). The question of how relevant the current methods of antiserum generation from in vitro-grown A. salmonicida components are for diagnosis in situ has to be addressed.

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