sensitivity limit of 10 10 cellswell was detailed for the Aer hydrophila system

Sensitivity limit of 10 10 cellswell was detailed for

This preview shows page 228 - 230 out of 594 pages.

sensitivity limit of 10^-10^ cells/well was detailed for the Aer. hydrophila system devised by Sendra et al. (1997). Rapid identification of Fla. psychrophilum and Fla. branchiophilum was achieved by ELISA, which detected >1 x lO"^ cells/ml from infected spleen (Rangdale and Way, 1995) and 1 x 10^ cells/ml from gills (=the threshold value) (MacPhee et al., 1995), respectively. By use of ELISA, a useful typing scheme was devised for Fla. psychrophilum, recognising seven serogroups with host specificity (Mata et al., 2002). Similarly, an ELISA was developed for V. vulnificus and field-tested, the results of which indicated a sensitivity of lO'^-lO^ cells/ well, and an ability to detect non-culturable cells (Biosca et al, 1997b). We have successfully married monoclonal antibodies to Aer. salmonicida with ELISA for a
Image of page 228
Diagnosis 207 test, which has proven suitable for use on fish farms. Indeed, experiments demon- strated that rehable diagnoses were achieved within 30min (Austin et ai, 1986). It is noteworthy that ELISA systems appear to be more sensitive than culturing for the detection of Aer. salmonicida (Hiney et al, 1994). An indirect ELISA has been effective at detecting Y. ruckeri (Cossarini-Dunier, 1985) and for determining the presence of antibodies to Edw. ictaluri in fish serum (Waterstrat et ai, 1989; Swain and Nayak, 2003). A development of this approach involved the use of tissue homogenisation (using 0.5% v/v Triton X-100 in 0.05 M PBS [pH 7.2]), filtration and then the ELISA (Earlix et al, 1996). This approach was used successfully to detect asymptomatic carriers, and permitted live bacteria to be filtered from 1 g quantities of tissue slurries, with a sensitivity of <10 CFU/g of tissue. The filter-ELISA system detected Edw. ictaluri in 80% of 98 channel catfish com- pared with a detection of 24% by culturing (Earlix et ai, 1996). A monoclonal-based ELISA was successful and specific in detecting Pisci- rickettsia in infected fish tissues (Aguayo et al, 2002). Immunohistochemistry Immunohistochemistry, based on avidin-biotin complexes, has identified V. sal- monicida in fixed tissues (Evensen et ai, 1991). Also, a peroxidase-antiperoxidase immunohistochemical technique appears to be useful for differentiating Y. ruckeri (Jansson et al., 1991). Complications with the interpretation of slides by interference with melanin and/or melanomacrophages resulted in the adoption of melanin- bleaching with 3.0 g/1 potassium permanganate for 20min followed by 1% (w/v) oxalic acid for 1.5 min before immunostaining, which removed some of the problems of interpreting the presence/absence of Ren. salmoninarum (Morris et al., 2002). Immunomagnetic separation of antigens A comparatively novel approach concerns the recovery of Aer. salmonicida cells with immunomagnetic beads coated with monoclonal antibodies to LPS coupled with culturing (Nese and Enger, 1993). Thus, the cells are recovered by serological pro- cedures for culturing on routine bacteriological media. Magnetic beads incorporating polyclonal antibodies with an enzyme immunoassay have found success for the rapid diagnosis of pasteurellosis, in which the commercial kit had a detection limit of lO"^ cells albeit with a problem of specificity, i.e. other photobacteria—namely
Image of page 229
Image of page 230

You've reached the end of your free preview.

Want to read all 594 pages?

  • Spring '20
  • Bacteria, representative, gram-negative bacteria

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern

Stuck? We have tutors online 24/7 who can help you get unstuck.
A+ icon
Ask Expert Tutors You can ask You can ask You can ask (will expire )
Answers in as fast as 15 minutes
A+ icon
Ask Expert Tutors