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2. Phagocytic activity: Phagocytosis is a common reaction of cellular defence and generally recognized as a central and important way to eliminate microorganisms or foreign particles. Phagocytosis can be assayed by incubating blood with a killed bacterial culture and examining stained smears for phagocytes containing bacteria [ 58 ].
The phagocytic activity are defined as phagocytic ratio (PR) and phagocytic index and are expressed as
Bactericidal activity: Bactericidal activity can be assayed by incubating macrophages with a live bacterial culture and then washing off the supernatant liquid, lying the macrophages and examine the numbers of live bacteria [ 59 ].
3. Oxidative radical production: A major way in, which neutrophil granulocytes contribute to nonspecific immunity, is by the production of oxidative radicals. Nitro-blue tetrazolium (NBT) reacts with oxidative radicals producing a dark blue color and is used to identify neutrophils actively producing them.
4. Myelo-peroxidase production: Activated neutrophils also produce myelo-peroxidase. The level of activation can be determined by incubating blood smears in an indicator reagent and examining cells under the microscope for degree of staining.
5. Immunoglobulin concentration: Some serum immunoglobulins are humoral antibodies and therefore heighten specific immunity, many others regulate nonspecific immunity.
6.
In vitro
measurement: Jeney and Anderson [
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] have described an
in vitro
method for screening substances for immunostimulation. In essence finally divided pieces of rainbow trout spleen are maintained in a tissue culture medium with a test substance and after 4 days cell suspension are prepared. For neutrophils the cell suspensions are treated with NBT and examined by spectrophotometry; for phagocytes aliquots of the cell suspension are shaken for 15 min. with a suspension of fixed sheep erythrocytes and then smears are made for microscopy.
7. In vivo measurement: In fish, specific immunity develops slowly and thus it is possible to assess immunostimulation by a challenge test with virulent bacteria, which rapidly kills large number of fish at a time. Any delay or reduction in mortality in treated fish compared to untreated group may be attributed nonspecific immunity systems.
Attributes of immunostimulants
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• Nontoxic to finfish and shellfish with no known side effects
• Enhance disease resistance against broad spectrum of pathogens
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