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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 [ Discount 100% Authentic Get 2790 Trainers light grey Choice Online UGWXm2fhZZ

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 [ 10 ] 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

• Safe for the environment and human health, biocompatible and biodegradable

• Promote a healthy body status by triggering the immune system of the host

• Nontoxic to finfish and shellfish with no known side effects

• Enhance disease resistance against broad spectrum of pathogens

• Reduce mortality due to opportunistic pathogens

• Prevent viral diseases

• Enhance the efficacy of antimicrobial substances

• Enhance the efficacy of vaccines and antibiotics

• Cheap, ecofriendly and easily available.

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