???????Fish Immunity and Nutrition

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???????Fish Immunity and Nutrition
Delbert M. Gatlin III Department of Wildlife and
Fisheries Sciences and Intercollegiate Faculty of
Nutrition Texas AM University System
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????Outline of Presentation

• 1) ???????General overview of immunology
• 2)????????????
• Nutrients affecting immunity and disease
  resistance
• 3)?????????? Non-nutrient immunostimulants
• ??? Glucans
• ??? Nucleotides
• ??? Prebiotics
• 4) ????? Summary and conclusions

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Verlhac and Gabaudan 1997
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????(??????)Innate (Non-specific) Immunity

• ??????,??First line of defense, includes
• ????(?????)Physical barriers (skin and mucus)
• ???? Humoral factors
• ???? Complement system
• ???????? Classical and alternative activation
• ??? Lysozyme
• ??? Lectins
• ?????? Cellular mechanisms
• ??????????? Macrophages and neutrophils
• ??? Chemotaxis
• ???? Phagocytosis
• ????????? Oxygen-dependent and -independent
  killing
• ????? Extracellular killing
• ????????????Natural killing cells and eosinophils
  

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??????Innate Immune Responses
V. Verlhac, ISNFF, Brazil 2008
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V. Verlhac, ISNFF, Brazil 2008
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?????Adaptive Immunity

• ???????????? Adaptive Immunity is
  antigen-specific
• ??????Components include
• ??(?????)IgM Antibodies (immunoglobulins) IgM
• ?B??????????????(??????)Produced by B
  lymphocytes recognize extracellular antigens
  (humoral antibody system)
• ???????T????(????)
• Cell mediated Immunity T lymphocytes (cellular
  system)
• T???????????????????? T lymphocytes recognize
  intracellular antigens expressed on the surface
  of body cells

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?????Adaptive Immunity
?????????????(????????)The two systems cellular
(T cells) and humoral (B cells) work together to
identify antigens (foreign proteins or
glycoproteins)
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V. Verlhac, ISNFF, Brazil 2008
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??????? Assessment of Immune Responses

• ???????? The immune response as quite complex
• ???????????? Measuring singular responses can be
  misleading
• ?????????????????????????
• The best measure of integrated immune responses
  is a live pathogen challenge
• ????????????,??????????????????
• There is good correlation between immune
  modulation and disease resistance when immunity
  is broadly characterized

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????? Nutrition and Immunity

• ???????????????????
• Nutrition is an important factor influencing
  immune responses and disease resistance
• ????/?? Feed quality/quantity
• ????? Nutrient availability
• ????? Anti-nutritional factors
• ?????? Immunostimulants
• ?????????????? Disease or stress correspond to a
  break in immunocompetence
• ???????????????????????????????????????
• Nutritional intervention can help manage disease
  by applying nutrition-based strategies which
  support optimal growth and immune system function

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?????????? Dietary Modulation of Immunity and
Disease Resistance

• ????????? Over-fortification of certain
  nutrients
• ??????????????
• Dietary supplementation of non- nutrient
  immunostimulants
• ?????????(?????)???????? Functions (substrate
  vs. signal) and actions of these various
  compounds is currently incomplete

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???????????? Nutrients Shown to Influence
Immunity and Disease Resistance of Fish
??/??? Protein/amino acids ??/??? Lipid/fatty
acids ???A/??? Vitamin A/astaxanthin ???C Vitamin
C ???E Vitamin E ? Iron ? Selenium ? Zinc
??? Arginine
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Nutrient allowance
Verlhac and Gabaudan 1997
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????????? Non-Nutritive Dietary Supplements

• ?????? Immunostimulants
• ????????? heterogeneous group including
• - ? -??? ?-glucans
• ??? nucleotides
• ??? chitin
• ??? peptidoglycan
• ??? lipopolysaccharides
• ???? levamisole
• ??? prebiotics

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• ???? Mechanisms of action
• ???? complement activation
• ??????? enhanced lysozyme production
• ??????????? increased oxidative capacity of
  phagocytes
• ?????????(????0.2 )??????,??????????Injection
  has generally produced more favorable responses
  than oral administration (0.2 of diet)
  immunosuppression may occur

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??????????????????? Cumulative Mortality of
Hybrid Striped Bass Exposed to S. iniae
a
b

b
b
glucan
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- disease and wound repair increase needs
V. Verlhac, ISNFF, Brazil 2008
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??? Nucleotides

• ????????????????????????????????????
• Enhanced resistance to infection by bacterial,
  viral, rickettsial and ectoparasitic organisms
  has been reported in salmonids.
• ???????????????????,??????????Alterations in
  non-specific immune responses not consistently
  reported although potential mechanisms of action
  include
• ???? complement activation
• ??????? enhanced lysozyme production
• ??????? activation of macrophages
• ???????????increase natural killer cell activity

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??????????????????? Cumulative Mortality of
Hybrid Striped Bass Exposed to S. iniae

Exp. 1
P 0.091
Exp. 2 P 0.017
Li et al. (2004) Fish Shellfish Immunol.
16561-569
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??? Prebiotics

• ?????????????????????????????Dietary constituents
  that alter the intestinal conditions to favor
  beneficial bacteria and stimulate immunological
  responses.
• ??? Fructooligosaccharide (FOS)
• ???? Mannanoligosaccharide (MOS)
• ????? Galactooligosaccharides (GOS)
• GroBiotic-A ???????????,??????????
• a mixture of partially autolyzed brewers yeast,
  dairy components and dried fermentation products
  

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???????????Hybrid striped bass-bacterial
challenge
S. iniae suspension 9.3105 CFU/ml
Survival ()
P lt 0.01
Li and Gatlin (2004) Aquaculture 231445-456
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??????-?? Hybrid striped bass weight gain



Plt0.1 Plt0.05
Li and Gatlin (2005) Aquaculture 248197-205
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???????????? Survival during Mycobacterium
challenge
P0.018
Li and Gatlin (2005) Aquaculture 248197-205
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?????????????????? Cumulative Mortality of Golden
Shiners Exposed to Flavobacterium columnare
a
b

b
b
Bacterial challenge by immersion after 16 wk
feeding
Sink et al. (2007) N. Am. J. Aquacult. 69305-308
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?????????????????????????? Cumulative Mortality
of Golden Shiners Exposed to Flavobacterium
columnare with or without Confinement Stress
Bacterial challenge by immersion after 10 wk
feeding
Sink and Lochmann (2008) N. Am. J. Aquacult.
69305-308
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?????????????????? Cumulative Mortality of
Rainbow Trout Exposed to Infectious Hematopoietic
Necrosis Virus
IP injection after 9 wk feeding
Sealey et al. (2007) N. Am. J. Aquacult.
69400-406
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????????? Evaluation of Prebiotics with Red Drum

• ?????????????????????????????????Two
  separate in-vitro experiments were conducted with
  sub-adult red drum to evaluate various prebiotic
  compounds including
• GroBiotic -A, ????????
• GroBiotic -A Brewers yeast and
  Fructooligosaccharide (FOS)

• ?????????????????????0, 24 and 48 h
• Incubate replicate tubes with GI tract inoculum
  and prebiotic anaerobically for 0, 24 and 48 h
• ??16S rDNA ??? Isolate 16S rDNA for extraction
• ??200?????????????????(DDGE)??
• Amplify a 200 base pair region and analyze using
  denaturing gradient gel electrophoresis (DGGE)
• DGGE??????????????DGGE pattern allows differences
  in
• microbial species to be
  distinguished.

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DGGE?????Dendogram of DGGE Analysis
85.7
90.5
82.7
94.9
80.6
72.0
Lactococcus sp..
81.0
8
96.5
90.8
86.2
Burr et al. (2008) J. Environ. Sci. Health, B
43253-260 .
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????Experimental diets
Ingredient Basal FOSa MOS TOS GBAb
??? Menhaden meal 29.2
?? Soybean meal 37.5
?? Dextrin 15.0
??? Menhaden oil 8.1
?? Vitamin premix 3.0
?? Mineral premix 4.0
CMC 2.0
??? Fructooligosaccharide (FOS) 0 1.0
???? Mannanoligosaccharide (MOS) 0 1.0
???????Transgalactooligosaccharide (TOS) 0 1.0
GroBiotic - A (GBA) 0 1.0
???Celufil 1.2 0.2 0.2 0.2 0.2
S 100.0 100.0 100.0 100.0 100.0
a Inulin
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???-??????Red drum crude protein digestibility

A
A
A
A
B
B
P0.0019
Burr et al. (2008) Aquaculture Res. In press
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???-?????Red drum lipid digestibility

A
AB
B
C
C
C

P0.0007
Burr et al. (2008) Aquaculture Res. In press
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????Experimental challenge

• ?4?,????????????????,?????????? On week 4,
  Amyloodinium ocellatum dinospores transferred
  into biofilter ? in situ challenge (Li et al.
  2005)
• ??????????2??Growth and survival monitored for 2
  more weeks
• ?????????,???????????????Clinical signs and
  effects recorded and histopathology of infested
  gills was evaluated (Texas Veterinary Medical
  Diagnostic Laboratory, TVMDL)

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??Results

• 6??????Survival after 6 weeks

A
A
AB
B
A
P 0.02
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??Results

• ????Feed efficiency

A
AB
B
B
B
P 0.02
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??Results

• ????? Serum lysozyme

A
A
A
AB
B
P 0.02
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??Results

• ????????????
• Intracellular superoxide anion production

A
A
AB
B
B
P 0.01
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??Results

• ?????? Other immune responses

??Ingredient ??units ??Basal FOS MOS TOS GBA
????????????Neutrophil oxidative radical production Pr gt F 0.09 mg/mL 3.8 4.2 3.7 3.8 3.9
???????????? Extracellular superoxide anion production Pr gt F 0.08 nmol O2 2.9 2.5 3.0 2.7 3.2
mg NBT diformazan/mL of blood
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GroBiotic-A ??????????? Summary of GroBiotic-A
Effects in Aquaculture

• ????????Observed effects to date
• ?????? Enhanced growth performance
• ???????? Improved immunity and disease resistance
• Increased nutrient availability in soybean-
• meal-based diets
• Changes in GI tract micobiotia
• Lactococcus spp. isolated from GI tract inoculum
  of red drum incubated with GroBiotic-A

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??Conclusions

• ????????????,????????????????The immune system of
  fish is relatively complex and vital to
  maintaining health and disease resistance
• ???????,?????????????????????????Dietary
  modulation of disease resistance holds promise as
  a relatively inexpensive and environmentally
  responsible alternative to chemical therapy for
  disease control
• ??????????????????????????????????????????????,???
  ??????????? Immunostimulants such as certain
  nucleotides and prebiotics have shown consistent
  improvements in fish production, immune responses
  and disease resistance of various fish species,
  thus enhancing the efficiency of aquacultural
  production

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??Acknowledgements

• The Southern Regional Aquaculture Center of the
  United States Department of Agriculture
• International Ingredient Corporation for
  providing GroBiotic-A and funding.
• Texas Veterinary Medical Diagnostic Laboratory
  for providing analytical support.
• Students and staff at the Texas AM Aquacultural
  Research and Teaching Facility