|
 

 

 
  
 


 

  



ë Disease problems have grown proportionally with
the intensive or expansive culture of aquaculture
species
ë Why?
1) Increased stocking densities (lower profit margins)
2) Infected carriers (largely broodstock)
3) Infected facilities (GMPs being followed?)
4) Poor nutrition (we are way behind)
5) Substandard water quality (traditional)
ë è    greater susceptibility via weakening of
greater
resistance under intensive culture conditions
 ÿ  
  

ë or fish, response to a foreign agent is rather similar to
that of mammals; shrimp, very rudimentary
ë Response can be highly specific (a specific antibody for a
specific antigen) is known as the  
  
 [
ë ÿhe immune system Ơscansơ the body to identify any
substance (natural/synthetic or living/inert) that it
considers foreign
ë Differentiates between Ơselfơ and Ơnon-
Ơnon-selfơ
ë Works with several types of white blood cells, located
throughout the body, that work together in a highly
integrated way
  


ë  

à any type of barrier within the host
that allows it to resist the pathogen
ë 
 
à attributed to


 
 
à
inherited ability to produce antibodies without
stimulation by antigens
ë  
à host is stimulated by

  
à
contact with antigens
ë  
à acquired through the use
  
à
of antibodies from other animals (vaccination)
ë we will add another term today,  


 

  
 


ë   
 

 
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ë è $



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èegins when macrophage
encounters this non-
non-self
entity (e[g[, virus)à
macrophage literally
Ơeatsơ the substance,
digests it and displays
pieces of the invader on
its surface[ ÿhese
pieces are antigens
antigens[[
Meanwhile, other viral
particles are at work,
infecting nearby host
cells[[
cells
Ñ
 
 


   
 
  
  

#
#
`ntigenic fragments alert
a specific type of ÿ
lymphocyte (Ơhelperơ
ÿ) to begin
choreographed attack
of intruder
Helper recognizes antigen
particles and binds to
the macrophage via
an antigen receptor
Helper ÿ cells are unique
to a specific antigen
 
  
  

%
%
ÿhis binding stimulates
production of
chemical substances
such as interleukin
interleukin--1
(IL--1), tumor necrosis
(IL
factor (ÿ ) by
macrophage
Helper ÿ cells generates
interleukin--2 and
interleukin
gamma interferon
(I -y)
`ll substances facilitate
intercellular
communication

 



&

ë ÿ  steps up production of IL- IL-1, it also

causes fever in homeotherms
ë ÿ  and IL-IL-1 are cytokines (cellular)
ë IL-
IL-1 also causes fever but additionally forms
immune cell clusters and stimulates the
helper ÿ cell to release ILIL--2
ë IL-
IL-2 causes ÿ cells to release gamma
interferon which, in-
in-turn, activates
macrophages
ë IL-
IL-2 also instructs other helper ÿ cells and
Ơkillerơ ÿ cells to multiply
  
  
  

'
'
`s mentioned IL-IL-2 instructs
helper ÿ
s and Ơkiller ÿ
sơ to
multiply
Proliferating helper ÿ
s release
substances that cause è
cells (another type of
lymphocyte) to multiply and
produce antibodies
Meanwhile, many invader cells
have been consumed by
macrophages, but other
Ơdaughterơ viral particles
have escaped and are
infecting other cells
  
  
  

(
(
Miller ÿ cells start shooting Ơholesơ
in the surface of infected host
cells
`ntibodies released by è cells bind
in a lock-
lock-and-
and-key fashion to
antigens on the surface of
invaders that have escaped
macrophages (`g (`g--`b
complex)[
complex )[
Makes it easier for macrophages
and special killer lymphocytes
to destroy unwelcomed
entities[
èinding of antibodies with antigens
signals release of a blood
component, complement
complement,, to
puncture virus membrane
(death)
  
  
  

)
)
inally, as the infection is
brought under control, yet
another type of ÿ cell, the
suppressor ÿ cell,
cell, tells è
cells, helper ÿ
s and killer
ÿ
s to turn off
Most immune cells die, but a few
remain in the body, called
memory cells
ÿhey will be able to respond
more quickly the next time
the body is invaded by the
same foreign substance
 
  
 
*
ë jultured finfish and shellfish account for
approximately 25% of world aquatic animal
production
ë With intensification comes a deterioration in culture
environment, leading to increased incidence of
disease
ë Poor water quality affects the fish immune system in
a negative way
ë ÿhe status of being immune is Ơan inherited ability to
resist infectionơ (Shoemaker et al[, 2000)
ë I[e[, recognition of Ơnon
Ơnon--selfơ or a foreign agent, with
subsequent response and memory in vertebrates
 
  
 
*
ë ish are the most primitive vertebrates, but had to
develop an immune system for protection
ë the only exception was cold water speciesà due to
low bacterial generation time at lower temperatures
ë those living under schooling conditions and in warm
environments needed a highly developed response
ë all fish pathogens contain antigens
antigensàà viral particles,
bacteria, fungi, toxins and animal parasites
 
  
 
*
ë Immune response in fish includesà
ƛ expansion of cells for the immune response
ƛ expression of the cells and molecules (e[g[,
antibody)
ƛ the coordination of the response by regulatory
substances
ë Study of fish immunity and disease resistance is
relatively young compared to mammals
ë Early work was largely comparative, now focuses
on understanding how immune system responds
to foreign agents or how innate resistance can be
selected for by breeding programs
   
 * *


+


  
 

  

 



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ÿ 


ë Most important immunocompetent organsà thymus,
kidney (head, trunk), spleen and liver
ë Immune tissues in these organs not well defined
(Manning, 1994)
ë ÿhymusàà develops ÿ
ÿhymus ÿ--lymphocytes (helpers, killers;
similar to other vert
s), indirect evidence
ë Midneyàà important in both immunity and
Midney
hematopoiesis, site of blood cell differentiation
ƛ Early immune response handled by entire kidney
ƛ With maturity, anterior used for immune response; posterior
for blood filtration, urinary activities
 
ÿ 


ë Midney (cont[)à
ƛ blood flows slowly through kidney and antigens
are Ơtrappedơ or exposed to reticular cells,
macrophages, lymphocytes
ƛ `nterior is where Ơmemoryơ occurs (Secombs et
al[, 1982)
ë Spleenà secondary to kidney, involved in
Spleenà
immune reactivity and blood cell formation,
contains lymphocytes and macrophages
ë Liveràà could be involved in production of
Liver
components of the complement cascade,
important in resistance; not real clear
 
ÿ 


ë Mucus and skinà
skinà natural barriers, has
molecules with immune actionsà
ƛ Lysozyme
ƛ jomplement
ƛ atural antibodies (`b) and immunoglobulins (Ig)
ƛ Specific antibodies tentatively reported in mucus
of Ictalurus punctatus (Lobb, 1987); Oncorhyncus
mykiss (St[ Louis
Louis--jormier et al[, 1984)
ƛ Zilberg and Mlesius, 1997) showed mucus
immunoglobulin elevated in I[ punctatus after
exposure to bacteria
 m 


   


1) on--specific immune cells
on
ƥ 

 
  
  à à most important
cells in immune response, produce cytokines (jlem et al[,
1985), primary cells involved in phagocytosis and first
killing of pathogens upon first recognition and subsequent
infection (Shoemaker et al[,1997)
ƥ m   àà primary cells in early stages of
inflammation (Manning, 1994), neutrophils produce
cytokines to recruit immune cells to damaged or infected
area; neutrophils are phagocytic in I[ Punctatus,
Punctatus, kill
bacteria by extracellular mechanisms
ƥ m$ 
àà use receptor binding to target cells
and lyse them; important in parasitic and viral immunity
 m 


   


2) Phagocytosisà most primitive of defense
Phagocytosisà
mechanisms, occurs in stages
* Movement by chemotaxis (directional) or
chemokinesis (non
(non--d) of phagocytes in response
to foreign object
* `ttachment via lectins
* Engulfment of the foreign agent (simple
movement into the phagocyte)
* Milling and digestion
ƥ Oxygen-independent mechanismsà low pH, lysozyme,
Oxygen-
lactoferrin, proteolytic/hydrolytic enzymes
ƥ Oxygen dependent mechanisms
 m 


   


3) onspecific Humoral Moleculesà
Moleculesà
Molecule jomposition Mode of `ction

Lectins Specific sugar-

sugar- Recognition,
binding proteins precipitation,
agglutination
Lytic enzymes jatalytic proteins Hemolytic and
lysozyme, etc[ antibacterial activity
ÿransferrin/lactoferrin Glycoprotein Iron binding

jeruloplasmin `cute--phase protein

`cute jopper binding

j-reactive protein `cute--phase protein

`cute `ctivation of
complement
Interferon protein Resistance to viral
infection
 m 


   


ë Lytic enzymes are antibacterial molecules that cleave
the  1,4 linkages n-n-acetyl muramic and n- n-acetyl
glucosamine in bacterial cell walls
ë Lysozyme (another enzyme) works on Gram- Gram-positive
bacteria, complement on Gram-
Gram-negative
ë `cute--phase proteins are serum proteinsà
`cute
ceruloplasmin responsible for binding of copper,
usually generated as the result of stress
ë utrition also influences levels of j
j--reactive protein
 m 


   


'  
à
à consists of 20 or more chemically
different serum proteins + glycoproteins having enzyme
function
ë originally named Ơcomplementơ because it was
considered a biological substance complementing the
action of antibody
ë Instead, antibodies actually activate a series of reactions
Ơcomplement cascade[ơ
in serum known as the Ơcomplement cascade[ơ
ë interacts with either a specific antibody, or acts non-
non-
specifically on surface molecules of bacteria, viruses and
parasites; both pathways exist in fish (Sakai, 1992)
ë `ctionàà clears antigenic molecules, immune complexes,
`ction
participates in inflammation and phagocytosis
  

*
ë  
à à the antibody response to foreign antigens
ë ish posses è-è-cells (surface immunoglobulin-
immunoglobulin-positive
cells), similar to mammals in structure
ë Surface IgM of è- è-cells serves as receptor for antigen
recognition and is of same specificity as the antibody
molecule that will be produced (Janeway and
ÿravers, 1994)
ë Unlike crustaceans, fish possess immunologic
memory (`rkoosh and Maattari, 1991)
ë ÿheir primary and memory response both use the
same IgM molecule, with eight antigen binding sites,
a potent activator of complement
 !
!   


*
ë Used to eliminate intracellular pathogens (e[g[,
bacteria, virus, parasites)
ë Relies on contact of the foreign invader with the
subsequent presentation of an antigen having the
same major histocompatability complex (MHj I or II)
to ÿ-
ÿ-helper cells (REM?)
ë Once ÿ-
nce ÿ-helper cells are stimulated, the produce
cytokines that result in stimulation of effector cells
(cytotoxic lymphocytes) or macrophages
ë jytokines stimulate aforementioned cells and also
recruit new cells to the area, activate them
ë Work quite well against bacteria, important against
Edwardsiella ictaluri (Shoemaker, et al[, 1999)
 *




   



 
  
 * 

,
 


Individuals may exhibit differences in innate
Genetics resistance and acquired immunity
ÿemperature, season, photoperiod
Environment
Water quality, pollution, density, handling and
Stress transport, breeding cycles
eed quality and quantity, nutrient availability, use
utrition of immunostimulants, antinutritional factors in feeds
`ge, species or strains, individuals
ish
Exposure levels, type (parasite, bacterial, viral),
Pathogen virulence
Ô Ñ ,*Ô 
    
 

 !"-**./*  
* 0Ô
%

Ô
 *


 

  
 
 
ë Resting fish body temperature is near ambient
ë pathogen generation time is temperature
dependent
ë fishes living in cold temperatures have little need
for an immune response
ë coldwater fishes do not produce
immunoglobulins
ë immune response slower at cold temperatures
(up to 28 days!)
*


 

  
 

ë Immune competency develops relatively
slowly in animals
ë mammals obtain antibodies through mother
s
milk for up to six weeks
ë not the case with fish
ë rainbow trout are found to be immune
competent at an early age (0[3g)
ë significanceàà immunization of very young fish
significance
is practical

  





 





ë Most immunizing substances developed for

fish have been 
 

ë these are killed, whole-
whole-cell suspensions of
pathogenic bacteria
ë some practical viral vaccines exist (e[g[, for
jj , see subsequent notes on viruses)
ë probably will take place through injection of
avirulent viral strains
ë immunization against animal parasites might
also eventually be possible


  

ë ÿypical response is of short duration
ë very dependent upon environmental
temperature
ë primary response to injection is usually only a
few weeks
ë secondary injections nine weeks after primary
have resulted in maintenance of protective
antibody  ,
, as in higher animals

#  
  
 

 
ë `s mentioned, fish and shrimp differ significantly in
their ability and degree to which they carry out this
response
ë the capacity to recognize, expand the specific
recognition, express specific recognition, and
coordinate defense is much lower in shrimp
ë mistakeàà often drug manufacturers and scientists
mistake
assume that fish and shrimp have the same immune
competency
ë thus, inappropriate decisions have been made on
how defense mechanisms might be enhanced in
shrimp
 
 
 
 
  
ë Shrimp blood is known as 
ë it contains both oxygen-
oxygen-carrying molecules



)) and immunoreactive molecules
( 



known as 


ë lectins are glycoproteins (sugar + protein) that
bind with the sugar portion of other molecules,
particularly foreign ones
ë these lectins have broad specificity, meaning they
will bind with a broad range of other molecules,
not just sugars
ë for example, they can bind with the sugar moeity
of lipopolysaccharides, or  !
!


 
 
 
 

 
ë Gram negative bacteria (e[g[, Vibrio sp[
sp[ and yeasts
which contain beta-glucans can be recognized by
lectins
ë they also happen to recognize viruses and other
infectious agents with surface glycoproteins
ë after recognizing the foreign agent, the lectin will
agglutinize it, rendering it ineffective
ë the specificity for binding by a lectin cannot be
increased as with antibodies
 
 
 
 

 
ë ÿhe only way the immune response in shrimp can be
enhanced is by putting more lectins in the bloodstream
ë after the infection is over, the cells that produce lectins
completely lack the ability to remember the infectious
agent
ë so, immune response in shrimp is not an acquired one
ë another characteristic of lectins is that once bound to a
sugar on the foreign agent, the complex is easily
phagocitized
ë the phagocytic cell is known as 

   
   

ë `s mentioned, the primary defense cells in shrimp
are called 
 
ë certain hemocytes have the ability to phagocytize
foreign cells, others to encapsulate and render
agents ineffective
ë the defense mechanisms of shrimp are thus more
primitive and singular in their ability to control
infection
ë this means that stress is more likely to negatively
impact shrimp defenses against infection
ë no backup systems available when primary system
fails!!
 
 
 
 

 
ë blocking attachment by use of drugs or
diets containing beta-
beta-glucans might
prevent the binding of foreign agents
ë along with lectins, shrimp have
& , an anti-
anti-bacterial enzyme
ë lipolytic enzymes against viruses
 è   
 

ë èacteria and fungi are dealt with by
appropriate measures (e[g[, similar for most
aquaculture animals)
ë Most work has dealt with bacterial pathogens
ë Relatively few parasitesà cuticular excretions
and molting get rid of them
ë Most problems lie with prevention and/or
treatment of viruses
    

ë `s mentioned, shrimp have both a cellular and
humoral response to virusesà
ƛ jertain proteins respond to -glucan (component of
bacterial cell wall)
ƛ Hemocytes attack bacteria, release compounds
causing browning reaction in the HP
ë èutƦ no antibodies generated!
ë o defense against viruses has to date been
described in any detail
ë jonclusionàà there must be some defense that
jonclusion
has been overlooked!
    

ë ÿhere is also little histological response to
virusesà blood cells don
t go to location
ë iral infections are persistent, remain evident
for life of shrimp
ë Despite having no set specific response to
specific viral pathogens, shrimp appear to
have a have a high tolerance to them
ë jase in pointà historical information on viral
epizootics in Southeast `sia
 ¦ -,

.
ë ur current management practice is to look
for SP
SP,, high
high--health animals for stocking
ponds
ë Most PL
s derived from new sources, not from
survivors
ë ÿhe history of each batch is important to
know!
ë Implicationàà perhaps SP animals are not
Implication
appropriate!
 m / 
ë If you sample a normal shrimp pond in SE `sia, 88%
of shrimp are infected with a virus
ë 53% have been infected with two to three viruses
ë Survival now (after multiple years in population) has
returned to a more or less normal level
ë Does this indicate resistance or tolerance
tolerance??
ë Resistance = no sign of pathogen in individual;
however, virus can be detected in tissues
ë jonclusionàà something different from resistance
jonclusion
ÿ 0

 

$
Shrimp viral response is an active process
ë Involves binding of viron to receptor site
that triggers some kind of Ơmemoryơ
ë èinding is not related to infection receptor
ë Memory causes reduced   
ë Subsequent binding turns off ability of virus
to induce death in host
ë Death is prevented, but not infection
ë iral replication can take place, but no death
`





 


    





  



 
 


0



 



ë Initialà brief and evolutionary with acute
Initialà
mortality via apoptosis, leads to intermediate
phase
ë Intermediateàà virus and host live together,
Intermediate
but without mortality; better host survivors
replicate so population is positively selected
for against virus
ë inalàà hard to find virus, mutual existence
inal
governed by genetic factors
 

 

ë Higher virulence is naturally selected

against
ë o resistance to infection = reduced or
low virulence
ë Point
Pointàà no pressure on virus to become
virulent
ë Point
Pointàà may increase competition for
new viruses to enter host!
 ¦ ...
ë Use survivors as a source of broodstock
ë Expose progeny to virus or tolerene to
develop tolerance (avirulent virus)
ë When? Possibly at Zoea 3 or earlier
ë How? ÿolerene developed specifically for
each virus
ë Implicationsàà for larval rearing, it means
Implications
introduction of a  
in proper form
0  

0 
1*
ë o clear response to
viruses
ë Survivors remain
infected
ë Pathogen persists
ë Survivors infectious to
others
ë ÿolerance is a normal
situation
ë o antibodies
ë Multiple active
infections are normal
Ñ

ë Specific response to
viruses
ë Survivors often don
t
remain infected
ë Pathogen removed from
body
ë May or may not be
infectious to others
ë ÿolerance not normal
ë `ntibodies present
ë Usually only one virus
at a time
Ñ