Infectious disease in aquaculture: Prevention and control

550 Pages·2012·4.2 MB·English

by  Brian Austin

Infectious disease in aquaculture
© Woodhead Publishing Limited, 2012
1
The innate and adaptive immune system
of fi sh
C. J. Secombes and T. Wang, University of Aberdeen, UK
Abstract: This chapter describes what is known about the main components and
responses of the innate and adaptive immune system of fi sh. The chapter fi rst
reviews the organs, cells and molecules of the immune system known in a few
economically important or model fi sh species. Molecular evidence suggests a
similar immune system exists throughout the jawed vertebrates yet marked
differences are also apparent. The innate parameters are at the forefront of fi sh
immune defence and are a crucial factor in disease resistance. The adaptive
response of fi sh is commonly delayed but is essential for long lasting immunity
and a key factor in successful vaccination.
Key words: fi sh immune organs, pattern recognition receptors (PRR), innate
immune responses, adaptive immune responses, immune regulation.
1.1 Introduction
Fish possess innate and adaptive immune defence systems. The innate
parameters are at the forefront of immune defence and are a crucial factor
(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)in disease resistance. The adaptive response of fi sh is commonly delayed
but is essential for long-lasting immunity and is a key factor in successful
vaccination. The massive increase in aquaculture in recent decades has put
greater emphasis on studies of the fi sh immune system and defence against
diseases commonly associated with intensive rearing of a few economically
important species. Such research has helped defi ne the optimum conditions
for maintaining immunocompetent fi sh in culture, for selection of fi sh stock
(breeding), as well as developing and improving prophylactic measures such
as vaccination, and use of probiotics and immunostimulation in the aqua-
cultured species.
However, there is great variation in disease susceptibility and immune
defence between different fi sh species, a refl ection of the extended time the
present day teleosts have been separated during the evolution of this fi sh
group. Thus the immune response described in one species may not be the
© Woodhead Publishing Limited, 2012
4 Infectious disease in aquaculture
same in other species. Indeed, the immune system is largely unknown in
most fi sh species, especially in newly aquacultured species, limiting the
development of immune control strategies against infectious disease. This
chapter will describe what is known about the main components of the
innate and adaptive immune system of fi sh.
1.2 Overview of immune cells and organs in fi sh
Vertebrates live in an environment containing a great variety of infectious
agents – viruses, bacteria, fungi, protozoa and multicellular parasites – that
can cause disease, and if they multiply unchecked they will eventually kill
their host. Thus vertebrates have evolved effective immune responses that
initially recognize the pathogens or other foreign molecules (antigens),
triggering pathways that subsequently elicit effector mechanisms to attempt
to eliminate them. The immune responses elicited fall into two main catego-
ries: innate (or non-specifi c) immune responses and adaptive (or specifi c)
immune responses.
Immune responses are mediated by a variety of cells and secreted soluble
mediators. Leucocytes are central to all immune responses, and include
lymphocytes (T cells, B cells, large granular lymphocytes), phagocytes
(mononuclear phagocytes, neutrophils and eosinophils) and auxiliary cells
(basophils, mast cells, platelets). Other cells in tissues also participate in the
immune responses by signalling to the leucocytes and responding to the
soluble mediators (cytokines) released by leucocytes such as T cells and
macrophages.
The cells involved in the immune responses are organized into tissues
and organs in order to perform their functions most effectively. These struc-
tures are collectively referred to as the lymphoid system, and are arranged
into either discretely encapsulated organs or accumulations of diffuse lym-
phoid tissue. The major lymphoid organs and tissues are classifi ed as either
(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0) primary (central) or secondary (peripheral). Lymphocytes are produced in
the primary lymphoid organs and function within the secondary lymphoid
organs and tissues.
In mammals, the thymus, foetal liver and bone marrow are the primary
lymphoid organs, where lymphocytes differentiate from lymphoid stem
cells, proliferate and mature into functional cells. T cells mature in the
thymus whereas B cells mature in the foetal liver and bone marrow. In the
primary lymphoid organs, lymphocytes acquire their repertoire of specifi c
antigen receptors, i.e. T cell receptor (TCR) and B cell receptor (BCR), in
order to cope with antigenic challenges that individuals receive during their
lifespan, with cells having receptors for autoantigens mostly eliminated
early in development. For example, in the thymus, T cells learn to recognize
self-MHC (major histocompatibility complex) molecules but if they react
to self-antigens presented by these molecules they are eliminated. It is
© Woodhead Publishing Limited, 2012
The innate and adaptive immune system of fi sh 5
worth noting that some lymphocytes develop outside the primary lymphoid
organs (Alitheen et al., 2010; Peaudecerf and Rocha, 2011).
The generation of lymphocytes in primary lymphoid organs is followed
by their migration into peripheral secondary lymphoid tissues. In mammals,
the secondary lymphoid tissues comprise well-organised, encapsulated
organs, such as the spleen and lymph nodes (systemic organs) and non-
encapsulated accumulations of lymphoid tissues. The spleen is responsive
to blood-borne antigens and lymph nodes protect the body from antigens
from skin or from internal surfaces. The lymphoid tissue found in associa-
tion with mucosal surfaces is called the mucosal associated lymphoid tissue
(MALT), and includes GALT (gut-associated lymphoid tissue) in the intes-
tinal tract, BALT (bronchus-associated lymphoid tissue) in the respiratory
tract, and lymphoid tissue in the genitourinary tract (Randall, 2010; Suzuki
et al., 2010).
In the secondary lymphoid organs, germinal centres (GC) are unique
structures in birds and mammals where the collaboration between pro-
liferating antigen-specifi c B cells, T follicular helper cells (Tfh), and the
specialized follicular dendritic cells (FDC) produces high-affi nity antibody-
secreting plasma cells and memory B cells that ensure sustained immune
protection and rapid recall responses against previously encountered
foreign antigens (Gatto and Brink, 2010). GCs develop in the B cell follicles
of secondary lymphoid tissues during T cell-dependent (TD) antibody
responses. The mature GC is divided into the dark and light zones on the
basis of their histological appearance. The antigen-specifi c B cells prolifer-
ate in these locations (Hauser et al., 2007) and undergo somatic hyper-
mutation, antibody class-switch recombination, and are then selected by
the FDC and Tfh. Therefore, the GC response endows a population of
antigen-activated B cells that secrete antibodies (or immunoglobulins,
Ig) with a high affi nity for the antigen and with a relevant Ig isotype,
resulting in a more effi cient clearance of the antigen (Good-Jacobson and
Shlomchik, 2010).
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1.2.1 The thymus
The term ‘fi sh’ refers to a heterogeneous group of organisms that include
the Agnathans (lampreys and hagfi sh – jawless vertebrates), Chondrich-
thyes (sharks and rays) and Osteichthyes (bony fi sh, that include the largest
group of fi sh the teleosts) (Nelson, 1994). In this chapter the term fi sh will
be used to refer to bony fi sh unless otherwise specifi ed. As in birds and
mammals, fi sh have cellular and humoral immune responses, and central
organs (Fig. 1.1) whose main function is involved in immune defence.
The thymus is considered a key organ of the immune system in jawed
vertebrates. It is thought to have evolved in early fi sh species as a thickening
in the epithelium of the pharyngeal area of the gastro-intestinal tract
(Bowden et al., 2005), and is identifi able in the Chondrichthyes and the
© Woodhead Publishing Limited, 2012
6 Infectious disease in aquaculture
Thymus Head kidney Caudal kidney Mucus/skin
Gills Liver Spleen Gut
Fig. 1.1 Immune tissues in teleost fi sh. The approximate sites of immune tissues
are superimposed onto a rainbow trout (Oncorhynchus mykiss).
Osteichthyes. It generally develops in the lamina propria of the gastroin-
testinal tract in pouches located at the base of the gill arches, and subse-
quently migrates to the underlying mesenchyme during ontogeny. In most
teleosts the thymus is located near the gill cavity and is closely associated
with the pharyngeal epithelium (Zapata et al., 1996). Although usually
found as a paired organ in most vertebrates, the thymus can appear as more
than one pair of organs in teleosts. For example, each gill chamber of cling-
fi sh, Sicyases sanguineus, has a pair of thymus glands with one taking up a
superfi cial position and the second located close to the gill epithelium
(Gorgollon, 1983).
The cells in the mammalian thymus can be divided into hematopoietic
cells (CD45+ cells, which include thymocytes, dendritic cells (DC), macro-
(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0) phages and B cells) that are transient passengers and resident stromal cells
(CD45− cells). CD45− cells include two lineages: thymus epithelial cells
(Keratin+) that originate from the pharyngeal pouch endoderm (third pouch
in the mouse) and mesenchymal cells (Keratin−), which are a mixture of cell
types that contribute to various structures of the thymus such as the capsule
or vasculature (Rodewald, 2008). The thymus is organized into the inner,
morphologically lighter zone, the medulla, and the outer, morphologically
darker zone, the cortex. In mammals T-cell progenitors enter through the
cortico-medullary blood vessels and can differentiate into NK cells, DC and
T cell lineages (De and Pal, 1998). Within the cortex are lymphocytes in a
stroma of cells of epithelial morphology, and macrophages. The structure of
the fi sh thymus is highly variable between species and within a species in
an age-dependent manner. In many fi sh species there is no clear cortico-
medullary differentiation as would normally be seen in mammals. Zonation
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The innate and adaptive immune system of fi sh 7
of the thymus has been observed in turbot (Scophthalmus maximus L.) and
halibut (Hippoglossus hippoglossus L.) but not in salmonids (Tatner and
Manning, 1982; Fournier-Betz et al., 2000).
Although zonation is absent in the young carp thymus, later on a complex
intermingling of cortex into the medulla occurs in developing carp, with
zonation becoming visible during the fourth week post-fertilisation
(Romano et al., 1999). The size of lymphocytes also can vary with species.
A comparative study of three fi sh species revealed that lymphocytes are
typically basophilic and 3–5 μm in diameter, whilst populations of darker
staining small lymphocytes (2–2.5 μm) are observed in later development
(Chantanachookin et al., 1991).
The thymus, kidney and spleen are the major (non-mucosal) lymphoid
organs of fi sh. In freshwater fi sh, the thymus is the fi rst organ to become
lymphoid, although prior to this the kidney can contain hematopoietic
precursors but not lymphocytes. However, in marine fi sh the order in
which the major lymphoid organs develop is kidney, spleen and then the
thymus (Zapata et al., 2006). Early development of the thymus in fi sh
has been studied in many diverse teleost species and has shown that the
development timeframe can differ from species to species even when
accounting for temperature effects on growth (Bowden et al., 2005). The
relationship between growth and development can be dynamic and physi-
ological age expressed as degree-days does not factor out all differences.
Thus at 5 days pre-hatching at 14 °C, the rainbow trout embryo already
possesses the rudiments of a thymus (Grace and Manning, 1980), whereas
a thymus is only seen at 28 days post-hatching in Atlantic cod (Schrøder
et al., 1998).
1.2.2 The bone marrow equivalent: the head kidney
Hematopoietic stem cells (HSCs) found in the mammalian bone marrow
(BM) are crucial throughout life for their ability to differentiate and gener-
(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)ate all hematopoietic lineages while maintaining the capacity for self-
renewal. This remarkable ability can be demonstrated in mice where a
single HSC can reconstitute all the immune cells of a lethally irradiated
animal, thereby maintaining a functional immune system throughout life.
The bone marrow in mammals is the site where B-cells originate and
develop from HSCs via progression through downstream multipotent pro-
genitors, lymphoid primed multipotent progenitors, common lymphoid
progenitors, B-cell progenitor intermediates and fi nally naive B cells
expressing rearranged surface bound Ig (see below) (Santos et al., 2011).
The bone marrow is absent in fi sh but the cephalic portion of the kidney
(head kidney or pronephros) is considered analogous to mammalian
bone marrow, at least in terms of hematopoiesis (Zapata, 1979). The trunk
kidney (mesonephros) is also hematopoietic, although it also contains renal
tissue.
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8 Infectious disease in aquaculture
The kidney in fi sh is often a Y-shaped organ that is placed along the body
axis above the swim bladder (Fig. 1.1). The lower part is a long structure
situated parallel to the vertebral column, most of which works as a renal
system. The active immune part, the head kidney, is formed by the two arms,
which penetrate under the gills. The head kidney has a reticulo-endothelial
stroma consisting of sinusoidal cells (endothelial and adventitial cells) and
reticular cells (macrophage-type reticulum and fi broblast-like reticular
cells) similar to those of the mammalian bone marrow (Meseguer et al.,
1995). The macrophage-type reticulum cells are characterized by their cyto-
plasmic processes and acid phosphatase positive lysosomes. The fi broblast-
like reticular cells are peroxidase negative and acid and alkaline phospha-
tase, glucose-6-phosphatase, beta-glucuronidase and ATPase positive, and
are joined by desmosomes and form an extensive network between the
hematopoietic parenchyma. The types of hematopoiesis described within
the fi sh head kidney include erythropoiesis, granulopoiesis, thrombopoiesis,
monopoiesis and lymphoplasmopoiesis (Abdel-Aziz et al., 2010). Erythro-
poiesis includes a number of developmental stages, including proerythro-
blasts, basophilic erythroblasts, polychromatic erythroblasts, acidophilic
erythroblasts and young and mature erythrocytes. The granulopoietic series
consists of cells with variable shape and size depending on the stage of
maturity, from myeloblasts to mature granulocytes. The lymphopoietic cells
include lymphoblasts, large lymphocytes, small lymphocytes and active and
inactive plasma cells, whilst the thrombopoietic series consists of thrombo-
blasts, prothromboblasts and thrombocytes. Melano-macrophage centres
(MMC) are also present in the head kidney, and are thought to function as
primordial GCs (Agius and Roberts, 2003; Saunders et al., 2010).
The fi sh head kidney is also an important endocrine organ, homologous
to mammalian adrenal glands, and contains aminergic chromaffi n and inter-
renal steroidogenic cells. The adrenal homologue and hematopoietic tissues
can be mixed, adjacent or completely separated, with the former lining the
endothelium of the venous vessels or being located in close proximity to
(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0) them (Gallo and Civinini, 2003). The fi sh adrenal homologue is under hor-
monal and neuronal control. Interrenal cells secrete corticosteroids and
other hormones that may play an important role in modulating stress
responses, osmoregulation and the immune response. Thus, the head kidney
is an important organ with key regulatory functions and the central organ
for immune–endocrine interactions and even neuroimmunoendocrine
cross-talk.
The fi sh head kidney appears to be the primary organ for antibody pro-
duction (Tian et al., 2009). The fi rst appearance of antibody secreting lym-
phocytes varies considerably among fi sh species. The fi rst appearance of B
cells, as defi ned by the expression of Ig, is later in marine species compared
to freshwater species, with larvae being 20–30 mm in length when Ig is fi rst
expressed, about a week after hatching in the case of rainbow trout and
channel catfi sh (Magnadottir et al., 2005).
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The innate and adaptive immune system of fi sh 9
1.2.3 The spleen
The spleen in mammals is the largest secondary immune organ in the body
and is responsible for initiating immune reactions to blood-borne antigens
and for fi ltering the blood of foreign material and old or damaged red
blood cells. These functions are carried out by the two main compartments
of the spleen, the white pulp (including the marginal zone) and the red pulp,
which are vastly different in their architecture, vascular organization and
cellular composition (Cesta, 2006). The spleen is also a major secondary
lymphoid organ in fi sh, although absent in Agnathans where spleen-like
lymphohematopoietic tissues occur in the intestine (Fänge and Nilsson,
1985; Press and Evensen, 1999). It contains the same elements as the other
vertebrates: blood vessels, ellipsoids, red pulp and white pulp. However, the
red and white pulp in fi sh is less clearly defi ned than in homeothermic
vertebrates. The pulp occupies the majority of the organ, and consists of a
reticular cell network supporting blood-fi lled sinusoids that hold diverse
cell populations, including macrophages and lymphocytes. The white pulp
is often poorly developed and typically has two main components: the
melano-macrophage accumulations and the ellipsoids. The spleen can also
be a major reservoir of disease and there is much interest in trying to
understand its role in protection against bacterial infection (Hadidi et al.,
2008) as well as in red blood cell regulation. The populations of lymphocytes
and macrophages capable of mounting an immune response are situated
close to sites of antigen trapping and often associated with accumulations
of melano-macrophages.
The melano-macrophages may form MMCs, bound by a thin argyrophilic
capsule and surrounded by white pulp, often in association with thin-walled,
narrow blood vessels (Agius, 1980; Press and Evensen, 1999). Fish MMCs
are typically located in the stroma of the haematopoietic tissue of the spleen
and kidney, and in the liver in some species, and may be primitive analogues
of the GCs of mammals and birds. GCs contain specialized FDC that inter-
act with antigen-specifi c B cells to produce high-affi nity antibody-secreting
(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)plasma cells and memory B cells (Gatto and Brink, 2010). An antibody,
CNA-42, usually employed for labelling FDC of higher vertebrates, can
label free melano-macrophages and splenic MMCs, and the key initiator
of antibody affi nity maturation (activation-induced cytidine deaminase,
AID) is expressed in cells that co-locate with melano-macrophages, sug-
gesting an evolutionary relationship between fi sh MMCs and mammalian
GCs (Vigliano et al., 2006; Saunders et al., 2010). The MMCs can retain
antigens for long periods, possibly in the form of immune-complexes (Agius,
1980; Press and Evensen, 1999), and increase in size or frequency in condi-
tions of environmental stress and during infection (De Vico et al., 2008;
Suresh, 2009).
The ellipsoids terminate in arterioles with a narrow lumen that runs
through a sheath of reticular fi bres, reticular cells and macrophages,
the ellipsoid. Ellipsoids appear to have a specialized function for plasma
© Woodhead Publishing Limited, 2012
10 Infectious disease in aquaculture
fi ltration and the trapping of blood-borne substances, particularly immune
complexes (Secombes et al., 1982; Press and Evensen, 1999). Blood-borne
substances are retained in the ellipsoidal wall and taken up by the rich
population of macrophages surrounding these vessels. The subsequent
migration of antigen-laden macrophages to MMCs has been described
(Press and Evensen, 1999). Ellipsoids occur in most fi sh but may be indis-
tinct or lacking in certain species (Fänge and Nilsson, 1985). Similarly,
splenic lymphoid tissue is poorly developed in some fi sh where diffuse
layers of lymphocytes surround arteries and MMCs, with scattered lympho-
cytes within the whole parenchyma. In contrast, in the icefi sh (Chaeno-
cephalus aceratus), a teleost which possesses practically no erythrocytes, the
dominant cells of the spleen parenchyma are lymphocytes and macrophages
(Walvig, 1958). Plasma cells secreting Ig are scattered throughout the white
pulp and isolated spleen lymphocytes stimulated in vitro produce plasma
cells (Bromage et al., 2004).
1.2.4 The gills
The fi sh gill is a multifunctional organ involved in gas exchange, ionoregula-
tion, osmoregulation, acid–base balance, ammonia excretion, hormone pro-
duction, modifi cation of circulating metabolites and immune defence
(Rombough, 2007). In fi lter-feeding species, such as the sardine (Sardina
pilchardus), the gills may also perform a feeding function. Agnathan hag-
fi shes have primitive gill pouches, while lampreys have arch-like gills similar
to the higher fi shes. In lampreys and elasmobranchs, the gill fi laments are
supported by a complete interbranchial septum and water exits via external
branchial slits or pores. In contrast, the teleost interbranchial septum is
much reduced, leaving the ends of the fi laments unattached, and the mul-
tiple gill openings are replaced by the single caudal opening of the opercu-
lum (Wilson and Laurent, 2002). The basic functional unit of the gill is the
fi lament, which supports rows of plate-like lamellae. The lamellae are
(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0) designed for gas exchange with a large surface area and a thin epithelium
surrounding a well-vascularized core of pillar cell capillaries. The lamellae
are positioned for the blood fl ow to be counter-current to the water fl ow
over the gills. The lamellar gas-exchange surface is covered by squamous
pavement cells, while large, mitochondria-rich, ionocytes and mucocytes are
found in greatest frequency in the fi lament epithelium.
Fish pathogens readily spread in the water, and the thin respiratory epi-
thelium of the gills represents an obvious entry for pathogens. For example,
infectious salmon anaemia (ISA) virus infection is believed to be estab-
lished fi rst in the gills before spreading to other organs (Rimstad and
Mjaaland, 2002). The physical barrier of the fi sh gills consists of the gill
epithelium, a glycocalyx layer and a mucus layer. The gill is a major organ
for antibody secreting cell production following direct immersion immuni-
zation (Dos Santos et al., 2001). Lymphocyte accumulations have been
© Woodhead Publishing Limited, 2012