marine or brackish water. It should be noted that many Pathogens and Parasites 227
species of Vibrio cause disease in aquatic organisms, but
the focus here will be primarily on the most common ERM is that multiple varieties of Y. ruckeri strains or
species causing vibriosis and include Vibro anguillarum b iotypes exist and may be linked to disease severity in
(also referred to as Listonella anguillarum), V. ordalii, salmonids. Outbreaks can cause significant impact and
and V. salmonicida (also referred to as Aliivibrio salmo- surviving fish can become asymptomatic carriers.
nicida). Vibrio anguillarum had major economic impacts However, similar to vibriosis, ERM is generally prevent-
on salmonid aquaculture prior to the implementation of able by the use of commercial vaccines. Prior to vaccine
commercial vaccines, which can be highly effective development, it was believed that ERM caused up to 35%
against vibriosis. Mortality in Atlantic cod (G. morhua) loss and an economic impact of approximately USD 2.5
aquaculture has also been reported due to V. anguil- million in Idaho’s Hagerman Valley.
larum, and early reports from Chile indicated outbreaks
with moderate mortality of up to 8% due to V. ordalii. ERM is readily confirmed through culture of Y. ruckeri
Vibrio salmonicida has been shown to accumulate rap- in general media followed by biochemical tests and
idly in the blood and colonise the intestine, which is or other confirmatory serologic methods or PCR. This
thought to result in release and spread in the environ- bacterium is motile and measures 0.5–0.8 × 1.0–3.0 μm.
ment. However, overall losses and impact to commercial Although it impacts rainbow trout and other salmonids
aquaculture can be kept low if vaccination procedures at colder temperatures (generally 15 °C or below) growth
are implemented effectively. is optimal between 22–25 °C. As mentioned, different
strains or biotypes exist. The Type I (Hagerman strain) is
Vibrio spp. can be diagnosed easily as it grows readily usually considered the most virulent. It has been found
on or in standard culture media. Differentiation at the that the biotype of Y. ruckeri can be linked to vaccine
species level is more difficult but biochemical tests and efficacy and understanding this is important when devel-
PCR based assays are available. When fish succumb to oping vaccination programs. Separate biotypes have
Vibrio a range of external and internal clinical signs can been found in Europe and North America, and recent
be observed. Mortality in unvaccinated fish is usually atypical biotypes found in Australia were linked to high
high and infection results in a bacteraemia with fish mortalities in Atlantic salmon (Bridle et al., 2012).
showing signs of lethargy, dark colouration, reduced
feeding response, and possible petechial haemorrhaging Acute infections with Y. ruckeri in young fish result in
near the belly and base of the fins. Anaemia is common heavy losses as it causes a septicaemic infection. More
and multiple organs can be impacted. chronic infections may appear in larger fish where clinical
signs can include dark colouration, blindness, and leth-
As mentioned, vaccination is the preferred method of argy. Ulceration and haemorrhaging in the mouth are
prevention for vibriosis; however, if outbreaks occur it is classic signs and reflect the name ‘red mouth’. This is
important to remove moribund and dead fish quickly observed regularly in rainbow trout but infections in
and diagnose early in case antibiotic treatment is needed. Atlantic salmon may not produce this classic sign. Often,
As more species are reared in marine environments, ERM results in a septicaemic condition where internal
there is concern that new problems involving vibriosis clinical signs may include bloody ascites, enlarged spleens,
will develop and require additional strategies for control as well as intestinal and muscular haemorrhaging.
and prevention.
Some antibiotics have been used to control mortality
11.3.7 Yersinia ruckeri following ERM outbreaks. However, the most effective
means of prevention and control for ERM is through the
Enteric redmouth (ERM) caused by the Gram‐negative use of commercially available vaccines. Development of
bacterium, Yersinia ruckeri is a major cause of disease in commercial immersion ERM vaccines was considered
aquaculture worldwide. ERM was first described in rain- the single most important management tool for the trout
bow trout in the 1950s in the Hagerman Valley of Idaho, industry in Idaho when introduced, and clearly limited
USA and referred to as Hagerman red mouth disease. the potential impact of ERM early on. Although vaccines
This disease may also be referred to as yersiniosis in are effective, it is important to implement good fish cul-
areas outside the USA and primarily impacts rainbow ture practices and minimise environmental stressors that
trout. Movements of fish and eggs are thought to have would contribute to reduced disease resistance in any
contributed to early spread of Y. ruckeri, and it was found aquaculture situation.
in Canada shortly after initial isolation in Idaho. In the
mid‐1980s it was reported in Europe and has since been 11.3.8 Renibacterium salmoninarum
found in Norway, Denmark, UK, France, Germany,
Italy, South Africa, and Australia. One difficulty linked to Renibacterium salmoninarum is a Gram‐positive bacte-
rial pathogen that causes bacterial kidney disease (BKD),
which was first described in Scotland in 1930. This dis-
ease is considered a problem in salmonids but has been
228 Aquaculture Prevention of BKD is best achieved through avoidance
of exposure to R. salmoninarum. For broodstock that may
reported in non‐salmonids such as ayu (Plecoglossus be potential carriers, it is important to utilise the screen-
altivelis) and Pacific hake (Merluccius productus), and it ing and egg culling methods described above. Movement
was found that sablefish (Anoplopoma fimbri) developed of fish or eggs into facilities should require prior inspec-
clinical disease when experimentally challenged with tion of fish or eggs to determine disease status. If R. sal-
R. salmoninarum. Most salmonid producing countries in moninarum is endemic to a region, it is possible that
Europe, Asia, as well as North and South America have outbreaks could occur. This may require antibiotic treat-
reported BKD outbreaks in farmed and/or wild fish. At ment of infected fish; however, few antibiotics have been
this time Ireland, Australia, and New Zealand are consid- successful. Use of erythromycin is common, but overall it
ered BKD free. Renibacterium salmoninarum is trans- has been relatively ineffective in young fish. A vaccine for
mitted both horizontally and vertically. This poses BKD is commercially available and developed for Atlantic
serious problems in aquaculture programs where brood- salmon. This is a live vaccine, but interestingly it consists
stock are highly valuable. To manage the problem of ver- of the bacteria Arthrobacter davidanieli, which is a closely‐
tical transmission, resource enhancement hatcheries in related soil bacteria that provides cross‐protection when
the Pacific north‐west, USA have implemented an effec- fish are injection‐vaccinated.
tive screening program for migrating adult Pacific salmon
collected for hatchery production. Kidney samples from 11.3.9 Streptococcus spp.
each female broodfish are taken at the time of spawning
and infection levels are quantified using a standardised With the expansion of tilapia (Oreochromis spp.) aqua-
enzyme‐linked immunosorbent assay (ELISA). If fish test culture globally, one of the major disease problems has
positive with moderate or high levels of R. salmoninarum involved streptococcal septicaemic infections. Although
the fertilised eggs from that female are culled from the most prevalent in freshwater fish, infections have also
population. This has resulted in substantial reduction in been reported in both wild and farmed marine fish. In
overall incidence of BKD in these hatchery programs. freshwater, the following bacterial species appear to be
the most problematic; Streptococcus agalactiae, S. iniae,
Renibacterium salmoninarum is considered a coryne- S. ictaluri, S. difficile, and S. shiloi.
form bacterium that is a non‐motile short rod measuring
0.3–1.0 × 1.0–1.5 μm. It is an intracellular bacterium and Streptococcal organisms are small Gram‐positive bac-
standard culture methods for identification are problem- teria that may occur in chains consisting of 0.3–0.5 μm
atic as it grows slowly under most conditions, taking cocci. They can be isolated from various organs and cul-
from 8–12 weeks to produce colonies at 15 °C. Improved tured on different nutrient media. They are considered
culture methods can shorten this to as few 5–7 days. non‐motile and those associated with infections in fish
This may improve diagnosis of R. salmoninarum in the have been divided into four primary groups based on
future; however, current confirmation of infections relies specific characteristics.
on serological or molecular methods.
Outbreaks have been reported in many fish species,
Cases and reports of BKD are widespread and in gen- but cumulative mortalities in tilapia have been reported
eral juvenile salmonids are severely impacted by BKD. as high as 50–60%. Clinical infection results in a variety
However, adult fish can develop clinical disease in both of external signs including, but not limited to exophthal-
freshwater and marine environments. Both wild and mia and petechial haemorrhaging near the operculum,
hatchery produced Pacific salmonids and Atlantic anus, mouth, and fins. Internally, the intestinal tract,
salmon can be affected by R. salmoninarum. Disease pyloric caeca, and liver may show petechial haemorrhag-
severity increases when smolts are transferred from ing. Once systemic, bacteria can move to various organs
freshwater to seawater most likely due to the physiologi- including the liver, heart, kidney, stomach, intestine,
cal stresses of the smoltification process. Coho salmon brain, eyes, and musculature. Outbreaks with S. agalac-
smolts infected with R. salmoninarum experienced tiae have been reported to cause mass mortality in tila-
much higher mortality when held in seawater compared pia. Streptococcal infections can also be a problem in
to siblings maintained in freshwater. catfish culture, and rainbow trout have been experimen-
tally infected.
Clinical BKD may include a number of signs including
darkening of the skin, lethargy, exophthalmia and abdom- Prevention and control of streptococcal infections is
inal distention. Spawning fish with heavy infections may difficult; however, it is important to maintain good water
show haemorrhaging at the base of their fins and inter- quality and minimise stress in an aquaculture setting.
nally can exhibit classic white‐grey granulomatous lesions Removal of moribund or dead fish is important and
in the kidney. The bacterium can be isolated from other should an outbreak occur, there have been reports of anti-
organs as well, and in some cases the infection can move biotics such as enteroflaxin and erythromycin‐doxycycine
into the musculature where tissue destruction and necro-
sis may occur.
being effective (Tung et al., 1985). In recent years, work Pathogens and Parasites 229
has been carried out to develop vaccines to protect fish
from major disease impacts. In tilapia, vaccines have been In general, Saprolegnia is opportunistic and can para-
shown to be efficacious when injection delivered, and sitise at temperatures ranging from 2–35 °C. In most
antibody mediated protection appears to be important. A aquaculture operations, S. parasitica is considered the
commercial vaccine consisting of inactivated S. agalactiae most prevalent and concerning. Costs associated with
(Biotype 2) is available for cultured fish but must be deliv- this pathogen are estimated to be in the tens of millions
ered via injection. Caution should be taken when handling of dollars in salmon and catfish culture facilities annually.
fish suffering from streptococcal infections as some Although S. parasitica is more common, S. diclina and
Streptococcus spp. are considered zoonotic and can cause S. ferax are considered more pathogenic than S. parasitica
infections in humans, which typically happens due to to Atlantic salmon eggs during incubation. New aquacul-
injuries to the skin while handling or preparing fish for ture species such as burbot (Lota lota), which require
cooking (see section 11.12). temperatures below 4 °C for egg incubation are highly
susceptible to Saprolegnia (Figure 11.10). It was shown
11.4 Fungi that without administration of chemical treatments,
mortality of both eggs and larvae due to Saprolegnia was
Fungi are considered eukaryotic organisms and are often near 100%.
referred to as water molds or oomycetes. They are micro-
scopic, filamentous, absorptive organisms that function Infection with Saprolegnia in fish will often start at the
as decomposers in ecological systems. They reproduce site of a previous injury or even a lesion resulting from
both sexually and asexually and can produce toxins that other infections. Saprolegnia will manifest and appear as
may be harmful to animals. These water molds may be cotton‐like tufts on the external surface of the lesion of a
referred to as pseudo‐fungal organisms and cause dis- fish or will rapidly spread once an infection is established
eases such as saprolegniasis and branchiomycosis, which on the surface of eggs. If fish are immunocompromised
are discussed in this section. in any way as a result of predisposing stress or other fac-
11.4.1 Saprolegniasis tors, infection with Saprolegnia may be enhanced (Cain
Saprolegnia disease (saprolegniasis) can develop and and Polinski, 2014).
impact cultured and wild fish or their eggs/embryos. In
general, Saprolegnia is considered an opportunistic Saprolegnia are considered ubiquitous in most fresh-
pathogen that feeds on necrotic tissue or organic debris. water environments and therefore fish or eggs will likely
When conditions are optimum Saprolegnia can cause be exposed in an aquaculture facility. Infections can be
infection in just about any fish species at any life stage. limited in fish by minimizing stress and maintaining opti-
However, infections are most prevalent in aquaculture mum culture conditions. Handling can cause physical
facilities during egg incubation or early larval rearing or injury, which predisposes fish to fungal type infection at
may become a problem in pre or post spawning salmonid these sites. For incubating eggs or newly hatched larvae,
broodstock.
Figure 11.10 Characteristic hyphae shown attached to incubating
Saprolegnia are classified as fungal‐like organisms and burbot eggs. Eggs clearly infected with Saprolegnia appear cloudy
with filamentous hyphae, sporulation, and a classic ‘cot- and are dead, while visibly uninfected eggs appear healthy with
ton tuft’ like appearance. However, some differences sep- developing embryos inside. Source: Reproduced with permission
arate them from true fungi and place them more closely from Dr M. Polinski.
taxonomically to heterokonts, which include organisms
such as diatoms and brown algae. Presumptive diagnosis
may rely on visualization of cotton‐like tufts and identifi-
cation of non‐septate filamentous hyphae. Confirmation
to species level is difficult by microscopy, but genetic
sequencing based on PCR identification is becoming
more common. In most cases Saprolegnia are rarely diag-
nosed to the species level since treatment procedures are
identical for all. Nevertheless, common species that
are known to infect fish or fish eggs include S. parasitica,
S. diclina, and S. ferax (see Cain and Polinski, 2014).
230 Aquaculture 11.5 Protozoans
Saprolegnia may develop if nutrients are present; there- Protozoans are among the most significant parasite
fore, it is important to clean all organic material including problems in bivalve and finfish aquaculture industries
dead eggs or egg shells as often as possible. Standard pro- where open water sources are utilised. They comprise a
cedures in most facilities utilise routine treatments of diverse group of unicellular eukaryotic organisms, many
eggs (and possibly larvae) with approved chemicals such of which are motile. Protozoans range from 10 to 52
as hydrogen peroxide, formalin, or in some cases sodium micrometers, but can grow as large as 1 mm, and can be
chloride. For fish, such as adult salmon, that may be heav- observed using a microscope. The protozoan cell has one
ily infected at sites where skin abrasions and damage or more nuclei, a set of cellular organelles and special
occur (common if utilizing wild broodstock returning to organelles serving vital functions such as locomotion,
spawning grounds), it is important to administer treat- food intake or invasion of the host organism. Some para-
ments as soon as possible. Malachite green was an early sitic protozoa have life stages alternating between prolif-
historic treatment and was quite effective; however, it is erative stages and dormant cysts. As cysts, protozoa can
no longer allowed as it was found to have carcinogenic survive harsh conditions, including exposure to extreme
and toxicological effects. In the US, formalin and hydro- temperatures or harmful chemicals, or long periods
gen peroxide are the most common chemicals used to without access to nutrients, water, or oxygen. At present
treat saprolegniasis. there are no commercial vaccines available for any fish‐
parasitic protozoa. This section provides an overview of
11.4.2 Branchiomycosis the most common groups that occur in aquatic animals
including flagellates, amoebae, haplosporidians, apicom-
Branchiomyces sanguinis and B. demigrans infections plexans, microsporans and ciliates (Figure 11.11).
cause a condition known as ‘gill rot’. Branchiomycosis is
known to impact cultured fish and has been linked to 11.5.1 Mastigophora (Flagellates)
severe losses in tilapia in Israel and in farmed catfish.
This condition can be linked to poor environmental con- The subphylum Mastigophora includes the dinoflagel-
ditions that affect gill health and provide an environment lates, blood parasitic trypanosomatids and ectoparasitic
that favours colonisation of Branchiomyces on the gills. bodonids (Figure 11.11a). This group is characterised by
This condition occurs at temperatures between 25–32 °C elongate flagella (singular: flagellum) which undulate to
and may manifest within 2–4 days following exposure if propel the cell through liquid environments. Flagella are
predisposing stressors are present. ‘whip‐like’ extensions of the cell membrane with an inner
core of microtubules. Most parasitic flagellates have a
To diagnose and distinguish between B. sanguinis and simple, one‐host life cycle and reproduce by longitudinal
B. demigrans, gill preparations should be examined binary fission.
under light microscopy. B. sanguinis infects the lamellar
capillaries and is characterised by a (0.2 μm) thin hyphal The dinoflagellate Amyloodinium ocellatum is a dan-
wall and spores of 5–9 μm in diameter, while B. demi- gerous agent of marine aquaculture fishes, causing fatal
grans is found in the parenchyma of the gills and is epidemics worldwide. Outbreaks can occur rapidly and
thicker with walls of 0.5–0.7 μm and spores measuring result in 100% mortality within a few days. Amyloodinium
12–17 μm. Gill rot may result in high mortalities and has a direct but tri‐phasic life cycle. The parasites feed as
clinical signs of disease include those typical of most gill stationary trophonts on the epithelial surfaces of the skin
problems. These include lethargy and gulping of air and gills. Trophonts attach to fish with an attachment
at the surface of the water. Examination of fish may disc that has filiform projections (rhizoids) that embed
show striated gills with pale areas of necrosis due to deep into the epithelial tissue of the host. After reaching
infection. maturity the trophont detaches from the fish skin and
forms a reproductive cyst, the tomont, in the substrate.
Unlike Saprolegnia it does not appear that Branchiomyces The tomont divides forming multiple free‐swimming
are ubiquitous in the environment and movement of individuals (dinospores) that can infect a new host.
infected fish should be prevented. Prevention of disease is Under optimal conditions that parasite can complete its
directly linked to good management practices that main- life cycle in less than one week. The severe disturbance of
tain high water quality and minimise other stressors in an the parasite on the epithelia can lead to infected fish rub-
aquaculture setting. Infected fish are considered carriers bing their body against objects or hard surfaces and
of these pathogens and should be separated from non‐ osmoregulatory problems. Infections can result in
infected fish. Treatment with formalin or copper sulphate hyperventilation, anorexia and mass mortality. The free‐
may be effective at controlling mortality due to branchio- swimming dinospore is suceptible to certain drugs, but
mycosis, but to limit the spread of these pathogens all
dead fish should be disposed of appropriately and ponds
dried and disinfected.
Pathogens and Parasites 231
(a) (b) (c)
(d) (e) (f)
Figure 11.11 Schematic diagram of representative protozoa that may be present in invertebrate and finfish aquaculture. (a) flagellates (e.g.,
Icthyobodo); (b) amoebae (e.g. Paramoeba); (c) haplosporidians (e.g., Bonamia); (d) apicomplexans (e.g., Perkinsus); (e) microsporidia (e.g., Thelohania);
and (f) ciliates (e.g., Cryptocaryon). Source: Reproduced with permission from Dr Kate Hutson, graphics by Eden Cartwright, Bud Design.
trophonts and tomonts are relatively resistant, making they bite fish, although some species may be transmitted
eradication challenging. Infection can be diagnosed by directly between hosts. Blood smears on stained slides
observing parasites through light microscopy and molec- show the presence of these parasites using light mic-
ular diagnostic tools have been developed. ropsy. Anaemia may result due to haemolysins released
from the parasites; lipids and proteins that cause lysis of
Ichthyobodosis is an important parasitic disease that red blood cells by destroying their cell membrane. Young
has caused severe loss among ornamental and farmed fish fish tend to be more vulnerable and mortality can result
worldwide. Infections by members of the genus from an infection.
Ichthyobodo (Figure 11.11a) have been reported from
more than 60 different host species in freshwater and sea- To avoid infection by parasitic flagellates there should
water. The disease is caused by heavy infections on the be high filtration and treatment of receiving water.
skin and gills. In the past, infections have commonly been Leeches and other blood‐sucking parasites such as
associated with a single variable species, Ichthyobodo gnathiid isopods can serve as vectors of flagellates that
necator. However, molecular studies have revealed that infect the blood system and should be eradicated to
the genus Ichthyobodo consists of several different species. reduce transmission. External flagellates may be treated
Two life stages are known, a kidney‐shaped free‐swim- with formalin, hydrogen peroxide or hyposalinity (for
ming stage and a sessile pyriform state which penetrates marine species) and some anaesthetics can cause flagel-
the epithelial cell lining. Infected fish may show discolora- lates to detach. High or low temperatures or salinity may
tion of the skin and hyperventilate. Osmoregulation of fish inhibit multiplication of flagellates but may not be feasi-
hosts may be impaired due to destruction and fusion ble for the host organism.
of gill lamellae and can cause considerably morbidity
and possibly mortality. Both life stages are susceptible to 11.5.2 Sarcodina (Amoebae)
formalin and oxidising agents. Most species of amoebae are free‐living although a small
number are parasitic in animals. In aquaculture amoebae
Trypanosomes are parasites of invertebrates and fishes can be problematic for crustaceans, echinoderms and
worldwide in freshwater and marine environments. They fish. Amoebae exhibit locomotion by the formation of
are normally found in the blood system, on the gills or in pseudopodia (false feet) or by distinct protoplasmic flow.
the digestive system. These parasites have a complex life Movement is also used by many species to engulf and
cycle, with several development stages within the intesti- ingest food items by phagocytosis. Amoebae are extremely
nal caecae of an intermediate leech host. Leeches may
transfer blood trypanosomes (e.g., Cryptobia spp.) when
232 Aquaculture Bonamiosis is a lethal infection of the haemocytes of
flat oysters caused by Haplosporidia in the genus
robust and survive under a wide range of challenging Bonamia (see section 10.5.1.1). This intrahaemocytic
environments. They reproduce by binary fission or multi- protozoan quickly becomes systemic with overwhelm-
ple fusion. ing numbers of parasites coinciding with the death of
the oysters. Infection in oysters rarely results in clinical
Paramobae perurans (the causative agent of amoebic signs of disease and often the only indication of the
gill disease) has become an issue for Atlantic salmon infection is increased mortality. In some cases, the dis-
farming worldwide and affects a range of farmed marine ease is accompanied by yellow discoloration and lesions
fish species. Presently amoebic gill disease is a major on the gills and mantle, but in most cases infected oys-
issue for salmonid aquaculture in Australia, Ireland, ters appear normal. Lesions can be detected by histol-
Scotland, Norway and the USA with 10% to 82% mortal- ogy and may occur in the connective tissue of the gills,
ity (see section 17.8). The severity of the disease is largely mantle, and digestive gland. Although the life cycle is
influenced by high salinity and high temperature. Clinical unknown, it has been possible to transmit the disease
signs include respiratory distress and lethargy, which are experimentally in the laboratory by cohabitation or
associated with grossly visible gill lesions. The case defi- inoculation of purified parasites. Bonamia ostreae has
nition of amoebic gill disease is based on histology, the been associated with considerable devastation of oys-
presence of hyperplastic lesions and associated amoebae. ter populations. It was first observed in France in 1979
The disease causes characteristic changes in the gill tis- and caused substantial destruction of Ostrea edulis
sue, including severe hyperplasia of lamellar epithelium populations before spreading through much of Atlantic
and an inflammatory response which leads to mortalities coastal Europe.
if left untreated.
Similarly, outbreaks of Haplosporidium nelsoni along
The most effective treatment for P. perurans is a fresh the mid‐Atlantic coast of the USA have devastated oyster
water bath for two hours which alleviates pathological populations and caused significant economic disruption
signs of infection. Gills are usually scored based on mac- of coastal communities. Oyster mortality associated with
roscopic examination to indicate the necessity for bath outbreaks can exceed 90%, producing significant finan-
treatment, but there are limitations with this method cial losses for oyster industries. Haplosporidium nelsoni
because it is based on subjective interpretation and expe- is believed to have been introduced to the USA from
rience. Baths are stressful to fish and impact production, Asia. It infects the Pacific oyster (Crassostrea gigas) in
because fish need to be starved prior to bathing. There is Asia, Europe and the west coast of the USA and the east-
evidence that a percentage of amoebae can survive and ern oyster (C. virginica) along the east coast of North
recover from a fresh water bath as they are able to form America and Canada. Mortalities are usually highest in
vacuoles to separate and then expel influxes of freshwa- the summer months, and also increase in higher salinity
ter (Lima et al. 2015). Excluding caged salmon from waters. The disease reduces the feeding rates of infected
upper cage depths where free‐living P. perurans tend to oysters and reduction in stored carbohydrates inhibits
be more abundant could be an effective management normal gametogenesis in the spring, with a reduction in
strategy to reduce the speed at which initial infections fecundity.
occur. Use of cleaner fish as a biocontrol may be limited
as many species are susceptible to amoebic gill disease. 11.5.4 Apicomplexa (Sporozoans)
11.5.3 Haplosporidia The phylum Apicomplexa is a large group of protozoan
parasites with over 8000 species described as parasites of
The protist phylum Haplosporidia comprises over 40 invertebrate and vertebrate hosts. Apicomplexans have a
described species with representatives infecting a range special cell organelle, the apical complex, which facili-
of marine mollusc hosts with some found in freshwater. tates invasion of the host cell (Figure 11.11d). They
They produce spores without the complex structures undergo cyclic development involving three divisional
found in similar groups (such as polar filaments or processes: merogony, gamogony and sporogony. Cell
tubules), but the spore stage has ornamentation consist- division can occur by fission or endogeny. Fish apicompl-
ing of tails or wrappings (Figure 11.11c). Haplosporid exans are divided into two major groups; coccidia are
spores have a single nucleus and an opening at one end, primarily intestinal parasites, while haematozoa are
covered with an internal diaphragm or a distinctive blood parasites which have stages in fish with spore for-
hinged lid. After emerging, it develops within the cells of mation in leeches or gnathiid isopods. Species are usu-
its host, usually a marine mollusc or annelid. They ally differentiated based on the morphology of the spore,
develop within the digestive system and undergo internal also termed the oocyst.
budding to produce multicellular spores. The dynamics
of haplosporidians in their hosts is seasonal and depends
on environmental parameters.
Several species of the genus Perkinsus are responsible Pathogens and Parasites 233
for causing disease in molluscs including oysters, mus-
sels, clams and abalone worldwide. Perkinsus olseni is the rather than translucent and clear. Alternatively, infec-
only species known to cause disease in the Asia–Pacific tions can be diagnosed by microscopic detection of cysts
region and occurs in abalone, clams and pearl oysters. It and spores in squash preparations or histology of the
was first described from the abalone, Haliotis rubra, in muscle. The most successful prevention of infection is to
southern Australia. Perkinsus olseni is included on OIE’s drain culture ponds, lime them and dry them before
list of notifiable diseases because infection can cause restocking. There are no drugs currently available to
widespread mass mortality. Clusters of Perkinsus cells treat infestations.
near the surface of the abalone appear as a white nodule
or micro‐abscess in the foot and muscle. This develops 11.5.6 Phylum Ciliophora
to form a brown spherical pustule up to 8 mm or more in
diameter. Transmission is direct from host to host and all Ciliated protozoa are among the most common external
life stages are infective, with parasite cells released from parasites of fish but can also infect invertebrates. Most
the host following host death and decomposition. ciliates have a simple life cycle and divide by binary fis-
Prevalence is highly variable depending on host and sion. Ciliates can be motile, attached, or found within the
environmental conditions, but it is often 100%, as deter- epithelium. Ciliates living in or on fishes range from
mined by histology or PCR. Infections can impede respi- harmless ecto‐commensals to dangerous parasites in fish
ration and other physiological processes including aquaculture. With few exceptions, ciliates possess cilia at
growth and reproduction, and stress from high tempera- some stage of their life cycle. Asexual reproduction
tures is believed to exacerbate the disease. Adaptive occurs by transverse binary fission. About 150 species
immunity is not known in oysters or other molluscs, so occur in fish, most as ectoparasites causing fouling, irri-
they cannot be immunised against Perkinsus species. tation and local lesions and sometimes penetrating
Selective breeding of disease‐resistant strains may confer wounds. Some are endoparasites and can damage of the
some protection against infection. tissues or organs. Ciliates can irritate the surface cells,
penetrate into deep tissue layers and ingest the cell debris
11.5.5 Microsporidia (Microsporans) produced.
Microsporidia or microsporans are obligate intracellular Some of the most important ciliates in captive freshwa-
parasites which lack mitochondria and form small uni- ter fishes include Ichthyophthirius multifiliis, Chilodonella
cellular spores. Microsporans proliferate in host tissues spp., the trichonids (including Trichodina, Trichodinella,
by merogony (asexual division) followed by sporoblas- Tripartiella, and Vauchomia spp.) and Tetrahymena spe-
togenesis and sporogony (spore formation). Mature cies. In fish confined to ponds, tanks or aquaria, ciliates
spores contain a unique coiled polar tube which everts that live freely in the water column, such as Trichodina
forcibly to inject the infective sporoplasm into host cells spp. and Chilodonella spp., can form dense populations
(Figure 11.11e). Infections may be disseminated through- on fish resulting in morbidity and mortality. Chilodonella
out the tissues or they may cause focal lesions, inflam- spp. have a voracious appetite for living cells and use a
mation and granulomas. specialised mouth organ, the cytostome, to graze on bac-
teria, diatoms, filamentous green algae and cyanobacte-
Microsporan species in the genus Thelohania (family ria present on biofilm substrates of fish gills and skin.
Thelohaniidae) cause ‘cotton‐tail’ disease in aquatic
crustaceans. Infections have been detected in most In marine environments, Cryptocaryon irritans and
freshwater crayfish species, including wild and cultured scuticociliates are considered among the most problem-
marron (Cherax tenuimanus), yabbies (Cherax destruc- atic ciliate parasites. Cryptocaryon irritans cause marine
tor) and redclaw (Cherax quadricarinatus). Heavily white spot disease in tropical and subtropical marine tel-
infected muscles become white in appearance and are eost fish (Fig 11.11f ). It commonly occurs in public
unmarketable. Mildly infected crustaceans may be aquaria and food fish farming and can rapidly proliferate
stunted in growth, while heavy infections may be fatal. and severely impair the physiological functions of its
Transmission is assumed to be direct, via water‐borne host’s skin, eyes and gills. Cryptocaryon irritans has a
transport of infective spores and/or ingestion. In infected quadriphasic life cycle including four developmental
individuals, mature spores may penetrate adjacent cells, stages; the theront, trophont, protomont and tomont.
injecting infective sporoplasm which subsequently The trophont penetrates the host epidermis and obtains
divides and ultimately forms new spores. Heavy infec- nutrition by feeding on sloughed cells. Subsequently, the
tions can be detected macroscopically by visual exami- parasite leaves a small wound and visible white spot or
nation of crayfish tails which are opaque in appearance, nodule where each parasite encysts. It becomes a free‐
swimming protomont when it exits the host and moves
along the substrate for a few hours. The protomont
adheres to the hard surface, sheds its cilia and encysts
234 Aquaculture one year. When ingested by an oligochaete (in freshwa-
ter) or a polychaete (in the sea), they invade the worm’s
forming the reproductive stage tomont, which divides intestinal tissues where they proliferate. Infected anne-
over 3–28 days. After several divisions, the tomont rup- lids release tri‐radiate actinospores that float in the water.
tures, releasing about 300 free‐swimming theronts which Actinospores penetrate the surface of the fish on contact
must find a suitable host within 48 h or they will die. and migrate to the site where the sporogonic plasmo-
dium develops. Infections by myxozoans may be asymp-
Scuticociliates are aggressive and invasive minute cili- tomatic, but some may cause tissue hyperplasia, unsightly
ates that are known to cause disease in marine fishes, cysts, erosive and necrotic lesions, enzymatic lysis of fish
including sea horses, flounders, turbots, bass, tunas, and tissue (i.e., myoliquefaction), deformities and even death.
crustaceans. In southern bluefin tuna (Thunnus mac- The most notorious species are known from the genera
coyii) sea cage culture in Australia, fish infected with Myxobolus, Sphaerospora, Ceratomyxa, Hexacapsula,
scuticociliates exhibited atypical swimming behaviour Kudoa, Unicapsula, Henneguya, Enteromyxum and
followed by rapid death in winter. It is hypothesised that Tetracapsuloides. Some fish‐borne myxozoan species
the parasites initially colonise olfactory rosettes and then (i.e., Kudoa spp.) can cause food poisoning in humans
ascend to the olfactory nerves to eventually invade the (see section 11.12).
brain where they cause encephalitis. Species of the
scuticociliate Mesanophrys live as scavengers on the exo- ‘Whirling disease’ is an ecologically and economically
skeletons of crabs and lobsters, but if they find a break in debilitating disease caused by Myxobolus cerebralis in
the exoskeleton they invade the haemocoel, multiply and commercially reared salmonids. Whirling disease afflicts
kill the host. juvenile fish (fingerlings and fry) and causes skeletal
deformation and neurological damage. Fish ‘whirl’ for-
11.6 Myxozoans ward in an awkward, corkscrew‐like pattern instead of
swimming normally, find feeding difficult, and are more
Myxozoans are obligate parasites that use invertebrate vulnerable to predators. The parasite has a complex life
and vertebrate hosts as part of their life cycle and can cycle, alternating between salmonid fish and the oli-
occur in marine and freshwater environments. They are gochaete host, Tubifex tubifex. The parasite infects its
found principally in the muscle, brain and gall bladder of hosts during its free‐swimming triactinomyxon stage
fish. Myxozoa form complex valved spores with polar when appendages on the parasite pierce the skin of salmo-
capsules containing extrudible filaments which are used nids and sporoplam is injected into the host, where it
for attachment to host cells (Fig. 11.12a, b). Their devel- migrates through nervous tissue to areas of cartilage
opment involves multicellular differentiation, which does around the brain. There it matures into an M. cerebralis
not conform to the unicellular definition of the protozo- spore containing the classic polar filaments found in the
ans, and recent molecular studies confirm that myxozo- myxozoans (Figure 11.12a). Mortality rates may be up to
ans are cnidarians (Chang et al., 2015). Their transmission 90% in infected populations, and those that do survive can
requires a developmental phase in an invertebrate host, develop deformities due to spores residing in their carti-
although rare cases of direct fish to fish transmission lage. Dead fish act as a reservoir for the parasite, which
have been reported. Myxospores released from the fish may be released into water and ingested by susceptible
can survive in the aquatic environment for more than
(a) (b)
Figure 11.12 Schematic diagrams of representative myxozoa that may be present in finfish aquaculture. (a) Myxobolus sp.;
(b) Kudoa sp. Source: Reproduced with permission from Dr Kate Hutson, graphics by Eden Cartwright, Bud Design.
T. tubifex, thus removing mortalities promptly is impor- Pathogens and Parasites 235
tant for managing fish health.
human consumption of fish flesh infected with Kudoa
Proliferative kidney disease (PKD) also causes signifi- can cause food poisoning (see section 11.12). Infection
cant losses among salmonid (Pacific salmon and rainbow prevalence can be monitored within affected farms by
trout) populations in Europe and western North PCR or examining fillets for manifestation of soft‐flesh,
America. The parasite that causes PKD, Tetracapsuloides but there are no external clinical signs to identify and cull
bryosalmonae, was named in reference to its two known fish infected with Kudoa in the skeletal muscle. Kudoa
hosts – bryozoans and salmonids. Various freshwater yasunagai infects the brain of various important marine
bryozoans are susceptible to infection and release spores aquaculture fishes including Japanese sea bass
into the surrounding water where they can infect fish. (Lateolabrax japonicas), sea bream (Pagrus major), olive
Massive numbers of spores can be produced from rela- flounder (Paralichthys olivaceus), tiger puffer (Takifugu
tively small volumes of bryozoans. The disease is often rubripes), yellowtail (Seriola spp.), and Pacific bluefin
seasonally dependent occurring at water temperatures tuna (Thunnus orientalis). Clinical signs of infection
above 15 °C. Parasites are initially prominent in the blood include abnormal swimming behaviour and skeletal cur-
sinuses of the kidney and provoke a strong inflammatory vature. In Japan, mortalities of juvenile kingfish Seriola
response. Mortality in uncomplicated cases of PKD is lalandi in net cages occurred approximately two months
generally 20% or less but often secondary pathogens or after being transferred from hatchery facilities, with more
unfavourable environmental conditions coincide with than 50% of fish developing scoliosis and abnormal swim-
peak periods of PKD and mortalities can reach 95–100%. ming behaviour including whirling or lying at the bottom
In fish that have experienced a full clinical episode of the of cages. Symptomatic fish had difficulty feeding and suf-
disease, a strong acquired immunity develops. No vac- fered from damaged skin from swimming into the cage
cines have been developed to control PKD. net. To date, there is no effective control method for
Kudoa spp. in aquaculture. Managing the production
Kudoa spp. can occur in the muscle, kidney, ovary schedule to avoid seasonal abundance of infective para-
and brain of a variety of marine aquaculture fishes sites may be possible, but further information is needed
(Figure 11.12b). Infections of the skeletal muscle are the on the life cycle (i.e., identity of the invertebrate host),
most common. In British Columbia, Canada, annual seasonality and infection dynamics.
costs to the Atlantic salmon aquaculture industry due to
K. thyrsites infections in the muscle can reach millions of 11.7 Platyhelminths
dollars. Infection may be characterised by visible cysts,
which can make fillets unsightly, unappetising, and there- Although some turbellarians have adopted a parasitic life
fore unmarketable (Figure 11.13), or alternatively they style, there are three major classes of platyhelminths that
may be microscopic intracellular pseudocysts. Parasite‐ are entirely parasitic. Two of these, the tapeworms
derived enzymes degrade the flesh post‐mortem, making (Cestoda) and the flukes, (Digenea), are endoparasites
fillets soft and watery. Recent evidence suggests that and the third group, the Monogenea, comprise a range of
skin and gill parasites of fishes.
Figure 11.13 A wild captured tuna, with obvious white cysts in 11.7.1 Turbellarians
the muscle, in this case evidence of infection with the parasite Turbellarian worms are predominantly free‐living mem-
Kudoa. Source: Reproduced with permission from Dr R. D. Adlard. bers of the Platyhelminthes but include several parasitic
or predatory species, some of which are relevant to aqua-
culture. Most turbellarians differ from all other platyhel-
minths in that they lack an oral sucker, move in a gliding
fashion using multi‐ciliated epithelial cells, and having a
direct life cycle. Most forms reproduce sexually, and, with
a few exceptions, all are simultaneous hermaphrodites.
Paravortex spp. are viviparous turbellarians known
from molluscs and fish and can cause diseases character-
ised by host death, acute focal dermatitis, and secondary
infections of Vibrio. Infections of Paravortex have been
observed in hatchery‐reared stonefish, Inimicus japoni-
cus, fry and cage‐cultured sea perch, Lateolabrax spp.,
236 Aquaculture crayfish, they do not pose any considerable health risk to
the animal, however, large numbers of adhesive eggs laid
greater amberjack, Seriola dumerili and red sea bream on the surface of crustaceans may lead to rejection or poor
Pagrus major in Japan and commercial clams, Ruditapes prices at market.
decussatus.
11.7.2 Cestodes
Polyclads are carnivorous micropredators that pose a Numerous cestode species can cause disease in fish,
considerable threat to cultured invertebrates including while fish‐borne cestodes in Diphyllobothrium can cause
sponges, corals, mussels, edible and pearl oysters, scal- disease in humans (see section 11.12). Cestodes, com-
lops and giant clams. Efforts to culture acroporid corals monly called tapeworms, are hermaphroditic endopara-
in aquaria can be severely compromised by polyclad flat- sitic flatworms characterised by the presence of the
worms which, if not removed, can eat entire colonies scolex (the head) which attaches the worm to the host. A
(Figure 11.14a). Their occurrence in culture facilities string of segments (proglotids) with sex organs is found
warrants careful scrutiny because live flatworms are beneath the scolex. They feed by absorbing nutrients
camouflaged against live tissue. The Acropora‐eating over their tegument because they have no mouth or
flatworm, Amakusaplana acroporae, exhibits relatively intestine. Cestodes are oviparous and have complex life
high fecundity and lays multiple batches of capsules on cycles (i.e., they require more than one host to complete
the coral skeleton (Figure 11.14b), with each capsule their life cycle). The adult worms produce eggs which are
containing up to seven embryos. The polyclad flatworm delivered to the environment with the faeces of the host.
Stylochus matatasi can reach nearly 6 cm and have been An intermediate host (usually a crustacean) ingests the
associated with the death of giant clams, Tridacna gigas. egg or free‐swimming larvae (coracidica) and becomes
Although they can sometimes be observed on the mantle infected. Some tapeworms exhibit a two‐host life cycle,
of clams, they can also damage the inside of the mantle while others may have three hosts, where the second
cavity and may not necessarily be detected until after a intermediate host is typically a fish.
clam has died.
The Asian tapeworm, Schyzocotyle acheilognathi is
Temnocephalans are generally considered as ectosym- an important pathogenic cestode of aquaculture in Asian
bionts rather than parasites. They are relatively small cyprinid fish. The parasite is indigenous to East Asia, but
(0.5–1 mm) flatworms and lay their eggs in gills and on the
shell of freshwater crustaceans. Adult worms move over
the surface of the crayfish, eating algae and other micro-
fauna. Although they are very common on freshwater
(a) (b)
Figure 11.14 (a) The Acropora‐eating flatworm, Amakusaplana acroporae, can eat entire coral colonies in culture. (b) Worms lay multiple
batches of small brown coloured capsules on the coral skeleton, with each capsule containing up to seven embryos. Source: Reproduced
with permission from Kat Dybala, Kate Rawlinson and Jonathan Barton.
has spread rapidly throughout the world through the Pathogens and Parasites 237
ornamental fish trade. It exhibits a simple two‐host life
cycle involving common copepod species as an interme- or all of their larval development in molluscs with the
diate host. Once established, it may endanger native fish exception of marine blood flukes (aporocotylids) which
populations. The parasite attaches to the gut wall using complete their larval development in terebellid poly-
its scolex where it can cause severe damage to the intes- chaete annelids. In some cases, trematode sporocysts
tinal tract, including pressure necrosis and haemorrhage. occupy the gonad and cause partial or full castration of
Heavily infested fish exhibit reduced growth, loss of con- the intermediate host. Cercariae emerge and may have a
dition and mortality. Indeed, 100% mortality can occur tail or other locomotory device to assist in infection of
in infected hatchery‐reared carp. Triaenophorus spp. the second intermediate host where they encyst as meta-
include other pathogenic cestode species in Eurasia, cercariae. Trematode life cycles can be broken in aqua-
causing epidemics and mortality in juvenile rainbow culture where it is feasible to exclude intermediate
trout in hatcheries and rearing ponds. This parasite molluscan hosts or polychaetes.
causes unsightly infestation in the muscles of the fish and
can impact marketability. Aporocotylid trematodes or ‘blood flukes’, cause lethal
infections in cultured salmonids and cyprinids in fresh-
Most aquaculture facilities use local natural water water, and scombrids, carangids, sparids and tetradon-
bodies as their source of water, and these can be naturally tids in marine environments. Detection of adult flukes
infected with tapeworms. To reduce infestation by ces- can be made in the blood vessels, while thin shelled eggs
todes, the water should be filtered (e.g., screen or sand can be detected in the gills using microscopy or histol-
filters) or alternatively ground water could be used if ogy. Adult parasites release eggs into the fish’s vascular
available. The use of low‐value fish in the diet can poten- system which may be sequestered in the gill, heart, kid-
tially introduce second intermediate contaminated fish, ney, liver, spleen, pancreas, or other organs, where they
so alternative feeds such as an extruded pellet diet can be cause inflammation and decrease the physiological and
used. An anthelmintic, praziquantel, originally synthe- mechanical efficiency of these organs. Freshwater and
sised to treat endoparasitic flatworm infections of mam- marine blood flukes use either gastropods or terebellid
mals may have an effect on fish cestodes but will not kill polychaetes as the intermediate host, respectively
necessarily kill the eggs or free‐swimming larvae. (Figure 11.15). Blood flukes are unusual because their life
cycle lacks a second intermediate host and an encysted
11.7.3 Trematodes Figure 11.15 Schematic diagram showing the life cycle of Cardicola
Trematodes exhibit complex life cycles and the adult forsteri infecting farmed southern bluefin tuna, Thunnus maccoyii.
stage is parasitic in vertebrates. Few adult trematode Adult blood flukes in the circulatory system lay eggs which lodge in
species are known to cause considerable harm to their the gills. Eggs embryonate and hatch to release micracidia that seek
definitive fish host with the most notable exceptions the second intermediate host (terebellid polychaete). Asexual
being mostly extra‐intestinal parasites such as the aporo- reproduction takes place in terebellid polychaetes, where infective
cotylid blood flukes and the cyst‐forming didymozoids. cercariae develop in sporocysts. Source: Reproduced with permission
Metacercariae (encysted larvae) infection can cause permission Dr. Kate Hutson, graphics by Eden Cartwright, Bud Design.
mortalities in farmed fishes with subsequent economic
loss (e.g., Bolbophorus damnificus; see section 19.3.4)
and ocular diplostomiasis (infection of the eye by meta-
cercariae of Diplostomum spp.) can cause blindness in
farmed salmonids and catfish. Some metacercariae in
fisheries and aquaculture products (fish and shellfish)
are a source of infections in humans and domestic ani-
mals (see section 11.12).
Trematodes are hermaphrodites and most adults are
dorso‐ventrally flattened with a sucker around the mouth
and an additional ventral sucker. Gastrointestinal trema-
todes release eggs with the host faeces that hatch into
free‐swimming, sometimes ciliated, miracidia. The
miracidia invade a suitable mollusc host where asexual
reproduction takes place. Specificity of individual trema-
tode species to the molluscan host appears to be quite
restricted, usually to a species. Trematodes undergo part
238 Aquaculture plexity of the haptor: the Monopisthocotylea have one
main part to the haptor, often with hooks or a large attach-
metacercaria. Thus, the life cycle depends on the prox- ment disc, whereas the Polyopisthocotylea have multiple
imity of definitive and intermediate hosts to the free‐ parts to the haptor, typically clamps. Monopisthocotyleans
swimming infective stages of the parasite. Freshwater tend to live on the gills, skin and fins where they feed on
snails easily establish in culture systems and in pond epithelial tissue and include species that cause harmful
farms, while the polychaetes may proliferate on support- infections in aquaculture (e.g., species in Gyrodactylus,
ing materials on sea cages (i.e., in the biofouling) or in Pseudodactylogyrus, Dactylogyrus, Neobenedenia and
sediment. Manipulating the proximity of the fish host to Benedenia). Polyopisthocotyleans are almost exclusively
its fluke’s intermediate host is desirable, and several gill‐dwelling blood feeders and heavily infected fish suffer
marine blood fluke life cycles for tuna have been resolved from anaemia (e.g., infections from species in Discocotyle,
recently which will assist to develop appropriate man- Heterobothrium and Zeuxapta).
agement methods (e.g., Cribb et al., 2011; Fig. 11.15).
Monogeneans have a direct life cycle (they require only
Infection of farmed fish by trematodes can be man- a host to complete their life cycle) and most are ovipa-
aged by minimizing interactions between farmed stock rous – they produce eggs that are released and hatch in
and wildlife (namely molluscs or polychaete worms). For the aquatic environment (Figure 11.16). Monogeneans
example, metacercarial infections can be prevented are hermaphrodites and are capable of reproducing in
when fish‐eating birds are excluded using nets and/or isolation. Eggs laid by parasites can bear long filamen-
water filtration eliminates snail populations. Fallowing tous strings that catch on aquaculture infrastructure
or farming sea caged fish in deeper water likely reduces (nets, filters and the substrate) so that the infective lar-
interactions with intermediate hosts that live on or in vae, or oncomiracidia, hatch close to fish. The egg casing
sediment. Oral administration of praziquantel is effec- is physically strong, but a detachable lid or operculum
tive against blood fluke infections in tuna, although does permits escape of the oncomiracidium. Most species
not seem to affect eggs. have oncomiracidia that are equipped with rows of cilia,
which enable them to swim and directly attach to a new
11.7.4 Monogeneans host. Once attached, they typically migrate over the body
Monogeneans are ubiquitous in finfish aquaculture. They to the final site of attachment. At high water tempera-
infect captive ornamental and food fishes in fresh or tures parasite generation time is reduced, rapidly causing
marine environments and can also infect elasmobranchs numbers to increase and if unmanaged, infections may
and aquatic turtles. Commonly termed ‘flukes’, they are kill captive fish.
primarily ectoparasites and have a characteristic posterior
attachment organ called a haptor. The Monogenea can be Outbreaks of Neobenedenia species have caused
generally divided into two subclasses based on the com- particular devastation in marine fish aquaculture.
Figure 11.16 Schematic diagram showing the life cycle of Neobenedenia girellae infecting farmed cobia, Rachycentron canadum. Adult
Neobenedenia attached to the body surface of fish lay eggs into the water column which may become entangled in sea cage netting.
Following a short embryonation period (usually 5–8 days at 25 °C), eggs hatch in close proximity to fish. Ciliated oncomiracidia can
swim and attach to fish where they feed on epithelium and grow to sexual maturity. Source: Reproduced with permission from Dr Kate
Hutson, graphics by Eden Cartwright, Bud Design.
Neobenedenia (considered here as a collection of Pathogens and Parasites 239
potentially undifferentiated species) has been reported
from around the globe in multiple aquaculture industries changes should increase in summer temperatures when
including tilapia (Orechromis spp.), grouper (Epinephelus eggs hatch rapidly. Treatment of monogenean infec-
spp.), jacks (Seriola spp.), puffer fish (Takifugu rubripes), tions commonly involves fresh water, hydrogen perox-
barramundi (Lates calcarifer) and cobia (Rachycentron ide or formalin baths. The eggs are generally resistant to
canadum; Figure 11.16). Neobenedenia are dorsoven- short exposure to these chemicals due to their proteina-
trally flattened, oval‐shaped and adults range from ceous shell, but they are susceptible to desiccation.
2–7 mm. A single worm is capable of producing ~3300 Praziquantel has been tested against a range of monoge-
eggs in its lifetime. Live worms are almost entirely trans- nean infecting fish with promising results, although if
parent, but bathing in dechlorinated fresh water kills delivered orally, highly medicated feed may be unpalat-
them and turns specimens white. Haptoral sclerites able to some fishes. Administration of bath or in‐feed
(sclerotised hook structures) penetrate host epidermis treatments requires strategically timed dual delivery for
and injure fish skin and large populations of worms graz- optimal results to kill adult parasite populations on fish,
ing on fish can erode the epithelium. In some circum- followed by a second treatment to kill immature para-
stances, oncomiracidia predominantly recruit to the eye sites that have recruited as larvae from eggs around the
and can cause blindness. Infestations appear to irritate farm. Cleaner organisms (fish, shrimp) show considera-
fish so that they rub their body or ‘flash’ against struc- ble promise for reducing infection intensity of monoge-
tures or other fish, presumably in an attempt to dislodge neans, but the risks of co‐culture need thorough
attached parasites. Lesions may worsen from secondary examination.
infection (bacteria, viruses and fungi) of parasite‐
inflicted wounds. 11.8 Nematodes
Some monogeneans are viviparous (e.g., Gyrodactylus) – Nematodes, or round worms, are long, slender and
they develop the embryo in their uterus, give birth to the cylindrical unsegmented endoparasites tapered at each
daughter which is fully developed and already carrying extremity. They infect freshwater, marine and brackish
the next generation in the uterus. This mode of repro- fish species. Some nematode genera include species that
duction, hyperviviparity, has been colloquially termed can spread between animals and humans, e.g.,
‘Russian Doll’ and permits exponential population Anguillicola, Philometra, Skrjabillanus and Anisakis.
increases on susceptible hosts. Parasites are spread from Pathology in fish normally occurs within the intestines
direct physical contact between hosts or alternatively but nematodes can be found in all organs. Nematodes
they can detach and survive for a few days before infect- have separate sexes and exhibit complex life cycles.
ing a new host. Transmission from dead hosts to live Adults have to complete their life cycle in a specific bird,
hosts may also occur. Gyrodactylus salaris, a freshwater fish or mammal species while their larval stages may be
parasite of salmonids, is believed to have been intro- able to survive in a large variety of intermediate hosts.
duced with infected salmon from Sweden to Norway Any disruptions to these cycles prevent the develop-
based on high demand for salmon for stocking and ment of the adult nematodes, therefore cultured fish
experimental purposes (Johnson and Jensen, 1991). The with low connectivity to the n atural environment (such
parasite was new to Norwegian stocks and fish were as land based recirculating systems) are less likely to
incredibly susceptible, with the parasite spreading to at develop nematode infections. Few studies have exam-
least 46 rivers in Norway resulting in catastrophic ined the potential effects of nematodes on the health
ecological and economic problems. Economic loss condition or fitness of fish hosts, but humans may
become infected with nematodes if consumed in raw or
related to diminished fish stocks, loss of angler tourism undercooked seafood (see section 11.12). Nematodes
and ongoing surveys and management measures. have the potential to limit market value of fish through
Gyrodactylus‐infected rivers are periodically treated consumer attitudes.
with the indiscriminate pesticide/piscicide rotenone One of the most invasive nematode species is
prior to salmon runs to increase spawning success, but Anguillicoloides crassus which is strictly parasitic to eel
this treatment is only possible in short rivers with favour- species in the genus Anguilla. This parasite is known to
able biological and geographic conditions. infect six eel species and has been recorded from 46 coun-
tries in four continents (Lefebvre et al., 2012). Human‐
Attention to farm husbandry can reduce monogenean mediated transfers for aquaculture are suspected to be the
reason for the rapid invasion of this species around the
infections on livestock. On sea cage farms eggs laid by globe. Losses have been reported in wild and farmed eels
oviparous species can entangle on nets and other foul-
ing material (Figure 11.16) so regular cleaning or chang-
ing of nets can reduce egg load. The frequency of net
240 Aquaculture water sources or adding chemicals to eliminate interme-
diate crustacean hosts. Clean feed sources should be
and the parasite is considered among the main threats to maintained, such as an extruded pellet diet. There is no
the survival of European and American eel species. It has effective drug against fish nematodes but drugs such as
been reported in South East Asia, Europe, the USA and flubendazole, levamisole, mebendazole, trichlorphon and
Africa, but so far has not been reported in Australian eels, triclabendazole have been trialed with varying results.
probably as a result of strict import legislation. The para-
site causes severe pathology in the hosts’ swim bladder 11.9 Acanthocephalans
including lesions, inflammation, haemorrhaging and
fibrosis (Figure 11.17). Eels infected with nematodes typi- Acanthocephalans or ‘spiny headed worms’ comprise at
cally exhibit reduced swimming performance, swim near least 500 species of endoparasites of freshwater and
the surface and are emaciated. marine fish, although only a few species are reported to
be problematic in aquaculture. They live in the host
Anguillicoloides crassus is trophically transmitted – it intestine where they attach using a retractable proboscis
depends on predator–prey interactions to complete its covered in numerous hooks. They have no mouth or
life cycle. Eggs leave the swim bladder via the pneu- intestine and absorb nutrients from the lumen. Worms
matic duct and pass through the intestine and hatch in have separate sexes and a complex life cycle. Adult female
water. Alternatively, they may hatch internally. Newly worms living in the intestine of fish copulate and eggs are
hatched second‐stage larvae attach to the substratum passed with the host faeces into the aquatic system. Eggs
by their caudal extremity and wiggle intensively, pre- are ingested by a free‐living intermediate crustacean
sumably to stimulate predation by aquatic invertebrates. host (e.g., amphipod or isopod) which becomes infected.
They can survive and remain infective for days. A range When the definitive fish host ingests the crustacean, the
of crustacean species may serve as intermediate hosts, parasite develops to the adult stage in the fish intestine.
including copepods and ostracods. Once ingested, that
larvae invade the haemoceoel where they grow and Epidemics caused by acanthocephalans have been
moult. Eels become infected when they eat infected reported in aquaculture hatcheries, particularly in sal-
aquatic organisms, including paratenic fish hosts (a monids and eels. The most well‐known genera include
host is not necessary for the development of a particular Acanthocephalus, Echinorhynchus, Pomphorhynchus
species of parasite, but serves to maintain the life cycle and Neoechinorhynchus. The attachment of the probos-
and can promote dispersal). Once ingested by the eel, cis to the mucosa elicits an inflammatory response at the
the parasite passes through the intestinal wall and site of attachment. Heavy infestations may cause leth-
migrates to reach the swim bladder wall within one argy, morbidity and deformation of the spinal column
week (Figure 11.17). and may result in reduced host absorption of nutrients
from the intestine. Prevention of intermediate hosts into
Disrupting the life cycle is one of the best preventative aquaculture systems will break the life cycle.
strategies for reducing nematode infections in aquacul-
ture systems. This can be achieved by filtering incoming
Figure 11.17 One of the most invasive nematode species is 11.10 Leeches
Anguillicoloides crassus which parasitises the swim bladder of
eel species in the genus Anguilla. Source: Reproduced with Leeches (Phylum Annelida) are segmented worms in
permission from Dr S. Klimpel and Dr S. Emde. the Subclass Hirudinea. Some medicinal species are
farmed for use in human medicine in a quirky aquacul-
ture industry called Hirudiculture. Not all leeches are
bloodsuckers and many leeches encountered living
freely in ponds and rivers are predators. Leeches can
infect a variety of aquaculture organisms including
crustaceans, molluscs, fish, frogs and soft‐shelled tur-
tles (note ‘oyster leeches’ are turbellarians) and occur in
freshwater, brackish and marine environments (Kearn,
2004). As micropredators, or temporary parasites, they
may leave their host after feeding and not reattach to a
new host until the last meal has been digested. In addi-
tion to crawling, where the anterior and posterior suck-
ers are used for locomotion, some leeches may swim.
Leeches are negatively phototactic, which prompts Pathogens and Parasites 241
them to seek out the benthos where they rest attached
to vegetation or submerged infrastructure. When hun- Figure 11.18 The marine leech, Zeylanicobdella arugamensis,
gry, they become sensitive to water turbulence and has been associated with mortalities of juvenile barramundi,
shadows generated from passing fish. The leech then Lates calcarifer. Source: Reproduced with permission from
stretches out like a rod and performs ‘searching’ move- D. B. Vaughan.
ments by swaying the body. Sensory structures includ-
ing simple eyes, papillae and sensilla also enable them to removable hard substrates can be introduced and periodi-
find prey or hosts. cally removed so that cocoons cemented to them can be
destroyed. Biological controls, such as cleaner fish, may be
When contact is made with a potential host the oral feasible.
sucker is attached and the posterior sucker released from
the substrate. Some leeches also have adhesive secre- 11.11 Crustaceans
tions that play a role in cementing them to the surface of
the host. Leeches are simultaneous hermaphrodites and Parasitic crustaceans have great economic importance as
adults will detach from the host in order to lay cocoons agents of disease in wild and farmed fish populations.
on a chosen substrate, including aquaculture structures These parasites can affect host survival and cause
such as moorings and nets. Cocoons contain a ring‐ unsightly changes in the flesh. Among them, the copep-
shaped compartment that is effectively sealed from the ods are most dominant in aquaculture.
environment which protects the developing embryo 11.11.1 Branchiurans
(Kearn, 2004). Cocoons are usually adhered to the sub- Branchiurans, commonly called fish lice, are temporary
strate and each typically contains a single egg. Hatching ectoparasites. Most branchiurans occur in fresh water,
is temperature dependent and young leeches can survive but a few species infect the skin of marine fish. Of the
for a week or more before their first blood meal. 150 or so species, about 125 belong to the genus Argulus.
Argulids range from 2 to 30 mm in length, with females
Leeches attach to their host using anterior and poste- typically larger than males, and most can be observed by
rior suckers and use their jaws to gain access to blood. naked eye on the surface of fish (Figure 11.19). They
They may prevent clotting while feeding by injecting exhibit a flattened, disc‐shaped body with a prominent
saliva that inhibits the host’s clotting enzyme, thrombin. pair of compound eyes. They bear a pair of conspicuous
Leeches can ingest several times their own weight in ventrally‐directed suckers (modified first maxillae)
blood at one meal and digestion is slow, which enables which is the principle mechanism of attachment. Each
the leech to survive for up to several months without the sucker is able to move independently of the other and the
host. Some leech species feed mainly from the fins, but parasite is able to move in a fast ‘walking’ motion over
feeding has been reported to occur all over the body sur- the host’s body. Parasites typically attach to the caudal
face, including the gill chamber. Clinical symptoms of peduncle, flank, caudal fin and pectoral fins with few
leech infection include anaemia, lethargy, body discol- occurring in the buccal or gill cavities.
ouration, fish scale loss, and frayed fins and restless
swimming. Severe infestations of leeches render aquatic Transmission is direct since they are excellent swim-
animals unmarketable due to unsightly clusters of worm‐ mers and will actively seek fish and attach themselves.
like parasites, frayed fins, haemorrhages and swelling at During the day, Argulus have been shown to hover,
attachment and feeding sites (Figure 11.18). Excessive almost motionless in aquaria before advancing towards
blood loss probably occurs with intense and prolonged
infestations and can result in mortality of aquaculture
stock. Moreover, leeches can serve as a vector for other
parasites and pathogens, including bacteria, viruses,
flagellate trypanosomes and digeneans.
The marine leech, Zeylanicobdella arugamensis, has
been associated with mortalities of juvenile and adult
grouper (Epinephelus coioides) in the Philippines and
mortality in grouper and barramundi fingerlings Lates
calcarifer reared in sea cages in Malaysia. In some
instances, between 80–100% of fish in sea cages may be
infected (Kua et al., 2014). Formalin bath treatments
(50 ppm) are effective for managing leeches attached to
fish, while draining and drying out of pond facilities desic-
cate leech cocoons and render them unviable. Alternatively,
242 Aquaculture invaded the world. It primarily infests goldfish and other
cyprinids, but has also been found on a range of other fish
Figure 11.19 A rainbow trout with a severe infestation of Argulus hosts. Argulus coregoni primarily infects salmonids but
on the body surface. Source: Reproduced with permission from can also be found on cyprinids and other hosts. Rainbow
Dr C. Williams, Environment Agency, UK. trout, Onchorhynchus mykiss, sampled at a commercial
fish farm in Central Finland in 2002 exhibited 100%
potential hosts. At night, the parasite switches to an infection prevalence with a range between 4 and 1309
active, widely searching strategy, actively swimming in A. coregoni individuals per fish (Bandilla et al., 2005).
long straight lines. Fish infected with Argulus are more
likely to obtain additional fish lice because infected To prevent introduction of argulids in aquaculture,
individuals swim erratically, providing greater attrac- incoming water can be filtered, and incoming fish quar-
tion for hovering parasites. The suckers used for princi- antined. In ponds, removable hard substrates such as
pal attachment are supplemented by a variety of hooks wooden slats can be introduced and periodically removed
and spines. Indeed, the ventral margins of argulids are so that eggs laid on them can be destroyed. Anti‐
typically covered in backwardly directed spines. Argulus crustacean substances or insecticides are commonly
attaches itself on the host facing into the current so that used to treat fish, but argulids rapidly develop resistance
these spines and hooks engage in the host’s skin and to chemical treatments. Their temporary parasitic
prevent it from sliding backwards or being dislodged. lifestyle could potentially enable them to avoid predation
Unlike copepods, argulids do not retain fertilised eggs by cleaner fish, although small fish are known to ingest
in strings or sacs but leave the host and attach their argulids.
eggs to hard surfaces, such as stones or aquaculture
infrastructure. The eggs are laid one at a time, side by 11.11.2 Isopods
side, in columns. The stage that hatches from the egg is Parasitic isopods comprise three major groups including
immediately parasitic and searches for a host on which the cymothoids, epicaridians and gnathiids. Most iso-
it will mature. pods seen on aquaculture fish are cymothoids (Family
Cymothoidae). Cymothoids are primarily ectoparasitic
Once attached to the host, they are believed to either on marine fish in warm waters but occur occasionally in
pierce the host’s skin and suck blood and other internal temperate systems. Their legs form grappling hooks for
fluids, or feed on mucus and skin sloughed off by the attachment to external surfaces or in the mouth cavity of
host. Infested fish become lethargic, cease feeding, lose their hosts. The female permanently attaches to the host
condition and may try to remove parasites by rubbing and releases eggs into a brood pouch or ‘marsupium’
against the substrate. In chronic infections the skin before they embryonate, hatch and undergo two moults
becomes opaque, ulcerative lesions develop and the fins before being released. After a short swimming period
become frayed. Mortalities are usually associated they need to find a fish host, or they will die. Attached
with hundreds of argulids per fish, likely as a result of a parasites use a mouth cone to tear at the fish’s flesh and
breakdown in epithelial integrity. Argulids have been pierce into the tissue to penetrate blood vessels or
implicated in the transfer of viruses and other parasitic sinuses. It has been speculated that cymothoids may
organisms. transmit viral diseases or allow viruses to enter through
damaged tissue from attachment or feeding activity.
Argulus species have been recognised as pests of Cymothid isopod‐inflicted mortalities in mariculture
farmed trout in Europe and carp in China for centuries. facilities are common and have been reported from
The most notorious species include A. coregoni and A. important farmed fishes including, but not limited to,
japonicus. Argulus japonicus is believed to have been seabass (Dicentrarchus labrax), gilthead sea bream
introduced with aquarium fish from Asia and has now (Sparus aurata), barramundi (Lates calcarifer), catfish
(Mystus gulio) and the goby (Oxyurichthys microlepsis).
Infections of hatchery‐reared barramundi (Lates calcari-
fer) in the branchial and anterodorsal regions by
Cymothoa indica resulted in skin lesions and have been
associated with lowered growth rates and mortality.
Parasites may be introduced to fish through wild zoo-
plankton used as food, consequently, infection could be
reduced by filtering wild zooplankton to remove the
infectious swimming larvae of C. indica, or by using
alternative live feeds.
Parasitic isopods of Bopyroidea and Cryptoniscoidea Pathogens and Parasites 243
(commonly referred to as epicarideans) are unique in
using crustaceans as both intermediate (pelagic cope- potential low host densities in wild environments and
pod) and definitive hosts (both benthic and pelagic spe- exhibit high fecundity and specialised dispersal stages,
cies). Among epicarideans, nearly all species in hence in confined, captive environments with high den-
Bopyroidea are ectoparasitic on decapod hosts. The sities of fish they can cause severe epidemics. The most
energy burden they impart on their host can have dra- infamous species, Lepeophtheirus salmonis, has two
matic changes for the host’s reproductive capability. infective larval stages (the chalimus stages) which attach
Indeed, female cultured giant freshwater prawns to fish using a frontal filament. This mode of attachment
Macrobrachium malcolmsonii, infected with Probopyrus is essential to maintain connection to the host, otherwise
buitendijkibut in the gill chamber do not produce roe. they would fall off during subsequent moulting to pre‐
When the parasites are removed females recover. adult stages. Adult females can produce six to eleven
broods of paired egg strings that contain 300 to 1000
Commercially significant mortalities of eels and mullet eggs, with potential for multiple paternity in a single egg
have been associated with infection by gnathiid isopods. sac. Egg development is closely related to seawater tem-
Gnathiids are temporary parasites of fishes. Only the perature with development taking longer at colder tem-
‘praniza’, the larval stage, parasitise the gills and body of peratures. Eggs hatch while still attached to the female,
fish while the adults are free‐living. Fishes confined in releasing a planktonic copepodid stage that is susceptible
cages can be attacked by a large number of pranizae at to the speed and direction of currents. Local conditions
night. Within 2 to 4 hours, the larva’s modified mid‐gut impact whether or not there is re‐infection of the site or
becomes engorged with host fluid and the isopod leaves contamination from neighboring farms. In Chile,
its victim. Consequently, some aquaculture farms may be Atlantic salmon (Salmo salar) and trout (Oncorhynchus
oblivious to the impact of infections. Gnathiid infections mykiss) are highly susceptible to infections of the caligid
should not be underestimated because they serve as vec- copepod Caligus rogercressyi which naturally occurs on
tors of some fish blood protozoa and may also spread several native marine fishes that occur near farms.
filarial nematodes. Female parasites produce two egg strings containing 29
eggs each but can produce up to 11 pairs of strings in
Scavenging cirolanid isopods have also been reported their short lifetime of approximately seven months.
to inflict mortalities in some captive fishes including bar-
ramundi (Lates calcarifer) stocked in sea cages. Cirolanids In most countries, strict lice control regimes have been
are not parasitic, so infestations are likely to result only put in place to reduce the release of salmon louse larvae
when fish are compromised by other injuries or disease. from aquaculture facilities into the environment to
negate negative impacts on wild migratory salmonids
11.11.3 Copepods and other farms. This usually involves ‘treatment trig-
gers’, whereby a certain number of female lice on fish
Copepods, commonly called fish lice, have considerable warrant synchronised delousing in the area. The cost of
economic importance in marine and freshwater fish managing salmonid sea lice in 2013 was USD 80 million
aquaculture. Commonly called fish lice, copepods are in medicines. However, parasites show resistance or are
one of the most diverse groups of metazoan ectoparasite developing resistance against most chemicals in use. This
of fishes and also infect a wide range of invertebrates. is largely because of limitations in dose efficiency because
They are typically small and inconspicuous but are treatments do not result in 100% of parasites being killed.
extremely abundant. Some copepods are mesoparasites Parasites that survive treatments respond quickly given
(partly embedded in their host) with highly modified high fecundity, short generation times and efficient dis-
anchoring structures (e.g., Lernaea cyprinacea) while persal. The industry is beginning to accept that the battle
caligid copepods have a cephalothorax that forms a against drug resistance cannot be won because drug
broad shield that acts like a suction cup to hold the louse development takes time and parasite populations
on to the fish. Copepods have free‐swimming and para- respond too quickly. There is now research emphasis in
sitic life stages. When attached to fish hosts they feed on determining the feasibility of vaccine development
mucus, epidermal tissue and blood, and untreated infec- against sea‐lice following elucidation of the salmon louse
tions can result in host death, elevated stress, immuno- genome (Torrissen et al., 2013). Meanwhile, some farms
suppression, secondary infections and increased risk of in Scotland, Norway, and Ireland use cleaner fishes
predation. Copepods may also be carriers of bacteria and (Labridae and Cyclopteridae) as a biological control as
viruses, probably obtained from their attachment to and they will pick off and consume sea lice from infected
feeding on contaminated fish. salmon. The successful use of cleaner fish depends on
providing ample shelter and clean cages, as cleaner fish
Sea lice cause multi‐million‐dollar losses to the salmon readily seek alternative feed sources if the nets are over-
farming industry worldwide. They are well adapted to grown. Several million wild‐captured cleaner fishes are
244 Aquaculture indicating that moving mussel farms into deep water off-
shore could reduce the incidence of infection.
routinely used in Norway, but there are now multi‐mil-
lion‐dollar research programs into farming certified dis- 11.12 Fishborne Zoonotic Agents
ease free labrids to supply farms. The use of cleaner fishes and Aquaculture
reduces or avoids the need to use parasiticides and the
farmed fish can be harvested without drug residues. Human contact with and consumption of fishes and
11.11.4 Pea Crabs shellfish presents potential disease risks from bacterial
Pea crabs are small parasitic crabs (Malocostraca: pathogens and metazoan parasites. Of the large numbers
Pinnotheridae) that live as parasites in the mantle cavity of species of pathogens and parasites that infect aquacul-
of bivalves (e.g., oysters, clams, mussels), abalone, inside ture organisms, only a few cause illnesses in humans.
the test of sea urchins and in the rectum of sea cucum- Nevertheless, the reported incidence of fish‐borne zoon-
bers. Pea crabs are completely reliant on their host for oses has increased in recent years because of increased
shelter and food. Adult female pea crabs have a soft‐ raw or undercooked fish consumption (e.g., ‘sushi’ and
shelled exoskeleton and are oval in shape with their eggs ‘sashimi’), improved diagnosis, and growth in aquacul-
tucked under their abdomens, giving them an almost ture development and the international seafood market.
spherical, pea‐shaped appearance. Adult males have a Negative impacts on human health from ingestion of
hard, chitinous exoskeleton and are generally smaller pathogens and parasites may be prevented by freezing or
than the female. Female pea crabs spend their entire adult heating infected fresh meat. Indeed, current European
lives within a single host, while adult males will leave Union legislation requires that all fishery products from
their host in order to find a mate. A pheromone‐based finfish or cephalopods that are intended to be consumed
mate location system is likely used by pea crabs to reduce raw must be frozen before consumption. This includes
the risks associated with the location of females. Male cold smoked fish where the smoking process does not
pea crabs attempt to enter a mussel hosting a female crab achieve a core temperature of 60 °C for at least one min-
by stroking the mantle to increase mussel valve gape. ute. This section provides a brief overview of primary
human zoonoses including bacteria and metazoan para-
Pea crabs can cause problems for bivalve aquaculture sites associated with fisheries, aquaculture and orna-
through reduced growth rates and end‐consumer com- mental aquaria. For further information please consult
plaints. Interestingly, the prevalence of pea crabs in pearl reviews by Lima dos Santosa and Howgate (2011), and
oysters can be as high as 85%, without apparent harm to Gauthier (2015).
the host. In contrast, the pea crab Nepinnotheres novaez-
elandiae infects approximately 5% of farmed greenlip There is substantial epidemiological and molecular
mussels, Perna canaliculus, in New Zealand, and causes evidence to support classification of bacteria species
30% reduction in meat yield of infected individuals which Clostridium botulinum, via ingestion, and Streptococcus
equated to estimated loss of USD 2.16 million annually iniae, Mycobacterium spp. and Vibrio vulnificus via inoc-
(Trottier et al., 2012; Figure 11.20). There are no known ulation, as fish‐borne zoonoses (Gauthier, 2015). A vari-
preventative methods for pea crab infections, although ety of other bacteria have been reported as potential
the prevalence of infection is higher in shallower water, fish‐borne zoonotic agents, but evidence is limited and
few molecular genetic analyses have been made to link
Figure 11.20 The pea crab, Nepinnotheres novaezelandiae, infects fish and human strains. Clostridium botulinum, which
approximately 5% of farmed green‐lip mussels, Perna canaliculus, occurs in the intestines of marine and freshwater fish
in New Zealand. Source: Reproduced with permission from species worldwide, has a potent paralytic neurotoxin
Dr. O. Trottier. which induces paralysis in humans. Disease in fishes
from C. botulinum has been reported in earthen pond
culture of salmonids and catfishes and human botulism
has been associated with consumption of contaminated
fish products, notably smoked fish in northern temper-
ate regions of the world. Streptococcus iniae has been
reported in a variety of fish hosts and has been impli-
cated in outbreaks with cellulitis related to processing
and handling raw or live fishes in the Unites States,
Southeast Asia, Canada and Hong Kong. Similarly,
Mycobacterium spp. cause granulomatous inflammation
of human skin, also known as ‘fisherman’s finger’. Pathogens and Parasites 245
Antibiotic therapy is generally effective for mycobacte-
ria, although surgical excision of lesions may be required. The World Health Organization and the Food and
Vibrio spp. are widely distributed in marine and estua- Agriculture Organization of the United Nations esti-
rine environments and can cause serious disease in cul- mated that more than 18 million people were infected
tured fishes. Vibrio vulnificus has been reported in with fish‐borne zoonotic trematodes in 2002. Human
humans that have handled fish, including eels. Infection infection by trematodes is particularly important in
is associated with gastroenteritis, septicemia and wound developing nations where many people are dependent on
infections and is of particular concern because of high freshwater fish as the major source of protein. Liver
case fatality rates (3.6%). Vibrio cholera, which produce flukes include three pathogenic species in the family
cholera toxin, has also been implicated in human disease Opisthorchiidae including Clonorchis sinensis (endemic
outbreaks associated with the consumption of shellfish to Japan, China, Taiwan and Southeast Asia), Opisthorchis
and has been reported in water used to house or trans- viverrini (endemic throughout Thailand, the Lao People’s
port ornamental fish. Democratic Republic, Vietnam and Cambodia) and O.
felineus (distributed throughout Europe). The life cycle
Fish protozoans are not known to be infective to involves a freshwater snail in which asexual reproduction
humans, but there is emerging evidence that myxozoan takes place and freshwater fishes (particularly cyprinids)
species can cause food poisoning. Since the year 2000, are intermediate hosts. Fish‐eating mammals, including
western regions of Japan have reported a foodborne dis- humans, dogs and cats, act as definitive hosts, in which
ease that causes vomiting and diarrhea within several sexual reproduction occurs. Adult parasites attach to the
hours after ingesting olive flounder (Paralichthys oliva- bile duct in humans and can lead to obstruction when
ceus), tuna (Thunnus spp.) or amberjack (Seriola dumer- there are high burdens. O. viverrini and C. sinensis are
ili). In October 2010, a particularly large outbreak was capable of causing cancer of the gall bladder and/or its
reported among individuals who consumed olive floun- ducts. Poor sanitation practices and inadequate sewer-
der sashimi that had been raised in aquaculture systems. age infrastructure can facilitate the spread of the parasite
Microscopic examination revealed that the fish were eggs in human faeces into bodies of fresh water.
infected with Kudoa septempunctata in the muscle.
Since then, other wild and farmed fishes associated with Anisakiasis is a disease caused by infection of larval
instances of food poisoning have been examined and ascaridoid nematodes whose normal definitive hosts are
confirmed positive for Kudoa species providing mount- marine mammals. Freshwater and marine fishes the sec-
ing evidence that these parasites are the most likely cause ond intermediate hosts and to acquire an infection they
of the diarrhea outbreaks. must consume infected prey. The risk of infection from
farmed fish can be substantially reduced by using extruded
Infection from cestodes belonging to the genus pellet diets. In addition, gutting fish soon after they are
Diphyllobothrium can be transmitted to humans by caught allegedly prevents migration of nematode para-
consumption of raw or undercooked freshwater and sites to muscles. The disease currently affects over 2000
marine fishes. The incidence of human infection with people per annum worldwide with most cases noted in
Diphyllobothrium tape worms has been increasing in Japan and sporadic cases in most other parts of the world.
urban areas of Japan and in European countries and recent Worms can only survive for a limited period in the gastro-
estimates indicate that approximately 20 million individu- intestinal tract of humans following ingestion. Acute gas-
als could be affected. Wild salmonids, which harbour the tric infections are manifested by gastric pain, nausea and
plerocercoid larva, appear to be the major transmitter of vomiting within hours of ingesting raw infected seafood.
this disease, although Diphyllobothrium larvae have also In chronic cases this may last from several weeks to two
years. Onset of intestinal anisakiasis occurs within seven
been reported in cultivated salmonids in several coun- days following ingestion with severe pain in the lower
tries. Diphyllobothrium exploits fish as its second inter- abdomen, nausea and vomiting. Removal of worms by
mediate host and infection in humans is incidental with gastrofibrescope is used for acute infections, but partial
resection may be required in chronic infections.
the natural definitive hosts including birds, bears, seals,
cats and dogs. The adult worm attaches to the mucosa of 11.13 Aquaponics
the ileum. Most infections are asymptomatic, but some-
times infections are associated with nausea, vomiting, Plants grown as part of aquaponic systems are subject to
abdominal pain, diarrhea, and discharge of the parasite’s the same pests and diseases that affect field crops,
although they are less susceptible to soil‐borne agents.
main body (strobila), which can be as long as 12 metres! A Plants can absorb and concentrate chemicals used to treat
recent surge of clinical cases highlights a change in the parasites and infectious diseases of fish and invertebrates
epidemiological trend of this tapeworm disease from one
of rural populations to a disease of urban populations
worldwide who eat seafood as part of a healthy diet.
246 Aquaculture research investments to overcome challenges associ-
ated with aquaculture vaccine development.
held in recirculated systems, so therapeutics should be ●● Protozoans are among the most significant parasite
avoided. It should also be kept in mind that salt water, problems in bivalve and finfish aquaculture industries.
which is commonly used to treat parasitic diseases of They comprise a diverse group of unicellular eukary-
freshwater fish, is deadly to plants. There are several alter- otic organisms, many of which are motile. Some para-
native approaches to reduce plant pest and diseases sitic protozoa have life stages alternating between
including biological controls, disease‐resistant cultivars, proliferative stages and dormant cysts which can sur-
barriers, traps and manipulation of the environment. vive harsh conditions. At present there are no commer-
cial vaccines available for any fish‐parasitic protozoa.
11.14 Summary ●● Metazoan parasites are extraordinarily diverse, with
various routes of invasion. Species with direct life
●● Viral outbreaks cause significant mortality and sub- cycles are most frequently observed in aquaculture,
stantial economic impact to aquaculture operations. although where farm environments provide suitable
Outbreaks have dramatically impacted commercial habitat for intermediate hosts, parasites with complex
and resource enhancement aquaculture due to heavy life cycles may be common. The most successful strat-
losses and or regulatory requirements mandating the egy to reduce metazoan parasite infestations is to
eradication of fish infected with specific viruses of break the parasite’s life cycle through strategically
concern. New and emerging viral pathogens of aquatic timed treatments.
species are continuously being discovered and charac- ●● Parasites can serve as a vector for other pathogens and
terised, and it is critical that aquaculture operations parasites such as bacteria and viruses. This typically
develop and adhere to strict biosecurity practices to occurs in blood feeding parasites such as leeches and
minimise the risk of viral outbreaks. crustaceans, which may have the capacity to infect sev-
eral individual hosts during their life time and facilitate
●● Bacterial diseases cause significant economic impact disease spread.
due to mortality or performance issues in fish that ●● Human contact and consumption of fishes and shellfish
survive an outbreak. The majority of bacterial dis- presents potential disease risks from bacterial patho-
eases in aquaculture are caused by Gram‐negative gens and metazoan parasites. The reported incidence
bacteria; however, some infections with Gram‐posi- of fish‐borne zoonoses has increased in recent years.
tive bacteria result in significant impact. Future con- Negative impacts on human health from ingestion may
trol methods will need to rely on vaccines and other be prevented by freezing or heating infected fresh meat.
non‐antibiotic methods which will require substantial
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the marine parasitic amoeba Neoparamoeba perurans. aquacultured New Zealand green‐lipped mussels, Perna
Journal of Fish Diseases, 39, 629–637. canaliculus. Aquaculture, 344–349, 23–28.
Shinn, A.P., Pratoomyot, J., Bron, J.E., Paladini, G., Brooker, Whittington, R.J., Becker, J.A. and Dennis, M.M. (2010).
E.E. and Brooker, A.J. (2015). Economic costs of Iridovirus infections in finfish – critical review with
emphasis on ranaviruses. Journal of Fish Diseases, 33,
95–122.
249
12
Prevention of Disease by Vaccination
Andrew Barnes
CHAPTER MENU 12.8 Research and Development Track for
12.1 Introduction, 249 Commercial Fish Vaccines, 267
12.2 A Beginner’s Guide to Fish Immunology, 250
12.3 Vaccinating Fishes, 260 12.9 Future Trends: Vaccination in the Age of Genomics, 269
12.4 Types of Vaccine, 260 12.10 Conclusions, 269
12.5 Routes of Delivery, 263 12.11 Summary, 270
12.6 Adjuvants, 266
12.7 Vaccination in Practice, 266 References, 270
12.1 Introduction advancing applied field that has been instrumental to
the goal of sustainable growth of aquaculture.
12.1.1 Preface
It is difficult to write about a field as complex as immu- 12.1.2 Definition of Vaccine
nology and infectious disease in a style that is approach- We are all familiar with the concept of immunisation
able to a very diverse, generally educated audience such through the vaccines we receive in our infancy. They pro-
as readers of this aquaculture textbook. In 1950, the tect us against a range of infectious diseases that were
composer Roger Sessions wrote in the New York Times once prevalent and often deadly prior to the introduction
“I remember a remark of Albert Einstein, which certainly of mass inoculation programs. Indeed, the only diseases
applies to music. He said, in effect, that everything to be completely eradicated in human history, smallpox
should be as simple as it can be, but not simpler.” I think and rinderpest, were thwarted through rigorous vaccina-
this phrase applies equally well to this chapter, and I tion regimes in humans and cattle respectively. There is a
almost certainly made the previous edition simpler at the certain poetry to the eradication of a disease in cattle by
expense of accuracy. I have attempted to remedy this in vaccination: The word vaccine is derived from the Latin
this revised version by improving the accuracy through vacca, a cow, and a vaccine, strictly defined, is a cowpox
the increase of important detail without sacrificing virus or lymph containing it. This is based on the successful
c larity. To this end, I have structured it as an abridged attempts by Edward Jenner to prevent infection with
narrative in the main body of the text, with important smallpox by administration of the closely‐related cowpox
details explained further in separate boxes and figures. virus. The term vaccine is now used more generally to
In this way the reader should be able to follow the logical define any preparation used to confer immunity to a dis-
flow of the vaccine response whilst filling in the neces- ease by inoculation and the principle relies on the recipi-
sary details from the boxes and by reference to more ent having an adaptive immune system which initiates a
comprehensive reviews. There have been many signifi- response to the components of the vaccine that results in
cant advances in fish immunology and fish vaccination the memory of those components. The immune system
since the previous edition of this book. I hope this revised of the vaccinated individual is then able to respond more
chapter will inspire readers to study further in a rapidly
Aquaculture: Farming Aquatic Animals and Plants, Third Edition. Edited by John S. Lucas, Paul C. Southgate and Craig S. Tucker.
© 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.
250 Aquaculture (Gordon et al., 2006), but also a more economically
sustainable industry. Many bacterial diseases of fishes
quickly and activate protective effector systems with are now successfully prevented through vaccination.
greater magnitude on subsequent encounters with the
same patterns or structures. The first question that I am asked when people find
out that I have worked in the aquaculture vaccines indus-
12.1.3 History of Fish Vaccines try for nearly 30 years is “Do you really inject every single
fish?” The answer is yes we do, but to understand why
Fish vaccination has a surprisingly long history, with the injectable vaccines are so effective whilst other modes of
reporting of successful specific protective immunity in delivery are generally less so, we need to consider the
trout against furunculosis in the US caused by ‘Bacterium processes involved in an effective, long‐term (lifetime)
salmonicida’ (Aeromonas salmonicida) as early as 1942 response to vaccination by the fishes. In other words, we
(Duff, 1942). Further early advances with vaccines need an understanding of fish immunology.
against bacterial diseases in fishes were made in the
1960s and 1970s when bacterins (killed bacterial 12.2 A Beginner’s Guide to Fish
preparations) were used to control Yersiniosis in trout Immunology
cultivated in the US for sport fisheries (Busch, 1978;
Ross and Klontz, 1965; Stevenson, 1997). Indeed, the Immunology is a fascinating and vastly complex field of
first commercial fish vaccine was introduced in 1976 by study and there are excellent textbooks that cover mam-
Colorado‐based Wildlife Vaccines Inc., granted a USDA malian and human immunology in detail (Murphy, 2011;
license for the use of vaccines against Y. ruckeri and Roitt et al., 2001). Fish immunology is a little different
Vibrio anguillarum. Mass vaccination programmes from human and mammalian immunology and is further
against bacterial diseases followed the rapid expansion of complicated by the vast diversity of teleost fishes. In fact,
the intensive Atlantic salmon industry in Norway and fishes are the most diverse extant vertebrate infraclass
Scotland. Vaccines against vibriosis in the late 1980s and with more than 26 000 extant species (compared to
early 1990s in Norway and Scotland were followed in the around 5000 mammals) and, whilst fishes share many of
mid‐1990s with efficacious vaccines against furunculosis the basic immune processes and receptors common to
caused by A. salmonicida and winter mortality caused by all jawed vertebrates (Flajnik and Du Pasquier, 2004),
Moritella viscosa. Successful control of these bacterial there are critical differences consistent with the diver-
diseases through vaccination revolutionised the Atlantic gence of the fishes from other vertebrate lineages more
salmon sector and brought forth a wealth of research and than 400 million years ago (Magadan et al., 2015). The
development in the field of fish immunology, pathology fishes are the most primitive animal infraclass to develop
and vaccination. specific adaptive immune memory based on immuno-
globulin (antibody‐like) receptors and specialist popula-
Perhaps the most significant indication of the success tions of cells called leucocytes (Table 12.1) and there are
of the vaccination programme is the decline in chemical some excellent recent reviews that outline the key
use in the Atlantic salmon industry: The Norwegian features of fish immunity and how they fit into an
Office of Statistics keeps excellent records of antibiotic evolutionary context (Fillatreau et al., 2013; Magadan
use that illustrate the remarkable effect of the advent of et al., 2015; Sunyer, 2012, 2013). For the purposes of this
bacterial vaccines on chemical usage in Norwegian sal- chapter, however, we will focus on the aspects of the
monid aquaculture (Statistisk Sentralbyrå). In the period immune system that we can exploit through vaccination
between the height of the furunculosis era in 1987 to the and what makes the fish immune system different from
statistics released for 2003, antibiotic consumption by mammals as it pertains to successful application of
the industry dropped by 98%. Currently average antibi- vaccination in farmed fishes. The immunology in this
otic usage is less than 0.001 g per kg of salmon produced, chapter should therefore be considered as introduction
down from 0.9 g /kg in 1987. Most of this reduction in (i.e., incomplete) and it is written as an overview for
use occurred between 1987 and 1994 and directly reflects those working in the broader fish health field and as a
the introduction of oil emulsion injectable vaccines basis for further reading for those wishing to study the
against winter mortality and furunculosis, coupled with field in depth. To help get the most out of this introduc-
the improved husbandry techniques implemented by tory applied immunology text, I have provided a table of
farmers to ensure the success of the vaccination pro- definitions for some of the specialist terms used in this
grammes (Grave et al., 1999; Sommerset et al., 2005). chapter (Table 12.1) and the cells of the immune system
This represents not only a phenomenal reduction in that we will focus on (Figure 12.1).
environmental impact and a greatly reduced potential
public health risk resulting from maintenance of antibi-
otic resistance determinants in the aquatic environment
Prevention of Disease by Vaccination 251
Table 12.1 Quick reference guide to immunological terms.
Term Definition
Antibody
Antigen Soluble, Y‐shaped immunoglobulin molecule with a hyper‐variable antigen‐binding (Fab) region and
Antigen presentation a constant (Fc) region that is conserved within, but different between, isotypes (See Box 12.3).
Chemokine Any molecule or part of a molecule that can be bound by antibody or MHC
Cytokine Translocation of antigens to the cell surface for screening by patrolling T‐lymphocytes.
Chemical signal released during inflammation that acts to attract key immune cells to the site of
Epitope infection by chemotaxis along increasing chemokine concentration gradient.
Immunogen Chemical signal released by cells during inflammation that acts on other cells (or may be self‐acting)
causing them to respond. Cytokine signalling is critical to initiation, systemic dispersal and
Immunoglobulin (Ig) amplification of the immune response.
Antigens are comprised of one or many epitopes. These are molecular patterns that are recognised by
Inflammation complementary paratopes on the MHC, TCR or antibody molecule.
Leucocytes Any molecule that may elicit an adaptive immune response by itself and has been used interchangeably
with antigen throughout the literature. However, an immunogen is always an antigen, but an antigen
Major may not necessarily be an immunogen as it is possible for antigens not to induce adaptive immunity.
Histocompatibility Evolutionarily conserved superfamily of proteins that contain at least one immunoglobulin fold or
Complex (MHC) domain. The fold structure is stabilised by disulphide bridges and is central to the ability of Ig family
molecules to specifically bind to antigens and receptors.
Paratope Critical to the innate immune response, a process that draws fluids and immune cells to the site of
infection (see Box 12.2).
Somatic Commonly known as ‘white blood cells’. These cells of the immune system are derived from the
recombination myeloid and lymphoid lineages. Lymphoid comprises B‐cells, T‐cells and natural killer (NK) cells.
The myeloid lineage comprises the granulocytes including neutrophils (phagocytic), basophils and
T‐cell receptors eosinophils that modulate inflammation. The agranular myeloid cells comprise monocytes and their
(TCR) derivatives, macrophages and dendritic cells.
Ig superfamily proteins critical to antigen presentation. MHC class I is found in almost every cell
and presents protein epitopes synthesised inside the cell on the surface where they are assessed by
CD8+ T‐lymphocytes. ‘Normal’ proteins are ignored. Abnormal proteins (e.g., from viruses) activate
the lymphocyte and it kills the cell. MHC class II is found only in antigen presenting cells (dendritic
cells, B‐lymphocytes and possibly macrophages). Antigens presented by MHCII enable maturation of
CD4+ T‐helper lymphocytes.
Molecular pattern that is exactly complementary in shape to an epitope. This permits very specific
binding. Paratopes are found in members of the Ig superfamily such as the groove of the MHC
molecules, the Fab region of antibody and the epitope recognition domain of the T‐cell receptors.
The process of selecting from and mixing (recombining) different gene segments during early cell
(somatic) development. This enables the Ig superfamily, for example, to introduce unprecedented
diversity into their antigen binding paratopes. These regions can form structures capable of binding
molecular shape in nature.
Another member of the immunoglobulin superfamily found on the surface of T‐lymphocytes that
recognises and binds antigen presented via MHC class I or II molecules on tissue cells and
B‐lymphocytes respectively.
12.2.1 What are the Cellular Processes (a salmon parr in this case) showing the major immune
Involved in Response to Vaccination? tissues and the development of adaptive immunity
In the photographs in Figures 12.2 and 12.3, barramundi following vaccination. The first responder cells are the
are being vaccinated by injection in the peritoneum with major immune sentinels; tissue‐resident macrophages
a small dose (usually 50–100 μL) of killed bacteria emul- and dendritic cells (DC) along with lymphocytes that
sified in oil. What happens when that emulsion of dead comprise a small population of leucocytes that are resi-
bacterial cells arrives in peritoneum of the fishes? Let us dent in the peritoneal cavity of the fish (Afonso et al.,
consider this in a model by reference to Figure 12.4, in 1997) (Figures 12.1 and Figure 12.4, Object 2). These will
which we see a schematic representation of a fish initiate the processing of the vaccine via innate immune
252 Aquaculture
Cell type Notes Figure 12.1 Quick reference guide to some cells
Dendritic cell of the adaptive immune system. Source:
Professional antigen presenting cells can present exogenous Reproduced with permission from Andy
Macrophage antigen by MHCII. Many DC subclasses in mammals and Barnes, 2017.
likely to be also in fish. Involved in T-cell screening in the
Neutrophil thymus during development to negatively select precursor
T-lymphocytes with TCR that self-recognise.
Monocyte
CD4+ The major tissue-resident phagocytic sentinel cells. These
T-lymphocyte cells engulf potential pathogens and destroy them with
CD8+ an array of toxic reactive agents. They are also M HCII+ so
T-lymphocyte may present antigen. Fc receptors enable rapid location and
destruction of pathogens bound by antibody.
B-lymphocyte
The major phagocytic sentinel cells of the blood. Similarly
to macrophages these cells destroy pathogens with
an array of toxic reactive agents. Short-lived (one to several
days) these cells are replenished from the pronephros.
Myeloid lineage pre-cursors. These cells originate in the
pronephros and can mature into macrophages and DCs.
During inflammation monocytes move to the site of
infection and may supplement tissue macrophages and DCs.
Progenitors originate in the pronephros, but they are
seeded to the thymus where they are selected to remove
self-recognising clones. CD4+ T-cells respond to antigen
presented by MHCII and orchestrate the adaptive immunity
by enabling maturation of B- and CD8+ T-cells.
The TCR of cytotoxic or killer T-lymphocytes binds antigen
presented by MHCI on any cell. Because MHCI presents
antigen of intracellular origin, these are the major antiviral
effectors. On activation, CD8+ T-cells pass cytotoxic granules
to infected host cells initiating programmed cell death.
B-lymphocytes have surface-located lg that binds exogenous
antigen. In fish, B-cells are phagocytic and express MHCII
so may present antigen. When they mature under direction
of CD4+ T-cells, they proliferate and differentiate into short-
lived antibody-secreting plasma cells and long-lived memory
cells. High numbers in the pronephros after injection vaccines.
Figure 12.2 Vaccination of fishes by injection: Fishes are accumulation of fluid and cells at the site of vaccination
vaccinated by intraperitoneal injection. In these barramundi, (Figure 12.4, Object 3). It is this influx of cells that will
vaccine is delivered by hand with repeat injection guns that can begin processing the vaccine for the development of spe-
be adjusted to give a precise dose of vaccine. Care is taken to cific immune memory. The resident tissue macrophages
ensure that needles are of appropriate size for the fish being and DCs may be supplemented by monocytes, drawn
vaccinated and are changed before they become blunt. to the site from the circulatory system, and these will
Source: Reproduced with permission from Andy Barnes, 2017. functionally mature directed by cytokines expressed by
macrophages, neutrophils and some tissues as part of
mechanisms (see Box 12.1), leading to mild inflamma- the inflammatory response (Figure 12.4, Object 3).
tion in the peritoneum at the site of vaccination (see Lymphocytes will also arrive via the circulatory system,
Box 12.2) (Figure 12.4, Objects 2 and 3). Neutrophils, the directed to the site by chemotaxis along chemokine gra-
primary immune sentinels of the blood (white blood dients; chemokines are also released by macrophages
cells), egressing from the locally‐permeablised vascular and neutrophils during the inflammatory response and
walls, will enhance the inflammation leading to further enable other immune cells to accurately locate the source
of the problem (Figure 12.4, Object 3). Phagocytosis
(ingestion) of vaccine components by macrophages,
neutrophils, B‐lymphocytes and DCs will follow. But the
fate of these ingested components will differ depending
upon the cell involved. Macrophages and neutrophils
are the primary effector cells of the immune system and,
ultimately, most invading organisms are destroyed
by these cells. We will return to these later. DCs are
s pecialists in antigen presentation, a pivotal process in
the development of immune memory of the antigen that
requires cooperation between multiple cell types
(Figure 12.4, Object 4). It is likely that this occurs in the
secondary lymphoid tissue of the fish, the spleen, with
Prevention of Disease by Vaccination 253
Figure 12.3 Vaccination of fishes by
injection: A typical small‐scale commercial
vaccination set up at a barramundi farm in
Queensland, Australia. The vaccination
table has recirculated water that contains
anaesthetic in the central reservoir, whilst
the channels on each side of the central
reservoir have flowing clean farm water
passing through them. Anaesthetised fish
are gently lifted from the central reservoir,
injected, then released into the channel
where the clean water flow transfers them
back to a recovery tank which can be seen
in the foreground of the photograph.
Source: Reproduced with permission from
Andy Barnes, 2017.
these loaded DCs migrating there via the circulatory of the vaccine. But how does this help to protect the fish
system. This takes some time; in barramundi at 28 °C, from reinfection? These helper T‐cells are now able
a substantial increase in DC markers is detected in the to ‘help’ with maturation of B‐lymphocytes (Fig. 12.1).
spleen about a week after vaccination (Zoccola et al., B‐lymphocytes are characterised by the expression of
2015). In the spleen, these antigen presenting cells immunoglobulin (Ig) or antibody on their surface
(APC) digest the vaccine components and present (Box 12.3 Immunoglobulins and antibody). Each naïve
antigenic epitopes as short peptides to immature helper B‐lymphocyte will express an Ig molecule that is specific
T‐lymphocytes by translocation of the antigen fragments for (and can bind to) a unique pattern. Thus, in a large
to the cell surface via their Major Histocompatibility population of B‐lymphocytes, there is capacity to recog-
Complex class II (MHCII) molecule (Figure 12.4, Object 4) nise and bind to a diverse array of molecular patterns.
(Fig. 12.5a). Once at the cell surface, epitopes in the groove Consequently, amongst the repertoire of B‐lymphocytes
of the MHCII molecule are recognised specifically by that arrive in the inflamed peritoneum after vaccination,
the T‐cell receptors (TCR) on the T‐lymphocytes. This, there will be a substantial cohort of cells that have Ig that
along with strict regulation by co‐stimulatory factors on can bind to differing components in the vaccine. In
the APCs, initiates clonal expansion (rapid cell division) fishes, B‐lymphocytes are phagocytic and can ingest
of this lineage of T‐cells expressing the TCR that matches bound intact antigen and present it via MHCII (Li et al.,
only the antigenic peptide presented by the MHCII of 2006). Since this discovery in fishes, it has been found
this APC. This proliferation is followed after several that mammalian B‐cells may also be phagocytic, so mod-
g enerations by maturation to CD4+ T‐helper cells that els of antigen presentation are currently under revision.
may be effector, memory or regulatory (Figure 12.4, In fishes, it is likely that, similarly to DCs, loaded B‐cells
Object 4) (Figure 12.5a). migrate via the circulatory system to the spleen and
elevated numbers of Ig + cells are detected in the spleen
So after a week or two (depending upon water around a week post‐vaccination (Dos Santos et al.,
temperature), our vaccinated fish has an expanded popu- 2001). Once in the spleen, the loaded B‐lymphocytes
lation of mature helper T‐cells that have T‐cell receptors can begin a conversation with the mature helper
specific for patterns contained amongst the components
254 Aquaculture 12
6 Haematopoeisis First
Lymphocyte responders
Immune selection
memory Posterior kidney
Peritoneum
Pronephros Thymus Swimbladder
Gill lamellae Liver Gut
Heart Spleen 4 3
Circulatory Proliferation Inflammation
system Differentiation
Blood capillary
Maturation
5
Peritoneum
Figure 12.4 Schematic representation of Atlantic salmon parr showing the major immune tissues and the progression of the response to
vaccination by intraperitoneal injection. The pronephros or head‐kidney is the major haematopoietic organ in bony fish (1) as they do not
have bone marrow. All of the major cells of the immune system originate here during development and through the life of the fish.
Lymphocyte precursors are selected in the thymus, which lies anterior to the head kidney and is connected, to remove those that
cross‐react with self epitopes. Macrophages and dendritic cells are derived from the monocyte‐lineage and it is these tissue‐resident
sentinels, seeded during early development, that are the first responders to infection or the microspheres of vaccine emulsion
(white spheres) in the peritoneum (2). Interaction with molecular patterns in the vaccine droplets results in phagocytosis and release of
cytokines (triangles) and chemokines (blue) (2), resulting in inflammation (3). During the inflammatory response, the tight junctions of the
vascular endothelium in local capillaries relax and the cells become sticky allowing leucocytes to attach and migrate through the cell
junctions, following the chemokine gradient to the site of the vaccine (3). Monocytes may mature to supplement the tissue‐resident DCs
and macrophages whilst neutrophils phagocytose vaccine particles, releasing cytokines and chemokines to regulate the inflammatory
response. DCs and B‐lymphocytes, loaded with vaccine antigens migrate to the spleen (4) where antigen presentation occurs with helper
T‐lymphocytes via MHCII and the T‐cell receptors, promoting proliferation and maturation of helper T‐cells and then proliferation and
maturation of B‐lymphocytes. Mature short‐lives plasma B‐lymphocytes enter the circulatory system and secrete antibody (IgM) that is
specific for epitopes in the vaccine resulting in the primary serum antibody response. Memory B and T‐cells migrate to the pronephros
and may reside there for long periods (6). These can initiate proliferation and maturation on further encounters with the vaccine antigens
(e.g., during infection) and mount a rapid and overwhelming secondary antibody response specific for those patterns. This is the basis of
immune memory (6). Source: Reproduced with permission from Andy Barnes, 2017.
T‐cells (Figure 12.4, Object 4). The instigation of these there is tightly choreographed cell communication
conversations is dictated by a mutual recognition of the between DCs (APCs), T‐lymphocytes and B‐lympho-
same molecular patterns by the Ig of the B‐cell and the cytes based on mutual recognition of the same molecular
TCR on the mature helper T‐lymphocyte. The TCR on patterns that were present in the vaccine; by the Ig on
the T‐lymphocyte will be able to recognise and bind the B‐cells; presentation by MHCII by B‐cells and by DCs
antigen presented by the B‐lymphocyte in the groove of and; recognition in this context by the TCR of the mature
its MHCII, antigen that was originally bound by the Ig on helper T‐lymphocytes – mature only because their TCR
the surface of the B‐cell and internalised. So we can see recognised that pattern when it was presented by the
Prevention of Disease by Vaccination 255
Box 12.1 Innate Immunity function throughout the animal kingdom and include the
Toll‐like receptors (TLR), C‐type lectin receptors (CLR) and
Vertebrates have both an innate and adaptive immune NOD‐like receptors (NLR). The response to each pattern
system that act together in a rapid ‘non‐specific’ defence mediated through these receptors is the same every time
(innate system) followed by longer term specific immune and often leads to inflammation (see Box 12.2). The initial
‘memory’ (adaptive system). The innate system is ‘always recognition (and usually destruction) of invading
on’, encoded in the germline and comprises both cellular m icroorganisms is orchestrated predominantly by the
and non‐cellular (humoral) components that respond professional phagocytic cells, the neutrophils in the blood
immediately upon insult (such as infection, vaccination or and mature tissue‐resident macrophages localised at the
envenomation). At its most basic, the immune system area of infection (Table 12.2). These cells release an array of
functions around three activities: cytokines (signalling molecules that act on nearby cells
with suitable receptors) and chemokines (soluble mole-
Pattern recognition – recognising there is a problem; cules that form a chemical gradient that immune cells can
Signalling – ensuring the correct systems are informed and follow by chemotaxis to the source of the insult), and initi-
ate inflammation. They are also the major effector cells
the information becomes systemic as well as localised; and will produce an array of toxic metabolites that kill
Effector – doing something about the problem (i.e., killing invading microbes. The defining principle of innate immu-
nity is that it is innate, encoded in the germline and the
the invading organism) and cleaning up afterwards. response to a particular pattern is always the same. One
can contemplate how quickly a pathogen with a genera-
Through millions of years of evolution, the innate immune tion time of a few tens of minutes may adapt through
function of animals has evolved to recognise and respond e volution to such a strong, constant selective pressure.
to conserved molecular patterns that are not usually The adaptive evolution of the host’s germline‐encoded
found in healthy higher animals. These include cell wall response is also constrained by generation time, but this is
components of bacteria such as peptidoglycan and likely to be years. The evolutionary playing field, in terms
lipopolysaccharide (LPS) (although many orders of fishes of innate immunity versus pathogen, is not a level one!
are not sensitive to LPS, a feature that differentiates their
innate immune system from that of mammals), glucans
and general signs of viral infection such as double‐
stranded RNA. These patterns are recognised by a range of
receptors that are largely conserved in both structure and
Box 12.2 Inflammation complement cascade, an ancient, antibacterial defence
mechanism of the innate immune system and a key partici-
The pro‐inflammatory response results in an influx of fluids pant in effective immunity and wound repair, along with
and cells to the site of vaccination: signalling molecules serum amyloid proteins that bind pathogens, coagulation
called cytokines (Table 12.2) are released from mac- factors and factors that restrict iron availability to patho-
rophages in response to the components in the vaccine. gens such as ferritin and hepcidins. There is also increased
Different cytokines act on different cells, but some act on release of serpins ‐ protease inhibitors that down‐regulate
the vascular endothelium (cells that line the blood vessels) inflammation – the inflammatory response must be strictly
causing the blood vessels to dilate, become more permea- regulated to prevent host damage and degenerative
ble (‘leaky’), and the cells of the endothelium to become d iseases. So the overall effect of inflammation is both local-
sticky, allowing circulating leucocytes to bind and migrate ised (the supply of leucocytes and fluids to the site of infec-
through between the cells to the site of the vaccine tion) and systemic in the up‐regulated production of acute
(or infection). The fluids leaking from the blood vessels phase proteins in organs such as the liver and their release
near the site of vaccination or infection cause the charac- into circulation, along with mobilisation of leucocytes into
teristic swelling associated with inflammation and contain circulation from the pronephros.
proteins that are useful in dealing with the infection or vac-
cine. These include immunoglobulin (lg), proteins of the
DCs and now able to bind the same pattern presented by by the T‐lymphocytes induce the B‐lymphocytes to
B‐cells. This is how the adaptive immune system is regu- proliferate, mature and differentiate. Proliferation of the
lated to be specific to the particular challenge. B‐lymphocytes, like the T‐lymphocytes, is clonal, thus
each new B‐lymphocyte will also carry the Ig paratope
Once the B‐ and T‐lymphocytes are bound via MHCII‐ on its surface that is specific for the antigen that t riggered
antigen‐TCR, signalling molecules (cytokines) secreted
256 Aquaculture the initial response. Most of the new B‐lymphocytes will
(a) be relatively short‐lived effector B‐lymphocytes or
Antigen presenting cell plasma cells. These cells enter the circulation and secrete
antibody with an antigen‐recognising domain that is
Bacterial identical to that of the Ig on the surface of that B‐cell
peptides lineage and that is consequently specific for the antigen
that bound the initial paratope on the surface (Figure 12.4,
Bacterial cells Object 5). This is how we derive the specific antibody
that we can detect in the blood serum as the primary
MHCII Cytokines immune response following vaccination. Memory is
induced because some of the new B‐lymphocytes differ-
(b) TCR Ig entiate into long‐lived ‘memory’ cells (in fishes these
may be long‐lived plasma cells that are seeded to the pro-
Immature CD4+ nephros and reside there for some time) (Figure 12.4,
T-helper cell Object 6). A simplified diagram of this cellular process is
given in Figure 12.5 a, b and c. These memory B cells
B-Iymphocyte can respond rapidly upon subsequent challenge by the
same antigen provided there is also a resident population
Maturation Cytokines of mature T‐helper cells with the correct TCR to initiate
(c) further proliferation, which of course there will be after
vaccination, as explained above. This leads to a rapid
Mature CD4+ TCR MHCII Bacterial increase in serum antibody on subsequent encounters
T-helper cell Co-stimulation peptides with the pathogen(s) that are contained in the vaccine
because there is already an expanded population of
Proliferation, maturation mature T‐helper cells and B‐cells specific for the pat-
and differentiation terns or epitopes of the pathogen. The specificity of the
primary antibody response, subsequent memory and
(d) To pronephros To circulatory rapid secondary response on subsequent challenge is due
system to the clonal differentiation and proliferation of the T
and B lymphocytes, that is, all of the new T‐helper and
‘Memory’ IgM B‐lymphocytes recognise the original antigen.
B-lymphocyte Plasma cells
Whilst we have considered the fate of a single molecu-
Figure 12.5 Simplified schematic of immune responses mediated lar pattern here, we have to be mindful that the vaccine
by MHCII (for external antigens). Antigen presentation by MHCII (and the pathogen from which it is derived) comprises
can only occur in specialist phagocytic antigen presenting cells many potential antigens and that many of these will be
(APC) such as dendritic cells, some macrophages and recognised by Ig on B‐cells, be presented by MHCII on
B‐lymphocytes in fishes. These cells engulf particles such as APCs and be recognised by TCRs, with each lineage of
bacteria and viruses when they are external to host cells, and B‐cells and T‐cells specifically recognising a different
present components on the surface via MHCII (a). Presentation by epitope or pattern. Thus the response described above
APCs via MHCII to the T‐cell receptors on immature helper T cells will occur across many lineages of lymphocytes that each
(b) results in their maturation. Mature helper T‐lymphocytes can recognise a different epitope on the pathogen: The
then activate of B‐cells that co‐recognise the same antigen response will be polyclonal – multiple lineages of lym-
(c) resulting in their differentiation and proliferation thereby phocytes proliferating in a clonal manner. Therefore the
increasing the number of B‐cells that recognise the original antibodies that result from this response will also be pol-
antigen. Effector B cells or plasma cells secrete antibody and this is yclonal, with Ig secreted by each lineage of B‐cells being
how we detect increasing specific antibody response to a specific specific for a different epitope, one per lineage. The mix-
antigen after vaccination (d). As effector B‐cells are short lived, the ture of antibody in circulation post‐response will conse-
quently recognise a broad range of patterns in the vaccine
and the pathogen from which it was prepared.
specific antibody response declines over time, but memory B cells
are long lived and can respond rapidly producing more antibody
on subsequent encounters with the same antigen (d).
Source: Reproduced with permission from Andy Barnes, 2017.
Prevention of Disease by Vaccination 257
Box 12.3 Antibody and Immunoglobulins
VL VH Translocon (e.g., Atlantic salmon heavy chain)
CL
CH1 V Cτ V D/JτCτ V Jτ Cτ V Dτ CDτ /JVτD/JCτ τ V D/Jμ∂CμAC∂A
VH
Fab CH1 IgH-A
FC CH2
VL IgH-B V JτCτ V D/Jτ Cτ V Cτ V D/Jμ∂CμBC∂B
CH3
CH2 CL Cluster (e.g., rainbow trout light chain)
CH3
IgL2(σ) VV JC V VV J C
IgL1(κ) V JC
n
Antibody is the most familiar member of the immunoglob- B‐lymphocytes such that each lineage carries an Fab para-
ulin (Ig) superfamily of proteins that also includes T‐cell tope assembled from differing combinations of V, D and J
receptors, Class I and II MHC and lymphocyte co‐receptors heavy, and V and J light loci. Additional diversity is intro-
such as CD4 and CD8, along with a diverse range of other duced by differing combinations of light and heavy chains
receptors found in many species ranging from simple in the Fab region of the complete antibody molecule.
sponges to man. All members have at least one domain Although the diagram of the gene loci in salmon and trout
called the immunoglobulin fold or Ig domain, a highly sta- above is highly simplified, you can see that the number of
ble groove that is central to their specific binding capabil- possible combinations is staggering! The FC region of the
ity. Antibodies are soluble immunoglobulin glycoproteins molecule also fulfils a critical functional role. The heavy
produced by B‐lymphocytes. The immunoglobulins of chain constant region comprises three Ig domains (CH1, 2
cold‐blooded vertebrates have been reviewed compre- and 3, inset) and a hinge and is conserved within, but dif-
hensively by (Pettinello and Dooley, 2014). The antibody fers between, antibody isotype. In fish there are three heavy
monomer is Y‐shaped and comprises two Ig heavy chains chain isotypes, IgM (Cμ), IgD (C∂) and IgT (CT) and this deter-
and two Ig light chains joined by disulphide bridges (see mines the effect of the antibody once it has bound to the
inset, shown in red). The heavy chain and light chain varia- antigen to form a complex because the different isotype FC
ble regions comprise a single Ig domain each (VL and VH regions interact with isotype‐specific FC receptors. IgD and
respectively) and are highly diverse in terms of amino acid IgM monomers are found on the surface of B‐lymphocytes.
sequence. It is the combination of the light and heavy chain Mature plasma B‐cells secrete IgM as a tetramer (four IgM
variable domains that makes up the antigen binding para- monomers linked at the FC region) and this constitutes the
tope or Fab region of the antibody. The extraordinary diver- systemic serum antibody response after vaccination. The
sity of structure amongst the Fab region, enabling tetrameric form of IgM is planar (flat) when in circulation
recognition of almost any possible molecular pattern, is and not bound to the antigen. During antigen binding, the
introduced because the variable domains of the heavy Fab regions are drawn towards the antigen resulting in a
chain are encoded by multiple genes from the variable (V), conformational change to a ‘staple’ form. This exposes the
diversity (D) and joining (J) loci of the IgH locus (or loci, IgM FC region allowing binding by specific FC receptors on
there are two in salmon). Those of the light chain are assem- macrophages and neutrophils. IgT is found on specialist B‐
bled from combinations from the Variable (V) and Joining lymphocytes in the mucosal epithelium of the gills, skin,
(J) of the IgL loci (See inset and (Edholm et al., 2011; and gut and probably fulfils a role similar to mucosal IgA in
Fillatreau et al., 2013; Hikima et al., 2011; Pettinello and mammals. This explains the partition between mucosal
Dooley, 2014; Yasuike et al., 2010). These loci are r earranged and systemic response (refer to main text and Figure 12.8).
by somatic recombination during the early development of
Source: Reproduced with permission from Andy Barnes.
12.2.2 How Does the Antibody Protect secreting it (primary immune response). It also has a popu-
the Fish? lation of multiple lineages of memory B and T‐helper cells
As a consequence of vaccination, our fish has an elevated that are specific for epitopes on the pathogen and can initi-
antibody level in its circulatory system that will last for as ate further clonal expansion of the B‐cell repertoire if they
long as the short‐lived plasma B cells are circulating and encounter the pathogen in the vaccine leading to a very
258 Aquaculture extracellular milieu of the peritoneum. This is very
effective against many species of pathogenic bacteria
rapid elevated antibody level (secondary immune response). that also invade the extracellular environment of the
What does the antibody do? As the response is polyclonal, host. In contrast, some bacterial pathogens and all
the mixture of antibodies will recognise many epitopes and viruses enter host cells and survive and replicate
the effector function of the antibody will differ depending within the host cells. These intracellular pathogens
upon the epitope that is bound but can be broadly sepa- require a different immune response because the major
rated into two categories, neutralizing and opsonising. antigen presenting cells, the lymphocytes and the pow-
Neutralising antibodies bind to epitopes that constitute erful immune sentinel/effector cells of the mac-
parts of molecules that provide life‐critical function to the rophages and neutrophils cannot enter host cells to
pathogen in a way that prevents them from functioning perform their function. Almost all non‐immune cells
resulting in inhibition or death of the pathogen. For exam- of the host tissues express the Major Histocompatibility
ple, they may bind and inhibit molecules involved in uptake Complex class 1 protein (MHCI). This, along with
of critical nutrients or minerals such as the iron uptake pro- ubiquitin (so named because it is found ubiquitously in
teins in the outer membrane of Aeromonas salmonicida all eukaryotic cells), constitutes part of the cells’ self‐
(Hirst and Ellis, 1994). Opsonising antibodies bind to checking mechanism during protein synthesis as part
epitopes on surface structures of the pathogen and enable of normal cell turnover. A subsample of proteins syn-
macrophages and neutrophils to easily detect and phagocy- thesised in the cell cytoplasm using the normal protein
tose the pathogen via cell‐surface receptors that are spe- synthesis machinery in every cell are checked and
cific, and have high affinity, for the conserved Fc part of the bound by ubiquitin. They are then degraded by a pro-
immunoglobulin molecule (see Box 12.3). The Fc region of tease called the proteasome and the resulting peptides
the IgM molecule that makes up the circulating antibody in loaded into the groove of the MHCI molecule for
fishes is not accessible to the receptors on macrophages and transfer via the endoplasmic reticulum and display on
neutrophils until the antibody has bound to its target, which the surface of the host cell. Cells that present normal
causes it to change shape exposing the Fc region. This pre- cell proteins in their MHC are ignored by cytotoxic
vents non‐specific activation of the macrophages and neu- (CD8+) T‐lymphocytes as ‘normal’. However, if a pep-
trophils by unbound antibody circulating in the fish tide o riginating from a non‐host source (for example a
(Box 12.3). Once the pathogen has been phagocytosed into viral protein as all viral proteins are synthesised by
a vacuole (the phagosome) it is killed and broken down by the host cell machinery) is displayed by the MHCI on
microbicidal compounds produced by the cells. In mac- the surface of the cell, it may be recognised by the TCR
rophages and neutrophils, oxidases in the membrane of the specific for that peptide‐MHCI complex as a rogue
phagosome surrounding the engulfed pathogen become cell. This interaction, along with co‐stimulatory factors
active and produce superoxide (an oxygen free radical) as a on antigen presenting cells, will activate the T‐cell ren-
progenitor of singlet oxygen, hydrogen peroxide, hydroxyl dering it cytotoxic. In this state the cytotoxic T‐cell
radical and peroxynitrite. Neutrophils produce hypochlor- can pass enzymes (granzymes) into the cytoplasm of
ous acid from hydrogen peroxide using myeloperoxidase the infected cell causing the cell to initiate programmed
(MPO). MPO is stored safely in granules within the cell cell death or apoptosis. The process is summarised
cytoplasm that degranulate releasing the enzyme upon acti- in Figure 12.6. The resulting dead cell and debris
vation after phagocytosis. Cooperation between mac- including any infecting virus particles are cleaned up
rophages and neutrophils in which MPO granules are by macrophages.
shared during the inflammatory response has been
observed in trout (Afonso et al., 1998), implying that Immune memory in this ‘cell‐mediated’ response
hypochlorous acid can be added to the repertoire of micro- requires interaction between activated T‐helper cells
bicidal compounds available to macrophages. In rapidly tar- and DCs with the CD8+ cytotoxic T‐cells. Activation of
geting pathogens for clearance in this way by macrophages CD8+ cells in this way leads to clonal proliferation of
and neutrophils, coupled with neutralisation or inhibition the CD8+ T lymphocytes resulting in high numbers of
of key processes in the bacteria, we can see how a high prev- long‐lived cytotoxic T‐cells patrolling the host with
alence of circulating specific antibody against a pathogen is TCR specific for the originating epitope. These fully
very effective at clearing it from the host. matured CD8+ T‐lymphocytes can be repeatedly stimu-
lated on encounter with host cells that display the
12.2.3 Immunity to Viruses and Other epitope in some of their surface MHCI molecules and
Intracellular Pathogens are very effective at clearing out infected host cells. A
So far we have considered how the immune system schematic representation of how various cells of the
of fish processes a vaccine that is delivered into the immune s ystem might be activated by a virus is shown
in Fig. 12.6.
Prevention of Disease by Vaccination 259
Antigen presenting cell Patrol for host cells
displaying viral peptides
Cytotoxic CD8+
in MHCI. Can be T-Iymphocytes
repeatedly stimulated
Kills infected cells
Viral displaying viral
peptides peptides in MHCI
Virus particles
MHCII Cytokines
TCR
Host cell
Virus replication
using host’s systems
Immature CD4+ Viral Proteins sampled
T-helper cell peptides during synthesis
Maturation MHCI MHCI
TCR TCR
Cytokines
TCR TCR Cytotoxic CD8+
Co-stimulation T-Iymphocyte
Mature CD4+ Immature CD8+ Proliferation and
T-helper cell T-Iymphocyte maturation
Figure 12.6 Simplified schematic of immune response mediated by MHCI (internally derived antigens such as viral proteins). Antigen
presentation by MHCI can occur in almost any cell but can only present antigens that originate from within the cell. Cells infected by
viruses or intracellular bacteria will present processed antigens from the infecting agent via MHCI. Presentation to T‐cells via MHCI will
lead to maturation of CD8+ cytotoxic T cells that kill the infected cell preventing further replication of the virus. However full maturation
that permits repeated stimulation of cytotoxicity requires interaction with mature CD4+ helper T‐cells. These must have been exposed to
viral antigen via MHCII for maturation. Source: Reproduced with permission from Andy Barnes, 2017.
12.2.4 How Does Understanding Immune 2) The development of a protective adaptive immune
Processes Help in Management of Fish response is based upon massive proliferation of mul-
Health? tiple lineages of specialist cells (T and B‐lymphocytes)
From the explanation given in this chapter, at least three and is therefore highly energy dependent. These new
key factors should be immediately apparent: cells will require synthesis of every component from
1) The development of adaptive immunity takes time. cell wall bilayer lipids to nucleic acids for genetic
material to proteins for enzymes and structural
There is little point in immunising a cohort of fishes s caffolds, plus fuel to live and carry out their function.
by vaccination and exposing them immediately to a Energy for this and all other processes in the fishes,
disease challenge. Therefore vaccination should including growth, is entirely derived from the
occur in advance of seasonal disease, or in a nursery digestible energy of the feed. Consequently, one will
environment that is reasonably well protected from expect to see a slight growth penalty in the week or
exposure to disease agents to provide sufficient time two post‐vaccination. This will be rapidly compen-
for specific lymphocyte proliferation and maturation sated in the months that follow. More importantly
to occur. however, the fishes require consistent feeding rates
260 Aquaculture
Table 12.2 Kinetics of immune response to vaccination in fish. The kinetics of immunity in fish is very important when considering how
and when to vaccinate. The process is temperature dependent, so water temperature must be taken into account in any calculations.
Kinetics are measured in degree days (°d), which is the water temperature in °C x time in days. Key factors to know are how soon your fish
will be protected after vaccination (the response time), and for how long (the duration of immunity or DOI). This depends on what type
of vaccine, how it is given and whether a booster vaccine is used. The table shows some indicative examples of response and duration
of immunity. These are general indications only and must be determined experimentally for each vaccine.
Response time Immersion Injection (aqueous) Injection
Time from delivery to full protection (aqueous) Primary ~500 °d (oil emulsion)
Booster ~150 °d
Duration of Immunity Primary ~500 °d Primary ~1500–3000°d Primary ~1000 °d
The length of time protection will last Booster ~150 °d Booster ~3000+ °d Booster not necessary
Primary ~ 1000 °d >8000 °d
Booster ~2000–3000 °d
with appropriate quality feeds during the weeks post‐ 12.3 Vaccinating Fishes
vaccination to ensure proper development of immu-
nity. This is not a time to try to save money by using During vaccination we are simply introducing an immu-
cheap or out‐dated, degraded feed or to experiment nogen and rely on the formation of immune memory as
with different feeding regimens or new diets. Care described in the preceding pages. The kinetics of the
should be taken with the timing of vaccination in immune response are very important as these determine
fishes that go off their feed at certain times of year. For how long it takes after vaccination until the fishes are pro-
example, barramundi and kingfish in Australia feed tected. It also determines how long the fishes will remain
poorly during the low water temperatures of mid‐ protected for (duration of immunity or DOI). Because
winter. In contrast, Atlantic salmon in Tasmania do most farmed fishes are ectothermic (tuna are one of sev-
not feed well in high summer water temperatures. It eral exceptions), kinetics of physiological processes are in
would be inadvisable to vaccinate at these times. part determined by water temperature. Thus when we
Local knowledge of farmers and veterinarians should measure immune kinetics in fishes, we must take this into
be applied. account and we do so by measuring immune kinetics in
3) The major effectors of the immune system, the pro- degree‐days (°d). This is simply the time in days multi-
fessional phagocytes (macrophages and neutrophils) plied by the water temperature in degrees Celsius. So if
produce microbicidal molecules that are very toxic, the primary response to vaccination takes 600 °d to
also to the host. The immune system works naturally develop peak primary serum antibody levels, that means
in a state of homeostasis with pro‐inflammatory it would take 20 days if the fishes are in water at 30 °C, but
signals balanced and regulated by complementary 60 days in a cold water species at 10 °C. The type of
anti‐inflammatory signals. Microbicidal reactive immune response we activate and the kinetics of this
oxygen is neutralised by host antioxidant enzyme response, therefore the success of the vaccination, will
systems. ‘Boosting’ the immune system by hypotheti- depend on a number of factors in addition to temperature
cally adding a pro‐inflammatory stimulus in the diet that are explored in this section. Some key elements of
or otherwise should be treated with a degree of scep- immune kinetics are summarised in Table 12.2.
ticism. The only ways in which the immune system
can be ‘boosted’ is by providing the best possible 12.4 Types of Vaccine
nutrition and a low stress environment. Selective
breeding of domesticated lines that are less stressed 12.4.1 Killed or Inactivated Vaccines
by the farm environment may also help with homo- In their simplest form, vaccines can be a killed version of
geneity of the response to vaccination across the an infectious agent. The agent is grown under culture in
cohort of fishes. appropriate nutrient medium for bacteria, or in tissue
culture using an appropriate cell line in the case of
Of course a fundamental contribution that our viruses, and then killed or inactivated by one of number
understanding of the fish immune system makes is of processes. The use of formalin is very common and
in the design, formulation and delivery of vaccines in
aquaculture.
quite effective for many killed bacterial vaccines and Prevention of Disease by Vaccination 261
formalin‐killed ‘bacterins’ still comprise the majority of
effective bacterial vaccines used commercially in aqua- organism is very difficult to grow in vitro. For example,
culture today. Killed vaccines constitute exogenous as viruses need to replicate inside host cells, viral v accines
antigens and are thus processed and presented via must be produced in cell culture and, for some fish
MHCII, eliciting a strong humoral immune response viruses, there are no permissive cell lines in which they
characterised by increase in circulating specific antibody can be grown successfully, or they may produce yields so
and subsequent immune memory via B‐lymphocyte low that they are not economically viable for vaccine
memory cell induction. They do not tend to elicit a production. Alternatively, some whole cell based prepa-
strong cellular immune response and this explains why rations may contain very toxic elements that are not
the majority of killed vaccines against viruses and inactivated by the treatment used to kill the vaccine, or
intracellular bacteria have low efficacy in practice. may contain highly antigenic components that do not
elicit protective immunity, but swamp any response to
12.4.2 Live, Attenuated Vaccines potential protective components that may make up a
very small proportion of the total components of the
Live, attenuated vaccines are simply live agents whose vaccine. The advance of molecular technologies has led
virulence (ability to cause disease) has been attenuated or to the development of subunit or recombinant vaccines,
reduced. This can be done by serial passage in vitro, with in which only the protective molecules, or the fragments
or without chemical agents such as the antibiotic of them that contain the protective epitopes are used in
rifampicin added to the culture medium. For example, the vaccine. This clearly requires extensive knowledge of
one live attenuated bacterial vaccine is prepared by the process of infection by the particular pathogen and
m ultiple (>16) serial passages on culture medium that which components are critical to the process and are also
contains the antibiotic rifampicin. In other candidate vac- accessible to the immune system. Once a candidate pro-
cines, key genes essential for virulence can be ‘knocked tein or part thereof has been identified, the simplest
out’ by site directed mutagenesis – using a nucleic acid means of producing the protein in substantial quantities
vector to specifically insert extra DNA (sometimes an is by cloning the gene into an expression plasmid and
antibiotic resistance gene) into the middle of the target expressing the protein in E. coli or in yeast. Generally
gene so that it can no longer be expressed by the patho- proteins derived from bacterial pathogens would be
gen. These attenuated strains no longer cause disease, or expressed in E. coli, whilst those from viruses may be
only very mild forms of it and are rapidly cleared by the expressed in yeasts to exploit the differences in eukary-
immune system. As they infect in the same way as the otic post‐translational modification – viral proteins are
original organism and replicate in the host, they tend to synthesised by the hosts cell machinery during infection
activate the same immune pathways. So, as a live virus or and fish are eukaryotes. The expression process is sche-
intracellular bacterium replicates inside the host cell, matically illustrated in Figure 12.7. Essentially expressing
components will be presented on that cell by MHCI and the protein in bacteria or yeasts and growing up a large
thereby initiate cellular immunity. This can get around volume of them is the simplest part of the process,
the problem of only activating humoral immune memory although not without its own difficulties. Purifying the
encountered when using killed vaccines. However, there recombinant protein from the final culture, and potential
is some concern over potential reversion to virulence in further ‘downstream processing’ such as refolding or
the field, the possibility of over attenuation (so that atten- conjugation to make it more visible to the immune sys-
uated strains are cleared too quickly without eliciting tem can add substantial extra difficulty, and therefore
immune memory) and, in the case of targeted mutagen- cost, to the process. This can make the cost of recombi-
esis, the release of live genetically modified organisms nant subunit vaccines prohibitive for fishes, which are
(GMOs) into the environment. Although there is a long relatively low value animals. A further disadvantage of
precedent for using live, attenuated vaccines, particularly recombinant vaccines is that, like killed vaccines, they
against viruses, in human medicine, the aforementioned are processed as exogenous antigens (presented by
concerns have meant that the live, attenuated approach MHCII) and therefore tend to only activate humoral
has met with very limited market or regulatory accepta- immune memory, generally making them less effective
bility in aquaculture. against viruses and intracellular bacteria. There is, how-
ever, one commercial vaccine based on a recombinant
12.4.3 Recombinant or Subunit Vaccines subunit of the infectious pancreatic necrosis virus
(IPNV) in Atlantic salmon. A recombinant VP2 protein
Both of the above technologies rely on the delivery of from the virus surface was included as a component of a
whole cells or viral particles to the animal in order to multivalent vaccine on sale for use in Norway through
gain immune protection. This can be problematic if the the late 1990s/early 2000s, although the level of
protection afforded by the IPNV component of this
v accine is not clear.
262 Aquaculture Product recovery
Cloning
Plasmid or
cosmid vector
Competent yeast Cell lysis and/or
or bacterial cell recovery from
culture medium
Transformation Fermentation Downstream
processing
Figure 12.7 Recombinant vaccine production process. The gene encoding the antigenic peptide or protein is cloned into an expression
vector – a plasmid with a very efficient promoter that drives expression of the gene in the host bacterium or yeast during fermentation.
The expressed protein is then recovered from the culture. This can be tricky as the protein may be secreted into the medium or
compartmentalised in the host cell in the cytoplasm, periplasm (space between the cell and outer membranes) or as inclusion granules
which can be very difficult to solubilise. All this must be determined experimentally before scale‐up and the amount of downstream
processing required to obtain the antigen in an effective form will determine the cost, and therefore market viability of the vaccine.
Source: Reproduced with permission from Andy Barnes, 2017.
12.4.4 DNA Vaccines vaccination is presented in Figure 12.9. The first com-
The DNA vaccine, sometimes called a nucleic acid or mercial DNA vaccine in any animal, Apex IHN, was
plasmid vaccine, is the most recent advance in vaccine licensed by Novartis Animal Health in 2005 for the
technology and has really been pioneered commercially prevention of infectious haematopoietic necrosis virus
in fishes. Like the recombinant vaccines, these vaccines (IHNV) in salmon in Pacific Canada. The vaccine com-
require in‐depth knowledge of the structure, virulence prised a plasmid expressing a major surface glycoprotein
and invasive processes of the target pathogen. Once a from the Rhabdovirus IHNV and is extremely effective.
potential candidate protein (such as a viral coat protein) In 2016 the first DNA vaccine licensed for fishes in
has been identified, the gene for that protein is cloned Europe was launched in Norway. This vaccine against
into a DNA vaccine plasmid vector (Figure 12.8). In salmonid alphavirus that causes pancreas disease in
essence this is very similar to the construction of the Atlantic salmon was initially developed by Aqua Health/
recombinant protein vectors. However, the critical dif- Novartis in the late 1990s/early 2000s and is indicative of
ference between recombinant and DNA vaccines is that the frustrating delays that can ensue through a combina-
the promoter (‘engine’ that drives expression of the tion of corporate, intellectual property and regulatory
desired gene) in the DNA vaccine is a promoter that is processes.
active in host (fishes or mammalian) cells, rather than in
bacterial or yeast cells. In commercial DNA vaccines this So why are DNA vaccines so effective in fishes?
promoter is derived from human cytomegalovirus Experiments with DNA vaccines against a similar rhab-
(CMV) and is incredibly efficient at driving gene expres- doviral disease in trout, viral haemorrhagic septicaemia
sion in eukaryotic cells. In the case of DNA vaccines it is virus (VHSV) have indicated that antibodies against the
the actual plasmid vector that constitutes the vaccine, virus can still be detected in DNA‐vaccinated fishes
rather than the expressed protein. Thus the plasmid vec- more than 2 years after injection, suggesting possible
tor is injected into the fish muscle and, in this process, a persistence of the vector in some fish cells. This seems
few muscle cells along the injection track become trans- hard to explain, as MHCI presentation of antigens on the
fected with the plasmid. The CMV promoter then drives surface of transfected muscle cells should result in their
expression of the target protein inside the host cell, mak- rapid eradication by specific CD8+ cytotoxic T‐cells and
ing it an endogenous antigen. Thus it is presented by macrophages. This appears to be what happens in labo-
MHCI and initiates the cellular immune system to acti- ratory mice and may explain the lower efficacy of DNA
vate maturation of CD8+ T‐lymphocytes that are critical vaccines in mammalian systems.
for good protection against many viruses and intracellu-
lar bacteria. A simplified theoretical response to DNA Are DNA vaccinated fishes genetically modified?
Certainly not! Although foreign DNA is actively
expressed by a few fish cells, the genetic material is never
Bacterial cell Fish cell (vaccine) Prevention of Disease by Vaccination 263
(fermentation) CMV promoter
by standard transformation. Thereafter the E. coli is
kan Gene of grown under appropriate selective conditions to main-
interest tain the plasmid in an industrial fermenter and the
Plasmid goes here! p lasmid is replicated at every cell division with the E.coli.
vaccine At the end of the fermentation, the bacterial cells are
construct Multiple lysed and the released plasmids are purified from the
cloning site lysate on industrial scale gel chromatography columns.
ColE1 BGH polyA 12.5 Routes of Delivery
Figure 12.8 A typical DNA vaccine vector showing the key Getting vaccines into fishes presents a number of prob-
elements of the plasmid construct. The elements to the left of lems, not least because of the numbers in which they are
diagram are active in the bacterial cell during fermentation and farmed and the low monetary value of the individual.
are associated with plasmid replication and maintenance. colE1 is This makes fish vaccines very similar to poultry vaccines
almost all of a very small bacterial plasmid characterised in E. coli in terms of the economics of use. Thus, it would seem
the 1970s and it contains all of the machinery necessary to sensible to use the easiest and least expensive mode of
replicate the plasmid and mobilise it into daughter E. coli cells delivery. Unfortunately, there is a trade‐off between low‐
when the bacteria divide. Kan is a kanamycin (antibiotic) cost/ease of use and actual efficacy in the fishes. In other
resistance gene that enables selection during fermentation. The words, the simplest and cheapest form of delivery may
transformed bacteria are grown in medium containing kanamycin not necessarily trigger the most appropriate immune
during the fermentation so that any daughter cells that do not response, or any response at all. In practice, vaccines may
have the plasmid are killed and won’t swamp the culture with be delivered to fishes via injection, immersion or bath-
empty E. coli. The elements to the right of the diagram are those ing, oral (on feed) and occasionally by spraying in an oil
expressed in the fish muscle cells when the plasmid is injected emulsion onto the surface of the water.
into them. The CMV promoter drives expression of any genes
downstream of it very efficiently in vertebrate cells. The multiple 12.5.1 Injectable Vaccines
cloning site immediately downstream of the promoter contains The vast majority of immunised fishes are vaccinated
many restriction enzyme cut sites so that vector can be cut with by injection into the peritoneal cavity (intraperitoneal
different restriction enzymes to allow directional cloning of the (IP) or intracoelomic injection). This may seem impos-
gene for the antigen of interest. It is important that the gene is sibly labour intensive when the sheer numbers of indi-
cloned into the vector in the correct orientation and in frame so viduals are considered, yet it has been adopted into the
that it will be correctly expressed and translated in the fish. In farm management cycle as routine. Use of repeat injec-
eukaryotes, messenger RNAs are polyadenylated (have a tail of tors that control the dose, and vaccination tables that
adenines) but they are not in bacteria. The bovine growth allow unvaccinated fish to remain anaesthetised, whilst
hormone polyadenylation signal (BGH polyA) after the multiple vaccinated fish gently flow to recovery tanks mean that
cloning site ensures that the gene being expressed has a polyA a few skilled individuals can vaccinate many tens of
tail and will be correctly translated in the fish. Source: Reproduced thousands of fishes per day. A typical small‐scale vac-
with permission from Andy Barnes, 2017. cination set up on a barramundi farm in Queensland is
shown in Figure 12.2. Occasionally, vaccines may be
incorporated into the fish genetic material or germline delivered via intramuscular (IM) injection. This is
and is therefore never passed on to progeny – the vacci- mandatory for DNA vaccines as host cells need to be
nated fish is genetically identical to the unvaccinated fish transfected by the plasmid and injection of DNA vac-
because its chromosomes remain unaltered. cines into the peritoneal cavity appears to be ineffec-
tive. Apex IHN is generally given as a separate shot IM
Producing plasmid in large quantity and of sufficient at the same time as the fishes receive their usual multi-
purity for vaccinating hundreds of thousands of fish valent IP injectable vaccine against a range of other
requires some substantial scale‐up for commercial use at diseases.
a price that is cost‐effective for farmers. Essentially the
DNA vaccine vector is produced in E. coli in a standard The reason farmers have adopted the apparently labo-
fermentation. The vector contains a bacterial origin of rious practice of IP injection is simply because it is very
replication and either an antibiotic resistance gene or effective. Good vaccines, delivered intraperitoneally to
complementary gene for an auxotrophic mutant to allow healthy fishes with minimum stress will generally protect
for positive selection in culture (Figure 12.8). The plas- throughout the lifetime of the fish, and any modern
mid vector construct is introduced into competent E. coli
264 Aquaculture
Secretion or Muscle cell
cell lysis Protein expression
Proteins sampled
during synthesis
MHCI DNA vaccine
TCR plasmids
Secretion or
cell lysis
Cytotoxicity
Cytotoxic CD8+ Antigen
T-Iymphocyte presenting cell
B-Iymphocyte TCR
MHCII TCR Cytokines
TCR CD4+ MHCII
Cytokines T-helper cell TCR
Cytokines
T & B-cell proliferation and maturation
Serum antibody response
Activation of immune memory
Figure 12.9 How DNA vaccination may hypothetically initiate both cellular and humoral immune responses in fishes. Immune pathways
involved in successful response to DNA vaccination in fishes are not yet clear. However, research suggests that a signal sequence is
important for full protection against rhabdovirus infections in salmonids suggesting the secretory pathway may be important, perhaps
enabling presentation of the target antigen by APCs via MHCII, in addition to MHCI presentation by the transfected muscle cell.
Source: Reproduced with permission from Andy Barnes, 2017.
vaccines will protect against five or more different fishes may seem much more appealing than intraperito-
d iseases from a single shot. This makes them very cost‐ neal injection, it does have a number of disadvantages.
effective over the farm cycle. Firstly, immersion tends only to stimulate the mucosal
immune system, with specific antibody secreting cells
12.5.2 Immersion or Bath Vaccines (B‐cells) detected only in the skin epithelium and gills
It is very difficult to vaccinate small fishes by injection, following immersion vaccination. These B‐cells express a
thus juvenile fish <10 g may be vaccinated by immersion unique class of Ig (IgT) analogous to IgA of the mucosal
in vaccine diluted in clean water from the hatchery. surfaces in mammals (Zhang et al., 2010). The contrast
Generally, immersions are very short with 60 s being the between the immune responses to injection and immer-
usual practice and no improvement is seen with longer sion is illustrated in the diagram (Figure 12.10). Without
immersion. Indeed gill irritation may occur with longer activation of systemic immunity, duration of immunity
immersions. Fishes may be lightly anaesthetised and net- may be very short and frequent revaccination is likely to
ted into a container with the diluted vaccine. After 60 s be required. This becomes a problem as the fishes grow
the fishes are removed, drained and returned to tanks for because they require a larger volume of water and there-
recovery. Only a defined biomass of fishes may be vacci- fore a much greater volume of vaccine, which becomes
nated per litre of vaccine because the antigen may expensive. Furthermore, the mucosal immune system
become depleted as batches of fishes are vaccinated in does not seem to protect at all against some pathogens
the same vaccine. This biomass has to be determined such as Lactococcus garvieae and Streptococcus iniae.
empirically by the manufacturer and included on the This is indicated very clearly in the experimental data
label indications for registration. Whilst immersion of shown in Table 12.3. Rainbow trout vaccinated against
L. garvieae by injection were highly protected in
Systemic Mucosal Prevention of Disease by Vaccination 265
Antibody secreting cells in: Antibody secreting cells in:
subsequent challenges (96–100% relative percent survival
Kidney Skin (RPS)). In contrast, when the same vaccine was given by
Spleen Gills immersion, no protection was achieved (Table 12.3). As a
High serum antibody titre High mucus antibody titre consequence, immersion tends to be used as a primary
vaccination to protect fishes that are too small to inject
Kidney Mucus through the hatchery, with injection subsequently used as
a boost prior to transfer to grow‐out.
Gills
12.5.3 Oral Vaccination
Peritoneal cavity Considered by many to be the ‘Holy Grail’ of fish vacci-
nation, the ability to deliver vaccines with feed would
IP injection Immersion seem to be the ideal solution: The farmer must feed the
fishes every day and sophisticated automated systems
Figure 12.10 Partition of mucosal and systemic immune response exist and are used commercially to deliver precise quan-
in fishes. Injectable vaccines stimulate systemic immunity based tities of feed to appetite. Why not simply include the vac-
on IgM, whilst immersion vaccines stimulate mucosal immunity cines in a batch of feed? The answer is that it doesn’t
based on IgT. The apparent separation of these two systems work. In spite of years of research throughout the 1990s
means that immersion vaccines offer short duration of immunity and 2000s, there is little compelling evidence to suggest
and are unsuitable for some infections. The ‘brick wall’ indicates that protective immunity can be elicited via the oral
that it is hard to activate systemic immunity by immersion or route in a practical manner in fishes. In part this comes
mucosal immunity by injection – the partition between the down to a degree of immune ‘tolerance’ to antigens that
systems seems to be quite rigid. Source: Reproduced with are ingested, to prevent food from generating a high sys-
permission from Andy Barnes, 2017. temic immune response and potential allergy. It is also
partly a result of the hostile environment of the gastroin-
Table 12.3 Difference in protection of rainbow trout from infection testinal tract; with the presence of digestive enzymes and
by Lactococcus garvieae when vaccinated by injection or extremes of pH, even the hardiest of antigens is likely to
immersion. Relative percent survival (RPS) is the percentage be digested long before it encounters any antigen pre-
of vaccinated fish that survive relative to challenged, unvaccinated senting cells. Moreover, the Peyer’s patches associated
control fish and is given as a percentage. A negative indicates that with immune processing in the mammalian gut have not
more vaccinated fish died than controls! Note how immersion been clearly identified in teleost fishes, although there is
vaccination (Imm) fails to protect the fish against this systemic lymphoid‐like tissue in the hindgut of some fish species.
pathogen, whilst injection (IP) with the same vaccine gives very This does not mean that immune protection via the oral
high protection. This is indicative of the different partitions route is impossible in fishes, simply that it is neither easy
of the immune system (mucosal and systemic) that are activated nor a certainty, and is likely to be highly pathogen‐
by the different routes of vaccination. dependent. Most research in the field of oral vaccines
has centred on the protection of the antigen against
Vaccine Batch Adjuvant Route of delivery RPS (%) digestive processes of the foregut and midgut in the hope
that if the antigen reaches the more absorptive hindgut
001/021 – IP 100.00 intact it will be taken up and processed. Of the large
001/036 – Imm number of potential ‘carriers’ that have been used to try
001/048 M1 Imm 3.05 to confer immunity on feed, a few are worthy of note.
001/048 M2 Imm Firstly, antigens have been successfully transported
001/048 P1 Imm −6.35 across the hindgut wall in trout and salmon by encapsu-
001/048 – Imm −23.83 lation in polylactide co‐glycolide microspheres (PLGA),
001/048 – IP −20.63 and PLGA particles have been found in the head kidney
(the major haematopoietic tissue) of the fishes after oral
3.07 administration. However, there is no evidence of any
protective immunity developing by this means. A com-
96.40 mercial oil‐based system for protecting antigens through
the gut has also been marketed. This essentially consti-
Source: Data from unpublished research by the author, 2001. tutes oil and a surfactant used to make an emulsion
c ontaining the antigen, which is then top‐dressed onto
feed and given to the fishes. Once again, evidence of
266 Aquaculture fishes that are sold round for the plate, market accept-
ance of oil‐adjuvanted vaccines has been slower.
protective efficacy is scant and the manufacturer gener-
ally recommends these formulations as boosters rather Side effects of oil‐adjuvanted vaccines have received a
than a primary vaccine. More recently, a system that lot of attention. In some cases, a localised immune reac-
makes the gut more amenable to vaccination has been tion may occur around the oil in peritoneum, resulting in
developed. In this proprietary system, a feed additive is melanotic adhesions that downgrade the market value of
given prior to vaccination that neutralises gut pH, inacti- the fillet or even make it unmarketable. Many compara-
vates gut proteases and increases gut wall permeability tive studies have been performed to try to determine
to permit antigen uptake. This appears to be an innova- why adhesions occur in some fishes and not others, and
tive approach but again data on consistently reliable effi- with some vaccines, but not others. Several hypotheses
cacy are not available and it has not been adopted have been put forward with explanations ranging from
commercially on farms in the decade following initial toxicity of the bacterin components (the adjuvants alone
publication. For some fish species, such as catfish, injec- do not seem cause adhesions), to poor practice during
tion is quite difficult. With the rapid growth of Pangasius injection (such as use of blunt or oversized needles) and
sp. catfish aquaculture in Asia and demand for upwards resulting infection of the injection pore. Regardless of
of 100 million doses of vaccine within this industry in the cause, customer dissatisfaction has prompted devel-
Vietnam alone, research into oral vaccination may enjoy opment of micro‐dose vaccines (given in very small
resurgence. However, as hatchery technology and prac- v olumes) by some companies, and these seem to reduce
tices continue to improve in Vietnam, the opportunity the problem.
for reliable injection vaccination using tried and tested
technology also increases. 12.6.2 Adjuvants for Other Types of Vaccine
Currently, there are no adjuvants routinely used for
12.6 Adjuvants immersion vaccines, although adjuvants that stimulate
systemic as well as mucosal immunity following immer-
Adjuvant roughly translates as ‘helper’. Adjuvants are sion vaccination would be extremely useful against some
substances added to vaccines to improve the immune diseases. With the advent of DNA vaccines, inclusion of
response. host cytokines within the vector so that they are
expressed along with the target antigen is an intriguing
12.6.1 Adjuvants for Killed Injectable concept. Cytokines are signalling molecules secreted by
Vaccines cells of the immune system in order to activate immune
Initially alum‐based adjuvants were favoured and sub- processes such as T‐cell maturation and macrophage
stantially improved immunity to simple killed bacterial activation (Table 12.1). Detailed knowledge of cytokine
vaccines. However, the advent of oil‐adjuvants revolu- mechanisms of action and discovery of the genes in the
tionised salmon aquaculture. Oil‐adjuvanted vaccines target species is an area of research that is progressing
are stable emulsions of various types prepared using the rapidly in fishes at present and has been reviewed
aqueous vaccine, a mineral or non‐mineral oil, and usu- (Secombes, 2008).
ally a small amount of surfactant to help the emulsion
form and remain stable. Most are formulated as water‐ 12.7 Vaccination in Practice
in‐oil emulsions with the aqueous component (usually
the antigens) encapsulated in the oil carrier as micro‐ The type of vaccine, presence or absence of an adjuvant,
droplets. The level of protection and duration of immu- and the route by which it is delivered can only be recom-
nity afforded with oil‐adjuvanted killed bacterial vaccines mended by vaccine manufacturers following extensive
against a range of bacterial diseases in salmon is unprec- experimental and field use of the product. Some indica-
edented, but the way in which oil adjuvants work is tors of how all of these factors interact and effect time to
unclear. They may improve antigen presentation or stim- protection and duration of immunity are presented in
ulate better cellular immunity, but one of the major Table 12.4.
modes of action appears to be a depot effect – the oil
emulsion remains in the peritoneum and prolongs The development of reliable vaccines, inclusion of
release of the antigen. Indeed tiny amounts of oil emul- adjuvants and a raft of supporting research into viru-
sion can occasionally be found within the peritoneum of lence of fish pathogens and immunity in teleost species
salmon during harvest. This is considered acceptable in have made a major contribution to the economic success
salmon as they are generally sold as fillets. However, in and improved environmental impact of fish farming.
Prevention of Disease by Vaccination 267
Table 12.4 Effect of vaccine type and route of administration on immune response and duration of immunity (DOI) in fish.
Killed Adjuvant Systemic Cell‐ Mucosal DOI
IP injection immunity Mediated immunity
IM Injection Oil Long
Immersion No Yes No No Modest
No Yes No No Short
Recombinant No No Yes
IP injection Oil Long
IM Injection No Yes No No Modest
Yes No No
Live attenuated No Long
IP injection No Yes Yes No Modest
Immersion Yes Yes Yes
In‐line Long
DNA cytokines Yes Yes No
IM injection
However, the technology would not work without care- teams or by experienced veterinarians after careful exper-
fully developed farming practices that have maximised imental laboratory and field validation. Provided hatch-
the performance of the products. It is imperative to vac- ery procedures have been followed, the label indications
cinate healthy fishes. In sick or stressed fishes, at best, for the vaccine adhered to and the fishes are handled
immunity will not develop and at worst, the vaccination carefully during and after the procedure the vaccine will
process will kill the fish. The need for healthy fishes for provide protection against the disease agents it contains.
vaccination coincided with significant improvements in
hatchery management and biosecurity. Hatcheries are 12.8 Research and Development
spotlessly clean, broodstock may be routinely tested for Track for Commercial Fish Vaccines
notifiable diseases, eggs are disinfected with ozone or
licensed biocides before being brought into the hatchery, Projects to develop and register new vaccine products
water is treated before flowing through the hatchery and have to be carefully managed to keep them progressing
personnel follow strict biosecurity procedures for entry and make sure that they don’t swallow up more money
and introduction of equipment. In this way, disease is than the final product market is worth. The research and
excluded from the hatchery until juveniles (now specific development stages for a typical commercial vaccine
pathogen free, SPF) are large enough to vaccinate. project are shown in Figure 12.11.
Subjecting the animals to stress during vaccination is 12.8.1 Research Phase
eliminated by careful fish handling and injection under The early phase of research or discovery phase is
general anaesthesia. The advent of highly selected, essentially screening and experimentation to find key
domesticated lines of fishes has also had a significant antigens, strains with particular properties or culture
impact on the practical use of vaccines. Domesticated conditions with promise for a defined problem within a
lines of salmonids are much harder to stress than their key market. The most promising candidates will then be
wild counterparts, evidenced by lower plasma cortisol taken forward for further research and evaluation to
levels. Coupled with the lower individual variability deliver proof of concept, preliminary safety and efficacy
amongst domesticated salmon, a more uniform response information and to allow some optimisation of growth
to vaccination with fewer non‐responders is generally and delivery conditions. At this stage, provisional pro-
achieved. Water temperature and size of fishes at duction outlines may be drawn up to enable potential
v accination are also taken into account to maximise the cost of goods to be estimated for internal rate of return
efficacy of the vaccine. calculations on the potential product. This is required
Generally, an optimum size range of fishes and opti-
mum water temperature range for safe vaccination will be
recommended by the vaccine manufacturers’ technical
268 Aquaculture
Sales product S STEWARDSHIP Figure 12.11 Commercial vaccine R & D
Commerical experience in major markets track. Flow diagram showing a typical
research and development track for a new
Documented product M REGISTRATION commercial vaccine for aquaculture.
Registration dossier & MARKET Source: Reproduced with permission from
Manufacturing dossier Andy Barnes, 2017.
Marketing platform INTRODUCTION
De ned product D2 CLINICAL DEVELOPMENT
Final formulation PRE-CLINICAL DEVELOPMENT
Key lable indications
Safety pro le
D1
STOP/GO Potential product EVALUATION
Proof of concept
R2
Preliminary safety and efficacy data
R1 Idea SCREENING
Promising antigen or strain with
activity in line with sector strategy
even at this very early stage, as the key decision on pre‐clinical development phase there must be a fully
whether to proceed into development needs to be made defined product.
at this point in the project. Research may seem expen-
sive, but once into the development phase, costs really Clinical development is the generation of all the
start to escalate. Moreover, researchers tend to become required documentation for registration in target coun-
very attached to projects and an objective decision to let tries, developing a manufacturing dossier and creating a
a project lapse if necessary is critical. Therefore, the mar- marketing platform in anticipation of product launch.
ket potential of the product needs to be established, the The registration dossier will include safety and efficacy
likelihood of high efficacy/ease of use needs to be clear, data determined under laboratory conditions (usually
and the feasibility of production scale‐up and registra- under some form of quality assurance scheme such as
tion also needs to be considered. Good Laboratory Practice or ISO). It will also include
clinical efficacy and safety in the field following granting
12.8.2 Development Phase of necessary animal test certificates, often conducted
Pre‐clinical development will involve laying down docu- under the reporting conditions of Good Clinical Practice.
mented Master Seed stocks of any organisms in the A marketing platform will be established using field and
product. The formulation of the end product must be laboratory efficacy data to support the product. The cul-
finalised (e.g., concentration, adjuvants etc.). The mination of this phase is the full registration and product
Production Outlines will be finalised and scaled‐up pro- launch.
duction batches made under good manufacturing prac-
tice (GMP). Quality control and quality assurance 12.8.3 Stewardship
methods and parameters must be set. The label indica- The project management plan does not stop with the
tions will need to be determined and any label claims product launch. Most vaccine companies have skilled
proven experimentally under controlled laboratory con- technical teams comprising scientists and veterinarians
ditions using GMP production batches. This will include that will steward the product through its lifespan in the
duration of immunity, time to onset of immunity, and field. Only extensive experience of the product in key
shelf life (it is important to store documented batches of markets will enable any problems to be identified and
vaccine early in this phase because in order to determine remedied. This is an‐ongoing process that requires a
shelf life you have to prove there is no loss of efficacy in good working relationship and flow of information
storage – it can take 18 months to claim a 12 month between the vaccine company and farmers through the
shelf‐life for a product). The safety profile must be also veterinarians and technical field team. Involvement of
determined and specified. Essentially, at the end of the the regulatory agencies will be mandatory should modi-
fications to licensed product be required.
12.9 Future Trends: Vaccination Prevention of Disease by Vaccination 269
in the Age of Genomics
12.10 Conclusions
The rapid advance in gene sequencing technology cou-
pled with equally rapid advance in bioinformatics tools A former colleague at Aqua Health Ltd (one of the pio-
for genome assembly and analysis has had a profound neering fish vaccine companies in the 1980s and 1990s)
effect in the field of biomedical sciences and, as a conse- once explained to me that vaccinating fishes was rather
quence, a revolution in human medicine is beginning. like buying an insurance policy: the farmer was buying
The dramatically falling costs of sequencing mean that it reassurance in case disease arose during the production
is now cost‐effective to apply genomics to aquaculture. cycle. To my mind, fish vaccines are very much better
In terms of vaccination there are several ways in which than that. An insurance policy is designed to minimise
the genomics revolution can be exploited. The first is the the risk that it actually has to pay out. In contrast a vac-
application of large‐scale genome sequencing to fish cine is designed (and rigorously tested) to guarantee that
pathogenic bacteria and viruses. Using an approach it will perform every single time – it always pays out. The
termed reverse‐vaccinology (Tettelin et al., 2006) it is quality of the science behind commercial fish vaccines
now possible to use bioinformatics tools to identify sur- therefore has to be excellent, as its validity will be scruti-
face and secreted antigens on pathogens very rapidly nised by farmers, health managers and veterinarians
from the genome sequences as potential vaccine targets. time and time again through the performance of the
By coupling this to large‐scale high‐throughput genome vaccines in the field. Peer‐review for academic scientific
sequencing and analysis of multiple (possibly hundreds) publication doesn’t even come close in terms of repeti-
of strains of the same pathogen, conserved epitopes tion and rigour.
across multiple strains may be identified in silico as can-
didate antigens that will protect across different sero- History shows that the advent of reliable oil emulsion
types. The potential to greatly improve vaccines, injectable vaccines was one of the key enabling tech-
particularly against highly antigenically diverse bacteria nologies in salmonid aquaculture. Their development
such as members of the genus Streptococcus that cause permitted expansion where previously there was con-
widespread losses in aquaculture is obviously high. straint by disease, and reduced chemical use for disease
Genomic analysis of the salmonid pathogen Yersinia control to negligible levels by exploiting the natural
ruckeri has already revealed novel diversity with implica- adaptability of the fish immune system. Vaccines for
tions for vaccine formulation (Barnes et al., 2016). There salmon now represent some of the most advanced tech-
is a caveat; whilst the time and cost taken to identify can- nologies and some of the most cost‐effective vaccines
didate antigens for recombinant and DNA vaccines has in existence. The perseverance and risk‐taking in
reduced dramatically, the time and cost of screening research and development by (at the time) very small
them all for efficacy in fishes most certainly has not! This biotech companies to get the technology to work, the
is a critical rate‐limiting problem. However there is per- open mindedness of regulatory agencies, and most of
haps a way in which genomics can also help here. As all the willingness of salmon farmers to change prac-
complete transcriptomes (assemblies of all the tran- tices to adopt the new technology were essential to this
scribed mRNA from target organs) become available for success story. Vaccine use and development has spread
more fish species, the use of transcriptomics to analyse into most regions where aquaculture is practised and to
the early onset of adaptive immunity in fishes immedi- most farmed finfish species over the last decade. There
ately post‐vaccination, paralleled with longer term anal- are some significant challenges ahead: In warm water
ysis of the fate of those animals post‐challenge may aquaculture strain variability amongst the pathogens
identify correlates of protection that can be detected and rate of antigen release by emulsions developed for
in the transcriptome very soon after vaccination. the longer farm cycle of cold water species is reducing
Transcriptomics permits us to ask the open question reliability of vaccines. Parasites, such as gill amoeba,
‘what is changing?’ in terms of gene expression because sea lice, and flukes, are a substantial problem in the sec-
we can analyse quantitatively the expression of every- tor. Vaccines are technically very challenging against
thing in a particular tissue such as blood, head‐kidney or parasites in any field and they will require innovative
spleen where previously we were constrained to a hand- solutions. Genomics and transcriptomics studies of
ful of genes for which we had sequence information. This both parasite and host will certainly play a role in
has potential to greatly reduce the time required for understanding the challenges presented by parasite
screening the effects of antigens. vaccines. But these challenges will be met and the
resulting technologies will be adopted by farmers
because, as history has demonstrated, the science will
be excellent and the price will be right.
270 Aquaculture (the time taken to establish an antibody response and
immune memory) is temperature dependent. Vaccine
12.11 Summary kinetics is therefore measured in degree‐days (time in
days x temperature in degrees C).
●● Teleost fishes have a highly evolved adaptive immune
system capable of being programmed to recall and ●● Different types of disease agent require different types
respond to previously encountered threats from bacte- and routes of vaccination, reflecting the partitions of
ria, viruses and parasites. Vaccination of fishes exploits the immune system that need to be activated. This
this adaptive immunity by establishing immune mem- means that the simplest route of administration or
ory of a particular pathogen in advance of exposure to type of vaccine is unlikely to be the most effective, as it
the disease in the farm environment. may not effectively activate the correct branch of the
immune system.
●● Immune memory is activated by strictly regulated
clonal expansion of specialist cells based on their ●● Most bacterial diseases can be effectively controlled
mutual recognition of patterns detected in the patho- through use of injectable vaccines. Advanced DNA
gen or vaccine. Some of these cells are long‐lived and vaccines that can activate cytotoxic T‐cell‐mediated
are stored in specialist immune tissues where they can immune memory critical for protection against some
initiate a response that is much faster and much greater viruses are now commercially established in aquacul-
than the original encounter of those patterns. ture. Mucosal immunity, which may be required for
improved resilience to ectoparasites such as gill
●● Vaccination establishes a primary antibody response amoeba and sea lice, can be established by immersion
over several weeks, and results in populations of B and vaccination but the duration of immunity is very short.
T‐lymphocytes specific for patterns in the vaccine that More research is needed on mucosal immunity.
can produce a very rapid and high antibody response
in the blood on subsequent exposure to the same path- ●● Rapid advances in genomics are accelerating discovery
ogen. Antibodies can kill or inhibit pathogens directly of protective antigens against bacteria and viruses but
or may label them for easy detection and elimination testing candidate vaccines in fishes is still a laborious
by patrolling phagocytic cells of the immune system, and rate‐limiting step in the development track.
protecting the fishes from the establishment of the Genomics may help to identify early correlates of pro-
disease. tection thereby reducing both testing time (and cost)
and the animal welfare implications of vaccine testing.
●● Because successful establishment of immune memory
requires large‐scale cell division, effective vaccination ●● Prevention of disease by vaccination is the most cost‐
is highly energy dependent and therefore considera- effective, environmentally friendly, safe and ethical
tion of feeding and growth during the vaccination way of reducing losses in aquaculture and a critical
period is important. Because most fishes are ectother- foundation to our future food security.
mic, the speed at which vaccination becomes effective
R eferences labrax, L.) systemic and mucosal antibody secreting
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Edholm, E.S., Wilson, M. and Bengten, E., 2011.
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273
13
Post‐harvest Technology and Processing
Allan Bremner
CHAPTER MENU 13.15 Concepts: Quality, Freshness, Shelf Life and
13.1 Introduction, 273 Quality Index, 285
13.2 Basic Characteristics, 274
13.3 Safety and Health, 274 13.16 Microbiology, Specific Spoilage Organism (SSO)
13.4 Nutritional Aspects, 275 and Other Spoilage Processes, 287
13.5 The Balance between Safety and Nutrition, 276
13.6 Aquaculture and Fisheries Products, 276 13.17 Freezing and Frozen Storage, 288
13.7 Harvesting, 276 13.18 Packaging, 290
13.8 Live Transport, 276 13.19 Quality Control, Quality Assuarance,
13.9 Muscle Structure: Rigor and Texture, 278
13.10 Stunning and Post-Mortem Processing, 280 HACCP and Risk Assessment, 293
13.11 Effects of Feed on the Product, 283 13.20 Traceability, Identification and Origin, 294
13.12 Specialised Niche Market Products, 284 13.21 Canning, 295
13.13 Flavours and Taints, 284 13.22 Smoking, 295
13.14 Texture, 285 13.23 Summary, 296
References, 296
13.1 Introduction biochemical composition, the accumulated results of
bacterial and enzymic action and the effects of processing:
Post‐harvest technology encompasses areas of harvest, ●● characteristic odours of a product are the result of
handling, slaughter, processing, packaging, manufacturing,
storage, transport, distribution and display, through to volatile compounds; and
the point of sale or the time of consumption. This ●● flavour is due to both volatile and non‐volatile
chapter can therefore only be considered as an intro-
duction to the topic and the reader should look also to compounds.
monographs and proceedings of conferences that are Inherent non‐volatile compounds of importance in this
listed in the References for further depth of detail regard are salts, amino acids and other non‐protein
(Sikorski et al., 1994; Sato et al., 1999; Kestin and Warriss, nitrogenous compounds and, in particular, the break-
2001; Bremner, 2002; Nesheim and Yaktine 2007; down products of the purine nucleotides. The levels of
Alasalvar et al., 2010; Boziatis, 2013; WHO/FAO 2012). these factors in aquaculture products can be affected by
harvesting and processing. Volatile components arise
The scope of post‐harvest technology is broader in from feeds; many are lipid based in origin and they are
aquaculture than in capture fisheries, since the selection, products of either enzymic or chemical oxidation (Haard
breeding, rearing, feeding and farming conditions can all and Simpson, 2000; Lindsay, 1990). Hence, both nutritional
be controlled to affect the properties of the product and practices, and processing and storage can have profound
must be recorded into cross‐referencing databases. The effects on the flavour and stability of the product.
appearance, odour, flavour and texture of aquaculture
products have a huge bearing on their marketability and The texture of the product is of considerable impor-
acceptability by the consumer. The odour and flavour of tance too, and harvest stresses and post‐harvest prac-
either raw or cooked product is a result of the inherent tices are factors that combine to affect textural
characteristics.
Aquaculture: Farming Aquatic Animals and Plants, Third Edition. Edited by John S. Lucas, Paul C. Southgate and Craig S. Tucker.
© 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.
274 Aquaculture chemical impurities. Since the environment of the
aquaculture production cycle is controlled for the most
In this chapter, emphasis is given to the interaction part, it could be expected that the product would be free
between the pre‐harvest practices and the post‐harvest from harmful organisms. It is the task of the industry to
product, since this is an area of vital concern to ensure this potentially beneficial situation is not eroded.
aquaculture and considerable research is needed. The However, even in the relatively controlled environment
material presented relates mostly to aquaculture products of brackish water ponds, cultured shrimp can be contami-
grown for human consumption. However, the consider- nated with bacteria such as Salmonella species and Vibrio
ations of harvest practices that involve low stress are cholerae. For example, of 304 samples of shrimp and
relevant to species reared for other purposes such as mud/water samples from 131 shrimp ponds, Salmonella
restocking of waterways for ecological, aesthetic or species were present in 16% of shrimp and 22% of mud/
recreational purposes. water samples, and V. cholerae occurred in 1.5% of
shrimp and 3.1% of mud/water samples.
13.2 Basic Characteristics
In some instances, aquaculture forms part of an
There are several basic characteristics that influence existing farming activity and runoff containing faecal
post‐harvest technology and processing, and these must material from terrestrial farm animals and contamination
be determined for each cultured species. These are: from waterfowl may introduce unwanted organisms.
●● the gross proximate composition (water, crude protein, The practice of fertilising ponds with manures and other
organic material (Chapters 2, 4, 9 and 18) can also
fat, crude fibre, ash, nitrogen‐free extract) including introduce unwanted organisms. Most manufactured
seasonal variations; feeds are likely to be free of problems, but intensive
●● the yields of edible product; culture practices that lead to wasted feed and high faecal
●● the composition of the edible product; loads provide an excellent environment for proliferation
●● the gross morphology of the edible parts; of undesirable bacteria.
●● alternative processing forms;
●● the distribution of by‐product and off‐cuts and their Failure to remove dead animals can even lead to
composition; outbreaks of botulism in fish as a result of cannibalism.
●● the chilled life of the product (0 °C); These ponds need to be completely drained and the soil
●● the frozen storage characteristics of the product; totally removed before the area is fit for use again.
●● the pattern of rigor mortis;
●● the inherent bacterial flora; Problems of harmful micro‐organisms are relatively
●● the nature of the spoilage bacterial flora; and less important in extensive systems or in situations in
●● the inherent nucleotide composition and its rate of which there is a high rate of water exchange. However,
change during storage. a number of problems can occur with seafoods from
Although all these characteristics are affected to some estuarine environments, which are partly due to the
extent by harvest regimes, selection, husbandry practices, nature of the animals involved. For example, because
feed and by a host of other factors, it is important that bivalve molluscs, such as oyster and mussels, are filter
they are determined. It is also important to determine feeders, they remove bacterial and viral particles from
the proportions of red to white muscle in a fish species, the surrounding water. This situation is worsened
the position of lipid deposits, the structure of the muscle because bivalves are often eaten raw and the whole ani-
and the skeletal structure. mal, organs and all, is consumed. Bivalves are readily
contaminated by organisms of faecal origin from raw or
13.3 Safety and Health improperly treated sewage (section 24.5.3.2). Prime
agents appear to be Norwalk or Norwalk‐like viruses
Aquacultured foods generally have a good safety record that cause viral gastro‐enteritis. Vibrio organisms are
(Nesheim and Yaktine, 2007; Alasalvar et al., 2010). Most also involved and one species, Vibrio vulnificus, can be
of the major food‐poisoning organisms of public health particularly virulent. Outbreaks often occur after rain-
significance, at least in the developed countries, do not fall, since the resulting decrease in salinity releases
occur in the aquatic environment unless it has been organisms from the sediments into the adjacent water
polluted by human activity. The runoff from farming, column where they can be ingested by the animal.
animal excrement, human sewage and sullage are the In addition, rainfall floods extra water into sewage
main sources of viral and bacterial contamination, systems causing flushing of untreated sewage.
and industrial contamination contributes metallic and
Another major cause for concern is the presence of
natural blooms of microalgae. In freshwater, blooms of blue‐
green algae may cause toxins which can be passed along
the food chain. In estuarine and marine environments,
paralytic shellfish poisoning (PSP), diarrhoeic shellfish Post‐harvest Technology and Processing 275
poisoning (DSP) and amnesic shellfish poisoning (ASP)
can occur with consumption of shellfish contaminated The application of more advanced methods of analysis
as a result of algal blooms (Nesheim and Yaktine 2007; for heavy metals has shown that many values recorded
section 24.5.3.1). When these occur, there is no option for fish in the older literature are in error and are far
other than to close the site to harvest. Monitoring pro- higher than the levels found by these more accurate
grammes and closures are now employed worldwide to methods.
prevent diseases from this source. Although the toxic
algal blooms are ‘natural’, there is growing evidence In general, farmed fishes are less likely to be infected
for the spread of these species in the ballast‐water of with parasites, but this cannot be taken for granted.
cargo ships. Many parasites inhabit portions of the fish, such as the
head, guts and belly flap areas, which are discarded
Other ‘natural’ water‐borne bacteria, such as during processing. Others, however, such as the anisak-
Aeromonas species, Vibrio vulnificus and Vibrio para- ids, are found in the flesh. These parasitic nematode
hemolyticus, have been associated with poisoning, worms are highly infective for humans but are destroyed
particularly in the summer months in temperate areas. thorough cooking and on freezing. More recently, the
The infective doses are not known for each organism, practice of eating raw fish or lightly preserved or pick-
but they are small. Consumption of raw shellfish must led fish dishes, such as ceviche, has become more com-
always be approached with care. mon in Western societies. It is therefore very important
that the product is monitored for the presence of
The process of depuration, that is treating the shellfish parasites.
for several days in clean, running water, has been adopted
in some areas (section 24.5.3.2). This process is partly 13.4 Nutritional Aspects
effective in reducing bacterial numbers, but viruses are
not easily removed. Far more effective is a properly The products of aquaculture are health giving and
designed and enforced quality assurance system in which nutritious, being good sources of vitamins, minerals
growing sites are selected to be free from problems in the and protein with a low total‐fat content and low glycae-
first instance and in which water quality is continually mic index. Marine products represent by far the best
monitored to ensure that no deleterious changes have natural source of the highly unsaturated fatty acids
occurred. (HUFAs) eicosapentaenoic acid (EPA) and docosahex-
aenoic acid (DHA). These fatty acids have important
These ‘shellfish sanitation’ programmes can be inte- roles in membrane functioning and as precursors for
grated into broader industry programmes involving biologically active compounds. They are considered
total quality management (TQM) and the hazard anal- beneficial in human nutrition in reducing plasma cho-
yses critical control point (HACCP) systems (Nesheim lesterol and triglyceride levels, and in reducing the risk
and Yaktine 2007). Schemes such as these are gener- of cardiovascular disease. They are used in treatments
ally a requirement for export. A general Code of for arthritis and other ailments (Nesheim and Yaktine
Practice for Aquaculture drawn up by the Codex 2007; FAO/WHO 2010). They are essential for develop-
Alimentarius Commission of the World Health ment of the brain and nervous system in infants, as a
Organization and the Food and Agriculture high proportion of DHA is found in the grey matter and
Organization provides a very good guide to practices nerve tissues.
required to ensure a safe product (FAO, 1995).
Recently the process of subjecting the shellfish to high As steps are taken to decrease the proportion of
pressure in a chamber is being commercially explored fishmeal and fish oil in aquaculture feeds (sec-
tion 8.6.5.1), so the levels of EPA and DHA in cultured
as a batch‐wise means of both significantly reducing fish will decrease unless alternative sources are found
bacteria, e.g., V. vulnificus, and aiding in releasing the such as adding the genes for them to plant crops. The
meat from the shell. The results of high pressure on level of these fatty acids is one of the major desirable
selling points for aquatic produce. Clearly, a balance
viruses are less well known so far. needs to be struck between maintaining high levels of
Chemical contamination invariably occurs through EPA and DHA and decreasing product costs by alter-
ing feed composition.
exposure to toxic wastes dumped or leached into the
aquatic environment. The potential for contamination Marine aquaculture products are a good source of
because of previous activities in the area or from adja- selenium, an essential element, which is a co‐factor
for the antioxidant enzyme glutathione peroxidase.
cent application must also be considered. However, Oysters and other shellfish are also good sources of
some natural accumulation of heavy metals, such as iodine.
mercury, can occur if mercuriferous ores (e.g., cinnabar)
are found in the region. Again, considerable attention
needs to be paid to the siting of aquaculture ventures.
276 Aquaculture ●● rises in blood cortisol;
●● an increase in metabolic rate;
13.5 The Balance between Safety ●● utilisation of glycogen resulting in:
and Nutrition
–– increases in lactate levels; and
The general consensus of medical and technical opinion –– decrease in tissue pH.
is that, in general, the benefits of eating aquaculture The provision of good aeration can help relieve this situ-
products far outweigh any potential risks (Nesheim and ation, but every effort must be made to reduce obvious
Yaktine 2007; FAO/WHO 2010). The broad range of stress factors during harvest (Robb, 2001). In rested fish,
nutrients and trace minerals, the contribution of the cortisol levels in the blood are generally below 10 ng/mL,
essential HUFAs, the low glycaemic index, the high but values of up to 10 times this level are common in
quality protein, and the high levels of free amino acids stressed or exercised fish. Similarly, lactate levels in the
and related non‐protein nitrogen products coupled with blood and muscle of rested fish are many times lower
ready digestibility make aquaculture products a most than in stressed or exercised fish. There is some evidence
nutritious natural food. that chronic stresses, such as those experienced during
harvest because of overcrowding or poor water quality,
13.6 Aquaculture and Fisheries Products result in a different mix of biochemical indicators from
those that may result in the imposition of a sudden stress
It is difficult to directly compare aquaculture products such as physical exertion due to capture.
with wild‐caught products. The variability in genetics, in In crustaceans and molluscs, the expenditure of energy
feed composition and in growth conditions makes com- in struggling for survival is met from arginine phosphate
parisons difficult to interpret except in the broadest and from glycogen. In crustaceans, the main end product
sense. Preferences for wild or farmed products will vary is generally lactate, but octopine is formed from the
greatly according to season and location of capture. In reaction of pyruvate with arginine in many bivalves and
general, aquaculture products tend to provide flesh that gastropods. In the gastropods, tauropine, alanopine and
in comparison with wild‐caught products is: strombine are similarly formed. Tauropine is formed in
●● softer in texture; abalone, and levels of these compounds have been
●● less strong in flavour; suggested as indicators of harvest and transport stress.
●● often of different hue; and
●● with a higher but more uniform oil content. 13.8 Live Transport
These differences are, in part, due to the fish being younger.
Many markets prefer their products to arrive live to
Atlantic cod reared in pens have higher contents of ensure freshness and to allow consumers to select their
sarcoplasmic proteins and different water‐binding own dish or, in some instances, to eat the product within
capacities compared with wild cod. Both factors affect seconds or minutes of death or, as in the case of shellfish,
the texture of the cooked flesh. Wild fish have to swim while still live (Figure 13.1). The returns for these markets
more than captive fish, and the myoglobin content of are often higher, but the risks are greater since the
their red muscle is often higher. Lower levels of free associated costs of transporting animals out of their
amino acids have been found in cultured coho salmon, natural environment can be considerable.
red seabream and ayu than in their wild‐caught counter-
parts. Similarly, other non‐protein nitrogen compounds Live Kuruma kreuruma shrimp (Marsupenaeus japonicus)
appear to occur in lower concentrations in cultured spe- fetch prices of approximately ¥13 500 per kilogram
cies. Variations in composition and distribution of tissue (ca. USD 120) in the Tokyo Tsukiji market (Figure 13.2).
constituents occur both with changing seasons and with This market is supplied by domestic sources, and from
maturation. There are also considerable seasonal varia- Taiwan, France and Spain. The overwhelming source,
tions in the mineral content of both wild and cultured however, is from China. Getting the product to the
fish, and textural changes are also apparent. market in a live state requires appropriate harvest
practices. The shrimp are active only at night and must
13.7 Harvesting be harvested then by the gentlest means possible. Simple
tangle nets or tunnel nets set near paddle wheels are
There are stresses during harvesting that may affect the appropriate. The shrimp are gently laid on to floating
final product. Crowding of fish in a net or in raceways wooden trays that stack one on the other and are trans-
during harvest leads to: ferred in these to a chilling tank to bring their tempera-
ture down to ~16 °C. The tanks are taken from the ponds
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