May | June 2014

Raindow Trout fry syndrome

The International magazine for the aquaculture feed industry
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I NCORPORAT I NG
F I SH FARMI NG T ECHNOL OGY
S
almonid aquaculture has suffered
from a number of different ailments.
Infectious Salmon Anaemia, caused
by the Isavirus from the ortho-
mixoviridae family, led to a major crisis of
the Atlantic salmon aquaculture industry
in Chile, dramatically reducing production
numbers.
Ectoparasitic conditions such as sea lice
infestations and amoebic gill disease are affect-
ing the salmon aquaculture industry through-
out the world, causing major economic losses.
Bacterial infections are also common, caus-
ing diseases such as Furunculosis or Bacterial
Kidney Disease.
One of the bacteria that causes problems
in salmonids is Flavobacterium psychrophilum.
The first time it was isolated, it was from the
kidney and external lesions of diseased coho
salmon in Washington, USA [3].
At that time, the disease was given the
name peduncle disease [12]. Infections with
F. psychrophilium have since been labeled
with many different names [3, 39, 26, 49,
7, 28, 25] but currently the names Bacterial
Cold Water Disease (BCWD) [17, 22] and
Rainbow Trout Fry Syndrome (RTFS) [40, 14]
are commonly used in the USA and Europe
respectively [1].
Until the 1980s the disease was only found
in North American salmonids at low water
temperatures (6-10C) [15]. Now it is found
throughout the world.
RTFS has been identified in North America
[17, 16], nearly every country in Europe [2, 43,
49, 53, 27, 38], Australia [44], Chile [52], Peru
[25], Japan [52, 19] and Korea [23]. It can now
infest non-salmonid hosts, like common and
crucian carp [24], European and Japanese eel
[24, 20], ayu [52, 23], pale chub [19], roach
[29] and tench [24].
The bacterium probably affects all types
of salmonids [36] but the most susceptible
species are rainbow trout and coho salmon
[11, 22, 8].
The most severe outbreak of disease
occurs in sac fry, where mortalities can reach
up to 50 percent. In fingerlings, mortalities can
range between 5-20 percent [15].
Furthermore, it has recently been noted
that it is capable of causing disease at much
higher water temperatures [15].
The bacteria
Flavobacterium psychrophilum are Gram-
negative bacteria cells that are flexible, weakly
refractile, slender rods with rounded ends [17,
41, 30, 36]. The disease starts with the pres-
ence of the pathogen in the water. Infected
fish shed bacterial cells into the water and
dead infected fish shed cells at much higher
numbers [31, 29]. The bacteria can also sur-
vive for several months in fresh water outside
the host [54, 51, 32, 35, 50]. Such infections,
from one fish to another or from environmen-
tal sources, are called horizontal pathogen
transmissions. Vertical transmissions are from
the parents to the offspring. This type of trans-
mission is also probable with RTFS [5, 47].
The bacteria have been found on the exterior
of the egg, the ovarian fluid and in milt [5,
47, 33]. Taking all this into consideration, it
is assumed that the bacteria can enter the
host via a combination of routes [36] and its
adhesive properties can help in the successful
colonisation of its tissues [36].
There are many environmental strains
of F. psychrophilum and not all of them are
pathogenic. There are at least three main
serotypes [27, 21, 48] and several distinct
genetic lineages with considerable genetic
RAINBOW
TROUT
fry syndrome
Table 1: Haemocytic activity of white leg shrimp
Parameter Control
Enro
10ppm
OS
500ppm
OS
1000ppm
OS
2000ppm
Pool SE
Month 1 Phagocytosis (%) 13.12 10.29 10.02 13.32 15.74 1.821
Phagocytic Index 2.05 0.77 0.72 1.96 2.43 0.7281
Prophenol Oxidase Activity 0.17 0.21 0.5 0.59 0.58 0.0689
Month 2 Phagocytosis (%) 10.34 12.89 10.91 11.38 13.82 2.6784
Phagocytic Index 1.15 1.16 0.98 1.19 2.6 0.6926
Prophenol Oxidase Activity 0.07 0.09 0.14 0.2 0.2 0.0228
Month 3 Phagocytosis (%) 44.95 52.44 69.71 77.76 82.09 5.5831
Phagocytic Index 30.01 34.6 62.21 81.61 82.35 13.9416
Prophenol Oxidase Activity 0.59 0.94 1.25 1.59 1.57 0.384
Aquaculture in its brief, modern
history has been hit by a number
of different diseases. Bacteria,
viruses and parasites can cause
serious problems for a fish farmer
by Kat Konstanti, International Technical Support Specialist, Meriden Animal Health
14 | INTERNATIONAL AQUAFEED | May-June 2014
FEATURE
and virulence variation [9, 29, 20, 45, 46, 37,
16, 38, 1]. This fact turns this disease into a
bacteriology minefield.
Treatment
The main method of treatment is use of
antibiotics. Oxytetracycline has often been
used around the world against RTFS [39, 4,
22, 28]. Amoxycillin and oxolinic acid have
widely been used In Europe [4, 6]. However
the development of antibiotic resistance
makes antibiotics an unsustainable method of
treatment [15].
A lot of effort has been put into develop-
ing a vaccine but little progress has been
made so far [15, 1]. Some experiments with
injectable vaccines, that have used either
formalin-killed or heat-inactivated bacteria,
have shown some promise but such vaccines
will be very impractical for farm scale fry
inoculations [15, 1]. Until a viable vaccine is
produced, the industry will have to rely on
the use of antibiotics for treatment, along with
good biosecurity and husbandry measures to
possibly avoid the outbreaks.
Barnes & Brown (2011) [1] suggested
that dietary research is warranted, such as an
evaluation of novel dietary ingredients in feeds
or improved feed formulations that lead to
changes in RTFS susceptibility.
Orego-Stim Aquatract L
Orego-Stim

is a natural feed additive

based on oregano, developed by Meriden
Animal Health. The oregano essential oil acts
as an immunity enhancer and growth promot-
er, with further benefits including antibacterial
and antimicrobial properties.
This section will be divided into two sub-
sections. The first outlines the mode of action
of oregano essential oil and the second dem-
onstrates its immunity-boosting properties
in aquaculture species. All the data in these
sections is obtained from studies conducted
by Meriden Animal Health.
Note: As the scope of this paper focuses
on the antimicrobial and antibacterial effects
of Orego-Stim

, growth promoting results

are not shown in the main text, however
results from commercial trials studying the
growth promoting effects of the product on
Rainbow trout, are shown in appendix 1 for
reference.
Mode of Action
Indirectly, Orego-Stim

acts as a growth
promoter and an appetizer. It improves feed
intake and digestion, boosts immunity and
has antioxidant and anticoccidial properties.
Primarily however, it is an antibacterial.
In general, gram-negative bacteria, in
contrast to gram-positive bacteria, have
both a cytoplasmic membrane and an outer
lipid cell membrane with a much smaller
peptidoglycan in between. As the ancestral
types of bacteria do not have the outer-
layer, it has been suggested that this extra
outer-layer in the gram-negative bacteria
has evolved as a protection against antibiot-
ics. This is a theory that could explain why
Table 2: Phagocytosis Activity of Catfish under normal conditions
Month Parameter Control
Enro
10ppm
OS
500ppm
OS
1000ppm
OS
2000ppm
Pool SE
Month 1
Phagocytosis (%) 42.55 54.55 23.72 56.85 70.31 5.59
Phagocytosis index (%) 20.31 44.62 33.57 45.61 87.12 14.02
Month 2
Phagocytosis (%) 9.54 10.57 14.23 14.22 17.00 4.84
Phagocytosis index (%) 1.36 1.45 4.06 4.19 4.18 2.21
Month3
Phagocytosis (%) 32.73 33.34 29.77 33.85 40.42 5.53
Phagocytosis index (%) 12.72 13.93 18.92 22.07 24.99 4.23
May-June 2014 | INTERNATIONAL AQUAFEED | 15
FEATURE
A/S
gram-negative bacteria are more resistant
to antibiotic treatment.
Unlike antibiotics, OS has a very primitive
mode of action. Orego-Stim contains unique
levels of Carvacrol and Thymol which are
natural phenols that act like disinfectants. The
active ingredients of Orego-Stim

can alter
the bacterias cell membrane, increasing its
permeability and resulting in water imbalance
and cell death. This inhibitory effect is not due
to the transfer of any chromosomes, neither
do they affect the cytoplasmic components
of the cells, thus there is no risk of increasing
bacterial resistance, like in the case of penicillin
or streptomycin (Hui, n.d).
Immunity boosting
Two studies conducted at the Kasetsart
University in Thailand, focused on determin-
ing the effectiveness of Orego-Stim

as
an antimicrobial agent against pathogenic
bacteria. The first study was conducted on
white leg shrimp, Litopenaeus Vannamei.
Five treatment groups were reared for three
months. During that time, three groups were
fed Orego-Stim

at different concentra-
tions (500ppm, 1000ppm & 2000ppm), one
group was treated with the commonly used
in-feed antibiotic, enrofloxacin (10ppm) and
a control group fed with the normal diet.
The phagocytic index and the prophenol
oxidase activity were measured monthly.
Phagocytes are cells that protect the animal
by ingesting foreign organisms such as bacte-
ria. Prophenol oxidase activity measures the
volume of enzymes produced which aid the
phagocytes in clearing pathogens from an
organism. Table 1 shows the results of the
study. It can be seen that the Orego-Stim

treated groups show
better immune func-
tion.
Following the three
months, the shrimp
where challenged with
the virulent gram-neg-
ative bacteria Vibrio
harveyi. The challenge
lasted for 10 days. The
bacteria were intro-
duced in two ways,
by bath treatment and
by oral administra-
tion. In both cases, the
vibrio bacteria count
on the intestine and
the hepatopancreas
in the Orego-Stim

2000ppm group was
equal or lower than
the control or the
enrofloxacin treated
groups. Furthermore,
the Orego-Sti m

2000ppm group exhib-
ited a significantly high-
er survival rate.
A study with the
same outline was con-
ducted on hybrid catfish
to evaluate the immune
status of the fish, studied
under two conditions; normal temperature
(27-32C) and low temperature stress (22-
24C). The results are shown in tables 2 and 3.
Appendix 1: Growth Promotion
Study on Rainbow Trout
A commercial trial conducted by a Denmark fish feed producer
studied the growth-promoting effects of Orego-Stim in rainbow
trout. Table 1 shows the results after the 49-day study.
It can be seen that the inclusion of Orego-Stim in the feed
increases the number of production while decreasing the FCR.
On this study, it was further calculated that (including the cost
of feed and Orego-Stim) the OS group added value of more
than 3000
Table: Commercial Trial Data on the growth promoting effects of
Orego-Stim on Rainbow Trout
Control
Orego-Stim
500ppm
Difference
Average Start Weigh (g) 64.6 67.6
Average Final Weight (g) 107.3 114.3
Weight Gain (g) 42.7 46.7 +9.4%
Starting Biomass (Kg) 1,250 1,250
Final Biomass (Kg) 2,087.5 2,212.5
Biomass Gain (Kg) 837.5 962.5 +14.9%
Production of Fish (Kg) 51,450 57,330 +11.42%
Feed Used for Production (Kg) 47,849 51,597 +7.8%
Feed Conversion Ratio (FCR) 0.93 0.90 -3.2%
16 | INTERNATIONAL AQUAFEED | May-June 2014
FEATURE
It can be seen that in both the normal
and stress conditions, the fish exhibit greater
efficiency to enhance phagocytosis activity and
percentage of phagocytosis, especially during
the first month.
Subsequently they were challenged with
the pathogenic bacteria Aeromonas hydroph-
ila. During the bath treatment challenge, the
groups treated with Orego-Stim

were as
effective as the group being treated with
antibiotics in preventing mortalities (100%
survival rate). However the Orego-Stim

1000-2000 groups were significantly more
effective in eliminating A. hydrophilia in the
intestines, without compromising beneficial
lactic acid bacteria.
Conclusion
Rainbow trout fry syndrome has been
plaguing the salmonid aquaculture industry
and has been causing severe economic losses
through mortalities and cost of treatments
which are becoming less effective as resistance
develops. Currently this disease is in the top 5
diseases that cause economic problems in the
UK aquaculture sector, possibly higher in the
EU and USA sectors.
The use of antibiotics is becoming less
and less sustainable. Certain antibiotics have
been banned in animal use in many countries
as they have been proved to be harmful for
human consumption. The antibiotics that are
available are becoming less effective from
overuse or misuse. Furthermore, even when
the antibiotics do work, their use requires
what is called a withdrawal period to
allow for any chemical residues that can
be attained in the flesh to dissipate. During
this time, the fish are vulnerable to further
infections.
As disease outbreaks will probably occur
during a farming cycle, alternative preventative
or therapeutic treatment options have to be
put in place to help the farmers protect their
livestock. With public opinion rapidly shifting
against the use of chemicals and medications
in their food, use of natural products that can
fulfil that job would be the best option for both
worlds.
With Orego-Stim

being this effective

in combating bacteria directly and boosting
the immunity of the organism, alongside its
growth promoting effects, further research on
its ability to prevent outbreaks of RTFS is war-
ranted. The easy in-feed application method
for the farmers, along with the increased food
consumption, the improved gut health and
the antibacterial properties that pathogenic
bacteria cannot defend against, all this turns
Orego-Stim

to the 100% natural additive

everyone has been waiting for.
References
Available at www.aquafeed.co.uk/ref4872
Table 3: Phagocytosis Activity of Catfish under Temperature Stress Conditions
Parameter Control
Enro
10ppm
OS
500ppm
OS
1000ppm
OS
2000ppm
Pool SE
Month 1 Phagocytosis (%) 23.55 26.62 28.50 41.24 41.60 4.51
Phagocytosis index (%) 8.95 8.23 11.43 27.64 27.04 6.60
Month 2 Phagocytosis (%) 5.05 5.66 5.82 5.97 8.48 2.14
Phagocytosis index (%) 0.41 0.50 0.53 0.55 1.23 0.40
Month3 Phagocytosis (%) 28.98 31.53 31.77 33.26 34.62 3.23
Phagocytosis index (%) 10.52 13.10 13.86 13.66 15.54 1.25
MORE INFORMATION:
Kat Konstanti
katkonstanti@meriden-ah.com
May-June 2014 | INTERNATIONAL AQUAFEED | 17
FEATURE
www.aquafeed.co.uk
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