PROBIOTICS IN SHRIMP CULTURE
Question:
I once read an article and there they said: maximal
limit of vibrio in farm is 100.000 cfu/ml. If content of vibrio is more than
100.000 cfu/ml, this is dangerous for penaeids. In my opinion, if the
content of vibrio is over 100.000 cfu/ml, it is easier to get white spot.
What do you suggest about that?
Bagus Satari
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COMMENTS 1:
We manufacture probiotics for aquaculture which have
a good record of protecting shrimp against Vibrio disease. In our experience
with Vibrio, the maximum safe limit depends on:
-
which
species of Vibrio are present, e.g. Vibrio harveyi
-
for a
given species, there is also strain-to-strain effects. For example, some V.
harveyi can exhibit virulence at levels as low as 100/mL, while others
become virulent only at much higher densities, e.g. 10,000/mL (or even
100,000/mL)
-
the
species, age and population density of the shrimp, and the quality of their
food
-
the
overall quality (chemical and microbial) of the water that the shrimp is
growing in
For this reason it is not possible to specify a
precise maximum limit for Vibrio, however you can safely say that if you
have 100,000 Vibrio/mL, your shrimp are in serious trouble.
There have been a number of replies about whether the
presence of Vibrio predisposes the shrimp to succumb to white spot; I think
it is safe to say that Vibrio disease is caused by a toxin that the
bacteria produce, and that the presence of this toxin is likely to
weaken the resistance of the shrimp to a range of other diseases, including
white spot. However, there can be other factors involved in triggering white
spot outbreaks, for example a sudden temperature drop.
Kevin Healey
Research and Development Manager
IAH Manufacturing
e-mail: khealey@iahp.com.au
URL: www.iahp.com.au
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COMMENTS 2:
Speaking about luminiscent bacteria, they have been
quite troublesome to many shrimp hatcheries in certain
seasons. Is the usage of probiotics a viable solution
for this problem?
e-mail: anilghanekar@yahoo.com
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comments 3:
Vibrio spp., and some Pseudomonas sp. have often been
typed as secondary infections resulting from WSSV outbreaks. I suppose the
opposite could be true as it is very difficult to determine if the pathogens
are cause or effect. Particularly in the case of the bacteria it is never
really clear if they attack and weaken the animals or simply multiply in
response to weak and dying animals and take the blame.
There has been a good deal of work with the filtered exotoxins (bacterial
cells removed with 0.01 micron filters) that Vibrio sp. produce and it is
indeed very toxic to all invertebrate species particularly larvae.
Probiotics, especially Bacillus sp. and Lactobacillus sp., offer very good
control of Vibrio sp. I am not a particular fan of using open ocean Vibrio
sp. as a probiotic, in light of the comments in the previous paragraph, but
there has been some demonstrated success with it against luminous Vibrio sp.
It strikes me as trying to use cobras to control mambas and can get out of
hand rapidly.
Phil Boeing
e-mail: pboeing@dc.rr.com
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comments 4:
What kind of toxins are we talking about, EXO or ENDO
(LPS) ?? or both?
e-mail: rretamales@easynet.net.ec
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comments 5:
I believe I said exotoxins in the posting, but in any
case that is what is most investigated. The effect of the supernatant of
specific CFU concentrations of various Vibrio sp. cultures on invertebrate
life stages. A lot of work was done years ago with bivalve larvae and during
the whole hatchery development programs for bivalves where totally axenic
algae cultures and feed regimes were of absolute necessity. I would
like to know about the endotoxin issue but it is a bit more complex to
investigate.
Phil Boeing
e-mail: pboeing@dc.rr.com
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Comments 6:
The work I've read about Vibrio toxins was done with
V. harveyi,
where the toxin was an extracellular protein (i.e. an exotoxin). It
has been separated from the cells that produced it and shown to be
toxic to shrimp. The production of the toxin is thought to be linked
to the mechanism for luminescence.
Kevin Healey
e-mail: khealey@iahp.com.au
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Comments 7:
I agree with Phil about the benefits of Bacillus spp.
as probiotics
for aquaculture; the key is their versatility which means they are
active in the water column and not just in the gut of animals. Also,
they can be delivered as spores, which are very robust, meaning that
they get to your farm in good condition. Lactobacillus spp are OK
gut probiotics (we use them to good effect with land animals) but I
don't believe that they do much for the water quality, and so they
should be included in the feed. Since they aren't spore formers they
need to be supplied in freeze dried form, but even so they can
easily lose activity during transport, storage, feed manufacturing,
etc.
Like Phil I have serious reservations about the use of Vibrio as
probiotics. The basic idea is to use a microbe that closely
resembles the pathogen (the microbe causing the disease), but isn't
itself pathogenic. This should make it a good and harmless
competitor for the pathogen, and if used right it can dominate the
ecological niche where the pathogen normally would flourish. This is
a classical biocontrol method: "competitive inhibition". The risk
is
that microbe chosen as the control agent might suddenly surprise you
by showing-up a previously-unknown pathogenic property of its own.
I've seen exactly this happen in agriculture, where the effect was
catastrophic.
Kevin Healey
e-mail: khealey@iahp.com.au
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Comments 8:
Yes, they can work well in hatcheries. It's best to
use them in all phases of hatchery operation right from the start of a run.
You also need to be careful with your algal cultures - once they move out of
their actively-growing phase they start to release protein (seen as foam)
and are likely to harbour Vibrio. Probiotics aren't used in the algal
culture as they can compete for nutrients. Artemia can also be a source of
Vibrio.
Kevin Healey
e-mail: khealey@iahp.com.au
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Comments 9:
May I ask that in hatcheries, probiotics are dealt
with Vibrio bacteria; how about fungi and protozoa? How would we treat them
if they were in the larval tank? I meant what kind of chemicals or drugs can
be used to kill fungi and protozoa, without affecting a probiotics
community?
M. Gia
e-mail: mgia004@yahoo.com
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COMMENTS 10:
We think there is a phenomenon called
"quorom sensing" where populations of microbe release certain
autoinducers (AI =homoserine lactone ??) that allow bacteria to communicate
with each other. This results in switching on of certain genes
when critical mass is achieved, example for production of toxins.
There have been suggestions that if we block the AI we may be able to
control/suppress luminescence vibrio as one option.
Robin Liew
e-mail: robinliew@myjaring.net
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Comments 11:
Please see my other recent postings on use of
Bacillus as
probiotics. Some Bacillus strains produce an enzyme which can
inactivate the autoinducer - this is called "quorum quenching".
Kevin Healey
e-mail: khealey@iahp.com.au
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Comments 12:
Are these AI-degrading bugs/products already
commercialised ? We have seen the effects in laboratory setting but
commercial application in large water body (???) not heard of any success.
Any comments on the possible usage of bacteriophages against lumi vibrio ?
Phages are host specific and in theory and concept-wise, should be able to
'kill' since they infect V.harveyi.
Robin Liew
e-mail: robinliew@myjaring.net
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Comments 13:
Not enough work was done on exotoxins produced
by vibrios and exoenzymes produced by beneficial bacteria. Some of the
aquaculture microbiologists are also of the opinion that exoenzymes produced
by bacilli have antiviral properties to act against white spot carriers. But
no clear data are available.
Vasudevan
e-mail: hiline@xlweb.com
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Comments 14:
Re the quorum quenching: This is an interesting area
of ongoing
research. My expectation is that at least some, if not all, of the
strains in our commercial probiotics will test positive, because (i)
they're effective against Vibrio & (ii) a number of Bacillus
strains have been shown to be active against autoinducer molecules &
(iii) the Bacillus strains we select for use as probiotics have such
a broad substrate range it would be surprising if they couldn't
degrade these amino acid derivatives. They don't need a specific
transport mechanism to take them up because they secrete their
enzymes out into the water, where the autoinducers are active.
It's worth re-stating that quorum-quenching is just one of the
weapons that a good Bacillus has in its arsenal against Vibrio. Its
a long story, but you can also include direct inhibition via
production of inhibitory metabolites, and competitive inhibition.
Then they help indirectly by lowering the stress on the shrimp by
improving the water quality and improving the pond environment(e.g.
by promoting the degradation of accumulated sludge). And then we can
consider their activity in the animal's gut, for example their
extensive array of degradative enzymes help in digestion of feed.
Re the phage: In principal, this could work, but I can see a lot of
practical problems. As you mention, phages are host specific, and
this carries right on down the the strain level. So the phage may
attack one strain of V. harveyi, but not another. Its an enormous amount of
work which involves constantly testing phage. Also, there are practical
matters to consider like how to manufacture the phage in high numbers, and
stabilise them so they get to your farm in good condition, and then to
work-up the methodology to use them effectively. And then maybe you'll
selectively knock-out the harveyi and just create a "biological
vacuum" for another pathogenic species of Vibrio to thrive in.
It's impossible to conduct practical aquaculture under sterile
conditions; bacteria will always be present, and some of them will
be pathogens. It makes sense to incoculate your pond or tank with
proven probiotic bacteria at the point of fill, so that the
pathogenic bacteria are actively managed right from the get-go.
Kevin Healey
e-mail: khealey@iahp.com.au
www.iahp.com.au
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Comments 15:
There is reason to anticipate that the probiotic
Bacillus species
will in future be proven to be active against "free" viruses in
the
water column - these are just particles with typically a lipid or
protein coat, and with attached docking proteins; all of these
classes of materials are substrates for Bacillus degradative
enzymes. From this it can be seen how probiotics could directly
protect shrimp against unattached virus particles brought-in by
water exchange. However, it’s hard to see how they can do so when the
Pl's arrive from the hatchery already infected with viruses, or when
the vector for infection is cannibalism of another infected shrimp.
In these cases the probiotics can help by maintaining a low-stress
pond environment, so that the shrimp have better resistance to
whatever level of virus challenge they are exposed to.
Kevin Healey
e-mail: khealey@iahp.com.au
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Comments 16:
May I inter-comment on my experiences with luminosis.
Pump the early log phase greenish brown Skeletonema
algae cells to the rearing tank. I mean to say, lower
the larval rearing tank water column by half and fill
the upper half with young greenish Skeletonema
cultures. Your larvae will not die because of
luminosis. The gut luminescence will reduce by 75 %.
Finally you will harvest the tank successfully.
Note : Greenish cells of Skeletonema means 28 hours
after inoculation outdoors.
Dr.Kitto
State of Kuwait
e-mail: ashakitto@yahoo.co.uk
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Comments 17:
As for Vibrio and WSSV I would like to ask why the
problems are seasonal?
A larval or PL tank has very good survival in spite of having high vibrio
and LB load how' that?
A pond in the 4th or 5th month of culture has high vibrio load but still the
crop comes out good.. why?
Generally there are not much of WSSV problems in summer months-why?
We need to be much focussed to really solve these issues..
Ramraj
e-mail: padlab@yahoo.com
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Comments 18:
What is the upper limit for probiotic use, is it
contra indicative at higher doses or just an unnecessary expense?
Anil Ghanekar
e-mail: anilghanekar@yahoo.com
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Comments 19:
We have never used antibiotics in our farms or
hatcheries, and make full use of probiotics
and probiotic principles, and have done so for the past 15 years and will
continue to do so Some years ago (mid 90's) we did some
very intensive research on the effectiveness of various bacillus
strains against vibrios and
viruses, including white spot, and there were clear beneficial results. We
took out patents in Australia and Thailand.
Adam Body
PO Box 39346
Winnellie, NT 0821
Australia
Telephone: 08 89886861
Fax: 08 89886859
e-mail: a.body@bigpond.com
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Comments 20:
On the contrary, in my experience, I still have not
used probiotics in my entire 21 years of experience in shrimp farming ( and
in different countries). Today, I am trying to test using it for 2 reasons;
perhaps for
future use, and just for curiosity since almost everybody is using and
talking about it.
In most cases of my culture problems, I just drain the stock at once and dry
it out, with some sterilizing applications of course. Sacrifices (which
includes financial losses, etc.) may vary too, just like when
you apply something to save the stocks. But I just feel it’s more
practical and it works.
e-mail: leonidoctala@yahoo.com
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Comments 21:
If you visit our website (www.iahp.com.au)
you will find datasheets
and applications bulletins for use of the probiotics in hatchery and
growout. These contain general usage rates that should be good
for most applications. As a rule of thumb, it’s good to monitor for
Vibrio, and if you find that numbers are growing, increase the
frequency or dose rate of the probiotics.
Kevin Healey
e-mail: khealey@iahp.com.au
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Comments 22:
In response to your question about why sometimes high
numbers of
Vibrio seem to be tolerated, I'd say that it depends firstly on how
virulent the Vibrio present actually are - I understand that this
can vary a lot between strains of Vibrio. Secondly, they seem to use
a group-signalling method to switch-on their virulence, so they can
be present, but still in their "benign" state. Lastly, you have to
consider the robustness of the shrimp / larvae. Some spawnings /
crops will be more robust than others, for a host of reasons,
including genetic variations, nutrition, water quality etc.
As to why disease outbreaks can be seasonal, well I'd like to know
too. I've heard that outbreaks of WSV can be triggered by rainfall
and / or a sudden temperature drop. Also, people tend to exchange
water when they have disease problems, this surely helps create
a "disease season" for their neighbours.
Kevin Healey
e-mail: khealey@iahp.com.au
URL: www.iahp.com.au
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Comments 23:
The Vibrio concentrations are seasonal most likely
due to different nutrient levels in the near shore environment. More rain
means more turbulence and more organic matter in the water = more bacteria
of all types. 98% of bacteria in seawater is associated with particulate
organic matter. Do a before and after any DE filter and check this for
yourself. Nutrients are seasonally responsible for algae blooms. With algae
comes Vibrio alginolyticus. With algae blooms comes the spawning season.
Zooplankton reproduction, dying unfertilized eggs, weak larvae, etc. Vibrio
anguillarum and others just love this opportunity. Sort of an annual
succession I have been living with for 25 years on three continents of
invertebrate hatchery work.
Now if you have good larvae you will have less culture problems all the way
through the culture phase. Since lipid is the primary energy reserve for
invertebrate larvae, the high lipid levels in certain spawns and well fed
broodstock will hold up far better against Vibrio sp. concentrations. I have
found high lipid eyed oyster larvae to contain TNTC Vibrio counts on TCBS
plates but show no ill effect what so ever. Same for shrimp larvae.
Start with a good egg. Check the lipid of the spawns with a lipid
specific stain like Oil Red "O" or Sudan Black. Throw out any eggs
or nauplii that do not show high colour. Do not mix bad spawns with good
spawns. You will have a lot less problems culturing the pathogens along side
the larvae.
Phil Boeing
e-mail: pboeing@dc.rr.com
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Comments 24:
Part of the key this would be using early-log phase
algal culture. When algae cultures enters stationary phase they start to
release protein (seen as foam) which is food for Vibrio. So with old algal
culture you can be inoculating your larvae with Vibrio, and also leaving a
film of Vibrio in your algal tank to seed the next algal culture you grow.
A question. What is your opinion about the best algae to use? You
mention Skeletonema, what about Chaetoceros?
Kevin Healey
e-mail: khealey@iahp.com.au
URL: www.iahp.com.au
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Comments
25:
Interested to read the postings which have raised
some very interesting points. It should be appreciated that many of the
virulence factors identified in a range of Vibrio sp. ( a good example being
V. cholerae) appear to be related to pathogenicity islands that have
presumably arrived via horizontal transfer from other bacteria or, in a
twist on the whole phage issue, by phage containing genes coding for
virulence factors integrating into the bacterial chromosome. Thus calling
all Vibrio sp. potentially 'bad' is probably an oversimplification of the
issue. Perhaps the virulent ones have been 'highjacked' by viruses? Phage
has also been shown to promote and transfer virulence in V. harveyi. I
know that many farmers swear by probiotics but, to be honest, really
convincing peer-reviewed papers on their potential efficacy in aquaculture
are limited (plenty of papers and technical reports suggest they show
promise though). There are also a range of potential food safety
issues related to their use. If you are in the EU, they need to be licensed
as either a feed additive (if the claim is as a 'gut flora' or zootechnical
improver), or, if the claim is health-related, then they will probably need
to obtain a veterinary medicine Marketing Authorisation (MA). Both
authorisations require a full product dossier submission (safety and
efficacy data) to the appropriate regulatory authorities by the
manufacturer. Addition to the culture vessel directly, rather than in feed,
may allow a way round this for some preparations (eg biofilter start up
cultures), but users should be careful. The EU feed additives regulations
talk about addition to 'drinking water' as well, showing how much
consideration was paid to aquaculture when they were framed! If the claim is
health related, then it will probably be classed as a veterinary medicine,
whatever the administration route. Similar rules govern the use of additives
and medicines in the US as well, so may be best to check with FDA.
Few references for those interested:
Munro J, Oakey J, Bromage E, Owens L (2003) Experimental
bacteriophage-mediated virulence in strains of Vibrio harveyi Diseases of
Aquatic Organisms 54:187-194
Faruque, S. H. , Albert, M.J. Mekalanos, J. J. (1998) Epidemiology, genetics
and ecology of toxigenic Vibrio cholerae. Microbiology and Molecular Biology
Reviews. 62(4) 1301-1314
Ruangpan L., Danayadol, Y. et al . (1999) Lethal toxicity of Vibrio harveyi to
cultivated Penaeus monodon induced by a bacteriophage. Dis. Aquat. Org.
35:195-201
David Verner – Jeffreys
e-mail: dvjeffreys@yahoo.com
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Comments 26:
I may be a fish producer, but the concepts are the
same. We have been avoiding anti-biotics for over a decade for
bacterial/fungal infections. Our methods are an indirect probiotics
approach using detailed operating procedures.
For example, when starting a new and cleaned larval tank the first
thing we do is contaminate it with sludge and slime from some
protozoan culture systems used to produce live food. These cultures
have no hosts for fish pathogens, but lots of food with lots of
decomposer species. Getting the surfaces precovered with
non-pathogens seems to help prevent pathogens from getting a hold on
the tank.
Another example is Artemia hatching with multiple tanks. We check to
see whether the hatch was clean without undesirable bacteria (via
bioassay) and then just lightly hose out the tank between batches.
If a tank becomes a problem, we bleach it and transfer some surface
material from one that is working better. This way you end up
selecting for the more beneficial species/strains of bacteria and
selecting against pathogenic species.
There is a whole host of little operating details that help determine
the status of the microbiological ecology in the system and it is the
status of that microbiological ecology that determines your
production levels. The "art" of aquaculture is
controlling the
microbiological ecology -- a lot of "black magic" in that we can't
even identify all the species of bacteria involved and can only
culture a fraction of the species.
Dallas E. Weaver, Ph.D.
e-mail: deweaver@surfcity.net
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Comments 27:
It is absolutely important to monitor vibrio cfu
count as this will determine your application rates. In general, for
remedial you have to use higher or very frequent dosage as oppose to
preventive. Agree with Kevin's comments. So it is not an issue blindly
throwing money into the ponds.
Robin Liew
e-mail: robinliew@myjaring.net
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Comments 28:
Re your comments about Vibrio, for sure the knowledge
about their mode of virulence is increasing, and it will be fascinating to
watch the progress. The cautionary note is that there is still some
unravelling yet to do, for example strains not harbouring known islands of
pathogenicity might still find ways to bite us.
Re the efficacy of probiotics, you mentioned many farmers swear by
them, and if you ask them why you'll get a sound and genuinely-
interesting answer. Most farmers are at least as sceptical as
scientists, but scientists can reserve their opinions until they
have satisfied themselves beyond all doubt by a huge body of
published evidence. Farmers wish they could too, but have to make
hard decisions to ensure the viability of their businesses. They
will only continue to use any product if they are convinced it's
worth the investment. One of problems with putting together
technical publications is the difficulty in simulating on-farm
conditions in controlled institutional environments. Another reason
is that while probiotics are an important part of a program of sound
farm or hatchery management, they're not magic. For example, if
sensible standards are relaxed because probiotics have been
introduced, then the customer might be disappointed with the result.
We have also been supplying probiotics for land animals since the
late '80's and the history and pattern of adoption is just the same.
Kevin Healey
e-mail: khealey@iahp.com.au
URL: www.iahp.com.au