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Aquaculture 164 Ž1998.

351–358

Control of luminous Vibrio species in penaeid


aquaculture ponds
)
D.J.W. Moriarty
Department of Chemical Engineering, The UniÕersity of Queensland, Qld 4072, Australia

Abstract

A crisis has arisen in the prawn industry in many regions with the onset of disease, with Vibrio
spp. being important major causal factors. The value of adding selected strains of Bacillus as
probiotic bacteria to control the Vibrio is shown by comparing farms in Indonesia using the same
water sources, which contained luminous Vibrio strains. The farms that did not use the Bacillus
cultures experienced almost complete failure in all ponds, with luminescent Vibrio disease killing
prawns before 80 days of culture were reached. In contrast, a farm using the probiotics was
culturing prawns for over 160 days without problems, by using Bacillus at abundances of about
1 = 10 4 to 1 = 10 5rml. The bacterial species composition was different in the pond water on the
two farms and demonstrates that it is possible to change bacterial species composition and
improve prawn production in large water bodies. Vibrio numbers, especially luminous Vibrio
numbers were low in ponds where a large abundance of specially selected Bacillus species was
maintained in the water column. Vibrio numbers were also low in sediments and no luminous
Vibrio occurred in sediments where the probiotic Bacillus were used. q 1998 Elsevier Science
B.V. All rights reserved.

Keywords: Prawn; Vibrio spp.; Probiotic bacteria

1. Introduction

A crisis has arisen in the prawn industry in many regions over the last few years with
the onset of disease. Pathogenic Vibrio spp. have been implicated as being one of the
major causes of these disease problems ŽRuangpan and Kitao, 1991.. Recently, V.
harÕeyi, a luminous species known to cause losses in hatcheries, has been found as a

)
Corresponding author. Biomanagement Systems, 315 Main Road, Wellington Point, Qld 4160, Australia.
Tel.: q61-7-3822-4833; fax: q61-7-3822-1526; e-mail: djwm@ozemail.com.au.

0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.


PII S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 1 9 9 - 9
352 D.J.W. Moriartyr Aquaculture 164 (1998) 351–358

major cause of disease in growout ponds ŽNithimathachoke et al., 1995.. To some


extent, these disease problems occurred because the interactions of microbes and their
effects on animals and their environment, at intensive production scales, have not been
well understood or have been treated only from a clinical pathology perspective. Often
the disease was treated rather than the underlying cause. The probiotic approach to
disease control has not been used very much in aquaculture, but it has excellent potential
Že.g., Olsson et al., 1992; Douillet and Langdon, 1994; Garriques and Arevalo, 1995..
The use of beneficial bacteria Žprobiotics. to displace pathogens by competitive
processes is being used in the animal industry as a better remedy than administering
antibiotics and is now gaining acceptance for the control of pathogens in aquaculture
ŽHavenaar and Hius in’t Veld, 1992.. These authors extended the definition of the term
‘probiotic’ from the use of live microorganisms in feed, to ‘‘a probiotic is a mono- or
mixed culture of live microorganism that, applied to animal or man, affect beneficially
the host by improving the properties of the indigenous microflora.’’ In their discussion,
they considered only human and land farm animals. In extending their definition to
aquaculture, it is clear that it also applies to the addition of live bacteria to tanks and
ponds in which the animals live, because these bacteria modify the bacterial composition
of the water and sediment. The health of animals is thus improved by the elimination of
pathogens or at least minimizing the effect of pathogens and by improving water quality.
Therefore, I argue that probiotics are used in aquaculture not only as feed supple-
ments, but also as water additives. This paper shows that it is feasible to displace the
luminescent Vibrio species present in pond water by adding selected Bacillus species.

2. Materials and methods

2.1. Farm sites

The farms were located about 60 km north west of Jakarta in Indonesia, and were
within 5 km of each other. Farm MM and Farm 1 used the same sea water supply canal.
Farm MM used the Detritus Management System ŽDMS. biotechnology, which con-
sisted in essence of adding certain Bacillus strains at high density and managing a stable
phytoplankton bloom throughout the grow out period.

2.2. Bacterial counts

Duplicate water samples were collected from ponds and were brought to the
laboratory on MM farm and processed immediately, or stored for a short time at 48C.
The water was first homogenized at 20,000 rpm for 30 s with a tissue homogenizer to
disperse particles, and then samples for viable plate counts were serially diluted in
filtered autoclaved pond water.
Sediment was collected in 40 mm dia. PVC tubes. They were pushed far enough into
the sediment to form a solid plug of clay to retain the sediment as the tube was pulled
out. The core was removed to make a corer. Two cores were collected from both the
D.J.W. Moriartyr Aquaculture 164 (1998) 351–358 353

edge and from the center of the ponds. The top 1 cm Ž4 cc in a 30 ml syringe. of
sediment was mixed with 50 ml filtered sterile water from the pond. Two ml subsamples
were homogenized at 20,000 rpm for 1 min with a tissue homogenizer and then were
serially diluted for plate counts.
Viable counts of Vibrio were made on TCBS agar from Oxoid using the spread plate
method ŽHerbert, 1990.. A comparison was made with dip slides on one set of samples.
Dip slides with TCBS agar Žsupplied by Biomanagement Systems, Darwin, Australia.
were tared, dipped into pond water for 2 s, drained briefly and weighed. The weight
increase of 100 " 10 mg indicated that 50 m l were absorbed per side of the dip slide.
Plates were incubated for 24 h and dip slides for 12 h at ambient temperature Ž30–358C..
Yellow and green colonies were counted. All colonies that appeared in these time
intervals were assumed to be Vibrio. Plates were viewed at intervals of 6 to 12 h for a
total period of 2 days in total darkness to determine the number of luminescent colonies.
The actual numbers of luminous Vibrio were probably higher, as colonies were not
plated separately onto luminous medium. One trial was carried out with replica plating
from TCBS to luminous medium. Replicas of several TCBS plates were made onto
nutrient agar made up in filtered pond water containing 3 grl glycerol, and numbers of
luminescent colonies compared.
The same colonies that were luminescent on the TCBS were luminous on the special
medium, and no further luminous colonies were found. Thus, the TCBS was accepted as
providing a reasonably quick indication of the presence of luminescent bacteria in the
routine monitoring of pond water and sediment.

2.3. Bacillus

The Bacillus used in the MM farm were the DMS series supplied by Biomanagement
Systems, Darwin, Australia. They were applied at intervals of 1 to 3 days to each pond,
at the rate of 50 to 100 l per hectare. The DMS 1000 and 1100 series used at the start of
the trials contained around 1 = 10 9 cellrml; they were selected for their ability to break
down organic matter rapidly, especially slime. The DMS-2000 series were selected for
their ability to produce antibiotics against luminescent Vibrio. All were applied at final
concentrations of about 1 = 10 4rml.
Several strains of Bacillus with antibiotic activity against luminescent Vibrio, now
marketed as a mixture under the trade name: PondPro-VC, were tried in the ponds.

3. Results

Total Vibrio numbers in source canal water Žsalinity 20 ppt. varied from 120 to
around 500rml and luminescent strains comprised about 1 to 10% of these ŽTable 1..
Results for total counts were similar with two different methods; spread plate counting
and dip slides. The precision of counts made with the dip slides was better than the
spread plate method.
354 D.J.W. Moriartyr Aquaculture 164 (1998) 351–358

Table 1
Total Vibrio and luminescent Vibrio ŽLum. Vibrio . numbers per ml in water from a pond Žat 90 days of
culture. and 2 sites on a source canal in west Java
Water source Plate count Dip slide
Vibrio Lum. Vibrio Vibrio Lum. Vibrio
Pond 2253"253 17"5 2302"119 18"6
Canal site 1 447"21 17"3 378"37 10"3
Canal site 2 121"19 1"1 138"14 16"5

A comparison of results for two different methods, standard petri dish plate counts and dip slides, is shown.
All colonies developing on TCBS agar were classified as Vibrio. Means and standard errors for 10 replicates
are shown.

3.1. Vibrio in ponds without the DMS system

Water in ponds and a reservoir on Farm 1 contained between 2000 and 6000
Vibriorml and sediment contained from 3000 to over 1 = 10 6rcc ŽTable 2.. Lumines-
cent strains numbered over 200rml in the pond water and reservoir and from 200rcc to
over 5 = 10 4rcc in the sediment ŽTable 2.. The luminescent Vibrio were typically
found in all farms in the water of that coastal area. For example, two other farms nearby
had 2% and 11% luminous Vibrio in the pond water; sediments were not analyzed
ŽTable 2..

3.2. Vibrio in ponds with DMS

In the pond water of MM farm that shared the same source canal as Farm 1, luminous
Vibrio were often found in the water of the ponds or when present, the abundance was
less than 100rml ŽTable 3.. Luminous Vibrio were usually absent until about day 60,
unless water was added from the canal, as in Pond 1 at day 40 ŽTable 3.. The numbers
of luminous Vibrio decrease with large additions of the probiotics marketed as PondPro-
VC ŽTable 3..

Table 2
Total and luminous Vibrio numbers per ml in pond water and sediment on farms that were not using probiotic
bacteria in west Java, Indonesia
Water source DoC) Water Sediment
Vibrio Lum. Vibrio Vibrio Lum. Vibrio
Farm 1 Pond 1 80 2576"366 205"81 3523"1300 164"51
Farm 1 Pond 2 80 5925"730 250"25 1.1"5=10 5 5.2"4.2=10 4
Farm 1 Reservoir 6333"683 250"67
Farm 2 Pond 1 60 2269"525 281"119
Farm 2 Pond 2 40 593"56 50"14
Farm 3 Pond 1 100 2106"2100 44"51

Values were not determined where no data are shown. Means and standard errors for 8 replicates are shown.
)DoC sdays of culture.
D.J.W. Moriartyr Aquaculture 164 (1998) 351–358 355

Table 3
Total and luminous Vibrio numbers per ml in pond water and sediment on Farm MM that was using the DMS
probiotic bacteria in west Java, Indonesia
Pond DoC) Water Sediment
Vibrio Lum. Vibrio Vibrio Lum. Vibrio
1 40 800"92 55"31 2.3"1.7=10 3 0
2 42 214"89 0 8.6"2.1=10 4 0
2 72 1000"170 19"12 1.5"04=10 5 0
3 19 4900"1700 0 5.8"2.7=10 5 0
4 142 2630"537 2"2 2.0"0.9=10 4 0
4 160 9800"1900 75"48 4.7"2.4=10 5 0

A large dose of the PondPro-VC bacteria was added shortly before DoC 142 in Pond 4. Means and standard
errors for 8 replicates are shown
)DoC sdays of culture.

3.3. Prawn production and surÕiÕal

Farm MM, which used the DMS probiotic technology, produced from 5 to 11 trha in
the first season of 1995 ŽTable 4.. In contrast, the farms that had high luminous Vibrio
abundance, and did not use the DMS probiotic program, experienced poor survival of
prawns, with crop losses often occurring around 60 to 80 days of culture. Production,

Table 4
Some typical values for prawn production in May 1995 from ponds on Farm MM, west Java with the Detritus
Management System ŽDMS. of probiotic bacterial technology
Pond
1 2 3 4 5 6 7
No.rm 2 41 41 41 44 40 41 44
Survival rateŽ%. 47 41 38 63 36 52 56
Days of culture 163 160 170 143 167 159 154
Mean size at harvest Žg. 37 35 43 43 37 36 48
Harvest Žkgrha. 6500 5300 6400 11,500 5300 7600 11,400

Table 5
Comparison of prawn production from ponds with and without the Detritus Management System ŽDMS. of
probiotic bacteria in the Philippines
Control DMS bacteria
2.
Area Žm 5000 5471
Stocking density ŽNorm2 . 46 42
Survival rate Ž%. 16 73
Days of culture 200 187
Mean size at harvest Žg. 39 28
Harvest Žkgrha. 3000 9500
356 D.J.W. Moriartyr Aquaculture 164 (1998) 351–358

when ponds were not aborted, was usually less than 5 trha. Detailed data were not made
available by the managers of these farms, but similar data were obtained for a farm in
the Philippines where luminous Vibrio were present ŽTable 5.. Survival and production
were lower in the control pond than in the pond with the DMS bacteria. A farm in the
same locality in eastern Negros, currently has 3 ponds at 170 days culture with the DMS
probiotics and a survival estimated at over 90%, whereas other ponds treated with
antibiotics to control Vibrio all experienced low survival ŽJ. Apostol, personal communi-
cation..

4. Discussion

4.1. Control of luminescent Vibrio

The high abundance of luminescent Vibrio is consistent with occurrence of disease


and poor or zero harvest results. Vibrio harÕeyi, a pathogen of P. monodon that causes
severe losses, is luminescent and a non-sucrose fermenter Ži.e., its colonies are green on
the TCBS agar. ŽBaticados et al., 1990.. The MM farm, which used the DMS probiotic
bacteria, had either a very low abundance or a complete absence of luminous Vibrio in
pond water and very good harvest results in 1995. This consistent and high productivity
occurred, even though the proportion of luminescent Vibrio in the pond water was high
in the sea water source, and the abundance of total Vibrio in the pond water was higher
than in the water source. Furthermore, luminescent Vibrio were completely absent at all
stages of grow out from the pond sediment in the presence of the DMS Bacillus species.
The low numbers of the luminescent Vibrio were correlated with the use of high
doses of the PondPro-VC, strains of Bacillus selected for inhibition of luminous Vibrio
on agar plates, and the high proportion of Bacillus to Vibrio in water and sediment. It
was found to be important to have regular additions of the Bacillus because where the
DMS program was not being used, Gram positive bacteria were present, but disease
problems occurred. In fact, Gram positive bacteria usually constitute at least 20% of the
total flora of sediment ŽMoriarty and Hayward, 1982.. Thus, it is not sufficient to rely on
natural populations to provide protection from Gram negative pathogens.
The data reported above indicate that the Vibrio populations were being affected by
the DMS Bacillus strains that were added. This change was beneficial to the prawns, as
they were protected from vibriosis. Prawns were healthier in ponds with DMS: harvests
were 5–11 trha, whereas Farm 1 and the other farms collapsed with vibriosis after 80
days ŽTable 4..
Unlike land animals, aquatic farmed animals are surrounded by a milieu that supports
their pathogens independently of the host animal, and so the pathogens can reach high
abundances around the animal. Vibrio grow attached to algae, and may reach high
population densities after being ingested with the algae and then excreted with lysed
algae in fecal pellets by zooplankton; they are gut bacteria in fish and prawn as well as
zooplankton ŽMoriarty, 1990.. In aquaculture ponds, where animal and algal population
densities are high, Vibrio numbers are also high compared to the open sea. The onset of
prawn disease due to exposure to these high numbers of Vibrio, especially when
D.J.W. Moriartyr Aquaculture 164 (1998) 351–358 357

pathogenicity has increased Žsee below. indicates that a defense is needed. Hence,
Bacillus spp. are used as probiotics to control Vibrio or preventing attachment.

4.2. Antibiotic use

Probiotics such as the use of Bacillus described here offer an alternative to antibiotic
therapy for sustainable aquaculture. There are severe problems with the use of antibi-
otics in aquaculture ŽBaticados et al., 1990.. If antibiotics are used to kill bacteria, either
strains of the pathogen or different bacteria survive because they carry genes for
resistance. These will then grow rapidly because their competitors are removed. Virulent
pathogens that then re-enter the hatchery tank or aquarium, perhaps from within biofilms
on water pipes or air lines or in the guts of the animals where they were protected from
the antibiotic, can then exchange genetic information with the resistant bacteria and
survive further doses of antibiotic. Thus, antibiotic-resistant strains of the pathogen
evolve very quickly.
There are several reasons why it is better to add Bacillus rather than antibiotics to
control Vibrio species. Many different antibiotic compounds are naturally produced by a
range of Bacillus species. Other bacteria are unlikely to have resistance genes to all
antibiotics at one time, especially if they have not been exposed to the Bacillus
previously. Bacillus secrete many enzymes that degrade slime and biofilms and allow
Bacillus and their antibiotics to penetrate slime layers around Gram negative bacteria.
Furthermore, Bacillus compete for nutrients and thus inhibit other bacteria from
growing rapidly. Thus any resistant bacteria cannot multiply readily and transfer
resistance genes. Bacillus also compete for space on surfaces—e.g., the gut wall—and
displace other bacteria if they are present in high numbers.
Because there are many different mechanisms involved in the probiotic process of
competition and exclusion, it is difficult for the pathogens to evolve all the necessary
resistance genes together. Competitive exclusion is one of the ecological processes that
can be manipulated to modify the species composition of a soil or water body or other
microbial environment. Small changes in factors that affect growth or mortality rates
will lead to changes in species dominance. We are still a long way from knowing all the
factors that control bacterial growth rates and even the complete species composition by
making use of the competitive exclusion principles ŽSmith, 1993..
Microbial ecology and biotechnologies have advanced in the last decade, to the point
that commercial products and technologies are available for treating large areas of water
and land to enhance population densities of particular microbial species or biochemical
activities. The practice of bioremediation Žor bioaugmentation. is applied in many areas,
but success varies greatly, depending on the nature of the products used and the
technical information available to the end user. The bacteria that are added must be
selected for specific functions that are amenable to bioremediation, and be added at a
high enough population density, and under the right environmental conditions, to
achieve the desired outcomes. Bioaugmentation and the use of probiotics are significant
management tools, but their efficacy depends on understanding the nature of competition
between species or strains of bacteria. They rely on the same concepts that are used
successfully from soil bioremediation and probiotic usage in the animal industry.
358 D.J.W. Moriartyr Aquaculture 164 (1998) 351–358

Acknowledgements

I am grateful to Mr. Trisno Suhendra, of Moisson Makmur farm, for providing access
to the farm and the use of his laboratory. I thank Mr. Diaz, Mr. Handoko and Mr. Deddi
at Moisson Makmur for their assistance with the laboratory work.

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