AU719544B2

AU719544B2 - A method of controlling a pathogenic bacterial population

Info

Publication number: AU719544B2
Application number: AU65750/96A
Authority: AU
Inventors: Adam G. C. Body; David J. W. Moriarty
Original assignee: BIOMANAGEMENT SYSTEMS Pty Ltd
Current assignee: BIOMANAGEMENT SYSTEMS Pty Ltd
Priority date: 1995-09-18
Filing date: 1996-09-18
Publication date: 2000-05-11
Anticipated expiration: 2016-09-18
Also published as: AU6575096A

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: "A METHOD OF CONTROLLING A PATHOGENIC BACTERIAL POPULATION" *S Sr

*SSSSS

The following statement is a full description of this invention, including the best method of performing it known to us: GH REF: P23988D/DAA:WW 2 FIELD OF THE INVENTION The invention relates to a method for controlling or reducing the numbers of pathogenic bacteria in a body of water. The invention has particular application to the field of aquaculture as well as the field of water quality control.

BACKGROUND TO THE INVENTION Aquaculture involves the high density farming of animals such as crustaceans or fish. In such environments spread of disease caused by bacteria can be rapid resulting in severe crop losses. Pathogenic Vibrio bacteria have been implicated as being one of the major causes of such disease problems (Ruangpan and Kitao, 1991). Recently V. harveyi, a luminescent bacterial species known to cause losses in shrimp hatcheries has been shown to be a major cause of disease in grow-out ponds (Nithimathachoke et al., 1995).

Various antibiotics have been used to control pathogenic bacteria in the aquaculture industry. There are, however, severe problems with the use of antibiotics in aquaculture (Baticados et al., 1990). A major concern is that indiscriminate use may give rise to antibiotic resistant strains of pathogens. It has been demonstrated that plasmids with genes for resistance to antibiotics 25 such as tetracycline, ampicillin, naladixic acid, streptomycin, trimethoprim, sulphonamides and chloramphenicol can be transferred between the Gram negative bacteria V. cholerae and E. coli and Aeromonas salmonicida, a known fish pathogen (Kruse and Sorum, 30 1994) .Indeed, it was found in that study that the rate of gene transfer between V. cholerae and A. salmonicida increased 100 times in the presence of tetracycline concentrations that were not high enough to kill the bacteria. The transfer of antibiotic resistance to a bacterial species such as E. coli which is common in the intestinal tract of animals, including humans is of particular concern.

Additions of bacteria have been made to aquaculture S:23988D 3 ponds by a limited number of farmers in recent years for the purpose of controlling ammonia build-up and decreasing amounts of slime and organic matter in the water, to thereby lessen sludge accumulation.

It has been reported that the addition of bacteria to an aquaculture pond for this purpose provided no beneficial effect (Scura, 1995). It has also been argued that one cannot change the bacterial species composition of an aquaculture pond on the basis of experimental results and that the addition of bacteria to aquaculture ponds is to be avoided (Boyd, 1995).

SUMMARY OF THE INVENTION The present inventors have now surprisingly found that Gram positive bacteria in sufficient numbers are able to control pathogenic bacteria in a water environment.

Accordingly, in a first aspect of the invention there is provided a method of controlling pathogenic bacteria in a body of water, comprising the step of adding an effective number of Gram positive bacteria from at least one Gram positive bacterial strain to the water, wherein the Gram positive bacteria are non-pathogenic and are capable of inhibiting or preventing the growth or replication of the pathogenic bacteria.

25 In addition, the method may comprise the step of supplementing the number of Gram positive bacteria initially added to the body of water with one or more further additions of the bacteria at selected intervals.

The method may be used to reduce the abundance of 30 pathogenic bacteria in, for example, ponds or dams used as a source of water for animals and so thereby reduce the possibility of diseases being contracted through consumption of the water. The method may also be used in "'*sewage treatment lagoons or ponds as well as a variety of different types of aquaculture. In particular, the invention has use in crustacean aquaculture, including shrimp, crab and crayfish farming, as well as in fish aquaculture. The method may be used in freshwater or S:23988D 4 salt water environments.

While the method may be used to control pathogenic bacteria in small bodies of water such as in hatchery tanks having a volume of from about 5 litres to about 20,000 litres, the invention finds particular application in substantially larger bodies of water such as aquaculture ponds which may be 0.2 to 5 hectares or more in area and have a depth of Im or greater.

In a second aspect of the invention, there is provided a body of water when treated by the method.

In a third aspect of the invention there is provided an animal when raised in a body of water treated by the method.

In a fourth aspect of the invention there is provided a method bf inhibiting infection of an animal by pathogenic bacteria in a body of water in which the animal is being raised, comprising the step of soaking feed material for the animal in an inoculum containing Gram positive bacteria from at least one Gram positive bacterial strain, prior to adding the feed material to the water, wherein the Gram positive bacteria are nonpathogenic and are capable of inhibiting or preventing the growth or replication of the pathogenic bacteria.

In a fifth aspect of the invention there is provided 25 non-pathogenic Gram positive bacteria in an inoculum and being capable of inhibiting or preventing the growth or replication of pathogenic bacteria, when used in a method of the invention.

The term "non-pathogenic" means that the at least 30 one Gram positive bacterial strain is not disease causing o*e.

in an animal farmed in the body of water, or in a particular animal which is intended to use the water or to consume the first mentioned animal or the water.

Typically, the at least one Gram positive bacterial strain will belong to the genus Bacillus and is preferably selected from the group of bacterial species consisting of B. polymyxa, B. subtilis, B. laterosporus, B. circulans, B. licheniformis and B. brevis. However, S:23988D it will be appreciated that the invention is not limited to the addition of one or more strains of these species to the body of water and that the Gram positive bacteria may be selected from genera other than Bacillus such as from within the Actinomycetes grouping.

The term "pathogenic bacteria" means one or more strains or species of bacteria present in the body of water that cause disease or stress in an animal farmed in the water, or in a particular animal that is intended to use the water or to consume the first mentioned animal or the water.

The pathogenic bacteria will usually consist of one or more strains of Gram negative bacteria and will typically belong to the genus Vibrio. Particular strains of pathogenic bacteria include strains from the species V. harveyi, V. cholerae, V. parahaemolyticus, V.

vulnificus and V. alginolyticus. Other pathogenic bacteria may include bacteria belonging to genera such as Aeromonas, Pseudomonas and Proteus amongst others.

It is an advantage of a method embodied by the invention that the number of pathogenic bacteria in a body of water can be controlled. By controlling the numbers of pathogenic bacteria the risk to animals farmed in the water contracting a disease caused by the 25 pathogenic bacteria can be decreased. It is a yet another advantage that by reducing the risk of disease, losses to farmers caused by death, slow growth or disfigurement of such animals as a result of disease can also be minimised.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is described in greater detail hereinafter with reference to preferred, non-limiting embodiments illustrated with reference to the accompanying drawings wherein: Figure 1 shows numbers of luminescent Vibrio bacteria in the water column of several ponds treated with additions of Gram positive bacterial strains S :23988D -6 selected for control of luminescent Vibrio compared to numbers in the water column of ponds not so treated.

Figure 2 shows numbers of total bacteria, Gram positive bacteria, total Vibrio and luminescent Vibrio bacteria in an aquaculture pond in relation to the addition of several bacterial supplements and saponin.

Figure 3 shows variation in abundance of total bacteria Gram positive bacteria, and total and luminescent Vibrio bacteria in the water column during shrimp grow out in an aquaculture pond in relation to additions of DMS-2006 bacteria and saponin.

Figure 4 shows variation in abundance of total bacteria, Gram positive bacteria, total and luminescent Vibrio bacteria in the water column during shrimp grow out in another aquaculture pond in relation to additions of saponin and DMS-2006.

Figures 5 and 6 show variations in numbers of total bacteria, Gram positive bacteria, spores and total Vibrio bacteria in the sediment of the ponds to which Figures 3 and 4 refer during the relevant monitoring periods.

Figure 7 shows shrimp harvests from aquaculture ponds treated with additions of Gram positive bacteria for controlling luminescent Vibrio compared to ponds not so treated.

25 Figure 8 shows the effect of additions of DMS-2003 bacteria on luminescent Vibrio strains in hatchery tanks.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION Gram positive bacteria from the genus Bacillus are able to decrease remarkably the numbers of Gram negative bacteria from the genus Vibrio in a water environment.

It is thought that this is achieved through competition with Vibrio bacteria for space and the production of a range of antibiotics that act on the Vibrio bacteria.

While the transfer of genetic information for virulence and resistance to antibiotics may readily occur between Gram negative bacteria as the work by Kruse and Sorum (1994) shows, the likelihood of such transfer is S:23988D significantly reduced or avoided by using Gram positive bacterial strains to control pathogenic Gram negative bacteria.

Antibiotics produced by Bacillus strains include cyclic oligopeptides, such as bacitracin, which can be produced by B. licheniformis, that inhibit bacteria cell wall synthesis, and linear or cyclic oligopeptides that interfere in bacterial membrane function such as polymyxin and circulin, which can be produced by B. polymyxa and B. circulans, respectively. Further antibiotics include basic peptides that inhibit protein synthesis, such as edeine which can be produced by B. brevis, and aminoglycosides that inhibit bacterial ribosomal function such as butirosins which can also be produced by B. circulans.

Bacillus strains also produce a range of extracellular enzymes that collectively degrade a wide range of nutritional and physical substrates used by Vibrio bacteria. Besides protease enzymes, Bacillus strains can also secrete nucleases and lipases. The .enzymes produced by Bacillus strains diffuse away from the cells whereas enzymes produced by many Gram negative bacteria are held in the periplasmic space or outer capsule of the bacteria. The hydrolysis of polymeric 25 compounds such as proteins, starch, cellulose and fats is a rate-limiting step that controls bacterial growth in aquatic ecosystems (Billen, 1990; van Wambeke, 1994).

Accordingly, bacteria that are efficient in hydrolysing substrates have a competitive advantage over those that '30 are less efficient. Hence, control of Vibrio bacteria by ~Bacillus strains is likely to be due to a combination of factors rather than solely the effect of released antibiotics on the Gram negative bacteria.

Bacillus strains that may be used in the present invention are readily available from a number of commercial depository institutions as indicated in Table 1.

S:23988D 8 .c *0* 0* *0 a TABLE 1: SOURCES OF SUITABLE GRAM-POSITIVE BACTERIA DMS UQM ATCC NCIMB Bacillus species 1003 411 8055 B. subtilis 1103 378 14580 9375 B. licheniformis 2002 B. laterosporus 2003 419 B. circulans 2004 1243 14880 B. licheniformis 2005 3563 15518 12846 B. circulans The codes used in Table 1 are as follows.

DMS: Biomanagement Systems Pty Ltd (PO Box 39346, Winnellie, Northern Territory 0821, Australia), UQM: The University of Queensland Culture Collection in the Department of Microbiology (Brisbane, Queensland, Australia), ATCC: American Type Culture Collection (Maryland, USA), and NCIMB: National Collection of Industrial and Marine Bacteria (Aberdeen, Scotland).

A description of the Bacillus strains referred to in Table 1 may be found in "Bergey's Manual of Determinative Bacteriology", 8th Edition, Baltimore, Williams and Wilkins (1974). Further descriptions may be found in "The Genera Bacillus and Sporobacillus", Norris J.R et al (1981) and "In The Prokaryotes", Starr M.P et 25 al. Berlin, Springer-Verlag. Reference may also be made to Priest F.G, "Systematics and Ecology of Bacillus in Bacillus subtilis and other Gram Positive Bacteria", A.L. Sonenshein, Ed. American Society for Microbiology, Washington D.C (1993).

While Bacillus strains can suppress a range of Vibrio bacteria, DMS-2002, DMS-2003 and DMS-2004 are particularly effective in suppressing V. harveyi, V. cholerae and V. parahaemolyticus. DMS-2005 has also been found to suppress V. harveyi. However, some strains may show no or little suppression of given pathogenic bacteria. Accordingly, it is necessary to screen particular strains in order to determine their effectiveness.

To determine which Gram positive strains can be S:23988D 9 used in the control of pathogenic bacteria a screening test can be carried out using conventional microbiological techniques. A screening method for Bacillus strains useful in aquaculture is described below as an example.

A selected Bacillus strain is streaked across 2%-4% tryptone soy agar medium using a flamed loop and incubated at 28-32 0 C for two days before being cross streaked with the selected pathogenic bacterium. The pathogenic bacterium is prepared by overnight incubation at about 30 0 C in a 2% NaCI nutrient broth prior to dilution in a sterile 2% NaCI solution. Cross streaking is performed with a flamed loop using 5-10Al of the test solution. The agar medium is then incubated for a further three days at 28-32 0 C prior to checking for clear zones.

The Gram positive bacteria can be added to the body of water in an inoculum solution. Generally, the concentration of the bacteria will be such that it provides about 1x10 4 cells/ml of the water and *preferably, between about 5x10 4 and 1x10 5 cells/ml.

For relatively small bodies of water such as hatcheries, the concentration of Gram positive bacteria in the inoculum is determined such that about 10ml of 25 inoculum is required per 1000 litres of water. However, for larger applications such as aquaculture ponds lower ratios can be used. In such instances, the inoculum may contain a nutrient mix (such is available under the brand name Activate from Biomanagement Systems, Winnellie, NT, 30 Australia) and be vigorously aerated prior to addition until the desired Gram positive bacterial concentration in the inoculum is reached. When adding the Gram positive bacteria to the body of water the inoculum is poured over as great an area as practical in order to maximise distribution of the bacteria.

While a single Gram positive bacterial strain can be added to the water it is preferable that a number of different Gram positive strains be used for broad control S:23988D I 10 of a range of pathogenic bacteria.

The added Gram positive bacteria will usually be soil and sediment bacteria that contribute to the food chain in the water environment. The advantage of using such bacteria is that their numbers, following addition to the body of water, soon decrease to an abundance that is typically found in nature.

Following the initial addition of the selected Gram positive bacterial strains further additions may be made in order to maintain the desired concentration of the Gram positive bacteria until abundance of the pathogenic bacteria have decreased to an acceptable value. The further additions are typically made at regular intervals and usually, 2 to 3 days apart. The number of bacteria added in each further addition are such that the concentration of the bacteria in the body of water is maintained above about 1x10 4 cells per ml of water.

Preferably, the numbers of the Gram positive bacteria are maintained at about 5x10 4 to 1x10 5 cells per ml.

One or more additions of other bacteria for maintaining water quality may also be made prior to and/or following the addition of the Gram positive bacteria selected for pathogen control. The other bacteria will also usually be Gram positive bacteria and oooQ typically, Bacillus strains. Generally, the other bacteria will be added to the water to a concentration of about 1x10 3 cells/ml and preferably, 1x10 4 cells/ml or greater.

The body of water may be mixed using any conventionally known means to maintain organic material in suspension and thereby minimise the deposition of organic sludge on the pond bottom. By avoiding the formation of a sludge layer the organic material is kept in the well-oxygenated water column where it provides a substrate for the added bacteria.

Treatment of feed material used during the farming of animals in aquaculture ponds may also assist in reducing or avoiding problems caused by pathogenic S:23988D 11 bacteria in the water. When feed material is added to an aquaculture pond it absorbs the pond water. Accordingly, it may also absorb pathogenic bacteria at that time which are subsequently ingested by the farmed animal. In order to inhibit infection of the animal with the pathogenic bacteria in this way, the feed material can be soaked in an inoculum containing Gram positive bacteria selected for their ability to inhibit or prevent the growth or replication of the pathogenic bacteria as described above, prior to the addition of the feed material to the pond. An inoculum for this purpose will generally contain about 1x10 8 cells/ml and preferably, 1xl10 0 to 1x10 11 cells/ml.

By absorbing the selected Gram positive bacteria the possibility of the feed material acting as a carrier once in the water for the pathogenic bacteria is decreased. Treatment of the feed material in this way also allows substantial numbers of the selected Gram positive bacteria to enter the gut of the farmed animal when the feed material is ingested. The ingested bacteria may then compete with other gut bacteria for space or otherwise reduce the numbers of other gut e **bacteria such as Vibrio species which are known to contribute to the pond flora through excretion from infected animals (Moriarty, 1990) The invention will now be further described with reference to a number of Examples.

EXPERIMENTAL.

*EXAMPLE 1 A comparison was made between two shrimp aquaculture farms located about 60 km northwest of *e C Djakarta, Indonesia and using the same sea water supply canal. Water in pond Al of farm A was exchanged at a rate of about 15% every few days with water from the 35 supply canal while water in ponds A2 and A3 was recycled e 9 daily via a reservoir. Fresh sea water was added to the reservoir from the canal at intervals of about one week to replace losses. Ponds Al to A3 were 0.44 ha, 0.49 ha S:23988D 12 and 0.23 ha in area respectively, with an average depth of about Im.

Additions of Gram positive bacteria for general water quality maintenance were routinely made to ponds in farm A to degrade excess shrimp feed pellets and minimise accumulation of faeces and organic sludge in the ponds.

Further additions of Bacillus strains selected for controlling Vibrio species using the screening technique described above were also made to the ponds when it was determined necessary. No additions of bacteria for water quality maintenance or for control of luminescent Vibrio species were made to ponds on farm B.

To prepare bacteria for addition to ponds on farm A, 50 ml of inoculum comprising the desired bacterial strain(s) were cultured in 20 litres of water pH 7 to 8 containing nutrients for 10 hours while being vigorously aerated. The water volume was then increased to 100 litres and 2 kg of crushed shrimp feed added prior to culturing the bacteria for up to a further 14 hours, again with vigorous aeration, to obtain a concentration of 1x10 9 cells/mi of bacteria for water quality control or 1x1010 cells/mi of the selected Bacillus bacteria for controlling luminescent Vibrio in the ponds, respectively. The resulting bacterial solutions were 25 added to ponds at the rate of 50 to 100 litres per i.: hectare.

Water samples were collected in duplicate centrally from ponds as well as near the edge of each pond and processed immediately or stored for a short time at 4 0

C.

Prior to counting, samples were homogenised using a tissue homogeniser at 20,000 rpm for 30 seconds to disburse particles. Homogenates were then serially diluted in filtered and autoclaved pond water for viable .,.plate counts.

Sediment was collected in 40 mm diameter PVC tubing. Two cores were collected from both the edge and from the centre of the ponds. The top 1 centimetre of sediment was mixed with 50 ml filtered and autoclaved S:23988D 13 pond water and 2 ml sub-samples homogenised at 20,000 rpm for one minute in a tissue homogeniser. Homogenates were again then subsequently serially diluted for plate counts using filtered autoclaved pond water.

Total bacteria were counted using an epifluorescence microscope after staining with acridine orange (Moriarty, 1987). Viable counts of Vibrio species were made on TCBS agar using a spread plate method (Herbert 1990). Plates were incubated for 24 hours at ambient temperature (30 0 C to 35 0 C) prior to counting yellow and green colonies. All colonies that appeared in this time interval were assumed to be Vibrio bacteria.

To determine the number of luminescent colonies the plates were viewed at intervals of between 6 to 12 hours for a total period of 2 days in total darkness.

TCBS agar contains sucrose and a dye that turns yellow when pH falls. Bacteria that ferment sucrose produce acid and so their colonies are yellow on TCBS agar. V. harveyi, a major pathogen of P. monodon is luminescent and is green on TCBS agar as it is a nonsucrose fermenter (Baticados et al, 1990). Many bacteria that ferment sucrose are not pathogenic to shrimp and so there is a generally positive correlation between the numbers of green colonies and disease in shrimp S25 aquaculture. There is also a good correlation between *e disease and the number of luminescent colonies.

To ensure the method of detection of luminescent Vibrio colonies was reliable, replicas of several TCBS plates were made using medium comprising nutrient agar.

The same colonies that were luminescent on the TCBS plates were also luminescent on the nutrient agar and no further luminescent colonies were found. Thus, the TCBS agar medium method was accepted as providing a reasonably quick indication of the presence of luminescent Vibrio in the routine monitoring of pond water and sediment.

Total Gram positive bacteria were counted with the method of Kwee et al. (1988), using a spread plate technique on a medium containing the fluorescent stain 8- S:23988D

I,'

14 anilino-l-naphthalene sulphonic acid (ANS) and standard plate count agar for fresh water bacteria (no added salt to suppress strictly marine bacteria) available from the Merck, USA. To prepare the medium, 25 mg ANS was mixed with 100 ml of standard agar in distilled water prior to the adjustment of the pH to 8.0 with 1M NaOH and autoclaving. The plates were viewed on a colony counter with yellow side illumination and vertical UV at 365 nm (Applethorn, Banksia Scientific, Brisbane, Australia).

Random checks of colonies that were both fluorescent and non-fluorescent were made with Gram staining to confirm the validity of the method.

Spores were counted on standard plate count agar after the first stage of the serial dilution was heated at 80 0 C for 10 minutes.

1.1 COMPARISON OF VIBRIO NUMBERS IN PONDS ON FARMS A AND B Total Vibrio numbers in source canal water (salinity 20 ppt) varied from 120 to about 500/ml and luminescent species comprised between 1 to 10% of these.

Total Vibrio numbers in the water and sediment of two ponds of farm B are shown below in Table 2. The percentages of the total Vibrio which were luminescent are also shown.

25 Table 2: Vibrio Abundance in Two Ponds on Farm B eeoc r r Total Vibrio Luminescent Luminescent Bac eria Vibrio Racteria Vihrio E acteria Pond 1 Pond 2 Pond 1 Pond 2 Pond 1 Pond 2 Watercells/ml 2000 6000 150 600 7.5 Sediment 6000 1x10 6 200 4x10 4 3.3 4 cells/cm 3 In water of ponds on farm A, total bacterial numbers of green coloured Vibrio comprised around 50% of total Vibrio abundance and there was an order of magnitude more Gram positive bacteria present resulting S:23988D I1' 15 from regular additions to the ponds. Luminescent Vibrio were generally not found in the water of ponds on farm A and when occasionally present were low in number (see Figure Luminescent Vibrio were not found in sediments of the ponds. In contrast, pond water in another farm located about 5 kms from farm B, which also received no bacterial additions, had 11% luminescent Vibrio in the water and Vibrio colonies that were green on TCBS agar generally comprised about two thirds of the total number.

1.2 DAILY RELATIVE BACTERIAL ABUNDANCES IN POND Al The bacterial community composition in pond Al of farm A was evaluated over a two week period. Gram positive bacteria were added to the pond water at intervals of 2 to 3 days. The results are shown in Figure 2, which also indicates numbers of Gram positive bacteria used. DMS-1000, DMS-1100 and DMS-2001 were added for water quality maintenance, while additions of DMS-2002 were made for control of luminescent Vibrio numbers. DMS-1000 comprised a mixture of B. subtilis, B. licheniformis and B. pasteuri, while DMS-1100 and DMS-2001 each comprised a different strain of B. subtilis. DMS-1000, DMS-1100 and DMS-2001 are all available from Biomanagement Systems Pty Ltd., Winnellie, Australia.

As shown in the figure, total Gram positive bacterial numbers changed by up to an order of magnitude on a daily basis, whereas the number of total bacteria and total Vibrio colonies remained relatively constant.

Luminescent Vibrio bacteria entered the pond when pond water was exchanged with source canal water. However, the luminescent Vibrio numbers decreased sharply after the addition of DMS-2002. Although a delay of two days was observed before luminescent Vibrio numbers decreased 35 after the initial addition of DMS-2002, that addition coincided with inflow of water from the source canal.

The additions of the Gram positive bacteria for water quality control had no effect on luminescent Vibrio S:23988D 16 numbers.

A significant increase in both total bacterial and Gram positive bacterial numbers occurred two days after tea seed cake (saponin) was used to kill fish in the ponds. The increase in Gram positive numbers of 2x10 5 /ml was about 1% of the total increase in bacterial numbers resulting from the addition of the saponin. Vibrio numbers did not increase at the same time as the numbers of Gram positive strains following the addition of the saponin.

1.3 RELATIVE BACTERIAL ABUNDANCES IN PONDS A2 AND A3 DURING SHRIMP GROW OUT Bacterial numbers were monitored in ponds A2 and A3 for periods of 70 and 120 days respectively, following addition of larvae of the shrimp P. monodon to the ponds.

DMS-2006, a mixture of Bacillus strains DMS-2003 and DMS-2004 capable of luminescent Vibrio control, was added following increases in the number of luminescent bacteria. The additions of DMS-2006 are indicated in Figures 3 and 4. Additions of Bacillus bacteria for water quality maintenance were also made regularly to the ponds during the monitoring period and for pond A2 were generally 10 litres/ha per day, while additions of litres/ha per day were made to pond A3 until day 90 and then 10 litres/ha per day for the remainder of the monitoring period. Bacterial numbers in the ponds were evaluated at 5 to 10 day intervals.

In pond A3 total bacterial abundance in the water was about 2x10 7 /ml on day one of the study rising to between 3 to 5x10 7 /ml after 60 days of culture Total Vibrio bacterial numbers were low at the beginning of the study and then fluctuated around 1x10 3 /ml. However, the number decreased dramatically in response to the addition of DMS-2006 between days 65 and 70 of the monitoring 35 period. Luminescent Vibrio were absent until day 60 and disappeared with the decrease in total Vibrio number following the addition of DMS-2006. As shown in Figure 3, the number of Gram positive bacteria in the water also S:23988D 17 increased markedly at this time. A decrease in luminescent Vibrio numbers was also observed toward the end of the monitoring period following a further addition of DMS-2006. The abundance of Gram positive bacteria varied between 1x10 3 and ix10 5 /ml of water during the study and again increased after saponin was added to the pond water. The increase occurred after day 30 and lasted until day 50. However, there was no corresponding increase in total Vibrio numbers or luminescent Vibrio numbers during that time.

Similar increases in Gram positive bacterial abundance were observed in pond A2 after saponin addition (see Figure Luminescent Vibrio numbers also decreased sharply while Gram positive numbers increased following the addition of DMS-2006.

Total bacterial numbers in the sediments of ponds A2 and A3 increased during the monitoring periods from around 1x10 8 to 5x10 9 /cm 3 (see Figures 5 and The figures also show that the number of Gram positive bacteria fluctuated around 1x10 7 /cm 3 while Gram positive spore abundance fluctuated around 1x10 6 /cm 3 Luminescent Vibrio were not detected in the sediment of pond A2 or A3 ":for the duration of the monitoring periods.

The results show that total Gram positive bacterial numbers in the ponds were significant relative to total bacteria, being around 1% in water and 1 to 5% in sediment. Accordingly, the bacterial additions were S.having a significant effect on species composition in the pond water; where Gram positive bacteria are not normally found in large numbers. Thus it was possible to modify the flora in the ponds. Modification of the flora can be maintained providing Gram positive bacteria are added regularly. The fact that abundance did not continually "increase in the water or sediment with the daily 35 additions demonstrates that the added Bacillus bacteria did not survive for long in those environments. The increase in total Gram positive bacterial numbers following the addition of saponin indicates that the S:23988D I I 18 bacteria were responding rapidly to changes in organic matter concentration and suggests that they were limited by the availability of organic carbon. As total Vibrio numbers did not increase with the Gram positive numbers it indicates they were being controlled by the added bacteria and could not compete for organic matter.

A high abundance of luminescent Vibrio bacteria in ponds is consistent with the occurrence of disease in shrimp cultured in the ponds and low to zero harvest results. As indicated on the right side of Figure 7 shrimp in ponds on farm B and those on other farms that were not treated with additions of Gram positive bacteria for water quality control and Bacillus strains selected for their ability to suppress luminescent Vibrio suffered from Vibrio disease at 30 to 80 days of culture. Many crops were lost entirely, and the maximum harvested was about 1 tonne per hectare. In contrast, ponds on farm A subjected to such treatment were free of Vibrio disease and able to culture shrimp for more than 160 days with harvests of between 5 to 11.5 tonnes per hectare as indicated on the left side of Figure 7.

EXAMPLE 2 .2.1 CONTROL OF VIBRIO BACTERIA IN HATCHERY TANKS The control of luminescent Vibrio bacteria by DMS-2003 was assessed in the presence of the 20 mysid stage P. monodon larvae (20 days) in 1 litre hatchery tanks containing sea water with a salinity level of ppt. The tanks contained no sand or sediment.

DMS-2003 was added to all hatchery tanks to a concentration of 1x10 4 /ml one day prior to luminescent Vibrio strains LV1 and LV2 isolated from farms A and B.

The Vibrio strains were added to the respective tanks at a concentration of between 2x10 5 /ml to 6x10 5 /ml. Vibrio counts were made at one day intervals on TCBS medium.

35 Two hatchery tanks were used as controls and contained Vibrio strain LV1 at 6x10 5 /ml and shrimp larvae only.

Two days after the addition of the Vibrio bacteria DMS-2003 was added to one control at the same S:23988D 19 concentration as the test tanks.

As shown in Figure 8, the numbers of the luminescent Vibrio strains in the test tanks had decreased significantly by day 3. A dramatic decrease was also observed for control B after the addition of DMS-2003. In contrast, luminescent Vibrio numbers in control A decreased much more slowly in the absence of DMS-2003.

Although the present invention has been described hereinbefore with reference to several embodiments, numerous variations and modifications are possible without departing from the scope of the invention which is defined in the following claims.

S:23988D 20 LIST OF REFERENCES CITED 1. Ruanqpan and Kitao, 1991. Vibrio Bacteria Isolated from Black Tiger Shrimp, Penaeus monodon Fabricius.

Journal of Fish Diseases 14: 383-388.

2. Nithimathachoke, Pratanpipat, P., Thongdaen., Withyachumnarkul. and Nash. G.

1995. Luminous Bacterial Infection in Pond Reared Penaeus Monodon. Asian Shrimp News 3rd quarter, Issue No 23. 1-4.

3. Baticados. Lavilla-Pitogo, C.R., Cruz-lacierda de la Pena. and Sunaz, 1990. Studies on the Chemical Control of Luminous Bacteria Vibrio harveyi and V.splendidus Isolated from Diseased Penaeus Monodon Larvae and Rearing Water. Dis. Aquat. Org. 9: 133-139.

4. Kruse. Sorum, H. 1994. Transfer of Multiple Drug Resistance Plasmids between Bacteria of Diverse Origins in Natural Environments. Applied and Environmental Microbiology. 60: 4015-4021.

Scura. 1995. Dry Season Production Problems on Shrimp Farms in Central America and the Caribbean Basin. In: Swimming Through Troubled Water.

Browdy, and Hopkins (Eds), Proceedings of the Special Session on Shrimp Farming, Aquaculture '95. World Aquaculture Society, Baton Rouge, pp 200-213.

6. Boyd, 1995. Chemistry and Efficacy of Amendments Used to Treat Water and Soil Quality Imbalances in Shrimp Ponds. In: Swimming Through Troubled Water. Browdy, and Hopkins J.S., (Eds), Proceedings of the Special Session on Shrimp Farming, Aquaculture '95. World Aquaculture Society, Baton Rouge, pp 183-199.

7. Billen. 1990. Delayed Development of Bacterioplankton with Respect to Phytoplankton: A Clue for Understanding their Trophic Relationships.

Arch. Hydrobiol. Beih. Ergebn. Limnol. 34, 191-201.

8. Van Wambeke. 1994. Influence of Phtyplankton Lysis or Grazing on Bacterial Metabolism and Trophic Relationships. Microb. Ecol. 27, 143-158.

9. Moriarty, D.J.W. 1990. Interactions of Microoganisms and Aquatic Animals, Particularly the Nutritional Role of the Gut Flora. pp.217-222.

In: R. Lesel Microbiology in Poecilotherms.

Elsevier Science Publishers, B.V. (Biomedical Division) S:23988D 21 Moriarty, 1987. Methodology for Determining Biomass and Productivity of Microorganisms in Detrital Food Webs. p.4-31. In: Detritus and Microbial Ecology in Aquaculture Moriarty and R.S.V. Pullin, Eds), ICLARM Conference Proceedings 13: 385 p. International Center for Living Aquatic Resources Management, Manila, Philippines.

11. Herbert, 1990. Methods for Enumerating Microorganisms and Determining Biomass in Natural Environments. Methods in Microbiology. 22: 1-39.

12. Kwee, Dommett, and Vos, 1988.

Fluorogenic Medium for Differential Gram-Negative, Gram-Positive and Total Bacterial Counts in Liquid Milks. Australian Journal of Dairy Technology.

43: 50-53.

*o S:23988D

Claims

1. A method of controlling pathogenic bacteria in a body of water comprising the step of adding an effective number of Gram positive bacteria from at least one Gram positive bacterial strain to the water, wherein the Gram positive bacteria are non-pathogenic and are capable of inhibiting or preventing the growth or replication of the pathogenic bacteria.

2. A method according to claim 1, wherein the at least one Gram positive bacterial strain belongs to the genus Bacillus.

3. A method according to claim 2, wherein the at least one Gram positive bacterial strain is selected from the group of bacterial species consisting of B. polymyxa, B. subtilis, B. laterosporus, B. circulans, B. licheniformis and B. brevis.

4. A method according to any one of claims 1 to 3 wherein the Gram positive bacteria are added to the water to an initial concentration of about Ixl0 4 cells/ml of the water or greater.

A method according to claim 4, wherein the Gram positive bacteria are added to the water to an initial *...concentration of between about 5x10 4 to about ie• 1x10 5 cells/ml of the water.

6. A method according to any one of claims 1 to wherein the method also comprises the step of making one or more further additions of the Gram positive bacteria the water at desired intervals.

7. A method according to claim 6, wherein each said further addition comprises the addition of sufficient said Gram positive bacteria to raise the concentration of the Gram positive bacteria in the water to about 1x10 4 per ml of water or greater.

8. A method according to claim 7, wherein each said further addition comprises the addition of sufficient said Gram positive bacteria to raise the concentration of the Gram positive bacteria in the water to between about 5x10 4 to about 1x10 5 cells/ml of the water. S:23988D 23

9. A method according to any one of claims 1 to 8, wherein the pathogenic bacteria consist of Gram negative bacteria.

A method according to claim 9, wherein the Gram negative bacteria belong to the genus Vibrio.

11. A method according to claim 10, wherein the Gram negative bacteria are luminescent Vibrio bacteria.

12. A method according to claim 10, wherein the Gram negative bacteria comprise one or more strains of bacteria selected from the group of bacterial species consisting of V. harveyi, V. cholerae, V. parahaemolyticus, V. vulnificus and V. alginolyticus.

13. A method according to any one of claims 1 to 9, wherein the pathogenic bacteria is a pathogen of shrimp.

14. A method according to any one of claims 1 to 13, wherein the body of water comprises a water source, a hatchery tank, an aquaculture pond or reservoir, or a treatment pond or lagoon.

A method according to claim 14, wherein the body of water comprises a hatchery tank or an aquaculture pond.

16. A method according to any one of claims 1 to further comprising the step of making one or more additions of other bacteria to the water for water *...quality maintenance.

S17. A method according to claim 16, wherein the other bacteria are other Gram positive bacteria.

18. A body of water when treated by a method as defined in any one of claims 1 to 17.

19. An animal when raised in a body of water treated by a method as defined in any one of claims 1 to 17

20. A method of inhibiting the infection of an eoom animal by pathogenic bacteria in a body of water in which the animal is being raised, comprising the step of soaking feed material for the animal in an inoculum containing Gram positive bacteria from at least one Gram positive bacterial strain, prior to adding the feed material to the water, wherein the Gram positive bacteria S:23988D i. I 24 are non-pathogenic and are capable of inhibiting or preventing the growth or replication of the pathogenic bacteria.

21. A method according to claim 20, wherein the Gram positive bacteria are present in the inoculum at a concentration of 1x10 8 cells/ml or greater.

22. A method according to claim 21, wherein the Gram positive bacteria are present in the inoculum at a concentration of about 1x101 0 to 1x101 cell/ml.

23. A method according to any one of claims 20 to 22, wherein the at least one Gram positive bacterial strain belongs to the genus Bacillus.

24. A method according to claim 23, wherein the at least one Gram positive bacterial strain is selected from the group of bacterial species consisting of B. polymyxa, B. subtilis, B. laterosporus, B. circulans, B. licheniformis and B. brevis.

A method according to any one of claims 20 to 24, wherein the pathogenic bacteria consist of Gram negative bacteria.

26. A method according to claim 25, wherein the Gram negative bacteria belongs to the genus Vibrio.

27. A method according to claim 25, wherein the Gram negative bacteria comprise one or more strains of bacteria selected from the group of bacterial species consisting of V. harveyi, V. cholerae, V. parahaemolyticus, V. vulnificus and V. alginolyticus.

28. Non-pathogenic Gram positive bacteria in the form of an inoculum and being capable of inhibiting or preventing the growth or replication of pathogenic bacteria, when used in a method as defined in any one of claims 1 to 17 or any one of claims 20 to 27.

29. A method of controlling pathogenic bacteria in a body of water comprising the step of adding at least one Gram positive bacterial strain capable of inhibiting or preventing the growth or replication of the pathogenic bacteria, substantially as hereinbefore described with reference to any one of the Examples. S:23988D 25 Dated this 18th day of September 1996 BIOMANAGEMENT SYSTEMS PTY LTD By their Patent Attorneys GRIFFITH HACK S:29.8