WO2000056166A1

WO2000056166A1 - Animal plasma supplement for aquaculture

Info

Publication number: WO2000056166A1
Application number: PCT/US2000/007611
Authority: WO
Inventors: Yukinori Takahashi
Original assignee: American Protein Corporation
Priority date: 1999-03-23
Filing date: 2000-03-22
Publication date: 2000-09-28
Also published as: AU3768900A; AR023135A1

Abstract

The present invention relates to a method of improving the survivability of aquatic animals exposed to disease. The invention includes the administration of a plasma product to the animal through the animal's feed. In a preferred embodiment, the supplement comprises up to 15 % by weight of the feed and may be administered in granulated or powdered form. The product is effective in decreasing the rate of mortality in aquatic animals following viral inoculation.

Description

TITLE: ANIMAL PLASMA SUPPLEMENT FOR AQUACULTURE

FTELD OF THE INVENTION

The present invention relates to the use of dried animal plasma or components purified therefrom as a disease prevention supplement (or immune stimulating agent) for aquaculture.

BACKGROUND OF THE INVENTION

Thailand is one of the fastest growing producers of black tiger shrimp (Penaeus monodoή). Over the past several years, Thailand's farmed shrimp production has been significant, with production reaching 248,000 metric tons in 1994. Limsuwan, C, 1997. Reducing the Effects of White-Spot Baculovirus Using PCR Screening and Stressors, AAHRI Newsletter. Vol. 6, No. 1.

In southern Thailand, yellow-head virus (YHV) first caused extensive losses in cultivation of black tiger shrimp in late 1992. Flegel, T.W. et al., 1997, Progress in Research on Yellow-Head Virus and White-Spot Virus in Thailand, In T.W. Flegel and I.H. MacRae (eds), Diseases in Asian Aquaculture III, Fish Health Section, Asian Fisheries Society, Manila. The same area was subsequently affected by widespread infection from white spot baculovirus (WSBV) beginning in 1994. The pond side losses caused by these two viral diseases are believed to be responsible at least in part for the 25% drop in shrimp production for the first half of 1995.

Losses from YHV have continued in shrimp populations, although the severity and frequency of outbreaks decreased sharply after the beginning of 1994. However, studies have shown that the occurrence of the virus in cultivation ponds is widespread in shrimp that show no gross signs of disease.

WSBV is one of the most virulent viruses reported to infect black tiger shrimp. Tapay, L.M. et al. 1997, Infection of White-spot Baculovirus-like Virus (WSBV) in Two Species of Penaeid Shrimp Penaeus stylirostris (Stimpson) and P. vannamei (Boone). In T.W. Flegel and I.H. MacRae (eds.), Diseases in Asian Aquaculture III. Fish Health Section, Asian Fisheries Society, Manila. It has been demonstrated to be similarly pathogenic and highly infectious for P. stylirostris (blue shrimp) and P. vannamei (white shrimp), the two penaeid species which are commercially cultivated in Hawaii and the Western Hemisphere.

Since its first appearance in 1995, WSBV has spread to most of the black tiger shrimp culture areas in Thailand. Limsuwan, Chalor, 1997, What kind of white spot kills shrimp?, AAHRI Newsletter, Vol. 6, No. 2. Histopathological examination of animals infected with WSBV reveals extensive cellular necrosis in the cuticular epidermis and mesodermal tissues. Clinical signs of WSBV include easily observed white spots of various sizes embedded in the shrimp's shell during the later stages of infection. Among the major diseases of shrimp, WSBV-caused disease is currently the most serious due to its widespread occurrence through culture areas along the coastal area of the Gulf of Thailand and the Andaman Sea. Shrimp cultured in low salinity water are also affected by WSBV.

In the past few years, mortalities from WSBV have been linked to outbreaks resulting from transport of disease by such vectors as wild shrimp and other Crustacea which enter the pond system during water exchange. Since shrimp farmers have changed to closed or semi-closed systems, reduced the amount of water exchange and have implemented proper pond management, the severity of infection has been reduced. However, WSBV still has the ability to cause problems to shrimp culture systems due to the presence of the virus in post-larvae.

Infection from Vibrio bacteria has been reported in black tiger shrimp since 1989 in the Samut Sakhorn area, where intensive shrimp farming in Thailand began.

Chanratchakool, Pornlerd, 1995, White Patch Disease of Black Tiger Shrimp (Penaeus monodoή), AAHRI Newsletter, Vol. 4, No. 1. Shrimp infected with Vibrio bacteria exhibit a dark reddish color on the shell with opaque muscle and exhibited loss of appetite. White spots or patches are also observed in some of the affected populations. Mortality usually occurs a few days after red discoloration. The principal causes of infection have been identified as pond bottom and water quality deterioration which stress the animal and lead to secondary bacterial infection.

Intensive research activity has been focused on the eradication of diseases caused by Vibrio bacteria and YHV and WSBV viruses since their discovery. Although the frequency and occurrence of YHV and WSBV outbreaks have decreased since 1994, losses have continued. Early attempts at preventing disease in shrimp and other crustaceans have included improving the pond environments and through the administration of antibiotics. These techniques, however, have not proven successful. Successful antibiotic treatment depends upon the uptake of the drug by the shrimp. However, stressed shrimp are unlikely to take medicated feed.

Methods of decreasing the stress factors of pond bottom and water quality deterioration have included improvement of the pond environment by water exchange, adjustment of feeding, limiting, aeration, and antibiotic treatment for the secondary infection. Unfortunately, however, proper pond management in and of itself has not been successful in eradicating these diseases.

More recently, prevention of YHV and WSBV have focused on the development of diagnostic techniques using selected probes derived from viral RNA and cDNA clones, including dot blot hybridization tests, in situ hybridization tests, and PCR amplification tests. These techniques have proven useful in testing broodstock animals, post larvae and other crustaceans as potential sources of the virus in the shrimp farming system. Since both viruses can be spread by infected water and by non-cultivated crustacean carriers, current preventative measures emphasize water management and exclusion of carrier species.

Despite such diagnostic testing, results show that some disease outbreaks occur in Thailand and elsewhere even in ponds stocked with post-larvae that tested negative for WSBV using these technologies. There are several possible reasons for this. First, randomly examined post-larvae samples do not necessarily represent the entire batch. Second, even virus-free shrimp can become infected if put in poorly managed culture systems. There is therefore a need in the art for an improved method of preventing disease in shrimp and other aquatic animals.

It is therefore a primary objective of the present invention to provide an improved method of preventing disease in aquatic animals which does not rely on the use of diagnostic techniques, including dot blot hybridization tests, in situ hybridization tests, and PCR amplification tests. It is a further objective of the present invention to provide an improved method of preventing disease in aquatic animals which does not require the administration of antibiotics or other medications.

It is yet a further objective of the present invention to provide an improved method of preventing disease in aquatic animals which is effective in preventing future outbreaks.

It is still a further objective of the present invention to provide an improved method of preventing disease in aquatic animals which may be administered in the animals' food source.

It is a further objective of the present invention to provide an improved method of preventing disease in aquatic animals which is easy and economical to use.

These and other objectives will become clear in the following detailed description of the invention.

SUMMARY OF THE INVENTION The present invention describes a method of preventing disease in aquatic animals through the administration of a dried plasma supplement. The supplement is prepared by separating plasma from animal blood, which is preferably porcine or bovine. The plasma is optionally concentrated, then dried to form a beige powdery substance.

The dried plasma supplement is fed to aquatic animals. Surprisingly, administration of the plasma supplement has been found to increase the survivability of the animals when challenged with various diseases, including those caused by the WSBV virus.

DETAILED DESCRIPTION OF THE DRAWINGS FIG. 1 is a graphical representation of the results of a study where Appetein and

AP920 were fed to shrimp for 7 consecutive days. The graph compares the rate of phagocytic activity (%) in the shrimp blood versus the number of days after dosing.

FIG. 2 is a graphical representation of the results of the study depicted in FIG. 2 measuring the number of latex beads destroyed versus the number of days after dosing. FIG. 3 is a graphical representation of the cumulative phagocytic activity index comprised of the data of FIG. 2 multiplied by the data of FIG. 3. FIG. 4 is a graphical representation of the melanin produced by the shrimp in the study depicted in FIG. 2-4. The graph compares the light absorption rate versus the number of days after dosing.

FIG. 5 is a graphical representation of the results of a trail whereby the diet of shrimp was supplemented with the composition of the invention, either in the form of Appetein (granular animal plasma) or AP920 (spray dried animal plasma in powder form). The graph compares the effectiveness of Appetein and AP920 versus control in increasing the survival rate (%) of shrimp tested.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of dried animal plasma or components purified therefrom as a disease prevention supplement (or immune stimulating agent) for aquaculture. According to the invention, marine animals fed a dried plasma supplement derived from non marine animals demonstrated increased survivability and immune activation upon exposure to traditional pathogens over those which were not fed the supplement.

The present invention contemplates use with any aquatic animal, preferably one which is used or maintained as a food source in aquaculture. In a preferred embodiment, the range of plasma supplement administered is up to 15 percent of the diet, however no critical range is observed and performance is increased with higher doses. In a preferred embodiment the disease prevention is directed to White Patch or White Spot disease and the aquatic animal food source is shrimp, although similar results have been observed in Grouper and would be expected for any aquatic animal.

The plasma is obtained by collecting blood from animals. The blood from any red blooded homeothermic animal (porcine, bovine, ovine, equine, avian) can be used to practice the invention. In a preferred embodiment the animal is a livestock animal which is slaughtered for its meat product. The blood, which is traditionally discarded or dried and processed as blood meal, may then be used for preparation of the compositions and implementation of methods of the invention. In a most preferred embodiment the blood is collected from pigs or cattle, with pig blood being most preferred. Generally, according to the invention, blood is collected, preferably at slaughter plants. In one embodiment, the blood may be held in a circulating stainless steel tank with anticoagulants such as sodium citrate or sodium phosphate to prevent clotting. Prevention of clotting is not essential to the invention as similar effects can be obtained with clot- removed serum or defibrinated plasma. Typically, the whole blood is then separated, preferably by centrifugation, although any other separation method may be used, into two parts, the cellular material (red corpuscles, white corpuscles, platelets, and other circulating precursor cells of the previous categories of cells) and plasma (or serum).

Plasma (serum) is composed of about 55-60% albumin, 25-30% globulin, 10% fibrinogen, and other proteins. As used herein the term "plasma" shall include the plasma portion of blood as well as any of the protein components which may be further purified therefrom. Purification of these components from plasma are methods known and commonly practiced by those of skill in the art. After separation, the plasma may be cooled to retard growth of bacteria and stored in an insulated tank until ready to dry. Plasma and/or the purified components of plasma, may then be further concentrated

(by membrane filtration). The concentrated product is next dried, preferably by spray- drying to form a beige powdery substance. Spray-drying should occur at temperatures low enough to prevent the complete denaturation of proteins but high enough to eliminate bacterial and viral contamination. Traditionally, a drier inlet temperature of approximately 375° to 400°F and an outlet temperature from the drier of 180-200°F will accomplish this objective. The resulting powdery substance will have a particle size of about 5 to about 30 microns. The powder may then be compacted or compressed (around 1200 to 1400 psi), ground and optionally may be screened or otherwise separated by size to increase homogeneity. The resulting particle size is at least about 50 microns. Preferably the size is greater than about 100 microns but less than about 2000 microns in diameter.

The granulated substance preferably comprises from up to 15% by weight of the base feed.

The dried plasma powder contemplated for use in this invention is comprised of high levels of amino acids. A typical amino acid assay of the powder by acid hydrolysis and subsequent column chromatography results in the following amino acid concentrations (grams per 100 grams of powder): Alanine 4.2

Arginine 4.7

Aspartic Acid 7.9

Cystine 2.8

Glutamic Acid 11.7

Glycine 3.0

Histidine 2.8

Isoleucine 2.9

Leucine 7.8

Lysine 6.8

Methionine 0.7

Phenylalanine 4.6

Proline 12.8

Serine 4.7

Threonine 4.8

Tryptophan 1.4

Tyrosine 3.6

Valine 5.3

Chemical and other properties if dried plasma include about 60-80% protein, 9% moisture, 5-20% ash, 2% fat, 50.0 ppm iron, 0.15% calcium, 1.50% chloride; 1.7% phosphorous, 0.09% potassium, aqueous solubility 88%.

Spray-dried animal plasma is commercially available from several sources including American Meat Protein Corporation product sold under the mark of AP 920™, or Appetein™.

According to the invention, plasma or the disease preventative agents isolated therefrom are fed to aquatic animal food sources to increase survivability upon challenge with various diseases such as white patch disease. The following examples are offered to illustrate but not limit the invention. Thus, they are presented with the understanding that various formulation modifications as well as method of delivery modifications may be made and still be within the spirit of the invention.

EXAMPLES

EXAMPLE 1

FIG. 5 and Table 1 depict the results of a trial done with supplementation of the diet of shrimp with the composition of the invention, either in the form of Appetein (granular animal plasma) or AP920 (spray dried animal plasma in powder form). As can be seen, the group that was fed 4% of Appetein had 73% survival rate despite virus inoculation with white spot disease. The control which was fed no supplement experienced as much as complete mortality 10 days after virus inoculation. The method for obtaining inoculum and challenge of the animals follows. First, shrimp were infected with white spot disease virus by injection. After the shrimp died they were frozen and stored at -80°C. The heads were then cut off the frozen shrimp and homogenized in water. The virus was separated out by centrifugation. The virus was inoculated in sea water and dipped live trial shrimp for 1 hour to make sure that the trial shrimps were infected. Shrimps used for the study were Kuruma Prawn or Penaeus Japonica. Similar results were observed with a trial to measure Phagocytic activity and phenoloxitarze activity.

Any traditional aquaculture feed may be supplemented according to the invention and such feeds are well known those of skill in the art. Appetein was used within the feed and diet composition comprised the following: moisture 8.05%, crude protein 59.38%, crude fat 7.12%, crude ash 17.55%, soluble CP 15.67%, VBN 0.08%. While not wishing to be bound by any theory it is postulated that there is a peptide or protein activator presence in the dried animal plasma that is acting as an "immune activator" rather than a specific IgG immune response. TABLE 1

Condition After Virus Inoculation

EXA MPLE 1 2

A. Phagocytic Activity (Please see FIGS. 1, 2, and 3)

FIG. 1 - Phagocytic Activity Ratio (%)

Appetein and AP920 contained diets were fed to shrimp for 7 consecutive days. On day 0, 3 and 7, shrimps' blood was collected (i.e. body fluid) and "dosed" latex beads (size = 1 micron) to simulate "antigen" to see how corpuscles attack or "eat" the latex beads out of total 100 beads. As can be seen from the table 4% Appetein had the highest percentage Phagocytic activity.

FIG. 2 - Average Inclusion (or Attack^ Count A measure of how many latex beads each corpuscle destroyed. Appetein at 4% was the highest. FIG. 3 - Phagocytic Activity Index

The index derived by multiplying Figures 2 and 3.

B. Phenol Oxidase Activity (Please see FIG. 4)

The inventors measured how much of "melanin" was produced by shrimp. Melanin has a function to wrap (i.e. eat) pathogenic organ function. So, if there are more melanin, then, higher immune-activation was achieved. As shown, the 4% Appetein achieved the highest rate of immune-activation.

As can be appreciated, the feeding of animal plasma to aquatic animals was effective in improving their resistance to disease. It is therefore seen that the invention accomplishes at least all of its stated objectives.

Claims

What is claimed is:

1. A method of stimulating the immune system of aquatic animals challenged comprising: administering to aquatic animals a supplement comprising animal plasma.

2. A method according to claim 1 whereby the supplement is administered through the animals' feed.

3. A method according to claim 2 whereby the supplement comprises up to 15% by weight of the animals' feed.

4. A method according to claim 1 whereby the supplement is spray-dried.

5. A method according to claim 4 whereby the particle size of the animal plasma is at least 50 microns.

6. A method according to claim 4 whereby the particle size of the animal plasma is less than about 2000 microns.

7. A method according to claim 1 wherein the source of the animal plasma is a livestock animal.

8. A method according to claim 7 wherein the source of the animal plasma is porcine or bovine blood.

9. A method according to claim 1 whereby the aquatic animal is selected from the group consisting of shrimp and grouper.

10. A method according to claim 9 whereby the disease is white spot disease.

11. A method of manufacturing a supplement for stimulating the immune system in aquatic animals comprising: collecting whole blood from animals; separating the plasma from the whole blood; concentrating the plasma to form a concentrated product; and drying the concentrated product.

12. A method according to claim 11 whereby the plasma is separated from the whole blood by centrifugation.

13. A method according to claim 1 1 whereby the plasma is concentrated by membrane filtration.

14. A method according to claim 11 whereby the concentrated product is dried by spray-drying.

15. A method according to claim 11 further including the step of: compressing the concentrated product following the drying step.

16. A method according to claim 15 whereby the concentrated product is compressed at about 1200-1400 psi.

17. A plasma feed product for increasing the survivability of aquatic animals exposed to disease comprising: aquatic animal feed; and animal plasma.

18. A plasma feed product according to claim 17 whereby the animal plasma comprises up to 15%) by weight of the product.

19. A plasma feed product according to claim 17 whereby the animal plasma is dried.

20. A plasma feed product according to claim 17 whereby the animal plasma is granulated or powdered.