AU2011218616A1 - Ostrich feed supplement - Google Patents

Abstract

The invention provides an ostrich feed supplement composition which includes at least one bacterial strain of Lactobacillus oris, Lactobacillus brevis, Lactobacillus johnsoni, Bifidobacterium pseudolongum subsp. globosum, and Enterococcus faecalls. The Lactobacillus oris is typically a strain which has been designated Lo5g, the Lactobacillus brevis is typically a strain which has been designated Lb512, the Lactobacillus johnsonii is typically a strain which has been designated Lj136, the Bifidobacterium pseudolongum subsp. globosum is typically a strain which has been designated Bg136, and the Enterococcus faecalis is typically a strain which has been designated P1.2. The bacteria can be obtained from the gastrointestinal tract or from faecal samples of healthy ostriches.

Description

Regulation 3.2 Revised 2/98 AUSTRALIA Patents Act, 1990 ORIGINAL COMPLETE SPECIFICATION TO BE COMPLETED BY THE APPLICANT NAME OF APPLICANT: University of Cape Town ACTUAL INVENTOR: REID, Sharon Joy ADDRESS FOR SERVICE: Peter Maxwell and Associates Level 6 60 Pitt Street SYDNEY NSW 2000 INVENTION TITLE: OSTRICH FEED SUPPLEMENT DETAILS OF ASSOCIATED APPLICATION NO(S): 2010/06209 - 31 August 2010 - ZA The following statement is a full description of this invention including the best method of performing it known to me: m:\docs\20111030\229096.doc 2 OSTRICH FEED SUPPLEMENT 5 FIELD OF THE INVENTION 10 This invention relates to a feed supplement for ostriches. BACKGROUND OF THE INVENTION 15 Ostrich farming is a rapidly growing industry world-wide. One of the major problems facing the industry is chick mortality, which can be as high as 50% in the first three months. A predominant cause of death is pathogenic infections of the gastrointestinal tract. Treatment with antibiotics often results in the development of resistant pathogens and prevents the invasion of naturally 20 occurring protective intestinal microflora in young birds, and there are also concerns about antibiotic residues in ostrich meat intended for human consumption. There is therefore a requirement for an alternative product to replace conventionally available antibiotics. 25 It has been shown from work with poultry, that neonatal nutrition enhances the normal development of the intestinal tract and stimulates immune development. Nutrition may therefore play a key role in the rearing of ostrich chicks, but knowledge of the nutritional requirements of ostriches is still not well defined.

3 Probiotics are live microbial feed supplements that, when administered in sufficient quantities, confer a health benefit to the host animal, generally by improving its intestinal balance. 5 It appears gut microbiota are often involved in resistance to disease in farm animals. The stressful conditions experienced by the young animal in modern farming practices causes changes in the composition and the activity of the gut microbiota. Probiotic supplementation can in some instances help to restore the balance and provide the type of microbiota found in more natural 10 environments, and bacterial probiotics have been shown to be effective in pigs, cattle and pre-ruminant calves. The reasons why probiotics successfully improve the health of some farmed animals is multifactorial. The primary effector of this beneficial effect is thought 15 to be due to competitive exclusion, whereby the colonisation of the gut by the commensals prevents the adhesion and successful establishment of the pathogenic strains. The majority of probiotics are members of the lactic acid producing bacteria (LAB), including Lactobacillus, Bifidobacterium and Enterococcus species. These bacteria produce a variety of substances 20 including organic acids, hydrogen peroxide or bacteriocins that are inhibitory to both gram-positive and gram-negative bacteria. Some LAB species have recently been shown to be essential for the normal development of the gut epithelium and the immune system, by modulating the innate defence systems of the gut. 25 However, not all LAB strains are effective feed additives, and probiotics do not appear to be effective in all animals. Research has shown that the effectiveness of the probiotic bacteria, and the health benefits these bacteria generate, is host-specific. For example, there are contradictory results concerning whether 30 the growth performance of poultry, and in particular chickens, is improved by probiotics (Arslan C., Turkish Journal of Veterinary and Animal Sciences, 2004, Vol. 28, pages 887-891; Gri L.R. et al., Arch. Geflugelk, 2008, Vol. 72(3), pages AL 4 136-139; Iji P.A., Australian Journal of Experimental Agriculture, 2008, Vol. 48, pages 1280-1283). The research on feeding ostriches, especially feed additives, is limited, but it would appear that poultry diets have not been successfully applied to ostriches, possibly because ostriches have different 5 diets when in the wild (e.g. different fibre consumption), different digestive systems and different nutrient utilisation. It is therefore likely that probiotics which are effective in poultry would not necessarily be effective in ostriches. 10 OBJECT OF THE INVENTION It is an object of this invention to provide a feed supplement that will at least partially alleviate the abovementioned problems. 15 SUMMARY OF THE INVENTION According to a first embodiment of the invention, there is provided an ostrich feed supplement composition comprising at least one of Lactobacilus oris, 20 Lactobacillus brevis, Lactobacillus johnsoni, Bifidobacterium pseudolongum subsp. globosum and Enterococcus faecalis. The Lactobacillus oris may be the strain designated as Lo59, the Lactobacilus brevis may be the strain designated as Lb512, the Lactobacillus johnsonii may 25 be the strain designated as Lj136, the Bifidobacterium pseudolongum subsp. globosum may be the strain designated as Bg136 and/or the Enterococcus faecalis may be the strain designated as P1.2. The composition may include at least two of, at least three of, at least four of or 30 all five of Lactobacillus oris, Lactobacillus brevis, Lactobacillus johnsonii, Bifidobacterium pseudolongum subsp. globosum and Enterococcus faecalis.

5 The composition may include approximately 10 9 cfu of the above bacteria. The composition may also comprise other additives and may be freeze-dried or 5 microencapsulated in an alginate bead. The composition may be mixed into an ostrich feed. The composition may be for improving the physical condition of ostriches and/or for reducing the mortality of ostriches. 10 According to a second embodiment of the invention, there is provided a method of preparing an ostrich feed supplement composition as described above, the method comprising the steps of: suspending bacterial cells of the bacteria of the composition in 5% 15 skimmed milk; and freeze-drying the bacterial cells or microencapsulating them to form alginate beads. According to a third embodiment of the invention, there is provided a method for 20 maintaining a group of ostriches, the method comprising the step of administering a composition as described above to the ostriches in the group. BRIEF DESCRIPTION OF THE FIGURE 25 Figure 1 is a graph indicating the weight gain by ostrich chicks during feeding trials using the feed supplement of this invention.

6 ) DETAILED DESCRIPTION OF THE INVENTION A feed supplement for ostriches is provided, which includes at least one bacterium selected from Lactobacillus oris, Lactobacillus brevis, Lactobacillus 5 johnsoni, Bifidobacterium pseudolongum subsp. globosum, and Enterococcus faecalis, to form a probiotic composition. The Lactobacilius oris is typically a strain which has been designated Lo59, the Lactobacillus brevis is typically a strain which has been designated Lb512, the Lactobacillusjohnsonii is typically a strain which has been designated Lj136, the Bifidobacterium pseudolongum 10 subsp. globosum is typically a strain which has been designated Bg136, and the Enterococcus faecalis is typically a strain which has been designated P1.2. The bacteria can be obtained from the gastrointestinal tract or from faecal samples of healthy ostriches, which represent the autochthonous microbiota of the ostrich. 15 Samples of these bacterial strains were deposited in terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Belgian Co-ordinated Collections of Micro organisms (BCCM/LMG), Gent, on 19 August 2011 and have been allocated 20 the accession numbers shown in Table 1. The composition of the invention can include any one of, any two of, any three of, any four of or all five of Lactobacillus oris, Lactobacillus brevis, Lactobacilius johnsoni, Bifidobacterium pseudolongum subsp. globosum and Enterococcus 25 faecalis, in equal or varying ratios of each strain. The composition can also comprise other additives, such as skimmed milk, alginate, antibiotics, cyanocobalamin, B-biotin, D-pantothenic acid, folic acid, L ascorbyl-2-polyphosphate, myo-inositol, niacin, p-amino-benzoic acid, 30 pyridoxine hydrochloride, riboflavin, thiamine hydrochloride or choline chloride. The composition can be formulated into a powder, liquid, pellet, tablet or bead, or any other suitable formulation for administering to an ostrich or mixing into 7 ) ostrich feed. The pellet, tablet or bead can include approximately 10 9 cfu of the above bacteria. The composition can result in the improvement of the physical condition of 5 ostriches, such as increased weight gain, the improvement of the health of the ostriches, e.g. fewer intestinal infections, and/or reduce the mortality of ostriches, and in particular ostrich chicks. The invention will now be described in more detail by way of the following non 10 limiting example. Example Probiotic bacteria are proposed to exert their beneficial effects in one or more 15 ways. Firstly, in order for them to arrive in the hindgut of the ostrich in a metabolically active, viable state, the bacteria have to be able to survive the passage through the extremely acidic stomach (pH ca. 2.0) and the small intestine where the concentration of bile can reach high concentrations. Probiotic bacteria should, therefore, have a good tolerance of acid and bile at 20 the concentrations encountered. Secondly, they should be able to colonise the host animal effectively in order to persist and produce a beneficial effect, and this means that they should be capable of adhering to either gut epithelial cells or to the mucous which lines the gut. The bacteria can also aid the digestion of the host animal by producing enzymes which help digest the material in the gut. 25 In the case of the ostrich, the diet consists largely of indigestible vegetable fibre, and the beneficial enzymes are therefore cellulases, xylanases and pectinases, all of which release shorter polysaccharide chains into the gut lumen, which may be taken up by the epithelial cells directly or else further digested by the birds' own enzymes. Lastly, some bacteria produce compounds which are 30 detrimental to potential pathogens, such as lactic acid, butyrate or other antimicrobial compounds which inhibit the growth of pathogens and allow the probiotics to out-compete the pathogens.

8 Isolation and selection of probiotic strains A survey of the culturable microbiota of the hindgut from healthy ostrich adults 5 and chicks of different ages reared on different feed supplements was completed. Different bacterial strains of Lactobacillus, Bifidobacterium and Enterococcus with probiotic potential were isolated from the gastrointestinal tract or from faecal samples from the ostriches. 10 The strains were characterised with respect to their fibre-degrading capabilities, their ability to withstand the harsh conditions of the gut and to colonise the gut, as well as their microbial safety (all results not shown). Five strains were selected for further testing as possible probiotic candidates (Table 1). 15 Table 1: Isolated strains selected for testing as an ostrich probiotic Strain BCCM/LMG Species name designation Origin Accession Number Lactobacillus oris Lo59 Farmed adult LMG-P-26662 Lactobacillus brev/s Lb512 Farmed adult LMG-P-26663 Lactobaci//us Lj1 36 Farmed adult LMG-P-26664 johnsoniae Bifidobacterium Bg1 36 Farmed adult LMG-P-26666 pseudolongum subsp. globosum Enterococcus P1.2 Wild adult LMG-P-26665 fae calls These strains are all anaerobes and can grow well in the gut of ostriches where the conditions are highly anaerobic. However, it was found that they are 20 reasonably aerotolerant and can survive in air for several hours. This means that they remain viable while they are being cultivated and prepared for administration to the ostriches, and would still be metabolically active in the ostrich gut.

9 ) Table 2 summarises the characteristics of the five selected strains. All of the strains were able to adhere to yeast and to autoaggregate - indicative of good cell adherent abilities. In addition, three of the strains showed excellent mucous adhesion. All strains showed good tolerance to acidic conditions and bile salts. 5 Three of the strains were found to inhibit the growth of indicator strains, including clostridia, which may contribute to enteritis in ostrich chicks. The five selected strains also all produce at least two extracellular enzymes, which may aid in nutrition by degrading indigestible compounds and releasing products which are digestible by the host. 10 Table 2: Probiotic characteristics for selection of strains Probiotic Characteristics Strain Yeast Mucous Antimicrobial Acid Bile Enzyme Ag tin binding Action' tolerance tolerance Production Lo59 + 68 S. typh, E. faec, R R cell, pect, phyt, C. bot prot Lb512 ++ 75 S. typh, S.aur, C. R R cell, xyl bot Lj136 +++ 41 nt R R amyl, cell, prot Bg136 ++ Nt none S R amyl, prot P1.2 ++ 18 E. coli, S. typh, C. R R amyl, cell, pect, perf, C. bot phyt, prot a: + symbols indicate increasing agglutination, as defined by size of cell aggregates determined by microscopy 15 b: % bound relative to L. rhamnosus GG c: Inhibition of growth of indicator strains Escherichia coli ATCC 25922 (E. coli), Salmonella typhimurium (S. typh), Staphylococcus aureus (S. aur), Enterococcus faecium vans (E. faec), Clostridium perfringens (C. perf) or C/ostridium botulinum (C. bot). d: S indicates sensitivity; R indicates resistance, or ability to grow at a pH of 3. 20 e: S indicates sensitivity; R indicates resistance, or ability to grow in the presence of 1 % ox bile. f: Production of amylase amyll), cellulose (cell), pectinase (pect), phytase (phyt), protease (prot) or xylanase (xyl). 25 Preparation of strains for feedstocks Collection and culture of faecal samples Five freshly voided faecal samples were collected from each group in sterile 30 plastic containers. A small portion of each sample was stored immediately in 10 anaerobic transport swabs (Oxoid), and the bulk of the faecal sample was immediately frozen and stored at -70"C. The faecal samples were processed immediately under strict anaerobic 5 conditions. Samples were diluted in anaerobic diluent, and plated on the following culture media: 1) BHI -to obtain the total culturable bacteria per gram of sample; 2) MRS medium with cysteine to select for Lactobacillus and Bifidobacterium species; 10 3) Tryptose Sulphite cycloserine (TSC, Oxoid) agar was used for Clostridium perfringens; 4) Salmonella-Shigella agar (SS) was used for Salmonella spp.; 5) McConkey agar (Merck) was used for Enterobacteriaceae. 15 A single, pure, isolated colony from each of the five selected strains was separately inoculated in 5 ml of MRS-Cys and incubated anaerobically overnight. The culture was Gram-stained to check for purity and I ml of the overnight culture was used to inoculate 500 ml of MRS-Cys broth which was incubated overnight. The cells were then harvested by centrifugation at 3840g 20 for 10 minutes at 44C (Beckman Coulter J2-21 M/E) before being anaerobically resuspended in 50 ml of 5% skimmed milk. The cells were then either microencapsulated or freeze-dried. Microencapsulation 25 The five different strains (50 ml of each) were mixed after resuspension, and then added to 250 ml of 3.6% alginate solution (Merck). The mixture was transferred into a 500 ml sterile funnel and slowly dripped into 1 L of 0.1 M Calcium chloride (CaCl 2 ) solution to form beads. The alginate beads were hardened for 45-60 minutes in the CaCl 2 before being washed in 0.85% saline 30 and sterile distilled water, respectively. The beads were dried at 30CC overnight, and sorted under sterile conditions to ensure that only beads of approximately 4 mm size were included before being packed in batches of approximately 500 in 11 20 ml sterilin tubes. Every week, three beads per selected isolate were individually dissolved in 1 ml 0.1 M Sodium Citrate buffer using a bead beater (LINEA TA 200S) for 10 5 seconds. Once dissolved, serial dilutions were plated on MRS-Cys plates for viability assessment. To ensure that the final products were free from possible laboratory contaminants, resuspended beads were plated on MacConkey agar, Bile 10 Esculin agar and BHI to detect E. coli, Enterococcus spp. and Clostridium spp., respectively. Random colonies were Gram stained to detect the typical cell morphology of each strain. The beads were stored in a dessicator. Fresh batches of beads were used every week and they were transported in cooler bags to the site of the feeding trial. 15 Freeze drying The skimmed milk suspension was transferred into a drying flask and shelled in ethanol for 45-60 minutes before being freeze dried (Balzers-Pirani gauge TiG 030) at 10 mbar pressure for 48 hours. The freeze dried samples were 20 transferred into 20 ml sterilin tubes and stored in a dessicator at room temperature. 0.1 g of the freeze-dried powder for each strain was resuspended in 1 ml Ringer's solution and suitable dilutions were plated on MRS-Cys plates for viability assessments. 25 Preparation of probiotic feed supplement Probiotic bacteria were incorporated into ostrich chick starter feed either as encapsulated beads or freeze-dried powder, such that the final concentration of bacteria was 10 7 gm- 1 feed.

12 ) Feeding Trial Different combinations of strains and individual strains were tested in preliminary feeding trials. The trials were monitored by establishing the basal 5 levels of selected bacterial groups in the faecal material of ostrich chicks, before and during probiotic supplementation, in order to determine the most effective probiotic candidates. One probiotic composition contained all 5 of the strains listed in Tables 1 and 2 above. 10 Trials were carried out at Elsenburg Agricultural Centre, Stellenbosch (South Africa). One hundred and eighty day-old ostrich chicks were randomly allocated to 12 groups with similar mass distributions, and were housed in separate pens. Three replicate groups were assigned to each treatment, and the four different treatments were with or without feed supplementation, and with or without the 15 growth promoter, Tylosin. Chicks had access to starter feed and water ad libitum. The feed supplement was administered immediately after grouping the birds (-109 cfu probiotic bacteria (one alginate capsule)) and again after 1 week. From week 2, the probiotic composition was provided at a level of 107 microorganisms per gram dried feed, or 300 mg alginate beads (- 6x10 9 cfu) 20 daily. Treatment continued for 9 weeks, with the birds being weighed weekly. Results The weight gain by the probiotic-fed birds was considerably increased (Figure 25 1). This shows that bacteria with good probiotic characteristics isolated from ostrich chicks, and in particular those listed in Table I above, can be used as a feed supplement which will improve weight gain and health in chicks. No detrimental effects on the chicks were observed. 30

Claims (18)

1. An ostrich feed supplement composition comprising at least one of Lactobacillus oris, Lactobacillus brevis, Lactobacillus johnsoni, Bifidobacterium pseudolongum subsp. globosum and Enterococcus faecalls.

2. An ostrich feed supplement composition according to claim 1, wherein the Lactobacillus oris is the strain designated as Lo59 (LMG-P-26662).

3. An ostrich feed supplement composition according to either of claims 1 or 2, wherein the Lactobaci/lus brevis is the strain designated as Lb512 (LMG-P 26663).

4. An ostrich feed supplement composition according to any one of claims 1 to 3, wherein the Lactobacillus johnsonii is the strain designated as Lj1 36 (LMG-P-26664).

5. An ostrich feed supplement composition according to any one of claims 1 to 4, wherein the Bifidobacterium pseudolongum subsp. globosum is the strain designated as Bg136 (LMG-P-26666).

6. An ostrich feed supplement composition according to any one of claims 1 to 5, wherein the Enterococcus faecalis is the strain designated as P1.2 (LMG P-26665).

7. An ostrich feed supplement composition according to any one of claims 1 to 6, which includes at least two of Lactobacillus oris, Lactobacillus brevis, Lactobacillus johnsoni, Bifidobacterium pseudolongum subsp. globosum and Enterococcus faecalis.

8. An ostrich feed supplement composition according to any one of claims 1 14 to 6, which includes at least three of Lactobacillus oris, Lactobacillus brevis, Lactobacillus johnsoni, Bifidobacterium pseudolongum subsp. globosum and Enterococcus faecalis.

9. An ostrich feed supplement composition according to any one of claims 1 to 6, which includes at least four of Lactobacillus oris, Lactobacillus brevis, Lactobacillus johnsonii, Bifidobacterium pseudolongum subsp. globosum and Enterococcus faecalis.

10. An ostrich feed supplement composition according to any one of claims 1 to 6, which includes Lactobacillus oris, Lactobacillus brevis, Lactobacillus johnsoni, Bifidobacterium pseudolongum subsp. globosum and Enterococcus faecalis.

11. An ostrich feed supplement composition according to any one of claims 1 to 10, which includes approximately 10 cfu bacteria.

12. An ostrich feed supplement composition according to any one of claims 1 to 11, which further comprises ostrich feed.

13. An ostrich feed supplement composition according to any one of claims 1 to 12, which is for improving the physical condition of ostriches.

14. An ostrich feed supplement composition according to any one of claims 1 to 12, which is for reducing the mortality of ostriches.

15. A method of preparing an ostrich feed supplement composition according to any one of claims 1 to 14, the method comprising the steps of: suspending bacterial cells of the bacteria of the composition in 5% skimmed milk; 15 freeze-drying the bacterial cells or microencapsulating them to form alginate beads.

16. A method for maintaining a group of ostriches, the method comprising the step of administering a composition according to any one of claims 1 to 14 to the ostriches in the group.

17. An ostrich feed supplement composition according to any one of claims 1 to 14, substantially as herein described with reference to the example.

18. A method according to claim 15, substantially as herein described with reference to the example. Dated this 25th day of August 2011. University of Cape Town Patent Attorneys for the Applicant PETER MAXWELL AND ASSOCIATES