GB2503235A

GB2503235A - Synergistic composition for minimising bacterial infection in animals

Info

Publication number: GB2503235A
Application number: GB1210854.4A
Authority: GB
Inventors: Stuart Charters; Nigel Denziloe Hemphill
Original assignee: QUEST VITAMINS Ltd
Current assignee: QUEST VITAMINS Ltd
Priority date: 2012-06-20
Filing date: 2012-06-20
Publication date: 2013-12-25
Also published as: GB201210854D0

Abstract

A synergistic composition is described comprising (i) oregano oil, (ii) thyme oil, (iii) ginger oil, (iv) cassia oil and (v) rosemary oil. The composition has been found to have utility against bacterial infections in animals, for example Campylobacter, Helicobacter, Salmonella, Coliforms or Listeria.

Description

Title: A composition, uses of a composition and methods of minimising bacterial infection in animals Descrirtion of Invention The present invention relates to a composition. In particular, the present invention relates to a composition for minimising bacterial infection in animals.

The present invention also relates to the use of a composition and methods of minimising bacteria in animals.

Animals, in particular livestock, suffer from bacterial infections. According to a (UK) Food Standards Agency survey of 2009, Campylobacter was present in 65% of the tested samples of raw chicken meat, sold in the UK. Salmonella was present in 6% of samples, whilst 0.5% of the samples contained S. enteritidis and S. typhimurium. Such infections are passed to humans, on eating infected meat, if meat is not fully cooked.

Bacterial infection in livestock has a detrimental effect on the efficiency of farms by, for example, reducing yield, affecting the producer's image and requiring the inclusion of medication, including antibiotics, in animal feed.

The inclusion of medication, including antibiotics, in animal feed is common.

However, such inclusion is expensive and can be seen by some consumers as off-putting because of potential cross-contamination of meat. The use of antibiotics in animal feed leads, long term, to increased bacterial resistance to antibiotics. Bacterial resistance to antibiotics comes with its own problems, including a continuing need for new antibiotics to be found and for the potential long term problem of untreatable bacterial infections in humans.

With the world's population growth ever increasing, demand for food keeps rising. At the same time, consumers and retailers seek cheaper food. At least these factors place heavy demands on increased efficiency in farming.

Minimising the use of artificial antibacterial agents in animal feed, whilst at the same time minimising bacterial growth in livestock, is preferable.

Dostofarm GmbH sell supplements for inclusion with animal feed. Dostofarrn GmbH's animal feed supplements, including oregano, are said to be beneficial in improving the taste of meat and stimulating the appetite of animals consuming the feed.

It is believed that the supplements of the present invention, when included in animal feed, act to minimise bacterial growth in the fed livestock. At the same time, it is believed that the supplements of the present invention do not act to alter meat flavour in the fed livestock.

Without wishing to be bound by theory, it is believed that the combination of certain oils in the supplements of the present invention provides a synergistic combination which, when included in animal feed, boosts efficiency by minimising bacterial infection.

According to a first aspect of the present invention, there is provided a composition for minimising bacterial infection, the composition comprising: oregano oil, thyme oil, ginger oil, cassia oil and rosemary oil.

Preferably, wherein the composition comprises 1-20 weight % oregano oil, or any intermediate weight % oregano oil.

Further preferably, wherein the composition comprises 0.1-3 weight% thyme oil, or any intermediate weight % thyme oil.

Advantageously, wherein the composition comprises 0.1-3 weight% ginger oil, or any intermediate weight % ginger oil.

Preferably, wherein the composition comprises 0.1-3 weight % cassia oil, or any intermediate weight % cassia oil.

Further preferably, wherein the composition comprises 0.1-3 weight% rosemary oil, or any intermediate weight % rosemary oil.

Advantageously, wherein the composition comprises: 1-20 weight % oregano oil; 0.1-3 weight % thyme oil; 0.1-3 weight % ginger oil; 0.1-3 weight% cassia oil; and, 0.1-3 weight % rosemary oil.

Preferably, wherein the composition comprises: 6.5-7.5 weight % oregano oil; 0.5-1.5 weight % thyme oil; 0.5-1.5 weight % ginger oil; 0.45-0.55 weight % cassia oil; and, 0.5-1.5 weight % rosemary oil.

Further preferably, wherein the composition comprises: 7 weight % oregano oil; 1 weight % thyme oil; 1 weight % ginger oil; 0.5 weight % cassia oil; and, 1 weight % rosemary oil.

Advantageously, wherein the composition further comprises a solvent.

Preferably, wherein the solvent is ethanol, propan-1-ol, propan-2-ol, benzyl alcohol, glycerol, hexane, acetone, hexyl alcohol, decyl alcohol, benzyl ethyl ether or mono-propylene glycol, or any possible combination of these solvents, preferably wherein the solvent is ethanol.

Further preferably, wherein the composition further comprises an emulsifier.

Advantageously, wherein the emulsifier is Bredol 697, lecithins, sodium alginate, agar, carageenan, locust bean gum, tamarind seed flour, guar gum, tragacanth, acacia, xanthan, polyoxyethylene (20)-sorbitan monooleate, cellulose, methyl cellulose, ethyl cellulose, hydroxypropylmethyl cellulose, glyceryl polyethyleneglycol ricinoleate, polyethyleneglycol ester of fatty acids from soya oil, sorbitan monolaurate, cassia gum, polyoxyethylene sorbitan monostearate (otherwise referred to as TweenTM), or sorbitan monostearate (otherwise referred to as SpanTM), or any possible combination of these emulsifiers, preferably wherein the emulsifier is Bredol 697.

Preferably, wherein the composition consists essentially of: 7 weight % oregano oil; 1 weight % thyme oil; 1 weight % ginger oil; 0.5 weight % cassia oil; 1 weight % rosemary oil; 67.5 weight % ethanol; and, 22 weight % Bredol 697.

Further preferably, wherein the composition is a supplement.

Advantageously, wherein the supplement is for addition to feed.

Preferably, wherein the composition is diluted in water.

Further preferably, wherein the composition is diluted in water in an amount of: 300m1! 1000 litres; 600m1/ 1000 litres; 900m1/ 1000 litres; 1200m1/ 1000 litres; or, 3000m11 1000 litres.

In a further aspect of the present invention, there is provided a composition according to the above, for use in therapy.

Preferably, for use in the treatment of bacterial infections in animals.

Further preferably, wherein the bacterial infection is Campylobacter infection, or Helicobacter, Salmonella, Coliforms or Listeria infection.

Advantageously, wherein the animals are livestock.

Preferably, wherein the animals are chickens, cows, goats, sheep, ducks, rabbits, pigs, horses, geese, partridges, ostriches, kangaroos, or any other animal.

In a further aspect of the present invention, there is provided a method of reducing bacterial infection in an animal, the method comprising: providing a composition according to the above; and, introducing the composition into the animal.

Embodiments of the invention are described below with reference to the accompanying drawings, in which: Figure 1 shows the percentage of cloacal swabs detected as being positive for Campylobacter (day 40) after using a supplement according to one non-limiting embodiment of the present invention.

Figure 2 shows the number of colony forming units (CFU) detected per cloacal swab (day 40) after using a supplement according to one non-limiting embodiment of the present invention.

Figure 3 shows the percentage of caecal samples detected as being positive for Campylobacter (day 42) after using a supplement according to one non-limiting embodiment of the present invention.

Figure 4 shows the number of colony forming units (CFU) detected per caecal sample (day 42) after using a supplement according to one non-limiting embodiment of the present invention.

Some of the components of the supplements of the present invention, together with their sources, are set out below.

Oregano oil Or/garium vulgare (CAS No.: 8007-11-2) is a plant species in the mint family.

Oregano oil contains, inter a/ia, carvacrol which is known to inhibit bacterial growth. Its antimicrobial mode of action is believed to occur in four steps: i) change permeability of cytoplasm membrane in bacterial cells; ii) dissipate membrane potential; Hi) inhibit AlP synthesis; resulting in, iv) bacterial cell death.

Thyme oil Thymus vulgar/s (CAS No.: 8007-46-3) is a medicinal herb. Thyme oil contains, inter al/a, thymol. Thymol is isomeric with carvacrol. Thymol is known to inhibit bacterial growth and was used to coat bandages before the advent of synthetic antibiotics.

Ginger oil Ginger is the rhizome of the plant Zingiber officina/e (CAS No.: 8007-08-7).

Ginger oil contains, inter a/ia, zingerone. Zingerone is known to inhibit bacterial growth in the gut of animals.

Cassia oil Cinnamomum cassia (CAS No.: 8007-80-5) is an evergreen tree with an aromatic bark and leaves, and is used as a spice. Cassia oil contains, inter a/ia, cinnamaldehyde, which has been shown to have antimicrobial effects.

Rosemary oil Rosemarinus officina/is (CAS No.: 8000-25-7) is a woody herb, in the mint family. Rosemary oil contains, inter a/ia, rosmarinic acid. Rosmarinic acid is known to have antibacterial effects.

Supplement formulations Supplements according to the present invention comprise the following essential oils: a. Oregano oil; b. Thyme oil; c. Ginger oil; d. Cassia oil; and, e. Rosemary oil.

One particularly preferred supplement, referred to herein as formulation A, has the following components: Formulation A Component Amount I weight % Oregano oil 7 Thyme oil 1 Ginger oil 1 Cassia oil 0.5 Rosemary oil 1 Ethanol 67.5 Bredol 697 22 Total 100 The source of each of the above components of formulation A, in this non-limiting example, was: Oregano oil: supplied by Aromatic Ingredients Ltd., Nottinghamshire, UK; Thyme oil: supplied by Sigma-Aldrich, UK; Ginger oil: supplied by Aromatic Ingredients Ltd., Nottinghamshire, UK; Cassia oil: supplied by Aromatic Ingredients Ltd., Nottinghamshire, UK; Rosemary oil: supplied by Aromatic Ingredients Ltd., Nottinghamshire, UK; Ethanol: supplied by Sigma-Aldrich, UK; and, Bredol 697: supplied by Akzo Nobel Surface Chemistry, Sweden.

In this specification, reference to an oil of a particular plant refers to an essential oil of the plant. Such oils are referred to with reference to their CAS numbers.

Ethanol was used as a solvent. Other alcohols, which are miscible with water, can be used as the solvent. Ethanol was selected as a preferable solvent because it is relatively cheap. Other possible solvents include, but are not limited to, propan-1-ol, propan-2-ol, benzyl alcohol, glycerol, hexane, acetone, hexyl alcohol, decyl alcohol, benzyl ethyl ether or mono-propylene glycol, or any possible combination of these solvents.

Bredol 697 is a non-ionic emulsifier used in animal feed. Other, non-limiting, examples of suitable emulsifiers include, but are not limited to, lecithins, sodium alginate, agar, carageenan, locust bean gum, tamarind seed flour, guar gum, tragacanth, acacia, xanthan, polyoxyethylene (20)-sorbitan monooleate, cellulose, methyl cellulose, ethyl cellulose, hydroxypropylmethyl cellulose, glyceryl polyethyleneglycol ricinoleate, polyethyleneglycol ester of fatty acids from soya oil, sorbitan monolaurate, cassia gum, polyoxyethylene sorbitan monostearate (otherwise referred to as TweenTM), or sorbitan monostearate (otherwise referred to as SpanTM), or any possible combination of these emulsifiers. An emulsifier is included so that the supplement is a homogenous mixture & more readily solubilised in the water for dosing. If the total oil content of the supplement is above around 20%, the mixture becomes too oily and separated into a non-homogenous mixture.

Supplements according to the present invention contain five common ingredients, namely, oregano oil, thyme oil, ginger oil, cassia oil and rosemary oil. It is believed that this combination of five ingredients provides a synergistic combination which acts to mitigate bacterial infection in animals, in particular livestock, more particularly chickens.

Provision of the supplements of the present invention to animals in their feed, as detailed below, acts to reduce bacterial infection. In particular, provision of non limiting example formulation A to chickens in their feed, as detailed below, reduces Campylobacter infection in their gut.

In vitro Comparative data An independent microbiological laboratory, Northern Hygiene Laboratories Ltd., carried out comparative tests of the efficacy of formulation A, and separately the individual oils of formulation A, in killing Campylobacter in vitro.

Campylobacter was grown on agar plates using standard microbiological techniques. Different oils, and combinations of oils, in differing dilutions in water, were placed on seperate agar plates carrying Campylobacter culture.

The results of the comparative tests indicate that the combination of at least oregano oil, thyme oil, ginger oil, cassia oil and rosemary oil provides a synergistic effect. This combination of oils provided for greater killing of Campylobacterin vitro than each of the oils individually. In addition, the results indicated that the sum of the kill effect on Campylobacter in vitro by each of the oils, individually, was less than the kill effect of the combination of the oils.

In vivo Trials With a view to confirming the efficacy of the non-limiting example formulation A of the present invention, the gut health of Campy/obacter-challenged birds was tested in vivo.

An independent research body, the Avian Science Research Centre, in Ayr, Scotland, carried out tests on an incremental dose of formulation A. 1. Feed The following quantities of feed (manufactured at Massey Feeds, Cheshire) were supplied by Flavex International.

Starter crumbs: 200kg Grower pellets: 600kg Finisher (roaster) pellets withdrawal: 2100kg The feed was a standard feed supplied to the chicken breeding industry.

2. Test Article Formulation A was supplied, and tested at different levels of concentration in water (treatments 1-6 shown in Table 1, below). Test product was added to drinking water as described in the Table 1.

Table 1. Test product dosage per treatment.

Treatment Description

1 Control -untreated (public water supply) drinking water 2 Formulation A added to drinking water from day 1 -42 @ 300m1/1 000 litres 3 Formulation A added to drinking water from day 1 -42 @ 600m1/1 000 litres 4 Formulation A added to drinking water from day 1 -42 @ 900m1/1 000 litres Formulation A added to drinking water from day 1 -42 @ 1 200m1/1 000 litres 6 Formulation A added to drinking water from day 1 -42 @ 3000m1/1 000 litres 3. Study Animals and Husbandry Conditions Animal Male broilers Breed (strain) Cobb 500 Animals per pen 10 Treatments 6 Replicates per treatment 8 Total animals 480 Average weight at Day 0 (g) 45 Average weight at Day 42 (g) 2477 Age at start of trial (d) 0 Age at end of trial (d) 42 Pen type Floor pens Lighting programme Initially 23 hours of light and 1 hour of dark which was changed progressively to 14 hours of light and 10 hours of darkness by day 6 and maintained thereafter Water An individual drinker was used in each pen.

Each drinker was attached to an independent water tank to which measured amounts of water were added. Water was provided ad libitum.

Feed Feed was provided ad /ibitum using a single tube feeder per pen.

Temperature The temperature was set at 32°C at the beginning and was reduced gradually to 26°C by 21 days of age and maintained thereafter Humidity Not controlled: ranged from 65 to 80%RH Vaccination programme None Welfare standards The trial was conducted according to the Codes of Recommendations for the Welfare of Livestock (Broilers).

4. Study Design and Ethical Approval The study was subjected to scrutiny by the Scottish Agricultural College's (SAC's) Animal Experiments Committee. The study design used is shown in

Table 2.

Table 2: The study design.

Treatment Description Pens Birds/p Birds

/treatment en treatment 1 Control -untreated (public water 8 10 80 supply) drinking water 2 Formulation A added to drinking 8 10 80 water from day 1 -42 @ 300m1/1 000 litres 3 Formulation A added to drinking 8 10 80 water from day 1 -42 @ GOOml/1 000 litres 4 Formulation A added to drinking 8 10 80 water from day 1 -42 @ 900m1/1 000 litres Formulation A added to drinking 8 10 80 water from day 1 -42 @ 1200m1/1 000 litres 6 Formulation A added to drinking 8 10 80 water from day 1 -42 @ 3000m1/1 000 litres Total 48 480 The study was a randomised block design (with pens blocked spatially in the house).

5. Management * Housing: The experiment was carried out in a multi-room controlled environment facility (House 5). Birds were housed in floor pens throughout the study.

* Environmental Control: The environment in the rooms used for this study has systems to control the lighting, temperature and ventilation.

Environmental conditions were recorded according to SAC SOP AH/ASRC/0O8/O1.

* Disease Control: Birds were inspected twice daily. The birds remained healthy throughout the study period and did not require any medication.

* Feed: Feed was manufactured by Massey Feeds (Cheshire). Commercial starter crumbs, grower pellets and finisher (roaster) pellets withdrawal feed was supplied by the client and were fed from days 0 to 12, 12 to 24 and 24 to 42 respectively.

* Biosecurity: Biosecurity followed the appropriate Scottish Agricultural College Standard Operating Procedure. In addition to SAC's standard biosecurity to minimise the risk of cross-contamination the following protocol was followed: boots were disinfected and overshoes and gloves changed between pens/treatments. Furthermore pens were entered only when required and then in order of treatment 6 followed by treatment 5 etc with pens in treatment 1 being entered last.

6. Procedures and records * Campylobacter Challenge At 17 days of age litter from a commercial flock that had tested positive for Campylobacter was added to all treatment pens. This method of challenging birds allows Campy/obacter that are present in field outbreaks to be presented to the experimental birds in a manner that more closely reflects the normal faecal/oral challenge route than would be the case say if the birds were gavaged with a laboratory strain of Campy/obacter.

* Feed Intake The feed was weighed at the beginning and at the end of each period on a pen basis and used to calculate average feed intake and FCR for the periods day 0 to 12, day 12 to 24 and day 24 to 42. In addition, feed was also measured on day 17, when birds were introduced to Campylobacter challenge.

* Body weight The birds were bulk-weighed at day 0, 12, 17, 24 & 42. The balances used were identified, tared and checked for accuracy before and during use. The data were collected over complete blocks by the same personnel.

Microbiology Samples were collected as described in Table 3 and were sent for microbiological examination during the experimental period (see Section 7 -Microsearch Laboratories Ltd).

Table 3. Samples collected for microbiological analysis.

Day Samples Total number of samples taken 16 12 x cloacal swabs per treatment 72 The 12 swabs per treatment consisted of one bird per pen and then two birds in four pens 16 4 x 50g litter per treatment 24 Each sample consisted of two pens of the same treatment bulked together 17 3 x bog litter challenged litter 3 12 x cloacal swabs per treatment 72 The 12 swabs per treatment consisted of one bird per pen and then two birds in four pens 4 x bog litter per treatment 24 Each sample consisted of two pens of the same treatment bulked together 12 x cloacal swabs per treatment 72 The 12 swabs per treatment consisted of one bird per pen and then two birds in four pens Day Samples Total number of samples taken 42 12 x caeca per treatment 72 (Birds were killed humanely and feathers were plucked and disinfectant was sprayed before dissection.) 42 2 x bird from Treatment 1 and Treatment B were 2 (whole dissected and whole tract was removed and following tracts) samples were collected: a) oesophagus, b) crop, c) proventriculus, d) gizzard, e) duodenum, 1) liver, g) small Intestine, h) caeca i) large Intestine * Meat Samples At day 40, breast meat from two birds per treatment (12 in total) were bagged & labelled for cooking & tasting to evaluate for herb taint by Flavex flavourists.

* Birds Four hundred and eighty broilers (Cobb 500) were used in this trial. Any sick and unthrifty chicks were culled, regardless of weight, prior to the initiation of the study.

* Allocation The pens were randomized.

* Observations Bird condition, house temperature, lighting, feeders and drinkers were checked and recorded daily on appropriate data capture form and were reported with a tick (1) and comments recorded if the preceding observations were abnormal.

* Mortality and Morbidity All pens were checked twice daily for dead and ill birds. Dead birds were sent for post-mortem.

* Statistical analysis of data The data obtained were subjected to Analysis of Variance (ANOVA) to determine the effect of experimental diets on broiler growth performance using a Genstat 11 statistical software package (IACR, FIothamstead, Hertfordshire, UK). Pen means were the units for statistical analysis. All statements of significance are based on the probability level of PcO.05.

* Bird Disposition At the end of the study, all birds were killed and carcasses incinerated.

* Raw Data The study investigator collated all the raw data in the electronic and hard copy and sent it to the monitor. Copies of all raw data will be archived at SAC.

7. Results * Environment The ambient temperature range was within the comfort zone of the birds and was similar between blocks (Appendix 2).

* Body weights and weight gain The mean body weights at day-old, day 12, 17, 24 and 42 are presented in Table 4. Experimental treatments had no effect (P>0.05) on the body weights compared to control treatment 1.

The overall body weight gain followed the pattern of the effects of the treatments on body weight (Table 5).

Table 4: Average body weight (kg/bird/period) at different experimental periods Treatment Day 0 Day 12 Day 17 Day 24 Day 42 1 0.045 0.335 0.570 0.910 2.513 2 0.045 0.334 0.571 0.909 2.469 3 0.045 0.329 0.559 0.897 2.436 4 0.045 0.335 0.568 0.897 2.492 0.045 0.338 0.559 0.922 2.501 6 0.044 0.335 0.565 0.903 2.453 P-value 0.829 0.646 0.817 0.843 0.838 LSD 0.002 0.010 0.023 0.044 0.133 There is a statistically significant difference when RcO.05; [SD -[east Significant Differences of Means. Treatment 1 = Control; Treatment 2 = formulation A @ 300g/1 000 litres; Treatment 3 = formulation A @ 600g/1 000 litres: formulation A @ 900g/1 000 litres; Treatment 4 = formulation A @ 900g/1 000 litres; TreatmentS = formulation A © 1200g/1 000 litres; Treatment 6 = formulation A © 3000g/1 000 litres Table 5: Average weight gain (kg/bird/period) in the different experimental periods Treatme Day 0-12 Day 12-17 Day 17-24 Day 12-24 Day 24-42 Day 0-42 nt 1 0.290 0.235 0.340 0.575 1.603 2.468 2 0.290 0.237 0.338 0.575 1.56 2.425 3 0.284 0.230 0.338 0.568 1.54 2.391 4 0.291 0.233 0.329 0.562 1.595 2.447 0.294 0.221 0.363 0.584 1.579 2.456 6 0.291 0.230 0.338 0.568 1.55 2.409 P-value 0.577 0.681 0.410 0.906 0.844 0.838 [SD 0.010 0.020 0.032 0.040 0.1144 0.132 There is a statistically significant difference when RcO.05; [SD -[east Significant Differences of Means. Treatment 1 = Control; Treatment 2 = formulation A © 300g/1 000 litres; Treatment 3 = formulation A @ 600g/1 000 litres: formulation A @ 900g/1 000 litres; Treatment 4 = formulation A @ 900g/1 000 litres; Treatment 5 = formulation A © 1200g/1 000 litres; Treatment 6 = formulation A @ 3000 gIl 000 litres * Feed Intake The average feed intake (AFI) per bird over the experimental period is presented in Table 6. No differences in AFI were observed between treatments during the different experimental period with the exception of day 12 to 17 when birds on formulation A had consistently reduced feed intake, but the differences were only significant (PcO.05) for birds on Treatment 2, 3 and 5 compared to Treatment 1. However, these differences in AFI were not reflected in the overall data (day 0 to 42) as indicated by the non-significant (P>0.05) differences between the Treatment groups.

Table 6: Average feed intake (kg/bird/period) in the different experimental periods Treatme Day 0-12 Day 12-17 Day 17-24 Day 12-24 Day 24-42 Day 0-nt 42 1 0.435 0.457° 0.802 1.259 3.914 5.435 2 0.433 o.4o3a 0.793 1.197 3.796 5.313 3 0.436 0.401a 0.777 1.179 3.815 5.312 4 0.433 0434abc 0.785 1.22 3.806 5.397 0.436 0.412 0.796 1.209 3.851 5.429 6 0.438 0448bc 0.778 1.226 3.768 5.324 P-value 0.981 0.031 0.888 0.326 0.725 0.862 [SD 0.014 0.041 0.050 0.072 0.195 0.275 There is a statistically significant difference when P<0.05; [SD -[east Significant Differences of Means. Different superscripts within same column means significant different at Pc0.05. Treatment 1 = Control-normal drinking water; Treatment 2 = formulation A @ 300g/1 000 litres; Treatment 3 = formulation A © 600g/l 000 litres: formulation A © 900g/1 000 litres; Treatment 4 = formulation A @ 900g/1 000 litres; Treatment 5 = formulation A © 1200g/1000 litres; Treatment 6 = formulation A @ 3000g/1 000 litres * Feed Conversion Ratio based on total production (FCRTP) Experimental treatments had no effect (P>0.05) on FCRTP during different experimental periods (Table 7).

Table 7: Feed Conversion Ratio based on total production (FCRTP) in the different experimental periods Treatment Day 0-12 Day 12-17 Day 12-24 Day 24-42 Day 0-42 1 1.522 1.995 2.236 2.474 2.291 2 1.504 1.768 2.128 2.449 2.246 3 1.545 1.812 2.139 2.493 2.284 4 1.503 1.877 2.174 2.418 2.247 1.498 1.903 2.097 2.449 2.246 6 1.532 1.998 2.178 2.465 2.276 P-value 0.182 0.268 0.273 0.749 0.457 LSD 0.042 0.425 0.120 0.100 0.061 There is a statistically significant difference when P<0.05; LSD -Least Significant Differences of Means. FCR TP = Total feed intake of period per pen / (total live weight of pen + total weight of dead birds in pen) -total live weight of pen in previous period. Treatment 1 = Control; Treatment 2 = formulation A @ 300g/1 000 litres; Treatment 3 = formulation A @ 600g/1 000 litres: formulation A @ 900g/1 000 litres; Treatment 4 = formulation A @ 9009/1 000 litres; Treatments = formulation A © 12009/1 000 litres; Treatment 6 = formulation A © 3000g/1 000 litres * Average Water Intake (AWl) The AWl during different experimental periods are presented in Table 8.

Experimental treatments had no effect (P>0.05) on the body weights compared to control (treatment 1). No differences (P>0.05) in AWl were observed during starter and grower phase. During finisher phase (day 24 to 42) significantly increased (RcO.05) AWl was recorded for birds in treatment 1 however, the differences were only significant compared to those in treatment 6. This difference in AWl was not reflected in the overall data (day 0 to 42 between the treatments.

* Water and Feed Ratio (W:F) The W:F ratio over the experimental period is presented in Table 9. No differences in W:F were observed between treatments during the study except for the period day 12 to 17 where birds on Treatment 6 had a reduced W:F ratio compared to highest ratio for birds on Treatment 2, 3 and 5. These differences in W:F ratio were due to the reduced AFI for birds in Treatment 2, 3. However, these differences in W:F ratio were not reflected in the overall data (day 0 to 42) as indicated by the non-significant (P>0.05) differences between the treatment groups.

Table 8: Average water intake (AWl) (kg/bird/period) in the different experimental periods Treatment Day 0-Day 12-17 Day 12-24 Day 17-24 Day 24-42 Day 0-42 1 1.079 0.918 2.497 1.579 8046b 11.27 2 1.045 0.874 2.365 1.491 7.524 10.72 3 1.136 0.870 2.358 1.488 7.584 10.85 4 1.096 0.897 2.426 1.529 7.369 10.78 1.053 0.878 2.394 1.497 7635ab 11.00 6 1.059 0.858 2.333 1.475 7.179a 10.38 P-value 0.787 0.267 0.278 0.399 0.041 0.192 LSD 0.1407 0.053 0.1475 0.107 0.517 0.679 There is a statistically significant difference when RcO.05; [SD -[east Significant Differences of Means. Different superscripts within same column means significant different at Pc0.05. Treatment 1 = Control-normal drinking water; Treatment 2 = formulation A @ 300g/1 000 litres; Treatment 3 = formulation A © 600g/1 000 litres: formulation A © 900g/1 000 litres; Treatment 4 = formulation A @ 900g/1 000 litres; Treatment 5 = formulation A © 1200g/1000 litres; Treatment 6 = formulation A @ 3000g/1 000 litres Table 9: Water and Feed ratio (W:F) in the different experimental periods Treatment Day 0-Day 12-Day 12-24 Day 17-24 Day 24-42 Day 0-42 12 17 1 2.48 2.02 1.98 1.97 2.06 2.08 2 2.41 217b 1.97 1.88 1.98 2.02 3 2.60 2.1 8b 2.00 1.91 1.98 2.04 4 2.53 2.08 1.98 1.94 1.93 1.99 2.42 219b 1.98 1.87 1.98 2.04 6 2.43 1.92a 1.90 1.89 1.90 1.95 P-value 0.871 0.037 0.273 0.287 0.336 0.279 [SD 0.346 0.189 0.087 0.097 0.142 0.110 There is a statistically significant difference when FcO.05; [SD -[east Significant Differences of Means. Different superscripts within same column means significant different at F<0.05. Treatment 1 = Control; Treatment 2 = formulation A @ 300g/1 000 litres; Treatment 3 = formulation A @ 600g/1 000 litres: formulation A © 900g/1 000 litres; Treatment 4 = formulation A © 900g/1000 litres; TreatmentS =formulation A © 1200g/1000 litres; Treatment 6 = formulation A © 30009/1000 litres.

* Mortality The overall percent mortality showed no differences (P>0.05) between different treatment groups (Table 10) Table 10: Percent Mortality over the trial period of 42 days.

Treatment Mortality (%) 1 16.2 2 11.2 3 13.7 4 6.2 10.0 6 16.2 F-value 0.483 LSD 11.73 There is a statistically significant difference when P<0.05; [SD -[east Significant Differences of Means. Treatment 1 = Control; Treatment 2 = formulation A @ SOUgh 000 litres; Treatment 3 = formulation A @ 600g11 000 litres: formulation A @ 900g11 000 litres; Treatment 4 = formulation A @ 900g11 000 litres; Treatment 5 = formulation A © 1200g11 000 litres; Treatment 6 = formulation A @ 3000 g/l 000 litres.

Microbiology Microbiology data were provided by Microsearch [aboratories [imited.

Litter and caecal samples taken at day 16 (prior to the addition of the contaminated litter) were negative for Campylobacter, confirming that there had been no inadvertent contamination of the environment or birds.

At day 40 cloacal swabs were taken from 12 birds per treatment and tested for the presence of Campylobacter and, where Campy/obacter were detected, the number of organisms present per swab were enumerated. As can be seen from Figures 1 and 2, as the concentration of the formulation A increased so there was a tendency for the number of birds that tested positive for Campylobacterto reduce and, for those birds that do test positive, for the number of Campylobacter isolated to be reduced. Cloacal swabs have the advantage of providing data without the need to euthanase the bird. However the most reliable method of assessing Campylobacter carriage is to take caecal samples post-mortem. Accordingly, at day 42, caecal samples were taken from 12 birds per treatment.

It can be seen (Figures 3 and 4) that the trends for both prevalence and numbers of organisms present in caeca that tested positive for Campylobacter reflected the cloacal swab data. That is to say, as the concentration of the formulation A increased so the prevalence and number of organisms present decreased. As can be seen from Table 11, the addition of formulation A at any of the concentrations used significantly reduces the number of viable Campylobacter present in the caeca with a tendency towards a linear dose response, the lowest number of organisms occurring in the Treatment with the highest concentration of formulation A. Table: 11: Effect of dietary treatments on the Campylobacter counts (Log 10 cfu/g) in the caeca (day 42).

Treatment Campylobacter counts (CFU) 2 8.901° 3 8.385° 4 70g4b 6 5373a P-value <0.001 LSD 0.689 There is a statistically significant difference when PcO.05 (within column differences are shown by different superscripts); LSD-Least significant difference of means. Tukey's t test was used to separate means.

* Organoleptic qualities A taste panel of sixteen staff at Flavex International Limited evaluated cooked chicken breast (Treatment 1 and 6) with skin on for taste and aroma. The panel were blind' to the treatments although the members were aware that the meat was from a trial using formulation A. There were no treatment specific comments (e.g. no comments that related to herb' or spicy taste or aroma), the only comments relating to how dry or moist the meat was (i.e. the cooking process).

8. Summary

Formulation A was successful in significantly reducing not only the prevalence of Campylobacterin the birds challenged but also in reducing the number of organisms within the caeca. The reduction in numbers of viable Campylobacter tended to follow a linear dose response with a reduction of over four log units when the highest concentration of formulation A was used compared with the control group. Based on work done by others, even a conservative two log reduction in carriage is considered to equate to an 80% reduction of human cases of Campylobacteriosis.

With products of this nature palatability can be a concern; that is to say if the birds find the product at all aversive then it can result in a reduction in intake of either food or water. However, in the in vivo study detailed above, the addition of formulation A, even at the highest dose of 3000g per 1000 litres of water, did not affect the intermediate or final body weights of the birds, there being no significant difference (P>0.05) between the body weights at day 42. Similarly there were no significant differences in average weight gain or feed conversion ratios (calculated as total production) between birds on the different treatments. While significant differences were recorded for feed intake in one period, days 12-17, the differences were non-specific in that they did not relate to product concentration or any other obvious variable. Although body weights were unaffected by the treatments there was evidence that the highest concentration of the product had a tendency to have a detrimental effect on water intake, the intake for Treatment 6 being significantly less than the control (Treatment 1) for the period 24-42 days. There was no detectable effect, however, on the water to feed ratio.

While, because of the experimental conditions, overall mortality was greater than would be expected in a commercial flock, the mortality did not differ significantly between the control and formulation A treatments.

In conclusion, addition of formulation A to the drinking water of broilers (chickens) challenged with Gampylobacter resulted in a reduction in prevalence and caecal counts of Carnpylobacter, with reduction of over four log units in birds fed the higher strength formulation A. There were no adverse effects on growth or growth related factors such as feed conversion ratio.

As can be seen from the above results, it is clear that a product in accordance with the present invention is able to mitigate bacterial infection in animals, in particular livestock, more particularly chickens.

It is expected that the above results can be extrapolated to other livestock, for example, cattle, pigs, sheep, goats, ducks, geese, pheasant and other animals.

When used in this specification and claims, the terms "comprises" and "comprising" and variations thereof mean that the specified features, steps or integers are included. The terms are not to be interpreted to exclude the presence of other features, steps or components.

The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

Claims

Claims 1. A composition for minimising bacterial infection, the composition comprising: oregano oil, thyme oil, ginger oil, cassia oil and rosemary oil.
2. The composition of claim 1, wherein the composition comprises 1-20 weight % oregano oil, or any intermediate weight % oregano oil.
3. The composition of claim 1 or claim 2, wherein the composition comprises 0.1-3 weight % thyme oil, or any intermediate weight %thyme oil.
4. The composition of any one of claims 1-3, wherein the composition comprises 0.1-3 weight % ginger oil, or any intermediate weight% ginger oil.
5. The composition of any one of claims 1-4, wherein the composition comprises 0.1-3 weight % cassia oil, or any intermediate weight % cassia oil.
6. The composition of any one of claims 1-5, wherein the composition comprises 0.1-3 weight% rosemary oil, or any intermediate weight% rosemary oil.
7. The composition of any one of claims 1-6, wherein the composition comprises: 1-20 weight % oregano oil; 0.1-3 weight % thyme oil; 0.1-3 weight % ginger oil; 0.1-3 weight% cassia oil; and, 0.1-3 weight % rosemary oil.
8. The composition of any one of claims 1-7, wherein the composition comprises: 6.5-7.5 weight % oregano oil; 0.5-1.5 weight % thyme oil; 0.5-1.5 weight % ginger oil; 0.45-0.55 weight % cassia oil; and, 0.5-1.5 weight % rosemary oil.
9. The composition of any one of claims 1-8, wherein the composition comprises: 7 weight % oregano oil; 1 weight %thyme oil; 1 weight % ginger oil; 0.5 weight % cassia oil; and, 1 weight % rosemary oil.
10. The composition of any one of claims 1-9, wherein the composition further comprises a solvent.
11. The composition of claim 10, wherein the solvent is ethanol, propan-1 -ol, propan-2-ol, benzyl alcohol, glycerol, hexane, acetone, hexyl alcohol, decyl alcohol, benzyl ethyl ether or mono-propylene glycol, or any possible combination of these solvents, preferably wherein the solvent is ethanol.
12. The composition of any one of claims 1-11, wherein the composition further comprises an emulsifier.
13. The composition of claim 12, wherein the emulsifier is Bredol 697, lecithins, sodium alginate, agar, carageenan, locust bean gum, tamarind seed flour, guar gum, tragacanth, acacia, xanthan, polyoxyethylene (20)-sorbitan monooleate, cellulose, methyl cellulose, ethyl cellulose, hydroxypropylmethyl cellulose, glyceryl polyethyleneglycol ricinoleate, polyethyleneglycol ester of fatty acids from soya oil, sorbitan monolaurate, cassia gum, polyoxyethylene sorbitan monostearate (otherwise referred to as TweenTM), or sorbitan monostearate (otherwise referred to as SpanhM), or any possible combination of these emulsifiers, preferably wherein the emulsifier is Bredol 697.
14. The composition of any one of claims 1-13, wherein the composition consists essentially of: 7 weight % oregano oil; 1 weight % thyme oil; 1 weight % ginger oil; 0.5 weight % cassia oil; 1 weight % rosemary oil; 67.5 weight % ethanol; and, 22 weight % Bredol 697.
15. The composition of any one of claims 1-14, wherein the composition is a supplement.
16. The composition of claim 15, wherein the supplement is for addition to feed.
17. The composition of any one of claims 1-16, wherein the composition is diluted in water.
18. The composition of any one of claims 1-17, wherein the composition is diluted in water in an amount of: 300m1/ 1000 litres; 600m1/ 1000 litres; 900m1/ 1000 litres; 1200m1/ 1000 litres; or, 3000m1/ 1000 litres.
19. A composition according to any one of claims 1-18, for use in therapy.
20. A composition according to any one of claims 1-18, for use in the treatment of bacterial infections in animals.
21. A composition for use according to claim 20, wherein the bacterial infection is Gampylobacter infection, or He/icobacter, Salmonella, Coilforms or Listeria infection.
22. A composition for use according to claim 20 or claim 21, wherein the animals are livestock.
23. A composition for use according to claim 22, wherein the animals are chickens, cows, goats, sheep, ducks, rabbits, pigs, horses, geese, partridges, ostriches, kangaroos, or any other animal.
24. A method of reducing bacterial infection in an animal, the method comprising: providing a composition accoiding to any one of claims 1-18; and, introducing the composition into the animal.
25. A supplement with the formulation of formulation A.
26. Any novel combination of features disclosed herein.