Bird feed and in particular poultry feed, comprising synthetic capsaicinoid derivatives and such feed for prophylactic use or treatment of <i>Salmonella </i>infection

Abstract

The invention relates to bird feed comprising synthetic capsaicinoids for prohylactic use or treatment of salmonella infection.

Claims

1. A poultry feed comprising at least one chemical compound of the formula I: ##STR00001## wherein R is phenyl; or tautomers or salts thereof, and wherein the feed comprises 5-20 mg/kg of the compound of formula I, or tautomer or salt thereof.

2. The poultry feed according to claim 1, wherein the feed is at least one feed selected from the group consisting of a seed, corn, a worm, a millet, an oat, and a peanut, and the feed is at least one form selected from the group consisting of a pellet, a slurry, drinking water, and an emulsion.

3. The poultry feed according to claim 1, wherein the poultry feed is selected from the group consisting of chicken feed, turkey feed, duck feed and goose feed.

4. The poultry feed according to claim 1, wherein the poultry feed is broiler chicken feed.

Description

FIGURES

(1) FIG. 1—Rank number of CFU/g values of control (blue dots) and the 20 mg/kg phenylcapsaicin treatment (red dots) (x-axis) plotted against CFU/g counts (y-axis) on day 20 (a) and day 29 (b)

EXPERIMENTAL

(2) An experiment in which the effect of phenylcapsaicin, supplemented at different dose levels to broiler feed, against Salmonella colonisation and translocation has been conducted. Besides the effect against Salmonella infection the effect of phenylcapsaicin on growth performance was studied.

(3) The study aimed to evaluate the effects of dietary inclusion at different dose levels of phenylcapsaicin, an analogue of natural capsaicin (1% solution in soy oil) on the intestinal colonisation and translocation of Salmonella enteritidis and growth performance of Salmonella challenged broilers. The experiment was carried out with 420 Ross 308 male broilers and lasted 30 days. The birds were housed in thirty three-tier battery cages (14 birds per cage at the start).

(4) Five experimental treatments were evaluated, each replicated six times. The experimental design is outlined in Table 1. Treatment I consisted of birds which were not challenged with Salmonella enteritidis and received the control diet. The birds of treatments II, III, IV and V were all challenged with Salmonella enteritidis PT4 and received a control diet or the control diet with 20 mg/kg, 28 mg/kg or 36 mg/kg phenylcapsaicin, respectively. The control diet used is found in Table 2.

(5) TABLE-US-00001 TABLE 1 Experimental design - Salmonella challenged broiles. Salmonella Number enteritidis Group of cages Dietary treatment challenge I 6 None (Control diet) No II 6 None (Control diet) Yes III 6 20 mg/kg phenylcapsaicin Yes IV 6 28 mg/kg phenylcapsaicin Yes V 6 36 mg/kg phenylcapsaicin Yes

(6) TABLE-US-00002 TABLE 2 Control diet used - Salmonelle challenged broiles Starter Grower 0-15 days 15-30 days Maize g/kg 317.6 191.3 Wheat g/kg 250.0 400.0 Soybean meal CP 480 g/kg g/kg 280.0 260.0 Rape seed meal CP 335 g/kg g/kg 50.0 50.0 Potato protein Ash >10 g/kg g/kg 10.0 0.0 Soy oil g/kg 52.0 65.0 Premix g/kg 5.0 5.0 Lime g/kg 16.0 14.0 Monocalciumphosphate g/kg 9.5 5.5 Salt g/kg 2.0 2.1 Sodiumbicarbonate g/kg 3.0 2.5 Natuphos 10000G (phytase - g/kg 0.05 0.05 enzyme) Hostazym NSP-enzyme g/kg 0.10 0.10 Clinacox g/kg 0.20 0.20 L-lysine HCl g/kg 1.75 1.75 DL-methionine g/kg 2.20 1.85 L-threonine g/kg 0.45 0.50 L-valine g/kg 0.15 0.15 Total g/kg 1000.0 1000.0 Calculated nutrients (g/kg) Crude protein g/kg 218 207 Crude fat g/kg 74 83 Crude fibre g/kg 29 29 Ash g/kg 61 54 Dig. Lysine g/kg 10.7 10.4 Dig. Methionine g/kg 5.0 4.6 dig M + C g/kg 8.0 7.6 Dig. Threonine g/kg 6.9 6.7 Dig. Valine g/kg 8.5 8.3 ME.sub.broiler.sup.1 MJ/kg 12.07 12.43 Ca g/kg 9.4 8.0 P total g/kg 6.0 5.1 Av. P g/kg 4.0 3.3 Na g/kg 1.7 1.6 Cl g/kg 2.0 2.1 K g/kg 8.9 8.5

(7) The broilers were inoculated in the crop on days 15 and 16 of age with on average 3.7×108 CFU nalidixic acid resistant Salmonella enteritidis PT4. Based on necropsy of five birds per cage on days 20 and 29 of age, Salmonella enteritidis counts in the caecal digesta (intestinal colonization) and spleen (translocation) were determined. Growth performance of the birds (body weight gain, feed intake and feed conversion ratio) was determined at 15 and 30 days of age.

(8) Measurements

(9) Test Product

(10) The soy oil with the 1% solution of phenylcapsaicin was analysed for the phenylcapsaicin dose.

(11) Experimental Diets

(12) Samples of each experimental diet (4 starter and 4 grower diets) were analysis on the inclusion level of phenyl capsaicin.

(13) Samples of each experimental diet (4 starter and 4 grower diets) were analysed for dry matter, ash, crude protein, crude fat (HCL), crude fibre, calcium and phosphorus.

(14) Droppings

(15) At 13 days of age (prior to inoculation) droppings were sampled and pooled per cage to determine the initial Salmonella enteritidis situation. Droppings per cage were collected over a period of 2 h on clean paper underneath each cage.

(16) Caecal Digesta and Spleen Sampling

(17) Salmonella enteritidis concentration in digesta of the caeca and of the spleen were measured to determine intestinal colonization and translocation, respectively, by sacrificing five random birds per cage on 20/21 and 29/30 days of age. For this purpose the birds were euthanized. During inoculation at day 15/16, in each pen five randomly chosen birds were marked with a green wingtag and five randomly chosen birds were marked with a blue wingtag. At 20/21 days the birds with a green wingtag were sacrificed and at 29/30 days of age the birds with the blue wingtag were euthanized. Birds were first anaesthetized by a mixture of Sedamun and Ketamine (5:3) via injection in the breast muscle (dosage: 1 ml/kg BW) and subsequently euthanized by an intravenous injection of T61 (an aqueous solution containing (in mg per ml) embutramide, 200; mebezoniumiodide, 50; tetracainehydrochloride, 5). After euthanizing the birds were weighed and thereafter the caecal digesta and the spleen were collected. After collection, the samples were immediately cooled at 2-8° C. prior to analyses for nalidixic acid resistant Salmonella enteritidis PT4.

(18) The concentration of Salmonella enteritidis PT4 in digesta and spleen was determined after decimal dilutions of the samples in a buffered peptone solution. The diluted samples were inoculated on Brilliant Green Agar plates containing 100 μg/l nalidixic acid. Plates were incubated during 24 h at 37° C. Typical Salmonella enteritidis PT4 colonies were counted and the concentration of the Salmonella enteritidis PT4 (cfu/g) in the original samples was calculated according to standard procedures.

(19) Growth Performance

(20) All broilers were weighed per cage at 0, 15 and 30 days of age to determine the body weight (BW).

(21) Feed intake (FI) was recorded per cage on day 15 and 24 of age.

(22) Mortality per cage was recorded daily.

(23) BW, body weight gain (BWG), FI, feed conversion ratio (FCR) and mortality were calculated over the following periods: 0-15 days, 15-30 days and the entire experimental period (0-30 days of age).

(24) Animal Health Observations

(25) Health of birds was checked daily and aberrations were recorded as well as probable cause of death for dead or culled birds. Temperature and humidity in the animal room were recorded.

(26) Statistical Analyses

(27) Growth Performance and Spleen Weight

(28) Statistical analyses on growth performance and spleen weight data was performed within the “Salmonella challenged” treatments (treatment II-V). Response parameters were statistically analysed by ANOVA using GenStat statistical software (17th edition, VSN International Ltd., Hemel Hempstead, UK), using row/tier as block factor, and diet as explanatory variable according to the statistical model given below:
Y=μ+row.sub.i/tier.sub.j+diet.sub.k+e.sub.ijkl
Where: Y=Response parameter μ=General mean row/tier=Row of battery cages (i=1,2) as block and within row tier (j=1,2,3) as sub-block diet=Effect of the diet (k=1 . . . 4) e=Error term

(29) The P-value of the treatment effect and the LSD (least significant difference (P=0.05)) were provided per response parameter. Treatment effects with a P-value≤0.05 were considered to be statistically significant.

(30) Salmonella enteritidis PT4 Counts

(31) To examine for differences in numbers (CFU/g) of Salmonella in the caecal digesta of chickens fed a diet containing 20, 28 or 36 mg/kg phenylcapsaicin compared to a control group on a diet without phenylcapsaicin. First a Pearson's chi-square test was performed on ranked (low to high) concentration values between the control and treatment group fed 20 mg/kg. Due to missing values, this analysis could not be done on the 28 and 36 mg/kg treatments. Since the broilers were kept in different cages, cages were first treated as random effect factors using a generalized linear mixed effects model (GLMM), applying the glmmPQL function from the MASS library of R, assuming Poisson distribution due to count data. Second, the data were analysed excluding the effect of cages by a generalized linear model applying the glm function from the MASS library of R, again assuming Poisson distribution. Analyses were performed on data from both sample days (day 20 and day 29; overall), and on data from day 20 and 29 separately. On day 20, the highest control value (1.3×CFU/g) and the two highest values in the 28 mg/kg treatment group (1.3×10′ and 1.2×10.sup.7 CFU/g) are considerable higher than the next highest value (1.1×10.sup.6 and 4.0×10.sup.6 CFU/g) for the control and 28 mg/kg treatment groups, respectively and were therefore considered to be outliers. Analysis were thus performed on the remaining data after excluding these data points.

(32) Results

(33) Spleen weight in Salmonella inoculated broilers at 20 and 29 d of age was numerically higher than spleen weight in broilers without Salmonella inoculation which implicates a successful Salmonella infection. Dietary supplementation of phenylcapsaicin at 20, 28 and 36 mg/kg did not affect the spleen weight at 20 and 29 days of age of the Salmonella inoculated broilers Broilers fed diets containing 20 mg/kg phenylcapsaicin numerically had lower Salmonella counts than control chickens on a diet without phenylcapsaicin (P values of 0.15 for both days (20 and 29 days of age) and 0.13 for day 29). The results are summerized in Table 3. The differences in ranked numbers are highly significant between the control and the 20 mg/kg treatment group, with the 20 mg/kg group having consistently lower Salmonella counts than the control group at both day 20 as well as day 29. The results are found in Table 4 and in FIG. 1. Caecal colonization of Salmonella enteritidis PT4 was not affected by dietary inclusion of 28 and 36 mg/kg phenylcapsaicin. These results implicate that most probably, the effective dose is in the range up to 20 mg/kg phenylcapsaicin instead of phenylcapsaicin dose levels over 20 mg/kg.

(34) TABLE-US-00003 TABLE 3 Summary statistics from the generalized linear mixed model (where cage was treated as random effect factors using a generalized linear mixed effects model (GLMM), applying the glmmPQL function from the MASS library of R) and generalized linear model (without controlling for the random effect of cage, applying the glm function from the MASS library of R) used to examine for differences in CFU/g counts between chickens fed a diet containing 20 mg/kg phenylcapsaicin and chickens fed a control diets without phenylcapsaicin. Outliers are defined as values > 1.0 × 10.sup.7 CFU/g. Std. t Input data R test error df value P Both days (day 20 and 29) All CFU/g counts glmPQL 3.521 106 0.223 0.82 glm 3.521 116 0.229 0.82 CFU/g counts; outliers masked glmPQL 1.507 105 1.469 0.15 glm 1.507 115 1.469 0.15 Day 20 All CFU/g counts glmPQL 1.444 10 1.240 0.24 glm 1.444 58 1.240 0.22 CFU/g counts; outliers masked glmPQL 0.468 10 0.822 0.43 glm 0.468 57 0.822 0.41 Day 29 All CFU/g counts glmPQL 1.580 10 1.646 0.13 glm 1.580 58 1.646 0.11

(35) TABLE-US-00004 TABLE 4 Summary statistics from the Pearson's chi-square test testing for differences in ranked numbers of Salmonella counts in caecal digesta Day χ.sup.2 df Probability level Day 20 21.6 1 0.001 Day 29 6.67 1 0.01

(36) In the Salmonella challenged birds, body weight (BW) and body weight gain (BWG) of broilers fed diets with 36 mg/kg of phenylcapsaicin was significantly lower than BW and BWG of broilers fed diets with 0, 20 or 28 mg/kg phenylcapsaicin. The lower body weight gain of broilers fed diets with 36 mg/kg resulted in a higher feed conversion ratio as compared to the other treatments with Salmonella inoculated broilers. Dietary inclusion of phenylcapsaicin had no effect on daily feed intake of Salmonella inoculated broilers.

(37) In the grower phase (15-30 days), after Salmonella challenge, dietary inclusion of phenylcapsaicin at 20, 28 and 36 mg/kg did not affect feed intake and feed conversion ratio of Salmonella inoculated broilers. A tendency towards a lower BWG was observed by increasing the dose level of phenylcapsaicin (P=0.099). Broilers who received the diets with the highest phenylcapsaicin dose (36 mg/kg) had numerically the lowest BWG and the highest FCR.

(38) Body weight (BW) and body weight gain (BWG) in Salmonella inoculated broilers fed diets with 36 mg/kg of phenylcapsaicin was significantly lower than BW and BWG in Salmonella inoculated broilers fed diets with 0, 20 or 28 mg/kg phenylcapsaicin. Dietary inclusion of phenylcapsaicin had no effect on daily feed intake and feed conversion ratio of Salmonella inoculated broilers

(39) Conclusion

(40) Dietary inclusion of 20 mg/kg phenylcapsaicin resulted in statistically significant differences in individually ranked caecal Salmonella enteritidis PT4 counts compared with the control treatment. This result can be an indicator of a Salmonella reducing effect of including 20 mg/kg phenylcapsaicin in broiler diets.