Lactylates for the prevention and treatment of infections caused by gram-positive bacteria in animals

Abstract

The present invention pertains to the use for preventing or treating intestinal infections caused by gram-positive bacteria in animals of an antibacterial compound selected from lactylate in accordance with formula 1,
R2—COO—[—CH(CH.sub.3)—COO].sub.n—R1  Formula 1
or a Na, K, Ca, Mg, Fe(II), Zn, NH.sub.4, or Cu(II) salt thereof, a glycolylate of formula 2,
R2—COO—[—CH2—COO].sub.n—R1  Formula 2
or a Na, K, Ca, Mg, Fe(II), Zn, NH.sub.4, or Cu(II) salt thereof, a lactate ester of formula 3,
HO—CH(CH.sub.3)—COO—R22  Formula 3
and/or a glycolic acid ester of formula 4,
HO—CH2—COO—R2  Formula 4
wherein R1 is selected from H, n stands for an integer with a value of 1-10, and R2 stands for a C1-C35 alkyl or alkenyl chain which may be branched or unbranched. The compound, which preferably is a lactylate or a Na, K, Ca, Mg, Fe(II), Zn, NH.sub.4, or Cu(II) salt thereof, is particularly useful in the treatment or prevention of Clostridia. An animal nutrition composition and a method for preventing or treating infections are also claimed.

Claims

1. A method for treating intestinal infections caused by gram-positive bacteria of the genus Clostridia in animals comprising feeding the animal with an effective amount of an antimicrobial compound in combination with one or more coccidostatic components, wherein the antimicrobial compound is selected from myristoyl lactylate and a Na, K, Ca, Mg, Fe(II), Zn, NH4, or Cu(II) salt thereof, and wherein the amount of antimicrobial compound is in the range of 0.001 to 0.5 wt % based on the total weight of each feed fed to the animal.

2. The method for treating intestinal infections caused by gram-positive bacteria in animals according to claim 1, wherein the animal is selected from cattle or poultry.

Description

EXAMPLE 1

Efficacy of a Mixture of Sodium Lauroyl Lactylate and Sodium Myristoyl Lactylate Against Necrotic Enteritis in Chicken

(1) The efficacy of a mixture of sodium lauroyl lactylate and sodium myristoyl lactylate against necrotic enteritis in chicken has been evaluated by Schothorst Feed Research in an experimental C. perfringens infection model which they have developed in which a coccidiosis infection is used as a pre-trigger for C. perfringens to colonise the small intestine and cause necrotic enteritis. A coccidiosis infection is initiated by a pathogenic Eimeria maxima and, on the peak of the coccidiosis infection, birds are inoculated with a C. perfringens strain that proved to be pathogenic to broiler chickens. A coccidiosis infection caused by E. maxima (resulting in lesions in the middle segment of the small intestine) followed by a Clostridium infection results in a highly reproducible model and an easy and accurate way of scoring for necrotic enteritis lesions, because lesions of E. maxima and Clostridium are easy to distinct while lesions of both pathogens do not occur in the same intestinal segment. The experiments are performed in cooperation with the Animal Health Service (GD).

(2) The experiment consisted of one treatment and two control treatments. All treatments consisted of six replicate cages with 19 broilers per cage. The treatments are given in Table 1.

(3) TABLE-US-00001 TABLE 1 Description of the treatments and diet codes Day 9 Day 14, Supplementation of Trt. Inoculum: 15 and 16 additive Remark 1. Saline Liver broth — Control 2. Eimeria C. perfringens.sup.2) — Control/ maxima Experimental 3. Eimeria C. perfringens test mixture (0.3% Experimental maxima mixture of 50 wt. % sodium lauroyl lactylate and 50 wt. % myristoyl lactylate) .sup.1)10,000 of sporulated oocysts of Eimeria maxima in 1 ml .sup.2)1 × 10.sup.8 cfu C. perfringens in 1 ml
Animals, Management and Procedures

(4) One day-old male Ross 308 broiler chickens were supplied by Probroed & Sloot B.V., the Netherlands. At day 0, broilers arrived at the laboratory facilities of the Animal Health Service (Deventer, the Netherlands) and were housed in digestibility cages after individual weighing. Based on a weight-class system 19 birds were allotted to 30 Schothorst litter floor digestibility cages, resulting in a similar mean weight per cage. Broilers were housed in these cages until the end of the experiment at day 20. At day 9, if no mortality occurred, the number of chickens was standardised to 17 and bird weight was measured again. First, birds with obvious visual aberrations were removed and second, birds were removed at random to decrease the number to 17. Lighting and temperature schedule throughout the experimental period was as follows, 22 hours of light followed by 2 hours darkness in the first period from day 0 to 9 followed by 18 hours of light and 6 hours darkness throughout the rest of the experiment. The ambient temperature was gradually decreased from 32° C. at the start to 25° C. at the end of the experiment.

(5) Feed was supplied for ad libitum intake from day 0 onwards with exception of the 5 hours prior to inoculations (days 9, 14, 15 and 16) and sections (days 15, 16 and day 20). Water was available for ad libitum intake throughout the experiment.

(6) Feed Composition

(7) The broilers were supplied a wheat/soybean meal-based starter diet from day of arrival until day 9. From day 9 onwards, a wheat/barley-based grower diet was fed until the end of the experiment (day 20). Grower feeds were fed as meals because of the necessity of homogenously mixing in the test products after feed production. Diets did not contain any coccidiostats or antimicrobial feed additives other than the test product. The nutrient composition of the experimental diets was according to Dutch standards to meet nutrient requirements of broilers (CVB, 2006).

(8) Inoculum

(9) At day 9, broilers were inoculated with either 1 ml saline or E. maxima (10.000 sporulated oocysts/chicken in 1 ml) after a 5 hours feed withdrawal period. From day 14 onwards, broilers were either inoculated with 1 ml liver broth (DIFCO) or C. perfringens once per day persisting three days after a 5 hours feed withdrawal. A detailed overview of the different treatments is presented in Table 1.

(10) The pathogenic C. perfringens strain was obtained from the Animal Health Service in Deventer, the Netherlands (approx. 10.sup.8 cfu in 1 ml). The strain was grown on an agar of sheep blood and the culture is typed by CIDC (Central Institute of Animal Disease Control in Lelystad) as C. perfringens producing type α and β2 toxins. Each day a freshly prepared inoculum was used.

(11) Lesion Scoring

(12) Clostridium perfringens: Gross and microscopic lesions generally occur in the small intestine, particular in the proximal site. The following scoring method was used:

(13) 0: no lesions

(14) 1: 1 to 5 small lesions (spots of less than 1 mm diameter)

(15) 2: more than 5 small lesions (spots of less than 1 mm diameter) or 1 to 5 larger lesions (spots of 1 to 2 mm diameter)

(16) 3: more than 5 larger lesions (1 to 2 mm diameter) or erosive zones

(17) 4: dead birds with positive necrotic enteritis diagnoses post mortem

(18) All birds were scored “blind”, i.e. the person scoring the birds for lesions did not have knowledge of the birds treatment.

(19) Measurements

(20) During the experiment the following parameters were measured:

(21) Individual body weight at day of arrival and means per cage at day 9 and day 20 of the experiment

(22) Body weight of the birds prior to necropsy

(23) Feed intake per cage in the periods from day 0 to 9 and daily feed intake from 9-20 days of age

(24) Coccidiosis lesions and necrotic enteritis lesions in the small intestinal mucosa of 24 birds per treatment at day 15, day 16 and day 20 of the experiment (total of 72 birds per treatment).

(25) Mortality per cage from 0 to 20 days of age.

(26) Daily records were kept of all routine study activities, health disorders and of mortality (with its most probable cause).

(27) Statistical Analyses

(28) Raw data were analysed for outliers. Significant outliers were excluded from the statistical analysis. The incidence of NE-lesions (% of affected birds) was analysed by Fisher Exact Test, whereas the severity of lesions and daily feed intake measurements were analysed by analysis of variance (ANOVA) using Genstat statistical software. Treatment means were compared by the least significant difference (LSD). P≤0.05 was considered to be statistically significant, whereas 0.05<P≤0.10 was considered to be a near-significant trend.

(29) Results and Discussion

(30) Incidence and Severity of Lesions

(31) Lesion Scoring at Day 15 (1 Day Post Infection)

(32) In Table 2, the percentage of positive scored birds (birds with NE lesions) is given as well as the mean lesion score of all positive scored birds. Because the mean lesion score of all examined birds, affected as well as unaffected, gives a more representative picture for the population, statistical analyses have been performed over these results (see the fifth column of Table 2). The severity of lesions in both positive and negative scored birds is indicated on a scale of 0 to 4 (see section “lesion scoring”).

(33) TABLE-US-00002 TABLE 2 Birds observed with NE (%) and the mean severity of lesions scored at day 15 (1 day p.i.). Positive Lesion birds Lesion severity Group Treatment Dosage (%) severity pos. birds).sup.1) 1 Negative control —  0.sup.a 0.0.sup.a 0.0 2 Positive control — 16.sup.ab 0.5.sup.b 3.0 3 Test mixture 0.3% 17.sup.ab 0.4.sup.b 2.5 .sup.a,bValues with no common superscript in a column differ significantly (P ≤ 0.05). .sup.1)Lesions severity of NE-positive scored birds

(34) A significant treatment effect was observed on the NE incidence. As expected, the lowest incidence was observed in the uninfected control treatment but results were comparable to the results of the treatments supplemented with the test mixture and unsupplemented infected control.

(35) Based on the ANOVA it was concluded that there was a significant treatment effect on the severity of necrotic lesions on day 15 (P<0.001). On lesion severity it was evident that lesions were more severe in the infected treatments, unsupplemented as well as supplemented, than the uninfected control treatment for there were no positive score birds in the latter. Among the infected treatments no statistical differences were observed.

(36) Lesion Scoring at Day 16 (2 Days Post Infection)

(37) In Table 3, the percentage of positive-scored birds and the mean lesion score of birds is given for day 16.

(38) TABLE-US-00003 TABLE 3 Birds observed with NE (%) and the mean severity of lesions scored in all necropsied birds at day 16 (2 days p.i.). Positive Lesion birds Lesion severity Group Treatment Dosage (%) severity pos birds).sup.1) 1 Negative control —  0.sup.a 0.0.sup.a 0.0 2 Positive control — 68.sup.b 2.1.sup.c 3.2 3 Test mixture 0.3% 41.sup.b 1.1.sup.b 2.7 .sup.a,b,Values with no common superscript in a column differ significantly (P ≤ 0.05).

(39) Comparing the results of NE incidence and lesion severity on day 16 to the results of day 15, it is clear that the severity of infection was higher 2 days post infection. Although again a significant treatment effect was observed on the NE incidence, it is evident that this is due to the difference between the uninfected control treatment and infected treatments, which is as expected, whereas among infected treatments there was no significant difference observed.

(40) A sharp distinction can be drawn on lesion severity 2 days post infection. The treatment supplemented with the test mixture resulted in a clear reduction in lesion severity compared to the infected unsupplemented control, although mean lesion scores were still higher than the uninfected control.

(41) Lesion Scoring at Day 20 (6 Days Post Infection)

(42) At day 20 no significant differences was observed between treatments. All treatments recovered from NE, at least based on macroscopical evaluation, with 0% incidence and obviously 0.0 for lesion severity.

(43) Mortality

(44) Mortality is one of the parameters to measure the severity of an infection with Clostridium in a flock. In this experiment the mortality was compared among treatments. Mortality was 14.6% in the infected control treatment (treatment 2) and 0% in the uninfected control. Supplementation of the test mixture resulted in a reduction in mortality (5.1%).

(45) Production Parameters

(46) Besides lesions scoring, production parameters like body weight and daily feed intake were measured during the trial period.

(47) Body weight of one day-old broilers was in all treatments approx. 47 grams. Because treatments from day 0 to 9 were similar, no differences in body weight gain and feed intake were observed in this period.

(48) In the infection period from day 9 to 20 both production parameters were significantly affected by the individual treatments. Body weight gain was highest in the uninfected control, as expected, while broilers in the infected unsupplemented treatments showed the lowest body weight gain. This resulted in a 30% lower final weight at day 20 (523 g versus 749 g). The infected supplemented treatment resulted in a significantly higher feed intake and body weight gain compared to the infected unsupplemented control. Reduction in production performance could be reduced with 10% showing a loss in final weight of approx. 20% when compared to the uninfected control group (approx 583 g versus 749 g). It was concluded that the test mixture significantly increased production performance during a subclinial Clostridium infection.

EXAMPLE 2

In Vitro Tests of Lactylates Against Clostridium

(49) Liquid cultures of Clostridium perfringens ATCC 13124 were grown in screw-capped tubes (100×16 mm) containing 10 ml brain heart infusion broth (Oxoid CM225, Basingstoke, United Kingdom) for 24 hours at 30° C. Brain heart infusion broth was prepared with varying amounts of lactylates. The pH of the media was adjusted to 6.0 with 9 M sulphuric acid using a Handylab pH 12 pH meter equipped with a Blueline 16 pH (micro) probe (no. 285129163). All media were sterilised by filtration using 0.45 μm cellulose acetate filters (Minisart syringefilter, sterile and non-pyrogenic, no. 16555, Sartorius, Göttingen, Germany) (9). 300 μl of each medium was transferred to a panel of a sterile Bioscreen honeycombe 100 well plate (Thermo electron Oy, Vantaa, Finland). Completed well plates were stored at −30° C. until further use.

(50) Well plates were inoculated with 3 μ1 culture using a sterile Hamilton repeating dispenser (Hamilton, Bonaduz, Switserland). The growth rate of the test organisms was determined at 30° C. with the Bioscreen C culture system (Oy Growth Curves AB Ltd, Helsinki, Finland). In order to assure low oxygen conditions the Bioscreen was placed inside an anaerobic cabinet equipped with a type M-12 oxygen sensor (In Vivo.sub.2 400 hypoxia workstation, Biotrace International Plc, Bridgend, United Kingdom). The oxygen tension was regulated at 0% oxygen using a Ruskinn gas mixer module (Biotrace International Plc). The Bioscreen C kinetically measures the development of turbidity by vertical photometry in up to 200 wells simultaneously. The optical density of the cultures was automatically measured at fixed time intervals at 420-580 nm using a wide band filter.

(51) Table 4 shows the MIC values for the various lactylates tested for Clostridium perfringens ATCC 13124 in brain heart infusion broth. In the parentheses the number of repeats is given. MIC stands for the Minimal Inhibitory Concentration, which is the lowest concentration where the increase in absorbance of a culture did not exceed the threshold value, which was defined as the average increase in absorbance value of the blanks plus three times the standard deviation.

(52) It appears that even at very low concentration lactylates are capable of suppressing the growth of Clostridium perfringens.

(53) TABLE-US-00004 TABLE 4 MIC values of different lactylates Lactylates MIC values (%) C8 lactylate  0.05% (2x) C10 lactylate  0.04% (2x) C12 lactylate 0.002% (2x) C14 lactylate 0.001% (2x) C16 lactylate 0.002% (2x) C18:1 lactylate  0.02% (2x) Mixture of 1:1 C10/C12 lactylate) 0.002% (3x) Mixture of 1:1 C12/C14 lactylate 0.001% (3x)

EXAMPLE 3

Dose-Response Studies and Prevention Studies of a Mixture of Sodium Lauroyl Lactylate and Sodium Myristoyl Lactylate Against Necrotic Enteritis in Chicken

(54) Analogous to Example 1, the influence of the dose of the compound was studied. Further, the use of the mixture on chicken which were not previously infected with Emeria and Clostridium was studied.

(55) The treatments performed are summarised in Table 5:

(56) TABLE-US-00005 TABLE 5 Description of the treatments Trt. Description 1. Uninfected 2. Uninfected + 0.3% test mixture 3. Infected.sup.1) 4. Infected + 0.6% test mixture 5. Infected + 0.3% test mixture 6. Infected + 0.3% test mixture (silica) 7. Infected + 0.15% test mixture 8. Infected + 0.075% test mixture 9. Infected + 0.038% test mixture 10. Infected + 0.019% test mixture 11. Infected + 0.010% test mixture 12. Infected + 0.005% test mixture The test mixture was made up of 50 wt. % of sodium lauroyl lactylate and 50 wt. % of myristoyl lactylate.

(57) The results may be summarised as follows.

(58) In this experiment a subsequent infection with E. maxima and C. perfringens resulted in an incidence of necrotic enteritis of 56% and an average lesion score of 1.6 during the first two days post infection. Supplementing bird diets with test mixture reduced the number of infected birds and a dose response effect was observed, showing the highest efficacy in the 0.6% and 0.3% treatments. Lesion severity was significantly reduced due to test mixture supplementation and the dose response effect was also strongly present on this parameter. Lesions were less severe in the treatments with the highest doses of test mixture. The test mixture supplemented in a pure form resulted in a somewhat better response than the test mixture supplied via a silica carrier. Supplementation of test mixture with 0.6% resulted in a significant reduction in mortality (4.6%) and was not significantly higher than the not infected control treatment. Results with 0.3% test mixture supported the results observed in the lesion scoring.

(59) With regard to production performance an effect was observed when comparing healthy birds with or without test mixture supplementation with each other. The provision of test mixture tended to increase body weight in the starter phase and grower phase. Supplementing infected birds with higher doses of test mixture resulted in the birds having a similar final weight (day 37) as birds that were not infected at all.

(60) It can be concluded that the test mixture, especially in a dose of 0.3 wt. % or higher is effective in preventing necrotic enteritis development in broiler by showing a lower incidence and lesions that were less severe.

(61) Although the present invention has been described with reference to preferred embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.