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Efficacy of a lactylate on production performance and intestinal health of broilers during a subclinical Clostridium perfringens infection
Efficacy of a lactylate on production performance and intestinal health of broilers during a subclinical Clostridium perfringens infection M. Lensing,* J. D. van der Klis,*1 T. Fabri,† A. Cazemier,‡ and A. J. Else‡ *Schothorst Feed Research, Meerkoetenweg 26, 8218 NA, Lelystad, the Netherlands; †Animal Health Service, Arnsbergstraat 7, 7400 AA, Deventer, the Netherlands; and ‡Purac Biochem BV, Arkelsedijk 46, 4206 AC, Gorinchem, the Netherlands that a LauL dose higher than 0.15% should be used to expect positive effects on lesion severity and mortality. None of the LauL doses led to a significant better response on growth performance. In a third trial, efficacy of LauL was compared with commercial products that limit bacterial activity in the intestinal tract (Aromabiotic Poul 60) or coccidiosis (chemical coccidiostat, Clinacox). None of the products were able to reduce the number or severity of lesions, and no effect on production performance was observed. Thus, despite the clear positive effect seen in experiment 1, and in experiment 2 with LauL doses higher than 0.15%, supplementing this lactylate to the diet does not consistently reduce C. perfringens colonization in broiler chickens because no such effects were observed in experiment 3. These results, however, provide a scientific basis for future studies to further investigate lactylates as potential additives to reduce the severity of necrotic enteritis in broilers in a C. perfringens challenge model.
Key words: Clostridium perfringens, lactylate, intestinal health, broiler 2010 Poultry Science 89:2401–2409 doi:10.3382/ps.2010-00942
INTRODUCTION Clostridium perfringens, an α-toxin producing grampositive bacterium, is an enteric pathogen for poultry. Clostridium infections may appear as an acute clinical or subclinical disease (Løvland and Kaldhusdal, 2001), with symptoms varying from loss in performance to mortality. In case of a subclinical infection, the duodenal-jejunal mucosa is damaged, which decreases nutrient digestion and absorption, reduces weight gain, and increases feed conversion ratio (FCR; Elwinger et al., 1992; Hofshagen and Kaldhusdal, 1992; Kaldhusdal et al., 2001; Hofacre et al., 2003). Factors that stimulate intestinal mucus production (e.g., gastrointestinal infections like coccidiosis) are considered predisposing fac©2010 Poultry Science Association Inc. Received June 13, 2010. Accepted August 15, 2010. 1 Corresponding author: [email protected]
tors for Clostridium infections because C. perfringens is a mucolytic bacterium (Collier et al., 2003). Coccidiosis infections can be controlled by coccidiostats, which indirectly prevent Clostridium infections. Some coccidiostats, like ionophores that belong to a class of antibiotics, can also be used to control Clostridium infections directly, because they control Clostridium growth in the gastrointestinal tract. However, a real alternative to antibiotics or coccidiostats that effectively fights off Clostridium in a direct manner has not yet been found. In search for such a product, fatty acids and fatty acid derivatives provide obvious candidates because they have long been known and used as antimicrobials (Kabara and Marshall, 2005). Fatty acid derivatives have detergent or surfactant properties, and it is these properties that most probably endow them with antimicrobial activity by way of interacting with the cell membranes of the microorganisms. Its efficacy, as an antimicrobial, is determined by the type of derivative and the fatty acid chain length and degree of
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ABSTRACT Clostridium perfringens, an α-toxin producing gram-positive bacterium, is an enteric pathogen for poultry. Because subclinical C. perfringens infections often result in damage of the intestinal mucosa, decreased nutrient digestion, and poor performance, efforts should be taken to find an effective strategy that controls overgrowth of C. perfringens. For this purpose, the efficacy of a sodium lauroyl lactylate (LauL) as a feed additive to prevent C. perfringens colonization in broilers was determined. First, the effect of LauL was compared with capric and lauric mono- and diglycerides (MDG) and capric and lauric free fatty acids in Clostridium-infected chickens. Clostridial lesion scoring at d 16 showed that MDG and LauL were both effective in reducing the severity of lesions. When taking into account results on BW gain and mortality, LauL was more effective than MDG. For this reason, a dose response study was made to determine the optimal dietary dosage of LauL. In this experiment, it was shown
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MATERIALS AND METHODS Experimental Design Experiment 1. The objective of this experiment was to test the efficacy of a C12–C14 lactylate (LauL) against a C10–C12 MDG (50:50 mix of C10–C12 fatty
acids bound to MDG) and C10–C12 FFA (50:50 mix of C10–C12 FFA) on Clostridium-infected chickens. A total of 600 one-day-old male Ross 308 broiler chickens were randomly assigned to 5 treatments with 6 replicated cages each. The experiment was carried out from 0 to 20 d of age. From day of arrival to d 9, chicks were fed a starter diet without any supplements and from d 9 onward, a grower diet was given. After standardization to 17 birds per cage at d 9, chicks were challenged with Eimeria maxima and C. perfringens. Control treatments were infected-nontreated (INT) or noninfected-nontreated (NINT). Three other infected treatments (IT) received the grower diet supplemented with 0.3% FFA (IT + FFA), 0.3% of MDG (IT + MDG), or 0.3% LauL (IT + LauL). Products were produced and supplied by Purac Biochem (Gorinchem, the Netherlands). Dosages were chosen based on results from in vitro studies in combination with the expected kinetics of the products in the intestinal tract. All treatments were randomly allocated to litter cages except NINT cages that were grouped together to prevent cross-contamination. Postmortem clostridial lesion scoring occurred at d 15 and 16 and procedures are described later. Remaining birds were used to collect data on growth performance. Birds were weighed per cage at d 0, 9, and 20 to determine BW gain (BWG) from d 0 to 9 and d 9 to 20. Feed intake (FI) per cage was recorded during the same periods but daily in the infection period from d 9 to 20. Based on FI and BWG, FCR was calculated on a cage basis. Mortality with its most probable cause was recorded from d 0 to the end of the experiment. Experiment 2. The objective of experiment 2 was to find the optimal dosage of LauL in a dose response study, being the most promising molecule from experiment 1, to fight C. perfringens. This experiment was similar to experiment 1 with regard to breed and sex, infection protocol, and response parameters. A total of 1,320 one-day-old Ross 308 male broiler chicks were randomly assigned to 11 treatments with 6 replicate cages each. Treatments were designed as noninfected and treated (NIT) with 0.3% LauL (NIT + 0.3 LauL) or without (NINT). The infected treatments were set up as a dose response starting with a 2 times higher dose as was used in experiment 1 (IT + 0.6 LauL) and stepwise lowering the supplementation with 50%, from 0.6% to 0.3% to 0.15%, toward 0% supplementation of LauL (INT). All diets with or without LauL supplementation were supplied from d 0 to 37 to see if a prolonged use of LauL before inoculation was more effective. Postmortem lesion scoring was performed on d 15, 16, and 17. Experiment 3. Experiment 3 was similar to experiment 2 with regard to breed, sex, the infection protocol, response parameters, and start and length of the dietary treatments. A total of 960 one-day-old broiler chickens were randomly allocated to 8 treatments with 6 replicate cages each. Treatments were designed with 2 controls, INT and NINT without supplementation of LauL or with 0.3% LauL (IT + 0.3 LauL or NIT
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saturation. Bayliss (1936) investigated the relation between the chemical composition of a soap and its germicidal properties. For the saturated soaps, he found the maximum antimicrobial activity with sodium myristate (C14:0) fatty acids. Among the esters of fatty acids, the naturally occurring glycerol esters are the most well known and widely used. In particular, monoesters with medium-chain fatty acids (MCFA) have been used as antimicrobials (Kabara and Marshall, 2005). Other less well-known esters of fatty acids are the lactylates; these are esters of lactic acid in which the C2-hydroxy group of lactic acid is esterified to a fatty acid. The main commercial use of lactylates is in the bakery industry, with C18 fatty acids (Boutte and Skogerson, 2004). Lactylates made with MCFA have been used in cosmetics (patent application EP0595528). Both components of lactylates, lactic acid and fatty acids, by themselves are known and used as antimicrobials in animal production. Lactic acid can be used to reduce Campylobacter and Salmonella contamination of broiler carcasses at processing (Byrd et al., 2001) and free fatty acids (FFA) such as MCFA have been shown to reduce Clostridium infections in broiler chickens (Jansman et al., 2006). In vitro, lactylates with different types of fatty acids have shown already to be potent inhibitors of C. perfringens (patent application EP02082739A1), with the lowest minimum inhibitory concentration of 0.001% (wt/wt) for a lactylate with C12 and C14 fatty acids. That means that in vitro they are 100- to 1,000-fold more effective inhibitors than FFA or lactic acid alone. The series of trials described here were made to test a sodium lauroyl lactylate (LauL; Puramix 30, Purac Biochem, Gorinchem, the Netherlands) containing C12 and C14 fatty acids under in vivo conditions. The first trial was used to compare the effects of a LauL to C10–C12 monoglycerides (mono- and diglycerides; MDG) and free C10–C12 MCFA (FFA) in Clostridium-infected chickens. Because LauL was the most promising molecule, a dose response study was made to determine the optimal dietary dosage. In a third trial, the efficacy of LauL was compared with commercial products that limit bacterial activity in the intestinal tract (Aromabiotic Poul 60; Vitamex, Drongen, Belgium; ABP) or coccidiosis (chemical coccidiostat; C) and consequently its effect on intestinal mucus production. This series of experiments was made to determine whether MCFA lactylates can be of help in reducing problems caused by C. perfringens in broiler chickens.
ANTI-CLOSTRIDIAL EFFECTS OF SODIUM LAUROYL LACTYLATE
+ 0.3 LauL), to test if LauL would benefit in healthy and infected chickens. The efficacy of 0.3% LauL in infected chickens was tested against 4 IT treatments with 0.2% LauL dosage (IT + 0.2 LauL), the combination of LauL with a chemical coccidiostat (Clinacox: 100 g/ ton, Janssen Animal Health, Beerse, Belgium; IT + 0.3 LauLC), a chemical coccidiostat alone (IT + C), and a commercial MCFA product (Aromabiotic Poultry: 1.6 kg/ton in starter and 1.2 kg/ton in grower phase, Vitamex, Drongen, Belgium; IT + ABP).
Birds and Housing
Experimental Diets Broilers were supplied a wheat-soybean meal-based starter diet for ad libitum intake from day of arrival until d 9. At d 9, a wheat-barley-based basal grower diet was fed until the end of the experiment (Table 1). Grower diets were fed as mash because of the necessity of homogeneously mixing in the test products after feed production. Diets did not contain any nonstarch polysaccharide enzymes, coccidiostats, or antimicrobial feed additives other than the test products. The nutrient composition of the experimental diets was according to Dutch standards to meet nutrient requirements of broilers (CVB, 2008).
Challenge To induce the onset of a C. perfringens infection, a mild E. maxima infection was used as pretrigger. Birds were orally inoculated at d 9 with 1 mL of E. maxi-
ma (Central Veterinary Laboratory, Weybridge, UK; ~10,000 sporulated oocysts per mL; 1 mL per bird) or with sterile saline after a 5-h feed withdrawal period. This inoculation with E. maxima has consistently shown to induce C. perfringens with an average of 50% of chickens scored positive for clostridial lesions. Moreover, reddish E. maxima lesions are distinctly different from white C. perfringens lesions. After the 5-d incubation time for coccidiosis to develop, birds were orally inoculated on d 14 with C. perfringens type A [~108 cfu/mL; 1 mL of liver broth (Difco, Detroit, MI) per bird] or with 1 mL of sterile liver broth after a 5-h feed withdrawal period to induce necrotic enteritis. After inoculations, birds had immediate access to feed. The pathogenic C. perfringens strain (code GD 5.11.53) was obtained from the Animal Health Service (Deventer, the Netherlands). The strain was grown on an agar of sheep blood and the culture was typed by the Central Institute of Animal Disease Control (Lelystad, the Netherlands) as C. perfringens producing type α and β2 toxins.
Pathological Parameters During the peak of Clostridium infection at d 15 and 16 (1 and 2 d postinfection), 4 random selected birds per cage were used for postmortem coccidial and clostridial lesion scoring in the duodenal and jejunal segment of the small intestine (48 birds per treatment). In experiments 2 and 3, a third scoring was done at d 17 (3 d postinfection). Birds were killed by an intracardial injection with a euthasate (T61; Intervet, Mechelen, Belgium). The abdomen was opened and the gastrointestinal tract was exposed. The gastrointestinal tract was segmented into the duodenum and jejunum (the segment from the gizzard to the 10 cm preceding Meckel’s diverticulum). All birds were scored blind (i.e., the person scoring for lesions had no knowledge of the bird’s treatment). The following method was used to score the C. perfringens lesions: 0 = no lesions; 1 = 1 to 5 small white lesions (spots of less than 1 mm in diameter); 2 = more than 5 small white lesions (spots of less than 1 mm in diameter) or 1 to 5 larger lesions (spots of 1 to 2 mm in diameter); 3 = more than 5 larger lesions (1 to 2 mm in diameter) or erosive zones; 4 = dead birds with positive necrotic enteritis diagnoses postmortem. Based on the clostridial lesion scores, the percentage of Clostridium-infected birds was calculated by the following equation: (sum of birds scored >0/total number of birds scored) × 100%. The percentage of birds affected indicated the incidence of C. perfringens. By categorizing the severity of lesions from 0 to 4, an average lesion score could be calculated for each treatment, giving an indication of the severity of the C. perfringens infection besides its incidence.
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One-day-old male Ross 308 broiler chickens were supplied by Probroed & Sloot B.V. (Meppel, the Netherlands). At d 0, broilers arrived at the poultry facilities of Schothorst Feed Research B.V. (experiments 1 and 3; Lelystad, the Netherlands) or the Animal Health Service (experiment 2; Deventer, the Netherlands) and were housed in 2-tier litter floor digestibility cages after individual weighing (surface area 0.90 × 0.65 m). Twenty birds were allocated to cages based on a weight class system such that mean weight per cage was similar at the start of the experiment. Broilers were housed in these cages throughout the entire experiment. At d 9, before first inoculation, the number of chickens was standardized to 17, and bird weight was measured before and after standardization. Lighting and temperature schedule throughout the experimental period was 22L:2D in the time period from d 0 to 9 and 18L:6D from d 9 to the end of the experiment. The ambient temperature gradually decreased from 32°C at the start to 25°C at the end of the experiment. Feed was supplied for ad libitum intake from d 0 onward with the exception of the 5-h feed withdrawal period before inoculations (on d 9 and 14). Water was available for ad libitum intake throughout the experiment. Broilers were not vaccinated during the experiment.
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Table 1. Composition of the basal diets in experiments 1, 2, and 3 Starter (d 0 to 9) Item
Experiment 1
Experiments 2 and 3
Experiment 1
Experiments 2 and 3
19.35 25.73 — 47.76 — — — 1.19 1.77 1.34 0.61 0.11 1.50 0.28 0.22 0.08 0.06 109 55 199 51 26 384 2,759 8.9 5.1 1.4 2.7 8.5 10.4 7.6 6.5
18.54 25.73 — 48.57 — — — 1.19 1.77 1.83 0.62 0.28 1.50 0.28 0.22 0.08 0.06 122 55 199 51 26 392 2,798 9.0 5.1 1.4 2.7 8.5 10.4 7.6 6.6
— 19.50 5.26 35.88 1.09 24.24 3.83 4.81 1.86 1.12 0.78 0.12 0.50 0.31 0.27 0.09 0.34 117 49 187 80 36 360 2,864 6.7 5.7 1.6 1.9 7.8 9.6 7.7 6.9
— 19.53 5.26 35.77 1.09 24.24 3.82 4.59 1.86 1.13 0.78 0.13 0.70 0.33 0.28 0.15 0.34 117 50 185 83 40 361 2,865 6.7 5.7 1.6 2.0 8.0 9.6 7.7 6.9
1Hipro
(Brazilian soybeans; Cargill, Amsterdam, the Netherlands). mineral-vitamin premix contained the following per kilogram: Ca, 185 g; Na, 80 g; Cl, 100 g; Cu, 1,200 mg; Fe, 4,500 mg; Mn, 7,000 mg; Zn, 3,700 mg; I, 100 mg; Se, 15 mg; vitamin A, 1,000,000 IE; vitamin D3, 200,000 IE; vitamin E, 2,500 IE; vitamin B1, 50 mg; vitamin B2, 500 mg; pantothenic acid, 800 mg; niacin, 4,000 mg; vitamin B6, 300 mg; folic acid, 100 mg; vitamin B12, 1,500 µg; biotin, 10,000 µg; vitamin K3, 125 mg; choline, 20,000 mg. 2The
Three in vivo experiments were performed in succession. The experimental protocols and all procedures complied with the guidelines of the Ethical Committee for Animal Experiments (Lelystad, the Netherlands).
Statistical Analysis The incidence of C. perfringens (% of birds scored >0) and mortality rate were analyzed by Fisher’s exact test, whereas the severity of lesions, measurements on production performance, and other parameters were analyzed by ANOVA using GenStat statistical software (GenStat version 12, Hemel Hempstead, UK). Raw data of BW and feed intake were analyzed for outliers. Outliers were marked significant when exceeding 2.5 × SD of the treatment mean and were excluded from the data set. Treatment means were compared by the least significant difference. The statistical model(s) used per experiment were as follows, based on the entire data sets or subsets for each experiment. The model used for experiments 1, 2, and 3 was as follows:
Yij = μ + Blocki + Treatmentj + eij, with Yij = response parameter; μ = mean; Blocki = effect of block (i = 1 to 6); Treatmentj = effect of treatment (j = 5, 11, and 8 for experiments 1, 2, and 3, respectively); and eij = residual error. The model used for experiments 2 and 3 was as follows: Yij = μ + Blocki + Dosej + eij, with Yij = response parameter; μ = mean; Blocki = effect of block (i = 1 to 6); Dosej = effect of dose (j = 9 and 3 for experiments 2 and 3, respectively); and eij = residual error. The model used for experiment 3 was as follows: Yijk = μ + Blocki + Coccidiostatj + Testk + Interactionjk + eijk, with Yijk = response parameter; μ = mean; Blocki = effect of block (i = 1 to 6); Coccidiostatj = effect of coc-
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Ingredient (%) Maize Soybean meal, Hipro1 Sunflower seed meal Wheat Wheat middlings Barley Maize starch Soybean oil Animal fat Limestone Monocal Salt Premix vitamins and minerals2 Premix Lys (HCl 79%) Premix Met (dl 99%) Premix Thr (l 98%) Natrium carbonate Calculated nutrient (g/kg) Moisture Ash CP Crude fat Crude fiber Starch AME broiler (kcal) Calcium Phosphorus Sodium Chloride Potassium Apparent digestible Lys Apparent digestible Met + Cys Apparent digestible threonine
Grower (d 9 to end of experiment)
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Table 2. The percentage of birds scored positive for Clostridium perfringens lesions and the lesion severity in the infected-nontreated (INT) group and noninfected-nontreated group (NINT) presented as mean values of lesion scoring at d 15 and 16 (1 and 2 d postinfection, n = 48) Item Experiment 1 NINT INT Experiment 2 NINT INT Experiment 3 NINT INT
Positive birds (%)
Lesion severity (scale 0 to 4)
P-value
0 42
0.0 1.3
<0.001
0 62
0.0 1.8
<0.001
0 83
0.0 2.2
<0.001
RESULTS General The INT groups in all experiments were scored positive for C. perfringens. As an average of lesion scoring performed at d 15 and 16, the INT groups resulted in 42, 62, and 83% of birds scored positive for clostridial lesions in experiments 1, 2, and 3, respectively. In NINT groups, all birds were scored negative. Also, clostridial lesions were more severe (P < 0.001; Table 2). Calculating the scores from 0 to 4 in all necropsied birds, a lesion severity of 1.3, 1.8, and 2.2 for the 3 successive experiments was determined.
Experiment 1 On d 15 and 16, the number of birds scored positive for C. perfringens was not affected by dietary treatments FFA, MDG, and LauL compared with the INT control (Table 3). The severity of lesions was not significantly affected by any of the treatments on d 15, whereas on peak of infection at d 16, dietary supplementation of MDG and LauL resulted in lesions that were less severe compared with the INT and IT + FFA groups (P < 0.001; Table 3). Three days later (6 d postinfection), no significant differences were observed between treatments (data not shown) as all treatments recovered from necrotic enteritis, based on macroscopical evaluation. A similar pattern was observed for mortality, which was higher in the INT control, FFA, and MDG treatment compared with the NINT treatment (P ≤ 0.05). The LauL treatment was intermediate, not being significantly different from any treatment (Table 3). Production parameters from d 9 to 20 were affected by dietary treatments. Body weight gain was highest in
the NINT control (Table 3), as expected, whereas the INT treatment had the lowest BWG. All dietary treatments, FFA, MDG, and LauL resulted in a higher FI and BWG compared with INT (P < 0.001).
Experiment 2 The percentage of birds scored positive for C. perfringens was significantly influenced by challenge and dietary treatments. No lesions were observed in NINT and NIT treatments, whereas 56% was infected in the INT control treatment (mean of d 15, 16, and 17; Table 4). Supplementation of 0.6% LauL (IT + 0.6 LauL) resulted in 35% of birds scored positive. The other LauLsupplemented treatments resulted in similar percentages of broiler scored positive for necrotic enteritis as INT. With regard to severity of lesions, birds supplemented with 0.6% LauL had less severe lesions than birds in the INT control (P < 0.001). A dose of 0.3% LauL did reduce lesion severity as well toward a near-significant reduction in comparison to the INT control group (P ≤ 0.10). All other LauL-supplemented treatments did not improve results on lesion severity (Table 4). A sigmoid dose response curve was fitted to the data, explaining 81% of the variation (Figure 1). It was shown that a dose higher than 0.15% should be used to expect any positive effect of LauL on lesion severity. A similar pattern was observed for mortality (data not shown), being highest in the INT control (18%) but reduced in 0.6% LauL to 5% (P ≤ 0.05) and 11% in 0.3% LauL (P ≤ 0.10). Production parameters were only affected by dietary treatments from d 9 to 20. Body weight gain was highest in the NINT control (574 g) and NIT + 0.3 LauL (591 g), as expected, whereas all infected treatments had a 150 to 200 g lower BWG from d 9 to 20 (data not shown). None of the LauL doses led to a significantly better response on final weight at d 37 (Table 4).
Experiment 3 Like in experiment 2, the percentage of birds scored positive for C. perfringens was significantly influenced
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cidiostat (j = yes, no); Testk = effect of test product (k = with or without LauL); Interactionjk = coccidiostat × test effect; and eijk = residual error. Effects were considered significant at P ≤ 0.05, whereas 0.05 < P ≤ 0.10 was considered to be a near significant trend.
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Table 3. The percentage of birds observed with necrotic enteritis and the severity of lesions scored in all necropsied birds at d 15 and 16 (1 and 2 d postinfection, n = 48) and the results of the remaining birds on BW gain (BWG) and feed intake (FI) from d 9 to 20 (experiment 1) Positive birds (%) Treatment1
Dosage (%)
Lesion scoring NINT INT IT + FFA IT + MDG IT + LauL SEM P-value
— — 0.3 0.3 0.3
NINT INT IT + FFA IT + MDG IT + LauL SEM P-value
— — 0.3 0.3 0.3
0b 16ab 23a 33a 17ab NA2 ≤0.05 BWG d 9 to 20 (g)
d 16
d 15
0b 68a 60a 58a 41a NA ≤0.05 FI d 9 to 20 (g)
539a 318c 377b 368b 375b 10.6 <0.001
741a 510c 567b 558b 554b 10.9 <0.001
0.0 0.5 0.5 0.5 0.4 0.18 0.30 Mortality d 9 to 20 (%)
d 16
0.0b 14.6a 14.8a 11.8a 5.1ab NA ≤0.05
0.0c 2.1a 2.0a 1.7b 1.1b 0.25 <0.001
a–cValues
with no common superscripts in a column differ significantly (P ≤ 0.05). = not infected and not treated; INT = infected and not treated; IT + FFA = infected and treated with 0.3% free fatty acids; IT + MDG = infected and treated with 0.3% mono- and diglycerides; IT + LauL = infected and treated with 0.3% lauroyl lactylate. 2NA = not available in Fisher’s exact test as a nonparametric statistical method. 1NINT
by challenge (P < 0.001). No lesions were observed in NINT and NIT treatments, whereas 80% was infected in the INT control treatment (mean of d 15, 16, and 17). Supplementation of LauL (0.3 or 0.2%), ABP, or C or the combination of 0.3% LauL and C did not reduce the number of infected birds when compared with the INT control. Lesion severity of infected birds treated with ABP had more severe lesions (2.5) than the INT control and 0.3 or 0.2% LauL treatments (varied from 2.0 to 2.2). Supplementation of C with or without 0.3% LauL gave intermediate results (2.2 and 2.3, respective-
ly), being lower than the ABP group but slightly higher than the LauL treatments and the INT control (data not shown). The highest mortality rate of 32.4% was observed in the ABP treatment, which was higher than in the 0.2% LauL or 0.3% LauLC group (P ≤ 0.05). Supplementing 0.3% LauL or C resulted in a similar mortality rate as the INT control (Table 5). Results of production parameters of the infected treatments are presented in Table 5. No differences in growth performance were observed between treatments.
Table 4. Birds observed with necrotic enteritis (%) and the mean severity of lesions scored in all necropsied birds at d 15, 16, and 17 (1 to 3 d postinfection, n = 72) and the final BW of the remaining birds at d 37 (experiment 2) Treatment1 NINT NIT + 0.3 LauL INT IT + 0.005 LauL IT + 0.010 LauL IT + 0.019 LauL IT + 0.038 LauL IT + 0.075 LauL IT + 0.15 LauL IT + 0.3 LauL IT + 0.6 LauL SEM P-value a–fValues
Dosage (%) — 0.3 — 0.005 0.010 0.019 0.038 0.075 0.15 0.3 0.6
Positive birds (%) 0c 0c 56ab 69a 51ab 58ab 54ab 60ab 57ab 43ab 35b NA2 ≤0.05
Severity (scale 0 to 4)
BW d 37 (g)
0.0f 0.0f 1.6abcd 2.0a 1.5bcd 1.7abc 1.6abcd 1.8ab 1.6abcd 1.2de 0.9e 0.17 <0.001
2,249 2,302 2,246 2,151 2,229 2,305 2,147 2,169 2,259 2,275 2,305 16.6 0.193
with no common superscripts in a column differ significantly (P ≤ 0.05). = not infected and not treated; NIT = not infected and treated with 0.3% lauroyl lactylate; INT = infected and not treated; IT + x LauL = infected and treated with x% lauroyl lactylate. 2NA = not available in Fisher’s exact test as a nonparametric statistical method. 1NINT
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Performance
d 15
Severity (scale 0 to 4)
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DISCUSSION
Figure 1. Estimation of the dose response relationship between the dietary lauroyl lactylate (LauL) concentration and the lesion severity (experiment 2). The closed dots indicate the infected-treated and open dot indicates the infected-nontreated group. Clostridial lesions were scored on a scale of 0 to 4 (0 = no lesions and 4 = most severe lesions and death due to necrotic enteritidis). Dots sharing no common letter differ significantly from each other (P ≤ 0.05). Color version available in the online PDF.
birds. This lower FI does not fully explain the 150 to 200 g loss in BWG after infection (assuming a FCR of 1.4). The stronger effect of the infection on BW may be related to reduced nutrient digestion and absorption after C. perfringens colonization as well as to inflammation. Bacterial pathogens, such as C. perfringens, are known to cause mucosal damage of the intestinal gut,
Table 5. Production performance of infected birds not treated (INT) or treated with test products (experiment 3)1 Treatment BW d 0 0 to 9 d Growth (g) Feed intake (g) FCR2 9 to 21 d Growth (g) Feed intake (g) FCR 21 to 30 d Growth (g) Feed intake (g) FCR 30 to 36 d Growth (g) Feed intake (g) FCR 0 to 36 d Final weight (g) Feed intake (g) FCR Mortality (%) a,bValues
INT
44.1 172 229 1.332 455 660 1.469 761 1,211 1.595 638 1,063 1.667 2,075 3,262 1.610 19.4ab
IT + 0.3 LauL
43.9 190 235 1.239 475 672 1.426 715 1,188 1.675 598 992 1.744 2,025 3,189 1.614 21.3ab
IT + 0.2 LauL
44.2 187 231 1.295 474 677 1.429 740 1,190 1.610 636 1,015 1.596 2,075 3,213 1.583 17.6b
IT + ABP
44.0 184 237 1.290 456 661 1.453 792 1,266 1.599 666 1,116 1.677 2,144 3,383 1.612 32.4a
IT + C
43.6 181 234 1.293 485 685 1.412 765 1,207 1.584 635 1,059 1.665 2,114 3,286 1.587 27.8ab
IT + 0.3 LauLC
SEM
P-value
43.7
0.096 2.2 2.3 0.0077 8.3 8.0 0.0153 12.4 15.6 0.0150 14.7 16.3 0.0277 26.2 35.7 0.0089 NA
0.205 0.241 0.966 0.014 0.610 0.868 0.836 0.401 0.610 0.441 0.850 0.314 0.807 0.558 0.532 0.866 ≤0.05
188 236 1.254 497 686 1.386 760 1,215 1.599 633 1,049 1.696 2,159 3,294 1.600 15.7b
with no common superscripts in a column differ significantly (P ≤ 0.05). = infected and not treated; IT + x LauL = infected and treated with x% lauroyl lactylate; IT + ABP = infected and treated with Aromabiotic Poul 60 (Vitamex, Drongen, Belgium); IT + C = infected and treated with Clinacox (Janssen Animal Health, Beerse, Belgium); IT + x LauLC = infected and treated with x% lauroyl lactylate and Clinacox. 2FCR = feed conversion ratio. 3NA = not available in Fisher’s exact test as a nonparametric statistical method. 1INT
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Colonization of C. perfringens in the intestinal tract of birds produces a strong inflammatory response, which leads to a severe damage of the intestinal epithelium (Van Immerseel et al., 2004). Consequently, this results in poor nutrient digestibility and growth performance. The negative effect of colonization of C. perfringens on health and growth performance was confirmed in all 3 broiler experiments of the current study. Experimental infection of 9-d-old broilers with E. maxima followed by an infection with C. perfringens at 14 d of age induced mild to severe clostridial lesions and pronounced reduction of performance from the day after infection until approximately 6 d after. Body weight gain in the healthy control (NINT) was in all experiments around 550 to 620 g from d 9 to 20 or 21, whereas BW gain was reduced by 150 to 200 g in the INT groups. A necrotic enteritis infection reduces growth performance that is usually caused by a decrease in feed intake during infection. A reduction in feed intake was also observed in these experiments. The effect of a clostridial infection on daily feed intake in experiment 2 is presented in Figure 2, which is a typical response for all 3 experiments. Although a mild coccidiosis infection was introduced at d 9, no difference in feed intake was observed until after the inoculation of C. perfringens at d 14. From d 14 onward, daily feed intake decreases until the peak of infection at d 16. After d 16, daily feed intake recovers but stays consistently lower than the healthy control group until d 19 (Figure 2). Between d 9 and 20, a 150 g lower FI was measured for the infected birds in comparison to the noninfected
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Figure 2. The effect of the subsequent infection of Eimeria maxima at d 9 and Clostridium perfringens at d 14 on daily feed intake between 9 and 20 d of age (experiment 2). NINT = noninfected-nontreated; INT = infected-nontreated. An asterisk (*) indicates significant effects of infection on a specific day (P ≤ 0.05). NS = no significant effects of infection. Color version available in the online PDF.
A chemical coccidiostat was preferred over an ionophore because the latter has strong antibiotic properties. Adding an ionophore to the diet could have killed off the inoculated C. perfringens even before causing intestinal damage, creating too big of a risk of failure of the infection model. In this third experiment, neither an effect of LauL nor a coccidiostat was observed. The absence of an effect of LauL and the coccidiostat is hard to explain but could have been caused by a stronger infection. The infection was worse in this study, resulting in 83% of birds showing an average lesion score of 2.2, whereas experiments 1 and 2 resulted in 42 and 56% of birds with lesions of 1.3 and 1.6, respectively (Table 2). The infection in the third study might have been too strong to find a preventive effect of LauL or a coccidiostat against an infection of C. perfringens. Although no effect of lactylate was seen in the third study, a slight positive effect was seen in the first 2 experiments. The positive effect of lactylate can be attributed to their chemical composition. Acyl lactylates are produced by the reaction between the acyl group of fatty acids from 6 to 22 carbon atoms and lactic acid in the presence of an alkali metal. This reaction produces the salts of the corresponding fatty acid ester of lactylic acid using the appropriate fatty acid and lactic acid. By controlling the fatty acid used and the degree of condensation of the lactylic acid component, different effects can be obtained. One of the characteristics of LauL is its strong bacteriostatic property. Sodium lauroyl lactylate has been shown to have a broad range of antimicrobial activity against bacteria, molds, and yeast (Marnett et al., 1966; Osipow et al., 1969). In these studies, bacteriostatic effects were shown against Staphylococcus aureus, Aspergillus terreus, and Aspergillus niger and several Bacillus and Saccharomyces species. In a Clostridium infection study performed in the Netherlands by the Product Board for Livestock Production, it was concluded that a mixture of C10 and C12 fatty acids had an anticlostridial effect, showing a reduction in the number of broilers infected and a milder lesion score (Jansman et al., 2006). Sodium lauroyl lactylate does contain C12 fatty acids as part of its structure and, based on these data, the mechanism of action for LauL could be attributed to the anticlostridial effect of C12 fatty acids alone. However, to attribute the anticlostridial effect of LauL only on the anticlostridial effect of C12 is too straightforward. Other chain lengths of fatty acids are just as bacteriostatic, but they are not as soluble in water as short- or medium-chain fatty acids, making it difficult or impossible to achieve the most active concentration in the cell. The intrinsic antimicrobial effect of the molecule may be different from its effective concentration in aqueous solution. The C12 fatty acids present in LauL are, therefore, a nice compromise between chain length and solubility but are by itself probably not the strongest antimicrobial. A lactylate, however, in combination with fatty acids with different chain lengths, has strong
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which impedes nutrient absorption and digestion (Van Immerseel et al., 2004). Besides that, the inflammatory response and activation of the immune system result in high energy costs and reduced efficiency of protein utilization that may lead to a reduced supply of nutrients for maintenance and growth. To be able to determine whether the lactylate used in the current study has potential to be effective against C. perfringens, the results on incidence and lesion severity were compared with the INT controls. In the first experiment, LauL containing C12–C14 fatty acids esterified to lactic acid was tested against C10–C12 MDG or C10–C12 FFA. Based on lesion severity and mortality rate, it seemed that this lactylate had the greatest potential to reduce negative effects of a C. perfringens infection. This led to a dose response study with LauL, which showed a clear significant reduction in lesion severity with higher concentrations of LauL in the diets, but did not reduce the number of birds scored positive for C. perfringens. From a sigmoid curve, it was concluded that only LauL doses higher than 0.15% were to be expected to improve lesion severity. In the third study, it was therefore decided to repeat a 0.3% dose. A 0.2% dose was also tested because the latter might have an effect but was not tested as such in experiment 2. Both doses of LauL could be used as dietary supplementation in practice and to determine their potential in the feed industry, efficacy of LauL was compared with some commercial products that are used in practice to control bacterial overgrowth directly or indirectly. Aromabiotic Poul 60 contains MCFA that have antibacterial properties and should have a direct effect on bacteria such as C. perfringens. Clinacox was used to see if LauL would have any advantages in comparison to or in combination with a chemical coccidiostat.
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REFERENCES Bayliss, M. 1936. Effect of the chemical constitution of soaps upon their germicidal properties. J. Bacteriol. 31:489–504. Boutte, T., and L. Skogerson. 2004. Stearoyl-2-lactylates and oleoyl lactylates. Pages 206–225 in Emulsifiers in Food Technology. R. J. Whitehurst, ed. Blackwell Publishing Ltd., Oxford, UK. Byrd, J. A., B. M. Hargis, D. J. Caldwell, R. H. Bailey, K. L. Herron, J. L. McReynolds, R. L. Brewer, R. C. Anderson, K. M. Bischoff, T. R. Callaway, and L. F. Kubena. 2001. Effect of lactic acid administration in the drinking water during preslaughter
feed withdrawal on Salmonella and Campylobacter contamination of broilers. Poult. Sci. 80:278–283. Collier, C. T., J. D. van der Klis, B. Deplancke, D. B. Anderson, and H. R. Gaskins. 2003. Effects of tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intestinal barrier function in a chick model of necrotic enteritis. Antimicrob. Agents Chemother. 47:3311–3317. CVB. 2008. Tables values. Animal Nutrition 2008: Feeding values of different feedstuffs and nutrient requirements for production animals. Central Bureau for Livestock Production, Lelystad, the Netherlands. Elwinger, K., C. Schneitz, E. Berndtson, O. Fossum, B. Teglöf, and B. Engström. 1992. Factors affecting the incidence of necrotic enteritis, caecal carriage of Clostridium perfringens and bird performance in broiler chicks. Acta Vet. Scand. 33:369–378. Hofacre, C. L., T. Beacorn, S. Collett, and G. Mathis. 2003. Using competitive exclusion, mannan-oligosaccharide and other intestinal products to control necrotic enteritis. J. Appl. Poult. Res. 12:60–64. Hofshagen, M., and M. Kaldhusdal. 1992. Barley inclusion and avoparcin supplementation in broiler diets. 1. Effect on small intestinal bacterial flora and performance. Poult. Sci. 71:959– 969. Jansman, A. J. M., C. M. F. Wagenaars, A. Schonewille, and H. Snel. 2006. Control of Clostridium and Campylobacter infections in poultry via natural antimicrobial feed components. (In Dutch). Pages 19–26 in Nutrition and Intestinal Health. Series of Research Reports 6. Product Board of Animal Feed, Wageningen, the Netherlands. Kabara, J. J., and D. L. Marshall. 2005. Medium-chain fatty acids and esters. Pages 327–360 in Antimicrobials in Food. CRC Press, Boca Raton, FL. Kaldhusdal, M., C. Schneitz, M. Hofshagen, and E. Skjerve. 2001. Reduced incidence of Clostridium perfringens-associated lesions and improved performance in broiler chickens treated with normal intestinal flora of adult fowl. Avian Dis. 45:149–156. Løvland, A., and M. Kaldhusdal. 2001. Severely impaired production performance in broiler flocks with high incidence of Clostridium perfringens-associated hepatitis. Avian Pathol. 30:73–81. Marnett, L. F., R. J. Tenney, and J. B. Thompson. 1966. US Patent 3,275,503. Method for the protection of loci susceptible to the growth of undesired micro-organisms. C. J. Patterson Co., assignee. Osipow, L. I., D. Marra, and N. Resnanski. 1969. Fatty acid lactylates. Pages 1–12 in Drug Chem. Ind., March, April, May 1969. Phillips, J. C., C. Topp, and S. D. Gangolli. 1981. Studies on the metabolism of calcium stearoyl-2-lactylate in the rat, mouse, guinea pig and man. Food Cosmet. Toxicol. 19:7–11. Van Immerseel, F., J. De Buck, F. Pasmans, G. Huyghebaert, F. Haesebrouck, and R. Ducatelle. 2004. Clostridium perfringens in poultry: An emerging threat for animal and public health. Avian Pathol. 33:537–549.
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antimicrobial properties. In the data given in patent EP2082739A1, the minimum inhibitory concentration values of C12 and C14 lactylates are the lowest. The minimum inhibitory concentration values for these lactylates tested for C. perfringens are, respectively, 0.002 and 0.001% in comparison to 0.04% for C10 lactylates. The mechanism of action is most probably due to the slow metabolization of the lactylate. Lactylates will break down on ingestion, releasing FFA and lactic acid, which will be metabolized as normal. There is some evidence that the metabolic degradation or intestinal absorption of lactylates may be delayed in comparison to lactic acid or FFA (Phillips et al., 1981). This delay may be sufficient to allow the lactylate to have a stronger effect on bacterial infection in the intestinal tract than either FFA or lactic acid alone. But even when it does hydrolyze, both of the molecules released (lactic acid and MCFA) still have some antibacterial activity. The combination between the strong antimicrobial lactylate, its bacteriostatic products in the form of MCFA and lactic acid, and its slow metabolization is the most probable explanation for the observed effects on clostridial lesion scores and mortality in broilers seen in experiments 1 and 2. Still, more attention should be paid to the molecule and its physiological effects because none of its expected properties did improve results in experiment 3. It can be concluded from this study that higher concentrations of LauL in broiler diets have a potential to reduce the severity of necrotic enteritis in broilers in an E. maxima and C. perfringens challenge model.
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Key words: Clostridium perfringens, lactylate, intestinal health, broiler 2010 Poultry Science 89:2401–2409 doi:10.3382/ps.2010-00942
INTRODUCTION Clostridium perfringens, an α-toxin producing grampositive bacterium, is an enteric pathogen for poultry. Clostridium infections may appear as an acute clinical or subclinical disease (Løvland and Kaldhusdal, 2001), with symptoms varying from loss in performance to mortality. In case of a subclinical infection, the duodenal-jejunal mucosa is damaged, which decreases nutrient digestion and absorption, reduces weight gain, and increases feed conversion ratio (FCR; Elwinger et al., 1992; Hofshagen and Kaldhusdal, 1992; Kaldhusdal et al., 2001; Hofacre et al., 2003). Factors that stimulate intestinal mucus production (e.g., gastrointestinal infections like coccidiosis) are considered predisposing fac©2010 Poultry Science Association Inc. Received June 13, 2010. Accepted August 15, 2010. 1 Corresponding author: [email protected]
tors for Clostridium infections because C. perfringens is a mucolytic bacterium (Collier et al., 2003). Coccidiosis infections can be controlled by coccidiostats, which indirectly prevent Clostridium infections. Some coccidiostats, like ionophores that belong to a class of antibiotics, can also be used to control Clostridium infections directly, because they control Clostridium growth in the gastrointestinal tract. However, a real alternative to antibiotics or coccidiostats that effectively fights off Clostridium in a direct manner has not yet been found. In search for such a product, fatty acids and fatty acid derivatives provide obvious candidates because they have long been known and used as antimicrobials (Kabara and Marshall, 2005). Fatty acid derivatives have detergent or surfactant properties, and it is these properties that most probably endow them with antimicrobial activity by way of interacting with the cell membranes of the microorganisms. Its efficacy, as an antimicrobial, is determined by the type of derivative and the fatty acid chain length and degree of
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ABSTRACT Clostridium perfringens, an α-toxin producing gram-positive bacterium, is an enteric pathogen for poultry. Because subclinical C. perfringens infections often result in damage of the intestinal mucosa, decreased nutrient digestion, and poor performance, efforts should be taken to find an effective strategy that controls overgrowth of C. perfringens. For this purpose, the efficacy of a sodium lauroyl lactylate (LauL) as a feed additive to prevent C. perfringens colonization in broilers was determined. First, the effect of LauL was compared with capric and lauric mono- and diglycerides (MDG) and capric and lauric free fatty acids in Clostridium-infected chickens. Clostridial lesion scoring at d 16 showed that MDG and LauL were both effective in reducing the severity of lesions. When taking into account results on BW gain and mortality, LauL was more effective than MDG. For this reason, a dose response study was made to determine the optimal dietary dosage of LauL. In this experiment, it was shown
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MATERIALS AND METHODS Experimental Design Experiment 1. The objective of this experiment was to test the efficacy of a C12–C14 lactylate (LauL) against a C10–C12 MDG (50:50 mix of C10–C12 fatty
acids bound to MDG) and C10–C12 FFA (50:50 mix of C10–C12 FFA) on Clostridium-infected chickens. A total of 600 one-day-old male Ross 308 broiler chickens were randomly assigned to 5 treatments with 6 replicated cages each. The experiment was carried out from 0 to 20 d of age. From day of arrival to d 9, chicks were fed a starter diet without any supplements and from d 9 onward, a grower diet was given. After standardization to 17 birds per cage at d 9, chicks were challenged with Eimeria maxima and C. perfringens. Control treatments were infected-nontreated (INT) or noninfected-nontreated (NINT). Three other infected treatments (IT) received the grower diet supplemented with 0.3% FFA (IT + FFA), 0.3% of MDG (IT + MDG), or 0.3% LauL (IT + LauL). Products were produced and supplied by Purac Biochem (Gorinchem, the Netherlands). Dosages were chosen based on results from in vitro studies in combination with the expected kinetics of the products in the intestinal tract. All treatments were randomly allocated to litter cages except NINT cages that were grouped together to prevent cross-contamination. Postmortem clostridial lesion scoring occurred at d 15 and 16 and procedures are described later. Remaining birds were used to collect data on growth performance. Birds were weighed per cage at d 0, 9, and 20 to determine BW gain (BWG) from d 0 to 9 and d 9 to 20. Feed intake (FI) per cage was recorded during the same periods but daily in the infection period from d 9 to 20. Based on FI and BWG, FCR was calculated on a cage basis. Mortality with its most probable cause was recorded from d 0 to the end of the experiment. Experiment 2. The objective of experiment 2 was to find the optimal dosage of LauL in a dose response study, being the most promising molecule from experiment 1, to fight C. perfringens. This experiment was similar to experiment 1 with regard to breed and sex, infection protocol, and response parameters. A total of 1,320 one-day-old Ross 308 male broiler chicks were randomly assigned to 11 treatments with 6 replicate cages each. Treatments were designed as noninfected and treated (NIT) with 0.3% LauL (NIT + 0.3 LauL) or without (NINT). The infected treatments were set up as a dose response starting with a 2 times higher dose as was used in experiment 1 (IT + 0.6 LauL) and stepwise lowering the supplementation with 50%, from 0.6% to 0.3% to 0.15%, toward 0% supplementation of LauL (INT). All diets with or without LauL supplementation were supplied from d 0 to 37 to see if a prolonged use of LauL before inoculation was more effective. Postmortem lesion scoring was performed on d 15, 16, and 17. Experiment 3. Experiment 3 was similar to experiment 2 with regard to breed, sex, the infection protocol, response parameters, and start and length of the dietary treatments. A total of 960 one-day-old broiler chickens were randomly allocated to 8 treatments with 6 replicate cages each. Treatments were designed with 2 controls, INT and NINT without supplementation of LauL or with 0.3% LauL (IT + 0.3 LauL or NIT
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saturation. Bayliss (1936) investigated the relation between the chemical composition of a soap and its germicidal properties. For the saturated soaps, he found the maximum antimicrobial activity with sodium myristate (C14:0) fatty acids. Among the esters of fatty acids, the naturally occurring glycerol esters are the most well known and widely used. In particular, monoesters with medium-chain fatty acids (MCFA) have been used as antimicrobials (Kabara and Marshall, 2005). Other less well-known esters of fatty acids are the lactylates; these are esters of lactic acid in which the C2-hydroxy group of lactic acid is esterified to a fatty acid. The main commercial use of lactylates is in the bakery industry, with C18 fatty acids (Boutte and Skogerson, 2004). Lactylates made with MCFA have been used in cosmetics (patent application EP0595528). Both components of lactylates, lactic acid and fatty acids, by themselves are known and used as antimicrobials in animal production. Lactic acid can be used to reduce Campylobacter and Salmonella contamination of broiler carcasses at processing (Byrd et al., 2001) and free fatty acids (FFA) such as MCFA have been shown to reduce Clostridium infections in broiler chickens (Jansman et al., 2006). In vitro, lactylates with different types of fatty acids have shown already to be potent inhibitors of C. perfringens (patent application EP02082739A1), with the lowest minimum inhibitory concentration of 0.001% (wt/wt) for a lactylate with C12 and C14 fatty acids. That means that in vitro they are 100- to 1,000-fold more effective inhibitors than FFA or lactic acid alone. The series of trials described here were made to test a sodium lauroyl lactylate (LauL; Puramix 30, Purac Biochem, Gorinchem, the Netherlands) containing C12 and C14 fatty acids under in vivo conditions. The first trial was used to compare the effects of a LauL to C10–C12 monoglycerides (mono- and diglycerides; MDG) and free C10–C12 MCFA (FFA) in Clostridium-infected chickens. Because LauL was the most promising molecule, a dose response study was made to determine the optimal dietary dosage. In a third trial, the efficacy of LauL was compared with commercial products that limit bacterial activity in the intestinal tract (Aromabiotic Poul 60; Vitamex, Drongen, Belgium; ABP) or coccidiosis (chemical coccidiostat; C) and consequently its effect on intestinal mucus production. This series of experiments was made to determine whether MCFA lactylates can be of help in reducing problems caused by C. perfringens in broiler chickens.
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+ 0.3 LauL), to test if LauL would benefit in healthy and infected chickens. The efficacy of 0.3% LauL in infected chickens was tested against 4 IT treatments with 0.2% LauL dosage (IT + 0.2 LauL), the combination of LauL with a chemical coccidiostat (Clinacox: 100 g/ ton, Janssen Animal Health, Beerse, Belgium; IT + 0.3 LauLC), a chemical coccidiostat alone (IT + C), and a commercial MCFA product (Aromabiotic Poultry: 1.6 kg/ton in starter and 1.2 kg/ton in grower phase, Vitamex, Drongen, Belgium; IT + ABP).
Birds and Housing
Experimental Diets Broilers were supplied a wheat-soybean meal-based starter diet for ad libitum intake from day of arrival until d 9. At d 9, a wheat-barley-based basal grower diet was fed until the end of the experiment (Table 1). Grower diets were fed as mash because of the necessity of homogeneously mixing in the test products after feed production. Diets did not contain any nonstarch polysaccharide enzymes, coccidiostats, or antimicrobial feed additives other than the test products. The nutrient composition of the experimental diets was according to Dutch standards to meet nutrient requirements of broilers (CVB, 2008).
Challenge To induce the onset of a C. perfringens infection, a mild E. maxima infection was used as pretrigger. Birds were orally inoculated at d 9 with 1 mL of E. maxi-
ma (Central Veterinary Laboratory, Weybridge, UK; ~10,000 sporulated oocysts per mL; 1 mL per bird) or with sterile saline after a 5-h feed withdrawal period. This inoculation with E. maxima has consistently shown to induce C. perfringens with an average of 50% of chickens scored positive for clostridial lesions. Moreover, reddish E. maxima lesions are distinctly different from white C. perfringens lesions. After the 5-d incubation time for coccidiosis to develop, birds were orally inoculated on d 14 with C. perfringens type A [~108 cfu/mL; 1 mL of liver broth (Difco, Detroit, MI) per bird] or with 1 mL of sterile liver broth after a 5-h feed withdrawal period to induce necrotic enteritis. After inoculations, birds had immediate access to feed. The pathogenic C. perfringens strain (code GD 5.11.53) was obtained from the Animal Health Service (Deventer, the Netherlands). The strain was grown on an agar of sheep blood and the culture was typed by the Central Institute of Animal Disease Control (Lelystad, the Netherlands) as C. perfringens producing type α and β2 toxins.
Pathological Parameters During the peak of Clostridium infection at d 15 and 16 (1 and 2 d postinfection), 4 random selected birds per cage were used for postmortem coccidial and clostridial lesion scoring in the duodenal and jejunal segment of the small intestine (48 birds per treatment). In experiments 2 and 3, a third scoring was done at d 17 (3 d postinfection). Birds were killed by an intracardial injection with a euthasate (T61; Intervet, Mechelen, Belgium). The abdomen was opened and the gastrointestinal tract was exposed. The gastrointestinal tract was segmented into the duodenum and jejunum (the segment from the gizzard to the 10 cm preceding Meckel’s diverticulum). All birds were scored blind (i.e., the person scoring for lesions had no knowledge of the bird’s treatment). The following method was used to score the C. perfringens lesions: 0 = no lesions; 1 = 1 to 5 small white lesions (spots of less than 1 mm in diameter); 2 = more than 5 small white lesions (spots of less than 1 mm in diameter) or 1 to 5 larger lesions (spots of 1 to 2 mm in diameter); 3 = more than 5 larger lesions (1 to 2 mm in diameter) or erosive zones; 4 = dead birds with positive necrotic enteritis diagnoses postmortem. Based on the clostridial lesion scores, the percentage of Clostridium-infected birds was calculated by the following equation: (sum of birds scored >0/total number of birds scored) × 100%. The percentage of birds affected indicated the incidence of C. perfringens. By categorizing the severity of lesions from 0 to 4, an average lesion score could be calculated for each treatment, giving an indication of the severity of the C. perfringens infection besides its incidence.
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One-day-old male Ross 308 broiler chickens were supplied by Probroed & Sloot B.V. (Meppel, the Netherlands). At d 0, broilers arrived at the poultry facilities of Schothorst Feed Research B.V. (experiments 1 and 3; Lelystad, the Netherlands) or the Animal Health Service (experiment 2; Deventer, the Netherlands) and were housed in 2-tier litter floor digestibility cages after individual weighing (surface area 0.90 × 0.65 m). Twenty birds were allocated to cages based on a weight class system such that mean weight per cage was similar at the start of the experiment. Broilers were housed in these cages throughout the entire experiment. At d 9, before first inoculation, the number of chickens was standardized to 17, and bird weight was measured before and after standardization. Lighting and temperature schedule throughout the experimental period was 22L:2D in the time period from d 0 to 9 and 18L:6D from d 9 to the end of the experiment. The ambient temperature gradually decreased from 32°C at the start to 25°C at the end of the experiment. Feed was supplied for ad libitum intake from d 0 onward with the exception of the 5-h feed withdrawal period before inoculations (on d 9 and 14). Water was available for ad libitum intake throughout the experiment. Broilers were not vaccinated during the experiment.
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Table 1. Composition of the basal diets in experiments 1, 2, and 3 Starter (d 0 to 9) Item
Experiment 1
Experiments 2 and 3
Experiment 1
Experiments 2 and 3
19.35 25.73 — 47.76 — — — 1.19 1.77 1.34 0.61 0.11 1.50 0.28 0.22 0.08 0.06 109 55 199 51 26 384 2,759 8.9 5.1 1.4 2.7 8.5 10.4 7.6 6.5
18.54 25.73 — 48.57 — — — 1.19 1.77 1.83 0.62 0.28 1.50 0.28 0.22 0.08 0.06 122 55 199 51 26 392 2,798 9.0 5.1 1.4 2.7 8.5 10.4 7.6 6.6
— 19.50 5.26 35.88 1.09 24.24 3.83 4.81 1.86 1.12 0.78 0.12 0.50 0.31 0.27 0.09 0.34 117 49 187 80 36 360 2,864 6.7 5.7 1.6 1.9 7.8 9.6 7.7 6.9
— 19.53 5.26 35.77 1.09 24.24 3.82 4.59 1.86 1.13 0.78 0.13 0.70 0.33 0.28 0.15 0.34 117 50 185 83 40 361 2,865 6.7 5.7 1.6 2.0 8.0 9.6 7.7 6.9
1Hipro
(Brazilian soybeans; Cargill, Amsterdam, the Netherlands). mineral-vitamin premix contained the following per kilogram: Ca, 185 g; Na, 80 g; Cl, 100 g; Cu, 1,200 mg; Fe, 4,500 mg; Mn, 7,000 mg; Zn, 3,700 mg; I, 100 mg; Se, 15 mg; vitamin A, 1,000,000 IE; vitamin D3, 200,000 IE; vitamin E, 2,500 IE; vitamin B1, 50 mg; vitamin B2, 500 mg; pantothenic acid, 800 mg; niacin, 4,000 mg; vitamin B6, 300 mg; folic acid, 100 mg; vitamin B12, 1,500 µg; biotin, 10,000 µg; vitamin K3, 125 mg; choline, 20,000 mg. 2The
Three in vivo experiments were performed in succession. The experimental protocols and all procedures complied with the guidelines of the Ethical Committee for Animal Experiments (Lelystad, the Netherlands).
Statistical Analysis The incidence of C. perfringens (% of birds scored >0) and mortality rate were analyzed by Fisher’s exact test, whereas the severity of lesions, measurements on production performance, and other parameters were analyzed by ANOVA using GenStat statistical software (GenStat version 12, Hemel Hempstead, UK). Raw data of BW and feed intake were analyzed for outliers. Outliers were marked significant when exceeding 2.5 × SD of the treatment mean and were excluded from the data set. Treatment means were compared by the least significant difference. The statistical model(s) used per experiment were as follows, based on the entire data sets or subsets for each experiment. The model used for experiments 1, 2, and 3 was as follows:
Yij = μ + Blocki + Treatmentj + eij, with Yij = response parameter; μ = mean; Blocki = effect of block (i = 1 to 6); Treatmentj = effect of treatment (j = 5, 11, and 8 for experiments 1, 2, and 3, respectively); and eij = residual error. The model used for experiments 2 and 3 was as follows: Yij = μ + Blocki + Dosej + eij, with Yij = response parameter; μ = mean; Blocki = effect of block (i = 1 to 6); Dosej = effect of dose (j = 9 and 3 for experiments 2 and 3, respectively); and eij = residual error. The model used for experiment 3 was as follows: Yijk = μ + Blocki + Coccidiostatj + Testk + Interactionjk + eijk, with Yijk = response parameter; μ = mean; Blocki = effect of block (i = 1 to 6); Coccidiostatj = effect of coc-
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Ingredient (%) Maize Soybean meal, Hipro1 Sunflower seed meal Wheat Wheat middlings Barley Maize starch Soybean oil Animal fat Limestone Monocal Salt Premix vitamins and minerals2 Premix Lys (HCl 79%) Premix Met (dl 99%) Premix Thr (l 98%) Natrium carbonate Calculated nutrient (g/kg) Moisture Ash CP Crude fat Crude fiber Starch AME broiler (kcal) Calcium Phosphorus Sodium Chloride Potassium Apparent digestible Lys Apparent digestible Met + Cys Apparent digestible threonine
Grower (d 9 to end of experiment)
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Table 2. The percentage of birds scored positive for Clostridium perfringens lesions and the lesion severity in the infected-nontreated (INT) group and noninfected-nontreated group (NINT) presented as mean values of lesion scoring at d 15 and 16 (1 and 2 d postinfection, n = 48) Item Experiment 1 NINT INT Experiment 2 NINT INT Experiment 3 NINT INT
Positive birds (%)
Lesion severity (scale 0 to 4)
P-value
0 42
0.0 1.3
<0.001
0 62
0.0 1.8
<0.001
0 83
0.0 2.2
<0.001
RESULTS General The INT groups in all experiments were scored positive for C. perfringens. As an average of lesion scoring performed at d 15 and 16, the INT groups resulted in 42, 62, and 83% of birds scored positive for clostridial lesions in experiments 1, 2, and 3, respectively. In NINT groups, all birds were scored negative. Also, clostridial lesions were more severe (P < 0.001; Table 2). Calculating the scores from 0 to 4 in all necropsied birds, a lesion severity of 1.3, 1.8, and 2.2 for the 3 successive experiments was determined.
Experiment 1 On d 15 and 16, the number of birds scored positive for C. perfringens was not affected by dietary treatments FFA, MDG, and LauL compared with the INT control (Table 3). The severity of lesions was not significantly affected by any of the treatments on d 15, whereas on peak of infection at d 16, dietary supplementation of MDG and LauL resulted in lesions that were less severe compared with the INT and IT + FFA groups (P < 0.001; Table 3). Three days later (6 d postinfection), no significant differences were observed between treatments (data not shown) as all treatments recovered from necrotic enteritis, based on macroscopical evaluation. A similar pattern was observed for mortality, which was higher in the INT control, FFA, and MDG treatment compared with the NINT treatment (P ≤ 0.05). The LauL treatment was intermediate, not being significantly different from any treatment (Table 3). Production parameters from d 9 to 20 were affected by dietary treatments. Body weight gain was highest in
the NINT control (Table 3), as expected, whereas the INT treatment had the lowest BWG. All dietary treatments, FFA, MDG, and LauL resulted in a higher FI and BWG compared with INT (P < 0.001).
Experiment 2 The percentage of birds scored positive for C. perfringens was significantly influenced by challenge and dietary treatments. No lesions were observed in NINT and NIT treatments, whereas 56% was infected in the INT control treatment (mean of d 15, 16, and 17; Table 4). Supplementation of 0.6% LauL (IT + 0.6 LauL) resulted in 35% of birds scored positive. The other LauLsupplemented treatments resulted in similar percentages of broiler scored positive for necrotic enteritis as INT. With regard to severity of lesions, birds supplemented with 0.6% LauL had less severe lesions than birds in the INT control (P < 0.001). A dose of 0.3% LauL did reduce lesion severity as well toward a near-significant reduction in comparison to the INT control group (P ≤ 0.10). All other LauL-supplemented treatments did not improve results on lesion severity (Table 4). A sigmoid dose response curve was fitted to the data, explaining 81% of the variation (Figure 1). It was shown that a dose higher than 0.15% should be used to expect any positive effect of LauL on lesion severity. A similar pattern was observed for mortality (data not shown), being highest in the INT control (18%) but reduced in 0.6% LauL to 5% (P ≤ 0.05) and 11% in 0.3% LauL (P ≤ 0.10). Production parameters were only affected by dietary treatments from d 9 to 20. Body weight gain was highest in the NINT control (574 g) and NIT + 0.3 LauL (591 g), as expected, whereas all infected treatments had a 150 to 200 g lower BWG from d 9 to 20 (data not shown). None of the LauL doses led to a significantly better response on final weight at d 37 (Table 4).
Experiment 3 Like in experiment 2, the percentage of birds scored positive for C. perfringens was significantly influenced
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cidiostat (j = yes, no); Testk = effect of test product (k = with or without LauL); Interactionjk = coccidiostat × test effect; and eijk = residual error. Effects were considered significant at P ≤ 0.05, whereas 0.05 < P ≤ 0.10 was considered to be a near significant trend.
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Table 3. The percentage of birds observed with necrotic enteritis and the severity of lesions scored in all necropsied birds at d 15 and 16 (1 and 2 d postinfection, n = 48) and the results of the remaining birds on BW gain (BWG) and feed intake (FI) from d 9 to 20 (experiment 1) Positive birds (%) Treatment1
Dosage (%)
Lesion scoring NINT INT IT + FFA IT + MDG IT + LauL SEM P-value
— — 0.3 0.3 0.3
NINT INT IT + FFA IT + MDG IT + LauL SEM P-value
— — 0.3 0.3 0.3
0b 16ab 23a 33a 17ab NA2 ≤0.05 BWG d 9 to 20 (g)
d 16
d 15
0b 68a 60a 58a 41a NA ≤0.05 FI d 9 to 20 (g)
539a 318c 377b 368b 375b 10.6 <0.001
741a 510c 567b 558b 554b 10.9 <0.001
0.0 0.5 0.5 0.5 0.4 0.18 0.30 Mortality d 9 to 20 (%)
d 16
0.0b 14.6a 14.8a 11.8a 5.1ab NA ≤0.05
0.0c 2.1a 2.0a 1.7b 1.1b 0.25 <0.001
a–cValues
with no common superscripts in a column differ significantly (P ≤ 0.05). = not infected and not treated; INT = infected and not treated; IT + FFA = infected and treated with 0.3% free fatty acids; IT + MDG = infected and treated with 0.3% mono- and diglycerides; IT + LauL = infected and treated with 0.3% lauroyl lactylate. 2NA = not available in Fisher’s exact test as a nonparametric statistical method. 1NINT
by challenge (P < 0.001). No lesions were observed in NINT and NIT treatments, whereas 80% was infected in the INT control treatment (mean of d 15, 16, and 17). Supplementation of LauL (0.3 or 0.2%), ABP, or C or the combination of 0.3% LauL and C did not reduce the number of infected birds when compared with the INT control. Lesion severity of infected birds treated with ABP had more severe lesions (2.5) than the INT control and 0.3 or 0.2% LauL treatments (varied from 2.0 to 2.2). Supplementation of C with or without 0.3% LauL gave intermediate results (2.2 and 2.3, respective-
ly), being lower than the ABP group but slightly higher than the LauL treatments and the INT control (data not shown). The highest mortality rate of 32.4% was observed in the ABP treatment, which was higher than in the 0.2% LauL or 0.3% LauLC group (P ≤ 0.05). Supplementing 0.3% LauL or C resulted in a similar mortality rate as the INT control (Table 5). Results of production parameters of the infected treatments are presented in Table 5. No differences in growth performance were observed between treatments.
Table 4. Birds observed with necrotic enteritis (%) and the mean severity of lesions scored in all necropsied birds at d 15, 16, and 17 (1 to 3 d postinfection, n = 72) and the final BW of the remaining birds at d 37 (experiment 2) Treatment1 NINT NIT + 0.3 LauL INT IT + 0.005 LauL IT + 0.010 LauL IT + 0.019 LauL IT + 0.038 LauL IT + 0.075 LauL IT + 0.15 LauL IT + 0.3 LauL IT + 0.6 LauL SEM P-value a–fValues
Dosage (%) — 0.3 — 0.005 0.010 0.019 0.038 0.075 0.15 0.3 0.6
Positive birds (%) 0c 0c 56ab 69a 51ab 58ab 54ab 60ab 57ab 43ab 35b NA2 ≤0.05
Severity (scale 0 to 4)
BW d 37 (g)
0.0f 0.0f 1.6abcd 2.0a 1.5bcd 1.7abc 1.6abcd 1.8ab 1.6abcd 1.2de 0.9e 0.17 <0.001
2,249 2,302 2,246 2,151 2,229 2,305 2,147 2,169 2,259 2,275 2,305 16.6 0.193
with no common superscripts in a column differ significantly (P ≤ 0.05). = not infected and not treated; NIT = not infected and treated with 0.3% lauroyl lactylate; INT = infected and not treated; IT + x LauL = infected and treated with x% lauroyl lactylate. 2NA = not available in Fisher’s exact test as a nonparametric statistical method. 1NINT
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Performance
d 15
Severity (scale 0 to 4)
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DISCUSSION
Figure 1. Estimation of the dose response relationship between the dietary lauroyl lactylate (LauL) concentration and the lesion severity (experiment 2). The closed dots indicate the infected-treated and open dot indicates the infected-nontreated group. Clostridial lesions were scored on a scale of 0 to 4 (0 = no lesions and 4 = most severe lesions and death due to necrotic enteritidis). Dots sharing no common letter differ significantly from each other (P ≤ 0.05). Color version available in the online PDF.
birds. This lower FI does not fully explain the 150 to 200 g loss in BWG after infection (assuming a FCR of 1.4). The stronger effect of the infection on BW may be related to reduced nutrient digestion and absorption after C. perfringens colonization as well as to inflammation. Bacterial pathogens, such as C. perfringens, are known to cause mucosal damage of the intestinal gut,
Table 5. Production performance of infected birds not treated (INT) or treated with test products (experiment 3)1 Treatment BW d 0 0 to 9 d Growth (g) Feed intake (g) FCR2 9 to 21 d Growth (g) Feed intake (g) FCR 21 to 30 d Growth (g) Feed intake (g) FCR 30 to 36 d Growth (g) Feed intake (g) FCR 0 to 36 d Final weight (g) Feed intake (g) FCR Mortality (%) a,bValues
INT
44.1 172 229 1.332 455 660 1.469 761 1,211 1.595 638 1,063 1.667 2,075 3,262 1.610 19.4ab
IT + 0.3 LauL
43.9 190 235 1.239 475 672 1.426 715 1,188 1.675 598 992 1.744 2,025 3,189 1.614 21.3ab
IT + 0.2 LauL
44.2 187 231 1.295 474 677 1.429 740 1,190 1.610 636 1,015 1.596 2,075 3,213 1.583 17.6b
IT + ABP
44.0 184 237 1.290 456 661 1.453 792 1,266 1.599 666 1,116 1.677 2,144 3,383 1.612 32.4a
IT + C
43.6 181 234 1.293 485 685 1.412 765 1,207 1.584 635 1,059 1.665 2,114 3,286 1.587 27.8ab
IT + 0.3 LauLC
SEM
P-value
43.7
0.096 2.2 2.3 0.0077 8.3 8.0 0.0153 12.4 15.6 0.0150 14.7 16.3 0.0277 26.2 35.7 0.0089 NA
0.205 0.241 0.966 0.014 0.610 0.868 0.836 0.401 0.610 0.441 0.850 0.314 0.807 0.558 0.532 0.866 ≤0.05
188 236 1.254 497 686 1.386 760 1,215 1.599 633 1,049 1.696 2,159 3,294 1.600 15.7b
with no common superscripts in a column differ significantly (P ≤ 0.05). = infected and not treated; IT + x LauL = infected and treated with x% lauroyl lactylate; IT + ABP = infected and treated with Aromabiotic Poul 60 (Vitamex, Drongen, Belgium); IT + C = infected and treated with Clinacox (Janssen Animal Health, Beerse, Belgium); IT + x LauLC = infected and treated with x% lauroyl lactylate and Clinacox. 2FCR = feed conversion ratio. 3NA = not available in Fisher’s exact test as a nonparametric statistical method. 1INT
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Colonization of C. perfringens in the intestinal tract of birds produces a strong inflammatory response, which leads to a severe damage of the intestinal epithelium (Van Immerseel et al., 2004). Consequently, this results in poor nutrient digestibility and growth performance. The negative effect of colonization of C. perfringens on health and growth performance was confirmed in all 3 broiler experiments of the current study. Experimental infection of 9-d-old broilers with E. maxima followed by an infection with C. perfringens at 14 d of age induced mild to severe clostridial lesions and pronounced reduction of performance from the day after infection until approximately 6 d after. Body weight gain in the healthy control (NINT) was in all experiments around 550 to 620 g from d 9 to 20 or 21, whereas BW gain was reduced by 150 to 200 g in the INT groups. A necrotic enteritis infection reduces growth performance that is usually caused by a decrease in feed intake during infection. A reduction in feed intake was also observed in these experiments. The effect of a clostridial infection on daily feed intake in experiment 2 is presented in Figure 2, which is a typical response for all 3 experiments. Although a mild coccidiosis infection was introduced at d 9, no difference in feed intake was observed until after the inoculation of C. perfringens at d 14. From d 14 onward, daily feed intake decreases until the peak of infection at d 16. After d 16, daily feed intake recovers but stays consistently lower than the healthy control group until d 19 (Figure 2). Between d 9 and 20, a 150 g lower FI was measured for the infected birds in comparison to the noninfected
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Figure 2. The effect of the subsequent infection of Eimeria maxima at d 9 and Clostridium perfringens at d 14 on daily feed intake between 9 and 20 d of age (experiment 2). NINT = noninfected-nontreated; INT = infected-nontreated. An asterisk (*) indicates significant effects of infection on a specific day (P ≤ 0.05). NS = no significant effects of infection. Color version available in the online PDF.
A chemical coccidiostat was preferred over an ionophore because the latter has strong antibiotic properties. Adding an ionophore to the diet could have killed off the inoculated C. perfringens even before causing intestinal damage, creating too big of a risk of failure of the infection model. In this third experiment, neither an effect of LauL nor a coccidiostat was observed. The absence of an effect of LauL and the coccidiostat is hard to explain but could have been caused by a stronger infection. The infection was worse in this study, resulting in 83% of birds showing an average lesion score of 2.2, whereas experiments 1 and 2 resulted in 42 and 56% of birds with lesions of 1.3 and 1.6, respectively (Table 2). The infection in the third study might have been too strong to find a preventive effect of LauL or a coccidiostat against an infection of C. perfringens. Although no effect of lactylate was seen in the third study, a slight positive effect was seen in the first 2 experiments. The positive effect of lactylate can be attributed to their chemical composition. Acyl lactylates are produced by the reaction between the acyl group of fatty acids from 6 to 22 carbon atoms and lactic acid in the presence of an alkali metal. This reaction produces the salts of the corresponding fatty acid ester of lactylic acid using the appropriate fatty acid and lactic acid. By controlling the fatty acid used and the degree of condensation of the lactylic acid component, different effects can be obtained. One of the characteristics of LauL is its strong bacteriostatic property. Sodium lauroyl lactylate has been shown to have a broad range of antimicrobial activity against bacteria, molds, and yeast (Marnett et al., 1966; Osipow et al., 1969). In these studies, bacteriostatic effects were shown against Staphylococcus aureus, Aspergillus terreus, and Aspergillus niger and several Bacillus and Saccharomyces species. In a Clostridium infection study performed in the Netherlands by the Product Board for Livestock Production, it was concluded that a mixture of C10 and C12 fatty acids had an anticlostridial effect, showing a reduction in the number of broilers infected and a milder lesion score (Jansman et al., 2006). Sodium lauroyl lactylate does contain C12 fatty acids as part of its structure and, based on these data, the mechanism of action for LauL could be attributed to the anticlostridial effect of C12 fatty acids alone. However, to attribute the anticlostridial effect of LauL only on the anticlostridial effect of C12 is too straightforward. Other chain lengths of fatty acids are just as bacteriostatic, but they are not as soluble in water as short- or medium-chain fatty acids, making it difficult or impossible to achieve the most active concentration in the cell. The intrinsic antimicrobial effect of the molecule may be different from its effective concentration in aqueous solution. The C12 fatty acids present in LauL are, therefore, a nice compromise between chain length and solubility but are by itself probably not the strongest antimicrobial. A lactylate, however, in combination with fatty acids with different chain lengths, has strong
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which impedes nutrient absorption and digestion (Van Immerseel et al., 2004). Besides that, the inflammatory response and activation of the immune system result in high energy costs and reduced efficiency of protein utilization that may lead to a reduced supply of nutrients for maintenance and growth. To be able to determine whether the lactylate used in the current study has potential to be effective against C. perfringens, the results on incidence and lesion severity were compared with the INT controls. In the first experiment, LauL containing C12–C14 fatty acids esterified to lactic acid was tested against C10–C12 MDG or C10–C12 FFA. Based on lesion severity and mortality rate, it seemed that this lactylate had the greatest potential to reduce negative effects of a C. perfringens infection. This led to a dose response study with LauL, which showed a clear significant reduction in lesion severity with higher concentrations of LauL in the diets, but did not reduce the number of birds scored positive for C. perfringens. From a sigmoid curve, it was concluded that only LauL doses higher than 0.15% were to be expected to improve lesion severity. In the third study, it was therefore decided to repeat a 0.3% dose. A 0.2% dose was also tested because the latter might have an effect but was not tested as such in experiment 2. Both doses of LauL could be used as dietary supplementation in practice and to determine their potential in the feed industry, efficacy of LauL was compared with some commercial products that are used in practice to control bacterial overgrowth directly or indirectly. Aromabiotic Poul 60 contains MCFA that have antibacterial properties and should have a direct effect on bacteria such as C. perfringens. Clinacox was used to see if LauL would have any advantages in comparison to or in combination with a chemical coccidiostat.
ANTI-CLOSTRIDIAL EFFECTS OF SODIUM LAUROYL LACTYLATE
REFERENCES Bayliss, M. 1936. Effect of the chemical constitution of soaps upon their germicidal properties. J. Bacteriol. 31:489–504. Boutte, T., and L. Skogerson. 2004. Stearoyl-2-lactylates and oleoyl lactylates. Pages 206–225 in Emulsifiers in Food Technology. R. J. Whitehurst, ed. Blackwell Publishing Ltd., Oxford, UK. Byrd, J. A., B. M. Hargis, D. J. Caldwell, R. H. Bailey, K. L. Herron, J. L. McReynolds, R. L. Brewer, R. C. Anderson, K. M. Bischoff, T. R. Callaway, and L. F. Kubena. 2001. Effect of lactic acid administration in the drinking water during preslaughter
feed withdrawal on Salmonella and Campylobacter contamination of broilers. Poult. Sci. 80:278–283. Collier, C. T., J. D. van der Klis, B. Deplancke, D. B. Anderson, and H. R. Gaskins. 2003. Effects of tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intestinal barrier function in a chick model of necrotic enteritis. Antimicrob. Agents Chemother. 47:3311–3317. CVB. 2008. Tables values. Animal Nutrition 2008: Feeding values of different feedstuffs and nutrient requirements for production animals. Central Bureau for Livestock Production, Lelystad, the Netherlands. Elwinger, K., C. Schneitz, E. Berndtson, O. Fossum, B. Teglöf, and B. Engström. 1992. Factors affecting the incidence of necrotic enteritis, caecal carriage of Clostridium perfringens and bird performance in broiler chicks. Acta Vet. Scand. 33:369–378. Hofacre, C. L., T. Beacorn, S. Collett, and G. Mathis. 2003. Using competitive exclusion, mannan-oligosaccharide and other intestinal products to control necrotic enteritis. J. Appl. Poult. Res. 12:60–64. Hofshagen, M., and M. Kaldhusdal. 1992. Barley inclusion and avoparcin supplementation in broiler diets. 1. Effect on small intestinal bacterial flora and performance. Poult. Sci. 71:959– 969. Jansman, A. J. M., C. M. F. Wagenaars, A. Schonewille, and H. Snel. 2006. Control of Clostridium and Campylobacter infections in poultry via natural antimicrobial feed components. (In Dutch). Pages 19–26 in Nutrition and Intestinal Health. Series of Research Reports 6. Product Board of Animal Feed, Wageningen, the Netherlands. Kabara, J. J., and D. L. Marshall. 2005. Medium-chain fatty acids and esters. Pages 327–360 in Antimicrobials in Food. CRC Press, Boca Raton, FL. Kaldhusdal, M., C. Schneitz, M. Hofshagen, and E. Skjerve. 2001. Reduced incidence of Clostridium perfringens-associated lesions and improved performance in broiler chickens treated with normal intestinal flora of adult fowl. Avian Dis. 45:149–156. Løvland, A., and M. Kaldhusdal. 2001. Severely impaired production performance in broiler flocks with high incidence of Clostridium perfringens-associated hepatitis. Avian Pathol. 30:73–81. Marnett, L. F., R. J. Tenney, and J. B. Thompson. 1966. US Patent 3,275,503. Method for the protection of loci susceptible to the growth of undesired micro-organisms. C. J. Patterson Co., assignee. Osipow, L. I., D. Marra, and N. Resnanski. 1969. Fatty acid lactylates. Pages 1–12 in Drug Chem. Ind., March, April, May 1969. Phillips, J. C., C. Topp, and S. D. Gangolli. 1981. Studies on the metabolism of calcium stearoyl-2-lactylate in the rat, mouse, guinea pig and man. Food Cosmet. Toxicol. 19:7–11. Van Immerseel, F., J. De Buck, F. Pasmans, G. Huyghebaert, F. Haesebrouck, and R. Ducatelle. 2004. Clostridium perfringens in poultry: An emerging threat for animal and public health. Avian Pathol. 33:537–549.
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antimicrobial properties. In the data given in patent EP2082739A1, the minimum inhibitory concentration values of C12 and C14 lactylates are the lowest. The minimum inhibitory concentration values for these lactylates tested for C. perfringens are, respectively, 0.002 and 0.001% in comparison to 0.04% for C10 lactylates. The mechanism of action is most probably due to the slow metabolization of the lactylate. Lactylates will break down on ingestion, releasing FFA and lactic acid, which will be metabolized as normal. There is some evidence that the metabolic degradation or intestinal absorption of lactylates may be delayed in comparison to lactic acid or FFA (Phillips et al., 1981). This delay may be sufficient to allow the lactylate to have a stronger effect on bacterial infection in the intestinal tract than either FFA or lactic acid alone. But even when it does hydrolyze, both of the molecules released (lactic acid and MCFA) still have some antibacterial activity. The combination between the strong antimicrobial lactylate, its bacteriostatic products in the form of MCFA and lactic acid, and its slow metabolization is the most probable explanation for the observed effects on clostridial lesion scores and mortality in broilers seen in experiments 1 and 2. Still, more attention should be paid to the molecule and its physiological effects because none of its expected properties did improve results in experiment 3. It can be concluded from this study that higher concentrations of LauL in broiler diets have a potential to reduce the severity of necrotic enteritis in broilers in an E. maxima and C. perfringens challenge model.
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