LyophilizedCarnobacterium divergens AS7 bacteriocin preparation improves performance of broiler chickens challenged withClostridium perfringens

Lyophilized Carnobacterium divergens AS7 bacteriocin preparation improves performance of broiler chickens challenged with Clostridium perfringens D. Józefiak,*1 A. Sip,† A. Rutkowski,* M. Rawski,* S. Kaczmarek,* M. Wołuń-Cholewa,‡ R. M. Engberg,§ and O. Højberg§ *Poznań University of Life Sciences Department of Animal Nutrition and Feed Management Wołyńska 33, 60-637 Poznań, Poland; †Poznań University of Life Sciences Department of Biotechnology and Food Microbiology Wojska Polskiego 48, 60-627 Poznań, Poland; ‡Poznań University of Medical Science Department of Cell Biology Rokietnicka 5D 60-806 Poznań, Poland; and §Aarhus University, Department of Animal Science, PO Box 50, DK-8830 Tjele, Denmark increased BW gain from d 29 to 42 (P = 0.048). The divercin supplementation increased the AMEn level (P = 0.015) and reduced digesta pH in crop and ileum (P = 0.004 and P = 0.042, respectively), but of nonchallenged birds only. Divercin supplementation, moreover, increased gizzard lactate concentrations (P = 0.003). The crop concentrations of lactate and succinate and the ileum concentration of lactate were increased by divercin supplementation (P = 0.005, P = 0.027, and P = 0.002, respectively) and C. perfringens challenge (P = 0.034, P = 0.053, and P = 0.0002, respectively). Divercin supplementation decreased villus heights (P = 0.0006) and crypt depths (P = 0.044) in noninfected birds, whereas in challenged birds, villus heights (P < 0.0001) were increased. In conclusion, the present study demonstrated a very complex response pattern of broilers exposed to C. perfringens challenge and dietary divercin AS7 supplementation, but it indicated that divercin AS7 may partly counterbalance the negative effects associated with C. perfringens.

Key words: bacteriocin, Clostridium perfringens, fermentation, histomorphology 2012 Poultry Science 91:1899–1907 http://dx.doi.org/10.3382/ps.2012-02151

INTRODUCTION Clostridium perfringens Type A is a major pathogen in the chicken gastrointestinal tract (GIT), producing toxins such as α-toxin and NetB, of which the latter is now considered the key virulence factor for development of necrotic enteritis (NE; Keyburn et al., 2008), one of the economically most devastating diseases for the broiler industry worldwide (Dahiya et al., 2006; Skinner et al., 2010). The bacterium is ubiquitous in ©2012 Poultry Science Association Inc. Received January 9, 2012. Accepted March 31, 2012. 1 Corresponding author: [email protected]

the environment and may thus be ingested with soil, feces, or dust. However contaminated feed or litter are considered the main sources of infection. Avirulent C. perfringens strains are often found in the GIT of healthy chickens where they may reside without harming the host bird. However, simultaneous blooming of virulent C. perfringens strains and colonization of the small intestine by Eimeria spp. may launch NE development. At present, NE is more or less controlled by the dietary addition of ionophores as anticoccidials, targeting both the parasite and C. perfringens. An alternative strategy to suppress C. perfringens colonization may be the dietary supplementation of bacteriocins, which are small peptides, lethal to bacteria other than the

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ABSTRACT The present study aimed to investigate the effects of Carnobacterium divergens AS7 bacteriocin (divercin AS7) on growth performance, digestibility, fermentation processes, selected microbial populations, and histomorphology in broiler chickens challenged with a mixture of 3 Clostridium perfringens isolates. In total, 480 one-day-old male Ross 308 chicks were randomly assigned to 4 experimental groups (12 replicate pens of 10 birds per treatment). The diets were either nonsupplemented or supplemented with a lyophilized preparation of divercin AS7. On d 18, 19, and 20, half of the birds were challenged twice a day with the C. perfringens mixture. The C. perfringens challenge did not influence broiler BW gain but impaired feed conversion ratio from d 29 to 42 (P = 0.023) and throughout the experimental period (P = 0.038). Moreover, the C. perfringens challenge resulted in decreased pH levels of crop, gizzard, and ileum contents (P < 0.05) and reduced the numbers of lactic acid bacteria in the ceca (P = 0.01). Divercin supplementation decreased broiler feed intake from d 14 to 28 (P = 0.001) but

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producing strain but with a rather narrow killing spectrum in comparison to antibiotics (Grilli et al., 2009; Svetoch and Stern, 2010). Bacteriocins are found in the GIT of animals, in soil, and in food, for example, fermented milk products, cheese, or meat (Cleveland et al., 2001). Moreover, there is evidence that bacteriocins can be isolated from the poultry GIT (Shin et al., 2008); however, so far, there is limited information available regarding the potential antimicrobial effect of these peptides against C. perfringens. Thus, the aim of the present study was to examine how dietary supplementation of lyophilized C. divergens AS7 divercin may affect broilers challenged with virulent strains of C. perfringens.

Birds and Housing In total, 480 one-day-old male Ross 308 chicks were randomly distributed to 4 experimental groups using 12 replicate pens per treatment and 10 birds per pen. The broiler chickens were kept in floor pens (1.2 × 0.8 m) over a production period of 42 d. The birds were given 23 h of light and 1 h of dark during the first week and then 19 h of light and 5 h of dark from d 7 to 21. From 22 to 42 d of age, there was 23 h of light and 1 h of dark. The basic diet provided was either nonsupplemented or was supplemented with a lyophilized divercin AS7 preparation, (0.2 g/kg) equivalent to 200 activity units (AU)/g of feed. The birds were either not challenged or were challenged with a mixture of 3 C. perfringens type A strains all isolated from diseased birds and all producing α-toxin and NetB (Abildgaard et al., 2010): strain 73.70560-e (PFGE type S11), strain 200302–11-Ba (PFGE type S48), and strain 97.73338–3 (PFGE type T7). The numbers in brackets refer to the pulsedfield gel electrophoresis types reported by Nauerby et al. (2003) and Abildgaard et al. (2009). The birds were individually challenged on d 18, 19, and 20, twice a day with a 12-h interval, applying an oral dose of 1 mL of physiological salt, containing the 3 C. perfringens strains in a concentration of 108 cells/ mL. The birds receiving no bacterial challenge were treated at the same time orally with 1 mL of physiological salt solution.

The feed intake and BW of the chickens were measured on d 28 and 42. The mortality was registered throughout experimental periods. At the end of the trial (42 d) from each experimental group, 21 randomly picked chickens (3 chickens from 7 pens) were killed by cervical dislocation. For digesta analyses (enumeration of bacteria, pH and organic acid concentrations), the contents of crop, ileum, and ceca from 3 birds per pen were pooled (7 replicate digesta samples per segment of approximately 10 g each). Approximately 5 g of the samples were used immediately for the culturing of bacteria and pH analyses. The rest of the samples were immediately frozen and stored at −20°C for later analysis of organic acids. For the evaluation of apparent metabolizable energy (AMEn) and total tract fat digestibility, 100 g of the excreta from each pen were collected at 42 d (n = 12). The excreta samples were immediately frozen, freeze-dried, and ground before further analyses. Approximately 15 mm of the ileum were collected from 10 broiler chickens from each treatment at 42 d. The ileum was cut close to the Meckel’s diverticulum distal to the end of jejunum and divided into 7-mm segments.

Diets and Feeding Program

Chemical Analyses

The composition of the experimental diet is shown in Table 1. The diet was formulated to stimulate the proliferation of the C. perfringens by use of viscous cereals (barley/wheat), animal fats (beef tallow/pig lard), and fishmeal. The diets were prepared in mash form, all raw materials were ground by disc mill (Skiold A/S, Denmark) at 2.5 disc distance, mixed without any heat treatment, and fed ad libitum to all birds. Until 14 d of age, all birds were fed the same basal diet without divercin supplementation. The divercin activity of the ly-

Feed samples were analyzed in duplicate for crude protein, crude fat, and crude fiber using AOAC (2005) methods 976.05, 920.39, and 2002.04, respectively. For all chemical analyses, samples were ground and passed through a 0.5-mm sieve. The concentration of titanium dioxide was determined according to the method described by Short et al. (1996), and the samples were prepared according to the procedure reported by Myers et al. (2004). Gross energy was determined using an adiabatic bomb calorimeter (KL 12Mn, Precyzja-Bit

Data Collection

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MATERIALS AND METHODS

ophilized product as well as the supplemented diets was quantified by 2-fold dilutions and expressed in activity unit (AU) per milliliter, as previously described by Ennahar et al. (2001). The divercin AS7 preparation was produced according to the technology elaborated by Sip et al. (1999) and was provided in lyophilized form. Lyophilization of the liquid divercin was performed in a lyophilizing cabinet (Christ 1825, Germany) for 5 d at a temperature of −55°C. The lyophilized liquid divercin was mixed under aseptic conditions with a carrier (Arbocel, powdered cellulose, JRS, Germany) at a ratio of 3:1 (wt/wt). The lyophilized divercin preparation was used in the diets at the expense of wheat in the amount of 0.2 kg/t, corresponding to approximately 200 AU/g of feed. In the last 5 d (37–42 d) of the experiment, 0.2% of the wheat was replaced by titanium oxide as an internal marker for the calculation of nutrient digestibility. The experiment complied with the guidelines of the Local Ethic Commission with respect to animal experimentation and care of animals under study.

LYOPHILIZED CARNOBACTERIUM DIVERGENS IN BROILERS Table 1. Composition of experimental diets Diet (1 to 42d)

Item  

32.7 25.0 21.5 3.0 5.4 6.0 3.0 1.1 0.50 0.40 0.28 0.21 0.03 0.10 0.26 0.20   12.95 21.5 10.0 3.45 0.85 1.3 0.55 0.93 0.81 0.42   21.8 3.9 10.1 12.9

1Provided

the following per kilogram of diet: vitamin A, 11.166 IU; cholecalciferol, 2.500 IU; vitamin E, 80 mg; menadione, 2.50 mg; B12, 0.02 mg; folic acid, 1.17 mg; choline, 379 mg; d-pantothenic acid, 12.50 mg; riboflavin, 7.0 mg; niacin, 41.67 mg; thiamine, 2.17 mg; d-biotin, 0.18 mg; pyridoxine, 4.0 mg; ethoxyquin, 0.09 mg; Mn (MnO2), 73 mg; Zn (ZnO), 55 mg; Fe (FeSO4), 45 mg; Cu (CuSO4), 20 mg; I (CaI2O6), 0.62 mg; Se (Na2SeO3), 0.3 mg. 2Replaced adequate amount of the wheat in each diet, from 37 to 42 d of broiler growth.

PPHU, Poland) standardized with benzoic acid. Nitrogen content was analyzed by the Kjel Foss Automatic 16210 (A/S N. Foss Electric, Denmark) and the crude protein content was calculated using a multiplication factor of 6.25. Fat content was determined using Soxtex System HT 1043, Extraction Unit (Foss Tecator Denmark).

Digestibility Calculations The apparent digestibility coefficients of fat were analyzed as described in detail by Józefiak et al. (2010b). The AMEn content of the experimental diets was calculated according to Hill and Anderson (1958) using titanium dioxide as an indigestible marker.

Microbiological Analyses The pH of pooled digesta samples from crop, ileum, and ceca was measured immediately after slaughter using a combined glass and reference electrode (Elmetron, Poland). For enumeration of bacteria, subsamples

of 2.5 g were diluted in 30 mL of sterile prereduced salt medium (Miller and Wolin, 1974) and the suspensions were homogenized for 2 min in CO2-flushed plastic bags using a stomacher homogenizer (Interscience, France). Subsequently, the samples were serially diluted in 10-fold steps using prereduced salt medium according to the technique of Miller and Wolin (1974). Lactic acid bacteria were enumerated by spread-plating on de Man, Rogosa, and Sharp agar (MRS; CM0361, Oxoid Limited, United Kingdom) and the total anaerobic bacteria on Columbia Blood Agar (CM0331, Oxoid Limited, United Kingdom) incubated for 48 h at 38°C in anaerobic jars (Anaerocult A, Merck 1.13829). The Clostridium perfringens were enumerated on Iron Sulfite Agar (42603, Biomerieux, Marcy l’Etoile, France) with d-cycloserine supplement (42619, Biomerieux) and incubated for 24 h at 38°C in anaerobic jars (Anaerocult A, Merck 1.13829). The concentration of shortchain fatty acids and lactic acid in the contents of the different gastrointestinal segments was determined by gas chromatography, as described earlier by Canibe et al. (2007), using a Hewlett Packard gas chromatograph (model 6890, Hewlett Packard, Agilent Technologies, Naerum, Denmark) equipped with a flame-ionization detector and a 30-m ZB-5 column with an internal diameter of 0.32 mm and coated with 5%-phenyl 95%-dimethylpolysiloxane with a film thickness of 0.25 μm.

Histomorphology The ileum samples were fixed immediately after preparation in freshly prepared formaldehyde solution, 40 g/L of formaldehyde prepared in 0.01 M PBS, pH = 7.4, and incubated for 12 h. Afterward, the samples were dehydrated in a series of alcohol solutions, placed in xylene, and then embedded within paraffin. At least 5 serial sections of 5 μm were cut from each block and were stained with hematoxylin and eosin. The stained material was examined under Axiophot OPTON light microscope with 5×5 magnification. The length of the villi was measured from the top of the epithelium villi to the junction with crypt. In the cross-sections, the lengths of all villi with a complete structure were measured. Destroyed villi were excluded from the experiment. Crypt depth and villus height were measured in 5 serial slides using a micrometer glass master (0.01 mm, PZO, Warsaw, Poland) and treated as the mean. This value was used in further statistical analysis.

Statistical Analysis Statistical analysis of the results was performed using the GLM of the SAS (SAS, 1990) according to the following general model: Yij = μ + αi + βj + (αβ)ij + δij, where Yij was the observed dependent variable; μ was the overall mean; αi was the effect of divercin; βj was

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Ingredient (%) diet  Wheat  Barley   Soybean meal   Beef tallow   Pig lard   Double zero rapeseed meal   Fish meal   Monocalcium phosphate   Mineral and vitamin premix1  Limestone   l-Lysine-HCl   dl-Methionine   l-Threonine   Sodium carbonate (Na2CO3)   Salt (NaCl)   Titanium oxide (TiO2)2 Calculated composition (%)   ME (MJ/kg)  CP   Crude fat   Crude fiber  Calcium  Lysine  Methionine   Methionine + cystine  Treonine   Nonphytate P Analyzed composition (%)  CP   Crude fiber   Crude fat   ME (MJ/kg)

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the effect of C. perfringens challenge; (αβ)ij was the interaction between divercin and C. perfringens challenge; and δij was the random error. In cases where the overall effect was significant (P < 0.05), means were compared pairwise (pdiff). Results are given as the least squares means with pooled standard error of the mean (SEM).

RESULTS Divercin AS7 Activity and Bird Performance

Fat Digestibility and AME The divercin supplementation increased (P = 0.015) the level of AMEn (Table 3). The lowest AMEn content was observed in infected broilers without divercin supplementation (Table 3). Neither the dietary divercin

Digesta pH, Organic Acid Concentrations, and Viable Bacterial Counts The pH values of the digesta in crop, gizzard, ileum, and ceca are shown in Table 4. There was a significant interaction between divercin supplementation and C. perfringens challenge regarding the pH of crop (P = 0.04) and ileal contents (P = 0.004). Divercin only reduced the pH of nonchallenged birds. The C. perfringens challenge resulted in decreased pH levels of crop, gizzard (P = 0.02), and ileal contents (P < 0.05) whereas in cecal content, the challenge tended to increase the pH (P = 0.054). In the contents of crop, gizzard, and ileum, the highest pH values were found in nonchallenged birds not receiving the divercin supplement. The concentrations of organic acids in the contents of different segments of the broiler chickens GIT are summarized in Tables 5 and 6. In the crop, lactate was the dominating organic acid, followed by acetate and succinate. The concentrations of lactate and succinate in the crop were higher when birds received divercin (P = 0.005, and P = 0.027, respectively) and after the bacterial challenge (P = 0.034 and P = 0.053, respectively). In the gizzard, the same 3 organic acids were detected; however, their concentrations were considerably lower as compared with crop digesta. In C. perfringens-challenged birds, the acetate concentration was higher (P = 0.005). The divercin supplementation increased the lactate concentrations in gizzard contents (P = 0.003). In ileum, additionally 2 acids (butyrate and formate) were detected (Table 6). Both divercin and C. perfringens challenge increased lactate concentrations. There was a significant interaction between the experimental factors regarding the concentration of lactate in ileal content (P = 0.036). Divercin increased the lactate concentra-

Table 2. The effect of divercin AS7 and Clostridium perfringens challenge on performance results of broiler chickens1 Performance Treatment Divercin2

Challenge3

− − + − − + + + Pooled SEM Model P Model RMSE Interaction terms   Divercin × challenge a,bMeans

14–28 d

29–42 d

1–42 d

BW gain, g

FI, g

FCR, g:g

BW gain, g

FI, g

FCR, g:g

BW gain, g

FI, g

FCR, g:g

1,041 1,008 1,042 1,005 9.7 0.384 62.0

1,584b 1,519b 1,664a 1,522b 15.2 0.003 84.1

1.52 1.51 1.60 1.52 0.01 0.328 0.1

1,279 1,296 1,244 1,320 10.9 0.124 67.2

2,345 2,343 2,388 2,381 15.7 0.679 102.7

1.84b 1.81b 1.92a 1.80b 0.01 0.0002 0.05

2,319 2,305 2,285 2,326 15.0 0.829 98.7

3,923 3,860 4,047 3,897 26.5 0.114 163.0

1.69b 1.68b 1.77a 1.68b 0.01 0.005 0.05

0.921

0.159

0.351

0.176

0.937

0.387

0.405

0.044

0.014

within a column with no common superscripts differ significantly (P < 0.05). 1Means represent 12 pens of 10 chicks each. 2Nonsupplemented or supplemented with a lyophilized divercin AS7 preparation (0.2 g/kg) equivalent to 200 divercin AU/g of feed. 3The birds were either not challenged or were challenged with a mixture of 3 C. perfringens type A strains producing α-toxin and NetB in dose of 108 cfu/mL.

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The estimated divercin AS7 activity was approximately 8% lower after lyophilization process than in the liquid preparation. The recovery of lyophilized divercin in the diets ranged in between 96 and 98%. In the period from 29 to 42 d, the divercin AS7 supplementation increased the BW gain of the broiler chickens (Table 2), in the range of 40 g (P = 0.048), but over the entire production period (1–42 d), no difference between treatments regarding BW gain was found (P > 0.05). The C. perfringens challenge did not influence the BW gain of the birds (P > 0.05). Divercin decreased the feed intake in the period from 14 to 28 d (P = 0.001). The C. perfringens challenge impaired feed conversion ratio in the period from 29 to 42 d and in the entire experiment (P = 0.023). There was a significant interaction between divercin supplementation and C. perfringens challenge regarding the feed conversion in the period from 29 to 42 d (P = 0.014) as well as for the entire period (P = 0.044). Divercin significantly improved the feed conversion of infected birds (P = 0.0002).

supplementation nor the C. perfringens challenge influenced the apparent fat digestibility.

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LYOPHILIZED CARNOBACTERIUM DIVERGENS IN BROILERS Table 3. The effect of divercin AS7 and Clostridium perfringens challenge on fat digestibility (%) and AME value (kcal/kg) of broiler chickens1 Treatment Divercin2

Crude fat digestibility

Challenge3

− + − + Pooled SEM Model P Model RMSE Interaction terms   Divercin × challenge

− − + +

AMEn

77.9 76.1 71.9 77.7 1.2 0.306 7.2

2,761a 2,798a 2,581b 2,715ab 29.0 0.030 150.4

0.148

0.362

a,bMeans

tions of nonchallenged birds. In the ceca, all detected acids (acetate, lactate, butyrate, iso-butyrate, valerate, succinate, and propionate) were found in numerically higher concentrations in the C. perfringens-challenged birds. However, only the concentrations of butyrate, iso-butyrate, valerate, and propionate were significantly higher (P = 0.008, P = 0.034, P = 0.0001, and P = 0.009, respectively). No effects of dietary divercin were observed in the ceca. The plate count results of the selected microbial populations are summarized in Table 7. Overall, the influence of the treatments on bacterial numbers was only marginal. There was an interaction between divercin supplementation and C. perfringens challenge considering LAB numbers in cecal content (P = 0.018), where divercin reduced these bacteria only in infected birds. Total counts of anaerobe bacteria capable to hydrolase sheep blood were not affected by the treatments. Clostridium perfringens were only quantified in ileal digesta. The counts were relatively low (3–4.5 log cfu/g)

and there was no effect of the bacterial challenge on C. perfringens numbers (P > 0.05), whereas the supplementation of divercin tended to reduce C. perfringens numbers (P = 0.109).

Histomorphology Significant interactions (P < 0.001) between divercin supplementation and C. perfringens challenge were observed with respect to villus height and crypth depth of the ileum epithelium (Table 8). Divercin decreased villus height and crypt depth in noninfected birds, whereas in challenged birds, divercin increased the villus height and crypt depth. The longest villi were observed in the nonchallenged and non-divercin-supplemented birds (P < 0.05), whereas in infected nonsupplemented birds, the shortest villi were measured. The depths of the crypts were smallest in nonchallenged birds supplemented with divercin and biggest in nonchallenged birds without divercin supplementation.

Table 4. The effect of divercin AS7 and Clostridium perfringens challenge on digesta pH of broiler chickens1 Treatment Divercin2 − + − + Pooled SEM Model P Model RMSE Interaction terms   Divercin × challenge a–cMeans

pH

Challenge3

Crop

Gizzard

Ileum

Ceaca

− − + +

4.8a 4.5b 4.5b 4.5b 0.04 0.001 0.1

3.6a 3.5ab 3.3b 3.3b 0.04 0.017 0.1

7.0a 6.2b 5.0c 5.8c 0.1 <.0001 0.3

6.1 5.9 6.2 6.2 0.05 0.093 0.2

0.040

0.518

0.004

0.151

within a column with no common superscripts differ significantly (P < 0.05). represent 21 chicks in 7 pooled replicates. 2Nonsupplemented or supplemented with a lyophilized divercin AS7 preparation (0.2 g/kg) equivalent to 200 divercin AU/g of feed. 3The birds were either not challenged or were challenged with a mixture of 3 C. perfringens type A strains producing α-toxin and NetB in dose of 108 cfu/mL. 1Means

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within a column with no common superscripts differ significantly (P < 0.05). 1Means represent 12 pens of 10 chicks each. 2Nonsupplemented or supplemented with a lyophilized divercin AS7 preparation (0.2 g/kg) equivalent to 200 divercin AU/g of feed. 3The birds were either not challenged or were challenged with a mixture of 3 C. perfringens type A strains producing α-toxin and NetB in dose of 108 cfu/mL.

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Table 5. The effect of divercin AS7 and Clostridium perfringens challenge on concentration (μmol/g of digesta) of the organic acids in crop and gizzard digesta of broiler chickens1 Interaction terms Item Treatment  Divercin2  Challenge3 Crop  Acetate  Lactate  Succinate Gizzard  Acetate  Lactate  Succinate

Pooled SEM

Inclusion or amount − −

+ −

− +

+ +

Model RMSE

Model P

 

Divercin × challenge  

14.6 65.5b 4.4b

17.9 90ab 5.6ab

17.3 82.6b 5.4ab

20.5 110a 7.2a

0.9 5.0 0.06

0.175 0.009 0.039

4.3 19.8 1.5

0.968 0.873 0.682

2.4b 9.6c 1.4

2.9ab 18ab 1.2

3.7a 14.2bc ND4

3.8a 21.2a 1.1

0.1 1.3 0.08

0.028 0.010 0.536

0.8 5.5 0.2

0.529 0.764 ND

a–cMeans

within a row with no common superscripts differ significantly (P < 0.05). represent 21 chicks in 7 pooled replicates. 2Nonsupplemented or supplemented with a lyophilized divercin AS7 preparation (0.2 g/kg) equivalent to 200 divercin AU/g of feed. 3The birds were either not challenged or were challenged with a mixture of 3 C. perfringens type A strains producing α-toxin and NetB in dose of 108 cfu/mL. 4ND = not detected. 1Means

In agreement with other studies, we observed the applied C. perfringens challenge model to impair feed utilization of the broiler chickens (Skinner et al., 2010) without inducing increased mortality of the birds (Olkowski et al., 2006; Gholamiandehkordi et al., 2007). Use of the lyophilized divercin preparation was observed to improve performance of the challenged birds, however, not to the same degree as in our previous work (Józefiak et al., 2010b, 2011), where we demonstrated dietary supplementation of divercin AS7 as a

liquid preparation to improve broiler performance similar to the use of the ionophore coccidiostat salinomycin. Broiler production results are usually closely related to diet digestibility and AMEn content. In the present study, divercin AS7 improved AMEn of the diets, whereas the C. perfringens challenge had no effect, partially agreeing with earlier findings also indicating a positive effect of divercin AS7 on AMEn values (Józefiak et al., 2010b; Józefiak et al., 2011). However, in the present study, C. perfringens-challenged birds had numerically the lowest AMEn value accompanied with the lowest apparent digestibility of crude fat.

Table 6. The effect of divercin AS7 and Clostridium perfringens challenge on concentration (μmol/g of digesta) of the organic acids in ileal and cecal digesta of broiler chickens1 Interaction terms Item Treatment  Divercin2  Challenge3 Ileum  Acetate  Lactate  Formate  Butyrate  Succinate Ceca  Acetate  Lactate  Butyrate  Succinate  Iso-butyrate  Valerate  Propionate a,bMeans

Pooled SEM

Inclusion or amount − −

   

Model RMSE

Model P    

   

Divercin × challenge

+ −

− +

+ +

   

5.4 23.3b 3.0 1.0 ND4

6.0 67.4a 1.8 0.6 2.1

5.0 73.6a 1.5 0.4 1.5

4.1 84.0a ND ND 1.6

0.3 5.9 0.4 0.1 0.2

0.308 <0.0001 0.408 0.566 0.588

1.7 18.3 1.0 0.3 0.8

0.306 0.036 ND4 ND4 ND4

53.1 4.4 14.3b 3.3 0.7b 1.3b 6.7b

45.9 2.2 13.2b 3 0.4b 1.1b 6.3b

54.4 2.2 18.3ab 3.7 0.7b 1.2a 8.9ab

59 10.2 20.5a 5.6 1.6a 2.1a 11.1a

2.2 2.4 1.0 0.6 0.1 0.1 0.6

0.213 0.775 0.049 0.490 0.003 0.001 0.037

10.4 7.1 4.7 3.1 0.3 0.3 2.9

0.178 0.588 0.392 0.395 0.016 0.271 0.286

within a row with no common superscripts differ significantly (P < 0.05). represent 21 chicks in 7 pooled replicates. 2Nonsupplemented or supplemented with a lyophilized divercin AS7 preparation (0.2 g/kg) equivalent to 200 divercin AU/g of feed. 3The birds were either not challenged or were challenged with a mixture of 3 C. perfringens type A strains producing α-toxin and NetB in dose of 108 cfu/mL. 4ND = not detected. 1Means

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DISCUSSION

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Table 7. The effect of divercin AS7 and Clostridium perfringens challenge on bacterial counts in digesta (log cfu/g of digesta) of broiler chickens1 Microbiology Treatment Divercin2

Lactic acid bacteria

Challenge3

− + − + Pooled SEM Model P Model RMSE Interaction terms   Divercin × challenge

− − + +

Crop

Total anaerobes

C. perfringens

Ileum

Ceca

Crop

Ileum

Ceca

Ileum

8.1 8.3 8.3 8.3 0.04 0.189 0.2

7.6 7.5 7.8 7.8 0.1 0.757 0.5

8.3a 7.9b 8.3a 8.5a

7.9 8.2 8 8 0.05 0.370 0.2

6.9 6.5 7.7 7.8 0.3 0.460 1.6

8.3 6.9 8.3 8.6 0.3 0.333 1.7

3.6 3 4.5 3 0.3 0.313 1.5

0.269

0.881

0.018

0.216

0.511

0.07 0.008 0.2

0.364

0.679

a,bMeans

The digesta pH reflects the activity of the gastrointestinal microbiota as well as digestion physiology (Engberg et al., 2002, 2004; Józefiak et al., 2004), and to our knowledge, the present observations are the first to show that C. perfringens challenge may affect pH throughout the GIT. This finding may explain some negative effects of C. perfringens colonization, such as impaired feed utilization, because all endogenous and exogenous enzymes are pH sensitive. Jia et al. (2009), on the other hand, challenged birds with a single dose of 108 cells of C. perfringens but observed no effect on digesta pH in the small intestine. In contrast to our earlier study (Józefiak et al., 2010a), the dietary inclusion of divercin AS7 in the present study reduced the pH of the digesta in crop and ileum. Using a divercin preparation that was lyophilized on a cellulose matrix may, however, have affected its mode of action, given that carbohydrates are known to affect the intestinal fermentation processes and thus digesta pH in the poul-

try GIT (Józefiak et al., 2004). Cellulose is an insoluble nonstarch polysaccharide, which is poorly fermented, but may increase enzyme activities, bile salt concentrations, and GIT functions, particularly of the gizzard (Hetland et al., 2004). The chicken GIT level of C. perfringens is typically highest in the period of 21 to 28 d of age, coinciding with typical outbreaks of clinical NE (Abildgaard et al., 2010). In the present study, C. perfringens was quantified at 42 d of age. Three weeks after the challenge, where the birds may have recovered regarding the GIT levels of the pathogen; the commensal microbiota and broiler performance in general may however still be affected, explaining some of the farm-associated problems experienced with subclinic forms of C. perfringens infections or NE, which are often underestimated but may affect production economy considerably. The observed ileal villus heights are in good agreement with recently reported values (Lumpkins et al.,

Table 8. The effect of divercin AS7 and Clostridium perfringens challenge on histomorphology of the ileum (μm) in broiler chickens1 Treatment Divercin2 − + − + Pooled SEM Model P Model RMSE Interaction terms   Divercin × challenge a–cMeans

Ileum Challenge3

Villus height

Crypt depth

− − + +

532a 426c 415c 462b 3.9 <0.0001 141.2

264a 187c 207b 255a 3.0 <0.0001 105.3

<0.0001

<0.0001

within a column with no common superscripts differ significantly (P < 0.05). represent 10 chicks from each treatment. 2Nonsupplemented or supplemented with a lyophilized divercin AS7 preparation (0.2 g/kg) equivalent to 200 divercin AU/g of feed. 3The birds were either not challenged or were challenged with a mixture of 3 C. perfringens type A strains producing α-toxin and NetB in dose of 108 cfu/mL. 1Means

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within a column with no common superscripts differ significantly (P < 0.05) 1Means represent 21 chicks in 7 pooled replicates. 2Nonsupplemented or supplemented with a lyophilized divercin AS7 preparation (0.2 g/kg) equivalent to 200 divercin AU/g of feed. 3The birds were either not challenged or were challenged with a mixture of 3 C. perfringens type A strains producing α-toxin and NetB in dose of 108 cfu/mL.

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Józefiak et al.

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2010), whereas earlier studies have shown much higher values, usually above 1 mm (Shakouri et al., 2009). Moreover, the dietary inclusion of divercin AS7 decreased villus height in nonchallenged birds but had the opposite effect in C. perfringens-challenged birds. Dietary divercin also caused a significant reduction of crypt depth in nonchallenged birds but not in C. perfringens-challenged birds. The presented results show long-term effects of C. perfringens challenge and may suggest that the effects of divercin are depending on the health status of the GIT. Many factors affecting villus height and crypt depth in broiler chickens have been reported, including feed allocation (Gonzales et al., 2003), genetic potential (Lumpkins et al., 2010), as well as different dietary factors (Langhout et al., 1999). However, it can be assumed that in most of these studies, corn-soybean meal-based diets were used. We used a relatively low-digestible and viscous diet, formulated to increase C. perfringens levels but suboptimal regarding bird performance. There is a close relationship between dietary factors and the development of the GIT, including microstructures and the indigenous microbiota. It can therefore be assumed that the reduced villus heights observed in the present study may be caused by the dietary challenge in accordance with the observations of fat digestibility and AMEn content of the diets as well as the findings of Mathlouthi et al. (2002), who compared performance, digestibility, and histomorphology results of broiler chickens fed corn- and rye-based diets; compared with the corn-based diet, the viscous, rye-based diet decreased performance, crude fat and protein digestibility, AMEn, as well as villus length, width, and surface. The observations of the present study illustrate a complex response pattern of C. perfringens challenge and divercin AS7 supplementation. The bacteriocin mode of action seems to depend not only on microbiota composition and GIT health status but also on the physical form of the applied compound; the effects obtained with divercin lyophilized on a microcrystalline cellulose carrier were thus different from those observed earlier with liquid divercin preparations. However, it can be concluded that divercin AS7 may reduce the negative effects related to a C. perfringens challenge by protecting broiler performance, improving AMEn content of the feed, and maintaining histomorphology of the GIT.

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