Effect of Dietary Protein Concentrates on the Incidence of Sub-clinical Necrotic Enteritis and...

METABOLISM AND NUTRITION

2010 Poultry Science 89 :34–43doi: 10.3382/ps.2009-00105

Key words: potato protein , fishmeal , soybean , necrotic enteritis

ABSTRACT An experiment was conducted to quantify the effects of 3 nutritionally complete (similar protein and energy) corn-based diets that contained different dietary protein concentrates (potato-CP 76%, fish-CP 66%, or a mixture of soy proteins, soybean meal-CP 48%, and full-fat soy-CP 36%) on the incidence of spon-taneously occurring subclinical necrotic enteritis (NE) in broiler chickens. A total of 1,260 birds were placed into 18 solid floor pens (70 birds per pen) and fed 1 of the 3 experimental diets from 15 to 31d of age. The weight gains and feed intakes of the birds fed the potato- and fish-based diets were lower (P < 0.001) than those of the birds fed the soy-based diets. Weight gain:feed intake ratio and mortality rate were not affected (P > 0.05) by dietary treatment The birds fed the potato-based diets had a higher incidence of necrotic lesions in the duodenum (P < 0.001) and proximal jejunum (P < 0.01) than those fed the soy-based diets. The chickens

fed the potato-based diet had a higher (P < 0.001) proportion of moderate to severe duodenal and distal ileal hemorrhages and liver lesions than the birds fed the soy-based diet. There was also a higher (P < 0.05) level of serum antibodies for Clostridium perfringens α toxin in birds fed the potato-based diet compared with the other 2 diets. The birds fed the fish-based diet had a similar (P > 0.05) incidence of subclinical NE in com-parison to the birds fed the soy-based diet, although there was a higher incidence of intestinal hemorrhagic lesions. The differences in incidence of subclinical NE were not consistent with the relatively small differences in amino acid content between the diets or in the con-tents of nonstarch polysaccharides. However, the potato protein-based diet had higher trypsin inhibitor activity and a lower lipid content that could have contributed to the increased incidence of subclinical NE.

Effect of dietary protein concentrates on the incidence of subclinical necrotic enteritis and growth performance of broiler chickens

M. W. C. D. Palliyeguru , S. P. Rose ,1 and A. M. Mackenzie

National Institute of Poultry Husbandry, Harper Adams University College, Newport, Shropshire, TF10 8NB, United Kingdom

INTRODUCTION Necrotic enteritis (NE) is a widespread and econom-

ically important bacterial disease in modern broiler flocks (Van Der Sluis, 2000). The subclinical form of the disease is more common than clinical outbreaks in broiler flocks (Kaldhusdal, 2000). The condition is not usually detected due to the absence of clear clinical signs; therefore, it is not treated and prevails unno-ticed apart from a poor growth performance (Lovland and Kaldhusdal, 2001), wet litter conditions (Williams, 2005), and the possible contamination of poultry prod-ucts for human consumption (Craven et al., 2001). The financial cost of NE has been estimated to be US $2.6

billion per year to the world’s poultry industry (Van Der Sluis, 2000).

In subclinical NE, the major pathological changes oc-cur in the small intestine (Gholamiandehkordi et al., 2007) and the liver (Lovland and Kaldhusdal, 2001). Intestinal Clostridium perfringens counts (Kaldhusdal and Hofshagen, 1992) and intestinal C. perfringensα-toxin levels (Hofshagen and Stenwig, 1992; Si et al., 2007) are also increased. The causative organism, C. perfringens, is ubiquitous and found in soil, dust, feces, feed, used poultry litter, the intestines of most healthy animals, and humans (Wages and Opengart, 2003). Clostridium perfringens also is a commensal bacterium of chicken intestines (Ewing and Cole, 1994) and so there are further predisposing factors that alter the intestinal balance in favor of the proliferation of the causative bacteria and allow them to migrate to the upper intestines. Apajalahti et al. (2001) identified the diet as the strongest determinant of the cecal bacterial community, so the diet composition may influence the susceptibility of broiler chickens to subclinical NE, al-

Received March 2, 2009. Accepted June 13, 2009. 1 Corresponding author: [email protected]

© 2010 Poultry Science Association Inc.

34

though there is a lack of direct experimental evidence. However, there is evidence that different dietary pro-tein sources affect the proliferation of C. perfringens within the cecum (Drew et al., 2004) and in the ileum (Wilkie et al., 2005) when birds are orally dosed with these bacteria. Also, studies have indicated that the dietary amino acid balance may influence the prolifera-tion of C. perfringens (Wilkie et al., 2005).

Most of the published experimental information on NE has reproduced the disease by dosing the birds with pathogenic strains of C. perfringens isolated from clini-cally diseased birds (McReynolds et al., 2007; Peder-sen et al., 2008). Although the various protocols dif-fer, most of these experiments involved keeping a small number of birds in cages with frequent dosing of the pathogen (Drew et al., 2004; Gholamiandehkordi et al., 2007). The majority of the published work that has examined dietary factors that predispose birds to NE has also used much higher inclusion rates of the in-dividual feedstuffs than would be used in proprietary feeds (Wilkie et al., 2005). All of these conditions are different from practical broiler chicken rearing meth-ods. However, Lovland et al. (2003) reported that a mild or subclinical NE initiates spontaneously without dosing the birds with pathogenic C. perfringens. This innate infection usually occurs when the appropriate predisposing factors (unmedicated diets, putative pre-disposing feeding regimens, housing birds on litter) are provided. This method of reproducing subclinical NE therefore provides the possibility of studying the effects of dietary factors under conditions that are directly re-lated to commercial production methods.

There is a need to determine whether dietary protein supply affects the incidence of subclinical NE in practi-cal broiler growth conditions. Dietary protein supply is a major variable in the formulation of poultry feeds and local and world price variations can result in large changes in the sources and quality of the protein used. Soy is the most frequently used protein concentrate in poultry feed formulations. Experimental evidence sug-gests that high contents of dietary fish meal increase the intestinal C. perfringens populations in pathogen-dosed birds (Drew et al., 2004). Wilkie et al. (2005) identified that birds fed diets with high potato protein inclusion rates also had higher counts of C. perfringens when compared with birds fed other plant protein di-ets. However, the effect of potato protein and fish meal, in comparison to soybean meal, on naturally occurring spontaneous subclinical NE has not been investigated. Therefore, the objective of this experiment was to iden-tify the effects of 3 different dietary protein concen-trates: potato protein concentrate (PPC), fish meal, or a mixture of soybean meal and full-fat soy in nutrition-ally complete diets, with similar protein contents (CP 21%), on the incidence of subclinical NE in male broil-er chickens reared in comparatively large flocks and in conditions close to practical production methods.

MATERIALS AND METHODS

Dietary Treatments and Experimental Design

Three practical broiler grower diets were formulated to be nutritionally complete for macronutrients (Avia-gen, 2007a) and the vitamins and minerals were pro-vided with a proprietary vitamin and mineral premix that either met or exceeded the NRC (1994) recom-mendations for broiler chickens between 15 to 31 d of age. Diets were corn-based, but a major proportion of the additional protein supply was provided by 1 of 3 protein concentrates: PPC (CP 79%) provided 58% of the total CP in the potato-based diet and fish meal (South American origin; CP 66%) provided 58% of CP in the fish-based diet. The soy protein-based diet was composed of a mix of soybean meal (dehulled; CP 48%) and toasted full-fat soy (CP 36%) and these 2 soy pro-teins together provided 61% of the total CP in the diet (Table 1). All 3 diets were formulated to have similar contents of calculated ME (3.1 mcal/kg), CP (21%), Lys (1.3%), Met and Cys (0.94%), and Thr (1.02%; Ta-ble 1). However the chemical analysis at the end of feed processing (mixing and pelleting) indicated that there were differences in amino acid, starch, lipid concentra-tions, and trypsin inhibitor activity between the treat-ment diets (Table 1). No antibiotic growth promoters or anticoccidial drugs were used in the diets. The diets were all provided as 3-mm-diameter pellets. Treatment diets were compared using 6 replicate pens for each treatment. Eighteen pens were used within 2 adjacent environmentally controlled rooms. The dietary treat-ments were randomly allocated to pens within 6 posi-tional blocks (3 per room).

Broiler Chicken Management and FeedingThe broiler chicken experiment was conducted un-

der the guidance of the Research Ethics Committee of Harper Adams University College. A total of 1,300 one-day-old male Ross 308 broiler chickens were reared in a solid-floored pen as a single flock in an environmentally controlled house. Adequate feeders and drinkers were provided for the age and the number of birds. For the first 15 d, birds were fed a proprietary, nutritionally complete broiler starter feed formulation that was un-medicated (without in-feed antibiotics or coccidiostat). On 15 d posthatch, the birds were weighed and 70 birds were randomly allocated to each of the 18 pens (alto-gether 1,260 birds were allocated to the experiment). The floor area of (1.5 m × 3 m) each pen was covered with wood shavings that comprised 4 parts new wood shavings to 1 part reused litter material from a previ-ous poultry flock that did not have a history of clinical NE but some subclinical NE and subclinical coccidiosis would have been expected. The birds were fed the ex-

PROTEIN CONCENTRATES AND NECROTIC ENTERITIS 35

perimental diets for the following 16 d. Feed and wa-ter were provided ad libitum during the experimental period. Feed levels were kept low in the hanging tube

feeders to minimize wastage. Light was provided for 23 h per day with controlled temperature and humidity. The growth and feed intakes of the birds were recorded

Table 1. Feed ingredients and calculated and determined chemical compositions (g/kg) of the ex-perimental diets

Item

Diets supplemented

Potato Fish Soy

Ingredients Corn 690 673 526.3 Sunflower seed meal 100 100 100 Dehulled soybean meal (CP 48%) — — 178 Full-fat soy (CP 36%) — — 120 Fish meal (CP 66%) — 192.5 — Potato protein concentrate (CP 79%) 158 — — Soy oil 10 10 30 l-Lys HCl 1 1 3 dl-Met 1 1 2.2 l-Thr — 1.5 2.5 Limestone 2 — 3 Monocalcium phosphate 13 — 10 Salt 5 1 5 Vitamin and trace element premix1 20 20 20Calculated chemical composition2

ME (kcal/kg) 3,134 3,132 3,129 Calcium 8.7 11.9 10.3 Phosphorus 5.8 7.5 5.9 Sodium 2.1 2.2 2.1 CP 217.0 219.2 211.9 Digestible protein3 176.8 183.1 190.1 Lys 13.1 13.2 12.9 Met 5.6 6.5 5.5 Cys 3.8 2.9 2.7 Met and Cys 9.4 9.4 9.3 Thr 10.2 10.2 10.2Determined chemical composition DM 874.5 869.5 869.2 Gross energy (kcal/kg) 3,923 3,876 3,947 Crude fat 24.6 41.6 48.6 Ash 49.4 51.4 57.4 Starch 406.6 386.1 456.5 Soluble nonstarch polysaccharides 84.3 58.2 96.0 Insoluble nonstarch polysaccharides 170.1 152.6 159.5 Total nonstarch polysaccharides 254.4 210.8 255.5 CP 206.1 215.5 212.2 Ala 10.4 12.5 9.8 Arg 10.2 11.6 13.4 Asp 19.6 17.3 20.0 Cys 3.5 3.1 3.7 Glu 29.7 33.7 38.2 Gly 9.4 11.3 9.0 His 5.2 6.1 5.5 Ile 8.6 8.2 8.4 Leu 19.3 17.0 16.8 Lys 11.6 12.2 11.6 Met 4.9 6.2 4.9 Phe 11.1 9.1 10.2 Pro 13.2 10.5 11.2 Ser 9.8 8.5 9.6 Thr 9.1 9.1 9.2 Trp 2.0 1.9 2.4 Tyr 6.8 5.5 5.9 Val 10.4 9.4 9.2

1Vitamin-trace mineral premix for broilers (Target Feeds Ltd., Whitchurch, UK) added per kilogram: 2,666 MIU of vitamin A (all-trans retinol), 150 mg of cholecalciferol, 1,300 IU of vitamin E (α-tocopheryl acetate), 150 mg of thiamin, 500 mg of riboflavin, 150 mg of pyridoxine, 750 mg of cyanocobalamin, 3 g of nicotinamide, 0.5 g of pantothenic acid, 75 mg of folic acid, 6.25 g of biotin, 12.5 g of choline chloride, 1 g of iron, 50 mg of cobalt, 5 g of manganese, 0.5 g of copper, 4 g of zinc, 50 mg of iodine, 10 mg of selenium, and 25 mg of molybdenum.

2Chemical composition was calculated with United Kingdom-derived nutrient composition tables (Premier Nu-trition, 2008).

3Digestible protein was calculated according to Lemme et al. (2004).

PALLIYEGURU ET AL.36

over the experimental period. Mortality was recorded daily.

Data CollectionLesion Scoring. On 27, 28, 29, and 30 d of age, 8

birds from each replicate pen (2 birds per day) were selected at random and killed by cervical dislocation and 5 mL of blood was collected in glass tubes from the jugular vein. The liver of each bird was examined for the presence or absence of lesions of hepatitis or cholangiohepatitis that were consistent with the patho-logical changes of NE (Lovland and Kaldhusdal, 1999; Sasaki et al., 2000). The intestinal tract was removed and the duodenal loop was separated and retained. Three further 8-cm sections were taken from the rest of the small intestine; the first of these sections was the proximal jejunum, the second section was either side of Meckel’s diverticulum (mid small intestine), and the third section was the terminal ileum. All 4 sections (in-cluding the duodenal loop) were immediately incised, washed in normal saline, and the mucosal surfaces were inspected and necrotic and hemorrhagic lesions were scored. Proximal jejunal sections were directly stored at −20°C. Sections (1 cm2) from the jejunum that had focal necroses were fixed in 10% formal saline and after 2 d, these sections were rinsed with water and placed in 70% ethanol for histopathological diagnosis. Duodenal lesions were scraped, smeared on glass slides, and 16 representative samples from each diet (4 samples per day) were directly plated (in duplicate) onto blood agar and incubated anaerobically at 37°C for 48 h.

A 5-point scoring system used by Gholamiandehkor-di et al. (2007) was planned to score intestinal lesions, but in the present experiment, all observed lesions were focal necroses with a size of 1- to 10-mm diameter and the rest of the intestinal sections had no necrotic le-sions. Therefore, only 2 scores were used: 0, no lesions and 1, focal necroses (1- to 10-mm diameter). Focal lesions varied from 1- to 2-mm white foci to around 10-mm-diameter mucosal depressions with yellowish green mucoid material laid in the periphery. When-ever such lesions appeared alongside the hemorrhagic lesions, these intestinal sections were recorded as ne-crotic lesions-positive regardless of the level of the other lesions. In addition, all of the intestinal sections were scored for hemorrhagic lesions regardless of the inci-dence of necrotic lesions. Sections with no hemorrhagic lesions were scored as zero, petechial hemorrhages (pin-point hemorrhagic foci of 1- to 2-mm diameter) of the intestinal mucosa were given score 1, purpura hemor-rhages (hemorrhagic foci of 3-mm diameter) as score 2, ecchymotic hemorrhages (blotchy or irregular hemor-rhages up to 1 to 2 cm in size) as score 3, coalesced paint brush hemorrhages as score 4, and extravasation as score 5.

Serum Antibodies for α Toxin. The collected blood was allowed to clot for 8 to 10 h at room temperature. The serum was then separated from the coagulum and

stored at −20°C. Eight serum samples from each pen were tested for antibody levels developed against the α toxin of C. perfringens using an indirect ELISA (Heier et al., 2001; Lovland et al., 2003). Each well of the Nunc immunoplates (F96 Maxisorp, 735-0083, Thermo Fisher Scientific, Rochester, NY) was filled with 100 µL of known antigen, phospholipase C type XIV (Sigma-Aldrich P 4039, St. Louis, MO), 10 µg/mL in sodium carbonate and bicarbonate coating buffer at pH 9.6. The plates were incubated at 4°C overnight to coat the wells with the antigen. The plates were then washed 3 times with PBS at pH 7.2, with 0.05% Tween 20 (PBST). A volume of 150 µL of 1% BSA in PBST was added to each well as a blocking buffer and incubated at 37°C for 2 h. The plates were then washed again in PBST and left for 1 h at room temperature to dry. The antigen-coated plates were individually sealed, packed, and stored at 4°C.

Serum samples were diluted at 1:250 in PBST and 50 µL of each sample was added into duplicate wells. The plates were left at 4°C overnight and then washed 3 times with PBST. Rabbit anti-chicken immunoglobu-lin IgY whole molecule conjugated with alkaline phos-phatase (Sigma-Aldrich A9171) was diluted (10−4) in blocking buffer and 50 µL of diluted anti-chicken anti-bodies was added to each well and then the plates were incubated at 37°C. After 2 h, the plates were washed with PBST.

The plates were incubated with 150 µL of para-ni-trophenyl phosphate (Sigma-Aldrich P 7998) for 1 h at 37°C. The reaction was stopped with 50 µL of 2 M NaOH. The optical density (OD) was read at 405 nm using a microplate reader (Bench Mark 170-6850, Bio-Rad, Hercules, CA). Resulting OD values were pooled for each pen.

Enumeration of C. perfringens. Clostridium per-fringens that were colonized on the mucosal surface of the proximal jejunum were quantified using the BIO K 086-C. perfringens antigen detection kit (Bio-X Diag-nostics, Jemelle, Belgium) in a double antibody sand-wich ELISA (McCourt et al., 2005). Four birds from each replicate pen were used to quantify C. perfrin-gens in the mucosa of the jejunum. Gut samples were thawed at room temperature and washed 3 times in PBS. Then the luminal surface of the proximal jejunum was scraped with a sterile surgical blade and half of the scraping was weighed into a microcentrifuge tube and diluted (2×) with dilution buffer (Bio-X Diagnostics) and mixed vigorously before being allowed to settle for 10 min. (The other half of the scraping was retained for coccidia oocyst counts.) Each sample was filled into 2 adjacent wells (one coated with specific monoclonal antibodies for C. perfringens and the other coated with nonspecific antibodies) of the ELISA plates. The plates were incubated at 21 ± 5°C for 1 h and then washed 3 times with washing solution (Bio-X Diagnostics). Sub-sequently, peroxidase-labeled anti-C. perfringens-specif-ic monoclonal antibodies were added to each well and then incubated at 21 ± 5°C for 1 h and washed 3 times.


Freshly mixed substrate (hydrogen peroxide) and chro-mogen (tetramethyl benzidine) was then added to each well. After 10 min, 1 M phosphoric acid was added to each well and the resulting OD was read at 450 nm using a microplate reader (Bench Mark 170-6850, Bio-Rad). A broth culture of C perfringens was used for se-rial dilutions for plate counts and at the same time for fixation in the dilution buffer (Bio-X Diagnostics) for ELISA (samples for ELISA were immediately frozen). After the plate counts (each dilution was counted 4 times), the ELISA was performed and a standard curve (r = 0.945) was created (Bench Mark 170-6850). This was used to convert the OD values into bacterial counts and the values were pooled for each pen.

Enumeration of Coccidia Oocysts. Eight birds from each replicate pen were sampled for coccidia oocyst counts in the proximal jejunum. Scrapings as taken for the C. perfringens counts were diluted (2×) with saturated NaCl and then placed into both sides of a McMaster counting chamber (Chalex Corporation, Wallowa, OR). The number of coccidia oocysts (all spe-cies found in the sample) was counted at 10 × 10 mag-nification. The counts were pooled for each pen before statistical comparison.

Feed Analysis. Dry matter content of the experi-mental feeds was determined by drying the samples to constant weight at 105°C in an oven. Ash content was determined by AOAC method 942.05 (AOAC Interna-tional, 2000). Nitrogen was determined with an auto-matic analyzer (Leco FP-528 nitrogen, Leco Corp., St Joseph, MI) by AOAC 968.06 (Dumas method) using EDTA as the standard and the protein content was calculated as nitrogen × 6.25. Gross energy in the feed was determined with an adiabatic bomb calorimeter (model 1261 isoperibol, Parr Instrument Co., Moline, IL) using analytical grade sucrose as the standard. The fat content was determined with the AOAC 920.39 method using a Soxtec 1043 extraction unit (Foss Ltd., Wigan, UK). Soluble and insoluble nonstarch polysac-charides (NSP) were determined using AOAC methods 991.43 and 985.29 (for soluble and insoluble polysac-charides, resistant starch, and lignin), AACC methods 32-07 and 32-05 (for total dietary fiber), and AACC method 32-21(for soluble dietary fiber; Megazyme K-TDFR assay kit, Megazyme International Ireland Lim-ited, Wicklow). Total starch contents of the diets were determined with AOAC method 996.11 for high amy-lose cereal starch and AACC method 76.13 for gelati-nized starch (Megazyme K-TSTA assay kit). Trypsin inhibitor activity was determined by measuring the in-hibition of milligrams of bovine trypsin for a gram of sample according to the Kakade method as modified by Smith et al. (1980). Crude protein and gross energy were determined in triplicate samples and all the other analyses were performed in duplicate.

Dietary amino acid samples were oxidized with a hydrogen peroxide-formic acid-phenol mixture. Excess oxidation reagent was decomposed with sodium meta-bisulphite. The oxidized sample was hydrolyzed with 6

M hydrochloric acid for 24 h. The hydrolysate was ad-justed to pH 2.20, centrifuged, and filtered. The amino acids were separated by ion exchange chromatography (Biochrom 20 analyzer, Amersham Pharmacia Biotech, Pittsburgh, PA) and determined by reaction with nin-hydrin using photometric detection at 570 nm (440 nm for proline). For tryptophan analysis, the samples were hydrolyzed with 4.2 M sodium hydroxide for 23 h. The hydrolysate was adjusted to pH 2.20, centrifuged, and filtered. The tryptophan contained in the hydrolysate was then detected by ion exchange chromatography (Biochrom 20 analyzer, Amersham Pharmacia Biotech) and determined by reaction with ninhydrin using pho-tometric detection at 570 nm (AOAC International, 2000).

Statistical Analysis. The effects of the protein sources on growth performance, serum antibody levels for α toxin, and the number of coccidia oocysts and C. perfringens in the proximal jejunum were compared using a randomized block ANOVA (GenStat Release 10.1, Lawes Agricultural Trust, Rothamsted Experi-mental Station, Harpenden, UK) using the pen as the experimental unit. Individual treatment differences were compared by a protected least significant differ-ence test using a probability of less than 0.05.

The data obtained for the incidence of intestinal ne-croses, intestinal hemorrhages, liver lesions, percentage of birds that had mucosal C. perfringens, and the pen mortality rates were compared using a nonparametric χ2 test.

RESULTSThe proximate nutrient compositions of the experi-

mental diets were approximately similar. However, the potato diet had higher trypsin inhibitor activity and lower crude fat content than that of fish or soy product-containing diets (Table 1). Trypsin inhibitor activities of potato-, soy-, and fish-based diets were 3.88, 1.51, and 0.82 mg/g, respectively. The soy diet had the high-est starch content among 3 diets. The fish diet had a lower soluble NSP content compared with the potato or soy diets. The insoluble NSP content was highest in the potato diet. The potato diet also had higher levels of valine, leucine, tyrosine, and phenylalanine, whereas the fish diet had high levels of glycine, lysine, and me-thionine. The soy diet had high Glu and Asp levels compared with the potato and fish diets (Table 1).

The weight gains and feed intakes of the birds fed the potato or fish diets were significantly lower than those fed the soy diets. Weight gain:feed intake ratio and mortality rate were not affected (P > 0.05) by dietary treatment (Table 2). The birds fed the potato diet had a higher (P < 0.01) incidence of necrotic le-sions in the duodenum and proximal jejunum compared with the birds fed the soy diet. The necrotic lesion in-cidence in the birds fed the fish diet was intermediate (Table 3). No necrotic lesions were identified in the mid or distal sections of the small intestine, in any of the


treatment groups, although pseudo-membrane forma-tion was observed in a large proportion of birds (data not presented). There was a higher (P < 0.05) level of serum antibodies for C. perfringens α toxin in birds fed the potato diet compared with the other 2 diets (Table 4).

Double hemolytic colonies on blood agar were con-firmed as C. perfringens with biochemical tests and all gram-stained smears predominantly had relatively large (4 to 6 µm long) gram-positive rods with blunt ends ex-isting as singles or up to 4 loosely attached chains or clusters. They were morphologically similar to C. per-fringens isolated on blood agar plates. Similar bacteria were also attached to damaged villi tips in histological examination of jejunal sections. Fifty-six percent of the birds had C. perfringens colonized on the mucosa of the proximal jejunum, but there were no (χ2 2 df = 2.14, P = 0.343) treatment differences (66.7, 45.8, and 54.2% for the potato, fish, and soy diets, respectively) in the percentage of birds that had C. perfringens colonized on the jejunum or in their counts (Table 4).

The birds fed potato or fish diets had a higher (P < 0.001) proportion of moderate to severe (scores of 3 to 5) duodenal hemorrhages (64 and 77%, respectively) than the birds fed the soy diet (53%) (Table 5). There were no treatment differences in hemorrhagic lesions in the proximal jejunum and the mid small intestine even though there was a relatively high overall inci-dence (Table 5). The birds fed the potato diet had a higher proportion (P < 0.001) of hemorrhagic lesions

than the soy- or fish-fed birds in the distal ileum of the small intestine (Table 5). The birds fed the potato diets also had a higher (P < 0.001) incidence of liver lesions (96%) than those fed the fish (54%) or soy (46%) diets (Table 3). Mortality tended (P < 0.1) to be higher in potato-fed birds (Table 2). Although none of the birds had clinical coccidiosis, the birds fed the soy diet had a higher (P < 0.01) number of coccidia oocysts than those fed the fish diet (Table 4).

DISCUSSIONThe growth performance of the birds in the experi-

ment was comparable with commercial levels (Avia-gen, 2007b). A proportion of the birds in all 3 dietary treatment groups had some evidence of the subclinical form of NE. This confirms the finding of Lovland et al. (2003) that a spontaneous C. perfringens infection, with some predisposing factors, is sufficient to reproduce subclinical NE in a proportion of birds in relatively large floor-reared flocks. At the beginning of the experi-mental period, changes in the environment (used litter mixed into fresh litter might have added some coccidia oocysts and C. perfringens) with simultaneous dietary changes would have predisposed the birds to subclini-cal NE, as described by Parish (1961). The mortality percentages found in the experiment were within the ranges reported in subclinical disease (Lovland and Ka-ldhusdal, 2001) and were within the range commonly found in commercial broiler flocks at this age.

Table 2. The growth performance of male broiler chickens from 15 to 31 d fed diets containing dif-ferent protein concentrates

Variable

Dietary treatments

SEM1Probability of

differencePotato Fish Soy

BW gain (kg) 0.950c 1.029b 1.113a 0.017 <0.001Feed intake (kg) 1.572b 1.633b 1.766a 0.036 0.005G:F (g:g) 0.606 0.629 0.632 0.014 0.372Mortality (%) 4.5 2.6 1.9 — 0.0722

a–cDifferent letter superscripts within rows indicate a significant difference (P < 0.05).1Data are means of 6 pens of 70 broiler chickens per pen.2Mortality was compared using the χ2 test: (χ2 = 5.27 with 2 df).

Table 3. Incidence1 of necrotic lesions of the duodenum and proximal jejunum and liver lesions in male broiler chickens2 fed diets containing different protein concentrates from 15 to 31 d and sampled at 27 to 30 d (posthatch)

Diet

Necrotic lesions in intestinal sections Liver lesions consistent with the pathological changes

of necrotic enteritis Duodenum Proximal jejunum

Potato 33a 33a 46a

Fish 23ab 25ab 26b

Soy 14b 17b 22b

χ2 (2 df) 30.39 10.69 30.39Probability of difference <0.001 0.005 <0.001

a,bDifferent letter superscripts within columns indicate a significant difference (P < 0.05).1Number of lesion-positive birds out of 48 birds sampled in each treatment.2Data are means of 6 pens of 8 sampled broiler chickens per pen (total number of sampled birds is 144 and 48

birds per each treatment).


The birds fed the potato protein diet, in comparison to the soy diet, had a significantly greater incidence of necrotic lesions in the duodenum and the jejunum (Table 3). The pseudo-membrane formation (mucosa covered with golden brown to greenish yellow loosely adherent material) that was observed in the distal and middle small intestine suggests that these birds might have had necrotic lesions in the distal and middle small intestine before sampling and they were recovering from these lesions. In addition to intestinal lesions, the birds fed the potato protein diet had a significantly greater incidence of liver lesions (Table 3), higher se-rum antibody levels for C. perfringens α toxin (Table 4), and a tendency for a relatively small increase in the flock mortality rate (Table 2). The proportion of birds that had C. perfringens colonized on the jejunal mucosa tended to be higher in the potato diet treatment. There was no evidence of an increased incidence of C. perfrin-gens organisms colonized on the gut wall, confirming the findings of McReynolds et al. (2004),who identi-

fied no relationship between the incidence of necrotic lesions and the total C. perfringens population of the jejunum.

Higher coccidia oocyst counts were found in the soy-fed birds compared with those fed the fish diet (Table 4). This indicates that the subclinical coccidiosis did not directly contribute to the pathological lesions in the intestine. However, coccidiosis is a factor that pre-disposes birds to NE (Al-Sheikhly and Al-Saieg, 1980; Shane et al., 1985) and has been identified to have a synergistic relationship with C. perfringens during the development of experimental NE (Park et al., 2008). Coccidia multiplication stages in the intestinal epithe-lium initiate gut mucosal damage but may then be ex-cluded from their proliferation sites when C. perfringens colonize the damaged intestinal epithelium further de-structing the enterocytes (Williams et al., 2003). Also, the diphtheritic membrane formation could impede the intraluminal dissemination of extracellular coccidial stages (Williams, 2005).

Table 5. Hemorrhagic lesion scores1 of the 4 sections of the small intestine in male broiler chickens fed diets containing different protein concentrates from 15 to 31 d and sampled at 27 to 30 d (post-hatch)

Diet

Lesion score in different sections of the small intestine

0 1 2 3 4 5

Duodenum Potato 0 5 10 13 10 10 Fish 0 1 10 21 12 4 Soy 2 15 14 12 4 1 χ2 = 35.38 (10 df) (P < 0.001)2

Proximal jejunum Potato 0 1 1 34 2 10 Fish 0 0 5 26 14 3 Soy 0 2 8 34 3 1 χ2 = 32.22 (8 df) (P < 0.001)Mid small intestine Potato 0 3 14 22 8 1 Fish 2 3 17 21 5 0 Soy 2 1 14 28 3 0 χ2 = 9.13 (10 df) (P = 0.52)Distal ileum Potato 4 1 8 18 16 1 Fish 10 5 14 9 10 0 Soy 4 1 27 13 3 0 χ2 = 33.93 (10 df) (P < 0.001)

1Hemorrhagic scores: 0 = no hemorrhagic lesions; 1 = petechial hemorrhages; 2 = purpura hemorrhages; 3 = ecchymotic hemorrhages; 4 = coalesced paint brush hemorrhages; 5 = extravasations.

2Calculated χ2 values and probabilities indicate a difference between the 3 dietary treatments in hemorrhagic lesion scores.

Table 4. Serum α-toxin antibody concentrations, coccidia oocysts, and Clostridium perfringens counts of proximal jejunal mucosa in male broilers fed diets containing different protein concentrates from 15 to 31 d and sampled at 27 to 30 d (posthatch)

Variable

Dietary treatments

SEMProbability of

differencePotato Fish Soy

α Toxin antibody level (optical density units), optical density at 405 nm 2.92a 1.90b 2.24b 0.2111 0.011Coccidia oocysts/g of mucosal scraping 1,593.0ab 954.0b 2,359.0a 259.51 0.006C. perfringens cfu/g of mucosal scraping (×107) 1.00 0.97 0.96 0.052 0.831

a,bDifferent letter superscripts within rows indicate a significant difference (P < 0.05).1Data are means of 6 pens of 8 sampled broiler chickens per pen.2Data are means of 6 pens of 4 sampled broiler chickens per pen.


The 3 diets used in the present experiment had a similar proximate nutrient composition, despite several nutritional differences. Amino acid imbalance has been suggested as a risk factor for subclinical NE (Wilkie et al., 2005; Dahiya et al., 2007a). Although there is evidence of an effect of dietary glycine on C. perfrin-gens counts in the small intestine and cecum when the birds are orally dosed with the bacterium (Wilkie et al., 2005; Dahiya et al., 2007a), in this experiment, there were no major differences in the glycine levels in potato (9.4 g/kg) and soy (9.0 g/kg) diets (Table 1). Bacterial degradation of aromatic amino acids, such as tyrosine, produces phenolic and aromatic compounds that are toxic to the intestinal epithelial cells (Smith and Mac-farlane, 1997); therefore, the higher levels of aromatic amino acids in the potato diet than in the other 2 diets could have contributed to the initial damage of the epi-thelial cells. The growth of C. perfringens under in vitro conditions needs 11 amino acids (Sebald and Costilow, 1975). Muhammed et al. (1975) suggested that some amino acids, such as methionine, stimulate the growth of the bacterium, but Dahiya et al. (2007b) found a re-duction of C. perfringens populations in the cecum and the ileum of the birds fed high Met-containing diets. However, in the present experiment, there was no dif-ference in Met levels between the soy and potato diets (4.9 g/kg), although the fish diet had a slightly higher level of Met (6.2 g/kg). The differences in amino acid balance between the diets do not appear to be corre-lated to the observed differences in subclinical NE.

Amino acid availability was not determined in the present experiment, but may have differed between the 3 treatment groups. The determined trypsin inhibitor activity was higher in the PPC-based diet than in the soy- and fish-based diet. A high trypsin inhibitor ac-tivity in PPC has been found in other studies (Lee et al., 1985). Therefore, it is possible that the PPC-based diet may have had a low protein digestibility and in-creased the protein entering the distal small intestine and ceca of the chickens when compared with the soy and fish diets. Taciak and Pastuszewska (2007) found that a PPC-based diet with low protein digestibility modified the cecal fermentation of rats, giving high-er levels of ammonia and butyrate instead of acetate and propionate in comparison to a soybean meal diet. High cecal butyric acid concentrations have also been observed in birds with subclinical NE (Mikkelsen et al., 2009).

The protease inhibitors in PPC can vary consider-ably depending on variety and quality of potatoes used (Jadhav and Kadam, 1998) and also with the method of processing (coagulant, coagulation temperature, and drying temperature; Knorr, 1980, 1982). Heat coagula-tion at low pH is the method mainly used for industrial purposes (Ralet and Gueguen, 2000; Lokra et al., 2008) and this method of processing can denature the proteins considerably, resulting in a very low nitrogen solubility at low pH (Knorr, 1982). However, the protease in-hibitors are less heat-labile, more soluble (at the whole

pH range in the intestine), and have lower molecular weights (5 to 25 kDa) when compared with the storage proteins of potato (Ralet and Gueguen, 2000; Lokra et al., 2009). Potato also contains high molecular weight (85 kDa) protease inhibitors, which can be cleaved into several functional, lower molecular weight fragments by intestinal trypsin (Walsh and Strickland, 1993). The high trypsin inhibitor activity in the PPC-based diet in the present experiment indicates that some protease inhibitor activity remained in PPC.

The dietary protease inhibitors could also have di-rectly influenced the incidence of NE by intensifying the activity of C. perfringens toxins. Trypsin inhibitors stabilize functional properties of phospholipase c (α toxin) for longer when they are directly bound with the toxin (Sof’ina and Rakhimov, 1998), which could facili-tate the destruction of phospholipid cell membranes. In addition, the high trypsin inhibitor content and some other possible protease inhibitors in the PPC-based diet could have inhibited pancreatic trypsin, which is able to cleave α (phospholipase c) (Sato et al., 1978; Baba et al., 1992) and β 2 (NetB) (Fisher, 2006) toxins to inactivate the toxic activities. These are 2 major toxins in C. perfringens toxin type A, which has been mostly implicated in the etiology of NE (Engstrom et al., 2003) Therefore, the higher trypsin inhibitor ac-tivity could have aggravated toxin-induced intestinal necroses in the birds fed PPC-based diets than in the birds fed the fish and soy diets.

The glycoalkaloid content of industrial PPC has been estimated to be 257 mg/kg (Lokra et al., 2008) and this exceeds the widely accepted safety limit of 200 mg/kg in humans (Smith et al., 1996). Glycoalkaloids are not heat-labile (Jadhav and Kadam, 1998) and also have the ability to disrupt cell membranes, which could damage the intestinal epithelium (Smith et al., 1996). Clostrid-ium perfringens usually colonize on damaged intestinal epithelium (Parish, 1961). Therefore, the glycoalkaloids together with subclinical coccidiosis could have predis-posed the C. perfringens damage, which subsequently exacerbated leading to necroses of the intestinal mu-cosa. Dietary potato glycoalkaloids are known to dam-age the cells in contact tissues such as liver and blood (Smith et al., 1996); therefore, when they accumulate in the liver, glycoalkaloids could have contributed to the high incidence of liver lesions observed in potato diet-fed birds when compared with the birds fed the other 2 diets.

The potato protein diet had a higher level of insoluble NSP but a low soluble NSP content compared with the soy diet. The corn content in the potato diet was higher than in the soy diet. Corn has a considerable content of insoluble NSP (70 g/kg) in the total NSP (76.3 g/kg; Meng and Slominski, 2005), which is probably the reason for the higher NSP level in the PPC-based diet. The published literature is contradictory on the effect of NSP. The inclusion of cereals with high levels of NSP such as wheat (Branton et al., 1987), barley (Ka-ldhusdal and Hofshagen, 1992), and rye (Craven, 2000)


increases the incidence of NE. Carbohydrase enzyme addition mitigates the negative effects of a C. perfrin-gens challenge (Jia et al., 2009). Conversely, Riddell and Kong (1992) could not demonstrate these effects and Branton et al. (1997) concluded that added com-plex carbohydrates and fiber in broiler rations reduced the intestinal lesions of NE. However, it is unlikely that the relatively small differences in NSP (Table 1) con-tributed to the incidence of subclinical NE in this ex-periment.

The oil content of PPC is very low (0.52 g/kg) and this resulted in a low oil content in the potato-based diet (Table 1). The low oil content in the potato diet could have been a contributory factor for the increased incidence of subclinical NE. High lipid intakes have been associated with an increased bile acid synthesis and excretion (Reddy et al., 1977). The antibacterial effect of bile salts has been demonstrated by Inagaki et al. (2006). However, Knarreborg et al. (2002a) identi-fied C. perfringens and Enterococcus faecium as the 2 bacterial species that had the highest bile acid hydrolase activity in the small intestinal flora of broiler chickens. Dietary soy oil has been shown to significantly reduce the population of C. perfringens in the ileal microflora of broiler chickens compared with dietary animal fat (Knarreborg et al., 2002b). In this experiment, the oil composition of fish meal would have differed from other 2 diets, but the soy and potato diets had similar sources (corn and soy) of dietary oils. Therefore, the oil compo-sition could not have contributed much to the disease incidence.

In conclusion, this experiment successfully reproduced spontaneously occurring subclinical NE in broiler chick-ens fed all 3 experimental diets. There was a significant increase in the incidence of subclinical NE in the birds fed potato protein within a nutritionally complete diet in comparison to soy- or fish-based diets. The differ-ences in NE incidence were not consistent with the rela-tively small differences in amino acid balance or NSP content, but the high trypsin inhibitor activity, low oil content, and possible heat-resistant toxic compounds of the potato protein diet could have contributed to the increased incidence of subclinical NE in the PPC-based diet-fed birds.

ACKNOWLEDGMENTS

Financial support provided by the Commonwealth Scholarship Commission is gratefully acknowledged.

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