High stocking density as a predisposing factor for necrotic enteritis in broiler chicks

Abstract

Stocking density is a management factor which has critical implications for the poultry industry. The aim of the present study was to investigate the effect of high stocking density as a predisposing factor in an experimental model of necrotic enteritis in broiler chicks. The experimental challenge model included an oral inoculation with 10-fold dose of attenuated anticoccidial vaccine and multiple oral inoculations with a specific strain of Clostridium perfringens. Two hundred and forty as hatched day-old broiler chicks were randomly allocated to four treatment groups according to the following experimental design: group N, with normal stocking density (15 birds/m²) and no challenge; group D, with high stocking density (30 birds/m²) and no challenge; group P, with normal stocking density and positive challenge; and group DP, with high stocking density and positive challenge. From each bird, the intestine, gizzard and liver were collected and scored for gross lesions. The intestinal digesta was collected for pH and viscosity determination. One caecum from each bird was taken for microbiological analysis. The statistical analysis and evaluation of the experimental data revealed significant interaction effects between “stocking density” and “challenge”, regarding gross lesion scores in intestine and liver, pH values in jejunum, ileum and caeca as well as C. perfringens counts in the caeca (P ≤ 0.05). High stocking density in challenged birds increased the gross lesion score in the intestine (P ≤ 0.05), contrary to unchallenged birds. It can be concluded that high stocking density affects unfavourably the welfare and gut health of broiler chicks, predisposes to necrotic enteritis in a subclinical experimental model and increases further its importance as a management factor for the poultry industry.

Introduction

Stocking density is defined as the number of birds or the total live weight of birds (kg) in a house at the same time per square metre of usable area (Council Directive, 2007). It is a key issue for the economical result of broiler production, because higher returns can be obtained as the number of birds per unit space increases. In addition, as the number of birds per unit of housing space increases, the cost of labour, housing, fuel and equipment per bird is decreased (Estevez, 2007).

Currently, consumers perceive stocking density to be one of the most important factors that influence animal welfare (Vanhonacker et al., 2008). However, animal welfare issues are considered controversial, because it is generally assumed that any improvements in the area of animal welfare will have a negative impact on farm profitability. The issue of stocking density lies at the heart of this controversy because limiting space allowances in production systems can have a major economic impact for industry as revenues per unit of space increase linearly with stocking density. However, if stocking density is too high, poultry health and welfare are compromised (Puron et al., 1995; Heckert et al., 2002; Bessei, 2006).

Excessively high stocking density in broiler chicks has been associated with growth retardation and increased proportion of downgraded poultry products, as well as with bird's disturbance, stress, lameness and greater risk of health-related problems (Pettit-Riley & Estevez, 2001; Sanotra et al., 2001; Heckert et al., 2002; Feddes et al., 2002; Dozier et al., 2006; Thaxton et al., 2006). These negative implications are the primary reasons for the implementation of European legislation that limits stocking density allowances (Council Directive, 2007) and increase further the importance of stocking density as a management factor for poultry industry.

It is almost impossible to prevent or eliminate all the negative consequences of high stocking density. Nevertheless, a better understanding of what occurs as the number of birds per unit of floor space increases will help us to increase our awareness of potential problems and to take appropriate preventive actions. The influence of stocking density on growth rate and leg problems acts through its influence on litter and air quality. High moisture content of the litter enhances microbial activity, which in turn leads to increase of temperature and ammonia in broiler houses (Bessei, 2006). Although the effect of high stocking density on the deterioration of microclimate and litter of poultry houses has been studied extensively (Dozier et al., 2005, 2006; Bessei, 2006; Estevez, 2007; Lovanh et al., 2007), its role in the pathogenesis of intestinal diseases and the mechanisms underlying these effects has not received adequate attention from the scientific community.

Necrotic enteritis is described as an intestinal disease of high economic impact, which affects the health and welfare of broilers and may even pose a threat to public health (Van der Sluis, 2000; Van Immerseel et al., 2004). It is one of the most important enteric diseases in poultry and may be present as an acute clinical or subclinical disease. Its cost to the global poultry industry is estimated at about $2 billion annually, while the occurrence of subclinical necrotic enteritis is estimated to result in a 12% reduction in body weight and a 10.9% increase in feed conversion ratio compared to healthy birds (Van der Sluis, 2000; Skinner et al., 2010).

Necrotic enteritis is a very complex enteric disease. Although the Gram-positive, spore-forming bacterium Clostridium perfringens is well recognized as the causative agent, a wide range of host factors, environmental factors and pathogen factors can influence the occurrence of an outbreak and the severity of the disease (Williams, 2005; Shojadoost et al., 2012). Any factor that alters the intestinal microbiota and/or changes the physico-chemical properties of the intestinal digesta will disturb the balance of the intestinal ecosystem and could affect the pathogenesis of necrotic enteritis in broiler chicks (Van Immerseel et al., 2004; Pedersen et al., 2008; Tsiouris et al., 2013). Therefore, it has become increasingly important to bring to light all the management practices, which could predispose to the outbreak of the disease, and to understand the mechanisms underlying to these effects.

The effect of stocking density on the growth rate, body weight and feed conversion ratio has been well documented (Puron et al., 1995; Heckert et al., 2002; Estevez, 2007; Simitzis et al., 2012). Although increased stocking density has been suggested by some authors as a predisposing factor for necrotic enteritis (McDevitt et al., 2006), to the best of our knowledge, there are no studies reported in the literature investigating the role of stocking density as a specific predisposing factor for necrotic enteritis. Hence, the objective of this study was to evaluate the effect of high stocking density in a well-established experimental model of necrotic enteritis in broiler chicks.

Materials and Methods

Strains and cultivation.

C. perfringens strain 56 was isolated from the intestine of a broiler chicken with severe necrotic enteritis lesions. It belongs to toxinotype A (no b₂ or enterotoxin genes) and, in vitro, produces moderate amounts of alpha-toxin. The strain carries the netB gene and has been used previously to induce necrotic enteritis (Gholamiandehkordi et al., 2007; Tsiouris et al., 2013). To facilitate the detection of the inoculated strain in experimental birds, rifampicin-resistant mutants were isolated with the gradient technique as described by Pedersen et al. (2008) using Brain Heart Infusion broth (02–599, Scharlau Chemie S.A., Barcelona, Spain) containing rifampicin in a gradient concentration from 0 to 100 μg/ml (R 3501-1G, Sigma Aldrich Chemie GmbH, Steinheim, Germany). Before inoculation, the bacterium was cultivated for 24 h at 37°C in Brain Heart Infusion broth in an anaerobic atmosphere (Anaerocult A, 1.13829.0001, Merck KGaA, Darmstadt, Germany).

The attenuated anticoccidial vaccine (Paracox-5, MSD Animal Health, Boxmeer, the Netherlands) was used at 10-fold dose on the 18th day of age by oral gavage, using an insulin syringe with a small plastic catheter adapted to its opening. The vaccine contains live, attenuated oocysts of Eimeria acervulina, Eimeria maxima (two lines), Eimeria mitis and Eimeria tenella.

Gumboro vaccination (Nobilis Gumboro D78, MSD Animal Health) was used as an additional predisposing factor to necrotic enteritis.

Birds, housing and experimental diets.

Two hundred and forty 1-day-old Cobb 500 as hatched broiler chicks were obtained from a local commercial hatchery and were randomly allocated into four treatment groups of 60 chicks each. Birds in each group were placed in a pen with deep litter of wood shavings, which were previously sterilized in an autoclave at 121°C for 20 minutes (Cyclomotic control, EA605A).

Each group was kept in a specially designed experimental room (Unit of Avian Medicine, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki), where the temperature, relative humidity and lighting were controlled, following the recommendations of the breeding company. The standard stocking density for control groups was 15 birds per m² (Council Directive, 2007), while in high stocking density groups it was 30 birds per m². The reduction of rearing space for high stocking density experimental groups was achieved by the use of wired fence in the middle of the pen.

The experimental rooms, prior to bird's allocation, were cleaned and disinfected with broad spectrum (CID 20 and VIROCID, CID LINES, Ieper, Belgium) and specific disinfectants (Neopredisan 135-1, Menno Chemie-Vertrieb Gmbh, Norderstedt, Germany) against Eimeria spp. and C. perfringens.

Broilers in all groups were fed a specially formulated three phase ration (starter 1–9 days, grower 10–16 days and finisher 17–24 days), which included large quantities of wheat and rye and has been described elsewhere (Tsiouris et al., 2014). In the finisher ration, fishmeal replaced the soybean meal as the protein source. No antibiotic growth promoters or anticoccidial drugs were used. Feed and water were available ad libitum throughout the trial.

Experimental design and evaluation of performance.

The bird experimentation received the approval of the Veterinary Directorate of Thessaloniki and was conducted in compliance with the National Institutes of Health guidelines, Greek Government guidelines and local ethics committee. All birds were vaccinated against Gumboro disease on the 16th day of age via drinking water, after removing the water for 2 hours.

A simple factorial analysis of variance (ANOVA) design was applied with the factors being “stocking density”, with two levels (normal and high stocking density) and “challenge” with two levels (negative and positive). Thus the following treatment groups were established: group N, with normal stocking density and no challenge; group D, with high stocking density and no challenge; group P, with normal stocking density and positive challenge; and group DP, with high stocking density and positive challenge as described below.

For the experimental induction of subclinical necrotic enteritis the birds were orally challenged, with 1 ml of C. perfringens containing 4 × 10⁸ colony forming units (cfu) three times daily (at 09:00, 13:00 and 17:00) on the 17th, 18th, 19th and 20th day of age, and with 10-fold dose of attenuated anticoccidial vaccine on the 18th day of age.

The increased stocking density was applied from the first day of age until the end of the experiment. In order to keep the rearing space and number of drinkers and feeders available per bird constant throughout the experimentation, there were adjustments after each sampling day.

Mortality was recorded daily. From each experimental group 15 birds per sampling day were removed on the 21st, 22nd, 23rd and 24th day of age. The birds were euthanized by exposure to a rising concentration of carbon dioxide in an airtight container and were subject to necropsy. The gastrointestinal tract was removed immediately and divided in its anatomical parts (gizzard, duodenum, jejunum, ileum, caeca).

The body weight was measured on a pen basis on the 1st, 10th, 16th, 17th and 21st day of age, while the feed consumption, the average daily weight gain and the feed conversion ratio were calculated for the periods of the 10th–16th day and the 17th–21st day.

Gross lesion scoring system.

The intestine, gizzard and liver were examined macroscopically and scored for gross lesions. In particular, the intestines were scored for necrotic enteritis lesions following a 0–6 scoring system described by Keyburn et al. (2008). Birds with an intestinal lesion score greater than 1 were classified as necrotic enteritis positive. The gizzards were scored for gross lesions by the use of a 0–2 scale, described by Novoa-Garrido et al. (2006). The livers were scored for gross lesions using a 0–2 scale, as described elsewhere (Tsiouris et al., 2013).

pH and viscosity.

After euthanasia, the digesta of the duodenum, jejunum, ileum and cecum from each bird were immediately collected in separate tubes (15 ml) and vortexed, in order to obtain a homogenous content from each anatomical part of intestine per bird. The pH of the duodenum, jejunum, ileum and cecum from each bird was measured using a digital pH-meter (pH 315i, WTW Wissenschaftlich-Technische Werkstätten, Weilheim, Germany).

The viscosity of intestinal digesta was determined according to Tsiouris et al. (2013). The homogenous contents of the jejunum and the ileum, from each bird, were transferred to separate Eppendorf tubes (1.5 ml) which were centrifuged at 3000 × g for 45 min, in order to separate the particles from the liquid phase. Supernatants (0.5 ml) from each tube were taken and the viscosity was measured in a Brookfield DV-II+ PRO Digital Viscometer (Brookfield Engineering Laboratories, Stoughton, MA, USA). Two readings were taken from each sample and were represented in units of centipoise (cP).

Bacteriological culture.

One caecum from each bird was used for microbiological analysis. The quantification of C. perfringens counts was carried out as described elsewhere (Tsiouris et al., 2013). The samples for bacteriological examination of C. perfringens were cultured anaerobically on 5% sheep blood agar (Columbia blood agar, 01-034, Scharlau Chemie S.A., Barcelona, Spain) containing C. perfringens selective supplement (SR0093 Oxoid Ltd, Cambridge, UK) for 24 h. Two to four mm wide, circular, transparent colonies with typical “target” haemolysis (an inner zone of L-haemolysis and an outer zone of partial haemolysis) were diagnosed as presumptive C. perfringens. In case of doubt, aerobic and anaerobic secondary cultivation on blood agar and microscopy of Gram stained smears were used. The counts of C. perfringens were calculated. The figures from the bacterial counts were recorded as cfu and were transformed to the common logarithm per gram of caecal content in each sample.

Parasitological and histopathological examinations.

In order to confirm the absence or presence of coccidia in non-vaccinated and vaccinated groups, respectively, parasitological examination of faeces with Faust method was applied on the 10th, 17th and 20th day of age and microscopical examination of smears from intestinal mucosa on the 21st, 22nd, 23rd and 24th day of age.

Tissue samples from the duodenum, jejunum (proximal to Meckel's diverticulum) and ileum (proximal to ileo-caecal junction) were fixed in 4% buffered formaldehyde for 48–72 h. Coronal sections from the samples were embedded in paraffin by a routine procedure. Dewaxed 3–5 μm thick sections were stained with haematoxylin and eosin.

Statistical analysis.

All analyses of the experimental data were based on the cumulative values received from all sampling days. Both parametric and nonparametric statistical methods were applied for the statistical analysis and evaluation of the experimental data. As all forms of parametric tests are based on the assumptions that the within-groups data are samples drawn from normally distributed populations with equal variances, both formal tests (Shapiro-Wilk and Lilliefors tests) and graphical displays (normal probability and detrended normal probability plots) were performed for assessing departures from Gaussian distribution. Levene's test was also used to test the null hypothesis that the error variance of a dependent variable is equal across groups. For accessing the assumptions of normality and stability of variances, data were also transformed to log_e, log₁₀ or sqrt (Zolman, 1993). A fixed-effect hypothesis model of ANOVA (Simple Factorial ANOVA) was performed to determine possible significant interaction effects between “stocking density” and “challenge”, regarding the overall mean gross lesion scores in the intestine, liver and gizzard, the pH values in duodenum, jejunum, ileum and caeca, the viscosity values of intestinal contents in jejunum and ileum, as well as the caecal counts of C. perfringens. Pairwise comparisons of main effects or simple effects of challenge or/and stocking density were performed using the Least Significant Difference test and based on respective estimated marginal means. All analyses were conducted using the statistical software program SPSS for Windows (v. 19.0). Significance was declared at P ≤ 0.05. Back-transformed mean values are reported in the results.

Results

The effects of stocking density and necrotic enteritis challenge on the body weight, average feed consumption, average daily weight gain and feed conversion ratio per treatment group are presented to Table 1. These data were collected and calculated on a pen basis, and had not been statistically analysed and evaluated. Therefore, the effect of stocking density and necrotic enteritis challenge on performance is only indicative. Neither high stocking density, nor challenge caused mortality in birds.

Table 1. The effect of stocking density and necrotic enteritis challenge on the body weight, average feed consumption, average daily weight gain and feed conversion ratio per treatment according to age.

	Treatment groups
	Negative control	High stocking density	Challenged	High stocking density and challenged
Age	Group N	Group D	Group P	Group DP
Body weight (g)
1 day	45	40	45	40
10 day	120	110	120	110
16 day	340	370	340	350
17 day	380	370	365	350
21 day	650	660	605	620
Average feed consumption (g)
Period of 10–16 day	607	442	583	482
Period of 17–21 day	607	464	559	440
Average daily weight gain (g)
Period of 10–16 day	37	43	37	40
Period of 17–21 day	67	73	60	68
Feed conversion ratio
Period of 10–16 day	2.76	1.70	2.65	2.01
Period of 17–21 day	2.25	1.60	2.33	1.63

The body weight of birds in all experimental groups as well as the average daily weight gain was rather similar. The challenge of birds reduced the body weight on the 21st day of age, but it had no effect on the average feed consumption and feed conversion ratio. On the contrary, the increased stocking density reduced the average feed consumption and improved the feed conversion ratio.

The effect of stocking density and necrotic enteritis challenge on the number of birds with macroscopic necrotic enteritis lesions and on the overall mean gross lesion scores of the intestine, liver and gizzard are presented in Tables 2 and 3, respectively. As shown in Table 2, none of the birds of groups N and D developed necrotic enteritis gross lesions in the intestine, while 87% of birds in group DP and 70% of birds in group P developed necrotic enteritis gross lesions. The overall mean score of necrotic enteritis gross lesions in the intestine in non-challenged groups was below 1. On the other hand, the overall mean score of necrotic enteritis gross lesions in the intestine in challenged groups was above 1, confirming the efficacy of the experimental model that was used for the reproduction of necrotic enteritis.

Table 2. The effect of stocking density and necrotic enteritis challenge on the number of birds with macroscopic necrotic enteritis lesions^a according to treatment group and sampling day.

	Treatment groups
	Negative control	High stocking density	Challenged	High stocking density and challenged
Age	Group N	Group D	Group P	Group DP
21 day	0/15	0/15	7/15	13/15
22 day	0/15	0/15	9/15	11/15
23 day	0/15	0/15	14/15	13/15
24 day	0/15	0/15	12/15	15/15
Total	0/60	0/60	42/60	52/60

^a Birds with intestinal lesions score greater than 1 were classified as necrotic enteritis positive.

Table 3. The effect of stocking density and necrotic enteritis challenge on the overall mean gross lesion scores of the intestine, liver and gizzard per treatment group (mean ± standard deviation).

Treatments		Gross lesion score
Challenge	Stocking density	Intestine	Liver	Gizzard
Negative	Normal Group N	0.64 ± 0.48^A,a	0.66 ± 0.78^A,a	0.79 ± 0.79
	High Group D	0.58 ± 0.49^A,a	1.50 ± 0.77^B,a	0.83 ± 0.55
	Total	0.61 ± 0.48	1.08 ± 0.87	0.81 ± 0.68^a
Positive	Normal Group P	2.18 ± 1.57^A,b	0.93 ± 0.78^A,a	1.00 ± 0.68
	High Group DP	3.20 ± 2.02^B,b	1.22 ± 0.86^A,a	1.10 ± 0.62
	Total	2.69 ± 1.87	1.05 ± 0.82	1.05 ± 0.65^b
Total	Normal	1.41 ± 1.39	0.80 ± 0.78	0.89 ± 0.74^A
	High	1.89 ± 1.97	1.38 ± 0.82	0.96 ± 0.60^A
	Total	1.65 ± 1.72	1.07 ± 0.85	0.93 ± 0.67
2-way interaction challenge × stocking density		S	S	NS
Effect of challenge		S	NS	S
Effect of stocking density		S	S	NS

^a,b Mean values in the same column for the same stocking density with a different superscript differ significantly (P ≤ 0.05). [Effect of challenge]

^A,B Mean values in the same column for the same challenge with a different superscript differ significantly (P ≤ 0.05). [Effect of stocking density]

Significant interaction effects between “stocking density” and “challenge” were observed, regarding the gross lesion scores in intestine and liver. In particular, high stocking density increased significantly the gross lesion score in intestine (P ≤ 0.05) in challenged birds in contrast to unchallenged birds (P > 0.05). Challenge of birds caused a significant increase in the gross lesion score in the intestine both in challenged and unchallenged birds (P ≤ 0.05). In addition, high stocking density caused a significant increase in the gross lesion score in liver in unchallenged birds (P ≤ 0.05), while challenge of birds caused a significant increase on the gross lesion score in gizzard (P ≤ 0.05).

Significant interaction effects between “stocking density” and “challenge” were observed, regarding the pH values of the jejunum, ileum and caeca (Table 4). The high stocking reduced significantly the pH values of duodenum digesta (P ≤ 0.05). Similar results were obtained in challenged birds (P ≤ 0.05). The pH of jejunum and ileum digesta was significantly lower in challenged birds, both for normal and high stocking density (P ≤ 0.05). Challenge of birds caused significant reduction of the pH of jejunum and ileum digesta in high density groups (P ≤ 0.05). The high stocking density was associated with a significant increase of the pH values of caecum digesta in unchallenged birds (P ≤ 0.05).

Table 4. The effect of stocking density and necrotic enteritis challenge on the pH values of intestinal contents per treatment group (mean ± standard deviation).

Treatments		pH
Challenge	Stocking density	Duodenum	Jejunum	Ileum	Caeca
Negative	Normal Group N	6.11 ± 0.15	5.97 ± 0.13^A,a	6.94 ± 0.31^A,a	6.00 ± 0.36^A,a
	High Group D	5.96 ± 0.19	5.96 ± 0.13^A,a	7.06 ± 0.31^A,a	6.50 ± 0.32^B,a
	Total	6.04 ± 0.18^a	5.96 ± 0.13	7.00 ± 0.31	6.27 ± 0.42
Positive	Normal Group P	6.03 ± 0.17	5.89 ± 0.13^A,b	6.64 ± 0.36^A,b	6.22 ± 0.47^A,b
	High Group DP	5.91 ± 0.018	5.77 ± 0.14^B,b	6.04 ± 0.35^B,b	6.35 ± 0.36^A,a
	Total	5.97 ± 0.018^b	5.83 ± 0.14	6.35 ± 0.47	6.29 ± 0.42
Total	Normal	6.07 ± 0.16^A	5.93 ± 0.13	6.79 ± 0.36	6.11 ± 0.43
	High	5.93 ± 0.18^B	5.87 ± 0.16	6.58 ± 0.60	6.43 ± 0.35
	Total	6.01 ± 0.18	5.,90 ± 0.15	6.69 ± 0.51	6.28 ± 0.42
2-way interaction challenge × stocking density		NS	S	S	S
Effect of challenge		S	S	S	NS
Effect of stocking density		S	S	S	S

^a,b Mean values in the same column for the same stocking density with a different superscript differ significantly (P ≤ 0.05). [Effect of challenge]

^A,B Mean values in the same column for the same challenge with a different superscript differ significantly (P ≤ 0.05). [Effect of stocking density]

The effect of stocking density and necrotic enteritis challenge on viscosity values of jejunum and ileum digesta are presented in Table 5. Challenge of birds resulted in a significant reduction of viscosity values both in jejunum and ileum digesta (P ≤ 0.05). Similarly, high stocking density groups showed significantly lower viscosity values (P ≤ 0.05).

Table 5. The effect of stocking density and necrotic enteritis challenge on the viscosity values of intestinal contents per treatment group (mean ± standard deviation).

Treatments		Viscosity (CP)
Challenge	Stocking density	Jejunum	Ileum
Negative	Normal Group N	8.48 ± 2.82	12.77 ± 3.53
	High Group D	8.16 ± 2.17	13.63 ± 3.65
	Total	8.30 ± 2.46^a	13.08 ± 3.53^a
Positive	Normal Group P	6.32 ± 2.,32	11.32 ± 4.40
	High Group DP	4.76 ± 2.36	10.81 ± 4.45
	Total	5.64 ± 2.44^b	11.07 ± 4.37^b
Total	Normal	7.31 ± 2.76^A	12.03 ± 4.01^A
	High	6.75 ± 2.80^B	11.82 ± 4.33^B
	Total	7.03 ± 2.78	11.94 ± 4.12
2-way interaction challenge × stocking density		NS	NS
Effect of challenge		S	S
Effect of stocking density		S	S

^a,b Mean values in the same column for the same stocking density with a different superscript differ significantly (P ≤ 0.05). [Effect of challenge]

^A,B Mean values in the same column for the same challenge with a different superscript differ significantly (P ≤ 0.05). [Effect of stocking density]

Significant interaction effects between “stocking density” and “challenge” were observed regarding the C. perfringens counts in the caeca (Table 6). High stocking density caused a significant increase on the C. perfringens counts in the caeca in unchallenged groups (P ≤ 0.05). Moreover, challenge of birds caused a significant increase of C. perfringens counts in the caeca, both in normal and high stocking density (P ≤ 0.05).

Table 6. The effect of stocking density and necrotic enteritis challenge on the caecal counts of C. perfringens per treatment group (mean ± standard deviation).

Treatments		Log10 C. perfringens (cfu)
Challenge	Stocking density	Caecum
Negative	Normal Group N	4.35 ± 0.98^A,a
	High Group D	5.45 ± 1.13^B,a
	Total	4.92 ± 1.19
Positive	Normal Group P	6.26 ± 1.12^A,b
	High Group DP	6.14 ± 1.39^A,b
	Total	6.20 ± 1.25
Total	Normal	5.36 ± 1.42
	High	5.80 ± 1.31
	Total	5.59 ± 1.38
2-way interaction challenge × stocking density		S
Effect of challenge		S
Effect of stocking density		S

^a,b Mean values in the same column for the same stocking density with a different superscript differ significantly (P ≤ 0.05). [Effect of challenge]

^A,B Mean values in the same column for the same challenge with a different superscript differ significantly (P ≤ 0.05). [Effect of stocking density]

Results of the faecal parasitological examination on the 10th and 17th day of age were negative in all groups, indicating the absence of Eimeria spp. infection. Thereafter, the faecal parasitological examination and microscopical examination of smears from intestinal mucosa were negative in unchallenged groups and positive in challenged groups.

The histopathological lesions of the duodenum, jejunum and ileum in birds challenged with C. perfringens and 10-fold dose of attenuated anticoccidial vaccine were compatible with necrotic enteritis lesions. Lesions were mainly located in the jejunum and ileum and to a limited extent in the duodenum. There was severe necrosis of the intestinal mucosa, with an abundance of fibrin admixed with cellular debris adherent to the necrotic mucosa, where large clusters of bacteria were detected. Villus fusion and shortening, as well as marked infiltration of heterophilic granulocytes were also observed.

Discussion

Stocking density is a management decision based on economics and welfare legislation. The main reason to apply high stocking density of broilers is to maximize the profit per square metre of poultry farm, by reducing costs per bird associated with labour, fuel, housing and equipment. In addition to the broiler performance, uniformity, product quality and profit considerations, stocking density also has important welfare implications. In particular, high stocking density increases the environmental pressures on the chicks and is associated with deterioration of performance, health and welfare of broiler chicks (Bessei, 2006; Dozier et al., 2006; Estevez, 2007).

In the present study, the challenge of birds decreased the body weight, while the high stocking density has no effect on the body weight. The reduction of body weight in challenged birds is attributed to the gut lesions and to the intestinal inflammation as a result of challenge with C. perfringens and Eimeria spp. (Gholamiandehkordi et al., 2007; Shojadoost et al., 2012). According to Estevez et al. (1997) and to Sørensen et al. (2000) there is decline in body weight when space per bird drops below 0.066 or 0.063 m². Although in the present study the space per bird was 0.033 birds per m², there was no reduction of body weight. However, if we express the stocking density in Kg per m², it is clear that the stocking density at the age of 24 days was not exceeding the 20 Kg per m², which is much lower than limit of the Council Directive (2007). In the present study, although the stocking density was 100% higher than control groups, the percentage of the area covered by the birds as well as the total body weight, was not so high, because the birds were on 24th day of age and weighed only 650 grams. The lack of effect of stocking density on body weight for the growing period that we observed in the present study is in agreement with previous studies (Bessei, 2006; Dozier et al., 2006).

Stocking density reduced feed consumption of birds and resulted in improvement of feed conversion ratio, since body weight was not affected. These results are in line with the findings of the study of Dozier et al. (2006), who observed a similar response to increasing stocking density during the early period of growth. The improvement in growth responses due to increasing stocking density is not clearly understood and might be related to metabolic heat production. As the stocking density is increased, the production of non-evaporative heat is increased, while the energy needed for heat production is decreased. In addition, during the neonatal period (approximately the first two weeks) chicks do not attain the homeothermic condition and are poorly insulated (Whittow, 1999). The saving of energy can be used for growth and may result in improvement of feed conversion ratio for these young birds. Moreover, we observed that an increased number of birds in high density groups were consuming spilled feed outside of the feeders, which improved further the feed conversion ratio. The feed spillage explains the high feed conversion ratio observed in control groups.

In challenged groups, the percentage of necrotic enteritis positive birds as well as the score of necrotic enteritis lesions in the intestine was significantly higher in the high stocking density group. Moreover, in unchallenged groups, the high stocking density was associated with a significantly higher number of C. perfringens in the caeca. The mechanisms underlying on the effect of high stocking density on the occurrence and severity of necrotic enteritis are not fully understood. One plausible explanation could be the immunosuppression that high stocking density causes. According to Heckert et al. (2002) there is a significant reduction in bursa weights and bursa weight/body weight ratios with increasing densities (ranging from 0.100 to 0.050 m² per bird), suggesting a higher degree of stress particularly for birds at densities above 0.066 m² per bird (15 birds per m²). The stress of birds reared under high stocking density can negatively affect the humoral immune system, compromising their ability to produce antibodies and to overcome infections.

Poultry litter, a mixture of poultry manure and different bedding materials, is an environmental ecosystem with a considerable range of characteristic microbial populations (Lovanh et al., 2007). Higher stocking density increases nitrogen and moisture level in the litter, downgrading the quality of poultry litter and creating a favourable environment for the microbial activity. Physico-chemical properties and microbiota of poultry litter have been shown to be factors that influence the intestinal ecosystem, since they affect colonization of broiler chicks by enteric pathogens, such as C. perfringens and Eimeria spp., facilitate horizontal transmission and increase pathogen shedding (Bessei, 2006; Burkholder et al., 2008; Guardia et al., 2011). The effect of stocking density on poultry litter quality and microbiota thus could be another plausible explanation for its predisposing role in necrotic enteritis.

According to Løvland and Kaldhusdal (1999), in cases of necrotic enteritis the high counts of C. perfringens in the intestine increase the risk of clostridia gaining access to biliary ducts and possibly the portal bloodstream through a damaged intestinal mucosa. In the present study, the challenge of birds with C. perfringens and E. maxima was associated with an overall higher score of gross lesions in liver as well as caecal counts of C. perfringens.

pH of intestinal digesta is one of the major gastrointestinal factors that influence the nutrient bioavailability and the intestinal microbiota, and vice versa (Pang & Applegate, 2007; Hajati & Rezaei, 2010). Therefore, the manipulation of digesta pH in broiler chickens could act as a tool to manage the potential for optimum gut health and maximum nutrient absorption (Gabriel et al., 2006; Morgan et al., 2014). Moreover, digesta pH has been used as a tool for the prevention of intestinal pathogens such as Salmonella spp, Campylobacter spp. and C. perfringens (Van Immerseel et al., 2006; McReynolds et al., 2007; Jansen et al., 2014). The main factors that determine the pH of intestinal digesta are the gastrointestinal secretions and the volatile fatty acids, produced by intestinal microbiota (Whittow, 1999). Thus, any factor altering the intestinal microbiota could have an effect on the pH of intestinal digesta.

Stocking density has a significant effect of on commensal bacterial microbiota of the digestive tract of young chickens and could affect the pH of intestinal digesta (Guardia et al., 2011). The results of the present study are in line with the above study, since high stocking density was associated with significant changes in the pH of intestinal digesta. The change in pH of intestinal digesta was probably the result of litter downgrading, which affects the intestinal microbiota. High humidity, warm temperatures and high pH favour the microbial proliferation in the litter (Bessei, 2006; Guardia et al., 2011). Moreover, litter downgrading has been shown to be correlated with higher concentrations of corticosteroids in the birds' faeces, suggesting that these birds are probably subjected to higher levels of stress, which also affects the intestinal microbiota (Dawkins et al., 2004).

In the present study, intestinal viscosities were rather high compared with field and experimental results reported in the literature (Bedford & Classen, 1992). The increased viscosity is caused by water-soluble non starch polysaccharides (NSPs) mainly from cereals used in the rations (wheat and rye). NSPs have been shown to cause digestive disorders and to predispose to necrotic enteritis in broiler chicks (Alzueta et al., 2003; McDevitt et al., 2006). The mechanisms by which viscosity affects nutrient digestion and absorption are rather complex. It is assumed that increased intestinal viscosity depresses nutrient digestibility by interference with the diffusion of digestive enzymes to their substrates and with the movement of digesta across the intestinal lumen. In addition, a higher intestinal viscosity increases the average retention time of the digesta, which is likely to create a favourable environment for bacterial activity, because the flow of digesta is reduced and the amount of undigested material in the intestinal tract is increased. This gives more time for the microbes to colonize the small intestine and results in greater competition with the host for nutrients (Alzueta et al., 2003; Shojadoost et al., 2012; Tsiouris et al., 2013).

Challenge of birds with coccidia and C. perfringens was associated with a reduction of the viscosity of jejunum digesta. Waldenstedt et al. (2000) already showed that coccidial infection decreases intestinal digesta viscosity. It is unclear whether the reduction in viscosity observed in the present study was to be attributed solely to the coccidia or whether C. perfringens also played a role. High stocking density did not affect the viscosity of intestinal digesta.

Although C. perfringens is the aetiological factor of necrotic enteritis in broiler chicks, it is isolated from 75% to 95% of healthy chicks. It colonizes mostly in caeca (10²–10⁴cfu/g; Shojadoost et al., 2012) and as a consequence, it can be found in the poultry litter (Novoa-Garrido et al., 2004). In the present study, high stocking density increased the caecal counts of C. perfringens in the unchallenged group, but had no significant effect in the challenged group. In chicken production, litter is a potential reservoir and transmission vehicle for pathogens and facultative pathogens. In particular, as the stocking density increases, the bacterial counts per square metre of poultry litter are increased (Burkholder et al., 2008; Guardia et al., 2011). This might contribute to the increased number of C. perfringens in caeca. According to Novoa-Garrido et al. (2004) acidification of litter results in significantly lower counts for C. perfringens in the caeca and the ileum in chickens. Moreover, the increased pH of caecal digesta of birds reared at high stocking density creates a favourable environment for the proliferation of C. perfringens (McReynolds et al., 2007; Garde et al., 2014). The high stocking density had no significant effect on caecal counts of C. perfringens, in challenged group, since was already high. This might be attributed to the challenge of birds with multiple oral inoculations with 4 × 10⁸cfu of C. perfringens, which was extremely high and masked the effect. A better understanding of the community structure and identity of the microbial populations in poultry litter will give us the ability to adjust the management practices that would reduce pathogen populations.

In conclusion, in the present study, stocking density had a significant effect on the viscosity of intestinal content, on the caecal C. perfringens and on the percentage of necrotic enteritis positive birds as well as on the severity of the necrotic lesions. The predisposing effect of high stocking density on necrotic enteritis increases its importance as a management factor and it is an additional criterion that amplifies its negative effect on the performance, health and welfare of broiler chicks. Further studies are needed in order to elucidate the effect of high stocking density on other intestinal pathogens and the mechanisms underlying these effects.

Acknowledgements

The authors express their gratitude to Professor F. Haesebrouck (Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Belgium) for generously and kindly providing the C. perfringens. Moreover, the authors are also grateful to veterinarians Sarakatsano Ioanni and Deligeorgi Ioanni for their excellent collaboration and assistance and to Mr Moulto Serafim for his technical support. Feed mills “Stravaridis S.A.” is thanked for feed formulation, while hatchery “Tzotzas-Koutsou S.A.” is thanked for the provision of day old chicks.