The effect of cold stress on the pathogenesis of necrotic enteritis in broiler chicks

Abstract

Cold stress is a physical environmental stressor with significant effect on the poultry industry. The aim of the present study was to investigate the effect of cold stress as a predisposing factor for 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. Birds were either challenged or not as described above, and either exposed or not to repeated cold stress (15°C for 12 h/day for 4 days). From each bird, intestinal gross lesions were scored and intestinal digesta pH and viscosity were measured. C. perfringens was counted in the caecum. The statistical analysis and evaluation of the experimental data revealed that the cold stress in challenged birds significantly increased the incidence and the severity of necrotic enteritis lesions (Ρ ≤ 0.05), while causing no lesions in unchallenged birds. Moreover, the cold stress caused a significant increase (Ρ ≤ 0.05) in the pH and C. perfringens counts in the caeca. The study provides evidence that cold stress increased the susceptibility to necrotic enteritis in a subclinical experimental model and thus should be regarded as a physical environmental stressor that could significantly affect the welfare, health and intestinal ecosystem of broiler chicks.

Introduction

Cold is one of the most important physical environmental stressors, which significantly affects the health and welfare of poultry (Hangalapura, 2006). It is one of the leading reasons for birds’ migration to more environmentally friendly regions of the world, in order to breed and feed their progeny (Dawson et al., 1983).

Fuel cost for heating of poultry houses is a major economic factor for the poultry industry and affects significantly the profit for the producer. It is estimated to be between 0.05 and 0.30 euro per bird, depending on the fuel price and the season as well as on the heat insulation and the heating system of the poultry houses. This cost makes cold one of the main barriers limiting the development of the poultry husbandry in cold regions of the world. Also, the heating systems in countries of the Northern Hemisphere during the winter season are insufficient to overcome the cold, thus resulting in cold stress for the birds, even in modern design poultry houses. Furthermore, with the increasing success of outdoor rearing systems, cold stress will become even more of an issue (Hangalapura, 2006; Campo et al., 2008).

Cold stress can increase the susceptibility of birds to a number of infectious and non-infectious agents (Sato et al., 2002; Huff et al., 2007). This has been documented, for example, for colibacillosis in turkey poults (Huff et al., 2007). Moreover, low temperature is one of the main triggers for pulmonary hypertension syndrome in modern fast-growing broilers (Sato et al., 2002). The mechanism underlying the predisposing effect of cold stress on disease susceptibility is not fully explained. According to Chen et al. (2012), cold stress can affect the function of the neuroendocrine, the anti-oxidation and the immune systems. In addition, birds raised under low temperature exhibited oedema, hyperaemia, haemorrhage and epithelial damage in the intestinal mucosa (Zhang et al., 2011).

Necrotic enteritis is described as a disease of high economic impact (Van der Sluis, 2000), which affects the health and welfare of broilers and also poses a threat to public health (Van Immerseel et al., 2004). It is caused by specific (NetB positive) strains of Clostridium perfringens, multiplying and releasing toxins in the intestinal tract of chicks. It is well accepted that necrotic enteritis is a multi-factorial disease in which the development of C. perfringens-induced necrotic lesions is triggered by the presence of one or more predisposing factors. Thus, any factor which predisposes to necrotic enteritis is regarded as very important.

Experience from the field and epidemiological studies have pointed out several such factors (Hermans & Morgan, 2007). These include environmental factors, nutritional factors and infectious agents. A number of these factors have been tested and confirmed under controlled experimental conditions. These include high stocking density (Tsiouris et al., 2015), presence of moderately high concentrations of deoxynivalenol in the feed (Antonissen et al., 2014) and coccidiosis (Williams, 2005). Surprisingly, chronic heat stress tends to protect the birds from necrotic enteritis (Calefi et al., 2014).

The effect of cold stress on the performance, hypertension syndrome, welfare and immune system in broiler chicks has been well documented (Sato et al., 2002; Hangalapura et al., 2003, 2004; Hangalapura, 2006; Özkan et al., 2007; Yang et al., 2014). However, the influence of cold stress in relation to necrotic enteritis in a well-established, experimental infection model has not been studied before. Hence, the aim of the present study was to evaluate the effect of cold stress on the pathogenesis 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 (Gholamiandekhordi et al., 2007; Tsiouris et al., 2013). To facilitate the detection of the inoculated strain in experimental birds, a rifampicin-resistant mutant was selected using 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 the chick's 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).

Birds and housing

Two hundred and forty 1-day-old Ross 308 broiler chicks were obtained from a local commercial hatchery and were randomly allocated into four treatment groups of 60 chicks each. There were two replicates per treatment group. 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 min.

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 (Aviagen, 2014). The standard stocking density for all groups was 15 birds/m² or 33 Kg/m² (Council Directive 2007/43/EC). The room temperature was measured at chick level. Following the recommendation of the breeding company throughout the rearing period, the temperature on day 17 was 25°C. In the experimental low temperature groups, however, the room temperature was reduced to 15°C from day 17 until day 20 for 12 h/day (09:00–21:00). Air conditioning devices and ventilation system were used, in order to decrease the room temperature to 15°C. Relative humidity of experimental rooms ranged between 50% and 70% from 2 weeks onward. Temperature and humidity were monitored in each room at two locations at bird level using a temperature—humidity record system (HOBO UX100-003 Temperature/Relative Humidity data logger, Onset Computer Corporation, Bourne, MA, USA). The experiment was conducted during the winter.

The experimental rooms, prior to birds' 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.

Experimental diets

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., 2013). In the finisher ration, fishmeal replaced the soybean meal as the protein source, in order to predispose to necrotic enteritis. No antibiotic growth promoters and anticoccidial drugs were used. Feed and water were available ad libitum throughout the trial.

Experimental design

The bird experimentation received the approval of Veterinary Directorate of Thessaloniki and was conducted in compliance with the National Institutes of Health guidelines, Greek Government guidelines and local ethics committee.

An experimental subclinical model for necrotic enteritis was used as previously described (Tsiouris et al., 2013). Briefly, all birds were vaccinated against Gumboro disease, while challenge of birds included both C. perfringens and 10-fold dose of attenuated anticoccidial vaccine.

The treatment groups used in this study were group N, which served as the negative control, group C, where birds were reared in low temperature, group P, where birds were experimentally challenged with C. perfringens and 10-fold dose of attenuated anticoccidial vaccine and group CP, where birds were both reared in low temperature and challenged, as described above.

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

Gross lesion scoring

The intestine was macroscopically examined and scored for gross lesions. In particular, the intestines were macroscopically examined and scored for necrotic enteritis lesions following a 0–6 scoring system described by Keyburn et al. (2008). Birds with intestinal lesions score greater than 1 were classified as necrotic enteritis positive.

pH

After euthanasia, the digesta of the duodenum, jejunum, ileum and caecum 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 caecum from each bird was measured using a digital pH-meter (pH 315i, WTW Wissenschaftlich-TechnischeWerkstätten, Weilheim, Germany).

Viscosity

The viscosity of intestinal digesta was determined as described elsewhere (Tsiouris et al., 2013). The homogenous content of the jejunum and the ileum, from each bird, was added to separate Eppendorf tubes (1.5 ml). The tubes were centrifuged at 3000 × g for 45 min, in order to separate the feed 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 examination

One caecum from each bird was used for microbiological analysis. The quantification of C. perfringens counts in caecum 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 millimetre 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 per gram of caecal content in each sample were calculated. The figures from the bacterial counts were recorded as colony forming units and were transformed to the common logarithm.

Parasitological examination

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 days of age and microscopical examination of smears from intestinal mucosa on the 21st, 22nd, 23rd and 24th days of age.

Histopathological examination

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

Both parametric and non-parametric statistical methods were applied for the statistical evaluation of the experimental results. For each variable, we used data for analyses from all sampling days and not from each sampling day separately. For assessing the assumptions of normality and stability of variances, data were transformed to log₁₀ or square root. In the case of normality and variance's homogeneity, a one-way analysis of variance was performed, to evaluate possible significant effects of treatment on the population of C. perfringens in caeca, as well as on the pH values of the duodenum, jejunum, ileum and caeca and viscosity values of the jejunum and ileum. Differences between mean values of specific treatments were evaluated using Duncan's new multiple-range test. Where assumptions about either variability or the form of the populations distribution were seriously violated, with or without transformed data, the Kruskal–Wallis non-parametric test was applied to evaluate treatment-dependent differences, while the non-parametric Wilcoxon rank sum test (Mann–Whitney U-test) was used to determine whether the medians score values of specific treatments were different enough to conclude that the respective samples were drawn from different populations. The Kruskal–Wallis non-parametric test was applied also to evaluate treatment-dependent differences on gross lesions’ scores in the intestine, while the non-parametric Wilcoxon rank sum test (Mann–Whitney U-test) was used to evaluate differences between median score values of specific treatments. The Chi-square procedure was also applied to test treatment-dependent associations of necrotic enteritis. All analyses were conducted using the statistical software program SPSS for Windows (v. 15.0). Significance was declared at P ≤ 0.05, unless otherwise noted (P ≤ 0.10). Back-transformed mean values are reported in the results.

Results

Neither cold stress nor challenge caused mortality in birds. The effect of cold stress, necrotic enteritis challenge model and its combination on the number of birds with macroscopic necrotic enteritis lesions and on the overall mean gross lesions score of the intestine is presented in Table 1. None of the birds of groups N and C developed necrotic enteritis gross lesions in the intestine, while 62% of birds (37/60) in group P and a significantly higher (P ≤ 0.10) percentage of birds in group CP (45/60) 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. Moreover, the overall median score of necrotic enteritis gross lesions in the intestine was significantly lower (Ρ ≤ 0.05) in group P compared to group CP, denoting that the two sets of data are from populations with different distribution functions.

Table 1. The effect of cold stress, necrotic enteritis challenge and its combination on the number of birds with macroscopic necrotic enteritis lesions as well as on the necrotic enteritis lesion score (x¯± SEM) per treatment group and age.

Age (days)	Treatment groups
	Negative control	Cold stressed	Challenged	Cold stressed and challenged
Necrotic enteritis positive birds
21	0/15	0/15	9/15	8/15
22	0/15	0/15	8/15	10/15
23	0/15	0/15	11/15	14/15
24	0/15	0/15	9/15	13/15
Total	0/60	0/60	37/60^A	45/60^B
Necrotic enteritis lesion score
Score	0.64 ± 0.07^a	0.40 ± 0.07^a	2.19 ± 0.23^b	3.79 ± 0.30^c

^{A, B} Necrotic enteritis frequencies in the same row with a different superscript differ significantly (P ≤ 0.05).

^{a, b, c} Necrotic enteritis scores in the same row with a different superscript differ significantly (Ρ ≤ 0.05).

Cold stress and challenge of birds had a significant (Ρ ≤ 0.05) effect on the pH and viscosity values of the intestinal digesta as well as on the C. perfringens caecal counts (Table 2). Cold stress caused a significant increase of the pH values of the caeca (Ρ ≤ 0.05). The challenge of birds caused a significant reduction of pH values of the jejunum and ileum (Ρ ≤ 0.05) and a significant increase of pH values of the caecum (Ρ ≤ 0.05), while the combination of cold stress and challenge of birds caused similar results, with the exception that this combination also significantly reduced the pH values of the duodenum (Ρ ≤ 0.05). The cold stress as well as the challenge of birds significantly reduced the viscosity of the jejunum digesta (Ρ ≤ 0.05), while the combination of challenge and cold stress reduced further the viscosity of the jejunum digesta (Ρ ≤ 0.05). Furthermore, cold stress was associated with a significant increase of the caecal C. perfringens counts (Ρ ≤ 0.05), while the challenge of birds as well its combination with cold stress increased further the caecal C. perfringens counts (Ρ ≤ 0.05).

Table 2. The effect of cold stress, necrotic enteritis challenge and its combination on the pH and viscosity values of intestinal contents and caecal C. perfringens per treatment group (x¯± SEM).

Anatomical Part	Treatment groups
	Negative control	Cold stressed	Challenged	Cold stressed and challenged
pH
Duodenum	6.12±0.03^ab	6.13±0.02^a	6.04±0.03^bc	6.00±0.03^c
Jejunum	5.97±0.02^a	6.03±0.03^a	5.89±0.02^b	5.87±0.03^b
Ileum	6.94±005^a	7.03±0.05^a	6.65±0.07^b	6.60±0.07^b
Caeca	6.00±0.06^a	6.27±0.07^b	6.23±0.08^b	6.25±0.07^b
Viscosity (cP)
Jejunum	8.48±0.55^a	7.64±0.45^b	6.33±0.42^b	5.18±0.45^c
Ileum	12.77±0.83^a	12.04±0.68^a	11.33±1.01^a	10.57±0.86^a
Log10 C. perfringens (colony forming units)
Caecum	4.36±0.17^a	5.45±0.20^b	6.26±0.19^c	6.70±0.18^c

^{a, b, c} Mean values in the same row with a different superscript differ significantly (Ρ ≤ 0.05).

The results of the faecal parasitological examination on the 10th and 17th days 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 groups N and C, and positive in groups P and CP.

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, diffuse coagulative 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

Cold stress is a physical environmental stressor which affects performance, health and welfare of broiler chicks and thus has a significant impact on the poultry industry (Sato et al., 2002; Hangalapura, 2006; Huff et al., 2007; Yang et al., 2014).

This study demonstrates that cold stress predisposes broiler chicks to necrotic enteritis. This can be concluded from the significantly increased number of necrotic enteritis positive birds and the more severe necrotic enteritis lesions, as well as from the higher caecal C. perfringens counts in challenged and cold stressed birds compared to challenged birds. The mechanisms underlying this effect have not been fully elucidated. One plausible explanation could be the immunosuppression of birds, when exposed to low temperatures (Regnier & Kelley, 1981; Gross & Siegel, 1988). Exposure of birds to low temperatures causes suppression of humoral (Gross & Siegel, 1988) and cellular immunity (Regnier & Kelley, 1981). However, according to Hangalapura (2006), cold stress of birds stimulates the cellular immunity, but not the humoral, although it increases the IL-12β and the IL-4. It is reasonable to suggest that the type and duration of cold stress treatments as well as the genetic background of the birds underlie these divergent results.

Cold stress can modulate immune responses via two potential pathways: a bioenergetic and an endocrine pathway. However, these two pathways may not be completely independent from each other. In particular, for the bioenergetic pathway both the thermoregulation and the immune system derive internal energy from the same source. During cold stress a substantial amount of energy is diverted from productive and immune functions to thermoregulation; thus feed intake will increase and immunosuppression will ensue, in order to compensate for heat production (Demas et al., 1997). Regarding the endocrine pathway, cold stress affects the function of the hypothalamic-pituitary axis, which results in the alteration of serum adrenal and thyroid hormone levels. As these hormones have immunomodulating effects, changes in their levels may affect the immune system either directly or indirectly (Madden & Felten, 1995; Hangalapura, 2006).

The response of birds to cold stress and challenge depends on the duration of cold stress and the timing of challenge in relation to the onset of cold stress (Zhao et al., 2014). If challenge is performed long after the onset of cold stress, the birds can adjust quickly to temperature variation, resulting in no effect. In particular, according to Shinder et al. (2002), the birds adjusted as soon as within 24 h after cold stress application, based on blood concentration of corticosterone. In the experimental design of this study, the room temperature was reduced daily (mimicking diurnal temperature changes) and cold stress was applied almost simultaneously with the challenge of birds.

The effect of cold stress as a predisposing factor for necrotic enteritis in broiler chicks, as observed in the present study, is in agreement with the results of Kaldhusdal & Skjerve (1996), who reported that prevalence of necrotic enteritis in Norway was higher during the cold season. However, the results of this study as well as the above observation are in contrast to the results of Long (1973) and Hermans & Morgan (2007), who reported that the prevalence of necrotic enteritis was higher during the warm season. This divergence indicates that the effect of season on the prevalence of necrotic enteritis does not depend only on temperature, thus making it difficult to separate the effect of cold stress in field conditions. Other factors, such as nutrition (e.g. mycotoxins level) and management (e.g. litter condition), which may also be affected by season, may be involved (Bryden, 2012; Aviagen, 2014).

The pH of the intestinal digesta depends on the feed composition and subsequently on the fermentation activity of intestinal microbiota (Guardia et al., 2011; Tsiouris et al., 2014). Thus, any drastic change in the intestinal microbiota could have an effect on the pH of intestinal digesta. In the present study, cold stress was associated with a significant increase in the pH of caecal contents as well as an increase in the population of C. perfringens in the caeca in unchallenged birds. Increased pH of caecal content favours the multiplication of C. perfringens (Juskiewicz et al., 2004; Lan et al., 2005). Challenge of birds as well as its combination with cold stress reduced the pH of duodenal jejunal and ileal digesta. The reduction of pH in the small intestine could be the result of a mixed challenge with Eimeria spp. and C. perfringens (Williams, 2005).

The viscosity of intestinal digesta was relatively high in all experimental groups. This is attributed to the feed formulation, which contained a high percentage of wheat and rye (Kaldhusdal & Skjerve, 1996). Wheat and rye are rich in non-starch polysaccharides, which are not digestible and result in increased viscosity (Riddell & Kong, 1992). 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.

In the present study, the cold stress increased the caecal C. perfringens counts significantly. The increased C. perfringens counts in caeca in the cold stress group could be the result of the increased pH of caecal digesta and the suitable environment for the proliferation of C. perfringens. In addition, there may be a direct effect of increased feed consumption (data not shown) and the reduced digestive efficacy, which subsequently increased the content volume in the lower intestinal tract. Since the requirements for growth of C. perfringens include more than 11 amino acids as well as many growth factors and vitamins, the specific ration, with high levels of animal protein and non-starch polysaccharides, provided the necessary growth substrate for the extensive proliferation of these bacteria (Van Immerseel et al., 2004). High populations of C. perfringens per se do not lead to an outbreak of necrotic enteritis. However, according to Kaldhusdal et al. (1999) the risk for necrotic enteritis outbreak increases as the population of C. perfringens in caeca is increased.

In conclusion, cold stress in broiler chicks increased the number of birds with lesions as well as the severity of necrotic enteritis lesions and the caecal C. perfringens counts after experimental challenge of birds with C. perfringens and 10-fold dose of live attenuated anticoccidial vaccine. The involved mechanisms are probably the immunosuppression and the alteration of intestinal microbiota of birds. The study provides evidence that cold stress increased the susceptibility of birds to necrotic enteritis in a subclinical experimental model and should be regarded as a physical environmental stressor that could significantly affect the performance, welfare, health and intestinal ecosystem of broiler chicks. Further studies are needed in order to elucidate the effect of cold stress on the intestinal microbiota, the gut associated lymphoid tissue and on the mechanisms underlying these effects.

Acknowledgement

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 Sarakatsanos Ioannis and Deligeorgis Ioannis for their excellent collaboration and assistance and to Mr Moultos 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.