Nutritional digestive disturbances in weaner rabbits

Animal Feed Science and Technology 173 (2012) 102–110

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Nutritional digestive disturbances in weaner rabbits夽 J.C. de Blas a,∗ , S. Chamorro a , J. García-Alonso a , P. García-Rebollar a , A.I. García-Ruiz b , ˜ a M.S. Gómez-Conde a , D. Menoyo a , N. Nicodemus a , C. Romero a , R. Carabano a b

Departamento de Producción Animal, Universidad Politécnica de Madrid, ETS Ingenieros Agrónomos, 28040 Madrid, Spain Nutreco Poultry and Rabbit Research Centre, 45950 Casarrubios del Monte, Spain

a r t i c l e

i n f o

Keywords: Gut microbiota Dietary composition Feed restriction Maternal effects Environment

a b s t r a c t Weaning is considered a stressful period for rabbits caused by abrupt changes of diet and environment. An immature immune system together with a transient decrease of nutrient digestibility places the animal in adverse conditions where digestive pathologies might take place. In recent years the European ban on antibiotic type growth promoters in animal feeds has even complicated weaning management for farmers because of the widespread and incidence of emerging animal diseases. Since the first outbreaks in 1997 the epizootic rabbit enteropathy (ERE) has become a threat to the industry as it can cause between 20 and 70% of mortality and up to 100% morbidity in European rabbit commercial farms. Although the aetiology of this disease remains unknown, proliferation of Clostridium perfringens could be a consequence of ERE and might be associated to the high mortality caused by this disease. Although, several antibiotics have been proven to be efficient against ERE, there is a need to find alternative solutions to control the disease in order to fulfil current EU legislation. Among them, nutritional management has become a priority, given its importance on digestive diseases. The present work summarises recent research related to the feed composition and management of young rabbits in farms affected by ERE, aiming to provide useful guidelines for nutritionists towards a preventive nutrition against this disease. Data dealing with maternal and environmental effects on ERE incidence and mortality are also discussed. © 2011 Elsevier B.V. All rights reserved.

1. Introduction ˜ et al., 2006, 2008). In a Weaning is a critical period for the development of digestive pathologies in rabbits (Carabano context of immature immune system, the separation from the mother and the substitution of milk with solid feed lead to stress, a reduction of intake of natural antimicrobials and a decrease of ileal digestibility of nutrients (and thus an increase of substrate flow to the fermentative area promoting microbial growth). Infectious digestive disorders account for a high proportion of all the rabbit diseases in the fattening period (Peeters et al., 1984; Marlier et al., 2003; Rosell, 2003). In most situations, these diseases have a multi-factorial origin that includes a combination of two or three different agents (virus, bacteria and coccidia) and affect animals between 5 and 8 weeks old (1 or 2 weeks after weaning). Rotavirus, Escherichia coli and Clostridium spp. are the most frequent virus and bacteria implicated in these illnesses although the prevalence of each one has been varying throughout the years. Before 1997, 70% of diarrhoeas

Abbreviations: ANF, anti-nutrional factors; CPE, Clostridium perfringens enterotoxin; ERE, epizootic rabbit enteropathy; MCFA, medium chain fatty acids; NDF, neutral detergent fibre; RFLP, restricted fragment length polymorphism. 夽 This paper is part of the special issue entitled Nutrition and Pathology of Non-Ruminants, Guest Edited by V. Ravindran. ∗ Corresponding author. Tel.: +34 14524850; fax: +34 15499763. E-mail address: [email protected] (J.C. de Blas). 0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.12.016

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Fig. 1. Effect of the use of a mixture of 100 mg/kg of apramicine sulphate and 120 mg/kg of tylosin in drinking water on fattening mortality and detection frequency of several potential harmful bacteria at ileal content of rabbits at 35 d of age (Chamorro et al., 2007).

were caused by strains of E. coli (mainly O103 serogroups) usually in combination with rotavirus or coccidia (Peeters et al., 1984; Camguilhem and Milon, 1989; Blanco et al., 1996, 1997). Mortality associated with these digestive problems was mostly moderate (5–13%). In these years, Clostridium spiroforme was identified as the responsible of iota enterotoxemia (Carman and Borriello, 1982). However, the prevalence of this disease in commercial farms was low and usually associated with previous digestive problems caused by virus or E. coli and the use of some antibiotics as clindamycine or lyncomycine (Licois, 1996). The epizootic rabbit enteropathy (ERE) outbreaks started in 1997 causing up to 70% of mortality in the fattening period. This disease remains widely spread in European rabbit farms and has focused most of the research in the past decade. Symptoms usually appear 2 weeks after weaning and include bloat, relatively low body weight, distension of stomach and small intestine, epithelial integrity disruption and either liquid or compacted caecal contents (Peréz de Rozas et al., 2005; Licois et al., 2006; Dewrée et al., 2007; Chamorro et al., 2010). The aetiological agent of ERE remains to be identified, as it has not been possible to reproduce the disease after inoculating specific pathogen free rabbits with isolated pathogens (Marlier et al., 2006). However, several works have reproduced experimentally the ERE symptoms by means of inocula originated from intestinal contents of ill animals or from dust collected in contaminated farms, in which bacterium Clostridium perfringens was detected (Licois et al., 2003; Marlier et al., 2006; Szalo et al., 2007). Moreover, several authors (Marlier et al., 2006; Szalo et al., 2007) have also shown that proliferation of Cl. perfringens could be a consequence of ERE and may be associated to the mortality caused by this disease, whereas other frequent digestive pathogens, as Cl. spiroforme, E. coli or viruses were not present (Licois et al., 2006). A study (Romero et al., 2009a) has shown that the average count of Cl. perfringens in the caecal contents of young rabbits was highly correlated with average diarrhoea mortality in the fattening period (R2 = 0.92; P<0.001). Also, high counts (>2 × 106 cfu/g) of Cl. perfringens were highly related with the appearance of the clinical signs of ERE (Romero et al., 2009a,b). Furthermore, the frequency of detection of Cl. perfringens in digestive contents (and also, that of Campylobacter spp. or Helicobacter spp.) assessed by RFLP techniques was positively related to ERE mortality in other studies (Chamorro et al., 2007, 2010; Gómez-Conde et al., 2007). Some strains of Cl. perfringens produce different pathogenic exotoxins (alpha, beta, epsilon and iota) and also an enterotoxin (CPE) that is produced during sporulation, that have been made responsible of the main symptoms and lesions in humans and several animal species (Songer, 1996; Petit et al., 1999; Van Immerseel et al., 2004). Sporulation occurs when a high concentration of vegetative cells (>106 ) is reached (Hatheway, 1990; EFSA, 2005). Several studies (Le Normand et al., 2003; Marlier et al., 2006; Cocchi et al., 2007) have detected the presence in digestive contents of rabbits affected by ERE of toxinotypes alpha and alpha-beta2, which were related to symptoms of liquid and compacted caecal contents, respectively. Parasitic lesions cause gut damage and stimulate the proliferation of Cl. perfringens in rabbits (Peeters et al., 1984) as occurs in poultry (Van Immerseel et al., 2004; Williams, 2005). Subclinical infections with Eimeria frequently takes place in rabbits (Peeters et al., 1984), and have been also linked to a higher incidence of ERE in commercial farms (Coudert et al., 2003; Marlier et al., 2006).

2. Use of medicated feeds Previous works have proven the efficacy of several antibiotics to control ERE incidence as Zn bacitracin in feed (Duperray et al., 2003) or in drinking water (Boisot et al., 2003b), tiamulin (Licois et al., 2006), tylosin (Bostvironnois and Morel, 2003) or the combination of tylosin with apramycin (Badiola et al., 2000). The positive effects have been explained by a lower sporulation of Cl. perfringens (Zn bacitracin, Elfadil et al., 1996), or by a reduction of the concentration of Cl. perfringens and other pathogens (Zn bacitracin, Agnoletti et al., 2007; and the combination of tylosin and apramycin, Chamorro et al., 2007; Fig. 1). Nevertheless, the continuous or preventive use of in-feed antibiotics has been largely contested (e.g., Mateos et al., 2002; Turnidge, 2004). Consequently, several nutritional strategies have been proposed to prevent ERE incidence.

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Table 1 Effect of age at weaning and dietary fibre level on the caecal concentration of vegetative cells of Cl. perfringens 14 d after weaning, and on fattening mortality in not medicated animals (according to Romero et al., 2009a). n

Caecal concentration of Cl. perfringens (cfu × 103 /g) Fattening mortality (%) Average mortality age (d)

32 162 162

Weaning (d)

NDF (g/kg)

SEM

28

42

330

420

285 0.267 42.8

166 0.141 54.7

216 0.196 48.2

236 0.220 49.2

Contrasts

– 0.035 0.74

1

2

0.001 0.05 0.001

0.01 NS NS

Significance of contrasts: 1 = weaning age; 2 = NDF content.

3. Formulation of fattening diets to control pathogen concentrations and toxin production in the gut 3.1. Optimal level of insoluble fibre A minimal content of insoluble fibre (320 g/kg neutral detergent fibre, De Blas et al., 1999), acid detergent lignin (37 g/kg, Perez et al., 1994; Nicodemus et al., 1999) and large particles (>0.315 mm, 20.6%, Nicodemus et al., 2006) in the growing rabbit diets is required to accelerate intestinal peristalsis, decrease total and caecal mean retention time of digesta, dilute dietary and ileal starch and protein contents and reduce total microbial and pathogen growth (Gidenne and Perez, 1994; García et al., 1995, 2000; Blas and Gidenne, 1998). It also decreases fattening mortality with respect to diets with an insufficient insoluble fibre concentration (<250–270 g/kg neutral detergent fibre, Gidenne et al., 2004a,b; Nicodemus et al., 2004) and increases resistance to enteropathogenic E. coli O103 strain (Gidenne and Licois, 2005). On the other hand, an excessive (>400 g/kg) neutral detergent fibre content in the diet leads to a decrease in feed efficiency and to an increase of fattening mortality (Gutiérrez et al., 2002a) and sanitary risk (mortality + morbidity, Feugier et al., 2006) in parallel to an impairment of mucosal structure (Alvarez et al., 2007) and a decrease of ileal mucosal enzymatic activity (Nicodemus et al., 2002; García et al., 2002b). A variation of dietary NDF concentration between these extreme values did not affect either Cl. perfringens caecal contents concentration 14 d after weaning or mortality in the fattening period (Romero et al., 2009a; Table 1). 3.2. Optimal level of soluble fibre The inclusion of moderate levels of soluble fibre (as fructans, galactans, ß-glucans, pectins and resistant starch, as described by Gidenne et al., 2010) in the fattening diet has been related to a reduction of ERE incidence and of Cl. perfringens frequency of detection and other pathogens in the digestive contents, improving the immune response and the feed efficiency, and reducing mortality after weaning (Gómez-Conde et al., 2007). According to Marounek et al. (1995), Gidenne et al. (2007) and Lavrencik (2007) the caecal flora of rabbits at 28 d of age is less developed than in adult animals and it is specialised in the fermentation of soluble carbohydrates. Consequently, the addition of digestible fibre to the diet might be useful to promote the growth of saprophyte bacteria in order to improve the competitive exclusion of pathogenic flora. In addition, the inclusion of soluble fibre in the diet might minimise the deterioration of intestinal villi and maintain the activity of the enterocytes in the post-weaning period, while the addition of lignified fibre led to structural atrophy, lower activity of intestinal cells and proliferation of Cl. perfringens (Chiou et al., 1994; Alvarez et al., 2007; Gómez-Conde et al., 2007, Table 2). Addition of soluble fibre also led to a decrease of caecal content pH and to an increase of volatile fatty acid production in the caecum (García et al., 2002a), which might contribute to reduce growth of E. coli as observed in Table 2 Effect of fibre source in diets containing 300 g/kg NDF on the integrity and activity of intestinal barrier, detection frequency of several potential harmful bacteria at caecum, and fattening mortality (Gómez-Conde et al., 2007). Beet-applepulp Neutral detergent soluble fibre (g/kg) Intestinal barrier (jejunum) Villus height (␮m) Crypt depth (␮m) Sucrase specific activity (␮glucose/g protein) Ileal flow of starch (g/d) Lamina propria (duodenum) Lymphocytes CD4+ (%) Lymphocytes CD8+ (%) Frequency of detection at caecum (%) Cl. perfringens Campylobacter spp. Fattening mortality (%)

Alfalfahay

Oathulls

120

94

72

722a 89.0a 8671a 0.5a

567b 115b 6495b 0.8b

493c 113b 8671b 1.2c

SEM

P

28.0 4.35 580 0.099

0.001 0.001 0.019 0.001

35.1 21.3

33.9 26.9

26.2 30.3

3.87 2.61

NS 0.074

5.70a 19.4 5.30a

2.87a 21.2 8.50ab

17.6b 37.8 14.4a

4.2 6.7 –

0.047 0.074 0.05

Values in a row not sharing a common letter (a–c) differ at P<0.05.

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Fattening mortality

0.4

0.3

0.2

0.1

0.0 4

5

6

Ileal flow of protein (g/d) Fig. 2. Effect of the apparent ileal flow of protein at the post-weaning period on the fattening mortality according to several experiments.

in vitro experiments (Prohaszka, 1980). From these results and other works made in commercial farms (Fabre et al., 2006; Margüenda et al., 2006), it can be deduced that besides to an optimal neutral detergent fibre level of around 340 g/kg, it is advisable the addition of about 100–120 g/kg of soluble fibre to the fattening rabbit diets. In the same way, several studies have shown a positive influence of the addition of oligosaccharides to the diet on the viability of fattening rabbits. A 20 g/kg inclusion of mannan oligosaccharides reduced the fattening mortality and promoted an increase in villi height, a decrease of caecal pH and a higher concentration of volatile fatty acids (Mourao et al., 2006). However, previous results obtained with the addition of other prebiotics in practical conditions were not fully consistent (Falcão-e-Cunha et al., 2007). 3.3. Reduction of ileal protein flow Protein and amino acid requirements are relatively high in young rabbits, not only for tissue accretion, but also for the renewal and growth of the intestinal mucosa (Lebas and Laplace, 1972). Minimal recommendations for crude protein and essential amino acids have been recently assessed by De Blas and Mateos (2010). Several amino acids, as threonine or glutamine, can play a role in preserving the intestinal mucosal integrity and function and to support normal immunological response around weaning. In this way, threonine is a major component of mucin proteins and glutamine is a primary energy source of enterocytes and immune cells playing an essential role in the repairing mechanisms of mucosa tissue (Windmueller and Spaeth, 1980; Newsholme et al., 1999). Recent work (Chamorro et al., 2010) indicates that dietary supplementation with 10 g/kg of glutamine modified ileal microbiota (with a decrease of the frequency of detection of several bacteria as Clostridium spp. and Helicobacter spp.), diminished the presence of Eimeria spp. at the jejunum, and reduced the mortality caused by ERE. The level and source of plant protein included in starter diets have also an influence on intestinal disorders, as a decrease of dietary protein content or an increase in ileal protein digestibility reduced the flow of protein towards the fermentative area and decreased the mortality due to ERE (Gutiérrez et al., 2000, 2003; García-Ruiz et al., 2006; Chamorro et al., 2007, Fig. 2). Other studies have indicated that an increase of the nitrogen flow reaching the terminal ileum incremented the populations of total anaerobic bacteria (García-Palomares et al., 2006), Cl. spiroforme (Haffar et al., 1988), E. coli (Cortez et al., 1992), and Cl. perfringens in rabbits (Chamorro et al., 2007) and other species (Drew et al., 2004; Zentek et al., 2004). The presence of amino acids in the intestinal lumen also enhances growth and toxin production from Cl. perfringens (Titball et al., 1999). Some essential and non-essential amino acids might also be required for toxin production (Nakamura et al., 1968; Muhammed et al., 1975). Otherwise, the association of coccidiosis and a high Cl. perfringens concentration in the gut of rabbits and other species has been related to mucosal damage and leak of plasma proteins which would favour proliferation of pathogens (Coudert et al., 2003; Van Immerseel et al., 2004; Williams, 2005; Chamorro et al., 2010). In this context it is relevant the information obtained in recent studies (García-Ruiz et al., 2005; Llorente et al., 2006, 2007), to characterise the true and apparent ileal digestibility of protein and amino acids of some common ingredients of rabbit diets. These data are useful not only for determining the amino acid supply, but also for estimating the amount of protein that remains available for microbial growth in the caecum. Endogenous nitrogen (digestive enzymes, mucoproteins, desquamated cells and urea) is other relevant source of protein for the gut microorganisms, which can account in rabbits for about 0.40–0.60 of the total ileal protein flow (García-Ruiz et al., 2004). This contribution is quite variable and influenced primarily by dry matter intake and secondarily by diet composition, as level of fibre or anti-nutritional factors (ANF), frequently present in legumes, might damage the intestinal mucosa and increase the flow of nitrogen towards the caecum. In this way, the replacement of low ANF protein sources (as animal protein or sunflower meal) with soybean meal protein impairs mucosal structure (Gutiérrez et al., 2000) and increases fattening mortality (Scheele and Bolder, 1987; Gutiérrez et al., 2003; García-Ruiz et al., 2006). The addition of hydrolysable tannins has also led to a decrease of mortality during fattening in an ERE environment (Maertens and Struklec, 2006). This result has been explained by the formation of non-degradable complexes of tannins with dietary protein and/or by the direct inhibition of microbial growth, as occurs in poultry with Cl. perfringens and Eimeria spp. (Dougald et al., 2008).

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Fig. 3. Effect of a feed restriction on fattening mortality (base 1 = mortality and feed consumption in control treatment ad libitum).

4. Weaning age and feeding management Another factor that might be potentially related to intestinal health in young rabbits is weaning age. Recent work has shown that suckling temporally limited enteropathogenic O103 E. coli mortality, reduced bacterial adhesion to the ileal epithelium and diminished faecal E. coli excretion (Gallois et al., 2007). Rabbit milk contains a high proportion of fat (about 130 g/kg) and of medium-chain fatty acids (MCFA), as caprylic (C8:0 ) and capric (C10:0 ), that account for more than half of the total milk fatty acids (Maertens et al., 2006). Recent work (Skrivanova et al., 2005; Gallois et al., 2008) indicated that short and medium-chain fatty acids have a significant bactericide effect against, respectively, Cl. perfringens and enteropathogenic O103 E. coli in in vitro conditions. Accordingly, it could be hypothesised that the inclusion of MCFA in the diet might help to reduce diarrhoea incidence during the fattening period. The high concentration of rabbits milk in these fatty acids might contribute to explain the protective effect that a delay of weaning age seems to exert on rabbit viability and to reduce E. coli O103 (Gallois et al., 2007) and Cl. perfringens (Romero et al., 2009a; Table 1) proliferation in faeces and caecal contents, respectively. However, several studies (Skrivanova et al., 2008; Gallois et al., 2008) have failed to demonstrate the protection against colibacillosis in rabbits supplemented with 10–20 g/kg MCFA in the diet. More work is needed to fully characterise the antimicrobial compounds present in doe’s rabbit milk. Late weaning also implies a better adaptation of the digestive and absorptive capability of young rabbits to the consumption of solid feed, as both the amylase and disaccharidase activities increases with age but remains low from 25 to 35 d (Corring et al., 1972; Dojana et al., 1998; Scapinello et al., 1999; Gutiérrez et al., 2002a). As a consequence, the flow of starch reaching the ileum in young rabbits increases with dietary starch content, which has been related to a higher sporulation and enterotoxin production by Cl. perfringens in in vitro studies (Labbe et al., 1976; Labbe, 1981) and to a higher diarrhoea incidence in the fattening period (Blas and Gidenne, 1998). In the same way, the supplementation of feed with amylases contributed to reduce fattening mortality in several studies (Gutiérrez et al., 2002b; Cachaldora et al., 2004). Otherwise, an increment of ileal nutrient flow caused by a ligature of the pancreatic duct led to an increase of microbial proliferation in the caecum (Catala and Bonnafous, 1979). A feeding restriction of at least 20% with respect to ad libitum intake (Fig. 3) reduced ERE incidence under experimental conditions (Boisot et al., 2003a; Foubert et al., 2008) and decreased fattening diarrhoea mortality and feed conversion rate in field experiments (Gidenne et al., 2009a,b). These results might be explained by a parallel increase in restricted animals of mean retention time of feed in the gut, a lower caecal pH and a higher caecal concentration of volatile fatty acids (Gidenne and Feugier, 2009), which together to a reduction of the nutrient overload of the hindgut might contribute to reduce pathogen proliferation in the digestive contents. 5. Maternal and environmental effects Several studies carried out in our group have indicated that litter has a consistent and highly significant effect on mortality during the fattening period, explaining around 20% of the total variability of the mortality rate (Fig. 4). The previous effect resulted from the fact that, as average, 50% of the losses occurred in only 14% of the litters, whereas in 50% of the litters no losses occurred. Furthermore, microbiological analyses carried out by RFLP method showed that the intestinal microbiota of rabbits from the same litter was more similar than that of animals from other litters of the same farm (García et al., 2005). These results suggest a transmission of immune competence to the young rabbits through the milk or the placenta. Litter effect might also be explained by a hypothetical pathogen contamination through the mother (or through the common ambiance). Changes in management, as the use of the batch breeding system, cleaning and disinfecting processes to improve environmental safety have been effective in reducing the incidence of ERE in field conditions (Licois et al., 2006; De Blas et al., 2007). The accumulation of spores and vegetative cells of Cl. perfringens in the environment can explain the variable incidence of ERE among commercial farms. Some preliminary results obtained in our group (Table 3) seem to confirm the existence of a relationship among environment, Cl. perfringens proliferation in the gut and ERE incidence.

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Fig. 4. Mortality occurrence in different fattening trials (number of rabbits/trial = 512, 733, 603, and 663) and proportion of its variability explained by the effect of litter (P<0.001 in all the trials; De Blas et al., 2007). Table 3 Effect of cleaning on the concentration of vegetative cells of Cl. perfringens in samples of dust taken in the fan of the farm, on the caecal concentration of vegetative cells of Cl. perfringens and on fattening mortality at 28–63 d of age in not medicated animals (De Blas et al., 2007). Trial

1 2 3 4 a b c

Cl. perfringens (cfu × 103 /g)

Fattening mortality (%)b a

Dust

Caecal content

0.7 250 680 1500

17 12c 57 130

3.6 2.1 17.2 33.3

Samples taken the 14th day after weaning (n = 20). n = 200, except for trial 4 where n = 471. Samples from caecotrophs instead of caecal contents (n = 36).

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