The role of an attenuated anticoccidial vaccine on the intestinal ecosystem and on the pathogenesis of experimental necrotic enteritis in broiler chickens

Avian Pathology ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: https://www.tandfonline.com/loi/cavp20 The role of an attenuated anticoccidial vaccine on the intestinal ecosystem and on the pathogenesis of experimental necrotic enteritis in broiler chickens V. Tsiouris , I. Georgopoulou , C. Batzios , N. Pappaioannou , A. Diakou , E. Petridou , R. Ducatelle & P. Fortomaris To cite this article: V. Tsiouris , I. Georgopoulou , C. Batzios , N. Pappaioannou , A. Diakou , E. Petridou , R. Ducatelle & P. Fortomaris (2013) The role of an attenuated anticoccidial vaccine on the intestinal ecosystem and on the pathogenesis of experimental necrotic enteritis in broiler chickens, Avian Pathology, 42:2, 163-170, DOI: 10.1080/03079457.2013.776161 To link to this article: https://doi.org/10.1080/03079457.2013.776161 Published online: 14 Apr 2013. Submit your article to this journal Article views: 899 View related articles Citing articles: 15 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=cavp20 Avian Pathology, 2013 Vol. 42, No. 2, 163
170, http://dx.doi.org/10.1080/03079457.2013.776161 The role of an attenuated anticoccidial vaccine on the intestinal ecosystem and on the pathogenesis of experimental necrotic enteritis in broiler chickens V. Tsiouris1*, I. Georgopoulou1, C. Batzios2, N. Pappaioannou3, A. Diakou4, E. Petridou5, R. Ducatelle6 and P. Fortomaris7 1 Unit of Avian Medicine, Clinic of Farm Animals, Faculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece, 2Laboratory of Animal Production Economics, Faculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece, 3Laboratory of Pathology, Faculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece, 4Laboratory of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece, 5 Laboratory of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, Aristotle University, Thessaloniki, Greece, 6Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820, Merelbeke, Belgium, and 7Laboratory of Animal Husbandry, Faculty of Veterinary Medicine, Aristotle University, 54627, Thessaloniki, Greece The objective of the present study was to investigate the effect of an attenuated anticoccidial vaccination on the intestinal ecosystem and on the pathogenesis of experimental necrotic enteritis (NE) in broiler chickens. Two hundred and forty 1-day-old Cobb 500 broiler chickens were randomly allocated to four treatment groups according to the following experimental design: control Group N; Group PN, where birds were vaccinated with anticoccidial vaccine; Group M, where birds were challenged with Clostridium perfringens and with Eimeria maxima; and Group PM, where birds were both vaccinated and challenged. From each bird, the intestine, gizzard and liver were scored for gross NE lesions. Intestinal digesta were collected for pH and viscosity determination. Samples from the gastrointestinal tract and liver were taken for microbiological analysis. Evaluation of the experimental data revealed that Group M had significantly higher overall mean NE intestinal lesions compared with Group PM. Viscosity values of jejunum digesta as well as pH values of the duodenum, jejunum and ileum digesta in Group M were significantly lower compared with Group PM. C. perfringens counts in the caeca of Group PM were significantly lower compared with Group M. The milder decrease of pH and viscosity values of intestinal content and the reduction of C. perfringens counts in the caeca in challenged and vaccinated birds may explain the lower score of NE gross intestinal lesions and may suggest a positive effect on intestinal ecosystem and a significant protective effect of attenuated anticoccidial vaccination against NE in a subclinical experimental model. Introduction Necrotic enteritis (NE) is caused by Clostridium perfringens. NE is one of the most common and economically devastating bacterial diseases in modern broiler flocks. It may be present as an acute clinical or subclinical disease (Van Immerseel et al., 2004). The occurrence of subclinical NE is estimated to result in a 12% reduction in body weight and a 10.9% increase in feed conversion ratio compared with healthy birds (Skinner et al., 2010). NE of broiler chickens represents a classical example of disease-syndrome, which is a consequence of imbalance of the intestinal ecosystem. For instance, a change in pH and/or viscosity of intestinal digesta affects the development of NE (McDevitt et al., 2006; Dahiya, 2007). Particularly, higher intestinal viscosity increases the average retention time of the intestinal content and the amount of undigested material in the intestinal tract, which gives C. perfringens more time and substrate to colonize the small intestine, to proliferate and to produce toxins (Waldenstedt et al., 2000). Conversely, the high concentrations of lactic acid produced by the bacteria can reduce the pH to levels that are low enough to inhibit the growth of C. perfringens (McReynolds et al., 2007). Despite our present understanding of the disease, and the identification of C. perfringens as the aetiological agent, the predisposing factors that lead to overproliferation of C. perfringens and the subsequent progression to disease are poorly understood. These predisposing factors are numerous, but many are illdefined and experimental results have been contradictory (Williams, 2005; McDevitt et al., 2006; Dahiya, 2007). In commercial poultry production, the most important and frequent predisposing factor to NE is the damage of intestinal mucosa caused by Eimeria spp. (Drew et al., 2004; Van Immerseel et al., 2004). In terms *To whom correspondence should be addressed. Tel: 30 2310 994555. Fax: 30 2310 994557. E-mail: biltsiou@yahoo.gr Received 29 November 2012 # 2013 Houghton Trust Ltd 164 V. Tsiouris et al. of prevention, the anticoccidial vaccination appears to be a promising alternative to the chemotherapeutic control of coccidiosis. The use of anticoccidial vaccines could also have adverse effects on the incidence of NE, due to the presence of mild subclinical lesions caused by coccidia (Williams, 2002). On the other hand, there is evidence for a protective role of anticoccidial vaccination against NE. Application of an attenuated anticoccidial vaccine before oral administration of Eimeria maxima infection followed by a mixed culture of C. perfringens strains, administered per cloaca, reduced the severity of NE gross intestinal lesions (Williams et al., 2003). Moreover, application of a multi-fold dose of anticoccidial vaccine reduces the mortality after challenge with C. perfringens, compared with chickens challenged with C. perfringens but not vaccinated (McReynolds et al., 2004). However, the mechanisms involved regarding the interaction between NE and anticoccidial vaccine and the latter’s effect on pH and viscosity of intestinal digesta, as well as on C. perfringens counts in the gastrointestinal tract, have not been studied before. Hence, the objective of the present study was to investigate the effect of an attenuated anticoccidial vaccine on the intestinal ecosystem and on the pathogenesis of NE in broiler chickens using a reproducible, well-established experimental model. Materials and Methods Strains and cultivation. C. perfringens strain 56 was isolated from the intestine of a broiler chicken with severe NE 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 NE (Gholamiandehkordi et al., 2007; Lanckriet et al., 2010). 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 mg/ml (R 3501-1G; Sigma Aldrich Chemie GmbH, Steinheim, Germany). Before the chickens’ inoculation, the bacteria were cultured for 24 h at 378C in Brain Heart Infusion broth in an anaerobic atmosphere (Anaerocult A, 1.13829.0001; Merck KGaA, Darmstadt, Germany). The challenge strain of E. maxima was the Weybridge strain, which is virulent and heterologous to the E. maxima lines contained in the attenuated anticoccidial vaccine (Williams et al., 2003). The parasites had been propagated once in 2-week-old Eimeria-free chickens (Shirley, 1996). Attenuated anticoccidial vaccine (Paracox-5; MSD, Hertfordshire EN11 9BU, London, UK) was used for oral vaccination of birds on the first day of age, using an insulin syringe with a small plastic catheter adapted to its opening. The vaccine contains live, attenuated oocysts of Eimeria acervulina, E. maxima (two lines), Eimeria mitis and Eimeria tenella. Nobilis Gumboro D78 (MSD, Hertfordshire EN11 9BU, London, UK), a commercial vaccine, was used as a predisposing factor to NE (McReynolds et al., 2004). It was provided via drinking water, after removing the waterlines for 2 h. Birds and housing. Two hundred and forty 1-day-old Cobb 500 broiler chickens were obtained from a local commercial hatchery and were randomly allocated into four experimental groups of 60 chickens. Birds in each group were placed in a cage with deep litter of wood shavings, which were previously sterilized in an autoclave at 1218C for 20 min (Cyclomatic control, EA605A, Erie, Pennsylvania, USA). Each group was kept in a specially designated experimental room (Unit of Avian Medicine, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Greece), where the temperature, relative humidity and lighting were artificially controlled, following the recommendations of the breeding company. The stocking density for each group was 15 birds/m2 or 33 kg/m2 (European Commission, 2007). 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 to 9 days, grower 10 to 16 days and finisher 17 to 24 days), which included large quantities of wheat and rye (Gholamiandehkordi et al., 2007). From day 17 onwards, the same isocaloric ration (finisher) was used with the exception that fishmeal replaced the soybean meal as the protein source, in order to predispose to NE. No antibiotic growth promoters and anticoccidial drugs were used. Feed and water were available ad libitum throughout the trial. Experimental design. All birds were vaccinated against Gumboro disease on the 16th day of age via drinking water. For the experimental induction of subclinical NE, the birds were orally challenged, using an insulin syringe with a small plastic catheter adapted to its opening, with 1 ml C. perfringens (4 108 colony-forming units) three times daily (at 09:00, 13:00 and 17:00 h) for four consecutive days, at the 17th, 18th, 19th and 20th day of age, and with 3 104 oocysts of E. maxima on the 18th day of age. The treatment groups used in this study consisted of: Group N, which served as control; Group PN, to which attenuated anticoccidial vaccine was applied; Group M, in which birds were experimentally challenged with C. perfringens and E. maxima: and Group PM, in which birds were both vaccinated with attenuated anticoccidial vaccine and experimentally challenged with C. perfringens and E. maxima (Table 1). From each experimental group, 15 birds per sampling day were removed at days 21, 22, 23 and 24. 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 in its anatomical parts (gizzard, duodenum, jejunum, ileum, caeca). Gross lesions scoring system. Intestine, gizzard and liver gross lesions were examined macroscopically and scored (Figure 1). In particular, intestines were examined macroscopically and scored for NE lesions following a zero to six scoring system described by Keyburn et al. (2006). Briefly, the scoring was as follows: 0 no gross lesions; 1  congested intestinal mucosa; 2 small focal necrosis or ulceration (one to five foci); 3 focal necrosis or ulceration (six to 15 foci); 4 focal necrosis or ulceration (16 or more foci); 5 patches of necrosis 2 to 3 cm long; and 6 diffuse necrosis typical of field cases. Lesion scores of two or more were classified as NE-positive. Gizzards were examined macroscopically and scored for gross lesions using a scale of zero to two, described by Novoa-Garrido et al. (2006). Gross liver lesions received a score 0 when no gross lesions were observed, a score 1 when liver congestion and/or gallbladder distention and wall thickening and/or bile discoloration were observed, and a score 2 when necrotic lesions were observed in the liver. Table 1. Experimental design to evaluate the role of an attenuated anticoccidial vaccine on the intestinal ecosystem and on the pathogenesis of experimental necrotic enteritis in broiler chickens. Experimental group N PN M PM Challenge Vaccination         Anticoccidial vaccine and necrotic enteritis 165 Figure 1. Gross intestinal, liver and gizzard lesion scoring system in the experiment of anticoccidial vaccination. See Materials and Methods for details of scoring system. Arrows indicate gross lesions. pH value determination. After euthanasia, the digesta of the duodenum, jejunum, ileum and caecum from each bird was immediately removed and placed in a plastic tube (10 ml capacity). Afterwards, each tube was vortexed in order to obtain a homogeneous mixture, in which pH was measured using a digital pH-meter (pH 315i; WTW WissenschaftlichTechnische Werkstatten, Weilheim, Germany). Viscosity value determination. A homogeneous mixture of the jejunum and ileum was placed in two Eppendorf tubes (1.5 ml). The tubes were centrifuged at 3000  g for 45 min 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, Massachusetts, USA). Two readings were taken from each tube and were represented in units of centipoise (cP). Bacteriological culture. Quantification of C. perfringens counts in the caecum was carried out according to Kaldhusdal et al. (1999). 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. Material for bacteriological examination of C. perfringens was taken aseptically from the liver and swab samples were taken from the gizzard, duodenum, jejunum, and ileum content and was done semi-quantitatively as anaerobic cultivation on 5% sheep blood agar plate (Columbia blood agar, 01
034; Scharlau Chemie S.A.) and C. perfringens selective supplement (SR0093; Oxoid Ltd, Cambridge, UK) for 24 h. Samples from challenged groups were plated on agar containing in addition 100 mg/ml rifampicin. Two to four millimetre wide, circular, transparent colonies with typical ‘‘target’’ haemolysis (an inner zone of clear haemolysis and an outer zone of partial haemolysis) were diagnosed as presumptive C. perfringens. In cases of doubt, aerobic and anaerobic secondary cultivation on blood agar and microscopy of Gram-stained smears were used. Results were determined semi-quantitatively using the fourth quadrant method, according to Ito et al. (2004). Briefly, the first quadrant was heavily streaked and a new sterile disposable swab was used to perform serial smearing on other quadrants. C. perfringens growth was examined after incubation, and scores from zero to four were given according to the absence of colonies and presence in the first, second, third and fourth quadrants of the Petri dish. Histopathological examination. Tissue samples from the duodenum, jejunum (proximal to Meckel’s diverticulum) and ileum (proximal to the ileo-caecal junction) were fixed in 4% buffered formaldehyde for 48 to 72 h. Coronal sections from the samples were embedded in paraffin by a routine procedure. Dewaxed 3 to 5 mm 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. All analyses were made on the cumulative values received from all sampling days. For accessing the assumptions of normality and stability of variances, data were transformed to log10 or square root. In cases of normality and variance’s homogeneity, one-way analysis of variance was performed to evaluate possible significant effects of treatment on gross lesions in the intestine, gizzard and liver, on the population of C. perfringens in the caeca, on the semi-quantitative analysis of C. perfringens in the gastrointestinal tract and liver, as well as on the pH values of the duodenum, jejunum, ileum and caeca and 166 V. Tsiouris et al. 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 population distribution were seriously violated, with or without transformed data, the Kruskal
Wallis non-parametric test was applied to evaluate treatment-dependent differences, while differences between mean values of specific treatments were evaluated using the non-parametric Wilcoxon rank sum test (Mann
Whitney U test). All analyses were conducted using the statistical software program SPSS for Windows (v. 15.0). Significance was declared at P 50.05, unless otherwise noted. Back-transformed mean values are reported in the results. Results Number of birds with macroscopic NE lesions according to treatment group and sampling day are presented in Table 2, while the overall mean grade of gross lesions scores in the intestine, gizzard and liver per treatment group are given in Table 3. Table 2 illustrates that the number of NE positive birds was higher in Group M compared with Group PM. Based on the results presented in Table 3, the overall mean grade of NE gross lesions in the intestine in unchallenged groups was lower than one, indicating that none of the birds in the unchallenged groups developed NE gross intestinal lesions. On the contrary, the overall mean in challenged groups was above one, indicating that birds of these groups developed NE gross lesions in the intestine. More specifically, Group M had a significantly higher overall mean grade of NE gross lesions in the intestine compared with Group PM. The overall mean grade of gross lesions in the liver in unchallenged groups was significantly lower than in challenged groups. Furthermore, between unchallenged groups as well as between challenged groups there was no significant difference. The overall mean grade of gross lesions in the gizzard was not significantly different between experimental groups. Viscosity and pH values of contents from different parts of the intestinal tract, as well as C. perfringens counts in the caeca per treatment group, are presented in Table 4. As Table 4 illustrates, pH values of the duodenum, jejunum and ileum digesta in challenged groups were significantly lower compared with the unchallenged ones. Furthermore, between unchallenged groups, pH values of duodenum digesta were significantly lower in Group PN compared with Group N, while pH values of the ileum digesta were the opposite. Between challenged groups, pH values of the duodenum, jejunum and ileum Table 2. Number of birds with macroscopic necrotic enteritis lesions according to treatment group and sampling day. Experimental group Sampling day N PN M PM 21 22 23 24 Total 0 0 0 0 0 0 0 0 0 0 5 11 12 11 39 3 7 12 7 29 Birds with intestinal lesions score greater than one (two or more) were classified as NE-positive. Table 3. Gross lesion scores in the intestine, gizzard and liver per treatment group. Experimental group Location N PN M PM Intestinea Liver Gizzard 0.6590.48A 0.6690.85A 0.7990.93A 0.5090.51A 0.5290.85A 0.5490.93A 4.6092.19B 1.2591.01B 0.5290.93A 3.0892.35C 1.0491.08B 0.7790.93A Data presented as mean9standard deviation. Mean values in the same row with a different uppercase superscript letter differ significantly (P50.05). digesta in Group M were significantly lower compared with Group PM. Viscosity values of the jejunum digesta in challenged groups were significantly lower compared with unchallenged groups. Between challenged groups, viscosity values of the jejunum digesta were significantly lower in Group M compared with Group PM. Viscosity values of the ileum digesta in challenged groups were significantly lower compared with unchallenged groups. Between unchallenged groups, viscosity values of the ileum digesta in Group PN were significantly higher compared with Group N. C. perfringens counts in the caeca in challenged groups were significantly higher compared with unchallenged groups. Between unchallenged groups, C. perfringens counts in the caeca were significantly lower in Group PN compared with Group N. Between challenged groups, C. perfringens counts in the caeca in Group PM were significantly lower compared with Group M. Overall mean grades of semi-quantitative analysis of C. perfringens counts in the gastrointestinal tract and liver per treatment group are presented in Table 5. According to the data in Table 5, C. perfringens counts in the gastrointestinal tract in challenged groups were significantly higher compared with unchallenged groups. Between unchallenged groups, C. perfringens counts in the jejunum were significantly higher in Group PN compared with Group N. Between challenged groups, C. perfringens counts in the duodenum, jejunum, ileum and liver were significantly higher in Group M compared with Group PM. The histopathological lesions of the duodenum, jejunum and ileum in birds challenged with C. perfringens and E. maxima were compatible with NE lesions. Lesions were located mainly in the jejunum and ileum and to lesser extent in the duodenum. There was severe necrosis of the intestinal mucosa, with an abundance of fibrin mixed with cellular debris adherent to the necrotic mucosa, in which large clusters of bacteria were detected (Figure 2). Villus fusion and shortening was observed (Figure 3). Cellular fragments were detected in the intestinal lumen as well as in the necrotic debris. In the lamina propria there was marked inflammatory reaction, which consisted of infiltration of heterophilic granulocytes and lymphocytes. Characteristic, also, was the presence of large numbers of rod-shaped bacteria individually and/or as clusters in the intestinal lumen, as well as in the intestinal mucosa and lamina propria (Figure 2). Furthermore, the presence of coccidia at different stages of multiplication (mainly schizonts) was detected in the intestinal mucosa (Figure 4). Anticoccidial vaccine and necrotic enteritis Table 4. 167 pH and viscosity values of contents in different parts of the intestinal tract, as well as C. perfringens counts in caeca per treatment group. Experimental group Location pH Duodenum Jejunum Ileum Caeca Viscosity Jejunum Ileum Log10 C. perfringens Caecum N PN M PM 6.1290.23A 5.9790.15A 6.9490.39A 6.0090.46A 6.0190.23B 6.0090.15A 7.0790.39B 6.1390.54A 5.7290.46C* 5.5490.54C 5.8790.93D 6.1290.46A 5.8990.31D* 5.8390.23B 6.4690.77C 6.2090.54A 8.4894.26A 12.7796.43A 8.5193.41A 15.0895.34B 3.2393.10B 5.6098.68C 4.5193.56C 7.97911.93C 4.3691.32A 3.2592.09B 6.0892.17C 5.1891.70D Data presented as mean9standard deviation. Mean values in the same row with a different uppercase superscript letter differ significantly (P 50.05 or *P50.10). Discussion The experimental model used in the present study for the reproduction of NE is a well-established and reproducible model that has been used in many research studies (Gholamiandehkordi et al., 2007; Timbermont et al., 2009; Lanckriet et al., 2010). It is a multifactorial model, using not only a specific diet formulation and a Gumboro vaccination, but also an oral inoculation of broilers with E. maxima and multiple oral inoculations with a specific strain of C. perfringens. The smaller decrease of pH and viscosity values of intestinal content and the reduction of C. perfringens counts in the caeca in challenged and vaccinated birds may explain the lower score of NE gross intestinal lesions and may suggest a positive effect on intestinal ecosystem and a significant protective effect of attenuated anticoccidial vaccination against NE in a subclinical experimental model. The protective effect of attenuated anticoccidial vaccination against NE observed in the present study is in agreement with the results of Williams et al. (2003) and McReynolds et al. (2004), who observed that anticoccidial vaccination reduced the severity of NE gross intestinal lesions and mortality, respectively. However, in a field study by Ernik & Bedrnik (2001) the anticoccidial vaccine predisposed to NE. In their study, in six successive flocks within the same poultry farm they reported the occurrence of NE, when coccidiostats drugs were replaced by the attenuated anticoccidial vaccine. The replacement of Table 5. Overall mean grade of semi-quantitative analysis of C. perfringens counts in the gastrointestinal tract and liver per treatment group. Experimental group Location Duodenum Jejunum Ileum Gizzard Liver N PN A 0.4791.16 0.1790.70A 1.5692.09A 0.0090.00A 0.0090.00A M A 0.6191.55 0.6191.31B 1.8392.25A 0.0890.39A 0.1490.70A PM B 2.4492.17 3.1791.70C 3.8690.54B 0.4290.77B 0.3690.93B 1.5691.94C 1.4791.94D 2.8691.70C 0.3390.70B 0.0090.00A Data presented as mean9standard deviation. Mean values in the same row with a different uppercase superscript letter differ significantly (P 50.05). the vaccine by the coccidiostats resulted in the nonoccurrence of NE. The contradiction in results probably arises from the fact that the present study was conducted in experimental conditions, while Ernik & Bedrnik’s (2001) study was performed in the field where the infection of birds was not controlled and the management practices, the ration and the genotype of birds, which also affect the pathogenesis of NE (Ross Tech, 1999), could be different between poultry flocks. Furthermore, in the present study the individual vaccination of the birds was more efficient compared with mass vaccination, and as a consequence gave a better protection against coccidiosis, which is one of the most important and frequent predisposing factors to NE (Drew et al., 2004). The protective effect of anticoccidial vaccine against NE could have several plausible explanations. Firstly, it could be attributed to the stimulation of non-specific and specific immunity mechanisms, as a result of local inflammation. It is well accepted that attenuated anticoccidial vaccination can cause mild coccidiosis lesions in intestinal mucosa (Williams, 2002). Eimeria spp., both wild and vaccine oocysts, exhibit a complex lifecycle composed of intracellular, extracellular, asexual, and sexual stages, so it is not surprising that host immune responses are also complex. Immune responses to this pathogen involve many facets of non-specific and specific immunity. The latter encompasses both cellular and humoral immune mechanisms (Dalloul & Lillehoj, 2005). The stimulation of local immunity results in a better preparation and more efficient defence of birds against intestinal infections, such as coccidiosis and NE. Secondly, the prevention of coccidiosis could be another explanation for the protective effect of the attenuated anticoccidial vaccine against NE. Intestinal damage will result in the release of plasma proteins into the lumen of the intestinal tract. Since the minimal requirements for growth of C. perfringens include more than 11 amino acids, besides many growth factors and vitamins, leaking of plasma to the intestinal lumen can provide a necessary growth substrate for extensive proliferation of these bacteria (Van Immerseel et al., 2004). The reduction of coccidiosis lesions in the intestinal mucosa limits the available nutrients for C. perfringens multiplication and toxinogenesis in the gastrointestinal tract. Finally, the prevention of coccidiosis lesions in the intestinal mucosa discourages the attachment of C. perfringens to 168 V. Tsiouris et al. Figure 2. Jejunum cross-section obtained from a 23-day-old broiler chicken, challenged with C. perfringens and E. maxima. Marked inflammatory reaction was observed, which consisted of infiltration of heterophilic granulocytes and lymphocytes. Moreover, the presence of large numbers of rod-shaped bacteria as clusters (2a) or individually (2b) was detected at the intestinal lumen and mucosa (haematoxylin and eosin). epithelium, its colonization and toxinogenesis (McDevitt et al., 2006; Timbermont et al., 2011). According to Løvland & Kaldhusdal (1999), in cases of NE increased numbers 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 increased the overall mean grade of gross lesions in liver as well as the counts of C. perfringens isolated from the liver. Moreover, the use of attenuated anticoccidial vaccine on the first day of age reduced the counts of C. perfringens isolated from the liver in birds that had been challenged with C. perfringens and E. maxima. This finding might be the result of a better prophylaxis against coccidiosis, and in particular against intestinal damage from coccidia multiplication. Another plausible explanation could be the reduction of C. perfringens counts isolated from the intestinal tract in birds vaccinated with attenuated anticoccidial vaccine on the first day of age. The challenge of birds with C. perfringens and E. maxima reduced the pH of the duodenum, jejunum and ileum digesta. This pH reduction of intestinal content may be due the mixed challenge used in the present experimental model for the reproduction of NE, and, in particular, the challenge with E. maxima (Williams, 2005). The pH of the caeca was unaffected, probably because the lesions of E. maxima are located in the small intestine (Johnson & Reid, 1970). Vaccination of birds with attenuated anticoccidial vaccine prior to challenge limited the pH reduction of the intestinal content, as birds were immunized against coccidiosis and developed less severe lesions. In the present study, the viscosity of intestinal content in all experimental groups was relatively high, as compared with results reported in the literature (Bedford & Classen, 1992). This could be attributed to the feed composition, and, in particular, to the large amount of wheat and rye, which are rich in watersoluble non-starch polysaccharides. The purpose of this specific feed composition was the predisposition to NE, as cereal grains rich in non-starch polysaccharides, which produce high intestinal viscosity, have been associated with the occurrence of NE (Kaldhusdal & Skjerve, 1996). A higher intestinal viscosity increases the average retention time of the intestinal content, which is likely to create a favourable environment for bacterial activity. In particular, the flow of digesta is reduced and the amount of undigested material in the intestinal tract is increased, which gives the microbes more time and substrate to colonize the small intestine (Waldenstedt et al., 2000). Although higher intestinal viscosity favours C. perfringens colonization to the small intestine, proliferation and toxin production (Waldenstedt et al., 2000), in the present study the challenged groups, which had severe NE intestinal lesions, had lower viscosity values compared with NE-negative groups. However, the viscosity of the intestinal digesta is only one of the numerous predisposing factors of NE (Williams, 2005; McDevitt et al., 2006). Eimeria spp. predispose to NE due to intestinal damage, which is caused by coccidial invasion and evolution of its lifecycle in the intestinal mucosa (Van Immerseel et al., 2004; Williams, 2005). Figure 3. Jejunum cross-section obtained from a 23-day-old broiler chicken, challenged with C. perfringens and E. maxima. The architectural structure of the intestinal mucosa was lost, while intestinal villi were segmented (3a) or fused (3b) (haematoxylin and eosin). Anticoccidial vaccine and necrotic enteritis 169 Figure 4. Jejunum cross-section obtained from a 23-day-old broiler chicken, challenged with C. perfringens and E. maxima. Aggregation of schizonts as well as infiltration of heterophilic granulocytes and lymphocytes was observed (haematoxylin and eosin). The plasma proteins, subsequently, are released into the lumen of the intestinal tract and provide to C. perfringens all the requirement nutrients for its growth and proliferation (Van Immerseel et al., 2004). Moreover, damaged intestinal mucosa encourages the attachment of C. perfringens to the epithelium, its colonization and toxinogenesis (McDevitt et al., 2006; Timbermont et al., 2011). These lesions lead to the reduction of viscosity of intestinal content, due to osmotic and absorptive changes in the intestinal tract (Waldenstedt et al., 2000). The attenuated anticoccidial vaccination limited the severity of coccidiosis lesions and subsequently limited the viscosity reduction in intestinal content. The attenuated anticoccidial vaccination of birds on the first day of age resulted in increased viscosity of the ileum content. However, according to Waldenstedt et al. (2000), coccidiosis markedly reduced intestinal viscosity. This disagreement could be attributed to the fact that vaccine oocysts are of low pathogenicity and do not cause such severe lesions as wild-type oocysts. Furthermore, the number of oocysts contained in the vaccine was much lower compared with field infection or experimental challenge (Waldenstedt et al., 2000; Holdsworth et al., 2004). The increased viscosity of the ileum content in birds vaccinated with attenuated anticoccidial vaccine was not followed by increased caecal counts of C. perfringens. The results obtained here confirm earlier reports (Waldenstedt et al., 2000), in which C. perfringens caecal counts did not change when viscosity of the intestinal digesta was increased by dietary supplementation with carboxymethyl cellulose. The predisposition to NE is probably influenced by the increase of nutrients availability to the clostridia, rather than by the effect of viscosity per se. According to Waldenstedt et al. (1999), vaccinated unmedicated birds had higher C. perfringens caecal counts than unvaccinated medicated birds. However, in the present study the attenuated anticoccidial vaccination reduced the C. perfringens counts in the caeca. This contradiction could be the result of the presence of anticoccidial drugs in the feed of control groups in the study of Waldenstedt et al. (1999). Anticoccidial drugs, mainly ionophores, possess both anticoccidial and anticlostridial action and can reduce C. perfringens counts in the intestinal tract (Lanckriet et al., 2010). To the best of our knowledge, the present data are among the first to describe a significant effect of the attenuated anticoccidial vaccination on the physicochemical conditions in the intestinal ecosystem and C. perfringens counts. Moreover, attenuated anticoccidial vaccination showed a significant protective effect against subclinical experimental NE in broiler chickens. These actions of the attenuated anticoccidial vaccine increase its importance, not only for the prevention of coccidiosis but also for its assistance with better control of NE and, in general, better gut health. Further studies are needed to define the mechanisms underlying these effects and the impact on other parameters of the intestinal ecosystem, such as microbiota and immunity. Acknowledgements The authors express their gratitude to Professor F. Haesebrouck (Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Belgium) and to Dr R. Marshall (Veterinary Laboratories Agency, Weybridge, UK) for generously and kindly providing the C. perfringens and E. maxima strain, respectively. Moreover, the authors are also grateful to PhD student Deligeorgi Ioanni and Mr Moulto Serafim for their excellent assistance and technical support. Feed mills ‘‘Stravaridis S.A.’’ is thanked for feed formulation, while hatchery ‘‘TzotzasKoutsou S.A.’’ is thanked for the provision of 1-day-old chicks. References Bedford, M. & Classen, H. (1992). Reduction of intestinal viscosity through manipulation of dietary rye and pentosanase concentration is effected through changes in the carbohydrate composition of the intestinal aqueous phase and results in improved growth rate and food conversion efficiency of broiler chicks. Journal of Nutrition, 122, 560
569. Dahiya, J. (2007). Nutritional strategies to control Clostridium perfringens in gastrointestinal tract of broiler chickens (PhD Thesis). Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Canada. Dalloul, A. & Lillehoj, S. (2005). Recent advances in immunomodulation and vaccination strategies against coccidiosis. Avian Diseases, 49, 1
8. Drew, M., Syed, N., Goldade, B., Laarveld, B. & Van Kessel, A. (2004). Effects of dietary protein source and level on intestinal populations of Clostridium perfringens in broiler chickens. Poultry Science, 83, 414
420. Ernik, F. & Bedrnik, P. (2001). Controlling coccidiosis in broiler growing. Poultry International, 40, 36
42. European Commission. (2007). Council directive 2007/43/EC of 28 June 2007 laying down minimum rules for the protection of chickens kept for meat production. Official Journal L, 182, 19
28. 170 V. Tsiouris et al. Gholamiandehkordi, A., Timbermont, L., Lanckriet, A., Van Den Broeck, W., Pedersen, K., Dewulf, J., Pasmans, F., Haesebrouck, F., Ducatelle, R. & Van Immerseel, F. (2007). Quantification of gut lesions in a subclinical necrotic enteritis model. Avian Pathology, 36, 375
382. Holdsworth, P.A., Conway, D.P., McKenzie, M.E., Dayton, A.D., Chapman, H.D., Mathis, G.F., Skinner, J.T., Mundt, H.-C. & Williams, R.B. (2004). World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for evaluating the efficacy of anticoccidial drugs in chickens and turkeys. Veterinary Parasitology, 121, 189
212. Ito, N., Miyaji, C., Lima, E., Okabayashi, S., Claure, R. & Graca, E. (2004). Entero-hepatic pathobiology: histopathology and semiquantitative bacteriology of the duodenum. Brazilian Journal of Poultry Science, 6, 31
40. Johnson, J. & Reid, W. (1970). Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Experimental Parasitology, 28, 30
36. Kaldhusdal, M., Hofshagen, M., Løvland, A., Langstrand, H. & Redhead, K. (1999). Necrotic enteritis challenge models with broiler chickens raised on litter: evaluation of preconditions, Clostridium perfringens strains and outcome variables. FEMS Immunology & Medical Microbiology, 24, 337
343. Kaldhusdal, M. & Skjerve, E. (1996). Association between cereal contents in the diet and incidence of necrotic enteritis in broiler chickens in Norway. Preventive Veterinary Medicine, 28, 1
16. Keyburn, A., Sheedy, S., Ford, M., Williamson, M., Awad, M., Rood, J. & Moore, R. (2006). Alpha-toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in chickens. Infection and Immunity, 74, 6496
6500. Lanckriet, A., Timbermont, L., De Gussem, M., Marien, M., Vancraeynest, D., Haesebrouck, F., Ducatelle, R. & Van Immerseel, F. (2010). The effect of commonly used anticoccidials and antibiotics in a subclinical necrotic enteritis model. Avian Pathology, 39, 63
68. Løvland, A. & Kaldhusdal, M. (1999). Liver lesions seen at slaughter as an indicator of necrotic enteritis in broiler flocks. FEMS Immunology and Medical Microbiology, 24, 345
351. McDevitt, R., Brooker, J., Acamovic, T. & Sparks, N. (2006). Necrotic enteritis: a continuing challenge for the poultry industry. World’s Poultry Science Journal, 62, 221
247. McReynolds, J., Byrd, J., Anderson, R., Moore, R., Edrington, T., Genovese, K., Poole, T., Kubena, L. & Nisbet, D. (2004). Evaluation of immunosuppressants and dietary mechanisms in an experimental disease model for necrotic enteritis. Poultry Science, 83, 1948
1952. McReynolds, J., Byrd, J., Genovese, K., Poole, T., Duke, S., Farnell, M. & Nisbet, D. (2007). Dietary lactose and its effect on the disease condition of necrotic enteritis. Poultry Science, 86, 1656
1661. Novoa-Garrido, M., Larsen, S. & Kaldhusdal, M. (2006). Association between gizzard lesions and increased caecal Clostridium perfringens counts in broiler chickens. Avian Pathology, 35, 367
372. Pedersen, K., Bjerrum, L., Heuer, O., Wong, D. & Nauerby, B. (2008). Reproducible infection model for Clostridium perfringens in broiler chickens. Avian Diseases, 52, 34
39. Ross Technology 98/36. (1999). In Team of Ross Breeders Limited (Ed.). Necrotic Enteritis and Associated Conditions in Broiler Chickens (pp. 1
6). Newbridge, Midlothian, EH28 8SZ, UK. Shirley, M. (1996). New methods for the identification of species and strains of Eimeria. In L. McDougald, P. Long, & L. Joyner (Eds.). Research in Avian Coccidiosis (pp. 13
35). University of Georgia, Athens, USA. Skinner, J., Bauer, S., Young, V., Pauling, G. & Wilson, J. (2010). An economic analysis of the impact of subclinical (mild) necrotic enteritis in broiler chickens. Avian Diseases, 54, 1237
1240. Timbermont, L., Haesebrouck, F., Ducatelle, R. & Van Immerseel, F. (2011). Necrotic enteritis in broilers: an updated review on the pathogenesis. Avian Pathology, 40, 341
347. Timbermont, L., Lanckriet, A., Gholamiandehkordi, A., Pasmans, F., Martel, A., Haesebrouck, F., Ducatelle, R. & Van Immerseel, F. (2009). Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers. Comparative Immunology, Microbiology & Infectious Diseases, 32, 503
512. Van Immerseel, F., De Buck, J., Pasmans, F., Huyghebaert, G., Haesebrouck, F. & Ducatelle, R. (2004). Clostridium perfringens in poultry: an emerging threat for animal and public health. Avian Pathology, 33, 537
549. Waldenstedt, L., Elwinger, K., Lunden, A., Thebo, P., Bedford, M.R. & Uggla, A. (2000). Intestinal digesta viscosity decreases during coccidial infection in broilers. British Poultry Science, 41, 459
464. Waldenstedt, L., Lunden, A., Elwinger, K., Thebo, P. & Uggla, A. (1999). Comparison between a live, attenuated anticoccidial vaccine and an anticoccidial ionophore, on performance of broilers raised with or without a growth promoter, in an initially Eimeria-free environment. Acta Veterinaria Scandinavica, 40, 11
21. Williams, R. (2002). Anticoccidial vaccines for broiler chickens: pathways to success. Avian Pathology, 31, 317
353. Williams, R. (2005). Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut integrity. Avian Pathology, 34, 159
180. Williams, R.B., Marshall, R.N., La Ragione, R.M. & Catchpole, J. (2003). A new method for the experimental production of necrotic enteritis and its use for studies on the relationships between necrotic enteritis, coccidiosis and anticoccidial vaccination of chickens. Parasitology Research, 90, 19
26.