Original Article
Campylobacter infection in chickens modulates the intestinal epithelial barrier function
Innate Immunity 2015, Vol. 21(2) 151–160 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1753425914521648 ini.sagepub.com
Wageha A Awad1, Andor Molna´r2, Jo¨rg R Aschenbach3, Khaled Ghareeb4,5, Basel Khayal1, Claudia Hess1, Dieter Liebhart1, Ka´roly Dublecz2 and Michael Hess1
Abstract Asymptomatic carriage of Campylobacter jejuni is highly prevalent in chicken flocks. Thus, we investigated whether chronic Campylobacter carriage affects chicken intestinal functions despite the absence of clinical symptoms. An experiment was carried out in which commercial chickens were orally infected with C. jejuni (1 108 CFU/bird) at 14 days of life. Changes in ion transport and barrier function were assessed by short-circuit current (Isc) and transepithelial ion conductance (Gt) in Ussing chambers. Gt increased in cecum and colon of Campylobacter-infected chicken 7 d post-infection (DPI), whereas Gt initially decreased in the jejunum at 7 DPI and increased thereafter at 14 DPI. The net charge transfer across the epithelium was reduced or tended to be reduced in all segments, as evidenced by a decreased Isc. Furthermore, the infection induced intestinal histomorphological changes, most prominently including a decrease in villus height, crypt depth and villus surface area in the jejunum at 7 DPI. Furthermore, body mass gain was decreased by Campylobacter carriage. This study demonstrates, for the first time, changes in the intestinal barrier function in Campylobacter-infected chickens and these changes were associated with a decrease in growth performance in otherwise healthy-appearing birds.
Keywords Campylobacter jejuni, chickens, intestinal permeability, intestinal barrier, Ussing chamber Date received: 14 November 2013; revised: 13 December 2013; accepted: 27 December 2013
Introduction Campylobacter jejuni is recognized as a leading cause of acute bacterial gastroenteritis in humans worldwide.1,2 Poultry is a major reservoir for C. jejuni and considered as the most common source for human infections as chickens can carry a high load of Campylobacter without clinical signs.3,4 Although C. jejuni is pathogenic to humans, it is believed that it is a commensal in avian hosts.5 Insights into the mechanisms underlying these differences in infection outcome following Campylobacter exposure are of crucial importance in understanding the interaction of such a microorganism with its host, in assessing the clinical and economic implications of Campylobacter shedding by chicken, and in taking rational preventive measures based on the latter assessments. The intestinal mucosa acts as a selectively permeable barrier, permitting the absorption of nutrients, electrolytes and water, while it also serves as a barrier of defence against pathogens by stimulating mechanisms that reduce the ability of pathogens and their toxins to
invade the mucosa.6–12 The mucosal permeability is adaptable and regulated in response to extracellular stimuli such as bacteria.10
1 Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria 2 Department of Animal Science and Animal Husbandry, Georgikon Faculty, University of Pannonia, Keszthely, Hungary 3 Institute of Veterinary Physiology, Department of Veterinary Medicine, Free University of Berlin, Berlin, Germany 4 Institute of Animal Nutrition and Functional Plant Compounds, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria 5 Department of Animal Hygiene, Behaviour and Management, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt
Corresponding author: Wageha A. Awad, Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterina¨rplatz 1, A-1210 Vienna, Austria. Email: [email protected]
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Man13 demonstrated in human cell culture models (T84 and Caco-2 cells) that several Campylobacter species have the ability to attach and invade intestinal epithelial cells and macrophages, to compromise the intestinal barrier integrity and physiology, to secrete toxins that kill host cells and to impair host cell functions. He also reported that attachment to the intestinal epithelium by Campylobacter increased the permeability of the intestinal epithelial barrier, decreased transepithelial electrical resistance, and resulted in a loss of tight junction proteins. Moreover, the common adhesive and invasive nature exhibited by several Campylobacter species implies that these mechanisms are one of the primary weapons used by different members of the genus Campylobacter to persist in and colonize the gastrointestinal tract. Nielsen et al.14 showed that Campylobacter concisus induced a time- and dosedependent decrease in tissue resistance, and increased permeability and apoptosis together with moderate changes in tight junction protein expression (claudin5) in HT29/B6 colonic epithelial cells, and this observation coincided with the ability of the bacterium to compromise barrier functions. In addition to affecting barrier function, enteric bacterial pathogens may also induce fluid and electrolyte secretion, and initiate inflammatory responses.15 We recently demonstrated that these pathological consequences of infection differ greatly between the intestinal epithelium of chickens compared to what is known for mammals and humans. In our previous study, exposure of jejunal and cecal epithelia from healthy chickens to living Salmonella Enteritidis or its endotoxin acutely decreased the intestinal ion permeability and induced (de novo) sensitivity to the inflammatory and neuronal mediator histamine.16 This indicated marked differences in the innate pathogen recognition processes between chickens and mammals, and may explain why chickens react to Salmonella and other invasive Gram-negative bacteria (e.g. Campylobacter) with subclinical carriage rather than overt disease. Whether and how the intestinal function is altered during the subsequent chronic carrier state, however, has not been investigated so far. Therefore, the aim of this study was to investigate the changes in passive ion permeability and histamine responsiveness in the chronic carrier state of C. jejuni infections. As such, this study can be considered as the first approach to obtain insight into the functional consequences of chronic Campylobacter colonization, using an integrative combination of in vivo and in vitro experiments.
Materials and methods Ethics statement The animal experiment was discussed and approved by the institutional ethics committee of the University of
Veterinary Medicine and Austrian Federal Ministry for Science and Research under the license number GZ 68.205/0227-II/3b/2011. All husbandry practices and euthanasia were performed with full consideration of animal welfare.
Birds and feeding Forty-eight 1-d-old broiler chickens (males and females) were obtained from a commercial hatchery (Ross-308, Geflu¨gelhof Schulz, Graz, Austria) and randomly divided into two groups (24 birds/group). The birds were housed on wood shavings and were provided with food and water ad libitum. The broilers were fed diets based on wheat, maize, barley, soybean meal, soybean oil, sunflower oil and a premix with vitamins, minerals, amino acids, salt and mono-calcium phosphate. The diet contained 22% crude protein, 8.5% fat, 3.3% crude fiber and 1.4% lysine. At 1 and 14 d of age, cloacal swabs were taken from all birds and directly plated on modified charcoal–cefaperazone–deoxycholate agar (CM0739; OXOID, Hampshire, UK) for Campylobacter determination (at 42 C for 48 h) to ensure absence of C. jejuni. One group was kept as infected and the other one as the non-infected control group. At 14 d of age each bird in the infected group was inoculated orally by a feeding tube (gavage) with 1 108 CFU/bird of the Campylobacter reference strain NCTC 12744 as previously described.17,18
Body mass of birds Body mass (BM) was determined at different time points and the body mass gain (BMG) was calculated as the difference between the final and initial bird mass during each of the weighing periods. Furthermore, feed intake over the course of the experiment was measured for control and infected birds and, consequently, feed conversion ratio (FCR) was calculated. In this experiment, six birds from each group were killed at each time point 7, 14 and 21 d post-infection (DPI) by bleeding of the jugular vein under anesthesia with a single dose of thiopental (1 mg/kg) injection into the wing vein.
Histomorphological examination of the small intestine For histomorphological examination (crypt-villus measurements), tissue samples were taken from jejunum close to the junction of Meckel’s diverticulum. The samples were fixed in 4% buffered formalin for 48 h. The processing consisted of serial dehydration, clearing and impregnation with wax. Tissue crosssections (three per bird, 5 mm thick) from each of the six birds per treatment were cut by a microtome and
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fixed on slides. Afterwards, deparaffination was performed in xylene (twice, 5 min each time) followed by rehydration in alcohol 100% (5 min), 96% (5 min) and 70% (5 min). A routine staining procedure was carried out using hematoxylin and eosin.19 The slides were examined with an Olympus BX43F light microscope (Olympus, Tokyo, Japan) fitted with a digital video camera (Olympus DP-26; Olympus). The images were analyzed using Olympus Stream image analysis software v. 1.7 (Olympus). The total of the intact welloriented, crypt-villus units were selected in triplicate for each intestinal cross-section per sample. The criterion for villus selection was based on the presence of an intact lamina propria. Villus height was measured from the tip of the villus to the villus-crypt junction, while crypt depth was defined as the depth of the invagination between adjacent villi. The villus width was measured at the basal and at the apical transverse of the villus. Villus surface area was calculated from villus height and width at half height, and the villus height to crypt depth (H:D) ratio was also calculated as previously described.20,21
Bacteriological investigation Concurrent with the killing of birds, contents of jejunum and cecum (1 g) were collected for C. jejuni enumeration. Samples were diluted 1:10 (mass:vol) in PBS (BR0014G; OXOID) and the mixture homogenized using an Ultra-Turrax (IKA, Staufen, Germany). Afterwards, 10-fold dilutions were made from the stock suspension, and each dilution was direct-plated on Campylosel agar (BioMerieux, Vienna, Austria). The plates were incubated microaerophilically at 42 C for 48 h. After incubation, typical Campylobacter spp. colonies were counted as CFU/g.
Ussing chamber analysis The intestinal segments were taken from the mid-jejunum, cecum and colon (two replicates of each segment/ bird) immediately after killing of birds (six birds/ group). The preparation of epithelia and mounting in Ussing chambers was done as previously described.22 The intestinal segments were harvested from the birds and placed into ice-cold buffer solution (contained in mmol/l: NaCl, 115; KCl, 5; CaCl2, 1.5; MgCl2, 1.2; NaH2PO4, 0.6; Na2HPO4, 2.4; L-glutamine, 1; Na-D/ L-lactate, 5; HEPES-free acid, 10; NaHCO3, 25; and mannitol, 10; pH 7.4) oxygenated with carbogen (95% O2/5% CO2). The intestinal segments were opened along the mesenteric border and washed free of intestinal content with buffer solution at 4 C. The underlying serosal layer was stripped off and the epithelial sheets were mounted in Ussing chambers. Epithelial sheets had an exposed serosal area of 1.1 cm2 and were incubated with 12 ml of buffer
solution on their mucosal and serosal sides under short-circuit conditions. The short-circuit current (Isc in mA/cm2) and tissue conductance (Gt in mS/cm2) were recorded. The basal measurements of Isc and Gt were taken after a stabilization period of 30 min (low/or no fluctuation of the measurements). Histamine, as a neural and immune mediator that commonly elicits secretion, was tested for its influence on Isc and Gt after infection. For this, histamine was applied basolaterally after 1 h of tissue incubation and the tissues were further incubated for at least 30 min. The effects of histamine application to the serosal side on the electrical variables are given as the changes in Gt or Isc (Gt or Isc) with reference to the value immediately before histamine addition.
Statistical analysis All data are presented as means and SEM. Following tests for normality (Kolmogorov–Smirnov’s test), Student’s t-test was used to detect significant differences between infected and control groups for performance, intestinal histology and electrophysiological responses in Ussing chambers. The mean of each bird was the experimental unit for histology data. Differences were considered significant at a level of P 0.05. All tests were performed using appropriate software (PASW statistics 20; SPSS, Chicago, IL, USA).
Results Campylobacter-associated change in BM and bacterial colonization during the entire trial All birds appeared clinically normal during entire trial. There were no signs of diarrhea, and fecal droppings looked normal in both control and infected birds. No mortality occurred over the course of the experiment. In addition, no differences were found between the BM of birds in the different groups prior to infection. Growth performance of infected broiler chickens, in terms of the average BM, numerically decreased (P < 0.1) at 7 and 14 DPI (510 61 and 1024 65 g, respectively) compared with the controls (620 26 and 1101 67 g, respectively). Later on, at 21 DPI, BM (1398 117 g) was significantly depressed by C. jejuni infection compared with the controls (1768 109 g; P < 0.05; Figure 1). In addition, no differences were observed in the average daily feed intake between control birds (106 g/bird/d) and infected birds (104 g/bird/d). However, the FCR of infected birds (1.81) was numerically higher than control birds (1.41), which may indicate that C. jejuni infection can affect feed efficiency. These results suggest that the lower slaughtering mass after C. jejuni infection may be due to the reduction in the feed efficiency of infected birds.
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(a) 2000
Control
C. jejuni
1800 1600
*
BM (g)
1400 1200 1000 800 600 400 200 0 14–d
21–d
28–d
35–d
Age (day)
(b) 1800 Control
C. jejuni
1600 1400
*
BMG (g)
1200 1000 800 600
*
400 200 0 BWG (14–21 d)
BWG (21–28 d)
BWG (28–35 d)
Overall BWG (14–35 d)
Figure 1. Effect of a Campylobacter infection at 14 d of age on BM and BMG of broilers. (a) Mean BM and (b) mean BMG from noninfected control chickens and Campylobacter-infected chickens receiving 108 C. jejuni (n ¼ 10). Data are means + SEM. Asterisks mark significant differences: *P < 0.05.
In the infected birds, at 7 DPI, C. jejuni colonization in jejunum and cecum were 0.69 103 3.51 and 0.11 106 0.69 CFU/g of contents, respectively. Later on, at 14 DPI, values of 0.26 105 1.42 and 0.67 108 4.74 CFU/g were recorded in jejunum and cecum, respectively. Furthermore, C. jejuni colonization in jejunum (0.66 105 5.50 CFU/g) and cecum (0.46 108 4.12 CFU/g) at 21 DPI was similar to 14 DPI. Campylobacter colonization was higher (P < 0.01) in the cecum and jejunum (P < 0.1) at 21 DPI than the colonization at 7 DPI. In contrast, C. jejuni was not detected in any of the control animals.
Influence of Campylobacter infection on intestinal histomorphology Intestinal histomorphometric analysis for the jejunum revealed that the C. jejuni colonization altered the small intestinal architecture (Figures 2–4). At 7 DPI, the villi in the jejunum were significantly (P < 0.001) shorter (662 mm) in the infected group than in the controls (889 mm) (Figure 3). Campylobacter infection also resulted in a decreased (P < 0.05) crypt depth and
villus surface area (121 mm versus 80 mm2) compared with controls (159 mm versus 110 mm2). These changes resulted in a significantly decreased apparent villus surface area (80 versus 110 mm2, P
Campylobacter infection in chickens modulates the intestinal epithelial barrier function
Innate Immunity 2015, Vol. 21(2) 151–160 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1753425914521648 ini.sagepub.com
Wageha A Awad1, Andor Molna´r2, Jo¨rg R Aschenbach3, Khaled Ghareeb4,5, Basel Khayal1, Claudia Hess1, Dieter Liebhart1, Ka´roly Dublecz2 and Michael Hess1
Abstract Asymptomatic carriage of Campylobacter jejuni is highly prevalent in chicken flocks. Thus, we investigated whether chronic Campylobacter carriage affects chicken intestinal functions despite the absence of clinical symptoms. An experiment was carried out in which commercial chickens were orally infected with C. jejuni (1 108 CFU/bird) at 14 days of life. Changes in ion transport and barrier function were assessed by short-circuit current (Isc) and transepithelial ion conductance (Gt) in Ussing chambers. Gt increased in cecum and colon of Campylobacter-infected chicken 7 d post-infection (DPI), whereas Gt initially decreased in the jejunum at 7 DPI and increased thereafter at 14 DPI. The net charge transfer across the epithelium was reduced or tended to be reduced in all segments, as evidenced by a decreased Isc. Furthermore, the infection induced intestinal histomorphological changes, most prominently including a decrease in villus height, crypt depth and villus surface area in the jejunum at 7 DPI. Furthermore, body mass gain was decreased by Campylobacter carriage. This study demonstrates, for the first time, changes in the intestinal barrier function in Campylobacter-infected chickens and these changes were associated with a decrease in growth performance in otherwise healthy-appearing birds.
Keywords Campylobacter jejuni, chickens, intestinal permeability, intestinal barrier, Ussing chamber Date received: 14 November 2013; revised: 13 December 2013; accepted: 27 December 2013
Introduction Campylobacter jejuni is recognized as a leading cause of acute bacterial gastroenteritis in humans worldwide.1,2 Poultry is a major reservoir for C. jejuni and considered as the most common source for human infections as chickens can carry a high load of Campylobacter without clinical signs.3,4 Although C. jejuni is pathogenic to humans, it is believed that it is a commensal in avian hosts.5 Insights into the mechanisms underlying these differences in infection outcome following Campylobacter exposure are of crucial importance in understanding the interaction of such a microorganism with its host, in assessing the clinical and economic implications of Campylobacter shedding by chicken, and in taking rational preventive measures based on the latter assessments. The intestinal mucosa acts as a selectively permeable barrier, permitting the absorption of nutrients, electrolytes and water, while it also serves as a barrier of defence against pathogens by stimulating mechanisms that reduce the ability of pathogens and their toxins to
invade the mucosa.6–12 The mucosal permeability is adaptable and regulated in response to extracellular stimuli such as bacteria.10
1 Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria 2 Department of Animal Science and Animal Husbandry, Georgikon Faculty, University of Pannonia, Keszthely, Hungary 3 Institute of Veterinary Physiology, Department of Veterinary Medicine, Free University of Berlin, Berlin, Germany 4 Institute of Animal Nutrition and Functional Plant Compounds, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria 5 Department of Animal Hygiene, Behaviour and Management, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt
Corresponding author: Wageha A. Awad, Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterina¨rplatz 1, A-1210 Vienna, Austria. Email: [email protected]
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Man13 demonstrated in human cell culture models (T84 and Caco-2 cells) that several Campylobacter species have the ability to attach and invade intestinal epithelial cells and macrophages, to compromise the intestinal barrier integrity and physiology, to secrete toxins that kill host cells and to impair host cell functions. He also reported that attachment to the intestinal epithelium by Campylobacter increased the permeability of the intestinal epithelial barrier, decreased transepithelial electrical resistance, and resulted in a loss of tight junction proteins. Moreover, the common adhesive and invasive nature exhibited by several Campylobacter species implies that these mechanisms are one of the primary weapons used by different members of the genus Campylobacter to persist in and colonize the gastrointestinal tract. Nielsen et al.14 showed that Campylobacter concisus induced a time- and dosedependent decrease in tissue resistance, and increased permeability and apoptosis together with moderate changes in tight junction protein expression (claudin5) in HT29/B6 colonic epithelial cells, and this observation coincided with the ability of the bacterium to compromise barrier functions. In addition to affecting barrier function, enteric bacterial pathogens may also induce fluid and electrolyte secretion, and initiate inflammatory responses.15 We recently demonstrated that these pathological consequences of infection differ greatly between the intestinal epithelium of chickens compared to what is known for mammals and humans. In our previous study, exposure of jejunal and cecal epithelia from healthy chickens to living Salmonella Enteritidis or its endotoxin acutely decreased the intestinal ion permeability and induced (de novo) sensitivity to the inflammatory and neuronal mediator histamine.16 This indicated marked differences in the innate pathogen recognition processes between chickens and mammals, and may explain why chickens react to Salmonella and other invasive Gram-negative bacteria (e.g. Campylobacter) with subclinical carriage rather than overt disease. Whether and how the intestinal function is altered during the subsequent chronic carrier state, however, has not been investigated so far. Therefore, the aim of this study was to investigate the changes in passive ion permeability and histamine responsiveness in the chronic carrier state of C. jejuni infections. As such, this study can be considered as the first approach to obtain insight into the functional consequences of chronic Campylobacter colonization, using an integrative combination of in vivo and in vitro experiments.
Materials and methods Ethics statement The animal experiment was discussed and approved by the institutional ethics committee of the University of
Veterinary Medicine and Austrian Federal Ministry for Science and Research under the license number GZ 68.205/0227-II/3b/2011. All husbandry practices and euthanasia were performed with full consideration of animal welfare.
Birds and feeding Forty-eight 1-d-old broiler chickens (males and females) were obtained from a commercial hatchery (Ross-308, Geflu¨gelhof Schulz, Graz, Austria) and randomly divided into two groups (24 birds/group). The birds were housed on wood shavings and were provided with food and water ad libitum. The broilers were fed diets based on wheat, maize, barley, soybean meal, soybean oil, sunflower oil and a premix with vitamins, minerals, amino acids, salt and mono-calcium phosphate. The diet contained 22% crude protein, 8.5% fat, 3.3% crude fiber and 1.4% lysine. At 1 and 14 d of age, cloacal swabs were taken from all birds and directly plated on modified charcoal–cefaperazone–deoxycholate agar (CM0739; OXOID, Hampshire, UK) for Campylobacter determination (at 42 C for 48 h) to ensure absence of C. jejuni. One group was kept as infected and the other one as the non-infected control group. At 14 d of age each bird in the infected group was inoculated orally by a feeding tube (gavage) with 1 108 CFU/bird of the Campylobacter reference strain NCTC 12744 as previously described.17,18
Body mass of birds Body mass (BM) was determined at different time points and the body mass gain (BMG) was calculated as the difference between the final and initial bird mass during each of the weighing periods. Furthermore, feed intake over the course of the experiment was measured for control and infected birds and, consequently, feed conversion ratio (FCR) was calculated. In this experiment, six birds from each group were killed at each time point 7, 14 and 21 d post-infection (DPI) by bleeding of the jugular vein under anesthesia with a single dose of thiopental (1 mg/kg) injection into the wing vein.
Histomorphological examination of the small intestine For histomorphological examination (crypt-villus measurements), tissue samples were taken from jejunum close to the junction of Meckel’s diverticulum. The samples were fixed in 4% buffered formalin for 48 h. The processing consisted of serial dehydration, clearing and impregnation with wax. Tissue crosssections (three per bird, 5 mm thick) from each of the six birds per treatment were cut by a microtome and
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fixed on slides. Afterwards, deparaffination was performed in xylene (twice, 5 min each time) followed by rehydration in alcohol 100% (5 min), 96% (5 min) and 70% (5 min). A routine staining procedure was carried out using hematoxylin and eosin.19 The slides were examined with an Olympus BX43F light microscope (Olympus, Tokyo, Japan) fitted with a digital video camera (Olympus DP-26; Olympus). The images were analyzed using Olympus Stream image analysis software v. 1.7 (Olympus). The total of the intact welloriented, crypt-villus units were selected in triplicate for each intestinal cross-section per sample. The criterion for villus selection was based on the presence of an intact lamina propria. Villus height was measured from the tip of the villus to the villus-crypt junction, while crypt depth was defined as the depth of the invagination between adjacent villi. The villus width was measured at the basal and at the apical transverse of the villus. Villus surface area was calculated from villus height and width at half height, and the villus height to crypt depth (H:D) ratio was also calculated as previously described.20,21
Bacteriological investigation Concurrent with the killing of birds, contents of jejunum and cecum (1 g) were collected for C. jejuni enumeration. Samples were diluted 1:10 (mass:vol) in PBS (BR0014G; OXOID) and the mixture homogenized using an Ultra-Turrax (IKA, Staufen, Germany). Afterwards, 10-fold dilutions were made from the stock suspension, and each dilution was direct-plated on Campylosel agar (BioMerieux, Vienna, Austria). The plates were incubated microaerophilically at 42 C for 48 h. After incubation, typical Campylobacter spp. colonies were counted as CFU/g.
Ussing chamber analysis The intestinal segments were taken from the mid-jejunum, cecum and colon (two replicates of each segment/ bird) immediately after killing of birds (six birds/ group). The preparation of epithelia and mounting in Ussing chambers was done as previously described.22 The intestinal segments were harvested from the birds and placed into ice-cold buffer solution (contained in mmol/l: NaCl, 115; KCl, 5; CaCl2, 1.5; MgCl2, 1.2; NaH2PO4, 0.6; Na2HPO4, 2.4; L-glutamine, 1; Na-D/ L-lactate, 5; HEPES-free acid, 10; NaHCO3, 25; and mannitol, 10; pH 7.4) oxygenated with carbogen (95% O2/5% CO2). The intestinal segments were opened along the mesenteric border and washed free of intestinal content with buffer solution at 4 C. The underlying serosal layer was stripped off and the epithelial sheets were mounted in Ussing chambers. Epithelial sheets had an exposed serosal area of 1.1 cm2 and were incubated with 12 ml of buffer
solution on their mucosal and serosal sides under short-circuit conditions. The short-circuit current (Isc in mA/cm2) and tissue conductance (Gt in mS/cm2) were recorded. The basal measurements of Isc and Gt were taken after a stabilization period of 30 min (low/or no fluctuation of the measurements). Histamine, as a neural and immune mediator that commonly elicits secretion, was tested for its influence on Isc and Gt after infection. For this, histamine was applied basolaterally after 1 h of tissue incubation and the tissues were further incubated for at least 30 min. The effects of histamine application to the serosal side on the electrical variables are given as the changes in Gt or Isc (Gt or Isc) with reference to the value immediately before histamine addition.
Statistical analysis All data are presented as means and SEM. Following tests for normality (Kolmogorov–Smirnov’s test), Student’s t-test was used to detect significant differences between infected and control groups for performance, intestinal histology and electrophysiological responses in Ussing chambers. The mean of each bird was the experimental unit for histology data. Differences were considered significant at a level of P 0.05. All tests were performed using appropriate software (PASW statistics 20; SPSS, Chicago, IL, USA).
Results Campylobacter-associated change in BM and bacterial colonization during the entire trial All birds appeared clinically normal during entire trial. There were no signs of diarrhea, and fecal droppings looked normal in both control and infected birds. No mortality occurred over the course of the experiment. In addition, no differences were found between the BM of birds in the different groups prior to infection. Growth performance of infected broiler chickens, in terms of the average BM, numerically decreased (P < 0.1) at 7 and 14 DPI (510 61 and 1024 65 g, respectively) compared with the controls (620 26 and 1101 67 g, respectively). Later on, at 21 DPI, BM (1398 117 g) was significantly depressed by C. jejuni infection compared with the controls (1768 109 g; P < 0.05; Figure 1). In addition, no differences were observed in the average daily feed intake between control birds (106 g/bird/d) and infected birds (104 g/bird/d). However, the FCR of infected birds (1.81) was numerically higher than control birds (1.41), which may indicate that C. jejuni infection can affect feed efficiency. These results suggest that the lower slaughtering mass after C. jejuni infection may be due to the reduction in the feed efficiency of infected birds.
Downloaded from ini.sagepub.com at HOWARD UNIV UNDERGRAD LIBRARY on February 13, 2015
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Innate Immunity 21(2)
(a) 2000
Control
C. jejuni
1800 1600
*
BM (g)
1400 1200 1000 800 600 400 200 0 14–d
21–d
28–d
35–d
Age (day)
(b) 1800 Control
C. jejuni
1600 1400
*
BMG (g)
1200 1000 800 600
*
400 200 0 BWG (14–21 d)
BWG (21–28 d)
BWG (28–35 d)
Overall BWG (14–35 d)
Figure 1. Effect of a Campylobacter infection at 14 d of age on BM and BMG of broilers. (a) Mean BM and (b) mean BMG from noninfected control chickens and Campylobacter-infected chickens receiving 108 C. jejuni (n ¼ 10). Data are means + SEM. Asterisks mark significant differences: *P < 0.05.
In the infected birds, at 7 DPI, C. jejuni colonization in jejunum and cecum were 0.69 103 3.51 and 0.11 106 0.69 CFU/g of contents, respectively. Later on, at 14 DPI, values of 0.26 105 1.42 and 0.67 108 4.74 CFU/g were recorded in jejunum and cecum, respectively. Furthermore, C. jejuni colonization in jejunum (0.66 105 5.50 CFU/g) and cecum (0.46 108 4.12 CFU/g) at 21 DPI was similar to 14 DPI. Campylobacter colonization was higher (P < 0.01) in the cecum and jejunum (P < 0.1) at 21 DPI than the colonization at 7 DPI. In contrast, C. jejuni was not detected in any of the control animals.
Influence of Campylobacter infection on intestinal histomorphology Intestinal histomorphometric analysis for the jejunum revealed that the C. jejuni colonization altered the small intestinal architecture (Figures 2–4). At 7 DPI, the villi in the jejunum were significantly (P < 0.001) shorter (662 mm) in the infected group than in the controls (889 mm) (Figure 3). Campylobacter infection also resulted in a decreased (P < 0.05) crypt depth and
villus surface area (121 mm versus 80 mm2) compared with controls (159 mm versus 110 mm2). These changes resulted in a significantly decreased apparent villus surface area (80 versus 110 mm2, P
