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Effects of immune stress on growth performance, immunity, and cecal microflora in chickens1
Effects of immune stress on growth performance, immunity, and cecal microflora in chickens1 X. J. Yang,2 W. L. Li,2 Y. Feng, and J. H. Yao3 Northwest A&F University, Yangling, Shaanxi, China 712100 significantly at 42 d of age in the chickens that were treated with LPS or CPM (P < 0.01). The proliferation of the peripheral blood mononuclear cell and the levels of serum IgG in the LPS-challenged chickens were higher than those in the control group chickens at 21 and 42 d of age, respectively (P < 0.05). Six clusters were identified at 21 d of age, but cluster 6 was a single sample. Only 5 clusters were identified at 42 d of age. The enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprints of the cecal samples from the no vaccination and the simplified vaccination groups clustered together with high coefficients. The ERICPCR fingerprints of the 3 cecal samples from the CPM and LPS treatment groups clustered together with high coefficients among them. The ERIC-PCR fingerprints of the microbial flora of the cecal contents revealed the potential effects of immune stress on the microbial populations of treated birds. These data suggest that broilers with simplified vaccinations or without vaccinations can achieve the same growth performance as broilers with general vaccinations, but immune stress can break the homeostasis of cecal microflora and impair intestinal mucosal immune function.
Key words: immune stress, growth performance, immunity, cecal microflora, chicken 2011 Poultry Science 90:2740–2746 doi:10.3382/ps.2011-01591
INTRODUCTION As the development of breeding technology in broiler chickens has improved, some new lines of broilers, such as Arbor Acres, Avian, Cobb, and Cobb Avian 48, have shown great improvement in growth performance. How©2011 Poultry Science Association Inc. Received May 5, 2011. Accepted August 12, 2011. 1 This work was supported by the National Nature Science Foundation of China (31001017), the Science & Technological Project of Shaanxi Province, China (2009K01-02, 2010ZDGC-02), the Natural Science Foundation of Shaanxi Province (No. 2010JQ3002), the Opening Program State Key Laboratory of Animal Nutrition (No. 2004DA125184F0809), and the Central University special fund basic research and operating expenses (No. QN2009020). 2 Contributed equally to this paper. 3 Corresponding author: [email protected]
ever, these new lines exhibit weaker immune function from emerging stress (Yalcin et al., 2001; Deeb and Cahaner, 2002). Immune stress is the loss of immune homeostasis by external forces; vaccination, drugs, transport, contamination, and other procedures involved in production could be considered external forces. In addition, many stress factors (disease, heavy vaccinations, ammonia gas, noise, feed density, temperature, and humidity) may also influence the immune systems of broilers (Campo and Davila, 2002; Mashaly et al., 2004; Yang et al., 2006; Shini and Kaiser, 2009; Rajkumar et al., 2011; Wilkinson et al., 2011). Immunizations have sometimes been administered in large doses at a high frequency. These vaccinations were considered necessary to reduce the mortality rate that occurs in modern poultry production; however, unsuitable vaccinations could lead to immune stress.
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ABSTRACT Immune stress is the loss of immune homeostasis by external forces. This study investigated the effects of different types of immune stress on growth performance, immunity, and the distribution of cecal microflora in broiler chickens. In total, 540 oneday-old Cobb 500 broilers were randomly assigned to receive 1 of 5 (n = 108 birds/group) treatments: 1) no vaccination; 2) simplified vaccination, which included the infectious bronchitis vaccine (H120), the inactivated avian influenza vaccine (AI), the live vaccine strain Clone-30 of the Newcastle disease virus (NDV), and the combined inactive vaccine for infectious bursal diseases and the Newcastle disease vaccine (ND-IB); 3) normal vaccination (simplified vaccination + second dose of ND-IB, H120, and AI); 4) lipopolysaccharide (LPS) stress (normal vaccination+LPS); or 5) cyclophosphamide (CPM) stress (normal vaccination+CPM).The results showed that the average BW and average feed intake decreased significantly after treatment with LPS or CPM (P < 0.05). Chickens that were challenged by LPS or CPM had a lower ileal CP digestibility than that of the control group (P < 0.01). Compared with the control group, the levels of secreted IgA decreased
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MATERIALS AND METHODS Chemicals and Vaccines For this experiment, LPS from Escherichia coli (L2880, Sigma-Aldrich Inc., St. Louis, MO) and cyclophosphamide (CPM; C0768, Sigma-Aldrich Inc.) were used. An attenuated strain of the infectious bronchitis vaccine (H120), the inactivated avian influenza (AI) vaccine (H5N2, N28 strain), the live vaccine strain Clone-30 of the Newcastle disease virus (NDV), and
the combined inactive vaccine for infectious bursal diseases and the Newcastle disease (ND-IB) were supplied by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin, China).
Birds and Experimental Design In total, 540 one-day-old broiler chicks (Cobb 500) were randomly assigned to 5 groups (9 replicates/ group; 12 birds/replicate). The birds were housed in a manner that isolated each group according to treatment. The birds were fed in galvanized 3-floor metabolism cages. The experimental design is described in Table 1. The birds were given access to mash feed and water ad libitum during the 42-d experiment. The birds in each group were fed the same diet during the same feeding period. The lighting program consisted of 24 h of light from d 0 to 3, 23 h of light from d 4 to 14, and 20 h of light from d 15 to 42. The temperature was controlled at 36°C from d 0 to 7, and then gradually reduced by 1°C per day to a final temperature of 26°C. All experimental protocols were approved by the Animal Care and Use Committee of the College of Animal Science and Technology of the Northwest A&F University (Shaanxi, China). As shown in Table 1, broilers were randomly assigned to receive 1 of 5 (n = 108 birds/group) treatments: 1) no vaccination; 2) simplified vaccination, which included H120, AI, NDV, and ND-IB; 3) normal vaccination (simplified vaccination + second dose of ND-IB, H120, and AI); 4) LPS stress (normal vaccination + LPS); or 5) CPM stress (normal vaccination + CPM).
Ileal CP and GE Digestibility Chromic oxide was included in the diet at 0.4% as an indigestible marker. The diet was fed to the chicks from d 16 to 21 and from d 37 to 42 with the diet being fed without the marker in between these 2 periods.
Table 1. Experimental vaccination and stress treatments on broiler chickens1 Treatment name (group number) Vaccine2 or chemical (day delivered) H120 (d 7) AI (d 10) NDV (d 12) ND-IB (d 15) ND-IB (2 times; d 25) NDV + H120 (d 30) Saline (d 16, 18, 20, 37, 39, 41), mL LPS (d 16, 18, 20,37, 39, 41), µg/kg of BW CPM (d 16, 18, 20, 37, 39, 41), mg/kg of BW 1The
No vaccination (1)
Simplified vaccination (2)
Normal vaccination (3)
LPS3 stress (4)
CPM3 stress (5)
— — — — — — 0.5 —
nasal drip SI nasal drip water vaccinate — — 0.5 —
nasal drip SI nasal drip water vaccinate water vaccinate water vaccinate 0.5 —
nasal drip SI nasal drip water vaccinate water vaccinate water vaccinate — 500
nasal drip SI nasal drip water vaccinate water vaccinate water vaccinate —
—
—
—
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dosage of the vaccine was chosen according to the manufacturer’s instructions; SI = subcutaneous injection. = infectious bronchitis vaccine; AI = inactivated avian influenza vaccine; NDV = live vaccine strain Clone-30 of the Newcastle disease virus; ND-IB = combined inactive vaccine for infectious bursal diseases and the Newcastle disease vaccine. 3LPS = lipopolysaccharide; CPM = cyclophosphamide. 2H120
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An increasing amount of research has been focused on the effects of immune stress on the health and growth performance of broiler chickens (Yang et al., 2008; Lai et al., 2011; Marcq et al., 2011). This research has shown that lipopolysaccharide (LPS) injection decreased feed intake and BW gain (Shini et al., 2008; Star et al., 2008; Yang et al., 2008; Lai et al., 2009). Therefore, to obtain higher effective poultry production, it is important to find a balance between nutrition and immune status of broiler chickens. Immune stress can influence organ growth, proliferation of lymphocytes, percentages of CD4+ and CD8+, profiles of chicken cytokines, and the immune response of antigen to antibody (Hangalapura et al., 2006; Li et al., 2007a; Shini and Kaiser, 2009; Shini et al., 2010). Research has shown that the immune response can affect growth performance, and that enteric diseases in commercial poultry contribute to losses in productivity, increased mortality, and the contamination of products for human consumption. The latter is related to changes in the cecal microflora. Previous studies have yielded widely varying results, and the mechanism of action is not yet fully understood. In this study, according to the feeding environment of broilers, we set up 5 different stress models to study the effects of immune stress on performance, immune function, and cecal microflora in broiler chickens.
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CP (or GE) = CP (or GE) concentration in digesta × [Cr2O3 concentration (mg/kg)]/[digesta Cr2O3 concentration (mg/kg)]. Apparent ileal CP and GE digestibility in the experimental diets were calculated using the following equation: Apparent ileal CP (or GE) digestibility = {[CP (or GE) concentration in diet – CP (or GE) output in ileum] × 100}/[CP (or GE) concentration in diet].
Immune Parameters Two birds from each replicate group were randomly weighed and killed at 21 d of age, and 2 birds from each replicate group were randomly weighed and killed at 42 d of age. The thymus, spleen, and bursa were removed from each bird. The organ weights were immediately measured following dissection and were expressed relative to BW (g of organ/kg of BW). The levels of serum IgG from 2 birds of each replicate group at 21 and 42 d of age were measured by ELISA (Bethyl Inc., West Chester, PA) according to the instructions of the manufacturer. The optical density at 570 nm was determined using an automated microplate reader (Bio-Rad, Richmond, CA). The blood was aseptically sampled from the wing vein and put into 5-mL heparinized vacutainer tubes. The blood was layered onto a 2-fold-volume lymphocyte separation medium (Dingguo Biotech Inc., Beijing, China) and centrifuged at 2,600 × g for 30 min at 48°C. The viable peripheral blood mononuclear cell (PBMC) was recovered and then washed in RPMI1640 (GIBCO, Invitrogen, Carlsbad, CA). The cells were cultured in 96-well microtiter plates and incubated for 48 h with 15 mg/mL of LPS at 40°C, 5% CO2, and
optimum humidity. The proliferation of the PBMC was measured by 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide colorimetric assay as described by Lee et al. (2008). The lysozyme concentration (LZM) was measured by trace turbidimetry according to the instructions of the manufacturer (Jiancheng Bioengineering Institute, Nanjing, China). The OD450 values were determined with an automated microplate reader (Bio-Rad).
Intestinal Secretory IgA Content One bird from each replicate group was randomly killed at 21 or 42 d of age. The intestinal chyme and mucous attachment near the vitelline diverticulum were collected into Eppendorf tubes, mixed with an equivalent volume of saline, and centrifuged at 1,000 × g for 5 min; the supernatant was conserved at −20°C. The levels of sIgA in the intestine of 1 bird from each replicate group at 21 and 42 d of age were measured by ELISA (Bethyl Inc.) according to the manufacturer’s instructions.
Enterobacterial Repetitive Intergenic Consensus PCR Fingerprints for Cecal Microflora Two birds from each replicate group were randomly chosen and killed at 21 or 42 d of age. The cecum was ligated, immediately collected, and then stored at −70°C. The enterobacterial repetitive intergenic consensus (ERIC)-PCR methods used for the cecal microflora were defined by Wei et al. (2004) and Ye et al. (2008). The sequence of the ERIC primers was based on work by Versalovic et al. (1991): E1 (ERIC1R), 5′-ATGTAAGCTCCTGGGGATTCAC-3′; and E2 (ERIC2), 5′-AAGTAAGTGACTGGGGTGAGCG-3′. Each 25-μL PCR reaction mixture contained 1.5 μL (0.6 μM) of each primer, 200 ng of total DNA, 12.5 μL of Taq DNA polymerase (1.25 U; Fermentas,), and 7.5 μL of PCR buffer. The PCR amplifications were performed in an automated thermocycler (Bio-Rad C1000 PCR) with the following program: 7 min at 95°C; 30 cycles of denaturation at 94°C for 1 min, annealing at 48°C for 1 min, and extension at 65°C for 8 min; and followed by a final extension at 65°C for 16 min. The concentrations were determined using a NanoDrop ND1000 spectrophotometer (Thermo Scientific Inc., Wilmington, DE), and then adjusted to 50 to 80 ng/µL. The size of the products was determined by 1% (wt/vol) agarose gel electrophoresis with 300 to 400 ng of the total PCR products loaded into each lane. The gels were stained with ethidium bromide and photographed with UVI (Bio-Rad Gel Doc XR). A dendrogram was constructed based on the Jaccard’s similarity coefficient with unweighted pair group method clustering using NTSYS-pc2.10 software (Exeter Software, Setauket, NY).
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The ileal digesta were collected from 1 bird from each pen on d 21 and 42. After the birds were killed, each body cavity was opened, the ileum removed, and the digesta gently flushed from the ileum into a labeled plastic container using distilled water from a 50-mL syringe. The collected digesta of 9 birds from each treatment were immediately stored at −20°C. The digesta samples were subsequently oven-dried (at 70°C), finely ground, and stored at −20°C until chemical analysis. The ground samples of digesta and the diets were subjected to CP analysis by 2300 Kjeltec (Foss Tecator Instruments, Hillerød, Denmark), and to GE analysis by a Parr adiabatic bomb calorimeter (Moline, IL). The chromic oxide content of the diets and digesta was measured by spectrophotometry. The CP and GE outputs in the ileal digesta were calculated using the following formula:
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Statistical Analysis The statistical analysis of the chicken data was performed by the procedure appropriated for a completely randomized design by the GLM procedure of SAS (SAS Institute Inc., Cary, NC). The differences among each treatment group were tested by Duncan’s multiplerange test. The differences due to the treatment were considered significant at P ≤ 0.05.
RESULTS Growth Performance
Immune Parameters There was no significant difference in the thymus index at 42 d of age among the 5 groups; however, the thymus index at 21 d of age decreased significantly in
Fingerprints for Cecal Microflora The ERIC-PCR fingerprints of cecal bacteria are shown in Figure 1. Each group produced banding patterns of various intensity containing 5 to 10 amplified fragments that ranged from 200 to 3,000 bp in size. All of the ERIC-PCR fingerprints shared 1 consensus fragment of approximately 750 bp (Figure 1). Groups 4 and 5 had fewer (approximately 2 to 3 bright bands) and smaller (approximately 700 to 800 bp) fingerprinting bands than those of the other groups. Using the agglomerative cluster approach outlined in unweighted pair group method clustering, 2 dendrograms were generated (Figure 2). The clusters differ significantly by cecum microflora fingerprints. Six clusters were identified at 21 d of age, but cluster 6 (Q23) was a single sample. At 21 d of age, subjects were grouped as follows: cluster 1, subjects Q11, Q12, Q17, and Q34; cluster 2, subjects Q24 and Q25; cluster 3, subjects Q32 and Q33; cluster 4, subjects Q52,Q53, and Q54
Table 2. Effects of different immune stresses on growth performance and nutrient digestibility in broilers1 Treatment name2 (group number)
Item
Period
FI3 (kg)
Wk 1 to Wk 4 to Wk 1 to Wk 1 to Wk 4 to Wk 1 to Wk 1 to Wk 4 to Wk 1 to 17–21 d 37–41 d 17–21 d 37–41 d
BW gain (kg) FCR3 GE (%) Ileal CP (%)
No vaccination (1) 3 6 6 3 6 6 3 6 6
1.04 2.96 3.99 0.75 1.51 2.25 1.39 1.97 1.78 78.11 77.12 75.59 84.96
± ± ± ± ± ± ± ± ± ± ± ± ±
0.01 0.07a 0.04a 0.02a 0.05a 0.03a 0.03 0.03 0.02 1.05 1.05 2.27 1.59a
Simplified vaccination (2) 1.01 2.96 3.97 0.74 1.49 2.23 1.37 1.99 1.78 77.53 78.41 67.13 83.47
± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.05a 0.03a 0.01a 0.04ab 0.02a 0.02 0.03 0.01 0.58 0.30 3.18 1.82a
Normal vaccination (3) 1.01 2.89 3.90 0.73 1.44 2.17 1.38 2.01 1.80 73.72 78.52 75.85 82.36
± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.04ab 0.03ab 0.01ab 0.03abc 0.01ab 0.02 0.03 0.01 5.99 0.23 1.15 1.03a
LPS stress (4) 0.97 2.74 3.71 0.68 1.36 2.04 1.42 2.03 1.82 78.66 75.41 72.52 78.62
± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.06b 0.03b 0.01c 0.03c 0.02c 0.02 0.04 0.02 0.77 1.42 3.00 2.74ab
CPM stress (5) 0.99 2.81 3.81 0.70 1.39 2.09 1.43 2.03 1.83 77.87 77.98 70.53 75.19
± ± ± ± ± ± ± ± ± ± ± ± ±
0.08 0.04ab 0.03ab 0.01bc 0.03bc 0.02bc 0.04 0.03 0.02 0.69 0.30 3.84 2.62b
P-value 0.355 0.026 0.018 <0.01 0.016 <0.01 0.548 0.477 0.197 0.729 0.063 0.226 0.010
a–cThe means in the same row with the same superscripts were not significantly different, and the means in the same row without the same superscripts were significantly different, with a difference of P < 0.05, the same as followed. 1The values are the means ± SE. The data are indicated as the ratio of GE and ileal CP, and the data for analysis are transformed by arcsine. 2Treatments: 1) no vaccination; 2) simplified vaccination, which included the infectious bronchitis vaccine (H120), the inactivated avian influenza vaccine (AI), the live vaccine strain Clone-30 of the Newcastle disease virus (NDV), and the combined inactive vaccine for infectious bursal diseases and the Newcastle disease vaccine (ND-IB); 3) normal vaccination (simplified vaccination + second dose of ND-IB, H120, and AI); 4) lipopolysaccharide (LPS) stress (normal vaccination + LPS); or 5) cyclophosphamide (CPM) stress (normal vaccination + CPM). 3FI = feed intake; FCR = feed conversion ratio
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The effect of different types of immune stress on feed intake (FI), BW gain, and feed conversion ratio (FCR) are presented in Table 2. Among the 3 vaccine groups (no, simplified, and normal vaccination), no differences were observed in FI or BW gain. However, compared with groups 1 and 2, the BW gain decreased significantly in chickens treated with LPS or CPM (P < 0.05 and P < 0.01, respectively), and the FI decreased significantly in chickens treated with LPS. The FCR showed no significant difference among the 5 groups (P > 0.05). There was no significant difference in the digestibility of GE. The CPM treatment decreased the digestibility of ileal CP compared with that of the other groups.
group 5 (Table 3). Compared with that of group 1, the spleen index increased significantly in chickens treated with LPS and decreased significantly in chickens treated with CPM (P < 0.01). The chickens in group 5 showed a lower bursa index than those of the chickens in the other groups (P < 0.01). The levels of sIgA decreased significantly in chickens treated with LPS and CPM stress (P < 0.01); they also showed higher levels of serum IgG than those of the chickens in the other groups at 42 d of age, whereas B-lymphocyte proliferation increased significantly in group 4 chickens (P < 0.05).
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Table 3. Effect of different immune stresses on immune organ growth in broilers1 Treatment name2 (group number) No vaccination (1)
Days of age
Item Thymus index (g/kg) Spleen index (g/kg) Bursa index (g/kg) LZM3 (μg/mL) sIgA (× 102 mg/g) IgG (g/L)
4.24 2.95 0.71 1.32 2.55 1.61 10.03 2.91 4.72 4.64 2.15 1.69 3.65 8.88
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.25 0.03a 0.13a 0.23a 0.18a 0.64 0.42a 0.04a 0.04a 0.28 0.36a 0.11a 2.82
3.93 3.48 0.68 1.13 2.80 1.64 9.46 5.01 4.57 4.68 2.22 1.93 3.84 8.15
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.20ab
0.53 0.05a 0.10a 0.33a 0.13a 0.84 0.66b 0.08a 0.06a 0.31 0.04ab 0.46a 2.43
Normal vaccination (3) 3.70 2.85 0.73 1.09 2.79 1.47 7.18 3.94 4.58 4.57 2.28 2.09 4.44 11.29
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.19ab
0.33 0.06a 0.11ac 0.19a 0.15a 0.72 0.61ab 0.12a 0.03a 0.31 0.09bd 0.25a 2.36
LPS stress (4) 3.50 3.13 1.22 1.85 2.10 1.59 7.15 2.38 4.21 4.28 2.41 2.54 7.11 9.51
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.35ab
0.33 0.16b 0.20b 0.25a 0.29a 1.85 0.44a 0.22b 0.15b 0.45 0.15c 0.96b 2.17
CPM stress (5) 2.96 2.11 0.59 0.71 1.17 0.58 6.08 2.45 3.56 3.87 1.77 2.28 1.79 8.69
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.24b
0.28 0.07a 0.10c 0.25b 0.09b 1.15 0.42a 0.20c 0.12b 0.67 0.13cd 0.42a 0.76
P-value 0.044 0.106 <0.01 <0.01 <0.01 <0.01 0.052 0.010 <0.01 <0.01 0.886 <0.01 0.024 0.413
a–dMeans
in the same row with no common superscript differ significantly (P < 0.05). are expressed as the stimulation index calculated as the optical density at 570 nm absorbance of the wells that were incubated with LPS divided by the optical density at 570 nm absorbance of wells that were incubated without LPS. 2Treatments: 1) no vaccination; 2) simplified vaccination, which included the infectious bronchitis vaccine (H120), the inactivated avian influenza vaccine (AI), the live vaccine strain Clone-30 of the Newcastle disease virus (NDV), and the combined inactive vaccine for infectious bursal diseases and the Newcastle disease vaccine (ND-IB); 3) normal vaccination (simplified vaccination + second dose of ND-IB, H120, and AI); 4) lipopolysaccharide (LPS) stress (normal vaccination + LPS); or 5) cyclophosphamide (CPM) stress (normal vaccination + CPM). 3LZM = lysozyme concentration; and PBMC = peripheral blood mononuclear cell. 1Values
; and cluster 5, subjects Q41, Q42, and Q43; cluster 6, Q23. At 42 d of age, 5 clusters were identified and subjects were grouped as follows: cluster 1, subjects H12, H13, H14, and H48; cluster 2, subjects H21, H23, and H25; cluster 3, subjects H41 and H46; cluster 4, subjects H35, H37, and H38; and cluster 5, subjects H55, H56, and H57. The ERIC-PCR fingerprints of the cecal samples from groups 1 and 2 clustered together with high coefficients, except for Q23. The ERIC-PCR fingerprints of the 3 cecal samples from groups 4 and 5 clustered together with high coefficients among them.
DISCUSSION Our results showed that immune stress decreased the FI and BW gain of the chickens (Table 2). It was reported that immune stress may decrease the growth performance of broilers (Eid et al., 2003; Malheiros et al., 2003; Lin et al., 2004; Virden et al., 2007). The current study showed that the injection of CPM inhibited the proliferation of B lymphocytes in the blood, which coincided with the immune organ index, such as the spleen and thymus (Table 3). Yang et al. (2006) reported that chickens inoculated with Eimeria tenella coccidia have higher IgG and sIgA levels. Our results showed that immune stress decreased growth performance. Hanna et al. (2000) reported that the gastrointestinal mucosal immune response mainly depended on sIgA-mediated humoral immunity. Our results demonstrated that immune stress had adverse effects on the mucosal immune function of broilers; both treatments of no vaccinations or simplified vaccinations can
maintain higher sIgA levels in broilers (Table 3). A combination of the effect of immune stress on growth performance and the immuno-modulator role of immune stress, which allocates nutrients toward immune responses, could possibly explain the depressed growth. A previous study showed that immune responses could affect growth performance (Rajapakse et al., 2010). Chicks are very susceptible to infections by enteric bacteria, and the gut mucosa is the first line of defense against the bacteria. Microflora play an important role in the intestinal defense system (Lee et al., 2010; Kim et al., 2011). Therefore, a change in cecal microbial communities becomes an important indicator of the intestinal nonspecific immune response. The cluster analysis described in this paper identifies genotypes among the 5 treatments (Figures 1 and 2). Berndt et al. (2007) reported that the capability of Salmonella serovars to enter the cecal mucosa affected both the level and character of the immune response in tissue. Although we did not identify the ERIC-PCR fingerprint consensus fragment in the bacterial strains, it may be assumed that immune stress can lead to a distribution change of cecal microflora. Some sets of bands are highlighted with yellow boxes (Figure 1) to demonstrate that higher similarities in the cecal microflora distribution existed between the no vaccination and the simplified vaccination treatment groups at 21 and 42 d of age, and higher similarities were found between the CPM and the LPS treatment groups at 21 and 42 d of age. These results suggest that chicken cecal microflora are at risk when chickens experience immune stress. A previous study has shown that a change in immune function
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PBMC3 proliferation (SI)
21 42 21 42 21 42 21 42 21 42 21 42 21 42
0.33a
Simplified vaccination (2)
STRESS AND CECAL MICROFLORA IN CHICKENS
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could serve as a growth promoter in broiler production by modulating the concentrations of intestinal microbial flora (Li et al., 2007b). Therefore, it is necessary consider the exact modulation mechanism of immune stress on the gut microflora in broilers. It can be concluded that broilers with simplified vaccinations or without vaccinations can achieve the same growth performance as broilers with normal vaccina-
tions in a controlled, rational feeding environment, but immune stress can interrupt the homeostasis of cecal microflora, impair intestinal mucosal immune function, and decrease humoral and cellular immune responses. It is generally recommended for broilers to be vaccinated only against the diseases to which they might be exposed. Vaccinating birds without a risk of exposure results in a growth cost to the bird.
Figure 2. Cluster analysis of the gel patterns based on the enterobacterial repetitive intergenic consensus PCR fingerprints of the cecal bacterial structure of chickens at 21 and 42 d of age. Note: Q1 to Q5 correspond to the cecal bacteria in chickens from groups 1 to 5 at 21 d of age, respectively; and H1 to H5 correspond to the cecal bacteria in chickens from groups 1 to 5 at 42 d of age, respectively. Treatments were as follows: 1) no vaccination; 2) simplified vaccination, which included the infectious bronchitis vaccine (H120), the inactivated avian influenza vaccine (AI), the live vaccine strain Clone-30 of the Newcastle disease virus (NDV), and the combined inactive vaccine for infectious bursal diseases and the Newcastle disease vaccine (ND-IB); 3) normal vaccination (simplified vaccination + second dose of ND-IB, H120, and AI); 4) lipopolysaccharide (LPS) stress (normal vaccination + LPS); or 5) cyclophosphamide (CPM) stress (normal vaccination + CPM). Color version available in the online PDF.
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Figure 1. The enterobacterial repetitive intergenic consensus PCR fingerprinting of the cecal bacterial genomes of broilers at 21 and 42 d of age. M = GeneRuler Ladder; lanes Q1 to Q5 correspond to the cecal bacteria in chickens from groups 1 to 5 at 21 d of age, respectively; and H1 to H5 correspond to the cecal bacteria in chickens from groups 1 to 5 at 42 d of age, respectively. Treatments were as follows: 1) no vaccination; 2) simplified vaccination, which included the infectious bronchitis vaccine (H120), the inactivated avian influenza vaccine (AI), the live vaccine strain Clone-30 of the Newcastle disease virus (NDV), and the combined inactive vaccine for infectious bursal diseases and the Newcastle disease vaccine (ND-IB); 3) normal vaccination (simplified vaccination + second dose of ND-IB, H120, and AI); 4) lipopolysaccharide (LPS) stress (normal vaccination + LPS); or 5) cyclophosphamide (CPM) stress (normal vaccination + CPM). Color version available in the online PDF.
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REFERENCES
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Berndt, A., A. Wilhelm, C. Jugert, J. Pieper, K. Sachse, and U. Methner. 2007. Chicken cecum immune response to Salmonella enterica serovars of different levels of invasiveness. Infect. Immun. 75:5993–6007. Campo, J. L., and S. G. Davila. 2002. Effect of photoperiod on heterophil to lymphocyte ratio and tonic immobility duration of chickens. Poult. Sci. 81:1637–1639. Deeb, N., and A. Cahaner. 2002. Genotype-by-environment interaction with broiler genotypes differing in growth rate. 3. Growth rate and water consumption of broiler progeny from weight-selected versus nonselected parents under normal and high ambient temperatures. Poult. Sci. 81:293–301. Eid, Y. Z., A. Ohtsuka, and K. Hayashi. 2003. Tea polyphenols reduce glucocorticoid-induced growth inhibition and oxidative stress in broiler chickens. Br. Poult. Sci. 44:127–132. Hangalapura, B. N., M. G. Kaiser, J. J. Poel, H. K. Parmentier, and S. J. Lamont. 2006. Cold stress equally enhances in vivo pro-inflammatory cytokine gene expression in chicken lines divergently selected for antibody responses. Dev. Comp. Immunol. 30:503–511. Hanna, M. K., B. L. Zarzaur, K. Fukatsu, R. C. Dewitt, K. R. Renegar, C. Sherrell, W. Yong, and K. A. Kudsk. 2000. Individual neuropeptides regulate gut-associated lymphoid tissue integrity, intestinal immunoglobulin A levels, and respiratory antibacterial immunity. J. Parenter. Enter. Nutr. 24:261–269. Kim, G. B., Y. M. Seo, C. H. Kim, and I. K. Paik. 2011. Effect of dietary prebiotic supplementation on the performance, intestinal microflora, and immune response of broilers. Poult. Sci. 90:75–82. Lai, H. T. L., M. G. B. Nieuwland, B. Kemp, A. J. A. Aarnink, and H. K. Parmentier. 2009. Effects of dust and airborne dust components on antibody responses, body weight gain, and heart morphology of broilers. Poult. Sci. 88:1838–1849. Lai, H. T. L., M. G. B. Nieuwland, B. Kemp, A. J. A. Aarnink, and H. K. Parmentier. 2011. Effects of repeated intratracheally administered lipopolysaccharide on primary and secondary specific antibody responses and on body weight gain of broilers. Poult. Sci. 90:337–351. Lee, K. W., S. H. Lee, H. S. Lillehoj, G. X. Li, S. I. Jang, U. S. Babu, M. S. Park, D. K. Kim, E. P. Lillehoj, A. P. Neumann, T. G. Rehberger, and G. R. Siragusa. 2010. Effects of direct-fed microbials on growth performance, gut morphometry, and immune characteristics in broiler chickens. Poult. Sci. 89:203–216. Lee, S. H., H. S. Lillehoj, E. P. Lillehoj, S. M. Cho, D. H. Park, Y. H. Hong, H. K. Chun, and H. J. Park. 2008. Immunomodulatory properties of dietary plum on coccidiosis. Comp. Immunol. Microbiol. Infect. Dis. 31:389–402. Li, M. O., Y. Y. Wan, and R. A. Flavell. 2007a. T cell-produced transforming growth factor-β1 controls T cell tolerance and regulates Th1- and Th17-cell differentiation. Immunity 26:579–591. Li, X. J., X. S. Piao, S. W. Kim, P. Liu, L. Wang, Y. B. Shen, S. C. Jung, and H. S. Lee. 2007b. Effects of chito-oligosaccharide supplementation on performance, nutrient digestibility, and serum composition in broiler chickens. Poult. Sci. 86:1107–1114. Lin, H., E. Decuypere, and J. Buyse. 2004. Oxidative stress induced by corticosterone administration in broiler chickens (Gallus gallus domesticus). 1. Chronic exposure. Comp. Biochem. Physiol. B 139:737–744. Malheiros, R. D., V. M. Moraes, A. Collin, E. Decuypere, and J. Buyse. 2003. Free diet selection by broilers as influenced by dietary macronutrient ratio and corticosterone supplementation.
1. Diet selection, organ weights, and plasma metabolites. Poult. Sci. 82:123–131. Marcq, C., E. Cox, I. M. Szalo, A. Théwis, and Y. Beckers. 2011. Salmonella Typhimurium oral challenge model in mature broilers: Bacteriological, immunological, and growth performance aspects. Poult. Sci. 90:59–67. Mashaly, M. M., G. L. Hendricks, and M. A. Kalama. 2004. Effect of heat stress on production parameters and immune responses of commercial laying hens. Poult. Sci. 83:889–894. Rajapakse, J. R. P. V., M. D. P. Buddhika, M. Nagataki, H. Nomura, Y. Watanabe, Y. Ikeue, and T. Agatsuma. 2010. Effect of sophy beta-glucan on immunity and growth performance in broiler chicken. J. Vet. Med. Sci. 72:1629–1632. Rajkumar, U., M. R. Reddy, S. V. R. Rao, K. Radhika, and M. Shanmugam. 2011. Evaluation of growth, carcass, immune response, and stress parameters in naked neck chicken and their normal siblings under tropical winter and summer temperatures. Asian-australas. J. Anim. Sci. 24:509–516. Shini, S., and P. Kaiser. 2009. Effects of stress, mimicked by administration of corticosterone in drinking water, on the expression of chicken cytokine and chemokine genes in lymphocytes. Stress 12:388–399. Shini, S., P. Kaiser, A. Shini, and W. L. Bryden. 2008. Biological response of chickens (Gallus gallus domesticus) induced by corticosterone and a bacterial endotoxin. Comp. Biochem. Physiol. B 149:324–333. Shini, S., A. Shini, and P. Kaiser. 2010. Cytokine and chemokine gene expression profiles in heterophils from chickens treated with corticosterone. Stress 13:185–194. Star, L., B. Kemp, I. van den Anker, and H. K. Parmentier. 2008. Effect of single or combined climatic and hygienic stress in four layer lines: 1. Performance. Poult. Sci. 87:1022–1030. Versalovic, J., T. Koeuth, and J. R. Lupski. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19:6823–6831. Virden, W. S., M. S. Lilburn, J. P. Thaxton, A. Corzo, D. Hoehler, and M. T. Kidd. 2007. The effect of corticosterone-induced stress on amino acid digestibility in Ross broilers. Poult. Sci. 86:338– 342. Wei, G., L. Pan, H. Du, J. Chen, and L. Zhao. 2004. ERIC-PCR fingerprinting-based community DNA hybridization to pinpoint genome-specific fragments as molecular markers to identify and track populations common to healthy human guts. J. Microbiol. Methods 59:91–108. Wilkinson, K. G. E., R. B. Tee, G. Tomkins, and R. Premier. 2011. Effect of heating and aging of poultry litter on the persistence of enteric bacteria. Poult. Sci. 90:10–18. Yalçin, S., S. Ozkan, and L. Turkmut. 2001. Responses to heat stress in commercial and local broiler stocks. 2. Developmental stability of bilateral traits. Br. Poult. Sci. 42:153–160. Yang, X. J., Y. M. Guo, X. He, J. M. Yuan, Y. Yang, and Z. Wang. 2008. Growth performance and immune responses in chickens after challenge with lipopolysaccharide and modulation by dietary different oils. Animal 2:216–223. Yang, X., Y. M. Guo, Z. Wang, and W. Nie. 2006. Effects of dietary supplementation with different oils on coccidiosis in chickens. Avian Pathol. 35:373–378. Ye, Y., Q. P. Wu, Y. H. Zhou, X. H. Dong, and J. M. Zhang. 2008. Analysis of major band of Enterobacter sakazakii by ERIC-PCR and development of a species-specific PCR for detection of E. sakazakii in dry food samples. J. Microbiol. Methods 75:392– 397.
Key words: immune stress, growth performance, immunity, cecal microflora, chicken 2011 Poultry Science 90:2740–2746 doi:10.3382/ps.2011-01591
INTRODUCTION As the development of breeding technology in broiler chickens has improved, some new lines of broilers, such as Arbor Acres, Avian, Cobb, and Cobb Avian 48, have shown great improvement in growth performance. How©2011 Poultry Science Association Inc. Received May 5, 2011. Accepted August 12, 2011. 1 This work was supported by the National Nature Science Foundation of China (31001017), the Science & Technological Project of Shaanxi Province, China (2009K01-02, 2010ZDGC-02), the Natural Science Foundation of Shaanxi Province (No. 2010JQ3002), the Opening Program State Key Laboratory of Animal Nutrition (No. 2004DA125184F0809), and the Central University special fund basic research and operating expenses (No. QN2009020). 2 Contributed equally to this paper. 3 Corresponding author: [email protected]
ever, these new lines exhibit weaker immune function from emerging stress (Yalcin et al., 2001; Deeb and Cahaner, 2002). Immune stress is the loss of immune homeostasis by external forces; vaccination, drugs, transport, contamination, and other procedures involved in production could be considered external forces. In addition, many stress factors (disease, heavy vaccinations, ammonia gas, noise, feed density, temperature, and humidity) may also influence the immune systems of broilers (Campo and Davila, 2002; Mashaly et al., 2004; Yang et al., 2006; Shini and Kaiser, 2009; Rajkumar et al., 2011; Wilkinson et al., 2011). Immunizations have sometimes been administered in large doses at a high frequency. These vaccinations were considered necessary to reduce the mortality rate that occurs in modern poultry production; however, unsuitable vaccinations could lead to immune stress.
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ABSTRACT Immune stress is the loss of immune homeostasis by external forces. This study investigated the effects of different types of immune stress on growth performance, immunity, and the distribution of cecal microflora in broiler chickens. In total, 540 oneday-old Cobb 500 broilers were randomly assigned to receive 1 of 5 (n = 108 birds/group) treatments: 1) no vaccination; 2) simplified vaccination, which included the infectious bronchitis vaccine (H120), the inactivated avian influenza vaccine (AI), the live vaccine strain Clone-30 of the Newcastle disease virus (NDV), and the combined inactive vaccine for infectious bursal diseases and the Newcastle disease vaccine (ND-IB); 3) normal vaccination (simplified vaccination + second dose of ND-IB, H120, and AI); 4) lipopolysaccharide (LPS) stress (normal vaccination+LPS); or 5) cyclophosphamide (CPM) stress (normal vaccination+CPM).The results showed that the average BW and average feed intake decreased significantly after treatment with LPS or CPM (P < 0.05). Chickens that were challenged by LPS or CPM had a lower ileal CP digestibility than that of the control group (P < 0.01). Compared with the control group, the levels of secreted IgA decreased
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MATERIALS AND METHODS Chemicals and Vaccines For this experiment, LPS from Escherichia coli (L2880, Sigma-Aldrich Inc., St. Louis, MO) and cyclophosphamide (CPM; C0768, Sigma-Aldrich Inc.) were used. An attenuated strain of the infectious bronchitis vaccine (H120), the inactivated avian influenza (AI) vaccine (H5N2, N28 strain), the live vaccine strain Clone-30 of the Newcastle disease virus (NDV), and
the combined inactive vaccine for infectious bursal diseases and the Newcastle disease (ND-IB) were supplied by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin, China).
Birds and Experimental Design In total, 540 one-day-old broiler chicks (Cobb 500) were randomly assigned to 5 groups (9 replicates/ group; 12 birds/replicate). The birds were housed in a manner that isolated each group according to treatment. The birds were fed in galvanized 3-floor metabolism cages. The experimental design is described in Table 1. The birds were given access to mash feed and water ad libitum during the 42-d experiment. The birds in each group were fed the same diet during the same feeding period. The lighting program consisted of 24 h of light from d 0 to 3, 23 h of light from d 4 to 14, and 20 h of light from d 15 to 42. The temperature was controlled at 36°C from d 0 to 7, and then gradually reduced by 1°C per day to a final temperature of 26°C. All experimental protocols were approved by the Animal Care and Use Committee of the College of Animal Science and Technology of the Northwest A&F University (Shaanxi, China). As shown in Table 1, broilers were randomly assigned to receive 1 of 5 (n = 108 birds/group) treatments: 1) no vaccination; 2) simplified vaccination, which included H120, AI, NDV, and ND-IB; 3) normal vaccination (simplified vaccination + second dose of ND-IB, H120, and AI); 4) LPS stress (normal vaccination + LPS); or 5) CPM stress (normal vaccination + CPM).
Ileal CP and GE Digestibility Chromic oxide was included in the diet at 0.4% as an indigestible marker. The diet was fed to the chicks from d 16 to 21 and from d 37 to 42 with the diet being fed without the marker in between these 2 periods.
Table 1. Experimental vaccination and stress treatments on broiler chickens1 Treatment name (group number) Vaccine2 or chemical (day delivered) H120 (d 7) AI (d 10) NDV (d 12) ND-IB (d 15) ND-IB (2 times; d 25) NDV + H120 (d 30) Saline (d 16, 18, 20, 37, 39, 41), mL LPS (d 16, 18, 20,37, 39, 41), µg/kg of BW CPM (d 16, 18, 20, 37, 39, 41), mg/kg of BW 1The
No vaccination (1)
Simplified vaccination (2)
Normal vaccination (3)
LPS3 stress (4)
CPM3 stress (5)
— — — — — — 0.5 —
nasal drip SI nasal drip water vaccinate — — 0.5 —
nasal drip SI nasal drip water vaccinate water vaccinate water vaccinate 0.5 —
nasal drip SI nasal drip water vaccinate water vaccinate water vaccinate — 500
nasal drip SI nasal drip water vaccinate water vaccinate water vaccinate —
—
—
—
40
dosage of the vaccine was chosen according to the manufacturer’s instructions; SI = subcutaneous injection. = infectious bronchitis vaccine; AI = inactivated avian influenza vaccine; NDV = live vaccine strain Clone-30 of the Newcastle disease virus; ND-IB = combined inactive vaccine for infectious bursal diseases and the Newcastle disease vaccine. 3LPS = lipopolysaccharide; CPM = cyclophosphamide. 2H120
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An increasing amount of research has been focused on the effects of immune stress on the health and growth performance of broiler chickens (Yang et al., 2008; Lai et al., 2011; Marcq et al., 2011). This research has shown that lipopolysaccharide (LPS) injection decreased feed intake and BW gain (Shini et al., 2008; Star et al., 2008; Yang et al., 2008; Lai et al., 2009). Therefore, to obtain higher effective poultry production, it is important to find a balance between nutrition and immune status of broiler chickens. Immune stress can influence organ growth, proliferation of lymphocytes, percentages of CD4+ and CD8+, profiles of chicken cytokines, and the immune response of antigen to antibody (Hangalapura et al., 2006; Li et al., 2007a; Shini and Kaiser, 2009; Shini et al., 2010). Research has shown that the immune response can affect growth performance, and that enteric diseases in commercial poultry contribute to losses in productivity, increased mortality, and the contamination of products for human consumption. The latter is related to changes in the cecal microflora. Previous studies have yielded widely varying results, and the mechanism of action is not yet fully understood. In this study, according to the feeding environment of broilers, we set up 5 different stress models to study the effects of immune stress on performance, immune function, and cecal microflora in broiler chickens.
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CP (or GE) = CP (or GE) concentration in digesta × [Cr2O3 concentration (mg/kg)]/[digesta Cr2O3 concentration (mg/kg)]. Apparent ileal CP and GE digestibility in the experimental diets were calculated using the following equation: Apparent ileal CP (or GE) digestibility = {[CP (or GE) concentration in diet – CP (or GE) output in ileum] × 100}/[CP (or GE) concentration in diet].
Immune Parameters Two birds from each replicate group were randomly weighed and killed at 21 d of age, and 2 birds from each replicate group were randomly weighed and killed at 42 d of age. The thymus, spleen, and bursa were removed from each bird. The organ weights were immediately measured following dissection and were expressed relative to BW (g of organ/kg of BW). The levels of serum IgG from 2 birds of each replicate group at 21 and 42 d of age were measured by ELISA (Bethyl Inc., West Chester, PA) according to the instructions of the manufacturer. The optical density at 570 nm was determined using an automated microplate reader (Bio-Rad, Richmond, CA). The blood was aseptically sampled from the wing vein and put into 5-mL heparinized vacutainer tubes. The blood was layered onto a 2-fold-volume lymphocyte separation medium (Dingguo Biotech Inc., Beijing, China) and centrifuged at 2,600 × g for 30 min at 48°C. The viable peripheral blood mononuclear cell (PBMC) was recovered and then washed in RPMI1640 (GIBCO, Invitrogen, Carlsbad, CA). The cells were cultured in 96-well microtiter plates and incubated for 48 h with 15 mg/mL of LPS at 40°C, 5% CO2, and
optimum humidity. The proliferation of the PBMC was measured by 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide colorimetric assay as described by Lee et al. (2008). The lysozyme concentration (LZM) was measured by trace turbidimetry according to the instructions of the manufacturer (Jiancheng Bioengineering Institute, Nanjing, China). The OD450 values were determined with an automated microplate reader (Bio-Rad).
Intestinal Secretory IgA Content One bird from each replicate group was randomly killed at 21 or 42 d of age. The intestinal chyme and mucous attachment near the vitelline diverticulum were collected into Eppendorf tubes, mixed with an equivalent volume of saline, and centrifuged at 1,000 × g for 5 min; the supernatant was conserved at −20°C. The levels of sIgA in the intestine of 1 bird from each replicate group at 21 and 42 d of age were measured by ELISA (Bethyl Inc.) according to the manufacturer’s instructions.
Enterobacterial Repetitive Intergenic Consensus PCR Fingerprints for Cecal Microflora Two birds from each replicate group were randomly chosen and killed at 21 or 42 d of age. The cecum was ligated, immediately collected, and then stored at −70°C. The enterobacterial repetitive intergenic consensus (ERIC)-PCR methods used for the cecal microflora were defined by Wei et al. (2004) and Ye et al. (2008). The sequence of the ERIC primers was based on work by Versalovic et al. (1991): E1 (ERIC1R), 5′-ATGTAAGCTCCTGGGGATTCAC-3′; and E2 (ERIC2), 5′-AAGTAAGTGACTGGGGTGAGCG-3′. Each 25-μL PCR reaction mixture contained 1.5 μL (0.6 μM) of each primer, 200 ng of total DNA, 12.5 μL of Taq DNA polymerase (1.25 U; Fermentas,), and 7.5 μL of PCR buffer. The PCR amplifications were performed in an automated thermocycler (Bio-Rad C1000 PCR) with the following program: 7 min at 95°C; 30 cycles of denaturation at 94°C for 1 min, annealing at 48°C for 1 min, and extension at 65°C for 8 min; and followed by a final extension at 65°C for 16 min. The concentrations were determined using a NanoDrop ND1000 spectrophotometer (Thermo Scientific Inc., Wilmington, DE), and then adjusted to 50 to 80 ng/µL. The size of the products was determined by 1% (wt/vol) agarose gel electrophoresis with 300 to 400 ng of the total PCR products loaded into each lane. The gels were stained with ethidium bromide and photographed with UVI (Bio-Rad Gel Doc XR). A dendrogram was constructed based on the Jaccard’s similarity coefficient with unweighted pair group method clustering using NTSYS-pc2.10 software (Exeter Software, Setauket, NY).
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The ileal digesta were collected from 1 bird from each pen on d 21 and 42. After the birds were killed, each body cavity was opened, the ileum removed, and the digesta gently flushed from the ileum into a labeled plastic container using distilled water from a 50-mL syringe. The collected digesta of 9 birds from each treatment were immediately stored at −20°C. The digesta samples were subsequently oven-dried (at 70°C), finely ground, and stored at −20°C until chemical analysis. The ground samples of digesta and the diets were subjected to CP analysis by 2300 Kjeltec (Foss Tecator Instruments, Hillerød, Denmark), and to GE analysis by a Parr adiabatic bomb calorimeter (Moline, IL). The chromic oxide content of the diets and digesta was measured by spectrophotometry. The CP and GE outputs in the ileal digesta were calculated using the following formula:
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Statistical Analysis The statistical analysis of the chicken data was performed by the procedure appropriated for a completely randomized design by the GLM procedure of SAS (SAS Institute Inc., Cary, NC). The differences among each treatment group were tested by Duncan’s multiplerange test. The differences due to the treatment were considered significant at P ≤ 0.05.
RESULTS Growth Performance
Immune Parameters There was no significant difference in the thymus index at 42 d of age among the 5 groups; however, the thymus index at 21 d of age decreased significantly in
Fingerprints for Cecal Microflora The ERIC-PCR fingerprints of cecal bacteria are shown in Figure 1. Each group produced banding patterns of various intensity containing 5 to 10 amplified fragments that ranged from 200 to 3,000 bp in size. All of the ERIC-PCR fingerprints shared 1 consensus fragment of approximately 750 bp (Figure 1). Groups 4 and 5 had fewer (approximately 2 to 3 bright bands) and smaller (approximately 700 to 800 bp) fingerprinting bands than those of the other groups. Using the agglomerative cluster approach outlined in unweighted pair group method clustering, 2 dendrograms were generated (Figure 2). The clusters differ significantly by cecum microflora fingerprints. Six clusters were identified at 21 d of age, but cluster 6 (Q23) was a single sample. At 21 d of age, subjects were grouped as follows: cluster 1, subjects Q11, Q12, Q17, and Q34; cluster 2, subjects Q24 and Q25; cluster 3, subjects Q32 and Q33; cluster 4, subjects Q52,Q53, and Q54
Table 2. Effects of different immune stresses on growth performance and nutrient digestibility in broilers1 Treatment name2 (group number)
Item
Period
FI3 (kg)
Wk 1 to Wk 4 to Wk 1 to Wk 1 to Wk 4 to Wk 1 to Wk 1 to Wk 4 to Wk 1 to 17–21 d 37–41 d 17–21 d 37–41 d
BW gain (kg) FCR3 GE (%) Ileal CP (%)
No vaccination (1) 3 6 6 3 6 6 3 6 6
1.04 2.96 3.99 0.75 1.51 2.25 1.39 1.97 1.78 78.11 77.12 75.59 84.96
± ± ± ± ± ± ± ± ± ± ± ± ±
0.01 0.07a 0.04a 0.02a 0.05a 0.03a 0.03 0.03 0.02 1.05 1.05 2.27 1.59a
Simplified vaccination (2) 1.01 2.96 3.97 0.74 1.49 2.23 1.37 1.99 1.78 77.53 78.41 67.13 83.47
± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.05a 0.03a 0.01a 0.04ab 0.02a 0.02 0.03 0.01 0.58 0.30 3.18 1.82a
Normal vaccination (3) 1.01 2.89 3.90 0.73 1.44 2.17 1.38 2.01 1.80 73.72 78.52 75.85 82.36
± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.04ab 0.03ab 0.01ab 0.03abc 0.01ab 0.02 0.03 0.01 5.99 0.23 1.15 1.03a
LPS stress (4) 0.97 2.74 3.71 0.68 1.36 2.04 1.42 2.03 1.82 78.66 75.41 72.52 78.62
± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.06b 0.03b 0.01c 0.03c 0.02c 0.02 0.04 0.02 0.77 1.42 3.00 2.74ab
CPM stress (5) 0.99 2.81 3.81 0.70 1.39 2.09 1.43 2.03 1.83 77.87 77.98 70.53 75.19
± ± ± ± ± ± ± ± ± ± ± ± ±
0.08 0.04ab 0.03ab 0.01bc 0.03bc 0.02bc 0.04 0.03 0.02 0.69 0.30 3.84 2.62b
P-value 0.355 0.026 0.018 <0.01 0.016 <0.01 0.548 0.477 0.197 0.729 0.063 0.226 0.010
a–cThe means in the same row with the same superscripts were not significantly different, and the means in the same row without the same superscripts were significantly different, with a difference of P < 0.05, the same as followed. 1The values are the means ± SE. The data are indicated as the ratio of GE and ileal CP, and the data for analysis are transformed by arcsine. 2Treatments: 1) no vaccination; 2) simplified vaccination, which included the infectious bronchitis vaccine (H120), the inactivated avian influenza vaccine (AI), the live vaccine strain Clone-30 of the Newcastle disease virus (NDV), and the combined inactive vaccine for infectious bursal diseases and the Newcastle disease vaccine (ND-IB); 3) normal vaccination (simplified vaccination + second dose of ND-IB, H120, and AI); 4) lipopolysaccharide (LPS) stress (normal vaccination + LPS); or 5) cyclophosphamide (CPM) stress (normal vaccination + CPM). 3FI = feed intake; FCR = feed conversion ratio
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The effect of different types of immune stress on feed intake (FI), BW gain, and feed conversion ratio (FCR) are presented in Table 2. Among the 3 vaccine groups (no, simplified, and normal vaccination), no differences were observed in FI or BW gain. However, compared with groups 1 and 2, the BW gain decreased significantly in chickens treated with LPS or CPM (P < 0.05 and P < 0.01, respectively), and the FI decreased significantly in chickens treated with LPS. The FCR showed no significant difference among the 5 groups (P > 0.05). There was no significant difference in the digestibility of GE. The CPM treatment decreased the digestibility of ileal CP compared with that of the other groups.
group 5 (Table 3). Compared with that of group 1, the spleen index increased significantly in chickens treated with LPS and decreased significantly in chickens treated with CPM (P < 0.01). The chickens in group 5 showed a lower bursa index than those of the chickens in the other groups (P < 0.01). The levels of sIgA decreased significantly in chickens treated with LPS and CPM stress (P < 0.01); they also showed higher levels of serum IgG than those of the chickens in the other groups at 42 d of age, whereas B-lymphocyte proliferation increased significantly in group 4 chickens (P < 0.05).
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Table 3. Effect of different immune stresses on immune organ growth in broilers1 Treatment name2 (group number) No vaccination (1)
Days of age
Item Thymus index (g/kg) Spleen index (g/kg) Bursa index (g/kg) LZM3 (μg/mL) sIgA (× 102 mg/g) IgG (g/L)
4.24 2.95 0.71 1.32 2.55 1.61 10.03 2.91 4.72 4.64 2.15 1.69 3.65 8.88
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.25 0.03a 0.13a 0.23a 0.18a 0.64 0.42a 0.04a 0.04a 0.28 0.36a 0.11a 2.82
3.93 3.48 0.68 1.13 2.80 1.64 9.46 5.01 4.57 4.68 2.22 1.93 3.84 8.15
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.20ab
0.53 0.05a 0.10a 0.33a 0.13a 0.84 0.66b 0.08a 0.06a 0.31 0.04ab 0.46a 2.43
Normal vaccination (3) 3.70 2.85 0.73 1.09 2.79 1.47 7.18 3.94 4.58 4.57 2.28 2.09 4.44 11.29
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.19ab
0.33 0.06a 0.11ac 0.19a 0.15a 0.72 0.61ab 0.12a 0.03a 0.31 0.09bd 0.25a 2.36
LPS stress (4) 3.50 3.13 1.22 1.85 2.10 1.59 7.15 2.38 4.21 4.28 2.41 2.54 7.11 9.51
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.35ab
0.33 0.16b 0.20b 0.25a 0.29a 1.85 0.44a 0.22b 0.15b 0.45 0.15c 0.96b 2.17
CPM stress (5) 2.96 2.11 0.59 0.71 1.17 0.58 6.08 2.45 3.56 3.87 1.77 2.28 1.79 8.69
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.24b
0.28 0.07a 0.10c 0.25b 0.09b 1.15 0.42a 0.20c 0.12b 0.67 0.13cd 0.42a 0.76
P-value 0.044 0.106 <0.01 <0.01 <0.01 <0.01 0.052 0.010 <0.01 <0.01 0.886 <0.01 0.024 0.413
a–dMeans
in the same row with no common superscript differ significantly (P < 0.05). are expressed as the stimulation index calculated as the optical density at 570 nm absorbance of the wells that were incubated with LPS divided by the optical density at 570 nm absorbance of wells that were incubated without LPS. 2Treatments: 1) no vaccination; 2) simplified vaccination, which included the infectious bronchitis vaccine (H120), the inactivated avian influenza vaccine (AI), the live vaccine strain Clone-30 of the Newcastle disease virus (NDV), and the combined inactive vaccine for infectious bursal diseases and the Newcastle disease vaccine (ND-IB); 3) normal vaccination (simplified vaccination + second dose of ND-IB, H120, and AI); 4) lipopolysaccharide (LPS) stress (normal vaccination + LPS); or 5) cyclophosphamide (CPM) stress (normal vaccination + CPM). 3LZM = lysozyme concentration; and PBMC = peripheral blood mononuclear cell. 1Values
; and cluster 5, subjects Q41, Q42, and Q43; cluster 6, Q23. At 42 d of age, 5 clusters were identified and subjects were grouped as follows: cluster 1, subjects H12, H13, H14, and H48; cluster 2, subjects H21, H23, and H25; cluster 3, subjects H41 and H46; cluster 4, subjects H35, H37, and H38; and cluster 5, subjects H55, H56, and H57. The ERIC-PCR fingerprints of the cecal samples from groups 1 and 2 clustered together with high coefficients, except for Q23. The ERIC-PCR fingerprints of the 3 cecal samples from groups 4 and 5 clustered together with high coefficients among them.
DISCUSSION Our results showed that immune stress decreased the FI and BW gain of the chickens (Table 2). It was reported that immune stress may decrease the growth performance of broilers (Eid et al., 2003; Malheiros et al., 2003; Lin et al., 2004; Virden et al., 2007). The current study showed that the injection of CPM inhibited the proliferation of B lymphocytes in the blood, which coincided with the immune organ index, such as the spleen and thymus (Table 3). Yang et al. (2006) reported that chickens inoculated with Eimeria tenella coccidia have higher IgG and sIgA levels. Our results showed that immune stress decreased growth performance. Hanna et al. (2000) reported that the gastrointestinal mucosal immune response mainly depended on sIgA-mediated humoral immunity. Our results demonstrated that immune stress had adverse effects on the mucosal immune function of broilers; both treatments of no vaccinations or simplified vaccinations can
maintain higher sIgA levels in broilers (Table 3). A combination of the effect of immune stress on growth performance and the immuno-modulator role of immune stress, which allocates nutrients toward immune responses, could possibly explain the depressed growth. A previous study showed that immune responses could affect growth performance (Rajapakse et al., 2010). Chicks are very susceptible to infections by enteric bacteria, and the gut mucosa is the first line of defense against the bacteria. Microflora play an important role in the intestinal defense system (Lee et al., 2010; Kim et al., 2011). Therefore, a change in cecal microbial communities becomes an important indicator of the intestinal nonspecific immune response. The cluster analysis described in this paper identifies genotypes among the 5 treatments (Figures 1 and 2). Berndt et al. (2007) reported that the capability of Salmonella serovars to enter the cecal mucosa affected both the level and character of the immune response in tissue. Although we did not identify the ERIC-PCR fingerprint consensus fragment in the bacterial strains, it may be assumed that immune stress can lead to a distribution change of cecal microflora. Some sets of bands are highlighted with yellow boxes (Figure 1) to demonstrate that higher similarities in the cecal microflora distribution existed between the no vaccination and the simplified vaccination treatment groups at 21 and 42 d of age, and higher similarities were found between the CPM and the LPS treatment groups at 21 and 42 d of age. These results suggest that chicken cecal microflora are at risk when chickens experience immune stress. A previous study has shown that a change in immune function
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PBMC3 proliferation (SI)
21 42 21 42 21 42 21 42 21 42 21 42 21 42
0.33a
Simplified vaccination (2)
STRESS AND CECAL MICROFLORA IN CHICKENS
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could serve as a growth promoter in broiler production by modulating the concentrations of intestinal microbial flora (Li et al., 2007b). Therefore, it is necessary consider the exact modulation mechanism of immune stress on the gut microflora in broilers. It can be concluded that broilers with simplified vaccinations or without vaccinations can achieve the same growth performance as broilers with normal vaccina-
tions in a controlled, rational feeding environment, but immune stress can interrupt the homeostasis of cecal microflora, impair intestinal mucosal immune function, and decrease humoral and cellular immune responses. It is generally recommended for broilers to be vaccinated only against the diseases to which they might be exposed. Vaccinating birds without a risk of exposure results in a growth cost to the bird.
Figure 2. Cluster analysis of the gel patterns based on the enterobacterial repetitive intergenic consensus PCR fingerprints of the cecal bacterial structure of chickens at 21 and 42 d of age. Note: Q1 to Q5 correspond to the cecal bacteria in chickens from groups 1 to 5 at 21 d of age, respectively; and H1 to H5 correspond to the cecal bacteria in chickens from groups 1 to 5 at 42 d of age, respectively. Treatments were as follows: 1) no vaccination; 2) simplified vaccination, which included the infectious bronchitis vaccine (H120), the inactivated avian influenza vaccine (AI), the live vaccine strain Clone-30 of the Newcastle disease virus (NDV), and the combined inactive vaccine for infectious bursal diseases and the Newcastle disease vaccine (ND-IB); 3) normal vaccination (simplified vaccination + second dose of ND-IB, H120, and AI); 4) lipopolysaccharide (LPS) stress (normal vaccination + LPS); or 5) cyclophosphamide (CPM) stress (normal vaccination + CPM). Color version available in the online PDF.
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Figure 1. The enterobacterial repetitive intergenic consensus PCR fingerprinting of the cecal bacterial genomes of broilers at 21 and 42 d of age. M = GeneRuler Ladder; lanes Q1 to Q5 correspond to the cecal bacteria in chickens from groups 1 to 5 at 21 d of age, respectively; and H1 to H5 correspond to the cecal bacteria in chickens from groups 1 to 5 at 42 d of age, respectively. Treatments were as follows: 1) no vaccination; 2) simplified vaccination, which included the infectious bronchitis vaccine (H120), the inactivated avian influenza vaccine (AI), the live vaccine strain Clone-30 of the Newcastle disease virus (NDV), and the combined inactive vaccine for infectious bursal diseases and the Newcastle disease vaccine (ND-IB); 3) normal vaccination (simplified vaccination + second dose of ND-IB, H120, and AI); 4) lipopolysaccharide (LPS) stress (normal vaccination + LPS); or 5) cyclophosphamide (CPM) stress (normal vaccination + CPM). Color version available in the online PDF.
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REFERENCES
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Berndt, A., A. Wilhelm, C. Jugert, J. Pieper, K. Sachse, and U. Methner. 2007. Chicken cecum immune response to Salmonella enterica serovars of different levels of invasiveness. Infect. Immun. 75:5993–6007. Campo, J. L., and S. G. Davila. 2002. Effect of photoperiod on heterophil to lymphocyte ratio and tonic immobility duration of chickens. Poult. Sci. 81:1637–1639. Deeb, N., and A. Cahaner. 2002. Genotype-by-environment interaction with broiler genotypes differing in growth rate. 3. Growth rate and water consumption of broiler progeny from weight-selected versus nonselected parents under normal and high ambient temperatures. Poult. Sci. 81:293–301. Eid, Y. Z., A. Ohtsuka, and K. Hayashi. 2003. Tea polyphenols reduce glucocorticoid-induced growth inhibition and oxidative stress in broiler chickens. Br. Poult. Sci. 44:127–132. Hangalapura, B. N., M. G. Kaiser, J. J. Poel, H. K. Parmentier, and S. J. Lamont. 2006. Cold stress equally enhances in vivo pro-inflammatory cytokine gene expression in chicken lines divergently selected for antibody responses. Dev. Comp. Immunol. 30:503–511. Hanna, M. K., B. L. Zarzaur, K. Fukatsu, R. C. Dewitt, K. R. Renegar, C. Sherrell, W. Yong, and K. A. Kudsk. 2000. Individual neuropeptides regulate gut-associated lymphoid tissue integrity, intestinal immunoglobulin A levels, and respiratory antibacterial immunity. J. Parenter. Enter. Nutr. 24:261–269. Kim, G. B., Y. M. Seo, C. H. Kim, and I. K. Paik. 2011. Effect of dietary prebiotic supplementation on the performance, intestinal microflora, and immune response of broilers. Poult. Sci. 90:75–82. Lai, H. T. L., M. G. B. Nieuwland, B. Kemp, A. J. A. Aarnink, and H. K. Parmentier. 2009. Effects of dust and airborne dust components on antibody responses, body weight gain, and heart morphology of broilers. Poult. Sci. 88:1838–1849. Lai, H. T. L., M. G. B. Nieuwland, B. Kemp, A. J. A. Aarnink, and H. K. Parmentier. 2011. Effects of repeated intratracheally administered lipopolysaccharide on primary and secondary specific antibody responses and on body weight gain of broilers. Poult. Sci. 90:337–351. Lee, K. W., S. H. Lee, H. S. Lillehoj, G. X. Li, S. I. Jang, U. S. Babu, M. S. Park, D. K. Kim, E. P. Lillehoj, A. P. Neumann, T. G. Rehberger, and G. R. Siragusa. 2010. Effects of direct-fed microbials on growth performance, gut morphometry, and immune characteristics in broiler chickens. Poult. Sci. 89:203–216. Lee, S. H., H. S. Lillehoj, E. P. Lillehoj, S. M. Cho, D. H. Park, Y. H. Hong, H. K. Chun, and H. J. Park. 2008. Immunomodulatory properties of dietary plum on coccidiosis. Comp. Immunol. Microbiol. Infect. Dis. 31:389–402. Li, M. O., Y. Y. Wan, and R. A. Flavell. 2007a. T cell-produced transforming growth factor-β1 controls T cell tolerance and regulates Th1- and Th17-cell differentiation. Immunity 26:579–591. Li, X. J., X. S. Piao, S. W. Kim, P. Liu, L. Wang, Y. B. Shen, S. C. Jung, and H. S. Lee. 2007b. Effects of chito-oligosaccharide supplementation on performance, nutrient digestibility, and serum composition in broiler chickens. Poult. Sci. 86:1107–1114. Lin, H., E. Decuypere, and J. Buyse. 2004. Oxidative stress induced by corticosterone administration in broiler chickens (Gallus gallus domesticus). 1. Chronic exposure. Comp. Biochem. Physiol. B 139:737–744. Malheiros, R. D., V. M. Moraes, A. Collin, E. Decuypere, and J. Buyse. 2003. Free diet selection by broilers as influenced by dietary macronutrient ratio and corticosterone supplementation.
1. Diet selection, organ weights, and plasma metabolites. Poult. Sci. 82:123–131. Marcq, C., E. Cox, I. M. Szalo, A. Théwis, and Y. Beckers. 2011. Salmonella Typhimurium oral challenge model in mature broilers: Bacteriological, immunological, and growth performance aspects. Poult. Sci. 90:59–67. Mashaly, M. M., G. L. Hendricks, and M. A. Kalama. 2004. Effect of heat stress on production parameters and immune responses of commercial laying hens. Poult. Sci. 83:889–894. Rajapakse, J. R. P. V., M. D. P. Buddhika, M. Nagataki, H. Nomura, Y. Watanabe, Y. Ikeue, and T. Agatsuma. 2010. Effect of sophy beta-glucan on immunity and growth performance in broiler chicken. J. Vet. Med. Sci. 72:1629–1632. Rajkumar, U., M. R. Reddy, S. V. R. Rao, K. Radhika, and M. Shanmugam. 2011. Evaluation of growth, carcass, immune response, and stress parameters in naked neck chicken and their normal siblings under tropical winter and summer temperatures. Asian-australas. J. Anim. Sci. 24:509–516. Shini, S., and P. Kaiser. 2009. Effects of stress, mimicked by administration of corticosterone in drinking water, on the expression of chicken cytokine and chemokine genes in lymphocytes. Stress 12:388–399. Shini, S., P. Kaiser, A. Shini, and W. L. Bryden. 2008. Biological response of chickens (Gallus gallus domesticus) induced by corticosterone and a bacterial endotoxin. Comp. Biochem. Physiol. B 149:324–333. Shini, S., A. Shini, and P. Kaiser. 2010. Cytokine and chemokine gene expression profiles in heterophils from chickens treated with corticosterone. Stress 13:185–194. Star, L., B. Kemp, I. van den Anker, and H. K. Parmentier. 2008. Effect of single or combined climatic and hygienic stress in four layer lines: 1. Performance. Poult. Sci. 87:1022–1030. Versalovic, J., T. Koeuth, and J. R. Lupski. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19:6823–6831. Virden, W. S., M. S. Lilburn, J. P. Thaxton, A. Corzo, D. Hoehler, and M. T. Kidd. 2007. The effect of corticosterone-induced stress on amino acid digestibility in Ross broilers. Poult. Sci. 86:338– 342. Wei, G., L. Pan, H. Du, J. Chen, and L. Zhao. 2004. ERIC-PCR fingerprinting-based community DNA hybridization to pinpoint genome-specific fragments as molecular markers to identify and track populations common to healthy human guts. J. Microbiol. Methods 59:91–108. Wilkinson, K. G. E., R. B. Tee, G. Tomkins, and R. Premier. 2011. Effect of heating and aging of poultry litter on the persistence of enteric bacteria. Poult. Sci. 90:10–18. Yalçin, S., S. Ozkan, and L. Turkmut. 2001. Responses to heat stress in commercial and local broiler stocks. 2. Developmental stability of bilateral traits. Br. Poult. Sci. 42:153–160. Yang, X. J., Y. M. Guo, X. He, J. M. Yuan, Y. Yang, and Z. Wang. 2008. Growth performance and immune responses in chickens after challenge with lipopolysaccharide and modulation by dietary different oils. Animal 2:216–223. Yang, X., Y. M. Guo, Z. Wang, and W. Nie. 2006. Effects of dietary supplementation with different oils on coccidiosis in chickens. Avian Pathol. 35:373–378. Ye, Y., Q. P. Wu, Y. H. Zhou, X. H. Dong, and J. M. Zhang. 2008. Analysis of major band of Enterobacter sakazakii by ERIC-PCR and development of a species-specific PCR for detection of E. sakazakii in dry food samples. J. Microbiol. Methods 75:392– 397.