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Influence of In-Feed Virginiamycin on the Systemic and Mucosal Antibody Response of Chickens

J. T. Brisbin^*, J. Gong^,1, C. A. Lusty^*, P. Sabour, B. Sanei, Y. Han, P. E. Shewen^* and S. Sharif^*,1

^* Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1; Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada, N1G 5C9; Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, Ontario, Canada, N1G 4Y2; and Nutreco Canada Agresearch, Guelph, Ontario, Canada, N1G 4T2

¹ Corresponding author: gongj{at}agr.gc.ca or shayan{at}uoguelph.ca

	ABSTRACT

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Subtherapeutic and prophylactic doses of virginiamycin are capable of altering the intestinal microbiota as well as increasing several growth parameters in chickens. In spite of the fact that the microbiota plays a role in shaping the host’s immune system, little information is available on the effects of in-feed antibiotics on the chicken immune system. The objective of this study was to examine the effects of an antibiotic, virginiamycin, on the development of antibody responses. Chickens were fed diets containing no antibiotics, along with either subtherapeutic (11 ppm) or prophylactic (22 ppm) doses of virginiamycin. Chickens were then immunized with keyhole limpet hemocyanin (KLH) and sheep red blood cells systemically, and with BSA and KLH orally. Although antibodies were detected against BSA in the intestinal contents of birds that were orally immunized, there was no difference among different treatment groups. Systemic IgG, and to a lesser extent IgM, antibody responses to KLH were greater (P < 0.05) in birds fed a diet containing 11 or 22 ppm of virginiamycin compared with control birds fed no antibiotic. No treatment effect was found in the sheep red blood cell-immunized birds. Results of the present study implicate virginiamycin in enhancing antibody responses to some antigens in chickens. Further studies are required to determine to what extent these effects on antibody response are mediated through changes in the composition of the microbiota.

Key Words: immune response • probiotic • dietary antibiotic • chicken • microbiota

	INTRODUCTION

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Dietary antimicrobials have been used extensively in the poultry industry as growth promotants and have been reported to have beneficial effects on animal growth, feed conversion efficiency, and the inhibition of pathogen growth (Stutz and Lawton, 1984; Gaskins et al., 2002). Virginiamycin, a streptogramin antibiotic, is commonly used in chicken feed at subtherapeutic concentrations as a growth promotant and at prophylactic concentrations for the prevention of necrotic enteritis caused by Clostridium perfringens. The impact of virginiamycin on different bacterial groups in the gastrointestinal tract of chickens has been shown to be most prominent in the ileal region, which is significant given the role of this region in nutrient absorption (Wise and Siragusa, 2007). Changes in the microbiota include changes in abundance of several bacterial targets, including species from the genus Lactobacillus (Dumonceaux et al., 2006). In particular, Lactobacillus salivarious is significantly decreased with virginiamycin treatment (Dumonceaux et al., 2006; Guban et al., 2006; Zhou et al., 2007)

It is generally thought that the improved growth performance observed in chickens fed antimicrobials is partly due to intestinal microbiota modifications whereby pathogens and bacteria that compete with the host for nutrients are eliminated or their population is reduced significantly (Gaskins et al., 2002). The importance of the role of the microbiota is evident because birds raised in germ-free environments do not demonstrate the typical growth enhancement when given in-feed antibiotics (Coates et al., 1955). Given the role of virginiamycin as a growth promotant, it can be assumed that the microbiota still present in chickens treated with this antimicrobial represent a population that is adapted to the antibiotic and that this population may have some positive impact on growth and feed efficiency.

The intestinal microbiota plays an important role in the antigenic stimulation and development of the gut-associated lymphoid tissues. In rabbits, the gut microbiota has been shown to be involved in shaping the immune system repertoire (Rhee et al., 2004). In addition, many other studies using germ-free animal models have shown that the gut microbiota is essential for the development of the mucosal immune system (Cebra, 1999; Bauer et al., 2006). Given the effects of administration of antibiotics on the destabilization of the chicken gut microbiota, especially during the development of the chicken mucosal immune system, it can be hypothesized that in-feed antibiotics could affect the quality and quantity of immune response, both locally and systemically. Thus, the objective of the current study was to evaluate the effect of subtherapeutic and prophylactic doses of virginiamycin on the systemic and mucosal immune response in chickens.

	MATERIALS AND METHODS

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Chickens and Housing

Newly hatched mixed-sex commercial broiler chicks were obtained from Maple Leaf Foods Inc. (New Hamburg, Ontario, Canada). Birds were maintained in floor pens on clean wood shavings at the Arkell Poultry Research Station (University of Guelph, Ontario, Canada). Chicks were provided with free access to water and food. The research complied with University of Guelph Animal Care Committee guidelines.

Experimental Design

To evaluate the systemic antibody response, chicks were randomly divided into 5 treatment groups: chicks in group I were given no in-feed antibiotics and were immunized (n = 12), group II chicks were given 11 ppm of virginiamycin and were immunized (n = 12), chicks in group III were given 22 ppm of virginiamycin and were immunized (n = 12), group IV chicks were given no antibiotic and were not immunized (n = 7), and group V chicks were given 11 ppm of virginiamycin and were not immunized (n = 7). In addition, a group of chicks that received no antibiotics but were hyperimmunized were used as a positive control (n = 3). Chicks in groups I, II, and III were immunized subcutaneously with 0.25 mL of 2% sheep red blood cells (SRBC; catalog no. A0422, PML Microbiologicals, Mississauga, Ontario, Canada) in PBS and 0.5 mL of PBS containing 100 µg of keyhole limpet hemocyanin (KLH; catalog no. H7017, Sigma, Oakville, Ontario, Canada) 14 d posthatch, followed by a secondary immunization 1 wk later. Phosphate-buffered saline was used as a placebo in those groups that were not immunized (IV and V). Blood samples were collected on the day of immunization as well as 7, 14, and 21 d post primary immunization. Birds in the hyperimmunized group were immunized 3 times, every 7 d starting at 2 wk of age, and were bled 4 d after the last immunization.

For an assessment of the mucosal antibody response, chicks were divided into 5 groups and administered antibiotics, as described above. Chicks in groups I, II, and III (n = 11 in each group) were immunized intraperitoneally with 0.5 mL of PBS containing 100 µg of KLH at 14 d of age and then via the oral route with 0.5 mL of PBS containing 100 µg of KLH 1 wk later. The same groups (I, II, and III) had ad libitum access to water containing 1.4 mg/mL of BSA (catalog no. BP1600-100, Fisher Scientific, Ottawa, Ontario, Canada) for 6 consecutive days, starting at 14 d of age according to the procedure introduced by Ameiss et al. (2004). Phosphate-buffered saline was used as a placebo in groups IV and V (n = 6 in each group), which were not immunized. Samples (gut contents and blood) were obtained 0, 7, 14, and 21 d post primary immunizations with KLH or 0, 7, 14, and 21 d after the start of BSA treatment. In addition, a group of chicks that received no antibiotics but were hyperimmunized were used as a positive control (n = 3). Birds in the hyperimmunized group were immunized intraperitoneally with KLH or BSA at 2 wk of age, followed by immunization via the oral route of either KLH or BSA twice at weekly intervals. Birds were bled 4 d after the last immunization. Blood and intestinal contents were collected as described.

Sample Collection

Blood samples were collected and kept at room temperature for approximately 2 h, then at 4°C overnight. Blood samples were centrifuged for 10 min at 580 x g, and serum was harvested and stored at –80°C. To evaluate the mucosal antibody response, 45 chickens were killed at different time points (described above), and intestinal samples were taken and processed as described previously (Haghighi et al., 2005). Briefly, intestines were trimmed of connective tissue and fat and cut into small pieces before mixing with 5 mL of PBS containing 100 µg/mL of trypsin inhibitor (catalog no. T-9003, Sigma). The mixture was vortexed thoroughly and centrifuged at 20,000 x g at 4°C for 30 min. After centrifugation, the supernatant was removed and stored at –20°C

Serological Analysis

A direct hemagglutination assay was performed to measure the total antibody (IgM and IgG) response to SRBC in serum according to the procedure of Haghighi and colleagues (2005). A positive result was recorded when at least 50% SRBC agglutination was observed.

Detection of antibodies against KLH in sera, and against KLH and BSA in intestinal contents was performed by using indirect ELISA assays. All secondary antibodies were purchased from MJS BioLynx Inc. (Brockville, Ontario, Canada) and were as follows, goat anti-chicken IgG HRP conjugated (catalog no. A30-104p), goat anti-chicken IgM HRP conjugated (catalog no. A30-102p), and goat anti-chicken IgA HRP conjugated (catalog no. A30-103p). The ELISA assays were performed according to the methods of Haghighi and colleagues (2005). Briefly, plates were coated with 100 µL of 120 µg/mL of BSA or 1 µg/mL of KLH in coating buffer and incubated overnight at 4°C. Plates were washed 3 times in washing buffer and blocked (PBS with 0.05% Tween 20 and 0.25% fish gelatin) at 37°C for 1 h. Serum samples were diluted 1:100 for the detection of IgG and 1:200 for the detection of IgM, whereas intestinal contents were diluted 1:20 for the detection of IgA. Plates were incubated for 1 h at 37°C with serum and intestinal contents for KLH ELISA and for 2 h at room temperature for BSA ELISA. Plates were washed 3 times for the detection of IgG. In the case of IgM and IgA ELISA, washings were done 3 times as described previously, except that the plates were incubated for 5 min in wash buffer. One hundred microliters of ABTS Peroxidase Substrate System (catalog no. 50-62-00, KPL, Gaithersburg, MD) was then added to each well, and the plate was incubated for 30 min at room temperature in the dark and, subsequently, absorbance was measured at 405 nm. Positive and negative control chicken sera were included in each plate.

To reduce background in the case of the intestinal contents, ELISA assays were performed with duplicate samples incubated in wells either with or without the antigens. The absorbance obtained from the uncoated wells was subtracted from the absorbance obtained from antigen-coated wells. In addition, to account for plate-to-plate variations, sample:positive ratios were determined. To do that, a positive control sample was included on each plate and the sample optical density was divided by the optical density of the positive control.

Statistical Analysis

Statistical analysis of data was performed by using SAS (SAS Institute, 2002). Duncan’s multiple range test was used to compare the antibody response among groups. Statistical significance was assessed at P = 0.05.

	RESULTS

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Systemic Antibody Responses to KLH and SRBC

To evaluate the effects of antibiotic treatment on the systemic immune response, antibody responses to KLH and SRBC were assessed. Immunization with SBRC induced specific anti-SRBC antibodies in the serum starting 7 d post primary immunizations and peaked by 14 d before decreasing at 21 d post primary immunization (data not shown). There was no significant difference in anti-SRBC antibodies generated among various treatment groups (P > 0.05).

Immunization with KLH induced anti-KLH antibodies of the IgG and IgM isotypes in the serum (Figure 1). The IgM response peaked at 7 d post primary immunization (Figure 1a), and birds treated with 22 ppm of virginiamycin had a significantly (P < 0.05) greater anti-KLH IgM antibody response than birds that received no antibiotics. Although the birds treated with 11 ppm of virginiamycin had a greater IgM response than the untreated birds and also had a lower response than the birds treated with 22 ppm of virginiamycin, the differences were not statistically significant.

View larger version (17K): [in this window] [in a new window]   Figure 1. Anti-keyhole limpet hemocyanin (KLH) antibodies in the serum. Mean optical density values are presented, and error bars represent the SEM. a) Anti-KLH IgM antibody response on 0, 7, 14, and 21 d post primary immunization. b) Anti-KLH IgG antibody response on 7, 14, and 21 d post primary immunization. Letters (a,b or a–c) indicate that the treatments were significantly different from nonimmunized controls at that particular time point (P < 0.05). Among the immunized groups, identical letters indicate that there was no statistical difference, whereas different letters represent a statistical difference between groups.

Mucosal Antibody Responses to KLH and BSA

Immunization with KLH resulted in no measurable anti-KLH IgG or IgA in the intestinal contents of chickens (data not shown). Likewise, feeding BSA in the drinking water for 6 consecutive days resulted in no measurable anti-BSA IgG antibody in the intestine (data not shown). Bovine serum albumin in the drinking water did generate an anti-BSA IgA response in the intestine; however, there was no statistical difference in anti-BSA antibodies generated among various treatment groups (P > 0.05; data not shown).

	DISCUSSION

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Virginiamycin, a commonly used antibiotic in poultry production, has been shown previously to alter the composition of the intestinal microbiota (Zhou et al., 2007). In this study, we investigated the effect of subtheurapeutic and prophylactic doses of this antibiotic on the systemic and mucosal antibody responses in broiler chickens. We observed that chickens immunized and treated with either dose of virginiamycin had an enhanced systemic antibody response to a soluble antigen, KLH, in their sera compared with immunized birds that did not receive virginiamycin. The mechanism whereby virginiamycin enhances the immune responsiveness is not known. However, it is possible that the observed increase in anti-KLH-specific IgG and IgM is related to changes in the composition of the intestinal microbiota of antibiotic-treated birds. It is also plausible that the microbiota of these birds was enriched in species of bacteria with probiotic properties. The ability of virginiamycin to alter the composition of the intestinal microbiota and increase or decrease the intestinal colonization by lactobacilli is well documented (Dumonceaux et al., 2006; Zhou et al., 2007). This is of interest, given that several bacteria from the genus Lactobacillus isolated from chickens were shown to have probiotic properties (Garriga et al., 1998; Gusils et al., 1999). The alteration of the microbiota through treatment with probiotics, for example, those containing Lactobacillus, has been shown previously to increase the systemic antibody response in chickens. Koenen and colleagues (2004) demonstrated that probiotic treatment enhanced the systemic antibody response to KLH, whereas Huang and coworkers (2004) demonstrated that some lactobacilli increase the IgA, but not the IgG, response to KLH. Haghighi and colleagues (2005) also demonstrated that chickens fed probiotic supplements had an increased IgM response to a systemically administered antigen.

Another potential explanation for our observation is a direct effect of virginmycin on the immune system. Antibiotics themselves have been known to modulate the immune response (Woo et al., 1999); however, it has been shown that virginiamycin is not absorbed when orally administered (Yamamoto et al., 2000). Therefore, although the direct modulation of the immune response by virginiamycin cannot be formally ruled out, it seems an unlikely explanation for our results.

The chickens immunized with SRBC and fed virginiamycin did not demonstrate a similar increase in antibody response compared with the chickens fed no antibiotic. Dafwang et al. (1985) also found that antibiotics had no effect on the antibody response to SRBC, whereas previous experiments by our group (Haghighi et al., 2005) demonstrated a significant increase in serum anti-SRBC antibody titer, specifically IgM, after treatment with a commercial probiotic product mainly containing Lactobacillus acidophilus. Aside from different immunization protocols used in these 2 studies, the discrepancy may be explained by the possibility that the composition of the microbiota of antibiotic-treated birds is different from that of probiotic-treated chickens. Hence, it is possible that the microbiota of probiotic-treated chickens is enriched for bacteria that stimulate an antibody response to a cellular antigen, such as SRBC, whereas there may be more bacteria in the microbiota of antibiotic-treated chickens that encourage a response to a soluble antigen. Importantly, it has been shown that different strains of lactobacilli contribute differently to an antibody-mediated immune response (Koenen et al., 2004). The dosage of the bacteria and the age and genetic background of the animal could also play a role.

When antibodies in the intestinal contents were compared among the BSA-immunized groups, there were no significant differences. There was, however, a tendency for the non-antibiotic-treated and immunized groups to have greater BSA-specific IgA in their intestinal contents. Although the mechanisms are unknown, there have been previous reports that changes in the microbiota composition induced by administration of probiotics may have no effects or may even reduce the specific antibody response in intestinal contents (Dalloul et al., 2003; Haghighi et al., 2005). This is perhaps related to the type of commensal bacteria that become predominant in the intestines of probiotic-treated birds.

The gastrointestinal tract contains an abundant and diverse microbiota that plays an important role in both the health and nutrition of the animal. The effects of the microbiota, especially the commensal bacteria with probiotic effects, on the immune system have been described previously. This study provides evidence that in-feed virginiamycin enhances antibody responses, at least systemically, to soluble antigens in broiler chickens. It remains to be determined, however, whether in-feed antimicrobials can influence immune responsiveness at the mucosal surfaces.

	ACKNOWLEDGMENTS

 
This study was supported by the Canadian Poultry Research Council (Guelph, Ontario), Poultry Industry Council (Guelph, Ontario, Canada), Natural Science and Engineering Research Council of Canada (Ottawa, Ontario), and Agriculture and Agri-Food Canada (Guelph, Ontario).

Received for publication April 18, 2008. Accepted for publication June 3, 2008.

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