Maternal antibody decay and antibody-mediated immune responses in chicken pullets fed prebiotics and synbiotics

INTRODUCTION

Newly hatched birds have immature and undeveloped immune system (Bar-Shira et al., 2003). Maternalderived antibodies (MDA) play a critical role in early protection against pathogenic organisms (Kramer and Cho, 1970). Maternal antibodies are immunoglobulins that are transferred from vaccinated or naturally infected breeder hens to the progeny through the egg, which provide passive immunity to progeny and protect them against infectious agents that cannot be cleared by the immature immune system (Mondal and Naqi, 2001; Hamal et al., 2006). It has been reported that progeny from hens with low antibody titers against E. coli are more likely to die after immunization with E. coli compared to the progeny of hens with high antibody levels (Heller et al., 1990). In addition, maternal antibodies reduce the growth-suppressive costs of an innate immune response toward pathogens during early development of the immune system (Soler et al., 2003; Brommer, 2004). However, this passive immunity has relatively short duration. The level of MDA peaks at 3 to 4 d posthatch, and then gradually decreases to undetectable levels at 2 to 3 wk of age (Sahin et al., 2001; Hamal et al., 2006). This makes birds vulnerable to infectious diseases, especially during wk 2 posthatch when the protection from MDA has declined, and the immune system of birds is not fully developed to respond properly to an early challenge.

Supplementation of diet with antibiotic growth promoters (AGPs) has been practiced in poultry industry for many years to protect birds against early infections caused by pathogenic enteric microorganisms (Dibner and Richards, 2005). However, the excessive use of antibiotics in animal feed can contribute to the development of antibiotic-resistant bacteria, which is a major public health concern (McEwen and Fedorka-Cray, 2002; Wegener, 2003). Therefore, there is intensive research in finding non-antibiotic or natural growth promoters to maintain health and protect birds against pathogens under commercial conditions in the face of a ban or limited use of AGPs (Huyghebaert et al., 2011; Ganan et al., 2012). Extension of protection provided by maternal antibodies or activation of humoral immunity by using antibiotic alternatives in diets can be used as a strategy to protect birds against early infection in post-antibiotic era.

Yeast-derived carbohydrates (YDC) and probiotics are among the alternatives, which are well known for their intestinal health benefits and potential immunomodulatory properties (McCracken et al., 1999; Panda et al., 2006; Yang et al., 2009). It has been reported that probiotics bacteria and cell wall polysaccharides present in YDC (β1,3–1,6-glucans and mannan) can function as microbe-associated molecular patterns (MAMPs), and modulate the immune system through pattern recognition receptors resulting in the production of cytokines, some of which (IL-4, IL-13, and IL-10) are involved in antibody production (Christensen et al., 2002; Shashidhara and Devegowda, 2003; Jawhara et al., 2012; Dibaji et al., 2014). The results of some studies have shown that supplementation of diet with YDC and probiotics may enhance humoral immune responses by increasing the antibody production (Guo et al., 2003; Haghighi et al., 2005; Haghighi et al., 2006; Ghosh et al., 2012). It was also reported that supplementation of diet with probiotic can extend the decay rate of maternal antibodies helping birds against early challenge caused by pathogens. In addition, a recent study in our group demonstrated that supplementation of diet with both YDC and synbiotics (SNB) modulated the innate immune response of pullet chicks, with the latter showing a more potent effect (Yitbarek et al., 2015). The present study therefore was conducted to investigate the effects of YDC and SNB on innate humoral immune response, decay rate of maternal antibodies, and antibody-mediated immune response against sheep red blood cells (SRBC) and bovine serum albumin (BSA) in pullet chicks.

MATERIAL AND METHODS

General Study Design

This study was approved and conducted under the guidelines of the University of Manitoba Animal Care Committee, and the birds were cared for in accordance with the recommendations established by the Canadian Council on Animal Care (CCAC, 1993). Three hundred day-old Lohmann chicken pullets were received from a local hatchery (Carltons Hatchery, Grunthal, Manitoba, Canada) and housed in cage units (Poultry Research Unit, University of Manitoba, Canada). Chicks were randomly allotted into 3 dietary treatments: chicks receiving unsupplemented basal diet (Control); chicks fed control diet supplemented with YDC; and chicks receiving control diet supplemented with SNB (YDC and Lactobacillus acidophilus, L. casei, Streptococcus faecium, Bacillus subtilis, and Saccharomyces cerevisiae). The chicks were housed in 30 cages with 10 replicates of 10 birds each assigned to each dietary treatment. Feed and water were provided ad libitum. To complete this study, 3 experiments were conducted.

Experiment 1

This experiment was designed to determine the decay rate of total IgG concentration in serum and decay rate of MDA against infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) in pullet chicks fed diets containing YDC or SNB. Blood samples were collected from 10 birds per treatment via the wing vein at d 0, 21, 28, 35, and 42 posthatch. Blood samples were clotted at room temperature for 2 h and centrifuged for 10 min at 580 × g, and serum was isolated and stored at –20°C until further analysis.

Determination of Total IgG, IBDV, and NDV Maternal Antibodies

Serum samples were analyzed for total IgG (Y) by sandwich enzyme-linked immunosorbent assay (ELISA) using chicken IgG ELISA quantitation and Starter Accessory Kits (Bethyl Laboratories, Montgomery, TX) following the manufacturer’s procedure. A microtiter plate reader (Soft Max Pro 3.1.1) was used to measure the absorbance at 450 nm and a 4- parameter logistic curve fit was developed using the chicken reference serum absorbance. Analysis for IBDV and NDV specific antibodies in sera were determined using specific infectious bursal disease and Newcastle disease commercial ELISA kits, respectively (IDEXX Laboratories Inc., Westbrook, ME), according to the manufacturer’s procedure.

Experiment 2

This second experiment was conducted to evaluate the effects of YDC and SNB on the decay rate of maternal and postvaccine antibodies against IBV in pullet chicks during the first 42 d posthatch. On d 1, the chicks were vaccinated with attenuated IBV (Massachusetts strain M41 3.0 log10EID50) before placement in experimental diets. Blood samples were analyzed at d 0, 21, 28, 35, and 42 posthatch. Blood samples were collected from 10 birds per treatment via the wing vein at d 0, 21, 28, 35, and 42 posthatch. Serum samples were analyzed using Infectious bronchitis-specific commercial ELISA kit following manufacturer’s procedure (IDEXX Laboratories Inc.).

Experiment 3

This experiment was designed to determine whether the inclusion of YDC or SNB can influence the antibody-mediated immune responses in chicken pullets following immunization with BSA and SRBC as model antigens. On d 28 and 35 posthatch, 10 birds per treatment were immunized and boosted respectively with 0.25 mL of 2% SRBC (Bethyl Laboratories Inc, Montgomery, TX) in phosphate-buffered saline (PBS) and 0.25 mL PBS containing 100 μg BSA (HyClone Laboratories, Logan, UT). The non-immunized group was fed the Control diet and injected with saline solution. To measure primary and secondary immune response to SRBC and BSA, 1 wk after each immunization, on d 35 and 42 posthatch, blood samples were collected from 10 birds per treatment. Serum sample were analyzed by hemagglutination assay for antibody response against SRBC and by ELISA for serum IgM and IgG response against BSA.

Hemagglutination Test for SRBC

A direct hemagglutination assay was performed to measure the total antibody response to SRBC in serum. Briefly, serum samples were heat treated at 56°C for 30 min. Then, 50 μL of PBS containing 0.05% of BSA was added into each well of a round-bottomed 96-well microplate. Serum samples were added in an equal amount of PBS in the first column of the plate and serially double diluted in the wells. Subsequently, 50 μL of 2% SRBC in PBS was added to each well and the plates were shaken for one minute followed by incubation for 24 h at 37°C. A positive result for agglutination titer was recorded when at least 50% of SRBC agglutination was observed.

Indirect ELISA for specific anti-BSA IgM and IgG

Detection of specific systemic antibody response of immunoglobulins IgG and IgM against BSA in sera was performed by indirect ELISA. Briefly, each well of a flat-bottomed 96-well microplate was coated overnight with 100 μL of coating buffer (0.1 M NaHCO3, pH 9.6) containing BSA (30 μg/mL) at 4°C. Wells were then washed 5 times with 200 μL of PBS with 0.05% of Tween 20 (P137 Sigma Aldrich Inc., St. Louis, MO) (PBST) as washing solution and were completely decanted between each washing step. Subsequently, the wells were covered by 100 μL of blocking buffer (PBST containing 0.25% of gelatine) and the plate was incubated for 2 h at room temperature. Washing was repeated, and was followed by addition of 100 μL of chicken serum (diluted 1:500 v/v in blocking buffer) to each well. Plates were incubated for 2 h at room temperature and then were washed 5 times with the washing solution. One hundred μL of detection antibody (goat anti-chicken IgG-Fc and IgM-Fc) conjugated with horseradish peroxidase (diluted in 50 mL of blocking buffer) was added to each well and incubated for 1 h at room temperature. Washing was repeated and was followed by addition of 100 μL of tetramethyl benzidine solution as substrate to each well and incubation for 15 min in the dark. Finally, stop solution was added to prevent further color development, and absorbance was measured at 450 nm using the microplate reader (Epoch, BioTek Instruments Inc., Winooski, VT).

Statistical Analysis

All data were analyzed using GLM procedure of SAS (SAS Institute Inc., Cary, NC). Differences among treatment means were tested using Scheffé’s multiple comparison. Results were expressed as least squares means and SEM. Probability values less than 0.05 were used as the criterion for statistical significance.

RESULTS

Sandwich ELISA results showed that inclusion of YDC alone did not cause any significant effect on IgG concentration; however, increased IgG concentration was observed in birds fed the diet supplemented with SNB on d 21 posthatch (Figure 1). No significant difference was observed for the decay rate of specific maternal antibodies against IBDV and NDV among treatment groups at wk 3, 4, 5, and 6 of age (data not shown). Birds fed diet containing YDC showed the lowest decay rate of specific maternal antibodies against IBV in wk 4; in other words YDC group showed the highest amount of maternal antibodies. However, in wk 6 posthatch, lower postvavaccine antibodies against IBV was observed in birds receiving YDC and SNB compared to the control (Figure 2). Regardless of dietary treatment, immunization with BSA significantly (P < 0.05) enhanced serum IgG and IgM response to BSA (Figure 3). However, regardless of immunization, supplementation of diet with YDC or SNB did not affect anti-BSA antibody response. The results of hemagglutination assay showed that immunization with SRBC increased total antibody response; however, dietary treatment did not affect the antibody response to SRBC (data not shown).

DISCUSSION

Humoral immunity is the component of immune system that is mostly mediated by antibody-releasing B lymphocytes (Scott, 2004). With increasing pressure on the poultry industry to cease or limit the use of AGPs, products or feed-additives that can extend protection provided by maternal antibodies or activation of humoral immunity can be used as a strategy to protect birds against early infection in post-antibiotic era. In pullet chicken, immunoglobulin Y is the functional homolog to human IgG and is considered the major antibody in blood circulation (Larsson et al., 1993; Sugita-Konishi et al., 1996). Immunoglobulin G is an important activator of the classical complement system that consists of a cascade of immune proteins

Maternal antibody decay and antibody-mediated immune responses in chicken pullets fed prebiotics and synbiotics - Image 1

Maternal antibody decay and antibody-mediated immune responses in chicken pullets fed prebiotics and synbiotics - Image 2

involved in pathogen elimination (Larsson et al., 1993; Sharma, 1997). In the present study, results for serum IgG concentration demonstrated that at d 1 posthatch, the level of total IgG antibodies was quite high, which is mostly associated with maternal antibodies. However, there was a general decline when measured again in wk 3, an observation that is in line with previous studies (Sahin et al., 2001; Ahmed and Akhter, 2003; Hamal et al., 2006). Supplementation of diet with SNB extends the IgG concentration in the serum of pullet chickens during wk 3 posthatch, suggesting that the synergistic effects between YDC and probiotics can lead to further modulation for the activation of acquire humoral immunity in pullet chickens. This can be very important for protecting pullet chickens against invading pathogens and can consequently reduce mortality at early stages of life when the protection from MDA has declined (Janardhana et al., 2009). In agreement with our results, Yitbarek et al. (2015) demonstrated that the supplementation of the diet with SNB increased the expression of IL-10 and IL-4, important cytokines involved in B-cell proliferation and differentiation to antibody-producing plasma cells. The lack of significant effect of either YDC or SNB on serum total IgG concentration after wk 3 is consistent with other studies in chickens and other animal species (Huang et al., 2004; Midilli et al., 2008), and might be explained by immune tolerance toward these products as non-pathogenic antigens. In the intestinal tract, unlike pathogens that induce persistent immune response, nonpathogenic antigens and commensal bacteria provoke transient noninflammatory response that is mainly mediated through polarization of naive T cells toward regulatory T (Treg) cells’ IL-10 production that induces differentiation of naive B cells to IgA-producing B cells (Clavel and Haller, 2007; Hooper et al., 2012). Unfortunately, in the present study, secretory IgA was not measured in the intestine. There is a possibility that immune tolerance toward YDC and SNB can lead to the production of intestinal IgA

Maternal antibody decay and antibody-mediated immune responses in chicken pullets fed prebiotics and synbiotics - Image 3

instead of IgG), which is considered a weak activator of complement systems (Cerutti and Rescigno, 2008).

There is some evidence regarding the positive effect of MDA on development of immune system through immunological imprinting (Gasparini et al., 2006; Grindstaff et al., 2006). The exact mechanism is still unclear; however, it has been reported that the B-cell repertoire may somehow model itself after antibodies in circulation (Fink et al., 2008). In the present study, supplementation of diet with YDC and SNB did not change the specific MDA concentration against IBDV and NDV. In contrast, Talebi et al. (2008) showed birds receiving diet-containing probiotics declined the decay rate of maternal antibodies against NDV and IBDV. The reasons behind this phenomenon remain to be elucidated; further research is required to clarify the immunomodulatory effects of YDC and SNB on the decay rate of specific maternal antibodies in pullet chickens.

It has been reported that the immunization of offspring with the same antigen to which their mother has been exposed does not lead to a strong innate immune response because of the maternally derived antigen specific antibodies that has been passed to progeny through the egg (Grindstaff, 2008). Immunosuppressive effect of MDA on postvaccine antibody-mediated immune response has been attributed to neutralization of vaccine virus by MDA (Siegrist et al., 1998). Naqi et al. (1983) demonstrated that birds carrying high levels of IBDV maternal antibodies showed no active immune response to IBDV vaccines, while birds with low maternal antibodies showed delayed response to vaccines. It is also reported that vaccination of birds carrying IBV maternal antibody at d 1 posthatching accelerate the rate of maternal antibodies decline (Mondal and Naqi, 2001). In addition, there is some evidence that vaccine adjuvants moderate the immune suppressive effects of MDA via enhancing immunogenicity (DeVries et al., 1990; Van Binnendijk et al., 1997). Therefore, considering the potential immune-adjuvant properties of YDC and SNB, in the second experiment we examined the interaction between maternal antibodies and diet on the development of postvaccine antibody response against IBV. The results demonstrated that diet containing YDC increased specific antibody response against IBV in wk 4 posthatching. At wk 4 of age, birds have already lost the maternal antibodies, and most of the antibodies in the blood circulation have been made in response to active immunization (vaccination). Therefore, increased specific antibody response against IBV in wk 4 may suggest immune adjuvant properties of YDC that can help birds to exert faster adaptive humoral immune response (unlike delayed response observed in the Control group) and elevated resistance against IBV. On the other hand, birds fed diet containing YDC and SNB had lower antibodies against IBV in wk 6 compared to the control group, suggesting the role of these products in regulating immune hemostasis that induces an immunological tolerance toward IBV, avoiding further activation of immune system (postvaccine immunity). This can be beneficial for birds because in the absence of a pathogen challenge, further activation of immune system could negatively affect performance (Klasing, 2007). In the present study, birds only got vaccinated at d 1 posthatch, when both the immaturity of immune system and high level of maternal antibodies in serum contribute to the postvaccine antibody-mediated immune response. Therefore, it would be interesting to evaluate the effects of YDC and SNB on postvaccine immunity following a replicated vaccine administration.

It has been reported that YDC and probiotics can modulate the immune response through PRRs expressed by antigen presenting cells and T cells (Shashidhara and Devegowda, 2003; Lebeer et al., 2010). On the other hand, during T-cell dependent B-cell activation, presentation of antigens such as SRBC and BSA by antigen-presenting cells (including B cells) to T helper cells happens through interaction between major histocompatibility complexes and T-cell receptors. This would lead to the mutual activation of B and T lymphocytes and production of antibodies (Parker, 1993; Obukhanych and Nussenzweig, 2006). Therefore, in the third experiment, we hypothesized that the supplementation of diets with YDC and SNB would lead to further activation of antibody-mediated immune response following immunization with T-cell dependent antigens, and the results demonstrated that the primary and the secondary serum IgG and IgM response against BSA were not affected by dietary treatments. In addition, no significant difference was observed for total antibody response against SRBC. In agreement with this result, a previous study in our group showed that supplementation of diet with YDC in broiler chickens did not influence antibody-mediated response following immunization with T-cell dependent antigens (Alizadeh et al., 2016). Furthermore, Haghighi et al. (2005) demonstrated that administration of probiotics to broiler diet did not result in a significant difference in IgG response against BSA. In contrast, the results of some studies showed that supplementation of diets with prebiotics and probiotics can enhance antibodymediated response against different antigens (Haghighi et al., 2005; Ghosh et al., 2012). The inconsistent results regarding the effects of probiotics and prebiotics on adaptive humoral immune response suggests that the immunomodulatory effects of these products cannot be generalized due to a number of factors such as genetic background of the host, the type of the probiotic and prebiotic used, immunization regimen as well as experimental conditions.

In conclusion, the results of this study demonstrated that administration of YDC and SNB in diets can extend serum IgG concentration of pullet chickens in wk 3 posthatch when birds have already lost the protection from maternal antibodies. However, the decay rate of MDA and specific adaptive humoral immune response were not affected by dietary treatments. In addition, combination of vaccination with YDC and SNB reduced postvaccine antibody-mediated immune response against IBV in wk 6 posthatch, suggesting the immunoregulatory properties of these products that prevent further activation of antibody-mediated immune response toward the same antigen to which their mother has been exposed.

This article was originally published in Poultry Science 2017, 96:58–64. http://dx.doi.org/10.3382/ps/pew244. This is an Open Access article under a Creative Commons License.

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