| Livestock Research for Rural Development 27 (11) 2015 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
This study was conducted to investigate the effects of probiotic supplementation on serum natural IgM levels binding keyhole limpet hemocyanin in indigenous chicken (IC). One hundred and fifty two months old chicken raised under low input-output system were sourced from small scale indigenous chicken farmers from Nyakach and Emineng sub counties of Kisumu and Baringo, Kenya respectively. The IC were of mixed sex, randomly divided into five treatment groups of 25 birds each. The treatments were 5 ml of Molaplus dissolved into 250, 500, 1000, 1500 and 2000 ml of drinking water. The birds were raised into group cages, and fed commercial grower mash for two months during the study period. A window of 14 days was left for immune stabilization, blood was then drawn from the web vein and serum separated immediately. Levels of IgM binding was assayed using an indirect ELISA technique.
IgM binding KLH was found but dietary probiotic supplementations did not significantly affect levels of IgM binding KLH in the serum. Probiotic supplementation in the diet did not further enhance KLH binding IgM in IC reared under village production system.
Keywords: immunoglobulin M, scavenging, supplementation
The indigenous chicken (IC) industry has become an important economic activity in Kenya and many countries. In large-scale rearing facilities, where chicken are exposed to stressful conditions, problems related to diseases and deterioration of environmental conditions often occur and result in serious economic losses. Prevention and control of these diseases have led in recent decades to a substantial increase in the chemical usage. The usage of these chemicals as a preventive measure has been questioned, there exist extensive documentation of the evolution of pathogens resistant to chemicals. The possibility of alternative to drug usage may be the use of probiotics and/ or breeding for disease resistance (Khobondo et al 2015b). Probiotics are being considered to fill this gap and already some farmers are using them in preference to antibiotics (Nava et al 2005).
Probiotic, meaning ‘for life’ in Greek, are defined as ‘a live microbial feed supplement, which beneficially affects the host animal by improving intestinal balance’ (Fuller 2001). The species currently being used in probiotic preparations are varied and many. These are mostly Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus salivarius, Lactobacillus plantarum, Streptococcus thermophilus, Enterococcus faecium, Enterococcus faecalis, Bifidobacterium spp. and Escherichia coli (Kabir 2009). Some other probiotics are microscopic fungi such as strains of yeasts belonging to Saccharomyces cerevisiae species (Fuller 2001). Probiotics may be composed of one or a combination of many strains.
Probiotics are used to help maintain a healthy microbial balance within the intestine to promote gut integrity and prevent enteric disease (Cox and Dalloul 2015). This is accomplished through three main mechanisms: competitive exclusion, bacterial antagonism, and stimulation of the immune system (Ohimain and Ofongo 2012). The manipulation of gut microbiota via the administration of probiotics influences the development of the immune response (McCracken and Gaskins 1999). It has been shown that probiotics stimulate different subsets of immune system cells to produce cytokines, which in turn modulate the immune response (Lammers et al 2003) and activate other cells. Amongst the subset of B cells, B-1 cells constitute the predominant subset of B cells in mammals (Islam et al 2004). While B-2 cells produce the majority of circulating specific antigen induced antibodies possessing high binding affinities. The antibodies secreted by B-1 cells called natural antibodies. They typically have low binding affinities and broad specificities (Parmentier et al 2004). Natural antibodies are usually produced without prior exposure to antigens (Khobondo et al 2015a). Foreign antigens like lipopolysaccharides (LPS), lipoteichoic acid, keyhole limpet hemocyanin, and bovine serum albumin (BSA) (Higgins et al 2007) that bind natural antibodies in the sera of unimmunized chickens have been found. In higher organisms including chicken, natural antibodies may be of isotype IgM, IgG, or IgA, but IgM is the predominant isotype. In mammals, B-1 cells are responsible for production of the natural IgM antibodies in serum (Khobondo et al 2015a) but in chicken B1 cells have not been defined yet. Natural IgM antibodies possess a wide range of activities, including regulation of immune response, induction of specific IgG antibodies, and protection against bacterial and viral infections (Nava et al 2005).
Natural antibodies in the chicken bind to antigens in a specific manner and the affinity of these interactions increases with age, suggesting a role for external stimuli. These roles can be exploited for breeding of disease resistance. Due to consumer concern on the use of antibiotics as growth promoters and prophylaxis in poultry diets, investigations evaluating the potential of dietary probiotics as substitutes for antibiotics do receive high priority and IC should not be exemptions. This is because, the IC are predominantly raised under extensive production system (Khobondo et al 2014). This prone the IC to various environmental challenges and disease causing pathogens hence the rationale of this study. Earlier, it was shown that that probiotics stimulate natural antibodies in poultry(Haghigi et al 2006).This experiment was carried out to determine possible effects of dietary probiotics on serum natural IgM levels in IC birds. The findings could be used to prevent disease burden or compliment breeding for disease resistance.
A feeding trial was conducted using one hundred and fifty (150) indigenous chicken sourced at two months from free range small scale farmers. The farmers were from Nyakach and Emening of Kisumu and Baringo countries, Kenya. The trial was done in a randomized complete block design with control. The birds were randomly allocated the 5 test diets with 25 replicates (25 birds/replicate) per treatment. 25 birds had no supplementation (control). The dietary treatments and water were offered ad libitum. The probiotic were added into drinking water by giving a specific concentration of 5 ml of Molaplus microbes (Molaplus LTD, Kenya) in different volumes (250, 500, 1000, 1500, 2000 ml) of the respective water once a day at 0900 hours. The Molaplus is a complex solution of various beneficial micro-organisms which are found naturally and are used in food manufacturing. When used in poultry production, they avail, chelated minerals, ant-oxidant, enzymes, vitamins, organic acids, lactic bacteria, yeast and phototropic bacteria (Molaplus.com). The experiment was done for a period of two months (60 days), thereafter IC were left for two weeks (14 days) for immunological stabilization, then blood sample was taken for natural antibody assay.
Blood samples (~2 ml in EDTA) from 150 IC was drawn from the wing vein of each bird and serum was separated immediately by centrifugation at 2000 rpm for 10 minutes for measuring IgM antibodies binding KLH. Isotype specific IgM antibody titers to keyhole limpet hemocyanin (KLH) in serum from the IC was determined by indirect enzyme-linked immunosorbent assay (ELISA). Briefly, 96 well plates were coated with 2 μg/ml KLH (MP Biomedicals Inc., Aurora, OH) and incubated overnight at 40C. Following washing with deionized water, the plates were incubated for 1.5 hours at 250C temperature with IC serum diluted 1:10 with dilution fluid (phosphate buffered saline (PBS) containing 0.5% horse serum and 0.05% Tween). Unbound serum was removed through washing. To detect IgM antibodies binding to KLH a 1:20,000 diluted affinity purified goat anti-chicken IgM (Fc specific), conjugated with horseradish peroxidase (GACh/IgM (Fc) /PO) antibody (Nordic Immunological Laboratories, Eindhoven, The Netherlands ) was added and incubated for 1.5 hours for 250C. After incubation with the conjugate and subsequent washing, 100 μl substrate-buffer (containing aquadest, 10% tetramethylbenzidine-buffer and 1.33% tetramethylbenzidine) per well was added and incubated for 10 minutes at room temperature. The reaction was stopped with 1.25M H2SO4. Absorbance levels per sample were measured with a spectrophotometer (mrc Scientific Instrument-UT- 6100, Israel) at a wavelength of 450 nm.
The data was analysed by using one way ANOVA using Proc GLM of SAS (SAS 2002). The following model was used;
Yij = µ+PRi+eij
Where y is the IgM absorbance level (titre value), µ is the overall mean, PRi is the fixed effect of treatment (i=2), e is the residual error.
Natural IgM antibodies binding KLH were detected in IC serum but, there was no significant difference between the IC fed probiotics and the birds not fed probiotics (control) (P ˃ 0.05). Neither was there significant difference in the titres of KLH binding IgM at 17 days after treatments (P ˃ 0.05) between the treatments (different inclusion levels of probiotic concentration). However, the control group (no probiotic fed) had higher LeastSquare Means than the birds fed probiotics of 1.753 and 1.730 respectively (Table 1).
| Table 1. The LSmeans and standard deviations (SD) of the dietary treatment of IgM titres binding KLH. The Control group showed higher IgM titres but not significant | |||
| Isotype | IgM | ||
| Parameter | LSMeans | Std error | p |
| Control | 1.75 | 0.121 | 0.873 |
| Probiotic | 1.73 | 0.065 | |
| LS means = least square means; S.D = standard deviation | |||
In accordance with previous findings, the present study revealed that serum antibodies (IgM binding KLH) are present in unimmunized IC. Since the IC likely did not encountered before, will not encounter KLH and cross reactivity with other antigen is unknown, this IgM antibodies bindin KLH can be regarded as natural antibodies. Natural IgM antibodies isotype are amongst the innate immunity (Vani et al 2008). Innate immunity as the first line of defense plays an important role in preventing or combating infection (Ehrenstein and Notley 2010). Natural antibodies have been detected in non-immunized cattle (van Knegsel et al 2007), humans (Ehrenstein and Notley 2010), rats, rabbits, fish, snakes and poultry (Sun et al 2011). In mammals, the Nabs are mostly produced by CD5+ B cells in the peritoneal cavity and intestines but also CD5- B cells (Casali and Notkins 1989) were described to produce Nab. Natural antibodies may arise independently of known antigenic stimulation. They are mostly poly-reactive, and poly-specific (Baccala et al 1989) with low binding affinity, and are generally encoded by the unmutated V genes in germ line configuration (Khobondo et al 2015 b). Evidence from various studies show that they are genetically controlled and inherited (Sun et al 2011). Most of Nab are of the IgM isotype class in lower vertebrates, fetus and neonates, but IgG and IgA Nab are also present as well in higher vertebrates (Marianne 2000). Production of Nabs may also be induced by the contact with non-pathogenic microbes, food, intestinal flora, probiotics and self-antigens (Quintana and Cohen 2004).
Most of Nab bind pathogen-associated molecular patterns (PAMP), eg lipopolysaccharide, lipoteichoic acid or peptidoglycan conserved along different genera and these serve as targets for identification of microbes by the innate immune system (Parmentier et al 2004). The levels of Nabs are likely dependent on several factors, amongst them the environment (Kachamakova et al 2006), genetic background (Ardia et al 2011) and age (Berghof et al 2010). Despite the plethora of data demonstrating the positive effects of probiotics on immune performance (Haghighi et al 2006), this study and some others have reported no significant enhancements due to probiotic supplementation (Rahimi et al 2011). It has to be kept in mind, however, that titres of IgM to KLH were measured 14 days after dietary treatment, so short term temporary effects could not be found. Also there was information on the IgG levels KLH. It is clear from the present study and other published research that responses to probiotic supplementation are inconsistent. Numerous investigations were done on possible factors that could influence the responses to these additives. For example, in broilers possible causes of variations in response to probiotic supplementation could be differences between strains, hybrids, age, sex, plane of nutrition, nutrient composition of the diet, microbial population of gastrointestinal tract, levels of inclusion of probiotics in the diet, duration of supplementation or other environmental conditions (Midili et al 2008). In this study involving IC, administration of probiotics did not significantly enhance serum IgM antibodies reactive to KLH. In this case, probiotic treatment resulted in the reduction of the mean reactive IgM antibodies in serum of the IC. These discrepancies could be due to a variety of factors including, but not limited to, strain(s) of bacteria utilized, composition and viability of the probiotic, preparation method, dosage, application method, frequency of application, overall diet, drug interactions, and condition of the animal (Huang et al 2004). The experimental design, source of birds and early stage production system in this study could be the cause of discrepancies in result. It is worth noting that the IC used in this study were naturally hatched and brood, the rearing was free range as well. This management system from incubation, hatching to the time they were sourced (2 months old) might have exposed them to these commensal microbes from the feces and environment. The chicken could have received a complete gut flora from the mother’s faeces and would infer immune response similar to probiotic microbes. The shell microbial contamination during incubation and hatching could play a role. The extensive system of IC production may have predorminantly exposed the IC to plethora of microbes, infested the gut with several microflora and consequently influenced Nab levels. These results are in agreement with the results of other studies in which probiotics (Huang et al 2004) or prebiotics (Franklin et al 2002) or combinations of probiotics and prebiotics (Midili et al 2008) were used in different animal species.
Several studies reported the role of probiotic in augmenting immune response (Cox and Dalloul 2015). There are evidence that probiotics stimulate production of natural antibodies ( Haghighi et al 2006) and different subsets of immune system cells to produce cytokines, which in turn play a role in the induction and regulation of the immune responses (Maasen et al 2000). It was found recentlyNab levels in elite improved breeds reflect different physiological health status (in this case enhanced survival ) as opposed to IC kept in confinement (in which Nab levels may signal a status of stress (Wondneneh et al 2015). Thus enhancement or decrease of Nab in birds may mimic sensitivity to stress or changing (dietary) conditions, indirectly reflecting the animals, condition to respond. In IC conditions due to husbandry may have been such that probiotics could not further enhance or decrease immune sensitivity.The induction of immune response and the preimmune antibody repertoire is a subject of debate. It is possible that resident dendritic cells (DCs) in the lamina propria, which directly sample the intestinal lumen and engulf commensal bacteria, could play a role (Yaman et al 2006). DCs express a repertoire of Toll-like receptors (TLRs) (Kabir et al 2005), and binding of structural components of commensal bacteria or probiotics to TLRs expressed on the surface of DCs may lead to activation and maturation of these cells (Apata 2008). Upon activation, DCs process and present antigens to other cells thereby promoting the activation and differentiation of different subsets of immune system cells, leading to the production of Th2 cytokines, such as interleukin 4 (IL-4), IL-10, and transforming growth factor, that are important for antibody production and isotype switching (Apata 2008). The former may have the implications on the Nabs level witnessed in this study. Most microbes used in probiotic may have shared PAMPs with the microbes already ingested by the chicken in the free range production system.
The authors are grateful to the Indigenous Chicken Improvement Programme (InCIP), Innovations For Livestock Industry (iLINOVA) Projects in Kenya financed by the European Commission and The National Commission for Science, Technology and Innovation (NACOSTI) for the financial support.
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Received 25 August 2015; Accepted 29 September 2015; Published 1 November 2015