Readers' Rating:

Rate this article

The ability of an animal to fight infection and disease is determined by the capacity of its immune systems to respond to invading pathogens. A number of factors have been shown to have a detrimental effect on the activity of the immune system. These include stress, environmental conditions, toxins, disease and nutrition (Husband and Gleeson, 1996). These factors, which can be minimised through good animal husbandry techniques, can result in animals becoming immunocompromised and therefore more prone to infectious diseases. It is therefore essential, in these cases, to maintain the activity of the immune system through the administration of immunostimulants.

The gastrointestinal tract is the major interface between the host and the environment and is therefore the main site for pathogenic challenge. For this reason it is essential that any enhancement of the immune response in animals manifest itself in the gastrointestinal tract in order to present a first line of defence against infection. The gastrointestinal tract is an important part of the immune system, which together with the respiratory and genito-urinary tracts, makes up a specialised compartment of the immune system termed the mucosal immune system.


Immunostimulatory capacity of yeast

It has been well documented that the dietary inclusion of yeast and its constituents increases the host animal’s ability to fight infection and disease (Killeen and Rosell, 1996) with mannanoligosaccharides (MOS) of the yeast cell wall being shown to be one of the main components involved. Mannanoligosaccharides have a number of different modes of action which include bacterial exclusion (Spring, 1996), neutralisation of mycotoxins (Devegowda et al., 1994) and immunostimulation (Newman, 1994).

Research in the last decade has concerned the capacity of MOS and other yeast preparations to stimulate the immune system when administered orally.

In vivo experiments have demonstrated that the material can activate both specific and non-specific immune responses. Savage et al. (1996) demonstrated an increase in mucosal IgA and systemic IgG in turkeys and Yoshida et al. (1995) showed an increased neutrophil activity in fish. These increases in activity have the potential to prime the host immune system against further pathogenic challenges (Verlhac et al., 1996).

Unfortunately, the study of the mechanics of mucosal immunity is a science which is still very much in its infancy and studies to date have been conducted on a limited number of species.

Accordingly, a number of studies were conducted to provide an insight into the mode of action of MOS and also to examine the effects in more varied animal species. The immunostimulatory capacity of a MOS-based product (Bio-Mos™, Alltech Inc.) was evaluated by performing a number of animal trials and investigating effects on the immune system.


Effects of MOS on immune response

Animal trials were conducted to investigate the effect of the oral administration of graded levels of MOS on the immune system of different species.

The purpose of these trials was fourfold. Firstly, they were designed to test whether or not MOS had an effect on the immune response. A second goal was to determine the optimum dose required to elicit a response.

Thirdly, species specificity of the response was determined; and finally an attempt was made to identify which immune response parameters were most affected by the preparation, allowing the development of a model system. Having undertaken these trials the information gained allowed the execution of comparative trials to investigate the ability of different yeast preparations to elicit an immune response.


TRIAL 1. DOSE RESPONSE TRIAL IN LABORATORY RATS

Materials and methods

Animals were blocked by weight and assigned to five groups, a control and four treatment groups (2, 4, 8 or 10 g MOS (Bio-Mos) per kg feed).

Experimental feeds were prepared by spraying the appropriate suspension of Bio-Mos (2, 4, 8 or 10 g in 100 ml distilled water) to 1 kg of feed. Control animals received feed sprayed with distilled water alone. Intestinal and blood samples were taken over a period of 32 days and a number of immune parameters were investigated. A lysozyme activity assay was performed to examine whether any alterations occurred in the activity of non-immune defence mechanisms.

Lysozyme is an integral part of the non-immune defence system, a system which consists of both physical and subcellular antimicrobial substances such as complement, lactoferrin and digestive enzymes. A change in lysozyme activity would indicate a potentiation of the host’s most basic defence mechanisms. The assay was based on the principle that lysozyme will, through its lytic action on bacterial cell walls, turn a turbid solution of Micrococcus lysodeikticus into a clear, soluble product. The procedure utilised was as described previously (Giles et al., 1990).

The level of activity of phagocytic cells in the blood was determined by executing an assay of oxidative radical production. The colorimetric assay recorded the amount of nitroblue tetrazolium (NBT) reduced to insoluble formazan by the oxygen radicals produced by the phagocytic cells. The methods were as previously described (Rumsey et al., 1994).

In order to investigate the ability of the non-specific immune system to clear the host of bacterial infection, an assay to determine killing ability was performed. The assay was based on the production of oxygen radicals by isolated neutrophils in the presence of indicator bacteria. The methods were as previously described (Rumsey et al., 1994).

Myeloperoxidase is expressed exclusively in neutrophils and as a result is used as an indicator of neutrophil activity in tissue samples. Intestinal tissue was monitored for enzyme activity to determine whether infiltration of tissue by neutrophils increased. The assay was performed as previously described (Schmid et al., 1996).

The level of the specific immune response was investigated by monitoring immunoglobulin concentrations in both blood (IgG) and intestinal (IgA) samples. A single radial immunodiffusion assay, using the method of Mancini et al. (1965), was used to determine the concentration of immunoglobulin.

The determination of intestinal antibody concentrations was performed on homogenates obtained for the determination of myeloperoxidase activity.

Samples were lyophilised and re-suspended to an appropriate volume in order to bring immunoglobulin concentrations to a readable level and were standardised against protein concentration.

Results were analysed by two-way ANOVA. If significance was demonstrated, results were then compared by Dunnett’s test of family error rate which produced confidence intervals for the difference between each treatment mean and the control mean. Results were also tested by one-way ANOVA where doses on each sampling time were grouped as treatment periods.


TRIAL 2. THE EFFECTS OF MOS ON THE IMMUNE RESPONSE OF DOGS FOLLOWING VACCINATION

Materials and methods

This trial was conducted to investigate if the oral administration of Bio-Mos enhanced the immune response of dogs post-vaccination. Dogs were randomly assigned to two groups, control and treatment. Dogs in the treated group received 2 g Bio-Mos per kg feed. All dogs were vaccinated against parvovirus, leptospirosis, adenovirus and distemper after a 1 week acclimatisation period on the experimental diet. Examinations were restricted to systemic immune responses and were monitored over a 9 week period.

In order to monitor the activity of the non-specific immune response, a glass adherent NBT assay was performed. The basis of the assay is that active neutrophils adhere to glass allowing them to be stained by NBT and enumerated. The assay method is essentially as described previously (Anderson et al., 1992) with slight modifications.

The specific immune response to vaccination was recorded by monitoring the level of circulatory IgG by the method earlier mentioned.

Results were tested by two-way repeated ANOVA followed by Fisher’s test of individual error rate, which provided pairwise comparisons between treatment means.


TRIAL 3. EFFECTS OF CELLWALL PREPARATIONS FROM DIFFERENT YEASTS ON IMMUNE RESPONSE OF RATS

Materials and methods

It has been well documented that yeast grown under different environmental conditions can have an altered cell wall structure (Polonelli et al., 1994). This trial examined whether growing two different strains of yeast, Saccharomyces cerevisiae and S. boulardii, under different environmental conditions would alter their ability to stimulate an immune response. Animals were divided into groups receiving the treatments as outlined in Table 1 and samples were taken after 21 days. The parameters measured were phagocytic activity and intestinal IgA levels by the methods mentioned earlier. Results were tested by one-way ANOVA followed by Dunnett’s test where applicable.


Table 1. Experimental treatments in Trial 3.*


*2 g preparation/kg feed.



RESULTS

Trial 1. Dose response in laboratory rats

A significant increase in IgA concentration on days 18 and 32 in response to Bio-Mos indicated stimulation of the mucosal immune response (Figure 1). This identifies intestinal IgA as an indicator parameter which could be used in comparative trials. Although a correlation between dose and response was not demonstrated, the increase in IgA level was shown to be significant when individual time versus dose treatments were grouped and compared to controls.

The lack of correlation between dose and response was most probably due to inter-animal variation. Nevertheless, the overall indication from these experiments was that dietary administration of Bio-Mos has the potential to confer protection against intestinal infection through the stimulation of mucosal IgA production. Although the specificity of the IgA was not investigated, evidence from the literature suggests that the induced IgA species are not solely directed against yeast cell wall antigens.

Previous studies using orally administered yeast in rats demonstrated the stimulation of a broad spectrum of intestinal IgAantibodies (Buts et al., 1990). An investigation of other immune parameters revealed no apparent stimulation of a systemic immune response (Figure 2). This is surprising as the preparation was shown to produce a significant increase in plasma IgG levels in turkeys (Savage et al., 1996). This discrepancy may simply be another indication of species specificity or may be related to different methodological procedure; nevertheless it does warrant further investigation.




Figure 1. The effect of graded levels of dietary Bio-Mos on intestinal IgA concentrations in rats. For all three sampling dates, pooled treatment means differed from the control (P<0.05).





Figure 2. The effect of dietary Bio-Mos on plasma IgG concentration.



Trial 2. Effects of Bio-Mos on the immune response of dogs following vaccination

As stated, the objective of this trial was to examine whether Bio-Mos had an adjuvant-like activity which would manifest itself in an increased immune response against vaccination. As expected, vaccination resulted in an increase in the number of circulating neutrophils (Figure 3). This response was more pronounced in animals receiving the experimental diet. Although no statistical significance could be attributed to this increase due to the low animal numbers, the result was encouraging as it indicated the potential for enhancement and also a second possibility for an indicator immune response.

In examining the specific response to vaccination, results showed a significant response after vaccination but no difference was apparent between control and Bio-Mos treated animals (Figure 4). It may have been more appropriate to measure IgM antibody levels or vaccine-specific antibodies; but unfortunately the appropriate antisera were not commercially available at the time of experimentation.


Trial 3. Effects of cell wall preparations from different yeasts on immune response of rats

The results of the first two trials established a model system by which different immunostimulants could be tested. Trials 1 and 2 determined that IgA and possibly neutrophil activity could be used as indicator parameters to compare the immunostimulatory capacity of the different yeast preparations. As a peak immune response was assumed to be around day 18 to 32 of treatment, sampling was restricted to this time period. A significant increase in phagocytic activity was seen in the animals treated with yeast preparations grown at an elevated temperature with numerical increases observed following administration of the other preparations (Figure 5).




Figure 3. The effect of dietary Bio-Mos on circulating neutrophil (NBT+ cells) numbers in dogs following vaccination.





Figure 4. The effect of the dietary Bio-Mos on the IgG responses of dogs following vaccination.





Figure 5. The effect of yeast cell wall preparations derived from yeast grown under different conditions on phagocytic activity in rats.





Figure 6. The effect of of yeast cell wall preparations derived from yeast grown under different conditions on intestinal IgA levels.



Increases seen in phagocytic activity were also reflected in intestinal IgA results (Figure 6). Although these increases were not significant, the most prominent increase was demonstrated in the animals treated with yeasts grown at a higher temperature.

It is reasonable to speculate that an increase in growth temperatures resulted in the production of heat shock proteins. These proteins are mannoproteins and can be deposited on the yeast cell wall (Polonelli et al., 1994).

Thus, the preparation may have a higher mannan content than the other preparations. Other possibilities include an actual change in mannan structure such as increased phosphorylation or altered side branching which could increase the immunogenicity of the oligosaccharide.

References

Anderson, D.P, T. Moritomo and R. deGrooth. 1992. Neutrophil, glassadherent, nitroblue tetrazolium assay gives early indication of immunization effectiveness in rainbow trout. Vet. Immunol. 30:419–429.

Buts, J.P., P. Bernasconi, J.P. Vaerman and C. Dive. 1990. Stimulation of secretory IgA and secretory component of immunoglobulins in small intestine of rats treated with Saccharomyces boulardii. Dig. Dis. Sci. 35:251–256.

Devegowda, G., B.I.R. Aravind, K. Rajendra, M.G. Morton, A. Buburanthna and C. Sudaharahan. 1994. A biological approach to counteract aflatoxicosis in broiler chickens and ducklings by the use of Saccharomyces cerevisiae cultures added to feed. In: Biotechnology in the Feed Industry. Proceedings of Alltech’s Tenth Annual Symposium (Eds. T.P. Lyons and K.A. Jacques). Nottingham University Press, Loughborough, Leics, UK. pp. 235–245.

Giles, S.J., M.R. Morley and F. Hazlehurst. 1990. Quantitative measurement of serum and plasma lysozyme: an automated method. Med. Lab. Sci. 47: 282–284.

Husband, A.J. and H. Gleeson. 1996. Ontogeny of mucosal immunity – environmental and behavioural influences. Brain Behav. Immun. 10:188–204.

Killeen, G. and V. Rosell. 1996. The potential of polysaccharide supplements in diets for livestock and pets. In: Biotechnology in the Feed Industry. Proceedings of Alltech’s Twelfth Annual Symposium (Eds. T.P. Lyons and K.A. Jacques). Nottingham University Press, Loughborough, Leics, UK. pp.149–158.

MacDonald, F. 1995. Use of immunostimulants in agricultural applications. In: Biotechnology in the Feed Industry. Proceedings of Alltech’s Eleventh Annual Symposium (Eds. T.P. Lyons and K.A. Jacques). Nottingham University Press, Loughborough, Leics, UK. pp. 97–103.

Mancini, G., A.O. Carbonera and J.F. Heremans. 1965. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochem. 2:235–254.

Newman, K. 1994. Mannan-oligosaccharides: Natural polymers with significant impact on the gastrointestinal microflora and the immune system. In: Biotechnology in the Feed Industry. Proceedings of Alltech’s Tenth Annual Symposium (Eds. T.P. Lyons and K.A. Jacques). Nottingham University Press, Loughborough, Leics, UK. pp. 167–174.

Polonelli, L., M. Gerloni, S. Conti, P. Fisicaro, C. Cantelli, P. Portincasa, F. Almondo, P.L. Barea, F.L. Hernando and J. Ponton. 1994. Heat-shock mannoproteins as targets of secretory IgA in Candida albicans. J. Infec. Dis. 169:1401–1405.

Rumsey, G.L., A.K. Siwicki, D.P. Anderson and P.R. Bower. 1994. Effect of soybean protein on serological response, non-specific defense mechanisms, growth, and protein utilization in rainbow trout. Vet. Immunol. 41:323–339.

Savage, T.F., P.F. Cotter and E.I. Zakrzewska. 1996. The effect of feeding a mannan oligosaccharide on immunoglobulins, plasma IgG and bile IgA of Wrolstad MW male turkeys. Poultry Sci. 75(Suppl. 1):43–145.

Schmid, R.A., M. Yamashita, K. Ando, Y. Tanaka, J.D. Cooper and G.A. Patterson. 1996. Lidocaine reduces reperfusion injury and neutrophil migration in canine lung allografts. Ann. Thorac. Surg. 61:949–955.

Spring, P. 1996. Effects of mannanoligosaccharide on different cecal parameters and on cecal concentrations of enteric pathogens in poultry. Ph.D. Dissertation Thesis, Swiss Federal Institute of Technology, Zurich, Switzerland.

Verlhac, V., J. Gabaudan, A. Obach, W. Schuep and R. Hole. 1996. Influence of dietary glucan and vitamin C on non-specific and specific immune responses of Rainbow trout (Oncorhynchus mykiss). Aquaculture 143:123–133.

Yoshida, T., R. Kruger andV. Inglis. 1995. Augmentation of non-specific protection in African catfish, Clarias gariepinus (Burch), by the long-term oral administration of immunostimulants. J. Fish. Dis. 18:195–198.

Readers' Rating:

Rate this article