There is a tendency to regard all micro organisms as harmful; to equate bacteria with germs. Nothing could be further from the truth. The number of non pathogenic species far exceeds the number of pathogenic species and many of the none pathogens are in fact useful, even essential for the continued existence of life on earth. One example of a beneficial group of micro organisms are those which inhabit the gastrointestinal tract of animals.
Composition of gut flora
There is, in the gut, a very complex population of micro organisms which interact with each other and with the host animal. Estimates put the number of different types of micro organisms in the gut at 400 and the total number of bacterial cells at 1014 ; a figure which far exceeds the total number of human beings in the world. Although the composition of the gut microflora is fairly constant and characteristic for each host species it can be affected by various factors such as:
age: the microflora of live young suckling mammal is different from that of the adult
diet: to some extent this will be responsible for the changes seen with age, but even between adults the composition of the diet can affect the composition of the gut microflora
environment: the conditions under which farm animals are reared differs from the natural conditions under which their wild counterparts developed. The physiological responses to the artificial nature of the domestic/farm environment may in turn affect the gut microflora
stress: the unnatural conditions of farm rearing product stresses which induce hormonal changes which can affect mucous secretion and flora composition of the gut.
medication: the use of antibiotics and other chemical antibacterial compounds either as growth promoters or as therapeutic agents can change the gut microflora in such a way as to allow the growth of pathogens.
Role of the gut flora
Why does the animal tolerate the presence, within its gut, of this vast number of micro organisms? It does so because it has evolved a symbiotic relationship in which the bacteria get food and a suitable environment for growth and the host animal acquires protection against some forms of disease. The evidence for this is as follows:
comparison of germ free and conventional animals with a complete gut microflora shows that the former are more susceptible to disease than are their normal counterparts.
oral administration of antibiotics and other antibacterial compounds increases susceptibility to disease. The difference is that the antibacterial compounds are suppressing the organisms which normally protect against disease, allowing the pathogens to grow.
animals with a deficient flora can have their resistance restored by administration of a faecal suspension from healthy adult animals of the same species. A good example of this effect is the faecal dosing of day old chickens hatched into a clean environment without the opportunity to acquire their protective flora from the mother hen. These chicks are more susceptible to colonisation with Salmonellae but after dosing with a faecal suspension from an adult chicken they become resistant.
Composition of probiotic preparations
While dosing with a faecal suspension is very effective, it risks introducing pathogens to the animal being dosed. To avoid this risk many groups of research workers throughout the world have attempted to produce a faecal suspension which is free from pathogens. Others have attempted to identify the particular organisms involved in the protective effect and restore the resistance by supplementing the diet with these known cultures. Preparations such as these are known as probiotics, a word first used in this context by Parker in 1974.
At this time Parker defined probiotics as: 'Organisms and substances which contribute to intestinal microbial balance'.
I later, in 1989, modified this to read: 'A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance'.
This revised definition stresses the importance of live cells as an essential component of the probiotic preparation. The most commonly used organisms in probiotic preparations are the lactic acid bacteria (lactobacilli, streptococci and bifidobacteria). These are found in large numbers in the gut of healthy animals and do not appear to affect them adversely. They are in the words of the America FDA, Generally Regarded as Safe (GRAS). Organisms other than lactic acid bacteria which are currently being used in probiotic preparations include Bacillus sp., yeasts (Saccharomyces cerevisiae Sac. boulardii) and filamentous fungi (Aspergillus oryzae).
These probiotic preparations may be presented in different ways depending on the animal receiving the supplement and the condition to be treated. Thus they may be in the form of powders, tablets, capsules, pastes or sprays.
Selection of Probiotic Organisms
The use of lactic acid bacteria as feed supplements goes back to pre-Christian times when fermented milks were consumed by humans. It was not until the beginning of this century that Metchnikoff, working at the Pasteur Institute in Paris, started to put the subject on to a scientific basis. He believed that the flora in the lower gut was having an adverse effect on the host and that these adverse effects could be ameliorated by consuming soured milk. In support of this he cited the observation that Bulgarian peasants consumed large quantities of soured milk and also lived to a great age; he was in no doubt about the causal relationship and subsequent events have, in part, confirmed his thesis.
He isolated what he called the 'Bulgarian bacillus' from soured milk and used this in subsequent trials. This organism was probably what became known as Lactobacillus bulgaricus and is now called L. delbrueckii subsp bulgaricus which is one of the organisms used to ferment milk and produce yoghurt. After Metchnikoff's death in 1916 the centre of activity moved to the USA. The American workers showed that the yoghurt organisms could not colonise the gut and reasoned that, if the effect was to be manifested in the gut then a gut micro organism was more likely to produce the required effect. Knowledge available at that time suggested the use of L. acidophilus and many trials were carried out using this organism. Encouraging results were obtained especially in the relief of chronic constipation. In the late 1940's interest in the gut microflora was stimulated by two research developments.
Firstly, the finding that antibiotics included in the feed of farm animals promoted their growth. A desire to discover the mechanism of this effect led to increased study of the composition of the gut microflora and the way in which it might be affecting the host animal.
Secondly, the more ready availability of germ-free animals provided a technique for testing the effect that the newly discovered intestinal inhabitants were having on the host.
This increased knowledge also showed that L. acidophilus was not the only lactobacillus in the intestine and a wide range of different organisms came to be studied and later used in probiotic preparations. Some of the more commonly used lactic acid bacteria are shown in Table 1
In the early days, the selection of strains for probiotic use was largely empirical.
However, recently with increased knowledge, attention has been paid to the features which are involved in colonisation of the gut. On the basis that the effect cannot be reproduced unless the organism is metabolising in the gut, it would seem to be rational to encourage its growth in the intestine. Factors which have been used in this respect are:
resistance to pH and bile acids which can be inhibitory in the gut.
ability to attach to the gut epithelial lining which aids in preventing the organisms being swept out of the gut by peristalsis.. The ideal probiotic would possess these characteristics together with the following features which the present knowledge of this subject suggest it would be desirable. It should be:
non-pathogenic and non-toxic
beneficial to the host aimal in some way
of high viability
stable on storage and in the field
able to survive in or colonise the gut
amenable to cultivation on an industrial scale
These are the characteristics which should be aimed at when developing new strains for probiotic use.
TABLE 1 Lactic acid bacteria used in probiotic products
LACTOBACILLI, STREPTOCOCCI, BIFIDOBACTERIA
L. acidophilusL. caseiL. delbrueckii subsp bulgaricus*L. brevisL. cellobiosusL. curvatusL. fermentumL. lactisL. plantarumL. reuteriS. cremorisE. faecium x
* previously L. bulgaricus § previously S. thermophilus x previously S. faecium
Probiotics for Farm Animals
Modern rearing methods which include unnatural rearing conditions and diets induce stress and can cause changes in the composition of the microflora which compromise the animals' resistance to infection. The aim of the probiotic approach is to repair the deficiencies in the microflora and restore the animals' resistance to disease (Fig. 1). Such a treatment does not introduce any foreign chemicals into the animal's internal environment and does not run the risk of contaminating the carcass and introducing hazardous chemicals into the food chain.
Probiotics are now replacing the chemical growth promoters for farm animals and claims have also been made for increasing resistance to disease. The benefits claimed for probiotics in farm animals are as follows:
increased growth rate
improved feed conversation
improved resistance to disease
improved milk yield and quality
improved egg production
The results obtained are sometimes variable but bearing in mind the different ways and conditions under which probiotics may be operating it is not surprising that they are sometimes not active. It should be remembered that probiotics are not a single entity; different probiotics contain different micro organisms which may behave differently. Even different strains of the same species may have different metabolic activities which affect the result when they are used as probiotics. Negative results may also be explained by the poor viability of the preparation. Although this is crucial to the outcome it is not always checked when trials are done. Other factors which may also explain variation in results include the growth phase of the animal, the type of dosing used and the hygienic condition of the housing. With all these possible variations it is not surprising that probiotics do not always give the desired result but the fact that significant results are obtained shows that using the right probiotic, under the right conditions and using the correct method of administration they do work and are an effective feed supplement for farm animals.
The use of probiotics as farm animal feed supplements Roy Fuller, BSc, MSc, PhD, CBiol, FIBiol.
The use of probiotics as farm animal feed supplements dates back to the 1970's. They were originally incorporated into feed to increase the animal's growth and to improve its health by increasing its resistance to disease. It was assumed that the effect of probiotics was linked to the gastrointestinal tract and effects on incidence of diarrhoea and other gut infections were expected. However, recent work in several different countries has indicated that the effects may be more general. The results obtained have shown that some of the bacteria used in probiotics (lactobacilli) are capable of stimulating the immune system. This exposes a whole new area of potential influence for probiotics in which it will be possible to influence disease situations in sites remote from the gut, as well as preventing intestinal disease. Suddenly the stories that farmers tell of probiotics protecting their calves against pneumonia are much more likely. The probiotic micro organisms in the gut can stimulate the immune system in two ways. They can either migrate through the gut wall as viable cells and multiply to a limited extent or antigens released by the dead organisms can be absorbed and stimulate the immune system directly. A third school of thought suggests that the lactobacilli are acting indirectly through an effect on the other components of the gut flora. It is the product of this change which induces the immune response. At present which of these methods is being used is not certain, but there appears to be some relationship between the ability of a strain to translocate and the ability to be immunogenic.
The improved immunity induced by the probiotic may be manifested in three ways:
Increased macrophage activity shown by the enhanced ability to phagocytose micro organisms or carbon particles.
Increased production of systemic antibody usually of the immunoglobulin classes 1gG and 1gM and interferon (a non specific antiviral agent)
Increased local antibody at mucosal surfaces such as the gut wall. These are usually 1gA.
There is now evidence that all of these types of response are occurring. The evidence gained mainly from experiments with laboratory rodents shows that lactobacilli (usually Lactobacilli acidophilus and L.Casei) when given by mouth increase macrophage activity with respect to a range of organisms different from the probiotic organism ie., it is a non specific response. For example L.casei can improve macrophage activity to Listeria monocytogenes. L.casei can also increase 1gA production in the gut. The increased antibody production was associated with protection against infection by Salmonella typhimurium and it is suggested that L. casei could be used as an oral adjuvant to prevent enteric infections. L.acidophilus is also effective in inducing production of intestinal antibody.
Using radio immunodiffusion assay it was shown that after feeding L.acidophilus for seven days there was a threefold increase in the total immunoglobulin in the gut. These effects are unlikely to be of use as therapy and will only operate effectively if given before the infective challenge. The effectiveness will also be dependent on the size of the dose. It is not clear what the optimal effective dose is. It is apparent that some organisms are more effective than others, and with the very limited amount of work done so far it would seem that L.casei is the best of those tried so far. Certainly in some respects it is better than L.acidophilus, L.bulgaricus and Streptococcus thermophilus. The latter two organisms are yoghurt starter bacteria and suffer from not being able to survive in the gut as well as intestinal strains like L.acidophilus and L.casei.
The advantage of the intestinal strains derives from the fact that live organisms appear to be necessary to get the maximum response. This would appear to be a direct function of the numbers suggested or the ability of live organisms to multiply and survive in the gut thus giving a longer period of continuous stimulation. The effect of lactobacilli on the immune system can also be measured by assessing the levels of certain enzymes in the macrophage. When L.bulgaricus and L.casei were compared in this way and given by the intraperitoneal and oral route, L.casei was equally effective by either route but L.bulgaricus showed less enzyme activity stimulation when given by the oral route than by the peritoneal route.
L.casei and L.acidophilus were tested singly and together by Perdigon for stimulation of phagocytic activity. It was found that the mixture was more effective than the strains given singly. This suggest that a mixture of bacteria was more effective and the individual effects of the component strains may be additive. In vitro experiments have shown that human peripheral blood lymphocytes stimulated by small quantities of yoghurt containing live lactic acid bacteria produced 3-4 times more interferon than the control cells. This effect was later confirmed in a clinical trial with human beings. In addition to the increase in interferon there was also an increase in the number of B lymphocytes, natural killer cells and concentration of 1gG. It is known that lactic acid bacteria adhere to the lymphocytes which produce interferon and this may be an essential part of the stimulation process.
The use of lactobacilli as immuno stimulators is in its early days but there is little doubt that they are effective in stimulating the immune response in several different ways. They may be particularly useful in animals like the pig where the neonate is immunologically immature and is totally dependent on its mother for antibodies. The administration of probiotics during the suckling period accentuates the maturation of the piglet's immune system and makes it better able to survive the trauma of early weaning. Similarly the early weaned calf benefits equally well from the same stimulation of the immune system.
More work has been done to make it possible to suggest ways in which this information, largely obtained from rodents, can be used most effectively in farm animals. A great deal more work needs to be done to elucidate the mechanisms of the effect and to determine what is the optimal dose and when is the best time to produce the cultures. That is for the immediate future. Looking beyond that we can see the use of genetic manipulation techniques to create new strains with better growth characteristics in the gut and improved probiotic effects. It may be possible to incorporate the antigens of pathogenic bacteria into the genome of lactobacilli or some other harmless gut commensal. In this way we could capitalise on the immune stimulating ability of the lactobacilli and use them to deliver the protective antigens and stimulate the lymphocytes to respond appropriately. Developments like this are still in the early stages and although it may be possible in the laboratory to produce such strains they must be able to express these newcharacters and remain stable under conditions of production and subsequently when growing in the intestine.
There are also safety guide-lines concerning the release of genetically manipulated micro organisms into the environment and these may also limit the speed at which developments of this sort proceed. But that should not blind us to the enormous potential benefits of this sort of approach, the on going work on the stimulation of the immune system by probiotics suggests that this approach must be correct.
PROBIOTICS FOR FARM ANIMALS Roy Fuller Intestinal Microecology Consultant Russet House, Ryeish Green, Reading, Berkshire
The use of probiotics for farm animals is still increasing. Time and cost considerations tend to limit the amount of experimental work which is undertaken. The work which has been done has yielded inconsistent results but the positive research findings which have been obtained, continue to confirm that under the right conditions probiotics can have beneficial effects on growth rate, feed conversion and resistance to disease. The recent results obtained in chickens, pigs and cattle with probiotics based on lactic acid bacteria and fungi are discussed and possible mechanisms considered.
The on-farm use of probiotics for cattle, pigs and chickens continues to expand but experimental work aimed at improving our knowledge of how they work, what are the optimal conditions for activity and development of more effective strains has not received the funding required to underpin the practical applications of probiotics. Consequently, few field trials have been done. Because of this lack of fundamental knowledge about the mechanism of the probiotic effect, those trials which have been done have yielded variable results.
A typical example is a trial done in Spain by Tortuero et al. (1995). They compared two probiotic preparations containing
Enteroccus faecium and Lactobacillus casei and
the two yoghurt starter organisms, Streptococcus salivarius subsp. thermophilus and L. delbrueckii subsp. bulgaricus.
In the first experiment the preparation containing E.faecium and L. casei increased the weight gain during the experimental period up to 21 days of age. The yoghurt-starter cultures also gave a positive response but only between 12 and 21 days of age. However, when the preparation containing E. faecium and L. casei was retested, it failed to improve the weight gain. The second test was done with pigs from split litters, whereas the first experiment was carried out with piglets distributed at random on to the two treatments. The significance of this is difficult at the moment to assess and no doubt there were other factors which varied between the two trials.
This sort of result is helpful in the sense that it confirms that under the right conditions a significant growth response can be obtained. But it also illustrates the kind of confusion that exists in this type of experimentation where attempts to repeat a positive result fail and, because of the difficulty of ensuring that all the relevant factors other than supplementation are constant, it is impossible to explain such a failure. There was a larger decrease in coliform count from log10 8.10 down to 6.85 in the group, showing the better improvement in growth rate. This group also showed an increase in the concentration of interleukin Z (from 3.74 ng/g down to 7.43 ng.g). Neither of these differences were statistically significant, however, they do agree with other studies showing affects on coliform count and immune status.
An extensive and well conducted poultry trial was published earlier this year (Nahashon et al. 1996). This measured a wide range of features including growth rate, feed conversion and egg production. The feed supplement used was a lactobacillus but no further information regarding species identification was given. The trial looked at effects occurring during the pullet phase (7-19 weeks) and during the egg-laying phase (20-59 weeks).
During the pullet phase, feed consumption and weight gain increased as a result of feeding the lactobacillus supplement, but for the layers there was no effect in weight gain. However, there was increased daily feed consumption and increased egg size. The quality of the eggs was not affected. In the past, several studies have shown positive effects on egg production but they were not statistically significant. To my knowledge, this is the first paper to describe an effect on eggs which is significant.
An interesting new approach to probiotic administration to chickens has been used recently. Embrex, in the States, have developed a device for inoculating eggs with vaccine. In collaboration with Probiotics International Ltd, UK, trials have been done with the multistrain probiotic, Protexin. Eggs at 18 days incubation were inoculated with Protexin into the airsac or amnion. This procedure had no effect on hatchability; in fact, the injected eggs had slightly increased hatchability and accelerated hatch date. The results so far available are very preliminary, but the mean figures from two trials indicate that there is an increase in body weight at 2 weeks of up to 8.7% depending on the dose and the rate of inoculation.
The work on probiotics for cattle has increased in recent years. In calves, studies have been conducted using saccharomyces cerevisiae, Aspergillus oryzae, various species of lactobacillus and Enterococcus faecium. In the last ten years, positive effects (not always statiscally significant) have been found for feed intake, weight gain, earlier weaning, decreased scouring, decreased faecal coliform count and reduced demand for antibiotic treatment.
It is interesting to note that benefits need not always be measurable in terms of increased growth rate or feed efficiency; as in the case of a study by Seymour et al. (1995) last year, the effect may be demonstrated by monitoring the days of fever experienced by the animal and the number of antibiotic treatments required to maintain it in good health.
In adult cattle the studies have used mainly the fungal probiotics. Using this type of preparation for beef cattle, recent trials have shown improvements in feed efficiency, dry matter intake. Analysis of all the published data on this subject indicated that the average increase in daily gain of cattle fed yeast culture was 7.3%. The corresponding figure for feed efficiency was 6.0% (Huber, 1996).
Numerous studies have been done with lactating cattle. Over several years the average increase in milk yield of cows treated with Aspergillus oryzae was 2.5%. Increases in milk yield have also been obtained by supplementation with yeast. Both types of supplementation have induced improved butterfat concentrations in milk.
Some information is now available on the mechanism of probiotic activity in cattle and some informed speculation can be made. In vitro and in vivo studies with fungal probiotics have shown an improvement in digestion of fibre. One of the curious features of the fungal probiotic effect is that it occurs without the probiotic organisms being able to multiply in the rumen. In vitro studies with rumen liquor suggest that the probiotic will survive for about six hours.
With the limited amount of knowledge available it has been suggested that fungal probiotics may produce their beneficial effects in thefollowing ways:
by stimulation of indigenous rumen fungi. There is some evidence that this might be occurring from in vitro studies which showed that Aspergillus oryzae can improve the growth of the rumen fungus Neosallimistic frontalis (Huber, 1990).
by increasing the number of cellulolytic bacteria in the rumen; There is also experimental evidence for this; (saccharomyces cerevisiae stimulated the growth of the rumen bacterium Fibrobacter saccinogenes (Dawson 1990).
by improved rumen metabolism resulting form decreased concentration of lactic acid which, in turn, is due to stimulation of lactic acid fermenting bacteria such as Selenomonas ruminantium and the consequent reduction in pH. However, this effect on pH is small and is considered by some workers to be unlikely to have any significant effect on rumen metabolism.
by removal of sugars, toxic ?metals?.... or molecular oxygen, all of which can inhibit the growth of cellulolytic bacteria.
A recent development which, although not strictly covered by the definition of probiotics but is closely related conceptually, is the use of specific substances to stimulate the growth of desirable bacteria already present in the gastrointestinal tract, particularly of the colon. The approach has been used for many years in the form of lactulose supplementation, but recently the emphasis has shifted to oligosaccharide substances based on various sugars and derived from various substrates. Although not totally specific they tend to stimulate the bifidobacteria much more than other groups of intestinal bacteria. Most of the published work relates to humans and significant changes have been obtained with respect to both composition and activity of the colon microflora (Gibson & Roberfroid, 1995). This type of product has been given the name Prebiotic.
A mannan oligosaccharide has been used in studies with turkey poults (Savage & Sakrzewska, 1995). Inclusion of the supplement at a rate of 0.11% of diet resulted in significant weight gains and improvement of feed conversion on poults up to 8 weeks of age. There were also significant increases in plasma IgG and bile IgA indicating an effect on the immune system (Savage & Zakrzewska, 1996). However, whether this is sufficient to provide protection against infection has yet to be determined. Interesting though these results are, the cost of the mannan oligosaccharide would probably make it, at present, an unlikely feed supplement.
The so-called prebiotics are an attractive development because they are non-viable. This removes from the manufacturers the problem of maintaining viability in a product which may have to tolerate large variations in moisture and temperature during its use on the farm. Several studies have shown how it is not always possible to rely on the product description which appears on the label. Not only may the viable count be low, the specification of bacterial content may also be misleading with the claimed component replaced by a completely different micro-organism. A non-viable preparation would remove these problems and would allow the use of genetic manipulation to improve yields without the attendant problem of release into the environment of genetically modified viable micro-organisms. However, it may not be possible, even when we know enough about the biotechnical basis of the probiotic effect, to reproduce the probiotic effect with a non-viable supplement. The unique quality of the probiotic is that it is designed to colonise and produce the probiotic agent at the required target site in the gut.
For example, it can grow and metabolise in the colon and produce a substance that would be readily digested if administered by mouth.
There is an enormous potential for the use of probiotics in farm animal feeds but, as with probiotics used in other contexts, we need to know more about the fundamental mechanisms of probiotic activity. The most effective way of achieving this end is by a thorough understanding of the gut microflora and its interaction with the host animal.
This is a very time consuming and expensive project and, in the present economic climate, is unlikely to be funded adequately. In the meantime, we must continue to gain information on the optimal conditions for the probiotic effect and attempt to produce more consistent results in field trials with farm animals.
Huber, J.T. The fungal and yeast culture story in lactating dairy cows, in Proc. South West Nutr. Manage. Conf., Tempe, AZ pp. 87-94 (1990)
Dawson, K.A. Designing the yeast culture of tomorrow: mode of action of yeast culture for ruminants and non-ruminants, in Biotechnology in the Feed Industry (ed. T.P.Lyons) Alltech Tecyhnical Publications, Nashville, Kentucky pp. 59-78 (1990)
Gibson, G.R, Dietary Modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 125, pp. 1401-1412 (1995) Huber, J.T. 1996 pers. comm.
Nahashon, S.N., Nakane, H.S. & Mirosh, L.W. Performance of single combe white leghorn fed a diet supplemented with a live microbial during the growth and egg laying phases. Anim. Fd. Sci. Technol. 57, pp. 25-38 (1996)
Savage, T.F. & Zakrzewska, E.I. Performance of male turkeys to 8 weeks of age when fed an oligosaccharide derived from yeast cells. Poultry Sci. 74 Suppl. 1) p.158 (1995)
Savage, T.F., Cotler, P.F. & Zakrzewska, E.I. The effect of feeding a mannan oligosaccharide on immunoglobulins, plasma IgG and bile IgA of Wrotstad M W male turkeys. Poultry Sci. 75 (Suppl. 1) p. 125 (1996)
Seymour, W.M. Norek, J.E. & Siciliano-Jones, J. Effects of colostrum substitute and of dietary brewers yeast on health and performance of dairy calves . J. Dairy Sci. 78, pp. 412-420. (1995)
Tortuero, F., Rioperez, J, Fernandez, E. & Rodriguez, M. L. Response of piglets to oral administration of lactic acid bacteria. J.Fd. Prot. 58 pp.1369-1374 (1995).
The contents of this entire page are made available for your informational purposes courtesy of Dr. Karl Frank, Webmaster of the site: www.albertaclassic.com/probiotics.php. Thank you for the permission to use the above articles by Mr. Roy Fuller. Don Perez