The microflora

All animals are born with a relatively sterile gut. Thus the newly born animal will be more easily colonised by pathogenic micro-organisms as there is no protective microflora to reduce the colonisation of pathogens at this stage of life.

Soon after birth the newly born animal acquires a complex collection of micro-organisms which populate its intestinal tract. This collection of micro-organisms is termed as the microflora. The gut microflora contains a variety of different bacteria and fungi of which there are typically ~400 different types of micro-organisms with a total population of ~ 10¹⁴ throughout the length of the intestinal tract.

Location in the digestive system

This complex collection of gut micro-organisms is distributed throughout the whole length of the gut. Within particular regions the organisms may be found in three niches:

(a) associated with gut wall. This can either take the form of direct attachment to the epithelium as in the case with lactobacilli in the crop, or entrapment in the mucous layer of the epithelium as happens in the caecum.

(b) attachment to food particles

(c) suspension in the liquid phase of the gut contents.

Composition

The composition of the flora varies in different regions of the intestine and is dependent on factors such as pH. The crop and small intestine tend to be dominated by lactobacilli with smaller numbers of other facultative anaerobes such as coliforms and streptococci. The posterior regions of the gut have large numbers of obligate (able to exist under only one set of environmental conditions) anaerobic bacteria. The caecum in particular, is favourable for the growth of anaerobes such as clostridia and bacteroides.

Role of gut flora in digestion

The microflora forms a symbiotic relationship with the host and benefits the host by aiding digestion by:

Producing various enzymes which are involved in the digestion/breakdown of large feed particles/polysaccharides such as cellulose.

The bacteria in the gut can also stimulate an immune response. For example, germfree animals have lower levels of gamma-globulin than do conventional animals with a complete gut flora.

The use of antibiotics can adversely affect the gut flora. When antibiotics are used, as a treatment for clinical disease, a proportion of the beneficial micro-organisms becomes disrupted and can lead to an increased susceptibility of the gut to colonisation of pathogenic bacteria. The consequent reduction in disease resistance is manifested by an increased vulnerability to salmonella and other pathogens colonisation of the gut which may lead to diarrhoea.

One factor that should be considered when dealing with animals is stress. There is also strong evidence that stress can affect the composition of the gut microflora. Stress can be described as a factor that stimulates homeostatic, physiological and behavioural responses in excess of the norm. The only accepted measure of the presence or absence of a stressor is the blood level of adrenal corticosteroids which becomes raised during stress which effects the peristaltic movement of the gut and the production of mucus within the gut. Stress also affects the intestinal microflora by reducing the concentration of lactobacilli and other Lactic Acid Bacteria (LAB) and increasing the concentrations of coliforms such as E. coli.

All stressful situations to which an animal is exposed contribute towards an increased intestine pH (more alkaline), and thus the gastrointestinal tract is likely to favour the development of pathogenic species such as E. coli at the expense of beneficial species.

Vaccination, antibiotic therapy, weaning, travel, rehoming or illness are a few of the factors that are considered as stresses that may result in a change of balance of gut flora in favour of pathogenic species.

Stress is also known to alter the protease content of saliva. As a result fibronectin and the autochthonous (commensal) bacterial population are lost from the oropharyngeal surface in stressed individuals. This autochthonous population is then rapidly replaced by a biofilm composed largely of Pseudomonas aeruginosa.

Stress can depress the immune response of animals and some antibiotics have been shown to depress the immune response significantly leading to a reduction in weights of the spleen and thymus. Withdrawal of the antibiotics in these cases can lead to the restoration of gut flora and a return to immune function. Antibiotics can also often reduce the lactobacilli population. Hence animals enduring stress exhibit alterations and breakdown of the regulatory mechanism in the gastrointestinal tract ecosystem allowing easier establishment of pathogens within the tract.

The word probiosis originated from Greek: pro (for) and biosis (life), and is therefore opposite in meaning to antibiosis, promoting the proliferation of bacterial species within the gastrointestinal tract. Probiosis is defined as 'the property of the normal adult flora to resist the overgrowth of component strains and the establishment of foreign strains' and is reinforced or re-established by probiotics. The concept of probiotics applied to preventative medicine is claimed to have originated from Metchnikoff. He postulated that the longevity observed in the Balkan people was due to the regular consumption of soured milk containing Lactobacillus bulgaricus.

The gut of the newly born animal is relatively sterile and is therefore deficient in the micro-organisms which normally populate the gut and provide resistance to disease. The intervention of a probiotic supplement establishes the gut microflora.

Probiotics have been shown to work by the following mechanisms:

• Competition for nutrients

Within the gut, beneficial as well as pathogenic micro-organisms will be utilising the same types of nutrients. Thus there will be a general competition for these nutrients to grow and reproduce. Hence, the more the gut is flooded with beneficial micro-organisms, the more competition is created between beneficial and pathogenic micro-organisms.

• Competition for adhesion sites

Adhering to adhesion sites along the wall of the gut is an important colonisation factor and many intestinal pathogens rely on adhesion to the gut wall to prevent them being swept away by peristaltic of food along the intestinal tract.

• Stimulation of immunity

Stimulation of antibody production (local and systemic). Increased macrophage activity. Increases gamma interferon levels.

• Direct antimicrobial effect

This can either operate via bacteriocins which are known to be produced by many species of lactic acid bacteria or by the production of organic acids which can either have a direct effect or operate by reducing the pH.

• Improvement in digestion

Probiotic micro-organisms act like and add to the healthy microflora by producing enzymes which aid the breakdown of polysaccharides molecules and hence utilise more nutrients form the diet. The microflora also produces vitamins which supply a secondary source to the host.

The role of the gut microflora can be exhibited by the example of germ free (GF) animals being more readily colonised and infected by gastrointestinal tract pathogens than conventional animals. For example, as few as 10 viable Salmonella enteritidis cells inoculated into the gastrointestinal tract of GF mice lead to the death of the animals within 5 to 8 days. The corresponding dose required to kill conventional mice was shown to be 10⁹ CFU.

The newly born animal shows greater susceptibility than the adult to oral infection by food-poisoning pathogens such as campylobacters and salmonellas. This can be attributed to the virtual absence of intestinal microflora.

Within a few hours of birth, the gastrointestinal tract of animals is rapidly colonised by facultative anaerobes, particularly coliforms and streptococci which may be present in the immediate environment. The value of probiotics is therefore to rapidly colonise the immature gut of newly born animals with beneficial micro-organisms. The fundamental rationale for the use of probiotics is to displace potentially pathogenic microbes or to supplement the beneficial CE action of existing non-pathogenic microbes in the intestines of an individual. This could be expressed more simply as an aim to establish and maintain a healthy intestinal environment.

To produce the desirable effect a minimum concentration of micro-organisms must be able to survive ingestion and grow in the intestine. However, the minimum effective dose of live bacteria cannot be easily identified. It has been suggested that once the concentration of a particular micro-organism fell to 10⁷ per g of faeces, it does not play a role in the ecosystem provided that it remains below this level at all times. This is supported by observations that the host animal can tolerate populations less than 10⁷ clostridia or enterobacteria per gram of intestinal contents. It is therefore postulated that a probiotic will be effective if it provides at least 10⁷ CFU and hence these levels have been adopted as a minimum dose.

• Safety - the micro-organism chosen as the components of a probiotic must be non-pathogenic and non-toxic
• Viability - a probiotic can only work if the micro-organisms contained within the probiotic remain viable during storage of the product and through the gut to ensure colonisation of these micro-organisms
• Minimum dose - the concentration of a probiotic must be such that inclusion rates provide 10⁷ - 10⁸ CFU per animal
• Quality insurance - it is essential that a probiotic has not become contaminated with any other micro-organism others than the particular probiotic micro-organisms chosen at any stage e.g. fermentation, of the manufacturing process or during storage.

Formula ProBiotic is a Functional Super Premium food for dogs that contains a live and highly concentrated probiotic bacteria which occur naturally in the gut of all healthy animals. These micro-organisms colonise the immature gut or re-establish the disrupted gut, thus promoting the mechanism of competitive exclusion against potential pathogenic bacteria.

The probiotic micro-organism, Enterococcus faecium, contained within Formula ProBiotic is sourced from the internationally recognised culture collection NCIMB (National Collections of Industrial and Marine Bacteria). The strain is grown in a fermentation chamber of 5000L capacity. It is then protected using cryoprotectants and then freeze-dried to form a powder before being blended with the other components in exact concentrations.

Enterococcus faecium is selected for its ability to perform specific functions within the digestive tract of the host. The justification for its inclusion in Formula ProBiotic is as listed below:

Enterococcus faecium

• Protection against enterotoxigenic E. coli diarrhoea (Jin et al., 2000; Underdahl et al. 1982; Wadstrom, 1984)
• Inhibition of Salmonella spp. including S. enteritidis, S. typhimurium, S. pullorum. (Audisio et al., 1999; Carina et al., 2000; Maia et al., 2001; Roach and Tannock, 1980) and Listeria (Audisio et al., 1999 and 2001).
• Prevents or shorten the duration of antibiotic-associated diarrhoea (Bergogre-Berezin, 2000; Elmer, 2001; Marteau et al., 2001).
• Production of antimicrobial substances (bacteriocins and lactic acid) active against gut pathogens (Audisio et al., 1999 and 2001; Carina et al., 2000).
• Immunostimulatory effect in humans (Agerholm-Larsen, 2000; Ferencik et al., 1999 and 2000).
• Growth in presence of bile salts and survival at gastric conditions (acidic pH) (Canganella et al., 1997; Nikoskelainen et al., 2000; Zacconi et al., 1992).
• Colonises the intestine (Nikoskelainen et al., 2000; Zacconi et al., 1992).
• Prevention of acute infantile diarrhoea and other diarrhoeal illnesses (Elmer et al., 1996)
• Proven efficacy in treatment of irritable bowel syndrome in humans (Gardiner et al., 1999).
• Increases cellulytic activity in caecum of chicken (Kumprecht et al., 1984)
• Reduction of cholesterol in humans (Agerholm-Larsen, 2000; de Roos and Katan, 2000) and mice (Zacconi et al., 1992).

Some of the ways in which Probiotics can benefit the host animal are:

• Reduction of the effects of stress
• Reduction of diarrhoea and other digestive upset
• Improved immunity and resistance to disease
• Reduction of Salmonella levels
• Improvement in digestion

Formula ProBiotic can be used at anytime in the animal's life but specific points of great importance are:

• immediately after birth to establish a correct microflora
• after changes of feed or housing
• pre and post anaesthesia
• during and after antibiotic or steroid therapy
• after vaccination
• in preparation for, during and after periods of stress

As the aim with probiotics is to restore and maintain normal gut function, they should be used whenever gut balance is upset. Situations likely to upset gut balance:

• Gut infections
• Vomiting and diarrhoea
• Antibiotic therapy
• Dietary changes
• Travel
• Stress
• Athletic competition
• Following surgery and anaesthesia
• Poor appetite

Strategic use of Formula ProBiotic during times of stress (vaccination, rehoming) nutritional changes (weaning), or after antibiotic use is very beneficial and an excellent way of introducing the concept of probiotic use.

There are no disadvantages to using Formula ProBiotic at any time!

There are no restrictions on the use of Formula ProBiotic. This product can be fed at any stage of production.

Formula ProBiotic contains micro-organisms commonly found in all healthy dogs all of which are GRAS (Generally Regarded As Safe) rated.

Formula ProBiotic has never been found to be toxic. Even more than 100 times the recommended level does not cause any problems. None of the ingredients used in the manufacture of Formula ProBiotic will cause irritations or allergic reactions.

Since the bacteria from Formula ProBiotic are inactive and inert in excreta there is no persistence of the additive or its residues in faeces. There are no known effects of Formula ProBiotic on methanogenesis. Formula ProBiotic is an inert material it will not persist in the environment. There are no known adverse effects of Formula ProBiotic on aquatic life, on soil fauna or terrestrial plants.

However, there are a great number of advantages:

• Completely safe and free from dangers of overdose
• Enhances the animal's own natural defence mechanisms and makes it better able to cope with opportunistic infection
• Highly effective under a wide variety of conditions

Whenever antibiotics are used, especially orally, the normal population of gut flora is disturbed and when antibiotic therapy is prolonged the effect on the gut can be profound. Probiotics will of course help to restore the normal population of gut microflora and this is one of the principle indications for their use.

Whilst the restoration of the gut microflora will be of most benefit when antibiotic therapy has finished, there is logic in giving probiotics at the same time as antibiotics. This is because the gut is a dynamic place with waves of microfloral activity occurring depending on the food present and the antibiotic concentration at any specific time. Therefore repeated boosts to normal microbial activity by using probiotics will be to the animals benefit.

The digestive tract of any animal will be colonised with many millions of micro-organisms. When the animal is healthy and disease free, the majority of these micro-organisms will be beneficial micro-organisms. However, even when the animal is healthy, there will still be potentially disease causing pathogen colonised within the 'healthy' gut. These pathogens are so low in concentration that disease will not develop until the animals' general health and immune status is affected.

Formula ProBiotic and other Probiotics have been shown to stimulate and maintain a high immune status of the animal and will hence help to prevent disease within an animal. Furthermore, the beneficial probiotic micro-organisms contained within Formula ProBiotic will act to Competitively Exclude potentially pathogenic micro-organisms within the gut. Formula ProBiotic, which contains Lactic Acid Bacteria, when colonised within the gut, will produce lactic acid which has a low pH which effectively produces the optimum conditions required for the growth of beneficial micro-organisms. This action helps to prevent the colonisation of coliforms such as E. coli which prefer a more alkaline pH.

We would recommend that Formula ProBiotic is stored in dry, cool and clean conditions, out of direct sunlight and kept sealed once the original container has been opened in order to exclude moisture and thermal decomposition.

Formula ProBiotic is stable at room temperature for 15 months. This has been determined by long term studies. The product is regularly sampled at point of manufacture for long term stability testing to be carried out.

Live yoghurt can be used to provide some live micro-organisms to the animal's digestive tract. However it has some distinct disadvantages when compared to Formula ProBiotic.

• The concentration of live micro-organisms present in the yoghurt is unknown but will tend to be relatively low and will depend on the freshness of the yoghurt and the conditions in which it has been stored. After opening, the microbial concentration can become depleted. Compare this with the high and guaranteed concentration of probiotic in Formula ProBiotic.
• The actual strains of micro-organisms contained within live yoghurts will tend to be chosen for taste and texture properties rather than a probiotic benefit. Hence the micro-organisms contained within the yoghurt may not produce any probiotic benefit.

The very acidic, low pH of the stomach in most animals is nature's way of attempting to remove some of the load of infection present in food. It is however not a completely effective process as is made apparent by the fact that the oral / gut route is the commonest way for infectious agents to enter the body.

The micro-organism contained within Formula ProBiotic is a Lactic Acid Bacteria (LAB). By definition these bacteria produce lactic acid which is acidic. This ensures the growth of the bacteria in an optimum acidic environment. By creating this acidic environment LABs are able to prevent the growth of coliforms such as E. coli as they prefer a more alkaline pH for growth.

Formula ProBiotic is also able to overcome the problem of stomach sterilisation by containing billions of micro-organisms so that some will always negotiate the pylorus and be available to colonise the gut. Also, the freeze-drying process, which is used to preserve the micro-organisms present in Formula ProBiotic, conveys an encapsulation which protects against stomach acid.

The strains of micro-organisms contained within Formula ProBiotic are fermented under strictly controlled conditions. Each strain has specific requirements for growth and parameters such as media used, pH, oxygen, temperature etc. These requirements are controlled by computer and by sampling. Additionally, testing at all stages of production and manufacture are carried out for contaminants.

Particular attention has been paid to the selection of the strains of micro-organisms contained in Formula ProBiotic in relation to ability to resist the action of bile, enzymes and acidity, the action of which can significantly affect viability.

Protection of the micro-organisms from adverse environmental conditions, both during and after production is important. Cryoprotectants are applied prior to freeze-drying. This gives additional protection against moisture, oxygen and heat and adds substantially to the shelf-life of the end product.