2021 Archives - Folium Science

FOLIUM Science’s Chief Scientific Officer Professor Martin Woodward shares his expertise on the importance of a healthy microbiome for animal health.

There is currently an increasing interest in the microbiology of the gut, why is that?

A little recognized fact is that the gut of any animal, bird or human contains more cells than the number of cells that make up the host animal itself. Furthermore, there are many different types of micro-organisms or bacteria that comprise what is described as the “gut microbiome”.

This array of micro-organisms play very important functions for the body, the most obvious of which is converting food into the nutrients needed for the growth and maintenance of the host animal. Ever since man began rearing animals for hunting, transport, companionship or food, it was recognised that providing the best available nutrition was good for the health and welfare of the animal. In the modern era much emphasis is placed on the diet of animals, whatever their role in our lives; thus resulting in the multi-billion dollar animal nutrition industry.

Surely diets are relatively simple?

There is a wonderful old adage that says ‘you are what you eat’ which has some semblance of truth about it. It is the host genetics that determine features and physical characteristics, but growth rates and health are totally dependent upon the right nutrients in the feed. It is essential to have the major building blocks (protein and amino acids) and energy sources (carbohydrates and fats) for growth and development but just as important are the trace elements and vitamins. Think about iron for the haemoglobin of red blood cells; without the presence of iron, oxygen uptake and its transport around the body is impossible.

The discovery that limes and lemons given to sailors on meagre rations of dried biscuits and grog prevented scurvy was one of the first examples of the impact of good (or bad) nutrition on health. The vitamin C provided, in this case by the citrus fruit is vital. The point is, it is essential to get a balanced diet that covers all bodily needs, and this is the role of the nutritionist.

OK, so the nutritionist has a very important role but what about the gut microbiome?

The number of different types of organism in the gut varies from one animal species to another and comprises anywhere from many hundreds to several thousand different species of bacteria, and this excludes the protists and viruses. The bacteria are the components of the gut that can aid nutrition and they can have many different roles For example, they can

provide enzymes to breakdown complex molecules to simpler ones that can be used for energy or building
breakdown unwanted substances such as toxins (detoxification)
produce short chain fatty acids especially butyrate that are used by the host gut cells as energy sources and thereby maintain the integrity of the gut, separating the gut contents form the body
produce many of the nutrients through their own metabolism that are essential for the host, the best example being the aromatic amino acids that animals cannot synthesize. This is not an exclusive list by any means but demonstrates the importance of healthy gut microbiome.

Ah yes, you mention a healthy microbiome but what happens when there are diseases especially those that can infect humans as well?

You raise a significant point. So far, we have talked about the bacterial component in terms of the positive effects they confer on the host. The phrase good/friendly/beneficial bacteria is often used to describe them.

However, not all bacteria are beneficial and many have evolved to colonize the gut to cause disease; we describe these as pathogens. Interestingly, a well-established healthy microbiome is very good at suppressing the effects of pathogenic bacteria. This was first identified and described in the late 1960s and early 1970s and called the ‘Nurmi Effect’ after the author of the paper. A healthy gut microbiome can competitively exclude some pathogens very effectively. However, stress or the use of antibiotics can disturb the composition of the gut microbiome and open the way for infection. The pathogens of real concern in animal farming and production are those that not only cause losses in productivity but also those that can enter the food chain and cause diseases in humans. The culprits are Salmonella, pathovars of Escherichia coli and Campylobacter amongst many others.

This sounds complex, how does Folium Science hope to prevent these productivity problems?

Of the complex issues raised here, perhaps the simplest to deal with is the infection of an animal by a pathogen. By definition, this is definitely not wanted in the gut of the host and many mechanisms can be employed to reduce or try to eliminate them. Farmers utilize barrier methods preventing access of potential sources of infection to the animals, rigorous cleansing and disinfection, vaccination and the use of probiotics amongst currently available options. None of these methods are fool-proof because, with the exception of vaccination, these are non-specific untargeted blanket measures. FOLIUM Science’s Guided Biotic technology precisely targets the specific pathogen of interest and only removes that single target leaving the microbiome otherwise unharmed and able to return into balance.

However, sustaining a healthy gut microbiome still remains one of the best barriers to infection and is the driver behind much of FOLIUM Science’s work, relating back to the principles of good nutrition and establishing a balanced microbiome. Our vision at FOLIUM Science is to develop systems that support the re-balancing of the microbiome after dysbiosis (unbalanced gut microbiome caused by disease). Here FOLIUM Science aims to develop novel metabolic interventions that, rather than knocking out a pathogen actually enhance the development of the beneficial bacteria.

There seems to be a lot of potential here and no use of antibiotics?

Yes the potential is huge and very exciting for FOLIUM Science. And yes, you have identified that our Guided Biotic technology can be used in animal production and farming to replace antibiotics. Not only that, the technology is already being developed to remove the genes that are responsible for encoding antibiotic resistance.

TRISTAN COGAN, HOLGER KNEUPER, HADDEN GRAHAM and MARTIN WOODWARD present a trial for a new CRISPR-based patented technology introduced into a vector Escherichia coli probiotic designed to selectively remove all Salmonella serovars from the bird gut.

Over the past few decades the meat, egg and milk sectors have faced the need to reduce the routine use of antibiotics in animal production, and the high incidence of food poisoning associated with animal product consumption. Approximately 130,000 tonnes of antibiotics were used in 2013 worldwide, with 75% of this in animals. Up to 90% of these antibiotics can be excreted into the environment via urine and feces, and approximately 400 resistance markers against 25 antibiotics can be found in chick caecal bacteria. Globally, around 700,000 human deaths per annum are attributed to antibiotic resistance and this is predicted by the FAO to increase to 10 million by 2050. With rising concern about the development of antibiotic resistance in human health, regulators, consumers and retailers have led the drive to reduce the sub-therapeutic use of antibiotics in animal feeds to zero. Endemic disease is re-emerging, adding costs to animal production systems and driving the need for alternative non-antibiotic interventions.

Food poisoning continues to be a problem across the world, with salmonellosis cases now increasing in many countries. Non-typhoidal
salmonellosis is reported to cause over one million infections, 19,000 hospitalizations and over 400 deaths annually in the US, with some Salmonella serovars in food showing antibiotic resistance. Although salmonellosis incidents are traditionally relatively low in Australia, recent egg-associated outbreaks have brought this back to the attention of the regulators and consumers. It is now possible to cause a targeted bacterium to self-destruct through the use of CRISPR, the biological sequences that make up the bacterial immune system. This technology is extremely precise, such that it can target a specific bacterium or a defined range of bacteria. This means that, unlike many antibiotics, it can be used to remove only the unwanted bacteria in the animal gut microbiome and leave beneficial gut flora unchanged, potentially enhancing the well-being of the animal. One way to induce bacteria in the animal gut to self-destruct is to introduce a suitable plasmid into the target organism(s) through conjugation via a probiotic included in the feed or drinking water. The
current trial looks at the ability of this technology, named Guided Biotics, to reduce Salmonella colonization in challenged broilers.

A non-pathogenic Escherichia coli strain was used as the vector in this trial, and was loaded with a plasmid including a CAS sequence and 3 target sequences specific to all Salmonella serovars. Ross 308 as-hatched birds (165) were obtained on day of hatch and housed under controlled biosecure conditions, with access to water and standard commercial
rations ad libitum.

Birds were dosed continually from day 1 with either:
1) No addition to water (45 birds)
2) Unmodified E. coli vector at 108
cfu/mL drinking water (45 birds)
3) Anti-Salmonella Guided Biotics
at 108 cfu/mL drinking water (45
birds)

In parallel, a group of 30 birds was dosed orally with 0.5 ml 105 CFU/ mL Salmonella Enteritidis strain FS26 on day 1. Birds were checked for Salmonella colonization at day 3 by
cloacal swab (ISO 6579-1:2017).
On day 5, three verified Salmonella[1]colonized birds (seeder birds, with 105 CFU/g in swabs) were marked and added to each of the test groups. Fifteen non-seeder birds from each group were euthanazed on day 12 (7 days post-mixing with seeder birds) and caecal contents were counted for Salmonella using both direct and enhanced methods. Caecal samples were serially diluted in PBS before plating onto XLD agar for direct counts, whilst for enhanced counts the samples were first incubated in Selenite Cystine broth for 18 hrs at 41o C before plating and counting (ISO 6579-1:2017). For the purpose of data transformation, samples
negative in either method were allocated a count of 1 CFU/g, while those negative in direct counts but positive in the enhanced method were allocated 500 CFU/g. Body weights of the remaining birds were monitored at day 42. Counts and weights were log transformed and statistical analysis conducted using GraphPad Prism. Data were assessed for normality of distribution using a D’Agostino and Pearson omnibus normality test and non-normal were analysed using a Kruskall-Wallis test with Dunn’s multiple comparison test post hoc. Differences were analyzed using Fisher’s exact test.
Results
All birds in the seeder group showed cloacal Salmonella counts of 105 CFU/g by day 3. By day 12 (7 days post introduction of seeder birds to test groups) all birds in the Control and E. coli vector-only groups were positive using the enhanced counts method, exhibiting caecal counts of 500-4,000,000 CFU/g.
Twenty two of these 30 birds were also positive with direct count. However, when the anti-Salmonella Guided Biotics was added to the drinking water, Salmonella was not detected in any birds with the direct method, and only 8 of the 15 birds tested were positive with enhanced counts. The Guided Biotics treatment reduced (p < 0.001) mean Salmonella counts by approximately log-3 (from log 4.12 to log 1.26, equivalent to 14,200 CFU/g to 18 CFU/g) and also improved 42-day liveweight by 15% (p = 0.02; Figure 1). Discussion The challenge method employed in this study is consistent with that often use in Salmonella vaccine tests and may be regarded as severe. All seeder birds were infected when introduced into the test pens, and the Salmonella shed to in-contact birds would be expected to be highly infective. This was confirmed by the universally high caecal counts in all Control birds 7 days after seeded-bird introduction. Conversely, the Guided Biotics, delivered by conjugation in the digestive tract, was able to stop Salmonella colonization in 8 out of 15 (53%) of the test birds. The average Salmonella count in caecal digesta was also reduced by approximately log-3 (thousand[1]fold) and the maximum Salmonella count lowered from 4 million CFU/g in Control birds to 500 CFU/g in Guided Biotics treated. The 15% increase in liveweight of birds fed the Guided Biotics relative to the Control birds further indicates the severity of the Salmonella challenge employed in this trial. The lack of any effect of the E. coli vector on colonization confirms that the Guided Biotics plasmid was essential for Salmonella reduction. This initial trial establishes the capability of Guided Biotics technology to specifically remove unwanted bacteria, in this case a single Salmonella serovar. The tested Guided Biotics is designed to target all known 2,400 Salmonella serovars, and laboratory trials have established efficacy across the main serovars involved in human food poisoning. Ongoing laboratory tests have also indicated that solutions for other unwanted bacteria, such as Clostridium perfringens and Avian Pathogenic E. coli, are feasible.
Furthermore, because the design of the targeting is specific, tests have confirmed that off-target killing of desirable or commensal bacteria can be avoided.
Conclusion
It is clear that this Guided Biotics technology has the potential to make a substantial contribution to the replacement of antibiotics in poultry production, reduce zoonosis incidents
and maintain bird performance in antibiotic-free diets.