Intro to GI microbiology, host and microbiome

what must we consider when investigating bacteriology of GI disease?

1. which samples

2. how to get said samples

3. homogenisation

4. dilution series

5. plating

2 disadvantages of visual examination?

• no viability count

• no information on what bacteria they are

what are 2 quantative methods to enumerate bacteria

• culture (depends on how it’s processed)
  molecular detection - using nucleic acid

3 methods of assessing presence of bacteria?

1. smears/slides

2. culture

3. sequencing

what don’t slides/smears work for when assessing presence of bacteria?

• rods and cocci

when using petri dishes for viable counts (culturing), what must we ensure about the sample and why?

• must ensure it’s mixed well to separate bacteria out → otherwise separate bacteria that are stuck together can form one colony rather than separate ones → underestimate

what are 4 general things to consider about sample collection?

1. get sample to lab quickly as possible

2. keep sample cool NOT frozen

3. avoid getting too much air in samples

4. get samples out of cadaver (especially intestinal ones!) as quickly as possible!!!!!!!!!!

how is the gut microbiota established? (5)

• foetus is microbiologically sterile until birth/hatching

• neonate acquires organisms from birth canal and immediate environment

• they compete for niche

• complexity increases

• stabilises

state 6 causes of impacts on gut microbiota

1. age of animal

2. stress level

3. use of drugs

4. genetics

5. diet

6. illness

how does stress affect GIT?

1. hormones and nervous changes in the gut

2. hormones trigger increased growth of bacteria

3. increased risk of disease

how can age affect infection risk?

• maternal immunity/bottle feeding/pre-ruminant

• acquired immunity

• weaning

• reduced immunity with increasing age

how can genetics affect gut microbiota composition?

• certain species we’ve genetically bred are more/less likely to be colonised by certain species therefore affecting what may cause infection

what genes are essential for colonisation/disease?

1. motility for target niches

2. surface adhesins

3. LPS

4. specific transporters for GIT specific nutrients

5. gene regulation for stress responses

6. invasion to sustain colonisation

how does the microbiota change in the GIT?

• microbioal populations change and reflect biology of the site

• at different sites we may find them at different locations: free living/attached to villus/deep in crypts

4 roles of the microbiota?

1. immune development

2. resistance to infection

3. role in nutrition

4. xenobiotic metabolism

how does the microbiota prime the immune system?

• microbiota exclude potentially harmful bacteria

• those intact with immune receptors (innate ones) train cells to promote immune tolerance

• mucous and other barrier protections e.g. AMPs get stimulated

what is competitive exclusion?

• existing flora present as competitive barrier to incoming bacteria, so can exclude and prevent colonisation/infection

why may we use competitive exclusion products?

• cane exclude and prevent colonisation/infection

what is the underlying principle of competitive exclusion?

• there’s competition for space, co factor elements, electron acceptors, carbon sources, nutrients

• some species are better adapted to this, will out compete others

• bacteria that don’t compete for the same thing won’t clash

what can some some species such as salmonella and campylobacter do to overcome competitive exclusion

• some produce inhibitors for other bacteria/things other species can grow on

• have many nutritional sources it can metabolise, therefore not affected as much by competition

why are antimicrobials not great?

• they can remove sensitive organisms from a particular niche → resistant bacteria repopulate

  • dysbiosis

why is penicillin so dangerous orally in rabbits?

• overgrowth of Clostridium spiroforme occurs → due to normal bacteria being kiilled

• may get diarrhoea

what are nutritional benefits of flora?

• fore and hindgut fermentation

• vitamin production - B12 in pigs!

why were antimicrobials used in growth promotion?

• reduces effect of flora on growth inhibition

  • it does increase growth rate and productivity

why is the use of antimicrobials in growth promotion banned in the EU and UK?

• antimicrobial resistance in bacterial flora

• we saw a reservoir of transferrable antibiotic resistance

How do bacteria act as xeonbiotics?

• gut flora can alter compounds

  • different compounds have different effects, could alter gut flora

how can the microbiota affect drugs/metabolites?

1. increase activity

2. inactivate

3. produce toxic metabolites and carcinogens

when the microbiota changes, what will this alter

• the metabolites that are produced - some which are good for the bothy, others which aren’t

what is the MGBA

• the microbiota gut brain axis

explain the MGBA

• it’s the complex communication between the central nervous system and the ENS and gut microbiota

• the metabolites produced by the microbiota may be able to enter the brain/affect local neurones → signalling and communication

• CNS via neurotransmitters and secretions can impact the gut microbiota

how do microbial structural components and metabolites function in the MGBA?

• they act on intestinal epithelium, then released into blood stream and cross BBB

how do pro-inflammatory cytokines produced by immune cells work in the MGBA?

• they stimulate the ENS and its sensory neurones or induce secretion of neuropeptides by entero-endocrine cells

what pathways are used for the MGBA

• blood circulation

• vagus nerve

what is the DI?

• dysbiosis index

what is the function of the DI?

• it’s a quantitative PCR assay that is used to assess feline/canine faecal microbiome individually

• can help us determine whether there’s any dysbiosis

why is it important to note what kind of gut tract an animal has in terms of their microbiota?

• monogastric, ruminant/foregut fermenter or hindgut fermenter

• the type of fermentation/GIT setup will affect the microbiota

• it’s the understanding that the microbiota will differ between species

state 9 things that can lead to dysbiosis?

1. abrupt diet changes

2. age

3. supplements

4. climate

5. exercise

6. transportation

7. antimicrobials and other medication

8. diseases such as salmonella, clostridium difiicile

9. indirect infection

what can we use to help equine dysbiosis (5)

1. probiotics

2. prebiotics

3. postbiotics

4. supplements

5. FMT - faecalmicrobial transplant

what is FMT and what is it used for?

FMT - faecal microbiota transplant

• focus for chornic diarrhoea cases

• stomach tubed

• avoid donor if recent antibiotics, parasites or high clostridia

what are genomes

• genetic material of animals, plants and bacteria

mitochondrial DNA:

1. describe the DNA within it

2. are all the proteins mitochondrai needs produced by its DNA?

3. how does it divide

4. where does mitochondrial DNA come from

1. single circular chromosome

2. no - most are produced by the nucleus

3. binary fission

4. comes from the egg only

how much of the mammalian genome is coding DNA?

• 4%

why are there so many non-coding repeptitive sequences?

• due to incorperation of invasive elements

why is the dog genome looked at a lot? which dog breed in particular has been used a lot?

• it’s looked at due to the similarity in size to the human genome and due to the amount of genetic diseases

• boxers have been look at a lot due to least amount of genetic diversity

what toxicosis is linked to genetics in dogs and what does this lead to

• copper

• the liver fails to expel dietary copper → build up which is toxic → illness and death

• autosomal recessive trait

what 3 states can a dog be in genetically with the copper toxicity gene?

• clear - 2 copies of normal gene

• carrier - one of the recessive geens

• affected - 2 copies of gene → copper toxicosis

How can DNA mapping be used in farming?

• sequencing can identify common single nucleotide polymorphisms

• these can be used to track breeding populations

• used to map traits of interest and localise where there may be genes of interest

for what species of commercial animals is sequencing of SNPs used for genetic mapping?

• poultry - to track positive traits in breeding

how can we compare genetic diversity of wild vs domestic species?

• can sequence mitochondrial DNA and analyse microsatellites and SNP markers

what do these mean:

1. transcriptome

2. proteome

3. metabolome

1. gene expression suggests what’s expressed

2. what’s expressed, doesn’t say if active

3. what’s happening

this is all in terms of genes

what can we assess in DNA for (6)

1. presence/absence in a population

2. polymorphisms

3. PCR detection of genes, SNPs

4. sequencing to identify gene presence + SNPs

5. individual/population variations

6. microbiome balance across populations of microbes

what can we use RNA to assess?

• presence/absence of a message

• qPCR - level of expression

• mRNA sequencing - broad expression profile

what is a proteome

• what’s translated and therefore likely active

what ethical issues are there with genome data?

• what does the data mean for our client?

• who holds the data?

• what’s it used for?

• effects on insurance?

• breed altering?

• disease prevention?

• better treatment selection?

• duties and rights of patients

• duties of information holders

2 approaches of qPCR?

1. uses a probe in the middle of the target

2. uses a chelating dye that only fluoresces when interrelated with dsDNA so more DNA product, more fluorescence

qPCR:

1. used for

2. why is it good

3. what does it improve

4. what is it capable to do

• quantify all groups of pathogens

• rapid, low cost, samples can be frozen

• improved pathogen + genetic disease detection

• test large numbrs of samples that might be received during outbreaks of livestock disease

3 applications of PCR

1. pathogen detection - meet threshold for detection limits

2. detection gene mutation

3. more comparative use of data against sample or other controls

give 3 example diseases detected by pathogen

• FIV

• feline coronavirus

• bovine resp disease

what is machine learning?

• subset of AI

• analyses large datasets, imrpoving its performance as it’s exposed to data

• builds models to make predictions/classifications on new, unseen data