Microbiology 2 Flashcards
Describe Caliciviridae
- Non-enveloped
- Icosahedral symmetry
- 180 identical protein molecules arranged in dimers forming 90 arch like structural units, form 32 cup shaped surface depressions
- Single molecule of linear +ve ssRNA
- 5’ end capped by covalently bound Vpg and 3’ end is poly adenylated
- Cytoplasmic replication
Describe the vesicular exanthema of swine virus
- Now extinct disease
- Virus present in marine mammals, act as reservoirs
- Acute disease characterised by vesicles in oral cavity, interdigital space and coronary bands
- Indistinguishable from other vesicular diseases
- Laboratory diagnosis - virus isolation, direct EM or PCR
Describe the causative pathogen of feline calicivirus
- Vesicirus of Caliciviridae family
- +ve RNA
- Small
- Non-enveloped
Describe the epidemiology of feline calicivirus
- Acute or sub-acute disease
- Incubation period 2-3 days
- Recover in 7-10 days when not complicated by secondary bacterial infections
Describe the clinical signs of feline calicivirus
- Conjunctivitis
- Rhinitis
- Tracheitis
- Pneumonia
- Vesiculation of oral epithelium
- Fever
- Lethargy
- Anorexia
- Stiff gait
- Rarely may get virulent systemic FCV: alopecia, cutaenous ulcers, subcutaenous oedema, high mortality
Describe the diagnosis of feline calicivirus
Virus isolation
Describe the causative agent of feline herpesvirus
- Herpesviridae
- Alphaherpesvirinae subfamily
- Feline herpes virus 1 (genus)
- dsDNA
- Large
- Enveloped
Describe the clinical signs of feline herpesvirus
- Sneezing
- Nasal discharge
- Dehydration
- Anorexia
- Pyrexia
- Ocular signs (chemosis, keratoconjunctivitis, corneal ulceration)
Describe the diagnosis of feline herpesvirus
- Virus isolation
- PCR
Describe the treatment of FHV and FCV
- Supportive and symptomatic
- Fluid therapy
- Broad spectrum antibiotics (prevent secondary bacterial infections)
- Tends to be self regulating disease, supportive treatment more than anything
How can FCV adn FHV be controlled?
- Inactivated and attenuated vaccines
- Quaratine for hospitalised cases
What are the 2 main causes of vesicular disease in cats?
- Feline calicivirus
- Feline herpesvirus
Describe feline rhinotracheitis
- Caused by feline herpes virus 1
- Sudden onset of sneezing, coughing, profuse nasal and ocular discharges, corneal ulcers
- acute disease similar to feline calicivirus infection
What are teh 6 genera of Piconaviridae and give the diseases they cause
- Apthovirus (FMDV)
- Enterocirus (SVDV)
- Cardiovirus (encephalomyocarditis)
- Rhinovirus (bovine rhinovirus 1-3)
- Hepatocirus (human hepatitis A)
- Parechovirus (human echovirus 22-23)
Give an example of an autoimmune disease of the oral cutaneous junction
- Pepmphigus vulgaris
- Usually dogs (rare)
- Antibodies directed against intracellular layers above basal cell layer
- Separation of epidermal cells
- Chemicals may give similar lesions
- Vesicles often burst before you can see them
What are the 4 bacterial forms
- Bacillus form (Lactobacillus acidophilus)
- Staphylococcus form (Staphylococcus aureus)
- Streptococcus form (Streptococcus equi)
- Spirulis (Leptospira, Campylobacter, H. pylori)
What are the different bacterial flagellal forms?
- Monotrichous
- Amphitrichous
- Lophotrichous
- Peritrichous
What is mean by monotrichous flaggella?
A single flagellum extending from one end of the cell
What is meant by amphitrichous flagella?
One flagellum extending from either end of the cell
What is meant by lophotrichous flagella?
A tuft of flagella extending from one or both ends of the cell
What is meant by peritrichous flagella?
Multiple falgella randomly distributed over the entire bacterial cell
What are fimbriae?
- Attachment pilus
- Strand of peptides attached to a bacterium
- Found on many Gram-ve and some Gram+ve bacteria
What are porins?
- Allow diffusion through outer membrane
- Specific membrane proteins forming a pore to allow diffusion
What are endospores?
- Highly resistant bodies produced by bacteria under specific conditions in order to survive
- Contain high levels of small acid soluble proteins
- Layered dehydrated structure
Describe the structure of the bacterial cell wall
- Made up of peptidoglycans
- Peptidoglycans are polysaccharides cross-linked with polypeptides
How can the cell wall of bacteria be targeted?
- Target hte peptidoglycans
- Beta-lactan antibiotics e.g. penicillin
- Lysosyme enzyme that cleaves disaccharides
Briefly outline the Gram stain process
- Crystal violet solution applied to sample
- Rinse
- Iodine
- Rinse
- Alcohol
- Safranin
- Rinse gentrly
Breifly outline the Acid-Fast staining process
- Ziel-Neelson carbolfuchsin added to slide, apply heat
- Acid alcohol to decolourise
- Wash with distilled water
- Counter stain with methylene blue
- Wash with distilled water
- Acid fast bacteria retain red colour, non-acid fast go blue
Why do mycobacteria stain red with Acid fast staining?
Mycolic acids in cell wall retain the carbolfuchsin stain
What are the 4 main biochemical tests used to identify bacteria?
- Catalase
- Oxidase
- Nagler
- Urease
Describe the catalase test
- Add hydrogen peroxide
- If bubbles then is positive
- Produce oxygen as catalase enzymes present to break down H2O2 into oxygen and water
Describe the oxidase test
- Add solution to paper, if turns blue is positive
- Detects presence of specific cytochrome-C-oxidases
- These protect agains oxygen radical damage
What is the Nagler test used for?
To identify organisms that liberate phospholipases
Describe the urease test
- Detects presence of urease enzyme
- Urea to ammonia and carbon dioxide
- Campylobacter like organisms
- Positive is pink
What is an anaerobe?
An organism which requires oxygen for life
What is an aerobe?
An organism which does not require oxygen for life, and does not survive in oxygen environments
What is a facultative anaerobe?
Organisms which prefer non-oxygen environments but can survive in presence of oxygen
What are micraerophilic bacteria?
Need oxygen to ferment carry out respiration, but high concentrations of oxygen are toxic
What is meant by enriched media?
A medium that has had something added to it that is needed by an organism to grow e.g. blood
What is meant by selective media?
A medium that has had something added and so will restrict which bacteria are able to grow on the plate e.g. bile salts, deoxychlorate, selective antibiotics
What are indicator media?
Media that have an idicator included in it in orde to identify colonies of bacteria e.g. a substrae is added that leads to a pH change when utilised and in turn leads to a colour change.
Often combined with selective media as done in MacConkey
Describe MacConkey agar
- Selective indicator
- Restricts which bacteria are able to grow through use of bile salts
- Lactose and neutral red pH indicator used to identify specific colonies
Outline the agglutination test
- Serological test
- Detects presence of serum agglutinins H and O
- H antigen is thread like portion of flagella
- O antigen is outermost portion of LPS on bacterial surface
- Positive result is where agglutination occurs
What is the Baltimore classification system for viruses?
Classification of viruses based on characteristics of genetic information e.g. dsDNA vs dsRNA etc
What types of infection can you get from viruses?
- Latent
- Persistent
- Lytic
- Transformation
What is a latent viral infection?
- The virus is present but not causing harm to cell, may later emerge in lytic infection
- E.g. Herpesvirus
What is persistend viral infection?
- Slow release of virus without cell death
- E.g. hepatitis
What is a lytic viral infection?
- Death of cell and release of virus
- e.g. FMD
What is a transformation infection?
- Transformation of normal cells to tumour cells
- E.g. Feline leukaemia virus, canine oral papilloma virus
Describe the key stages in viral infection
- Acquition into host (e.g. breathed in)
- Attachment and entry
- Initiation of infection at primary site
- Incubation as virus amplified
- Translation or immediate replication using host cell machinery
- Assembly of new virus particles
- Release from cell via cytolysis or budding
What methods can be used to detect viruses?
- Detection of viral proteins
- Detection of virus genetic material
- Serology
- Live virus culture
- Electron microscopy
How can viruses spread in the body?
- New virus made is released into body by cell
- Can be enveloped
- Taken up by other cells
Why are soe infections localised and others more general?
- Ability of virus to either infect only one cell types or more than one
- Some cell types are found everywhere, others only in specific areas
- Depends on surface proteins cells express as these are needed to viral attachment
What features are used to identify filamentous fungi?
- Made up of hyphae
- Fuzzy appearance
- Often have conidia at top that release spores
- Multicellular
What is a dimorphic fungus?
One that can grow as yeast or hyphal form depending on the environmental conditions (temperature)
- e.g. Cryptococcus neoformans
Describe the main features of a yeast
- Asexual division by budding
- Can also reproduce sexually
- Spores formed more like gametes
- Round shape of cells
- Form neat blobs rather than fuzzy shapes
What is the importance of the gut flora?
- Protects against establishment of pathogens
- Important in physiological health of animal
- Role in resistance to infection (exclusion and competition)
- Role in nutrition (metabolism)
- Role in bowel cancer
- Role in immune development (primes immunity)
- Role in evolution
What is meant by competitive exlusion?
- Competition for nutrition, electron acceptors and a carbon source
- Nutrition is not as readily available
- Is the use of desired microbial cultures that out-compete pathogens from colonising specific niches
What are some of the nutritional benefits of gut flora?
- Fore-gut fermentation in ruminants
- Hind-gut fermentation e.g. horses
- Vitamin production e,g in pigs Clostridium butyricum synthesises B12
What samples can be used to study the gut flora?
- Faecal
- Post mortem (fresh/not fresh)
- Samples during surgery
- In vivo analysis
What are some qualitative methods of gut flora study?
- Direct examination
- EM
- Microscopy
What is a disadvantage of using qualitative methods for gut flora study?
Most pathogens look the same so is only useful if have easily distinguishable pathogen
What are the disadvantages of using quantitative methods for gut flora study?
- Need to carry out in same conditions as where pathogen would be found
- May be better to use medium selecting for suspected cause of disease rather than total count
What are the quantitative methods of gut flora study?
- Total bacterial count
- Viable count
Describe total bacterial counts
- Direct count
- Dilute and count no of bacteria in faecal matter, live and dead, independent of growth requirements
- Does not show what is present
- Can carry out DNA analysis if want to know presence of specific organism
Describe viable bacterial counts
- Samples, can be variety
- Homogenise with buffer
- Supports pathogens, then dilute
- Dilution series and plating
- Use selective media to ensure desired bacteria present
- Combine with indicator to detect presence of particular groups
- Likely to have mixture of commensals otherwise
How can the presence of bacteria be assessed?
- Smear of sample with simple staining
- Direct counting by microscope
- Colony counting on selective media
What factors affect adult floral composition?
- Establishment
- Floral differentiation
- Disruption to flora
Describe how establishment affects adult floral composition
- Foetus microbiologically sterile
- Host supplied receptors for adhesins and niches for specific organisms
- Organisms have different receptors providing tissue specificity and different biochemisty allowing growth in specific niches
- Acquires organisms from surface parts of birth canal and immediate environment
- Immediate environment contaminated with organisms excreted from dam and other animals
- Once ingested compete for gut (the niche)
Describe how floral differentiation in the gut occurs
- Between species (anatomy, cell biology, diet, physiology, mucous composition)
- Within species (age, use of drugs, diet, stress, genetics, illness)
- Change over time maternal immuntiy/bottle feeding/pre-ruminant, antibacterial effects, acquired immunity, weaning, old age, complexity increases over time)
Describe how floral disruption affects the floral composition in the gut
- Antimicrobials remove sensitive organisms from particular niche
- Space that is left repopulated
- Antibiotics can aid colonisation by undesirable organsisms adn pathogens
- Remove competition by commensals, pathogens able to take over
What are the host factors that affect colonisation as the first stage of disease?
- pH
- Peristalsis
- Mucus and mucus integrity
- Bacterial competition
- Immune defence
- Antimicrobial products
- Host genetics and immune stimulation
What are the bacterial factors that affect colonisation as the first stage of disease?
- Surface structures for adhesion
- Nutrient acquisition
- Adaptation to pH, anaerobic conditions, temperature change
- Temperature only important in heterotherms
- LPS and membrane integrity for tolerance of harmful factors
- Motility to get to target niches
- Specific transporters to take up GI specific nutrients
- Toxins
What are the constituents of the normal flora of the oral cavity?
- On buccal surface, tongue and teeth: Streptococci, Pasteurellaceae, Actinomyces, E coli, Neisseria spp, Simonsiella
- In gingival crevice: Bacteroides, Fusobacterium, Peptostreptococcus, Porphyromonas, Preyotella
What are the constituents of the normal flora of the oesophagus?
- No define flora
- Contaminated by organisms similar to those in saliva
Describe the normal flora of the stomach
- Hostile to organisms
- Some pathogens endemic in number of populations
- Problems in neonates where environment not fully developed
Describe the normal flora of the rumen
- Complex structure of organisms
- Mostly obligate anaerobes
Describe the normal flora of the alimentary canal
- Bacteria (anaerobes, Enterobacteriaceae, Streptococci, Enterococci, Lactobacillus)
- Protozoa
- Fungi/yeasts
- Spiral organisms
Describe the normal flora of GI associated organs (liver, gall bladder, pancreas)
- No normal flora
- Some transient asymptomatic bacteraemia
- Clostridia spores in liver only germinate if oxygen tension drops very low and allows for germination
Describe DNA replication in prokaryotes
- Circular DNA
- Fast
- Single origin of replication
- 2 bidirectional replication forks
Describe DNA replication in eukaryotes
- Linear DNA
- Slow
- 100s of origins of replication
- Requires DNA polymerase and other enzymes
- Replication forks move in both directions
- Occurs in synthesis (S) phase of cell cycle
- Replication “bubbles”
Describe the polymerase chain reaction
- Rapid, simple method for copying and amplifying specific DNA sequences
- Repetitive cycles of DNA melting, annealing and synthesis
- Need to know sequence of short reigion of DNA on each end of larger sequence that needs to be copied and amplified
- Used to design 2 synthetic DNA oligonucleotides
- Each is complementary to sequence on one strand of DNA double helix at opposite ends of region to be amplified
- Serve as primers for in vitro synthesis
- Different length strands of DNA produced
What is agarose gel electrophoresis used for?
- To separate DNA or RNA molecules produced by PCR by size
- Negatively charged nucleic acid molecules move through agarose matrix with electric field
- Shorter move faster, migrate further than longer strands
- Visualisation by adding fluorescent dye that intercalates into DNA strands
Outline PCR use in diagnostics for infectious and non-infectious diseases
- Detection of bacteria
- Equine coronavirus
- Cancer diagnosis
- Feline panleukaemia virus
What are plasmids?
- Small self replicating circles of DNA
- Range in copy number per cell
- Partition genes so when bacteria divide both cells gain plasmid
- Not all bacteria have plasmids
- Can transfer between relateed bacteria carrying range of properties
What is often contained within plasmids?
- Transfer genes
- Replicating genes (regulate and copy number)
- Resistance genes
- Virulence genes
Describe structural arrangement of bacterial chromosomes
- Haploid
- Circular DNA
- Longer than typical prokaryote
- To fit in cell is organised into DNA-protein complex called nuleiod
- Supercoiling DNA using DNA gyrase
- DNA binding proteins (histones) used to further package
What is contained within bacterial nucleoids?
- Large amount of RNA polymerase
- RNA
- Many different regulatory proteins
- Not structural role
- Reflect main data transfer within cell
Describe bacterial transposons
- Used in transposition, can be self transmissible
- Sequences of DNA that can move to different positions within genome
- Intefrate into and replicate as part of genome
- Insertion can disrupt the genes they insert into
- Have accessory genes associate in them
- Sometimes carry additional genes e.g. antibiotic resistance
- Some transposon shown to transfer genes but most piggy back on other transfer systems
What is transposition?
The movement of chunks of DNA within the chromosome
What are insertion sequences?
- Type of transposon
- Known as IS elements
- Short DNA sequence that acts as simple transposable element
- Small relative to other transposable elements
- Only code for proteins implicated in transposition activity
- Different from transposons which carry accessory genes
What are the processes involved in bacterial gene transfer?
- Transformation
- Conjugation
- Transduction
Describe transformation in bacterial gene transfer
- Uptak eof naked DNA
- DNA contacts bacteria, taken up
- Some bacteria are naturally competent and can tak eup DNA without treatment, competence mechanisms
- Others require treatment to become competent
Describe conjugation in bacterial gene transfer
- Transfer through cell to cell contact
- Horizontal gene transfer (donor to recipient)
- Donor must have conjugative or mobilisable genetic element (plasmid or transposon)
- most conjugative plasmids have systems ensuring recipient cells does not already have similar element
- Move whole blocksof properties
- Most commonly antibiotic resistance, virulence factors
Describe viral transduction in bacteria
- Viruses infecting bacteria called bacteriophages
- Each virus specific for specific species of bacteria
- Need to bind to specific receptor
- Then inject genome and infect bacteria
How does the evolution of bacteria take place?
- Rearranging DNA (transposition and recombination)
- Deletion of genes
- Insertion of new genes transformation, conjugation, transduction)
- Mutation of genes already present
Explain the role of bacteriophages in bacterial evolution
- Viruses that infect bacteria
- Different types of bacteriophage
- Lytic or temperate
- Lytic undergo lytic cycle in bacteria (infect, replicate, lyse)
- Temperateintegrate in genome or act like plasmid, undergo lysis if triggered
- If temperate integrate when reform virus can take host DNA, when next infect can trasnfer genes to new bacterium
What are the possible outcomes of entrance of DNA into bacteria?
- Degradation by non-specific nucleases (no recombination)
- Degradation by specific restriction endonucleases (DNA restriction)
- Integration with genome (DNA recombination)
Outline genetic recombination in bacteria
- Sequences of DNA from 2 seperate sources integrate
- Can lead to inheritable changes in bacteria
- May be host spots for recombination
- Can have imperfect matches
- May be homologous or non-homologous
Define homologous genetic recombination
Aligment to similar sequences, crossover between aligned DNA strands
Define non-homologous genetic recombination
Between DNA sequences that contain no sequence homology
How is gene transfer restricted?
- Restriction system to prevent constant uptake of DNA
- Controlled by restriction endonucleases
- Degrade DNA not made in own cell
- Acts at specific sites
- Bacteria methylates own DNA where own restriction nuclease would cut so do not destroy own genome
What occurs in transcription?
DNA -> mRNA
What occurs in translation?
mRNA -> protein
What changes can occur to the DNA within bacteria?
- Point mutation
- Transposiion
- Re-arrangement
- Deletion
What is point mutation in DNA?
Single base changes, can have dramatic effect or no effect
What happens in rearrangement of bacterial DNA?
- Inversions, reversals
- CCross over and DNA strands interact
- Copes, end up with jumbled strand or cut due to deletion
- Cannot replicate from this
What is deletion in DNA?
Removal of a sequences of bases
Outline the methylation of DNA and CpG in post-translation events of bacteria and the effect on mammalian cells
- Methylation patterns of DNA different between kingdoms
- DNA from bacteria has stimulatory effect on mammalian immune cells
- Depends on presence of unmethylated CpG dinucleotides in bacterial DNA
- Mammalian DNA has low frequency of CpG
- Mammalian CpG mostly methylated
- Host immune responses stimulates as bacterial CpG DNA binds TLR9
- Leads to strong Th1-like inflammatory response
- CpG DNA therefore has potential as adjuvant
What are CpG sites of DNA?
CpG sites are regions of DNA where cytosine nucleotide occurs next to guanine nucleotide
