Microbiology Flashcards
1
Q
What does ELISA stand for?
What does ELISA detect?
A
Enzyme Linked Immunosorbent Assay
Technique used to detect specific molecules in samples
2
Q
What are antibodies?
What do antibodies do?
A
Secreted by B-lymphocytes when matured to plasma cells
Recognise and bind to molecules (antigens) on foreign particles, marking them for destruction by T-lymphocytes
3
Q
What are the uses of ELISA in veterinary medicine?
A
Disease detection
Detection of illegal drugs
Detection of hormones
4
Q
What is antibody detecting ELISA?
A
Uses antigens to detect a specific antibody in sample
5
Q
What is antigen ELISA?
What are the two types?
A
Uses specific capture antibodies to detect specific antigen in sample
Sandwich and competitive
6
Q
What is sandwich ELISA?
What is competitive ELISA?
A
Uses an antibody pair to detect specific antigen
If antigen is small and has only one epitope the sample antigen is made to compete with a labelled antigen for antibody binding sites
7
Q
What does epitope mean?
A
Binding site for antibody
8
Q
How do rapid immunomigrations (RIM) work?
A
Proteins absorbed onto strip of tissue such as nitrocellulose
Sample applied to one end of strip and migrates along by capillary action
(sample may need to be pre-treated (eg. diluted) prior to application
9
Q
Why in blood RIM tests to the lines show up as red?
A
Pigment in blood is red due to haemoglobin, therefore the line shows up red due to the pigment
10
Q
What is agglutination?
What can it indicate?
A
Process where antibodies clump together cells or particles
Presence of antibodies against bacteria or red blood cells
11
Q
What does latex bead agglutination assay detect?
A
Detect antibodies or antigens in body fluids including saliva, urine, cerebrospinal fluid, or blood
12
Q
What is haemagglutination?
What common assay uses this?
A
Agglutination of red blood cells
Direct Coombs’ Tests - identifies the presence of non-agglutinating antibodies on the surface of erythrocytes
13
Q
What must be included to make an immunoassay valid?
A
A positive and negative control
13
Q
What is a positive control?
A
Confirms if the procedure is performing as intended
- allows for confidence in the diagnostic test results, confirms that negative results are accurate, and assists in any protocol adjustments or optimizations that may be necessary
14
Q
What is a negative control?
Why is this useful for assays?
A
Known to not contain the biomolecule of interest
- adds validity to any positive results
It will show if any solutions of the assay are contaminated
15
Q
In a sandwich ELSIA, what would happen if the blocking step was left out?
In a sandwich ELSIA, what would happen there was no washing steps after the detection antibody was added to the well?
A
All wells, including the negative control wells, would show uniform overdevelopment
Same answer again!
16
Q
In antibody detecting ELISA what will the enzyme linked antibody attach to?
A
The constant region of the patient antibody
17
Q
List the 5 steps involved in indirect ELISA (measure antibody concentrations in various types of samples):
A
18
Q
List the 5 steps involved in sandwich ELISA (measure antigen concentrations in various types of sample):
A
19
Q
Why are wells blocked in ELISA?
A
Blocked prior to sample addition to prevent non-specific binding
20
Q
Is ELISA qualitative or quantitive?
A
Both
21
Q
What is herd health?
How is it managed/maintained?
What is the objective?
A
Monitoring and control/prevention of (endemic) diseases of UK farmed species (Infectious diseases, metabolic diseases, etc), but also to optimse fertility, nutrition and production
- Monitoring and appraising; a continual active process (keep records)
- A regular veterinary visit
- An overview of preventive systems
To optimise cow health and welfare as well as farm sustainability and profitability
22
Q
What is limit detection when talking about ELISA?
A
The minimal concentration that will be detected positive by the ELISA test (manufacturer should provide this number)
23
Q
What is sensitivity when describing diagnostic tests?
What is the sensitivity of a veterinary diagnostic test?
What type of test characteristic is sensitivity?
How would you know a tests sensitivity?
A
The test’s ability to identify positive results
Probability of a positive test, given that the patient truly has the disease
(if the test had 95% sensitivity and 100 cows where infected 95 would test positive and 5 would be false negatives)
Prevalence-independent test characteristic
The manufacturer should provide the sensitivity of the test.
24
What is a tests specificity?
What is the specificity of a veterinary diagnostic test?
What type of test characteristic is specificity?
How would you know a tests specificity?
The test's ability to identify negative results
The probability of a negative test given the patient if uninfected/healthy
(100 animals, 50 uninfected, specify of 98% = 49 of the 50 would test as negative and 1 would be a false positive)
Prevalence-independent test characteristic
The manufacturer should provide the specificity of the test.
25
What is seroconversion?
How can seroconversion be assed?
Interpret the serology results from paired samples:
Development of detectable specific antibodies to microorganisms in the blood serum as a result of exposure, infection or immunisation
Single sample cannot indicate when exposure occurred or if it was associated with a clinical disease so paired samples are taken 10-14 days apart (depends on disease)
26
If there is acute infectious disease suspected why would multiple paired sample be taken around 4 weeks apart (time really depends on pathogen)?
Due to expectation that a rising in antibody titre will be demonstrated
27
What values for IBR ELISA indicate negative, inconclusive and positive results?
What value of increase in a paired sample is considered significant?
0.15< = negative
0.15-0.25 = inconclusive
>0.25 = positive (previous exposure or recurrence)
Increase in more than 0.2
28
What values for RSV ELISA indicate negative, inconclusive and positive results?
What value of increase in a paired sample is considered significant?
0.15< = negative
0.15-0.24 = inconclusive
>0.24 = positive
Increase in more than 0.2
29
What values for BVD ELISA indicate negative, inconclusive and positive results?
0.2< = negative (no exposure of consistently infected)
0.2-0.3 = inconclusive
>0.3 = positive (previous exposure and seroconversion)
30
What values for mycoplasma bovis ELISA indicate negative, inconclusive and positive results?
What value of increase in a paired sample is considered significant?
0.25< = negative
0.25-0.29 = inconclusive
>0.29 = positive
Increase in more than 0.3
31
When screening using ELISA for IBR in cattle a few cattle are identified as latently infected (not an active infection), what prognosis would they be given?
(positive, inconclusive, negative?)
Seronegative
32
Why is an understanding of microbiology in veterinary science important?
For prevention and treatment
For sterilisation and biosecurity
For epidemiology and recognition of emerging diseases
Antimicrobial stewardship
Due to the growing resistance in antibiotics
So that you cam make empirical decisions (ability to justify why)
33
Order these microorganisms from largest to smallest:
Virus
Bacteria
Fungi
Prion
Fungi
Bacteria
Virus
Prion
34
What type of microorganism are the following and give the types you can find:
fungi
bacteria
virus
prion
Fungi
- eukaryotic
- yeast and mould
Bacteria
- prokaryotic
- gram -ve, gram +ve, acid fast, mycoplasma
Virus
- non-cellular
Prions
- non-cellular single proteins
- TSE
35
What're the basic structural components of bacteria and what are their main functions?
Chromosome (haploid, circular DNA compacted by DNA gyrase and proteins))
Plasmid (independent transfer DNA molecule)
Cell wall (defines morphology and protects)
Cell membrane (Passive and Active transport)
Flagella (protein tails, not all have them)
Capsule (protection, adherence, interferes with phagocytosis)
Pili (adhesion and plasmid transfer)
36
What is a counter stain?
Second Stan that contrasts parts that haven’t been stained by first stain
37
What allows the visualtsation of microorganisms?
Staining as they are translucent (gram stain most common)
38
What does the halo around these bacteria indicate?
Capsules
39
What are the structural differences between these bacterium cell walls?
Gram -ve
Gram +ve
Acid fast (Gram +ve with mycolic acids)
Mycoplasmas
Gram -ve
- thin cell wall with polysaccharides sticking out
Gram +ve
- capsule formation
Acid fast (Gram +ve with mycolic acids)
- other molecules in cell wall
Mycoplasmas
- no cell wall only membrane
40
Some bacteria can develop an endospore, why is this beneficial?
Great for survival
- hard to penetrate
- resistant to high temperatures
- resistant to common agents that would kill vegetative cells
41
If mycoplasmas are closely related to gram +ve why do they not stain as gram +ve?
They do not have a cell wall
42
How does having no cell wall impact mycoplasmas?
No structural morphology - pleomorphic
Fragile
Live in close association with host cells relying on their homeostatic mechanisms to maintain their environment
43
What are cocci and what colour do they stain?
Gram positive bacteria
Stain purple
44
What are rods and what colour do they stain?
Gram negative bacteria
Stain pink
45
Gram Positive and Gram Negative (clinical relevance)
1. Which is more sensitive to antibiotics?
2. Why is gram -ve having a higher lipid content important?
3. Which forms spores (relevance for disinfecting)?
4. Which has toxins (for neutralising)?
1. gram +ve
2. Slows entry of certain antibiotics
3. gram +ve
4. Both
46
What is the stain used to identify acid-fact bacteria?
Modified Ziehl-Neelsen stain
(Stain -1 Ziehl-Neelsen carbol fuchsin
De-stain with an acid alcohol
Counter stain with methylene blue
Those that maintain the red colour are defined as acid fast.
Can be used on smears or tissue sections)
47
How does bacteria grow?
What is a colony?
What is the name given to the generation of bacteria?
Binary fission - DNA copied and then splits into daughter cell
Group of bacteria based off of 1 bacteria cell
The doubling time (time for 1 to dive into 2)
48
How do you isolate bacteria into single colonies on a solid media?
Aseptic technique, sterile media and flaming loop to spread bacteria until separated
49
What are the 3 methods that can be used to measure bacterial growth?
- direct counting using a microscope (cannot distinguish viable from non-viable cells)
- colony counting (viable count)
- absorbance in liquid culture (optical density - more bacteria = more light absorbance)
50
You count the colonies from the 50µl (5 x 10µl) of the 10-4 dilution and then calculate up to how many would be in 1 ml.
How many cfu/ml in the original sample?
32 in 10-4
0.05 ml
1/0.05 = 20
32 x 20 = 640
51
What are the 4 phases of growth in a liquid culture?
1. Lag phase - bacteria adjust to medium and then start to metabolise and grow
2. Log/Exponential phase - bacteria grow so growth rate is determined
3. Stationary phase - bacteria at limit of resources, cannot exceed population, growth rate = death rate
4. Death phase - medium exhausted of resources sp no new growth but bacteria still dying
52
What is...
an aerobe
an anaerobe
a facultative anaerobe
a microaerophile
Aerobe - requires oxygen to grow
Anaerobe - cannot grow in presence of oxygen
Facultative anaerobe - can grow with and without oxygen
Microaerophile - can cope with low amount of oxygen
53
What impact does pH have on bacterial growth?
What are neutrophiles, acidophiles and alkaliphiles?
Most media is buffered at pH 7, buffer important so that during growth acidic metabolites produced don't inhibit growth
Most pathogens are neutrophile (most vet pathogens) so they balance the pH in a closed environment so they can grow
Acidophiles and alkaliphiles are found in extreme environments and aren't pathogenic
54
What impact does temperature have on bacterial growth?
Bacteria that infect animals are mesophilic and can be adapted to the temperature of the animal and have flexibility in their range which can allow survival even when not at optimum temperature (ie. outside host, cold slows growth but doesn't always stop it)
55
What impact does osmolarity (water potential) have on bacterial growth?
Bacteria membrane is semi-permeable so it can accumulate or lose solutes to balance water potential (prefers slightly positive inflow) so changing environment to alter bacterias water potential can disrupt the cell physiology
56
What is the differences between fastidious and non-fastidious bacteria?
What are chemoheterotrophs?
Fastidious = requires specific supplements to grow
Non-Fastidious = can grow from basic chemicals
Chemoheterotrophs = bacteria that use organic chemicals and carbon as sources of energy (most vet pathogens are this)
57
What are the key features f the following types of growth medias?
Minimal
Nutrient
Enriched
Selective
Indicator
Selective Indicator
Transport
Minimal - basic salts
Nutrient - all basic requirements
Enriched - additional organism specific supplements
Selective - added supplements for specific bacteria
Indicator - indicators react to specific bacteria
Selective Indicator - combination of the two
Transport - protects organisms in sample on way to lab
58
What are the 3 main ways of eliminating unwanted bacteria?
Sterilisation
- eliminates and kills
Washing
- removes and reduces risk
Disinfection
- does not kill all pathogens
59
What are virulence factors?
What is a virulence gene and how have they evolved?
Molecules that allow bacteria to adhere, invade, evade host defence, cause tissue damage, replicate or persist in host
Gene encoding a virulence factor - horizontal transfer of the gene by plasmids, transposons and phage transduction
60
Fill in the missing steps for the process of bacterial infection:
61
What can bacteria adhere to?
What features allow them to adhere?
Cells, secretory products (mucous), structural components (teeth), other bacteria
Pili and adhesive macromolecules imbedded in their membrane
(also potentially capsules, flagellum)
62
Once bacteria adheres to a host cell, how do both respond?
Bacteria will express proteins that enable survival
Host cells try to reduce impact and spread of infection
63
What are the 2 main strategies bacteria use to survive in the host cell?
1. Resistance to host defence (complement resistance, avoiding phagocytosis, protection against antibody recognition)
2. Active subversion of host defence
- killing phagocytotic cells
- changing host cell
64
What strategy does Streptococcus equi use to survive once it has invaded its host?
Avoiding phagocytosis (extracellular bacteria) by masking binding sites with it's M protein so phagocytes cannot bind and killing neutrophils through lysis.
65
What is Streptococcus equi?
Strangles
Enters horses mouth or nose, travels through lymph nodes in head and neck, end in guttural pouch where it colonises as it is protected from immune system and can survive long term after recovery.
66
How to bacteria get inside cells?
Through phagocytic cells (take up bacteria but risk of killing bacteria)
OR
Forced phagocytosis (manipulate cell to phagocytose bacteria)
67
How do bacteria move inside of cells?
Manipulation of host cytoskeleton (actin propel them forwards, microtubules)
68
Hw do bacteria obtain nutrients from their host cells?
Manipulation of host metabolism (poor nutrition inside cell)
69
How do bacteria move between cells without host defect picking up on them and killing them?
1. Actin tail
2. Attach to microtubules during cell division to go into daughter cells
70
Why is inflammation beneficial for bacteria in a host cell?
Increases the host cells glucose uptake which the bacteria can directly use
71
What does MacConkey agar show?
What are its selective agents?
If the bacteria is lactose positive (agar stays red and bacteria goes pink/red) as it decreases the pH
If bacteria lactose negative (agar goes white/colourless)
Bile salts and crystal violet dye
72
What does blood agar show?
Haemolysis types:
Alpha - incomplete haemolysis so produces a green zone
Beta - complete haemolysis so clear zone
Gama - no haemolysis so stays red
73
What does XLD agar show?
What are the selective agents?
How alkaline or acidic the bacteria is
Sodium deoxycholate
74
What does antimicrobial disk diffusion show?
If the bacteria has any antibiotic resistance helping with determining treatment paths
Growth up to dial indicates resistance
Clear circle around disk indicates sensitivity
75
What are the two staining methods that can be used for bacteria?
Gram stain (more preferable)
Dip Quick - can also be used for clinical smears (like blood)
76
Can endospore be stained?
Endospores have stain resistance cortexes
So to stain them they must be steam heated with a primary stain called stain-malachite green which has a low affinity of the cellular material and is water soluble so can be washed off and the cell counter stained with saffron.
77
What is nutrient agar?
A medium with all the basic (general) requirements for growth but without specific supplements.
78
What type of upper respiratory swabs would you take from dogs and cats?
Nasal swab
Oropharyngeal swab
Tonsillar swab
79
What are the 5 steps involved in taking a nasal swab from cats and dogs?
1. Prepare the swab- open packet and remove cap from sample tube
2. Gently take the animal’s muzzle and lift the head up
3. Gently insert the swab into the nares, keeping to the medial side of the nares
4. Do not insert the swab more than a few millimetres
5. Immediately insert the swab into the sample tube and label it.
80
What are the 5 steps involved in taking a tonsillar swab from a dog?
1. Prepare the swab- open packet and remove cap from sample tube.
2. Ask an assistant to open mouth and retrain dog.
3. Vigorously swab both tonsils.
4. Avoid the tongue and heavy saliva contamination.
5. Immediately insert the swab into the sample tube and label it.
81
What are the 5 steps involved in taking an oropharyngeal swab from a cat?
1. Prepare the swab- open packet and remove cap from sample tube.
2. Ensure cat is restrained
3. Use one hand to tilt head upwards whilst opening mouth
4. Roll swab across the region of the oropharyngeal near the tonsils
5. Immediately insert the swab into the sample tube and label
82
What upper respiratory swab can be taken from a horse?
What pathogen is frequently being investigated?
Nasopharyngeal
Streptococcus equi
83
What restraint is required to obtain a nasopharyngeal swab in a horse?
How do you know how far to pass the swab?
Standing physical restrain with headcollar, stocks or stable. Twitch but only if needed. Horses that may respond badly to an attempt may need to be sedated.
Swab should stimulate a swallow when it reaches the caudal soft palate and resistance will be met
84
Where are upper respiratory swabs taken in birds?
Tracheal swabs through the glottis
Nasopharynx through the choana (small birds)
85
What kind of transport media is used to preserve aerobic and anaerobic bacteria? (not for those needed for PCR)
Charcoal transport media
86
Advantages of upper respiratory swabs:
1. How valuable is the information they give?
2. How long does it take to get a sample?
3. How easy are they to obtain?
4. What is the impact on the animal you are taking the swab from?
5. Is any specialist equipment required?
6. Are swabs expensive?
1. valuable as identifies if bacteria or a virus is present
2. very quick
3. Yes, if proper restraint used
4. Mild discomfort and stress during procedure
5. No
6. No
87
What are the most important PRRs?
Toll-like receptors (TLRs)
They are transmembrane receptors (signal travels from outside of cell into cell/inside of vacuoles into cell)
- found in a wide range/all cell types
- have a wide range of roles
88
What is the bodies front line response?
The innate immune system (can differentiate between good and bad microorganisms)
89
How do host cells recognise pathogens?
They express specific receptors call PRRs that recognise PAMPS which are specific 'danger signals that pathogens express
90
What does PRRs stand for?
What does PAMPS stand for?
Pathogen recognition receptors
Pathogen associated molecular patterns
91
What is the main PAMP recognised in gram negative bacteria?
What is the main PAMP recognised in gram positive bacteria?
LPS - found in the cell wall
Peptidoglycan - found in the cell wall
92
What happens when a PAMP (ligand) is recognised by a PRR?
The activation of transcription factors lead to a more specific host response to that pathogen!
93
How do TLR work?
Leads to an inflammatory response
94
What does TLR4 recognise (most common)?
How is it found in a cell?
How does TLR4 play a role in endotoxic shock?
LPS (and therefore gram negative bacteria)
Paired (always 2 together) and bound to other molecules to activate it
No TLR4 receptors = resistance to endotoxic shock
95
TLRs are transmembrane receptors, where can they be found?
Embedded in lipid bilayers:
- Outer cell membrane for extracellular microbial compounds
- Vesicle membranes for cytoplasmic microbial compounds (in the cell)
96
What does TLR2 recognise?
TLR2 is a homo-/heterodimer, what does this mean?
What host response is TLR2 most important for?
Lipoprotein and lipopeptides in pathogens
TLR2 can interact with a wide variety of other TLR and non-TLR receptors so that more pathogen can be recognised (homo is two of the same proteins and hero is two different)
Recognising gram positive bacteria
97
As well as bacteria, what can TLR respond to?
PAMPS on viruses, protozoas and infectious disease
97
What is commensal bacteria?
Good bacteria found in places like the gut and the upper respiratory. tract. They are essential in developing homeostasis on the mucosal surfaces, support immune development, compete against pathogens, play a role in nutrition.
98
Why can commensal bacteria pose an issue to TLR receptors?
They have very similar PAMPS to pathogenic bacteria so limited contact between commensals and host receptors is vital.
99
EXAMPLE: How is limited contact between TLRs and commensal bacteria achieved in the gut lumen?
TLRs found on the basal surface on the lumen. When pathogenic bacteria gets in it damages the lumen causing it to come into contact with TLRs so tat the inflammation response can still occur.
100
What clinical syndrome arises if the host response is not properly controlled to achieve a healthy balance between pathogen destruction and immunopathology?
Sepsis
101
What is bacteremia/septicemia?
Presence of viable bacteria in the blood stream
102
What is SIRS and what does it stand for?
SIRS = systematic inflammatory response syndrome
Systematic response to an array of severe clinical insults (includes infection, hypoxia, trauma, etc.)
103
What is shock?
SIRS induced hypotension (reduced bp) unmanageable by fluid resuscitation in association with hypoperfusion (low blood flow) leading to reduced oxygen levels in organs/tissues (ischemia)
104
What is sepsis?
What is severe sepsis?
SIRS due to infection
Sepsis associated with organ dysfunction, hypofusion or hypotension
105
What is MODS and what does it stand for?
MODS: multiorgan dysfunction syndrome
Altered organ function in acutely ill patients requiring intervention to maintain homeostasis
106
If bacteria and/or bacterial products rich the blood stream instead of the localised inflammatory response containing and resolving it, what happens?
Patient will go into SIRS which leads to MODS
(so host response outweighs the effects of the direct toxicity of the bacterial infection)
107
When SIRS occurs with infection, what will the result always be and what are the complications that lead to this?
Sepsis
1. localised infection turns systemic (affects entire body)
2. mucosal barrier break down so commensal bacteria gets into blood stream
108
When mediators* are activated due to infection or other causes, what level will lead to a beneficial effect and what level will cause detrimental effects like SIRS?
*protein + lipid mediators, reduced oxygen species, anti-inflammatory mediators
Beneficial = low systemic levels of mediators
Detrimental = high systematic evils of mediators
109
Why is therapeutic intervention risky and difficult with a patient in sepsis?
There a a fine balance between preventing detrimental effects without depressing the beneficial effects
110
What are re main interventions that can be taken with sepsis?
- early diagnosis
- treatment of primary disease process
- supportive care
111
What therapeutic intervention are being used in human medicine?
1. removing circulating LPS to prevent receptor activation
2. preventing action of pro-inflammatory cytokines (less successful)
3. non-steroidal anti-inflammatory drugs (doesn't prevent shock)
^ these are all early stage interventions, after it becomes systemic you can only hope that supportive therapy will work
- ventilation, fluids, nutrition
112
What happens in the sepsis uncontrolled cascade?
- capillary endothelium activated in response to infectious agent
- inflammatory response activated (neutrophil adhesion etc)
- endothelium permeability causes leakage
In lungs this causes acute lung injury and respiratory distress
- red blood cells struggle to deliver oxygen to organs = tissue hypoxia
- thrombin formation causes fibrinogen to become fibrin which clumps with platelets and RBCs in the capillaries
- hypotension caused by decreased vascular tone diminished venous return, release of myocardial depressant factors
This often leads MODS, SIRS, shock and eventually patient death
113
Which term describes SIRS in association with infection?
Bacteraemia
Septicaemia
Sepsis
MODS – multiorgan dysfunction syndrome
Sepsis
114
Verotoxic E coli does not cause disease in adult cattle because...
Verotoxin is neutralized by lysosomes
115
Why does Listeria haemolysin/listeriolysin O destroy phagosome but not host cell membrane?
It is rapidly inactivated & degraded in cytoplasm
116
Anthrax belongs to which group of toxins?
A-B toxins
117
Which elements allow horizontal gene transfer (movement of genes between bacteria)?
Plasmids
Transposons
Bacteriophage
118
What is a virus?
Small infectious agent that replicates by infecting the cells of its host organism
These are called obligate cellular pathogens!
119
How large are viruses?
What are they mainly composed of?
Are they specified?
20 - 300 nm
Nucleic acid (genome) surrounded by a protein coat (capsid)
Yes, they can only infect specific cells that support viral replication = this is called virus tropism! (cell must have appropriate receptors and cell physiology)
120
What are the 3 virus shapes?
- icosahedral (foot + mouth)
- helical (rabies)
- complex (poxvirus)
121
What can be found on the virus protein coat?
Embedded surface proteins that bind to and help the virus to enter the host cell
122
What is the envelope found on enveloped viruses made of?
Where is the envelope derived from?
Made of lipids
Derived from budding from the host cells membranes (can be plasma, nuclear or internal organelles membrane)
123
Which are more stable, enveloped or non-enveloped viruses?
Non-eveloped
124
What types of virus genomes are there?
What does the genome influence?
DNA or RNA
Single or Double stranded
Negative* or Positive** sense
*reverse of positive, cannot code for protein
**can directly code for virus protein
How viruses make their mRNA
125
What are phylogenetic, how are they relevant to viruses?
Phylogenetics is the study of evolutionary relationships between biological entities
Uses genetic comparisons to work out how related viruses are
126
What are the basic functions of the following virus proteins:
Structural proteins
- capsid
- nucleocapsid
- envelope glycoproteins
Non-structual proteins
Accessory proteins
Structural proteins
- capsid = protest nucleic acid + helps binding to target cell
- nucleocapsid = fold up virus genome
- envelope glycoproteins = in lipid membrane of enveloped viruses mediate recognition of host cells + facilitate entry
Non-structual proteins = mediate genome replication, suppress immune system + change cellular environment for better cell replication
Accessory proteins = special functions
127
What is the basic virus replication cycle?
1. viral entry into body to find host cell
2. uncoating, inserts genome into cell
3. reaches nucleus and replicates via transcription
4. translation in cytoplasm to make proteins
5. virus assembles in cell membrane and new virus bud off the host cell
128
Can viruses be grown in the lab?
Extremely hard
- slow
- very specialised so need specific equipment etc.
129
Where do viruses get polymerase enzymes for replication of their genetic material?
The cell unless not provided in compartment where cell replicates (DNA virus - nucleus, RNA virus - cytoplasm) so virus will have to supply itself (so most RNA viruses)
130
How do dsDNA (double stranded) viruses replicate?
Replicate in nucleus
Uses host cells RNA polymerase - makes RNA and proteins
Encodes own DNA polymerase - makes virus genome
Envelopes and buds from cell
131
How do ssDNA (single stranded) viruses replicate?
Need active host DNA polymerase to replicate genome and host RNA polymerase to make mRNA (can only replicate in actively dividing cells eg. in bone marrow)
132
How do positive sense ssDNA (single stranded) viruses replicate?
Can make protein directly as RNA acts like mRNA
Genie replicated by viral polymerase
VERY FAST
133
How do negative sense ssDNA (single stranded) viruses replicate?
Genome cannot directly make mRNA
So, has to make positive sense copy to act as mRNA - must have own polymerase to do this (doesn't exist in host cells)
Genome then replicated by own viral RNA polymerase
(occurs in cytoplasm)
134
How do retroviruses replicate?
Enter cell
Virus RNA copied into DNA (has own reverse transcription enzyme as this is not normal)
cDNA travels to nucleus, chromosome cut and cDNA integrated (placing own genome into host cells DNA)
This is permanent, the genome will be in the cells DNA for life - can cause tumours
135
Which is more error prone, RNA or DNA virus synthesis and replication?
RNA synthesis - rapid but high proportion of genomes will have errors
DNA has proof reading and correction that's to their DNA polymerase system
136
What are some of the effects viruses can have on cells?
- transform normal cells to tumour cells
- lytic infection
- persistent infection
- latent infection that will eventually become lytic
137
What are the 2 different consequences that patients can end up with when they have a viral infection?
1. Clinical disease
- cell death + tissue damage
- host response (discharge, diarrhoea etc.)
- cancer
2. Sun-clinical infection
- no clinical signs
- can still be transmitted to other animals (nightmare for disease control)
- latent virus
- undetected effects (like fertility issues)
138
What are the 3 main factors that have an effect on the clinical outcome of viral infection?
- the virus
- the host
- the environment
139
When does genetic variation occur in viruses?
During replication of its genetic material
140
What are the two mechanisms during genetic replication of viruses that can cause genetic variation?
1. spontaneous mutation - mistake made during replication (more common in RnA)
2. Gene transfer between two viruses (usually related) in the same cell leading to recombination or reassortment of their genome = hybrid genomes (rare)
- Recombination can also happen between virus and host cell nucleic acid = host immune genes
- Reassortment happens in viruses with segmented genomes
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What is a prion?
Where are they usually found?
How do they spread?
A proteins thats tertiary structure - how it folds - has gone wrong forming a large protein aggregate (sites there taking up space and doing nothing)
In the CNS but before that, the spleen, lymph cells and nerve ends
By producing more misfiled proteins
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What is the disease prions cause in sheep?
What is the disease prions cause in cows?
How are prions spread?
Sheep = scrapies (came before BSE)
Cows = BSE
Puncture contact or indigestion
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How ca you distinguish between different bacteria?
- Morphology
- Motility
- Cell wall composition – i.e. Gram type
- Respiration (Aerobic/ Anaerobic/ facultative anaerobe/ microaerophilic)
- Colony morphology on specific plates
- Growth on specific media
- Haemolysis
- Substrate utilisation (i.e. what can the bacteria metabolise)
- Presence and absence of specific enzymes (i.e. catalase, oxidase).
- Tolerance to compounds
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What are the two techniques can be used for biochemical separation if cultures did not identify the bacteria?
1. biochemical tests (most common)
2. molecular techniques - looks at DNA sequences (new, not as common in vet med currently)
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What are the 6 biochemical tests that can be used to identify bacteria?
1. Haemolysis
- bacterias ability to lyse erythrocytes
- on blood agar looking at lyse of RBCs to access iron
2. Catalase Test
- shows bacteria respiration
- decomposition of hydrogen peroxide to H2O + O2 (fizzes when O2 produced) protecting cells from oxidative damage
3. Oxidase Test
- shows bacteria respiration
- identifies bacteria producing cytochrome c (enzyme) oxidase
- colour change blue from reduction of test compound
4. Coagulase Test
- identifies presence of bound coagulate or clumping factors
- bound coagulase causes fibrinogen in plasma to precipitate
5. Urease Test
- pH indicator as urease breaks down urea to CO2 + ammonia
- red = alkaline, yellow = acidic
6. Fermentation Assays
- ability to ferment different sugars
- look at pH change (indicator = phenol red), yellow means pH is low
- MacConkey agar
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What does selective media contain that allows it to only grow the isolate that you want?
Antimicrobial compounds that inhibit the growth of certain groups of bacteria
eg. enteric bacteria is resistant to bile salts so selective media with bile salts should kill the other bacteria so you are only left with enteric
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What is typing of bacteria?
What does it allow?
Separating bacteria within the same species
Linkage of bacteria derived from the same linage
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What are the 3 main typing methods with some examples?
Recognition of variable surface structures
- serotyping
- phage typing
DNA variation
- sequencing
- PCR
- Multi locus sequence typing (MLST)
Other
- mass spectrometry
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What is phage typing?
Using a virus that infects and lyses bacteria and seeing the effect that they have on bacteria of a same species, identifies structural differences within the species. The pattern of susceptibility leads to a phage type.
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What is serotyping?
Use of antibodies that recognise variable structures as the antibody-antigen interaction is very specific
An example is the latex bead agglutination test:
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What is the difference between serology and serotyping?
Serology: measures antibodies - uses sera from patients to measure if the patient has produced antibodies to a specific pathogen
Serotyping: uses antibodies - defines closely related bacteria by using antibodies known to bind to the antigens of that bacteria
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What is PCR?
Cyclic amplification of a specific piece of DNA using primers (small piece of DNA specific and complementary to target gene)
Allows:
- identification of pathogen
- quantification
- product can be sequenced
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What is multi locus sequence typing?
Method to classify bacterial/fungal isolates to allow epidemiological; studies/outbreak tracing
- 7 housekeeping genes in every strain
- allelic profile made
- any difference in sequence identified by allelic profile between isolates is a new allele and is given a different sequence type
154
How does mass spectrometry work?
- Isolate picked
- Suspended in buffer
- Sample put in mass spectrometer and shot with laser to produce +ve charged ion
- Ion accelerator sent to detector
- Protein profile made and matched against known bacterias
155
What is the purpose of the lungs defence system?
To exclude of remove foreign agents
156
In the respiratory tract there are two types of defence, non-specific and specific. What is the difference between the two?
Non-specific - removal of articles without recognitions
Specific - recognition of particles by the immune system
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How have animals adapted to prevent pathogen entry to defend the lungs?
Low head position to promote rest. tract drainage
Size, position and distribution of hairs within the nares
Narrow passages, turbulence and partial deposition within the nasal cavity
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How does swallowing help with lung defence?
Swallowing foreign particles is an easy way to destroy them thanks to stomach acid, so the nasal cavity has ciliary motion towards the larynx so that the particles are deposited here and swallowed
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How does the lung trap foreign particles for defence?
Mucous
- sits on top of cilia to trap particles
- contains antibacterial lysosomes which start the killing process
Cilia
- moves particles in mucous in a wave like motion to larynx for swallowing
Two combined = mucociliary escalator
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What cells produce mucous in the respiratory tract?
Goblet cells (viscous)
Submucosal/subepithelial cells (serous)
Club cells (in alveoli - serous)
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Sneezing and coughing are defence mechanisms of the lungs, what is the purpose of both and what do they indicate?
Both to remove foreign objects and/or irritants
Sneeze indicates upper respiratory tract
Cough indicates lower respiratory tract
162
What antimicrobial compounds are part of the lung defence system?
Defensins
- produced by macrophages, neutrophils and epithelium and kill bacteria, fungi and enveloped viruses by integrating into membrane and forming a pore = lyse
Lysozyme
- in mucous, anti-bacterial enzymes that break down bacterial cell walls
163
What are the main draining lymph nodes of the head/respiritory tract?
- parotid
- mandibular
- retropharyngeal
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What are the main draining lymph nodes of the thorax/respiritory tract?
Trachealbronchial
165
What is the difference between MALT and BALT?
MALT - mucosal associated lymphoid tissue
BALT - bronchus associated lymphoid tissue
MALT - aggregations of lymphocytes under ciliated epithelium
BALT - aggregations of lymphocytes under non-ciliated epithelium
166
What leucocytes would be found in healthy alveolar?
What would be found in an unhealthy alveolar?
- macrophages
- lymphocytes
- few neutrophil
- low mucous level
- macrophages
- lymphocytes
- frequent neutrophils
- excess mucous
- haemosiderophages (macrophages that have digested RBCs - indicates bleeding)
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What are two diagnostic tools (often used in race horses) that asses respiratory health by allowing sampling of the cells present?
Bronchioalveolar wash
Tracheal wash
168
What leucocytes are produced in the innate immune response of the rest. tract?
What molecules?
- macrophages
- neutrophils
- basophils
- eosinophils
- mast cells
- inflammatory mediators
- complement
- lysozymes
- surfactants
- defensives
- cytokines
169
What is produced in the adaptive immune response in the respiratory tract?
Antibodies and lymphocytes
170
What is the fate of inhaled particles in the respiratory tract?
- Trapped = swallowed
- Stimulation of receptors and reflexes = swallowed or expelled
- Stimulation of immune response and antimicrobial agents = killed
171
What is a bronchiolar spasm?
What causes a bronchiolar spasm?
Sudden constriction of smooth muscle walls of bronchioles
Irritants
Infection
Allergy (inflammation)
172
Why do allergic reactions cause bronchospasm?
They activate T and B cells. B-cells lead to type 1 hypersensitivity reactions. IgE antibodies are now formed. If comes into contact with allergy again mast cells and basophilic degranulate releasing histamine + other inflammatory mediators which cause smooth muscle in bronchioles to constrict!
173
What treatment strategies are required for the following outcomes:
Promote bronchodilation
Prevent bronchoconstriction
Reduce inflammation
Stimulate mucociliary action
Reduce allergic reaction
Prevent infection
Promote bronchodilation = agonist drug
Prevent bronchoconstriction = antagonist drug
Reduce inflammation = corticosteroid
Stimulate mucociliary action = mucokinetic
Reduce allergic reaction = hyposensitisation
Prevent infection = vaccination
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Apart from inflammatory mediators, what also plays a role in bronchiolar spasm?
The CNS activating the parasympathetic NS and therefore the vagus nerve which causes smooth muscle contraction and therefore bronchiolar constriction
175
Are fungi cells unicellular or multicellular?
They can be both
Mould = multi
Yeast = uni
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What do fungi cell walls contain?
What is fungi's relationship with oxygen?
What does it mean that fungi are heterophobic?
How do they reproduce?
What is their appearance usually like as colonies on agar?
Chitin and other polysaccharides
Aerobic
They obtain nutrients from dead organic matter or living organisms (parasites)
Both sexually and asexually
Shiny and moucusy
177
What drugs are fungi resistant to?
Antibacterial drugs
178
Label the fungi structure:
179
What are the 3 types of fungal reproduction?
Mould - hyphal growth
Yeast cells - division/budding
Some are dimorphic, they can grow in yeast or hyphal forms
180
Why are sporulation bodies important?
What are the two main types of asexual spores?
Can be used for identification in clinical samples
1. Conidia
- dermatophytes (skin infecting fungi)
---> macroconidia
---> microconidia
2. Sporangiospores
181
What media is typically used for fungi growth?
How long do you have to culture the sample before you can confirm a negative result?
Sabouraud dextrose media - specialised agar with lots of antibiotics to prevent bacterial contamination
4 weeks
182
How can you differentiate fungi?
- Look at colony characteristics
- Examine spore structure
- Features of vegetative hyphae
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If fungi invades tissue biopsy and histopathology will be done which is then looked at under the microscope.
What is a common stain used?
Periodic acid-Schiff reaction (PAS) (common H&E won't always stain)
(serology can also be used when trying to indentify)
184
Clinical
What type of fungi causes ringworm?
What type of fungi is malassezia pachydematis caused by and what is it associated with?
What type of fungal filamentous mould infection invades the nasal mucosa and turbinate bones in dogs?
Dermatophycytes that invade hair, skin, nails
Yeast, canine ear and skin infections
Aspergillus species
185
Fill in the blanks:
186
How does the indicator work for the following two bacteria on MacConkey agar?
Salmonella sp.
E. coli
Salmonella sp. - transparent + colourless as not lactose fermenting
E. coli - pink/red as lactose fermenting
187
How does the indicator work for the following two bacteria on XLD agar?
Salmonella sp.
E. coli
Salmonella sp. - red with black zone as xylose fermenting then undergo alkaline reversal to turn yellow
E. coli - yellow colonies as ferment xylose
188
Reorganise the steps of PCR so that they are correct:
189
What would the ideal vaccine achieve?
Elicit both antibody ad cellular responses - generating memory B (prevent virus binding to cell with neutralising antibodies) and T cells (attack and kill infected cell(CD-8 positive)) for future rapid response
190
What are attenuated viruses found in vaccines?
What are subunit vaccines?
What are virus vectored vaccines?
Weaker forms of the virus (not dead) - risk of disease if mutation occurs
Where the non-advantageous parts of the organism are removed - so it uses selected proteins
Where a safe virus is used as a 'vector' to deliver the genes from a pathogenic virus - no risk of disease
191
What are DNA vaccines?
What are RNA vaccines?
DNA - DNA encoding viral proteins that inset virus DNA into cells to produce virus proteins and therefore an immune response
RNA - RNA encoding virus protein that work the same as above but are more stable but cheap and quick to make
192
What type of vaccine is non-infectious and is delivered with adjuvant?
Inactivated virus vaccine
193
What is microbiota?
Combined microorganisms in particular environment (most not harmful)
194
What determines if bacteria is a commensal or pathogen?
1. location on host
2. acquisition of virulence genes
3. change in gene expression
4. host specific
5. host immune system
195
What is carriage?
A pathogen carried by a recovering or unaffected host without clinical signs (host immunity can control but not clear)
Strangles/Streptococcus equi is an example
196
What are 5 common bacteria found in the upper respiratory tract?
- streptococcus species (strangles)
- actinobacillus
- pasturella multocida (snuffles in rabbits)
- bordetella bronchiseptica (kennel cough)
- escherichia coli
All can be found in healthy respiratory tracts, it is when an overwhelming amount is found is when they should become a concern
197
If a respiratory tract infection is suspected what imaging/visual should be done?
- endoscopy/rhinoscopy
- radiography
- ultrasoundography
198
If a respiratory tract infection is suspected what sampling should be done?
- haematology
- tracheal aspiration (upper)
- bronchial lavage (lower)
- swabs
these can the lead to cytology like microscopy and culturing
199
What is the name given to commensals that that turn against the host under certain environments and host conditions?
opportunistic pathogens
200
When the host and virus interact, they determine ..?.. depending on the pathogenicity of the virus and susceptibility/resistance/tolerance of the host
- wether infection will occur
- the type of infection established
- the outcome of the virus-host encounter
200
In healthy animals, should the upper and lower respiratory tract be highly colonised with commensals?
The upper respiratory tract should be
The lower respiratory tract shouldn't be, it should be so low it's almost undetectable
201
What are the main effects viruses have on host cells that help them with replication and spread?
- morphological (lysis)
- functional (start or stop cell division)
- biochemical (induction of immune response, activate cell signalling pathways)
- metabolic reprogramming (to support viral replication and rapid cell growth)
- immunological (resistence or infection)
202
Viruses are obligate intracellular microorganisms, what des this mean?
They rely on the host cell machinery to survive and replicate their genome
203
What is viral tropism?
the ability of a given virus to productively infect a particular cell (cellular tropism), tissue (tissue tropism) or host species (host tropism)
204
What is innate immunity?
What is adaptive immunity?
Innate - non-specific and rapid response against viral infection (NK cells, type 1 interferons/ cytokines)
Adaptive - not 1st response, develops after previous exposure to a virus and is specific to that pathogen and related strains (T-cells+ antibodies)
205
Which innate immune cell plays a crucial role in host cell defence against viral infection?
How are they activated?
What/how do they prevent?
type 1 interferons which are a type of cytokine
through the activation of PAMPS
Viral protein synthesis and lead to apoptosis
206
What is an example of interferons being used as therapy against viral infection?
Against FIV infected cats
- stimulates innate immunity, decreases clinical signs, decreases co-infections
207
What does adaptive immunity rely on?
The binding of viral molecules or antigens to specific receptors on T+B lymphocytes
208
How do viruses exploit the host cell machinery?
Counteract host antiviral defenses
Hijack host cell resources
Shutdown host cell function
209
How can antibodies block viral binding and therefore entry into cells?
They bind to the surface of the virus, surrounding it and preventing it from interacting with the host cell receptors.
210
The influenza virus (RNA virus) is good example of a virus that hijacks host cell machinery to suppress host gene expression, give an example of how it does this:
Its viral polymerase captures host RNA polymerase II to initiate transcription. It then expresses endoribonuclease PA-X that takes over RNA splicing to selectively target host RNA's (like mRNA) for destruction so the host ribosome is available for viral proteins.
211
The influenza virus (RNA virus) is good example of a virus that hijacks host cell machinery to increase viral protein translation, give an example of how it does this:
The non-structural protein NS1 enhances translation at the level of initiation by binding to mRNAs and ribosomes which increases recruitment of ribosomes to the mRNA.
212
When viruses hijack host cell machinery they can form viral replication factories, what is the purpose of these?
What are they?
Enhance viral propagation
Specialised membranous compartments
213
Enveloped viruses bud off of heir host cells, what do non-enveloped viruses do?
Lyse and kill the cells by making pores in the surface or making the membrane more permeable as they need to exit through a gap in the membrane
214
What are the 5 different laboratory diagnosis's that can be done for suspected viral infection?
- detect and measure by isolating in cultured cells
- using antibody detection of viral antigens or proteins
- amplification and sequencing of viral nucleic acid (PCR)
- detection of virus proteins-specific antibody response (ELISA)
- serological tests to detect IgM, IgM or total antibody (positive serology ELISA)
215
A more practical ELISA set is a snap test, what can it be used for?
To detect antigens in serum, plasma or whole-blood
