Micro prac Flashcards
Describe how virus neutralisation tests work
POSITIVE SAMPLE - anti-viral Ab’s present, add viral Ab’s to cultured cells -> anti-viral Ab’s inhibit viral attachment and replication, no damage to cells
NEGATIVE SAMPLE
anti-viral Ab’s absent, add viral Ab’s to cultured cells where the virus can replication -> causes visible damage
What are 3 features of immunohistochemistry
- Labelled antibody
- Binds to antigen
- Localises site of replication or expression within the tissue
What are the 3 steps of PCR
- Melting: denaturing of the DNA duplex at a high temperature to yield single stranded DNA.
- Annealing: primers anneal to the single stranded target DNA sequence.
- Elongation: DNA polymerase extends the primers by adding dNTPs to the phosphate backbone.
What are 4 steps in quantitative (real time PCR)
Uses fluorescent dyes or probes to detect DNA amplification
Detect fluorescence after each cycle
Fluorescence exceeds background threshold
Allows quantification of target in original sample
Microsporum canis what is it, reservoir species, macroculture feature, clinical significance and what agar used to grow it
A fungal zoophilic dermatophyte
Reservoir -> cats, dogs, horses, humans
Macroculture -> colonies are flat, spreading, white to cream coloured, bright golden yellow to brownish yellow
Clinical significance -> ringworm in humans and skin lesions in animals
Agar -> Malt Extract Agar
Aspergillus Fumigatus what is it, reservoir species, macroculture features, clinical significance and what agar used to grow
A fungus
Reservoir -> almost everywhere on every substrate
Macroculture -> A. fumigatus -> grey-green
Clinical significance -> most common cause of invasive and non-invasive aspergillosis
Agar -> Sabouraud’s agar
Virus what need to grow in and what results in
Require Living cells (cell or tissue cultures)
Generally cause cytopathic effects (CPE) -> cells look sick
Equine Herpes Virus-1 what is the most common cells cultured within and what the cells look like in an uninfected and infected culture
Kidney cells derived from equine foetuses (EFK)
Uninfected culture -> typical mosaic-like pattern (EFK), cells grown as a monolayer
Infected culture -> virus “plaque” formed by virus growth leading to lysis of the infected cell
- the foci of the infection slowly spreads or enlarge until the entire cell culture is wiped out
Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus agalactiae what agar grown on (characteristics, what used for) and what look like in a culture
Grown on Sheep Blood Agar -> non selective medium and therefore allows the growth of a wide variety of organisms, detect sample purity, colony morphology and haemolysis
Pseudomonas aeruginosa -> blue/green pigment and sweet odour
Staphylococcus aureus -> smaller colonies compared to psudomonas
Streptococcus agalactiae -> are small colonies that are often beta-haemolytic (complete lysis of sheep blod cells in the vicinity of the colonies)
Pseudomonas aeruginosa and Staphylococcus aureus what can also be grown on (characteristics, what used for) and what can’t grow on it
Nutrient agar -> non-selective medium, less nutritious than blood agar, doesn’t support the growth of the same variety of organisms
-> easier to see the blue/green pigment of pseudomonas aeruginosa
Strep. Agalactiae isn’t supported by nutrient agar
E. coli and Salmonella typhimurium what agar grown on (characteristics and what grown on that agar), and the differences in look
MacConkey agar -> used to isolate and enumerate coliforms (Gram negative organisms of GIT of mammals)
Selective Differential Medium -> selective ingredient bile salts -> inhibit organisms that do not tolerate the presence of bile
Organisms that grow on that agar differentiated by their effect on neutral red (pH indicator) indicator of lactose fermentation
E. coli -> Yes lactose fermentation - acid reaction PINK COLONIES
Salmonella -> no lactose fermentation - alkali reaction, TRANSPARENT/PALE COLONIES
Salmonella what is the other agar that it can be grown on other than MacConkey agar and what does it look like
XLD agar -> used for the isolation and presumptive indentification of salmonella
On XLD agar salmonella colonies are black, indicative of H2S production
Methicillin resistant Staphylococcus aureus (MRSA) what agar is it grown on (characteristics and features, what grow) and what does it look like
Mannitol salt agar (MSA) is a selective-differential medium
- high salt concentration selects organisms that tolerant these conditions (surface of the skin), differentiated by their ability to utilise mannitol and detected by inclusion of pH indicator
MRSA - colonies appear opaque and are surrounded by a yellow halo
MRSA II culture can also be used -> typical colonies look rose and mauve in colour
-> antimicrobial susceptibility plate tests for the resistance and susceptibility of the isolate to range of antimicrobial agents -> growth within annular radius of less than 6mm indicates resistance
List the steps of the gram stain and the result
1) Apply crystal violet
2) leave for 1 minute -> Rinse
3) Apply Gram’s iodine
4) leave for 1 minute -> rinse
5) Acetone/alcohol -> rinse
6) apply Dilute Carbol fuchsin
7) leave for 1 minute
8) Rinse and blot dry
Gram positive -> PURPLE -> thick peptidoglycan retain satin
Gram negative -> PINK -> thin peptidoglycan, stain is lost
Using the microscope to look at bacteria, what are the 2 different conditions, objective lens, oil or non-oil, condenser up or down
1) Higher magnification ie gram smear
- 100x objective lens
- immersion oil
- condenser up
2) Lower magnification -> not for fine observations of bacteria
- x4, x10 and x40 objective lenses
- no immersion oil
- condenser down
What is the best way to test for EHV4 and EHV1 viruses and how use
Indirect ELISA
1) determining the epidemiology of EHV4 and EHV1
2) Identify horses that are latently infected carriers of EHV1 so managed on stud farms
3) Determine the cause of abortion where no foetal tissue samples can be obtained -> acute and convalescent serum
EHV4 and EHV1 what family of viruses, features and what cause
EHV4 -> Rhinopneumonitis
- respiratory disease in young horses
- treat with vaccine
EHV1 -> most important cause of viral abortion in mares can also lead to respiratory disease in foals, endemic in populations,
- can lead to abortion storms
1. respiratory spread from other horse 2. reactivation of latent virus -> keep mares separated
Aborting mare is infectious for 1-2 days from reporudctive tract (if infecting stallion) or 2 weeks from respiratory tract
Alphaherpesvirinae -> enveloped dsDNA, inactived by heat and pH, lifelong latent infection -> continours or periodic shedding
List the 6 steps of an indirect ELISA
1) Coat well with specific antigen (virus)
2) Block unused sites with unrelated Ag
3) Add test sera (Primary antibody present - Ab)
4) add enzyme-conjugated secondary Ab directed against Primary Ab
5) Add substrate that results in colour presentation of the secondary-primary Ab-antigen complex
6) read absorbance
<0.1 negative
0.1-0.2 - indeterminate (repeat test)
>0.2 positive
Acute (a) and convalescent (c) serum what are the 4 scenarios with indirect ELISAs
1) Absorbance below 0.2 -> negative with no change between a and c
2) If above 0.2 in acute and increasing in con then been exposed recently
3) If -ve and then +ve then again exposed recently
4) If +ve and decreasing or the same in con then not exposed recently
+ve >0.2
Direct ELISA List the steps
1) Coat wells with specific Ag (virus)
2) Block unused sites with unrelated Ag
3) Antibody conjugated with enzyme is added and incubated with the antigen
4) Add substrate that results in colour presentation of the secondary-primary Ab-antigen complex
6) read absorbance
<0.1 negative
0.1-0.2 - indeterminate (repeat test)
>0.2 positive
Sandwich ELISA what type of ELISA and the features
- capture antibody is used first and sticks to the ELISA plate
DIRECT - detection antibody is conjugated to an enzyme
INDIRECT - detection antibody is unlabeled and the secondary detection antibody is conjugated to an enzyme
How to graph results for ELISA and what occurs with the plateau and how to tell if animal infected recently
Absorbance (directly related to concentration of antibodies) VS Dilution of the plasma samples
Plateau -> saturation of the antigen by antibodies, this point depends on the assay
THEREFORE if doing calculations needs to be on the non-plateau areas
If infected recently then look at amount of absorbance if 4 fold increase between acute and convalescent of IgG concentration -> recent immune response
Streptococcus agalactiae, Staphylococcus aureus and Pseudomonas aeruginosa, clostridium gram positive or gram negative and shape
Staphylococcus aureus -> gram positive cocci in chains or diplococci
Streptococcus agalactiae -> gram positive cocci in clusters
Pseudomonas aeruginosa -> gram negative bacilli
Clostridium -> gram positive bacilli
Agglutination tests, when does it occur, when used and advantage and disadvantage
When Ab combines with its specific antigenic determinant or epitope on the surface of particulate Ag, agglutination occurs
Blood typing, pleuropneumonia of cattle, pullorum disease in chickens
Advantage -> animal can be held until results are obtained
Dis -> occurrence of significant number of false negatives and false positives
Haemagglutination assay what does it measure and the 3 scenarios
Measurement of antibody
1) No antibodies present -> so cells settle to the bottom -> red dot
2) Antibodies present -> Antibody binds to the RBCs creating a raft (murky)
3) Too many RBCs for the amount of antibodies present -> some of the RBCs settle to the bottom (red dot)
ELISA controls
1) Negative control for the serum (serum without antibodies)
2) Antigen control (for each antigen testing for)
Haemagglutination assay control
1) Control without serum (no serum control) so just has RBCs -> to test whether RBCs are viable
Zinc Sulphate precipitation what does it test and therefore when most commonly used in the field and normal values
Determining the amount of Ig transferred is based on the observation that Ig may be selectively precipitated from serum by ZnSO4
- generally used to detect failure of maternal Ig transfer -> thoroughbreds
Normal serum contains 80g/l protein of which 10 to 30g/l are Ig proteins
Interpretation of Zinc Sulphate precipitation test
Compare the relative turbidity of the sample (degree of precipitation) to the negative control.
Greater than 8 g/l
-> precipitate either suspended or largely at the bottom of the vial, very large amounts of the immunoglobulin may produce precipitate that settles to the bottom
4-5 g/l
-> light particulate precipitate with little or no precipitate settling to the bottom of the vial
- A more sensitive and quantitative assay could be used to determine or could just treat the foals
Less than 4 g/l
-> clear, hazy, or even milky without flocculent or particulate precipitate in the vial - TREAT with colostrum
Electrophoresis function, what does it detect and how used
Serum proteins may be separated by electrophoresis
Tracing obtained usually identifies at least 6 major protein fractions: albumin, alpha1, alpha2, beta1, beta2 and gamma globulin’s
Used as first order assessment of Ig content of serum, also diagnostic significance in detecting abnormalities of a variety of other serum proteins
Monoclonal gammopathy what characterised by and when generally seen with
Electropherograms which have the production of a large amount of a single Ig protein
- Tumors (Cancers) of lymphocytes in domestic species are common and generally caused by viruses, involves the transformation of either T or B lymphocytes. When lymphocytes are transformed can produce high levels of certain Ig proteins
Polyclonal gammopathy what characterised by and when generally seen with, give example
Electropherogram in which there is an increase in all major immunoglobulin classes
- result from reactive and chronic inflammatory processes resulting from severe chronic infections (viral such as feline infectious peritonitis and feline enteric coronavirus) and chronic immunological diseases and some neoplasms
List 4 physical and 2 biological quantitative assays of viruses
Physical 1) Ag capture ELISA 2) electron microscopy 3) haemagglutination, 4) quantitative PCR • Biological – requires viral replication, therefore viable virus and viable host cells 1) plaque assays 2) end point titration
What is a bacteriophage and what occurs to a cell culture when they are present
Viruses that parasitise bacteria and are the most abundant life-form on the planet
The release of bacteriphage from a bacterial cell results in lysis of that cell, therefore bacterial cell culture will contain small clearings, or holes called plaques which represent colonies or clones of bacteriophage
What is the most common method used to assay bacteriophages, what is the units and how to calculate
Plaque-count technique -> 10 fold dilutions of the phage are more and pipetted into melted agar maintained at 45-50degrees for 18 hours, sensitive bacterium is added and poured onto nutrient agar plate
One plaque originates from infection of one bacterial cell from one bacteriophage (PFU - plaque forming units) -> counting the number of plaques allows estimation the number of phage particles per ml of the original suspension
Virus titration in cell culture what can you detect and what also called
Determining how much virus is in the samples you have been supplied with -> serial dilution to an end-point
Also called end point titration assays
When virus isolation is required what are the two important factors and examples
1) Temperature -> optimal conditions -80degrees
- less than optimal conditions -15, +4, +20, +50 degrees
2) Time -> get to the lab in as short a time as possible after collection
Feline herpesvirus 1, feline calicivirus and feline parvovirus enveloped or non-envelopes and what type of nucleic acid and important features
Feline herpesvirus - 1
Enveloped and dsDNA, latent infection, inactived pH and heat, acute respiratory disease
Feline calicivurs
Non-enveloped, ssRNA, relatively resistant to heat and detergent, kittens most susceptible, upper respitaory tract transmitted via aerosol or direct contact, oral nasal route
Feline parvovirus - panleukopenia virus
Non-enveloped and ssDNA, highly resistant to environment, infect rapidly dividing cells (GIT, foetus -> cerebellar hypoplasia, bone marrow -> panleukopaenia and fever)
How to identify virus growin in a viral growth cell culture
The cytopathic effect (CPE) -> the morphological changes in the cells, produced by viral growth in monolayer cell cultures is sometimes characteristics enough to allow immediate grouping of the virus
End point titration assays what results from low dilutions, high dilutions and intermediate dilutions and what is the comparison calculation and how to calculate
Low dilutions – all cultures infected
High dilutions – no cultures infected
Intermediate dilutions – some infected and some uninfected cultures
• Titre expressed as 50% tissue culture infective dose (TCID50)
• Reciprocal of the dilution at which
• exactly 50% of the cultures are infected, and
• 50% are uninfected
End point titration assays what results from low dilutions, high dilutions and intermediate dilutions and what does the titre represent and how to calculate
Low dilutions – all cultures infected
High dilutions – no cultures infected
Intermediate dilutions – some infected and some uninfected cultures
• Titre expressed as 50% tissue culture infective dose (TCID50)
• Reciprocal of the dilution at which exactly 50% of the cultures are infected, and 50% are uninfected
End point titration assays what will you see at the end and what does that mean
Need to stain the plate
If virus grows the cell monolayer will be destroyed by the virus (clear area)
In wells where there is no viable virus (beyond the end point of the dilution series) the cell will appear healthy and still form a monolayer (purple/blue wells)
TCID50 define and what does it mean
If the TCID50 is detected at 10^-6 then what does the original sample must have contained
One TCID50 is the volume of virus sample that will infect 50% of inoculated cells, therefore, if a virus sample is diluted to contain 1 TCID50/unit volume and 4 wells are inoculated with 1 volume of this, 2 should be infected and 2 will be uninfected
Original sample must have contained 10^6 or 1,000,000 TCID50s
Virus titration assays what controls present
1) Cell control -> to make sure the host cells are viable
2) Control virus -> only present if determining the effect a certain variable has on the amount (viability) of virus
List and describe the 4 ways viral activity may be assessed
1) Animal inoculation -> use natural host or laboratory animals -> may not be economically practical and depending on virus different routes of inoculation are used
2) Chick embryo -> endpoint titration conducted by inoculating embryonated hen eggs infectivity based on
- haemagglutinating activity (Newcastle, influenza). death of embryo, pock count following
3) Tissue culture -> used in 3 general ways for titration of virus
1. tissue culture infection dose determination
2. plaque counting for those viruses which cause lysis of the cells
3. plaque counting for those oncogenic viruses which cause focal areas of cellular proliferation
4) Direct virus counting -> possible with electron microscopic procedures
Serum-Virus Neutralisation what does it measure, what type is used in the practical and what used for
Looking for antibodies against viruses only -> antibodies that neutralize and prevent infection
Beta procedure -> titrates the antibody/serum sample and incubates these dilutions with a known (constant) amount of virus -> in order to accurately compare the antibody concentrations in different samples
Serum-Virus Neutralisation test what are the 4 controls and what are they there for
1) Positive serum control = a characterised serum containing antibodies to the virus of interest
2) Negative serum control = a characterised serum known not to contain any antibodies to the virus being assayed
3) Uninoculated cell culture control = cells that are incubated in normal cell culture media only, without serums or viruses (ensure healthy cells)
4) Virus control = the virus used is retitrated to ensure the correct concentration is included in the test
What is the standard amount of virus used in serum-virus neutralisation tests, how measured and what if not correct
Standard is 100 TCID50
Measured via virus control -> ensure that it is 100 TCID50
If the virus is less than 100 TCID50 then will get a higher antibody titire than actually there
If the virus is more than 100 TCID50 then will get a lower antibody titre than actually there
serum-virus neutralisation tests what are the two types and what each type measures for
1) Determine Ab titres
- need known virus and susceptible cells
2) Determine unknown virus
- need known antibody and susceptible cells
What are the 5 sites of inoculation of an embryonated egg and function
1) Amniotic sac -> where the embryo lies
2) Chorioallantoic membrane -> highly vascularised membrane that is the respiratory organ of the embryo
3) Yolk sac (chlamydias) - attached to the embryo and contains nutrients for developing embryo
4) Intravenous inoculation
5) allontic sac - separates the chorioallantois from the amnion
Why is it important that flocks supplying the fertile cells for inoculation has never been infected with the virus of interest and what is this called
Anitbodies from the mother will be present in the yolk sac
Hen cannot be vaccinated or exposed to virus of interest
Need to be specific pathogen free
What is the most common area in the embryonated egg to inoculate, why and what done there
Allantoic inoculation is the easiest
o Get a high titre, high volume recovery
o Used for influenza A (Orthomyxoviridae) virus and NDV (Paramyxoviridae)
vaccine production
Endpoint titration and hameagglutination assays what endpoint when determining virus and antibody concentration
VIRUS
Endpoint titration -> end point = 50% well with CPE (infectious dose TCID50)
Haemagglutination -> last dilution with haemagglutination (Haemagglutinating units (HAU))
ANTIBODY
Endpoint titration -> end point = last dilution of serum without CPE
Haemagglutination -> last dilution of serum inhibiting the haemagglutination
What 2 viruses inoculated from embryonate eggs can then be assessed and identified via haemagglutination and why
A number of avian viruses such as influenza (Orthomyxoviridae) and Newcastle Disease (Paramyxoviridae) are able to attach to surface of RBC and form a lattice -> HAEMAGGLUTINATION
What is one HAU defined as and how calculate Haemagglutination titre of the antiserum
1 HAU = haemagluttination unit defined as the amount of virus present in the last dilution of sample to cause complete haemagglutination -> minimum concentration at which the virus will haemagglutinate the RBCs
Reciprocal
If this occurs at 1/320 dilution then the HA units of the original sample is 320 HAU
What is the difference between haemagglutination and haemagglutination inhibition and what does each measure in terms of unknown virus and how used to determine antibody
Haemagglutination -> when testing a haemagluttinating virus can determine the concentration of that virus in terms of HAU
Haemagglutination inhibition -> If serum containing antibody specific for a virus is present it will inhibit that virus from binding and haemagglutinating those RBCs -> identification of the virus
ANTIBODY
haemagglutination can also be used to test serum for the presence of antibody
Staph Aeurus screening what does it look like on 3 different plates
1) SBA- usually have a yellow colour and are haemolytic
2) MSA- opaque colonies surrounded by a bright yellow zone
3) CHROMagar MRSA II- pink colonies with a pale background
List the 4 controls used in the ELISA and why
1) Negative control -> ensures there isn’t any colouration (absorbance) for other reasons either then presence of proteins
2) EHV1 positive -> ensure that antibodies present will result in absorbance above 0.2
3) EVH4 positive -> ensure that antibodies present will result in absorbance above 0.2
4) Variations of EHV strain (antigens) also within other columns -> ensures that there is ability to identify the correct virus -> would expect EHV1 positive to bind to EXHV variation if not maybe wrong variation
Neonatal isoerthryolysis what occurs and how to test for with haemagglutination
o Mare exposed to foreign RBC antigens of stallion -> mare develops antibodies against this ->mare has another foal with
same RBC antigen as stallion -> foal suckles -> IgG against antigen of RBC of foal/stallion comes into contact -> haemagluttination
o If the mother tests positive to haemagglutination of the stallions
blood and hence potentially its foals blood, colostrum from the mare cannot be given, as this may cause neonatal isoerthrolysis.
As a result, other colostrum should be given
What would happen if a suspension of bacteriophage was injected into a rabbit and three weaks later serum
from rabbit was added to each of the tubes in the test five minutes before the addition of 3 drops of 18 hour
culture of host bacterium
There would be antibodies developed against the bacteriophage (as it is a virus)
- Hence if there was enough antibody in the serum to bind to the bacteriophage, there may be no plaques
seen
- However, if the serum was diluted so some plaques could remain unbound by the phage antibodies,
than the bacteriophage would still be able to cause plaques
List 3 bacteria of veterinary importance that can be identified via phage typing
Pseudomonas aeruginosa, E coli, Salmonella, Clostridium difficile
Newcastle disease - Avian Paramyxovirus 1 what nucleic acid, 3 important molecules, which one for virulence, transmission, 3 main virulence groups, diagnosis and treatment
ssRNA -> survives in environment
Haemagglutinin and Neuraminidase and Fusion protein (important for the virulence)
Virus replicates in epithelia of respiratory and GIT (hence why see signs here) -> faces and respiratory tract
1) Velogenic (viscerotropic -> 90-100% m and haemorrhagic intestinal, neurotropic 10-20% m with nervous signs
2) Mesogenic - mortality in young birds
3) Lentogenic - mild disease
DIagnosis -> tracheal or cloacal swabs
Treatment -> test and slaughter or vaccine if endemic
Avian influenza nucleic acid, virulence, replication, clinical signs, control and diagnosis
Enveloped - fragile
RNA - 8 segments with haemaglutinin and neuromnidase important
- low and high pathogenic straings
Low -> respiratory, mild
High -> sudden death, diarrhoea, laboured respiration
Control -> asymptomatic wild waterbirds
DIganosis -> swabs, nasal tracheal and cloahal, PCR
