Plenary Flashcards
Why do we need specified laboratories?
- for regulations or rules
- when there are suspected zoonosis
- Eradication - contorlling the free status
- certifications: free status, SPF herd (spesific pathogen free), transport, competitions
- when pathological findings are not sfficient to establish a diagnosis (influenza)
- herd diagnosis: vaccination programs, endemic virus present in the herd
What are the two methods of laboratory diagnosis
- Direct virus demonstration
- the whole virus is isolated, including its components: proteins, NA
- Indirect processes
- Antibody detection from the infected animals
What decides what kind of sample we take, how much and how it is sendt?
the aim of the examination
What are some things to consider when the sample is being collected and transported?
- the sample type and the timing of the sample
- unambigous mark (no missunderstandings)
- accompanying letter
- period and circumstances of the sample’s shipping to a diagnostic institute
What is the most important part of the accompanying letter?
it should include a suitable case history together with the sample
What are some thing that an accompanying letter should include?
- name, number, address of the vet and the owner
- personal data of the animal: ID, name, age, sex, vaccination history
What should be included in the case history on an individual animal level?
Time of disease onset
symptoms
course of the disease
What should be included in the case histoy at the herd level?
time of disease onset in the herd
symptoms
course of the disease
What kind of sample material can be submitted?
swabs: conjunctival, nasal, faecal
blood: EDTA, heparinized, serum
Organs
What should each lab inform the customers about?
- ehich diagnostic investications they do, what kind of tests
- prices
- what samples that should be sendt
- the best way to submitt the test
- time frame for results
- diagnostic report with a good explanation
What kind of sampeling should be done when usind direct methods?
Cadaver: tissue, organs
body fluids
non coagulated blood
what kind of samples should be sendt when doing indirect method investigations?
coagulated blood, serum
body fluids
secretions
incase of dead animal: heart blood, bloody liver (juice)
What samples should be taken in case of herd diagnosis?
there should be a statistical sample collection: depending on how many animals are present and how many is showing symptoms
in individual diagnosis what should be sendt?
paired sera, once from the early phase and then from 2-3 weeks later, to compare the antibody content
What are the categories of symptoms?
respiratory symptoms
gastroenteric symptoms
neurological symptoms
vesicula and skin disease
abortion
What samples should be sendt in case of respiratory symptoms?
Conjuctival + nasal swabs
EDTA-blood
in case of dead animal: lung + lymph, spleen, kidney, liver
What samples should be sendt in case of gastroenteric symptoms?
Live: faeces, vomit
dead: GI samples, liver, lymph, lung, spleen, kidney
What samples should be sendt in case of neurological symptoms?
live: CSF, conjunctival, nasal swabs, EDTA-blood
dead: Brain + spinal cord, lung, liver, spleen, kidney
What samples should be sendt in case of vesicular and skin diseases?
vesicular fluid, skin, lesions, biposia, swab
(some should be frozen)
What samples should be sendt in case of abortion?
fetus or organs
placenta, amniotic sac
Blood of the dam should always be included
What is important when sending samples for serological detections?
sera pair investigation
- acute phase sera or plasma + 2-4 weeks later sera or plasma
Some important things about shipping
as fast as possible/personally
arrival within 24h - depending on the type of sample
temp should be 4 degrees
appropriate wrapping
accurate address
During direct demonstration of viruses, how are the viruse isolated?
the virus will be injaculated and propagated it needs 2-3 weeks to be diagnosed
this is an old method but is very important, used to identify new viruses and mutant detection.
what is the prerquisite of in vitro propagation?
the infective virion –> early phase –Z virus shedding
How are organ pieces processed?
homogenized and then diluted with a phosphate buffer + antibacteria in 1:10 dilution
1st centrifugation: cell debris, quartz sand (1000 x g 10 minutes)
2nd centrifugation: purification (3000 x g 10 minutes) or filtration
How are swabs processed?
they are rinsed for 1-2h
1st centrifugation: cell debris, quartz sand (1000 x g 10 minutes)
2nd centrifugation: purification (3000 x g 10 minutes) or filtration
How are buffy coat processed?
it is centrifuged to separate it from the non-coagulant blood (hemoglobin is toxic for cell cultures)
WBC survives longer than RBS in water - heamolytic resistance or buoyant densisty
How are faeces and semen proccesed?
faeces: diluted and put in i mixing machine to be diluted
Semen: the cells needs to be be broken
What cells should be used when making a primary monolayer cell culture?
organs which are rich in epithelial cells
actively dividing cells - young animals
kidney testicle, thymus, embry
they should be removed aseptically and processed within a few hours
how are organs used for cell cultures proccesed in aseptic circumstances?
the outer membranes are removed, and then it is cut into small pieces
Trypsin should be added to spearate the cells by digestion
the cell-containing suspension is repeatedly removed and replaced with trypsin solution
then the trypsin effect is blocked in an ice bed
then sedimaentation of the cells by centrifugation and the removal of trypsin
suspension of cells in culturing medium
the cells are counted: 200 000 cells/ml
cell suspension is tranferred into a steril culturing flask
then it is incubated at 37 degrees 3-5 days
What is MEM?
Minimal Essential medium: the optimal environment isotonic, isoionic, isoosmotic, nutritive, antibiotics, antimycotics, indicator foeatal, calf serum
Why is calf serum added to the cell culture?
it is a protein source + mediator for cellular division
it is taken from colostrum free calves 5-10% to mkae cells grow, 2% to keep the cells alive
What are some examples of sterile culturing flasks?
Plate, Roux-flask, petri dish
What is meant by inoculation?
the introduction of a virus into a culture medium
What is subculturing?
passage
the medium is removed along with the cells from the wall of the flask
diluted, and put into a fresh culturing flask
Secondary culture is formed
with fresh, homogenous and in an increased amount
How many passages are possible and why?
2-3x
the cell culture changes in subsequent passages, so the sensitivity might decrease
What is cellular cloning?
when a single cell is cultivated and a permanent cell line is produced
What types of cell cloning is there?
Diploidic and aneuploidic (tumour)
what are the advantages of celllar cloning?
they are genetically homogenous, making them comparable
unlimited number of passages
long term storage
What are the disadvantages of cellular cloning?
the sensitivity for infections varies
contamination may occur
there may be presence of active oncogenes
Why do we use adsorption when we inoculate cell-cultures or embrionated egg?
to avoid CPE caused by toxins
When do we suspen in cell-cultures or embrinated eggs?
when viruses needs dividing cells
What is meant by co-cultivation?
isolation of cell-associated and latent viruses
simultaneous precessing and mixing of virus-infected and healthy cells
What is a white blood cell culture?
a buffy coat culture
used for WBC specific virus propagation (ASF)
What do we use suspension cultures for?
Vaccine production
wha tis microcarrier cultures?
a support matrix that allows cells to adhere on the surface (microcarrier beads)
the aim is the increased surface
What is shell vial assay
the virus is forced to find a cell
first it is inoculated
centrifuged
then the virus will sediment to the cell receptors after 16h incubation
mab staining to see the signs of virus
What are the requirements of the egg in terms of inoculation?
embryonated
SPF
white shelled - easier transillumination
What must be done to the egg before inoculation?
transilluminated - check embryo development and the localisation
disinfection (iodine, ethanol, formaldehyde)
Drilling through the shell (needle, lancet, electric drill)
Where on the egg should the inoculation happen?
depends on the virus and on the age of the embryo
What and when can viruses be injected in the yolk sack?
picorna, reo, adenoviruses
5-7 days
What and when can viruses be injected in the allantoic cavity, amniotic cavity
orthomyxo, paramyxo, coronavirus
9-12 days old embryo
What and when can viruses be injected into chorio-allantoic membrane?
pox, herpes virus
10-13 days old embryo
What and when can viruses be injected in intravenous inoculation?
orbivirus
16-17 days old embryo
What do we do with the egg after it has been inoculated?
seal it with paraffin or glue
incubate it for 33-37 min
and control it
How do we control the inoculated egg?
- Transillumination
- egg necropsy after 4-5 days
- check for dwarfism, distorition, death
- CAM: pock (size, inflammation, haemmorrhage, necrosis
- haemagglutination test on the allantoic fluid
Examination of the cell culture?
Check daily for CPE
if there is no CPE: Blind-passage or auxillary examinations should be performed: EM, HA, IF, IP
isolation, then plaque isolation, purification and then gain the virus strain
Examination of the embryonated egg?
daily examination, take samples from the allantoic fluid, CAM and embryo
Auxillary examinations: HA, EM, histopathology
What are the diagnostic approaches in regards to experimental infection of living animals?
rebies, african horse virus, arbovirus: intracerebral inoculation of suckling mice
classical swine fever - african swine fever differentiation
How are vaccines produced with use of living animals?
vaccines are produced from the organs of infected animals: rabbit haemorrhagic disease, classical swine fever
Vaccine controls on living animals?
harmless, efficient
How can we release the viruses from the infected cells (ø quartz)?
Mechaniacal method: thawing (3x) - ice crystals may be formed by freezing at 20 degrees
sonication - ultrasound (heat generation)
detergents (for nucleic acid incestigation) lipid in cell membrane will be opened
What is rough purification?
Centrifugation or filtration (450nm pore size)
What are the 5 methods of concentrating the viruses
- Precipitation
- Adsorption
- Ultrafiltration
- Dialysis
- Pelletisation
mention some concentration and purification methods (examples)
affinity chromatography
Density gradient ultracentrifugation
With what method can we precipitate viruses?
we precipitate the proteins, with:
(NH4)2SO4, PEG 6000, ethanol, resolve in buffer
With what method do we use adsoprtion for concentrating viruses?
they will be adsorbed on a filter/gel
non-specific chromatography
AL(OH)3, Ca3 (PO4)2
How do we concentrate viruses with ultrafiltration?
With hydrostatic pressure, the pore sizes are smaller than the diameter of the viruses
How do we concentrate viruses with dialysis?
Osmotic pressure
through a semi-permeable membrane
how do we concentrate viruses with pelletisation?
they will be centrifuged in a high speed for a long time
can achieve 1000x concentration
What is affinity chromatopgraphy?
virus spesific antibodies are bound to the chromatography column matrix and will be adsorpted
rinsing
elution with a buffer causing a pH change
What is density gradient ultracentrifugation
based on stokes law.
it is a powerful technique for separating complexes based on their molecular masses.
it will sediment viruses in dense solutions
How will de buoyant density be after density gradient ultracentrifugation?
nucleic acid/protein/lipid ration (incomplete virions are lighter
what is the difference between preparative ultracentrifugation and analytical ultracentrifugation
- Preparative u.c. –> for purification
- Analytical u.c. –> for measuring buoyant density
Virus identification: electron- microscope investigation
we can recognise the virions by their morphology, this is an advantage incase the virus does not replicate in the cell-cultures. the size and the shape of the virus is detected
What are the metho of electron-microscopic investigation?
- Ultra thin section
- Negative contrast method
- Immune-electron microscopic method
How does ultra thin section work
an ultr thin section is created from the organs
fixation: glutaraldehyde
embedding: durcupan resin
tungsten or uranium salts treatment (electron absorbent)
How does the negative contrast emthod work?
a diluted sample is treated with contrast material and dries onto the grid (the contrast material will not bind)
How does the immune-electron microscopic method work?
a diluted sample is centrifuged
the immune serum will be added to the sample and there will be precipitation
centrifugation will precipitate the virus in the sediment
What are some physico-chemical tests to investigate virus identification?
Electron-microscopic investigation
Chloroform resistance
halogenic uridin derivates
acridin-orange staining
chloroform resistance testing?
enveloped vs non-enveloped
halogenic uridin derivates?
DNA inhibitors
Acridin-orange staining
ssNA vs dsNA
Virus antigen detection methods
immunofluorescence
immunoperoxidase
complement fixation
agar gel immune diffusion test (AGID)
counter current immune electro-forezis (CIEF)
Radiou immuno assay (RIA)
Enzyme linked immunosorbant assay (ELISA)
How does immunofluorescence test work?
virus-spesific antibody is labelled with fluorescence staining, and after it binds to the infected cell, the cell culture is investigated using fluorescent microscope: the infected cells will appear with yellowish-green spots
How does the immunoperoxidase test work?
virus spesific antibodies labelled with peroxidase enzymes are added to the surface of the cell culture, the antibodies will bind to these cells, and after adding the substrate of the enzyme, there will be a colour change: revealing the prescense of the virus
How does complement fixation test work?
the antigen is released off the cells
FMD: the hemolysin lyses the sheep RBC in the presence of the complement
How does ELISA work?
enzyme linked immunosorbant assay
direct/indirect
competetive (discriminating/multispecies antibody detection)
sanwich - FMD
What are the microscopic investigations (virus identification)?
CPE, tissue specificity, type of pocks, haemadsorption
What are the EM investigations? (id of virus)
the dimension, size, shape of the virion, its symmetry, surface
what are the physico-chemical investigations of vegetative viruses?
enveloped/nonenveloped
DNA/RNA virus
Single/double stranded nucleic acids
how do we investigate group-specific antigens?
AGP, IPA, IF, IEM, ccIEF, ELISA, RIA, HA
How do we determine the serotype (id of virus)
virus neutralisation test, haemagglutination-inhibition test
How do we determin the subtype, variant?
clinical signs
investigation of nucleic acids
investigation with monoclonal antibodies
What is the theory behind nucleic acid hybridization?
when there is heating of dsDNA the double helix will split, and when it is cooled doen the complementary threads will rejoin
What are the different steps of nucleic acid hybridization?
A probe: labeled oligonucleotide (DNA or mRNA) complementary to the viral genome
Labeling: isotope or enzyme
sample + probe
heating then cooling
washing (removal of unbound probe)
autoradiography or substrate
What is the DNA microarray technique?
DNA samples are bound to glass slides or membrane filters (DNA chip)
measure te expression levels of large numbers of genes simulteneouslt or to genotype multiple regions of a genome
- hybridization with fluorescently labeled probes
- laser scanning
- computer analysis: identification, typing of viruses, comparison of virus strains, genetic relationship
What is PCR?
Polymerase chain reaction
used to amplify a single or a few copies of a DNA
What is needed during the PCR?
Template
primers
free deoxy-nucleotides
thermo-resistant polymerase
- Heating and cooling in cycles
might need reverse trancription if it is RNA
How do we detect the DNa after PCR
we need atleast 2 copies of the fragment
we can use agarose-gel electrophoresis, NA hybridization
What is real-time PCR?
There will be fluorescent labeling
laser detection of amplification products, computer analysis
quantification
What is sequencing?
is the process of determining the nucleic acid sequence – the order of nucleotides in DNA.
what is the name of the methods used for sequencing?
Sanger’s method: polymerization
polyacrylamide gel-electrophoresis
What is needed for sequencing?
Template
primer 6 deoxy-nucleotides
labeled dideoxy nucleotides
polymerase enzyme
this will lead to the polymerization of the complementary thread
What are dideoxy nucleotide?
chain elongation inhibitors of DNA polymerase used in sanger method for DNA sequencing
What is the aim when titrating viruses?
Diagnostics
vaccine production
experimental animal infection
What are the physical assay methods of titration?
Direct particle counting - EM
¨Haemagglutination
What are some Biological assy methods of titration?
Determination of the infective titre (endpoint method)
Plaque assay
Focus assay
Pock formation
Why and how do we do Endpoint dilution (infective titer)
In vitro propagation and identification
Method: serial tenfold dilution of the virus suspension, inoculation of the cell-cultures with each dilution. Incubation ( the period of inoculation is characteristic to the virus)
What does it mean when we have CPE in 50% of the inoculated cell-cultures?
we have an infective titer
it is the highest dilution of the virus, which cause CPE in the 50% in the inoculated cell-cultures, characteristic to the virus suspension
What is the virus concentration unit?
1 tissue culture infective dose
TCID50/ml
What is the virus concentration unit called when it is performed in embryonated eggs?
EID50: pock assay
What is the virus concentration unit when it is performed in experimental animals?
LD50
What are the two methods of calculation of virus concentration?
Reed-Muench method
Spearman-Karber method
What is the essence of Spearman-Karber method?
easier than the Reed-Muench method, no need of lot of numbers, only the data of dilution which is the next higher dilution of the dilution with 100% response (CPE)
How to perform plaque counting (infectiv titer)?
A serial tenfold dilution of certain virus suspension inoculation of the cell cultures from each dilution
adsorption: 1 hour 37 degrees
it is covered with semisolid maintance: agar or CMC
incubate at the time characteristic to the virus
local CPE in th einoculated cell- cultures
stained with vital stain
What are the vital stains of plaque counting called?
Gentian purple
Evans blue
Janus green
What is the unit used to evaluate the plaque counting?
the concentration of the virus suspsension given in plaque forming units = PFU/ml
How do we get the infective titer of the virus suspenion, using plaque counting?
At the inoculated cell-cultures containing less than 10 plaques we take the average of the plaque, multiply it with the negative reciproc of the dilution level
this gives the infective titer of the suspension
How do we do hemagglutinating titer determination?
The sample is put on a micro-haemagglutination plate known as takatsy plates
it is performed a twofold dilution of the virus suspension
added washed erythrocytes of the proper species (virus dependent)
then its incubated at the proper temperature for the virus
What is meant by hemagglutinating titer?
The highest dilution of the virus suspension, in which we can observe hemagglutination
What is meant by hemagglutinating specturm?
the different hemagglutinating viruses have different hemagglutinating spectrum, meaning different viruses react to diffrent RBC form different species, have different incubation time and at different degrees
what does the physical assay count?
the number of particles , regardless infectivity
what does the biological assay detect? (pros/cons)
only infecting viruses
incomplete/defective particles are not detected
purification may damage virions
successful infection may also depend on the cell metabolic stage
What are indirect virus demonstration`?
Using serological methods to indicate the presence of antibodies
What are the advantage of serological methods?
higher chance of demonstration
it is cheap
what are the disadvantages of serological methods?
they cannot differentiate the antibodies between
- maternal
- vaccine
- seroconversion
what is the principle of virus neutralization?
the sera is heat activated to inactivate non-spesific antibodies. and in th epresence of blocking antibodies reacting with antibody receptors of viruses. the virus is not able to adsorb to the cells
giving us an serotype-spesific result
What is the constant virus varying serum dilution method?
method for antibody detection
How to perform: Constant virus varying serum dilution method?
serial twofold dilution from the sera
100 TCID50 (or EID50 or PFU) viruses
incubation 1h 37°C –> antibodies neutralize the viruses
inoculation of the cell-cultures with each dilution
incubation 37°C for several days
CPE
what determines the serum neutralizing titer?
the highest dilution of the serum where there is 50% CPE
How to perform: rapid fluorescent focus inhibition/fluorescent antibody VN test?
(RFFIT/FAVNT: eg. rabies)
- ) VN (20 or 48 h incubation, virus concentration: FFD50 / TCID50)
- ) addition of fluorescent labelled virus-specific antibody: residual virus titer shows the antibody content of the serum sample
How to perform: indirect immunfluorescent test? (ilFA)
- ) VN
- ) addition of fluorescent labelled host Ig-specific antibody: the fluorescence shows the antibody content of the serum sample
how to perform: constant serum varying virus dilution method?
Neutralization index calculation for virus identification
- two serial tenfold dilution of the given virus suspension
- adding
- negative serum
- positive serum
- incubation 1h 37°C
- inoculation of the cell-cultures from each dilution
- incubation 37°C for several days
- CPE
How to evaluate constant serum varying virus dilution method?
Neutralization index= virus+negative serum titer/virus+positive serum titer >2
What is the plaque reduction test?
See if there are fewer plaques compared to the control viruses.
the degree of the reduction may be 50% (rabies), 75% (equine arteritis)
what is the plaque reduction titer of the serum?
the highest dilution of the serum where 50% of the cell cultures the plaque formation is inhibited
How is haemagglutination inhibition performed and for what type of viruses?
only for hemagglutinating viruses
serial twofold dilution of the serum sample
4-8 HAU of virus to each serum dilutions
incubation 1h at a temperature characteristic to the virus
addition of washed erythrocytes (1%)
what is the goal of serum hemagglutination inhibition titer?
the highest dilution where there is no HA - not enough antibodies to block the HA protein
what is the validity of the serum hemagglutination inhibition titer?
a virus between 4-8 HAU
actual titer of the known positive serum did not change more than one dilution level since the previous test
What are the two types of antibody detecting ELISA?
cELISA: competitive ELISA
iELISA: indirect ELISA
What are the advantages of competitive ELISA
advantage: „multispecies” kits, discriminative ELISA-s (e.g. IBR)
antibody in the serum binds / no antibody
secunder antibody: peroxidase labelled, specific to viral antigen
binding / no binding („competition”)
binding: color change – sample is negative
no binding: no color change: sample is positive
What is the indirect ELISA
- antibody in the serum binds / no binding
- secunder antibody: peroxidase labelled, host Ig-specific
- binding: color change – serum sample is positive
- no binding: no color change – serum sample is negative
