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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?

  1. the sample type and the timing of the sample
  2. unambigous mark (no missunderstandings)
  3. accompanying letter
  4. 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


course of the disease


What should be included in the case histoy at the herd level?

time of disease onset in the herd


course of the disease


What kind of sample material can be submitted?

swabs: conjunctival, nasal, faecal

blood: EDTA, heparinized, serum



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


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



What samples should be sendt in case of respiratory symptoms?

Conjuctival + nasal swabs


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?


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?


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


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?



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?


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

  1. Precipitation
  2. Adsorption
  3. Ultrafiltration
  4. Dialysis
  5. 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


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?

  1. Ultra thin section
  2. Negative contrast method
  3. 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



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


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?


DNA/RNA virus

Single/double stranded nucleic acids


how do we investigate group-specific antigens?



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?



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



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?


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?


vaccine production

experimental animal infection


What are the physical assay methods of titration?

Direct particle counting - EM



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



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?



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



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)

1.) VN (20 or 48 h incubation, virus concentration: FFD50 / TCID50)

2.) 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)

1.) VN

2.) 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