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1

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

2

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

3

What decides what kind of sample we take, how much and how it is sendt?

the aim of the examination

4

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

5

What is the most important part of the accompanying letter?

it should include a suitable case history together with the sample

6

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

7

What should be included in the case history on an individual animal level?

Time of disease onset

symptoms

course of the disease

8

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

time of disease onset in the herd

symptoms 

course of the disease

9

What kind of sample material can be submitted?

swabs: conjunctival, nasal, faecal

blood: EDTA, heparinized, serum

Organs

10

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

11

What kind of sampeling should be done when usind direct methods?

Cadaver: tissue, organs

body fluids

non coagulated blood

12

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)

13

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

14

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

15

What are the categories of symptoms?

respiratory symptoms

gastroenteric symptoms

neurological symptoms

vesicula and skin disease

abortion

16

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

17

What samples should be sendt in case of gastroenteric symptoms?

Live: faeces, vomit

dead: GI samples, liver, lymph, lung, spleen, kidney

18

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

19

What samples should be sendt in case of vesicular and skin diseases?

vesicular fluid, skin, lesions, biposia, swab

(some should be frozen)

20

What samples should be sendt in case of abortion?

fetus or organs

placenta, amniotic sac

Blood of the dam should always be included

21

What is important when sending samples for serological detections?

sera pair investigation

- acute phase sera or plasma + 2-4 weeks later sera or plasma

22

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

23

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.

24

what is the prerquisite of in vitro propagation?

the infective virion --> early phase --Z virus shedding

25

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

26

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

27

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

28

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

29

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

30

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

31

What is MEM?

Minimal Essential medium: the optimal environment isotonic, isoionic, isoosmotic, nutritive, antibiotics, antimycotics, indicator foeatal, calf serum

32

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

33

What are some examples of sterile culturing flasks?

Plate, Roux-flask, petri dish

34

What is meant by inoculation?

the introduction of a virus into a culture medium

35

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

36

How many passages are possible and why?

2-3x

the cell culture changes in subsequent passages, so the sensitivity might decrease

37

What is cellular cloning?

when a single cell is cultivated and a permanent cell line is produced

38

What types of cell cloning is there?

Diploidic and aneuploidic (tumour)

39

what are the advantages of celllar cloning?

they are genetically homogenous, making them comparable

unlimited number of passages

long term storage

40

What are the disadvantages of cellular cloning?

the sensitivity for infections varies

contamination may occur

there may be presence of active oncogenes

41

Why do we use adsorption when we inoculate cell-cultures or embrionated egg?

to avoid CPE caused by toxins

42

When do we suspen in cell-cultures or embrinated eggs?

when viruses needs dividing cells

43

What is meant by co-cultivation?

isolation of cell-associated and latent viruses

simultaneous precessing and mixing of virus-infected and healthy cells

44

What is a white blood cell culture?

a buffy coat culture

used for WBC specific virus propagation (ASF)

45

What do we use suspension cultures for?

Vaccine production

46

wha tis microcarrier cultures?

a support matrix that allows cells to adhere on the surface (microcarrier beads)

the aim is the increased surface

47

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

48

What are the requirements of the egg in terms of inoculation?

embryonated

SPF

white shelled - easier transillumination

49

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)

50

Where on the egg should the inoculation happen?

depends on the virus and on the age of the embryo

51

What and when can viruses be injected in the yolk sack?

picorna, reo, adenoviruses

5-7 days

52

What and when can viruses be injected in the allantoic cavity, amniotic cavity

orthomyxo, paramyxo, coronavirus

9-12 days old embryo

53

What and when can viruses be injected into chorio-allantoic membrane?

pox, herpes virus

10-13 days old embryo

54

What and when can viruses be injected in  intravenous inoculation?

orbivirus

16-17 days old embryo

55

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

56

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

57

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

58

Examination of the embryonated egg?

daily examination, take samples from the allantoic fluid, CAM and embryo

Auxillary examinations: HA, EM, histopathology

59

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

60

How are vaccines produced with use of living animals?

vaccines are produced from the organs of infected animals: rabbit haemorrhagic disease, classical swine fever

61

Vaccine controls on living animals?

harmless, efficient

62

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

63

What is rough purification?

Centrifugation or filtration (450nm pore size)

64

What are the 5 methods of concentrating the viruses

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

65

mention some concentration and purification methods (examples)

affinity chromatography

Density gradient ultracentrifugation

66

With what method can we precipitate viruses?

we precipitate the proteins, with:

(NH4)2SO4, PEG 6000, ethanol, resolve in buffer

67

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

68

How do we concentrate viruses with ultrafiltration?

With hydrostatic pressure, the pore sizes are smaller than the diameter of the viruses

69

How do we concentrate viruses with dialysis?

Osmotic pressure

through a semi-permeable membrane

70

how do we concentrate viruses with pelletisation?

they will be centrifuged in a high speed for a long time

can achieve 1000x concentration

71

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

72

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

 

73

How will de buoyant density be after density gradient ultracentrifugation?

nucleic acid/protein/lipid ration (incomplete virions are lighter

74

what is the difference between preparative ultracentrifugation and analytical ultracentrifugation

•Preparative u.c. --> for purification

•Analytical u.c.  -->  for measuring buoyant density

75

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

76

What are the metho of electron-microscopic investigation?

  1. Ultra thin section
  2. Negative contrast method
  3. Immune-electron microscopic method

77

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)

78

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) 

79

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

80

What are some physico-chemical tests to investigate virus identification?

Electron-microscopic investigation

Chloroform resistance

halogenic uridin derivates

acridin-orange staining

81

chloroform resistance testing?

enveloped vs non-enveloped

82

halogenic uridin derivates?

DNA inhibitors

83

Acridin-orange staining

ssNA vs dsNA

84

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)

85

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

86

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

87

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

88

How does ELISA work?

enzyme linked immunosorbant assay

direct/indirect

competetive (discriminating/multispecies antibody detection)

sanwich - FMD

89

What are the microscopic investigations (virus identification)?

CPE, tissue specificity, type of pocks, haemadsorption

90

What are the EM investigations? (id of virus)

the dimension, size, shape of the virion, its symmetry, surface

91

what are the physico-chemical investigations of vegetative viruses?

enveloped/nonenveloped

DNA/RNA virus

Single/double stranded nucleic acids

92

how do we investigate group-specific antigens?

AGP, IPA, IF, IEM, ccIEF, ELISA, RIA, HA

93

How do we determine the serotype (id of virus)

virus neutralisation test, haemagglutination-inhibition test

94

How do we determin the subtype, variant?

clinical signs

investigation of nucleic acids

investigation with monoclonal antibodies

95

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

96

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

 

97

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

 

98

What is PCR?

Polymerase chain reaction

used to amplify a single or a few copies of a DNA

 

99

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

100

How do we detect the DNa after PCR

we need atleast 2 copies of the fragment

we can use agarose-gel electrophoresis, NA hybridization

101

What is real-time PCR?

There will be fluorescent labeling

laser detection of amplification products, computer analysis

quantification

102

What is sequencing?

is the process of determining the nucleic acid sequence – the order of nucleotides in DNA.

 

103

what is the name of the methods used for sequencing?

Sanger's method: polymerization

polyacrylamide gel-electrophoresis

104

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

 

105

What are dideoxy nucleotide?

chain elongation inhibitors of DNA polymerase used in sanger method for DNA sequencing

106

107

What is the aim when titrating viruses?

Diagnostics

vaccine production

experimental animal infection

108

What are the physical assay methods of titration?

Direct particle counting - EM

¨Haemagglutination

109

What are some Biological assy methods of titration?

Determination of the infective titre (endpoint method)

Plaque assay

Focus assay

Pock formation

110

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)

111

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

112

What is the virus concentration unit?

1 tissue culture infective dose

TCID50/ml

113

What is the virus concentration unit called when it is performed in embryonated eggs?

EID50: pock assay

114

What is the virus concentration unit when it is performed in experimental animals?

LD50

115

What are the two methods of calculation of virus concentration?

Reed-Muench method

Spearman-Karber method

116

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)

117

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

118

What are the vital stains of plaque counting called?

Gentian purple

Evans blue

Janus green

119

What is the unit used to evaluate the plaque counting?

the concentration of the virus suspsension given in plaque forming units = PFU/ml

 

120

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

121

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

122

What is meant by hemagglutinating titer?

The highest dilution of the virus suspension, in which we can observe hemagglutination

123

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

124

what does the physical assay count?

the number of particles , regardless infectivity

125

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

126

What are indirect virus demonstration`?

Using serological methods to indicate the presence of antibodies

127

What are the advantage of serological methods?

higher chance of demonstration

it is cheap

128

what are the disadvantages of serological methods?

they cannot differentiate the antibodies between

  • maternal
  • vaccine
  • seroconversion

 

129

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

130

What is the constant virus varying serum dilution method?

method for antibody detection

131

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

132

what determines the serum neutralizing titer?

the highest dilution of the serum where there is 50% CPE

133

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

134

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

135

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

 

136

How to evaluate constant serum varying virus dilution method?

Neutralization index= virus+negative serum titer/virus+positive serum titer >2

137

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)

138

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

139

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%)

140

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

141

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

142

What are the two types of antibody detecting ELISA?

cELISA: competitive ELISA

iELISA: indirect ELISA

143

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

144

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