1. Plenary/practical

Question 1

lab investigations are necessary when

Answer 1

1. herd diagnosis to find endemic viruses
2. notifiable diseases
3. suspected zoonosis
4. eradiction programs
5. if in need of certification of free status (free from vertain diseases)

Question 2

zoonosis

Answer 2

natural transmission of disease btw. animals and humans

rabies

Question 3

the accompanying letter should include

Answer 3

1. location and contact information
2. case information
3. epidemiological information
4. submitted samples

Question 4

types of submittet sample material

Answer 4

swabs
blood (EDTA, heparinized, coagulated, serum)
organs

Question 5

direct detection methods, what type of samples

Answer 5

detection of the pathogen usually in early/acute phase

cadavers, tissues, organs, secretions, anti-coagulant treated blood

Question 6

indirect detection methods, what type of samples

Answer 6

antibodies in late/chronic phase

- coagulated blood or serum, milk, liqour, ventricle and organ secretion

Question 7

serological investigations used when

Answer 7

indirect method
paired samples: early and late phase ( to prove the infection)

min. antibody titer incr. of 4* should be considered significant

Question 8

ante-mortem sampling and post-mortem sampling of respiratory signs

Answer 8

ante-mortem: swabs, EDTA blood

post-mortem: lung and mediastinal lymphnodes

Question 9

ante-mortem sampling and post-mortem sampling of gastro-enteric signs

Answer 9

ante-mortem: faeces or faecal swabs, EDTA blood

post-mortem: parts of intestines, mesenterical lymphnodes

Question 10

ante-mortem sampling and post-mortem sampling of neurological signs

Answer 10

ante-mortem: EDTA blood, conjuctival/nasal swabs, liqour cerebrospinalis
post-mortem: parts of brain and spinal cord (lung, liver, spleen, kidney)

Question 11

ante-mortem sampling and post-mortem sampling of skin/mucosal diseases

Answer 11

ante-mortem: pieces of affected skin, papillomas, sarcoids, vesicular wall, vesicular fluid
post-mortem: same as ante-mortem

Question 12

in case of abortion, samples needed

Answer 12

foetus, placenta, amniotic sac, and most importantly a blood sample of the dam

Question 13

delivery of samples

Answer 13

courier or personal delivery
within 24h, EDTA blood max 6h
4 degrees celcius

Question 14

direct virus methods

Answer 14

1. virus isolation
2. antigen tests: ELISA, haemagglutination, peroxidase, IF:immuno flouresence
3. protein detection: western blot,
4. NA detection: nucleic acid hybridization, PCR

Question 15

Indirect virus methods

Answer 15

serological methods

ELISA, virus neutralization test, indirect IF, HAI (haemagglutination inhibitation)

Question 16

cultivation methods

Answer 16

in vitro cell tissue cultures
inoculation of embryonated eggs
experimental infection of animals

Question 17

cells used for production of primary monolayer cell culture

Answer 17

from organs rich in epithelial cells: kidney, testicles, thymus

and from young animals: embryo, new born, few days old

Question 18

how are cells from tissue samples seperated

Answer 18

digestion using trypsin-EDTA solution several times -> 0 degrees to inactivate trypsin -> centrifugation -> supernatant is removed

Question 19

MEM

Answer 19

minimal essential medium, aka culturing medium

• isotonic, isoionic, isosmotic (salts, buffer systems)
• nutritive (aa’s, carbs)
• usually contain foetal calf-serum (FCS) –> protein source and mediator of cellular divisions
  
  • –> growth medium: 5-10/, maintenance medium: 2%
• must be supplemented with antibiotics and antimycotics to prevent bacterial and fungal infection in cell culture, and indicators to detect the metabolism of the cells

Question 20

optimal cell conc. of cell culture

Answer 20

200 000 cells/mL

Question 21

incubation of cell culture

Answer 21

37 degrees celcius, 5% CO2 - cover bottom within 3-5 days, then contact inhibitation

Question 22

maintinance medium vs. growth medium

Answer 22

maintinance medium is deprived of FCS which slows down cell ageing, primart cell culture survive for 2-3 weeks

Question 23

how are secondary cell cultures made

Answer 23

aka. subculturing process, passage

cells removed from flask with trypsin -> fresh growth medium with more space, divided

Question 24

(dis) advantages to secondary cell cultures

Answer 24

more homologous
smoother cells
fresh culture with dividing activity
incr. the amount of cells

!but: less susceptible to viral infections

Question 25

why can we usually only subculture 3-4 times

Answer 25

fibroblasts will outgrow the epithelial cells, which are less susceptible to viral infections

lower mitotic activity after some divisions

Question 26

diploid cell cultures

Answer 26

• used to make permanent cell lines

- one well contains one cell -> colonies with single cell origin

Question 27

disadvantages of diploid cell cultures

Answer 27

less susceptible to some viral infections

virus cannot choose among different cells

Question 28

aneuploid cultures

Answer 28

made of tumor cells having high mitotic activity, usually premordial cell type

less susceptible to viral infections

only used in vaccines if they are deactivated later for safety

Question 29

inoculation

Answer 29

artificial induction of immunity

absorption and suspension methods are usually used

Question 30

absorption method

Answer 30

• used when sample is toxic for the cell culture
• incubation for 30 mins with the toxic supernatant, the virus will absorb to the surface of host cells, but toxins have not had enough time to harm the cells yet

Question 31

suspension mehod

Answer 31

used in toxin-free samples or when we expect viruses in the sample which does not have viral polymerase enzyme -> dividing cells are needed for their multiplication

Question 32

egg inoculation

Answer 32

33-37 degrees celsius, checked everyday for virus multiplication