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Microbiology 4 Flashcards

1
Q

List the important enteric viruses of veterinary species

A
  • Reoviridae (Rotavirus)
  • Parvoviridae (Parvovirus)
  • Coronaviridae (Coronavirus and Torovirus)
  • Paramyxoviridae (Paramyxovirus)
  • Flaviviridae (Pestivirus, BVDV)
  • Astroviridae (Astrovirus)
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2
Q

Describe the structure of Reoviridae

A
  • Icosahedral
  • Non-enveloped
  • Outer, middle, innre capsid
  • Core contains segmented genome (dsRNA)
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3
Q

Describe the epidemiology of Rotavirus

A
  • Encodes own polymerase
  • Cytoplasmic replication
  • Segmented genome (reassortment)
  • Multiple strains, no cross protection
  • Very stable in environment
  • Wide pH range, temp range
  • May persist after outbreak if poor disinfection
  • Major cause of diarrhoea in young farm animals
  • Faecal oral transmission
  • Minimal infectious dose
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4
Q

Describe the pathology of rotavirus

A
  • Infects enterocytes at end of villi
  • Villi shorter
  • Unable to absorb lactose due to loss of lactase
  • Impaired Na transport, reduced water absorption
  • Decreased digestion and absorption of milk in upper SI
  • Undigested milk in lower SI and LI = bacterial overgrowth, osmotic diarrhoea, watery scour
  • Pasty/watery diarrhoea
  • Secondary infection with E. coli, other viruses, coccidia
  • Dehydration
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5
Q

How is rotavirus diagnosed?

A
  • Sample faeces/gut contents
  • Detection of viral antigen - ELISA or latex aggluniation
  • Detection of viral RNA - polyacrylamide gel electrophoresis
  • Post mortem examination
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6
Q

Describe the structure of Coronaviruses

A
  • +ve sense ssRNA
  • Enveloped
  • Spike proteins on envelope
  • Contains nucleocapsid (ssRNA adnn protein)
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7
Q

Describe the epidemiology of Coronaviruses

A
  • Enteric and respiratory pathogens
  • Cause of common cold in man
  • Difficult to grow in lab
  • Commonly mutate
  • Survive well in environment
  • Tolerate low pH
  • Destroyed by common disinfectants
  • Lots of strains
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8
Q

Describe the pathogenesis of Coronaviruses

A
  • Infect cells in middle of villi

- TGEV, CCoV, FCoV virus spike proteins bind to aminopeptidase N (highly expressed in mature enterocytes)

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9
Q

Describe porcine coronavirus

A
  • TGEV (transmissible gastroenteritis virus)
  • EDV (porcine epidemic diarrhoea virus)
  • Other strains exist but these are teh enteric ones
  • Can be distinguished by serology
  • TGEV highly contagious, young pigs, diarrhoea/vomiting, rapid dehydration, high mortality
  • EDV - similar to TGEV, less severe
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10
Q

Describe coronavirus in cattle

A
  • BVC (bovine corona virus)
  • Scour 4d-3wk of wage
  • Dehydration, acidosis, depression, fever
  • Recovery in 4-5d
  • Winter dysentery in housed adult cattle
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11
Q

Describe canine coronavirus

A
  • Canine enteric CoV (CCoV)
  • Mild self-limiting diarrhoea
  • Novel genotypes may produce more severe disease (spontaneous mutation)
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12
Q

Describe feline coronavirus

A
  • Enteric virus but pathogenesis produces systemic sings
  • Biologically distinct biotypes: feline enteric coronavirus and feline infectious peritonitis
  • Following infection most will have transient infection, can shed for many months, some will acquire carrier state and shed for life
  • Minority will get FIP
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13
Q

Describe feline infectious peritonitis

A
  • FIP
  • Cahnges in virus lead to change of strain
  • Mutation of virus, stress, viral load
  • Dry or wet FIP
  • Wet: chest full of sticky yellow fluid
  • Dry: CNS fluid affected
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14
Q

Describe Torovirus

A
  • Rare
  • Equine: uncommon cause of diarrhoea
  • Bovine: diarrhoea in newborn calves
  • Feline: diarrhoea and third eyelid syndrome
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15
Q

Describe the structure of Flaviviridae (Pestiviruses)

A
  • +ve ssRNA
  • Enveloped
  • Icosahedral capsid
  • Cytoplasmic replication
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16
Q

What diseases are caused by Pestiviruses?

A
  • Bovine viral diarrhoea
  • Border disease in sheep
  • Classival swine fever
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17
Q

Describe bovine viral diarrhoea

A
  • Most important viral disease of cattle
  • Diarrhoea
  • Decreased fertility/milk yield
  • Abortion, congenital defects, stunted calves (transplancental)
  • Immunosuppression
  • Mucosal disease
  • 2 genotypes: BVDV-1 and BVDV2
  • Isolates fo both associated with mild and severe disease
  • 2 biotypes
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18
Q

What are the 2 biotypes of BVDV?

A
  • Non-cytopathic
  • Cytopathic
  • Exist for both genotypes 1 and 2
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19
Q

Describe NCP BVDV

A
  • Major cause of BVD
  • For mucosal disease need NC then CP
  • Can cause persistent infections
  • Can cross placenta, immune system sees as self, immunotoleratn, if survives then shed NCP
  • can survive without getting BVD but can get mucosal disease
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20
Q

Describe mucosal disease of BVDV

A
  • Infrequent consequence of BVDV infection
  • Develops only in persistently infected animals
  • Presence of NCP and antigenically related CP virus
  • Mutation of NCP virus in PI animal, uperinfection of PI animal with another CP virus
  • CP viruses show marked tropism for GALT
  • Severe diarrhoea, invariably fatal
  • Marked mucosal haemorrhage
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21
Q

Describe the structure of Parvovirus

A
  • Icosahedral capsid
  • Non-enveloped
    Small, linear ssDNA genomoe
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22
Q

Describe the epidemiology of parvovirus

A
  • Infect and kill actively replicating cells

- Persist for long periods in environment

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23
Q

Describe the pathogenesis of parvovirus

A
  • Infect and kill actively replicating cells
  • Degrade villi
  • INfect progenitor cells at base of crypts
  • Villi tip cells turnover normally but not replaced
  • Stunted villi, malabsorption, maldigestion
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24
Q

What are the 3 important enteric parvoviruses?

A
  • Feline panleukopaenia virus (FPV)
  • Canine parvovirus (CPV)
  • Porcine parvovirus (PPV)
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25
Q

Describe the epidemiology feline parvovirus

A
  • AKA feline infectious enteritis (FIE) or feline panleukopaenia
  • Faecal oral transmission
  • Persists in environment up to a year
  • Lots in faeces
  • Lymph nodes of naso- and oro-pharynx
  • Spread to other tissues
  • Needs rapidly dividing cells to propagate
  • Infects intestinal cells and bone marrow
  • Young or older unvaccinated most affected
  • Infected pregnant queens problematic
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26
Q

Describe the pathogenesis of feline panleukopaenia

A
  • Decreased WBC count

- Killing of lymphoid and myeloid stem cells

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27
Q

Describe the pathogenesis of feline infectious enteritis

A
  • Killing of stem cells in crypts
  • Dehydration
  • Can be fatal
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28
Q

Describe feline cerebellar hyperplasia

A
  • Infection in neonata kittens
  • High risk secondary bacterial infection
  • Perinatal infection
  • Cerebellum controls coordination adn balance = wobbly kittens
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29
Q

Describe the diagnosis of feline parvovirus

A
  • Faeces contain a lot of virus
  • Detect viral antigen (ELISA) or viral DNA (PCR)
  • Eidence of exposure to virus by detection of antibody in blood
  • Supportive evidence through marked leukopaenia
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30
Q

What are the types of canine parvovirus?

A
  • CPV-1 (minute virus, mild diarrhoea)

- CPV-2 (serious pathogen of dogs)

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31
Q

Describe canine parvovirus 2

A
  • Infection of actively dividing cells
  • Generalised neonatal disease
  • Myocarditis in neonatal puppies
  • Bone marrow = leukopaenia
  • Intestinal villi = enteritis = vomiting/diarrhoea
  • Intestinal adn mesenteric lymphoid tissues = immunosuppression
  • Mortality can be high even with appropriate treatment
  • Myocarditis can follow infection if survive
  • Due to myocardial necrosis, inclusion bodies in myocardial cells
  • Inflammatory cells increase and fibrosis occurs
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32
Q

What are the important genera of Paramyxoviridae?

A
  • Morbillivirus
  • Rubulavirus
  • Respirovirus
  • Pneumovirus
  • Metapneumovirus
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33
Q

Describe the virology of Paramyxoviridae

A
  • Large enveloped virus
  • -ssRNA
  • Sensitive to heat, detergent, desiccation
  • Cytoplasmic replication
  • Release by budding
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34
Q

What diseases are caused by Morbillivirus?

A
  • Measles
  • Canine distemper
  • Rinderpest
  • Peste des petits ruminants
  • Phocine morbillivirus
  • Cetacean morbillivirus
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35
Q

Describe canine distemper

A
  • Young dogs especially susceptible
  • Direct contact transmission
  • Replicates in URT
  • Spread to tonsils/lymph nodes
  • Viraemia and systemic spread to epithelia +/- CNS
  • does not only infect GIT
  • Pyrexia, depression
  • Ocular and nasal discharge
  • Cough
  • Vomiting, diarrhoea
  • Hyperkeratosis of nose/pads
  • Solid immune response = recovery
  • Poor immune response = development of neurological signs
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36
Q

Describe Rinderpest

A
  • Cattle plague
  • Highly infectious
  • Respiratory and alimentary tract
  • High morbidity and mortality
  • High fever
  • Nasal discharge
  • Ocular diascharge
  • Excess salivation
  • Oral and nasal erosions and ulcerations
  • Diarrhoea, with mucus, blood and debris
  • Dehydration followed by death
  • Mild signs if disease becomes endemic
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37
Q

Describe Peste de petit Ruminants

A
  • Goat plague
  • Similar to Rinderpest
  • Mucosal erosions and profusediarrhoea
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38
Q

Describe Newcastle disease

A
  • Avian Rubulavirus
  • Notifiable
  • Avian paramyxovirus-1
  • Chickens, ducks, pheasants, geese, turkeys
  • Shed in all excretions and secretions(aerosol)
  • Stable for weeks on carcasses
  • COmmon in wild birds (inapparent infection)
  • Mild conjunctivitis in humans
  • Strains vary in virulence and tropism
  • Deterined by F glycoprotein
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39
Q

What are the strains of Newcastle disease?

A
  • Lentogenic
  • Mesogenic
  • Neutropic velogenic
  • Viscerotropic velogenic
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40
Q

Describe lentogenic Newcastle disease

A
  • Mild, inapparent infection

- Confined to it and respiratory tracts which trypsin like proteases are present

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41
Q

Describe mesogenic Newcastle disease

A
  • Mild respiratory disease

- Some death in young birds

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42
Q

Describe neutropic velogenic Newcastle disease

A
  • Acute, severe, fatal with respiratory and nervous signs
  • Can be cleaved in many tissues by furin-like (ubiquitous) proteases due to altered cleavage site
  • Contain multiple basic AAs
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43
Q

Describe viscerotropic velogenic Newcastle disease

A
  • Severe
  • Fatal with haemorrhagic intestinal lesions
  • Respiratory disease
  • Can be cleaved in many tissues by furin-like (ubiquitous) proteases due to altered cleavage site
  • Contain multiple basic AAs
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44
Q

Describe the virulence factors of Newcastle diseae virus

A
  • F glycoprotein
  • Haemagglutinin/neuramidase (HN) enables virus attachment to cell receptor
  • Fusion protein (F) enables fusion and entry
  • F protein cleaved to become active by host cell proteases (forms F1 and F2)
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45
Q

Describe the diagnosis of Newcastle disease

A
  • Egg inoculation and testin shows presence
  • Agglutination tests
  • Virulence by intracerebral inoculation and demonstation of multiple basic AAs at F protein cleavage site
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46
Q

Describe Astroviruses

A
  • Mild self limiting diarrhoea in many species

- More severe in ducks where lethal hepatitis may occur

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47
Q

Describe the common features of enteric virus infection

A
  • Small infectious dose
  • Short incubation and life cycle
  • Lytic
  • Large amount shed in faeces
  • Tough, able to survive pH of stomach and environment
  • Secndary bacterial infection common
  • Multiple viral infections
  • Diarrhoea
  • May have vomiting (dogs and cats usually)
  • Death from dehydration can occur
  • Maternally derived antibody protects newborn
  • Disease as antibody levels fall or if there is failure of sufficient colostrum transfer/low antibody levels
  • Occurence of disease influenced by amount of virus, viral virulence, host resistance
  • High stocking density leads to outbreaks
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48
Q

Describe the common feature of enteric viral pathogeneis

A
  • Denudation of microvilli
  • Shortening, flattening and atrophy of villi
  • Leads to malabsorption
  • Food left in gut as not absorbed by SI leads to bacterial overgrowth
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49
Q

Describe the control of Rotavirus

A
  • Colostrum important
  • All-in, all-out management
  • Good hygiene
  • No vaccination for pigs in UK, available for cattle and horses
  • Aim to vaccinate dam to increase colostral antibodies
  • Can feed scour to sow during pregnancy but very risky
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50
Q

Describe the control of Coronavirus

A
  • Removal of infected animals
  • Maintain clean herd and housing
  • Vaccine available for some (reduce, do not eliminate disease)
  • Early weaning prevents
  • Separate age groups
  • Virus changes a lot, cannot vaccinate against all types
51
Q

Describe the control of bovine viral diarrhoea

A
  • Eliminate persistently infected animals from herd
  • Maintian closed herd
  • Maintain herd immunity so noo naiive cattle infected
  • Vaccination
  • Deliberate infection risky
52
Q

Describe the control of FPV

A
  • Maternally derived antibody wanes after 8 weeks
  • Vaccinate
  • Prevent exposure of kittens to FPV in environment
  • Vaccination before colostrum protection wanes
53
Q

Describe the control of CPV-2

A
  • Live attenuated and inactivated vaccines
  • Annual boosters
  • Additional vaccines in outbreak
  • Maternally derived antibody can interfere with vaccine so need multiple vaccinations or high titre vaccine
  • Virus hardy - transmitted by faecal oral route and fomites
  • Bleach effective at killing
  • Isolate pups until vaccination complete
54
Q

Describe the control of Newcastle disease

A
  • Biosecurity
  • Slaughter in event of sever outbreak with velogenic virus
  • Vaccinaion in most commercial flocks
  • Live attenuated and inactivated
55
Q

What viral protein of Coronaviruses is important in mediating binding of TGE virus to a target cell?

A

Spike rpotein

56
Q

Describe the binding and entering of rotaviruses to their target cells

A
  • VP4 activated by trypsin (cleaved) to activate the virus, promotes cell entry
  • VP8 adn VP5 remain non-covalently associated
  • Cell entry multistep process
  • Cleaved VP4 binds to sialic acid, integrins and other surface proteins in order to gain entry
57
Q

Explain the role of the NSP4 viral protein in the pathogenesis of diarrhoea in rotaviruses

A
  • Disrupts tight junctions
  • Enterotoxigenic function
  • Inhibits Na+-D-glucosesymport activity of the sodium-glucose linked transporter protein SGLT1
  • Allows paracellular flow of waer and electrolytes
58
Q

Give a basic outline of how Salmonella infects the host animal

A
  • Initial interaction of Salmonella with enterocyte
  • Bacterial internalisation
  • Intracellular survival
  • Dissemination in the host
59
Q

Describe the initial interaction of Salmonella with enterocytes

A
  • Salmonella interacts with enterocyte

- Delivers Sop proteins into cell cytoplasm via TTSS-1 adn Sip dependent pathway

60
Q

Describe Salmonella internalisation

A
  • Promotes own invasion
  • Sip, SopE and other Sops induve enterocyte membrane ruffling
  • Promotes bacterial invasion
61
Q

Describe Salmonella intracellular survival

A
  • Bacteria reside within membrane bound vesicles
  • Continue translocation of TTSS-1 secreted effeectors
  • Replication of Salmonella within veiscles promoteed by TTSS-2
  • Intracellular SopB protein affects inositol phosphate signalling events
  • Transient increase of Ins(1,4,5,6)P4 can antagonise closure of chloride channels, influencing net electrolyte transport (and so fluid secretion)
  • Salmonella infected cells secrete chemokines and prostagalndins, recruit inflam cells to foci of infection
  • Release of some chemokines and prostaglandins affected by intracellular activity of Sops
  • Salmonella interacts with inflammatory cells, stimulates release of proinflammatory cytokines, enhance inflammatory response
62
Q

Describe Salmonella dissemination in the host

A
  • Salmonella infected epithelial cells release pathogen elicited epithelial chemoattractant (PEEC) cross apical membrane
  • Stimulates PMN transepithelial migration between enterocytes
  • Infiltrating inflam cells phagocytose Salmonella, fate of tehse unclear
  • Salmonella infected enterocytes become extruded from the villus, shedding of infected cells into intestinal lumen resulting in villues blunting and loss of absorptive surfaces
  • Some infected cells migrate to drainig lymphatics, carrying Salmonella to systemic sites
63
Q

What is the function of type 1 TTSS?

A

Used for invasion of cells by Salmonella

64
Q

What is the function of type 2 TTSS?

A

Used for survival and replication of Salmonella within vesicles

65
Q

What are SOPs?

A
  • Salmonella outer proteins
  • Effector proteins function to alter host cell physiology and promote bacterial survival in host tissues
  • Contribute to formation of Salmonella induved filament formation
66
Q

What are SIPs?

A
  • Salmonella invasion proteins
  • Translocated into cell by TTSS-1, bind to actin, stimulate actin polymerisation and counteracts F-actin destabilising proteins (SipA)
67
Q

Explain the difference between S. gallinarium and S. typhimurium and poultry

A
  • T not host adapted, can get subclinical infections
  • G similar to Pullorum
  • Lesions throughout lungs and focal necrosis of liver and spleen
  • Sudden deaths
  • Characteristic findings: enlarged, friable, bile-stained liver and enlarged spleen
68
Q

Hwo does Salmonella survive inside cells?

A
  • Formaiton of Salmonella infuced filaments
  • Play role in intracellular replication
  • Effector proteins protect intracellular organism from bactericidal compounds e.g. reactive oxygen intermediates
  • Protect from phagolysosome fusion
69
Q

Why has the incidence of transmissible gastroenteritis virus decreased?

A
  • Reduced stocking density
  • Improved hygiene
  • Vaccine available (reduces incidence, will not eliminate)
70
Q

How may diarrhoea be caused?

A
  • Single strains of virulent pathogen
  • Mixed infections
  • Toxins
  • Response of host to pathogen or irritants
  • Disruption to nromal physiology
  • Direct effects of the pathogen
  • Stress e.g. shipping fever
71
Q

What is the role of diarrhoea?

A
  • Removal of infection
  • Stops build up of pathogen/toxins
  • May aid dissemination of pathogen
  • Product of mucosal damage/effects
  • Combination of all
72
Q

In bacterial infections, what may cause dsisease?

A
  • Adherence
  • Secretion of exotoxin
  • Invasion of target cells leading to cell death
  • Invasion of host cells and lymphatics leading to systemic disease
  • Triggering of inflammatory response
73
Q

How to type III secretion systems work?

A
  • type 1 and 2
  • 1 used for invasion
  • 2 used for intracellular survival
  • Tubes that span inner and outer membrane of host cell
  • Inject effector proteins through them
74
Q

How does viral infection cause disease?

A
  • Attachment
  • Invasion
  • Replication
  • Release of virus
  • Immune response to either viral infection or cell damage
  • Macrophage migration to the area
75
Q

What are the pathogenic mechanisms of diarhhoea?

A
  • Hypersecretion
  • Villous atrophy
  • Infiltrative and proliferative distortion of mucosa
  • Mucosal necrosis
  • May be combination or partial effects
76
Q

Describe how hypersecretion leads to diarrhoea

A
  • Functional disturbance of intestinal epithelial cells
  • Increased outflor or decrease in reabsorption of water
  • ETEC
  • Water moved into intestines thorugh specific action of crypt cells
77
Q

Describe the specific action of crypt cells in hypersecretion diarrhoea

A
  • Pump Cl- into crypt space
  • Crypt cells actively draw water into intestine
  • Cl- ions attract Na+ into crypt space, increases local osmotic pressure
  • increased osmotic pressure, water pulled into intestine
  • Some enterotoxins can trigger this
  • Hypersecretion of water
  • Lock system ON
78
Q

How can villous atrophy cause diarrhoea?

A
  • Destruction of epithelial cells on villi or in crypts
  • Leads to malabsorption
  • More water present in lumen
  • Villi stunted or fused
  • Attaching and effacing E coli, rotavirus, canine parvovirus
79
Q

Describe how infiltrative and proliferative distortion of mucosa can lead to diarrhoea

A
  • Chronic progressive immuno-inflammatory disease of adult ruminants
  • Packed with cells, lose villous distinction
  • Mucosa distorted
  • Flat surface rather than villi, lose surface area
  • Unable to resorb water
  • Recruitment of large numbers of macrophages and T-lymphocytes in lamina propria and sub-mucosa
  • Crypt compression and villous distortion
  • Reduce absorptive capacity
80
Q

Describe how mucosal necrosis can lead to diarrhoea

A
  • Infection leads to cell death
  • Secerity and extent depends on virulence of pathogen
  • Majority of diarrhoea from reduced absorption
  • Brachyspira hyodysenteriae, Clostridium perfringens typeC, some necrotoxigenic species fo E. coli and Salmonella
81
Q

Describe Lawsonia

A
  • Curved Gram -ve rod
  • Obligate intracellular pathogen
  • Microaerophilic
  • Cannot be grown on inert media, requires growth in enterocytes
  • Implicated in proliferative enteropathy f pigs and foals
  • Diagnosis by clinical signs, demonstration in mucosa or faeces by immunofluorescence or PCR, staining of tissue sections, serological tests
82
Q

What can molecular techniques be used for in veterinary medicine adn research?

A
  • Pathogen detection
  • Epidemiology
  • Diagnosis of genetic diseases
  • Quality trait loci (meat/wool quality)
  • Forensic
  • Public health (food testing for animal protein etc)
83
Q

List some importnat molecular techniques used in veterinary diagnostics and surveillance

A
  • Quantitative PCR
  • Single nucleotide polymorphisms
  • Variable numbers of Tandem Repeats
  • Restriction Fragment Length Polymorphisms
  • Random Amplification of Polymorphic DNA
  • Multi Locus Sequence Typing
84
Q

Outline the use of vectors in recombinant DNA technology

A
  • DNA (gene) cloning
  • Isolate and specifically amplify copies of a unique piece of DNA
  • Amplify targeted pieces of DNA using PCR
  • Specifically and reproducibly cut DNA into defined pieces (restriction enzymes)
  • Joing individual pieces of DNA together (DNA ligase)
  • Replicate specific pieces of DNA (cloning vectors derived from plasmids of phage)
85
Q

What are important feature of a plasmid to be used for DNA cloning?

A
  • Plasmid should have lots of different sites that restriction enzymes can be used at
  • Want to cut and slot into a specific place
86
Q

What steps are needed to amplify genes other than dsDNA by PCR?

A
  • mRNA is expressed part of genoome at any one time
  • Cannot be cloned directly
  • Complementary DNA (cDNA) strand produced to allow integration into dsDNA plasmid
  • From mRNA make complementary ssRNA stranf
  • Destroy original mRNA strand
  • Then make complementary to the first ssRNA strand
87
Q

What are the important features of a cloning vector?

A
  • Efficient means of introducing vector inot host cell (transformation, conjugation)
  • Vector must be able to replicate within host cell so that many copies of the vector can be obtained
  • Means to isolate vector DNA away from host DNA
  • Restriction enzymes in vector that can be used for cloning an insert DNA
  • Selectable marker on vector to indicate presence of vector in host cell
  • Unique restriction enzyme sites within selectable or screenable marker useful
88
Q

Describe bacteriophage derived vectors for DNA cloning

A
  • Similar to plasmids
  • Carrier molecules for DNA
  • Can carry larger pieces of DNA
89
Q

Describe M13 based vecors for DNA cloning

A
  • Single stranded circular DNA
  • Infection via F-pili
  • Converted to replicative form after infection (dsDNA)
90
Q

Describe cosmid vectors for DNA cloning

A
  • Plasmids that contain cohesive ends of gamma phage and gamma DNA sequences required for packagin DNA into gamma particle
  • Plasmid origin of DNA replication
  • At least one selectable marker
  • Unique restrictoin enzyme sites for insertine foreign DNA
  • Can be used to clone larger pieces of foreign DNA
91
Q

Describe YAC vectors for DNA cloning

A
  • Yeast artificial chromosomes
  • 2 copies of yeast telomere, yeast centromere, yeast ARS and appropriate selectable markers and unique restriction enzyme recognition sites
  • Allows cloning in eukaryote
  • Permits cloning of very large pieces of DNA (megabase range)
  • Some problems with insert instability
92
Q

Describe BAC vectors for DNA cloning

A
  • Bacterial artificial chromosome
  • Based on F-plasmid
  • Permits cloning of up to 300kb inserts
  • More stable than YACs
93
Q

Describe single nucleotide polymorphisms

A
  • Polymorphism: Having multiple alleles of a gene within a population, usually expressing different phenotypes
  • SNP: site in DNA occupied by different nucleotide pair among significant fraction of individuals in a population
  • e.g. susceptibility to scrapie
94
Q

Describe QPCR

A
  • Quantitative PCR (real time)
  • Amplify and quantify targeted DNA molecule by measuring fluorescent signal at each PCR cycle
  • Use of dsDNA dyes
  • Use of fluorescently labelled probes (or TaqMan probes)
  • Multiplex qPCR
95
Q

Describe the use of dsDNA dyes in qPCR

A
  • Bind to all dsDNA in PCR, causes fluorescence of dye

- Intensity of fluorescence proportional to amount of dsDNA (not specific to one PCR product)

96
Q

Describe the use of fluorescently labelled probes or TaqMan probes in qPCR

A
  • Based on properties of Taq DNA polymerase (DNA polymerase, 5’-3’ exonuclease - cleavage of dye labelled probes during PCR)
  • Intenstiy of fluorescence proportional to number of PCR products
  • More expensive but probe specific to target DNA
97
Q

Describe multiplex qPCR

A
  • Diagnostic test virulent/benign D nodosus

- Muliple fluorescent probes in same assay

98
Q

What are the applications of qPCR?

A
  • Pathogen detection
  • Quantification of gene expression
  • Detection of gene mutations
99
Q

Describe Variable Number of Tandem Repeats PCR technique

A
  • Location in genome where short nucleotide sequence organised as tandem repeat
  • Tandem repeat is patten of 2 more nucleotides repeated and repetitions are directly adjacent to each other
  • Between areas that code for proteins there are regions with little importance - repeat nucleotides
  • Often show variations in length between individuals
  • Generally identified by PCR
  • DNA finger printing
100
Q

Describe Restriction Fragment Length Polymorphisms

A
  • RFLP
  • Identification of differences in seuence when they occur in or near a restrictoin site
  • Presence or absence of restriction site
  • E.g. genotyping scrapie genotypes
101
Q

Describe Random Amplification of Polymorphic DNA

A
  • RAPD
  • Genotyping approach
  • Sequence info not required
  • DNA fragments from PCR amplification of random segmented of genomic DNA with single primer of arbitrary nucleotide sequence
  • 10-mer primers will or will not amplify segment of DNA depending on positions thatare complementary to primer’s sequence
  • Mutation affedting primer binding will not produce PCR
  • Different pattern in gel
102
Q

Describe Multi Locus Sequence Typing

A
  • MLST
  • Classify bacterial/fungal isolates
  • Epidemiolgoical studies/outbreak tracing etc
  • 7 housekeeping genes
  • Each strain has them
  • Allelic profile
  • If sequence different to all others, considered new allele and given new allele number
  • Each unique allelic profile give one digit sequence type (ST)
103
Q

What is meant by the term genome?

A

The complete set fo genes or genetic material present in a cell or organism

104
Q

What is the coding region of a genoome?

A

The portion of a gene’s DNA or RNA that is composed of exons (only a few % of total genome)

105
Q

What is the non-coding region of a genome?

A
  • Involved in regulating activity of coding regions

- Much of this has no known function

106
Q

What are the repetitive sequences of a genome?

A
  • Repeated base sequences
  • Most likely due to incorporation of invasive elements such as DNA transposons, retrotransposons and other repetitive elements
107
Q

What are pseudogenes?

A

Defective copies of functional genes (truncated, fragments)

108
Q

What are the transposable elements of a genome?

A
  • Sequences of DNA that can move around to different positions within a genome
  • Consists of DNA transposon, LTR-retrotransposons and non-LTR-retrotransposons
  • Integrate into adn replicate as part of genome via transposition
  • Interspersed reptitive non-coding DNA
  • Can induce mutations by inserting near or within genes
109
Q

Describe the mitochondrial genome

A
  • Single circular chromosome
  • Not all proteins necessary for mitochondrial function encoded by mitochondrial genome
  • Most coded by genes in cell nucleus and corresponding proteins imported into mitochondrion
  • Divide by binary fission
  • Comes from egg only
110
Q

Explain how genes are identified in genomic data

A
  • Genetic mapping of genome
  • Based on genetic techniques to construct maps showing positions of genes
  • Cross breeding examination of historied
  • RFLP, SSLP, SNP
  • Restriction maps f genome and fluorescent in situ hybridisation
  • Identify genome promoter, operator sequences, start codons, genes, stop codons, mutations, polymorphisms, repeats
111
Q

What is a transcriptome?

A
  • RNA copies of the active protein coding genes
  • All the RNA present in a tissue (or cell) at any one time
  • Very little of genoome actually encodes for proteins
  • Most of it is transcribed
112
Q

Describe RNAi

A
  • RNA interference
  • RNA mediated silencing of translation
  • double stranded areas of RNA processed to small RNA molecules
  • bind by base pairing to target nucleic acid
  • Direct several effector proteins to this nucleic acid
  • Protein containing complex named RNA induced silencing complex (RISC)
  • Usually results in inhibition of transcription or translation
113
Q

What is meant by epigenetics?

A
  • Heritabe changes in genome function that occur without alteration to DNA sequence
  • Methylated DNA best studied epigenetic mark
  • Present in animal’s offspring
  • Not all inheritance if medelian
  • Stressors across animals lifetime result in changes in methylation pattern
  • Epigenetic marks may be lost in cancerous cells resulting in uncontrolled replications
  • Methylation adn epigenetics very dynamic process
114
Q

What is meant by maternal/genomic imprinting?

A
  • Pattern of epigenetic marks you inherit from your parents
  • Most epigenetic marks erased during embryogenesis
  • Ones left over imprinted
  • Erasure and resetting important in returnign cell to stem cell state
115
Q

What is pre-mRNA

A

The transcribed gene including introns and exons

116
Q

What is mRNA?

A

Messenger RNA, genes with introns spliced out

117
Q

What is rRNA?

A

Ribosomal RNA, structural component of ribosome. 80% of RNA in the cell

118
Q

What is tRNA?

A

Transfer RNA, adaptor molecule in protein translation

119
Q

What is miRNA?

A

Micro RNA, regulates mRNA stability, translational role

120
Q

What is piRNA?

A

Piwi interacting RNA, transposon regulation

121
Q

What are CisNATs?

A
  • Cis-natural antisense transcripts.

- Modulate transcript levels

122
Q

What is snRNA?

A
  • Small nucelar RNA
  • Aka uRNA
  • Role in pre-mRNA splicing
123
Q

What is sno-RNA?

A
  • Small nucleolar RNA

- Role in rRNA and tRNA maturation

124
Q

Describe the mechanisms of RNA mediated control of transcription and translation

A
  • RNAi: inhibits transcription or translation

GI (31 decks)

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