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Veterinary Bacteriology & Mycology > Vaccines & Diagnostic Technology > Flashcards

Vaccines & Diagnostic Technology Flashcards

1
Q

What are live vaccines? Killed vaccines? Subunit vaccines? Live recombinant vaccines? DNA vaccines?

A

LIVE: contains an alive, attenuated pathogen that has limited growth and pathogenicity in the host

KILLED: contains chemically inactivated whole cells of pathogens

SUBUNIT: contains pieces of a pathogen (outer surface protein)

LIVE RECOMBINANT: contains a gene for antigen expressed in a microbe

DNA: contains a gene-encoding antigen expressed directly in immunized host

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2
Q

What are 3 reasons why we should be cautious with live attenuated vaccines?

A
  1. pathogen has the ability to revert back to a state of virulence
  2. can cause mild symptoms of disease
  3. induces a longer Th1 response (cellular)
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3
Q

What are 3 reasons why killed vaccines are not as common?

A
  1. not very effective
  2. too many antigens
  3. tend to stimulate only a short-lived Th2 response (humoral)
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4
Q

What are some major drawbacks to using subunit, live recombinant, and DNA vaccines?

A

SUBUNIT: useful, but often expensive and need to be mixed with adjuvants to be effective

LIVE RECOMBINANT: regulatory hurdles and public negative views

DNA: still needs refinement and a way to avoid gene silencing by host

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5
Q

How are attenuated live vaccines typically developed?

A

target mutations in a gene to see if it is a virulence factor —> excise/induce mutation —> see if new mutant is as virulent as the original

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6
Q

How are subunit vaccines typically produced?

A

overexpression of genes for antigen purification

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7
Q

How can transposons be used to define virulence factors?

A
  • use a plasmid containing a transposon to transform a pathogen and screen for clones
  • observe the ability of individual clones to cause infection
  • if not affected, gene interrupted is not a virulence gene
  • if affected, gene interrupted is a virulence gene
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8
Q

How are genes overexpressed for antigen purification?

A

once the antigen is identified, the gene encoding for it can be cloned and moved to a high copy number plasmid under the control of a regulated and efficient promoter to allow high levels of expression

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9
Q

How can a vaccine be developed to be effective against multiple diseases?

A

overexpression of an antigen from one pathogen and placed in another

(transform Brucella to also expressing pag gene codes for part of anthrax toxin that antibodies are able to neutralize = Brucella and Anthrax vaccine)

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10
Q

What is the current 9-step bacterial diagnostic routine?

A
  1. obtain clinical sample
  2. direct examination (stain smear, fluorescent Ab)
  3. inoculation of culture media (blood agar, MacConkey’s)
  4. incubation (aerobic, anaerobic)
  5. macroscopic/microscopic study (Gram stain)
  6. inoculation of differential media
  7. incubation
  8. read differential tests
  9. identification of genus
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11
Q

What are the main 3 advantages of using DNA-based tests?

A
  1. high sensitivity - can detect the presence of a single organism
  2. high specificity - can detect specific genotypes, determine drug resistance, and predict/confirm virulence
  3. speed - quicker than traditional culturing for certain organisms
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12
Q

In what 6 instances are DNA-based tests most likely used?

A
  1. indicate culture identity and confirmation
  2. when a sample has low numbers or difficult to isolate and grow (Mycobacterium, fungi, viruses)
  3. organisms present in small volume specimen (intra-ocular fluid, forensic samples like a swab)
  4. molecular epidemiology to identify point sources for hospital and community-based outbreaks
  5. zoonotic and highly infectious agents to minimize hazards of handling (Brucella, Franciscella, Coccidiodes)
  6. non-viable organisms (tied up in immune complexes)
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13
Q

What is a major con to using serology tests for identification over DNA-based tests?

A

host must make an antibody response first, which can take up to 10 days

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14
Q

What are 4 disadvantages of using a DNA-based test?

A
  1. expensive, including supplies and equipment
  2. so specific that you must have good clinical data to support infection by that organism before testing is initiated
  3. will miss new organisms unless sequencing is done (not practical in clinic)
  4. may be a problem with mixed culture infections - need to assay for all organisms causing the infection symptoms
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15
Q

What type of tool is the Polymerase Chain Reaction (PCR)? What are the 3 major steps?

A

an exponential amplification tool for a specific part of DNA for identification purposes - NOT diagnostic

  1. denaturation - unwind DNA
  2. annealing - primer binds to complement DNA sequence
  3. extension - Taq DNA polymerase extends primer 5’ to 3’ to synthesize cDNA
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16
Q

What is the causative agent of Brucellosis? What does it cause? How can it be diagnosed? What 3 aspects make it difficult to diagnose/treat?

A

Gram-negative, cocco-bacillus genus Brucella

abortion in livestock caused by Brucella abortus

INDIRECT: serum serology, antibodies to O-side chain
DIRECT: culture, identification

  1. all 8 Brucella species grow slowly
  2. biochemical phenotyping requires multiple tests
  3. vaccine strain RB51 can cause abortion if given to pregnant cows
17
Q

How is real-time PCR different than standard PCR?

A

fluorogenic version of PCR that detects the accumulation of amplification during the reaction; the data is then measured at the exponential phase of the PCR reaction, not via agarose gel separation and sequencing

18
Q

What enzyme is important for real-time PCR?

A

5’ nuclease that displaces the fluorogenic reporter from the DNA
reporter will only fluoresce if the primer has bound the DNA molecule and replication has displaced it and released it from its inhibitor

19
Q

What is the cycle threshold in a real-time PCR graph?

A

how many cycles of replication is necessary to get a positive fluorescent signal

20
Q

What are 3 advantages of RT-PCR?

A
  1. extremely fast, threshold time can be as little as 10-20 mins
  2. can multiplex the analyses so that multiple signals can be generated (detection of different serotypes, species, etc.)
  3. can readily be automated to handle large sample numbers
21
Q

What is the most accurate, fast, and least expensive diagnostic method available for bacteria and fungi that can be cultured? What is the major advantage with respect to results?

A

MALDI-TOF (Matrix-Assisted Laser Desorption Ionization - Time of Flight) Mass Spectrometry

computerized with a specific software - no human interpretation needed

22
Q

What are the main 4 major advantages to PCR-based technology? 2 drawbacks?

A
  1. sensitivity
  2. speed
  3. no need for culture
  4. specificity
  5. expense
  6. technical expertise
23
Q

How does recombinant DNA allow for vaccine enhancement?

A
  • overexpression of genes relatively easily for purification and in attenuated pathogens
  • targeted gene mutations very easily
  • creation of stable, non-disease producing DNA vaccines

Veterinary Bacteriology & Mycology (47 decks)

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