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General Microbiology Flashcards

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1
Q

Why is it useful to determine if bacteria is gram positive or gram negative?

A

Gives us an idea of what type of antibiotics to use.

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2
Q

What does gram stain help us do?

A

Differentiate between the two major groups of bacteria (gram positives and gram negatives)

It is the first step in identification of bacterial species and demonstrates a basic difference in cell wall structure

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3
Q

What are acid- fast stains used for?

A

To identify acid fast genetically engineered bacteria (mycobacteria). Hot pink. Grow inside macrophages so tend to grow in clumps (hundreds of organisms in a clump)

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4
Q

What is Giemsa used for?

A

Used to ID organisms in blood such as Malaria

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5
Q

What is dark stain used for?

A

Enables you to Look just at refracted light

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6
Q

What does diameter of a virus tell you?

A

Possibly the family the virus is in

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7
Q

What is a good way to identify and study a bacteria or virus?

A

Grow or culture in a cell free media, broth or agar, in a cell type

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8
Q

What are obligately intracellular parasites?

A

Only grow in animal cells. (can grow them in complex media, often containing serum)

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9
Q

How are chick embryos used?

A

As cultures. They are excellent for influenze and hard to grow viruses. Some bacteria and viruses can only be cultured using chick embryos.

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10
Q

What are pathogen free animals?

A

Relatively pathogen free animals that are used to study different viruses and parasites. Some are only able to be studied in this manner. Can use natural host or artificial host (mice are common)

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11
Q

Tests to detect microbial proteins or carbohydrates

A

Toxin antitoxin tests, labelled antibody test, ELISAs, neutralisation or inhibition assays, haemagglutination, latex aggulination

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12
Q

Pathogenesis (questions you might ask when considering pathogenesis)

A

How does the agent cause disease? How does the agent enter the host? How does the agent cause disease in the host (clinical signs)? How does the agent respond to infection? How does the agent get out of the host to infect new animals?

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13
Q

Virus taxonomy

A

Taxonomy based on type of nucleic acid, strategy of viral replication, morphology of virion, sequence analysis of the viral genome

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14
Q

Light microscopy- what is this essential for? What can it not be used for?

A

Essential for ID of bacteria and fungi. VIRUSES are not visible by light microscopy- electron microscopy for viruses.

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15
Q
A

a. Bacillus
b. Coccobacillus
c. Coccus
d. Chain of bacilli
e. Chains (Streptococci)
f. Clumps (Staphylococci)
g. Curved (vibrio)
h. Filamentous bacillus
i. Diplococci
j. “Chinese letters”
k. Branching
l. Spirillum

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16
Q
A

Bacillus anthracis

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17
Q
A

Streptococcus agalactiae

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18
Q
A

Bacillus anthracis

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19
Q

Gram negative or gram positive?

A

Gram positive because

Capsule is much thicker.

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20
Q

Gram negative or gram positive?

A
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21
Q
A

Helical, polyhedral, complex virus

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22
Q

3 special types of media

A

Enrichment media, selective media, indicator or differential media

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23
Q

Properties used in bacterial ID

A

Structural characteristics (cell shape and size), colony morphology

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24
Q

What does phenol red tell us?

A

Fermentation of various carbohydrates. Gas production

Yellow = acids produced by fermentation

Red= no acid produced

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25
Q

Virus structure: nucleocapsid and envelope?

A

Nucleocapsid: nucleic acid surrounded by protein coat (capsid)
Icosahedral or helical shape of capsid (protein coat)

Envelope: acquired by budding through cellular membranes, Glycoproteins on surface of envelope

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26
Q
A

Helical, polyhedral, complex

Helical ex. RNA: Paramyxo, Rhabdo, Corona

Complex: Poxvirus

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27
Q
A

Icosahedral i.e. DNA: Herpes, Parvo. RNA: Hepadna, Reto, Calici, Flavi, Picorna

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28
Q

What are the components and properties of enveloped viruses?

A

Components: lipids, proteins, glycoproteins

Properties: Labile in the environment, sensitive to acid, detergent, dying and heat, modifies cell membrane, released by budding and cell lysis

Must be in moist conditions, spread in large droplets, secretions, transfusions, etc, do not survive adverse conditions (GIT), do not need lysis to spread within host, antibody alone may not provide immunoprotection (Cell mediated immunity- a response that does nto involve antibodies, but rather involves the activation of phagocytes, antigen specific cytotoxic T lymphocytes, and the release of various cytokines in reponse to an antigen)

e.g. Herpes, Pox, Flavivuris, Togavirus, Coronavirus, Paramyxovirus

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29
Q

What are the components and properties of non-enveloped viruses?

A

Components: proteins

Properties: stable in the environment, insensitive to acid, detergent, drying and heat, released by cell lysis

Can be spread easily, infective after drying, survive adverse conditions (inside GI), resistant to detergents, antibody may provide immunoprotection

e.g. Parvo

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30
Q

How do we diagnose viral infection?

A

Embryonated egg inoculation (lots ot time, labour, and difficult, but for an infectious virus)

Cell culture (time, labour, difficult, but for an infectious virus)

Polymerase Chain Reaction (rapid, specialized equip, quantatative, doesnt need infectious virus)

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31
Q

Dimorphic

A

Fungi that can exist as mold/hyphal/filamentous form or as yeast. An example is Penicillium marneffei: At room temperature it grows as a mold, at body temperature it grows as a yeast.

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32
Q

Spores- sexual and asexual

A

Spores can generate another individual of the species. Any viable cell may be called a spore. Asexual or sexual reproduction.

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33
Q

Culture of fungi

A

Room temperature, fequently prolonged, aerobic, selective media, identification often morphological or cultural (colour, fruiting bodies). Easy to culture- think of yoghurt.

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34
Q

Identification of fungi in tissue- what is the method? what stain do you use?

A

Wet mounts of tissue samples, histological specimin stained with periodic acid Schiff (PAS) or silver

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35
Q

What do you need to consider when transporting a sample to the lab?

A

Obligate aerobe, obligate anaerobe, microaerophilic (needs oxygen but poisoned by high concentrations of oxygen), facultative anaerobe, aerotolerant, enveloped or non-enveloped

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36
Q

3 ways of antibody detection

A

Neutralisation assay, ELISA, HAI (hemagglutination assay)

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37
Q
A
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38
Q

Endospores

A

An endospore is a dormant, tough, non-reproductive structure produced by a small number of bacteria. Main purpose is to ensure the survival of the bacteria through tough environmental condition. IMPERVIOUS TO STAINS.

Source of the organism. Bacillus and Clostridium species. No metabolic activity. Resistant to heat, drying, disinfectants, and radiation. Impervious to stains.

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39
Q

Broad spectrum antimicrobial

A

Targeted is better. WIth the broad spectrum, the bacteria that survive will have plenty of room to spread out and take hold.

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40
Q

Viral replication

A

*Attachment: viral proteins bind to cell surface receptors

*Uptake of virus: by receptor of mediated endocytosis or by fusion with plasma membrane

*Uncoating: making viral genes available for transcription

*Early viral genes: shut down cellular protein synthesis, regulate expression of viral genome and production of enzymes (polymerase)

* Late viral genes: express structural proteins for virus assembly

*Enveloped viruses: acquire their envelope by budding through the plasma membrane, the cytoplasmic membranes or the nucleus membranes

* Non-enveloped viruses: accumulate in cytoplasm and are released when the cell lyses

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41
Q

How do you grow something that doesn’t replicate itself (virus)?

A

In a live host, in an egg (amniotic cavity, yolk sac, allantois), in cell culture.

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42
Q

Bacterial pathogenesis

A

*Bacterial adhesion: particularly important on mucosal surfaces

* often associated with pilli or fimbriae (what the bacteria uses to attach)

* Motility: flagellae (either polar or peritrichous)

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43
Q

Virulence factors of bacteria

A

* Exotoxins- damage cell membrane, disrupt intracellular processes e.g. tetanus toxin by Clostridium tetani and botulinum toxin by Clostridium botulinum, Bacillus anthracis.

* enzymes- elastase, collagenase, proteinase, coagulase e.g. Pseudomona aeruginosa- cause damage to host tissues like DNAses which break down DNA

* Leucotoxins- disrupt the phago-lysosome (cytoplasmic body -fusion of a phagosome with a lysosome- after fusion the food particles contained within the phagosome are usually digested by the lysosome)

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44
Q

How is the complement activated?

A

By virus infected calls, bacterial, or bacterial products.

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45
Q

Dissemination of virus within the host

A
  1. Local spread- from cell to cell in close proximity to site of entry. Contiguous infection from adjacent cells.
  2. Haematogenous spread- how does the virus get into the bloodstream? bite of an arthropod vector, iatrogenic- contaminated need, transfusion, etc, Primary replication- drainage to regional lymph nodes, thoracic duct, systemic circulation. Virus can be free in plasma or cell associated. (spin plasma or look at cells- making a diagnosis).

Primary replication- low titre (i.e. with enteroviruses- in intestinal epithelium)

Secondary replication- high titre (i.e. with enteroviruses- in lymphoid tissues, CNS)

  1. Tissue invasion- mechanism that promote movement from vascular space to tissues is poorly understood. Often associated with infection of endothelial cells and/or passive transport across the endothelial cells.
  2. Neural spread- virus moves to the nerve supplying the site of primary replication. Immune evasion strategy.
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46
Q

What is vertical transmission?

A

Mother to offspring- transmitting in utero or in ovo. Transmission across placenta, in birth canal, in colostrum/ milk. Cause embryonic death, mummification, resorption (time of gestation or congenital defects).

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47
Q

What is horizontal transmission?

A

Direct contact- licking, rubbing, biting, sexual contact OR indirect contact (fomites)- feed and water containers, bedding, dander, tack, clothes, etc.

Airborne, arthropod-borne transmission, iatrogenic transmission, nosocomial transmission, zoonotic

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48
Q

What are some host defence mechanisms of the respiratory tract?

A

* Goblet cells secrete mucus

* Gastric pH 2-3

* Proteases secreted by gastric and pancreatic cells

* Bile salts

*Mucus containing IgA

* Mucocilliary clearance: mucus propelled and impinging particles propelled orad by ciliated columnar epithelial cells

* Humoral and cellular immune mechanisms operate locally

*IgA- in mucus (local immunity) tonsillar lymphoid tissue, alveolar macrophages, other phagocytes

** can establish local (confined to cells linging intestinal lumen) or systemic (cross intenstinal mucosa and invade underlying tissues and spread within the body) infections

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49
Q
A
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50
Q

What are some characteristics of GI viruses?

A

Acid stable, resist degradation of bile salts, resist proteolytic inactivation. Parvo for example- infects the crypt cells. Outer protein coat of rotavirus virion is digested by trypsin in the small intestine. Coronaviruses are enveloped (exception to every rule)- but they are still GI viruses.

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51
Q

Genitourinary tract

A

HIV, HSV-2, HPV. No effective physical barrier. Condoms. Bacterial cystitis.- urine flushing a protective mechanism.

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52
Q

What are three other sites of entry and examples?

A

Mammary gland- acute mastitis- via teat canal during or after milking.

Umbilicus- neonatal sepsis.

Conjunctiva- direct inoculation, usually local infection, very rarely spreads systemically.

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53
Q

Routes of entry

A

* Skin

* resp tract

* intestinal tract

* oropharynx

* conjunctiva

* urogenital tract

* mammary gland

* umbilicus- neonatal infections

54
Q

Toxicoses

A

No entry required.

Botulism, tetanus, mycotoxicoses

Toxins survive passage through intestine

Direct effect on intestine or systemic effect

55
Q

Mucosal surfaces

A

Most common route of infection. IgA- blocking adherence, export of invading organisms, intracellular neutralisation, opsonisation (flags it for the rest of the immune system). Good immune response prevents infection and therefore prevents disease. Primary vs. secondary responses.

56
Q

Iceberg effect (describe)

A
57
Q

Clinical signs vs. symptoms

A

Clinical signs are abnormalities detected during a physical examination (objective observations) while symptoms are a feature that indicates a conditon of disease, particularly a feature that is apparent to the patient

58
Q

Infection vs. disease

A

Infection is the growth (detection) of infectious agents in the body. Disease refers to organ or body system dysfunction (some loss of function).

59
Q

Infectious disease vs. contagious disease

A

Infectious disease is caused by infectious agents. Contagious disease is an infectious disease the can spread from one animal to another animal.

60
Q

Making a microbiological diagnosis.

A

Take the history (single or multiple cases, duration, clinical signs, case definition, any treatment?), sample collection (sample from lesion affected, body system, aseptic collection technique, commensal or normal flora, sample methodology), transport to lab (obligate aerobes, obligate anaerobes, microaerophilic, facultative anaerobes, aerotolerant, enveloped or non-enveloped), diagnostic tests (agent: culture, electron microscopy, PCR, haemagglutination inhibition OR Ab detection: neutralisation assay, ELISA, HAI).

CONSIDER: Normal flora of animal

61
Q

How do you detect an antibody?

A

Agglutination, precipitation, complement fixation, neutralisation, immunoassay

62
Q

How do you detect an agent?

A

Culture (bacterial or viral), PCR, Agglutination, immunoassay, immunofluorescence, immunohistochemistry, FACS

63
Q

How do you detect CMI (cell mediated immunity)?

A

Hypersensitivity, Blastogenesis assays, Cytokine assays

64
Q

Agglutination Tests

A

Antigen binding to antibody results in agglutination. No indicator system required. Mostly bacterial diseases. This is also the basis of the blood typing test.

65
Q
A

Agglutination test for antibody

66
Q
A
67
Q
A
68
Q
A

Virus neutralisation test. Relies on agent propagation in cell culture. Cell monolayer. Liquid growth medium. Filtered sample.

Distermper, Parvo, etc. Unknown sample (with or without Abs)

Cell controls- so if cells aren’t growing well then assay isn’t valid

If cells are happy then we look at serum assay

then we look at virus titration to see if it neutralizes a known amount of virus. Look at the virus conc. (do we have the right amount?)- then we can read it

69
Q

Convalescent serum sample vs. acute

A

Serum from an animal or person who has recuperated from a particular infection vs. serum from a patient with a current (acute) infection for which bacterial or viral specific immunoglobulins (usually IgM) are measured.

70
Q

When might you use an embryonated egg inoculation?

A

Infectious virus but it is time consuming, labour intensive, and technically difficult.

71
Q

What are the three steps of PCR?

A

Melting- denaturing of the DNA duplex at a high temp to yield single stranded DNA

Annealing- primers anneal to the single stranded target DNA sequence

Elongation- DNA polymerase extends the primers by adding dNTPs to the phosphate backbone

72
Q

Sample collection and storage

A

*collect lesions on live animals (dead animals undergo autolysis- early after death is better)

*minimize contamination

*Collect samples before antimicrobial administration

*Refrigeration

*Appropriate ID and OHS approved container/media

73
Q

ELISA

A

ENZYME LINKED IMMUNOSORBENT ASSAY

For the detection of antibody-antigen interactions.

For an ELISA designed to detect an antibody, the plate is coated with virus or viral antigen that is inactivated. Serum samples to be tested for the presence of antibody to the virus are then added to the virus- coated wells to allow for any specific anti-viral antibody to the serum to bind the virus. The remainder of the ELISA test is then the application of a colorimetric method that allows detection and quantification of the amount of antibody that is bound to the virus.

* the enzyme acts as an amplifier, even if only few enzyme linked antibodies remain bound, the enzyme molecules will produce many signal molecules. The more primary Ab is present in the donor serum, the more secondary Ab + enzyme will bind and the faster color will develop.

e.g. Detection of antibodies for EHV4 and EHV1 (alphaherpesvirinae family)- closely related but cause different dieases. If you used the whole virus for EHV4 and EHV1 detection, you could not tell the test results apart for which virus. You must use smaller regions (glycoproteins in this case) that are strongly immunogenic and contain type specific epitopes. ELISA is the only test that can tell them apart.

74
Q

Ideal vaccine

A

Easy to administer, highly efficacious, one vaccination would provide life long protection, minimal side effects, safe to administer to all animals (young, old, pregnant), DIVA capacity (differentiated infecting vaccinated animals)

75
Q

Non-replicating vaccines vs. Replicating

A

Non-replicating (need multi doses- no memory response)- inactivated viral vaccines, bacterins (inactivated bacteria), toxoids (inactivated toxins), recombinant proteins, direct DNA

Replicating vaccines- attenuated viral vaccines, recombinant vector vaccines (more technologically advanced- what proteins are actually important? What gene encodes that protein? Use those. Etc.)

76
Q

Active vs. Passive Immunity

A

Immune response (memory cells) vs. Colostrum.

77
Q

Attenuated/ Live vaccine

A

Extremely effective but quite a few issues associated. Can cross the barrier into the foetus in a pregnant animal. Unsafe for unhealthy, young, or old animals.

78
Q

Killed vaccine- drawback?

A

At least 3 times of vaccinating 4-6 weeks later. Especially a problem if disease is imminent and need for rapid onset of immunity.

79
Q

DIVA

A

(Differentiation between Infected and Vaccinated Animals)

DIVA vaccines carry at least one epitopes less than the microorganisms circulating in the field. An accompanying diagnostic test that detects antibody against that epitope allows us to make that differentiation. Used to be called “marker” vaccines.

**Glycoproteins on the pathogen’s envelope. When animal gets infected they mount a response to all of these proteins. It matters because if you are thinking about controlling the outbreak- then you cannot differentiate the animals who have been vaccinated or infected with blood tests- they would look the same.

80
Q

Non-replicating/ inactive vaccines advantages and disadvantages

A

Most common in veterinary medicine

Advantages: safe and successful technology

Disadvantages: reduced immunogenicity, boosters required, adjuvants required.

* Antibody mediated (TH2) response

*subunit vaccines and recombinant proteins

81
Q

Toxoids

A

Circulating IgG is protective

Tetanus and botulinum toxins

Exotoxins are the issue and IgG is protective against

82
Q

Example of Recombinant DNA vaccine ex

A

Hendra virus. G protein: Binds to host cell receptors. Initiates infection. So vaccine binds the G protein receptor instead.

83
Q

Herd Immunity

A

Sufficient immune animals in herd to reduce the spread of infection, fewer suceptible, reduced excretion.

84
Q

Blanket Vaccination

A

All animals in a particular geographic area that are susceptible will be vaccinated

85
Q

Ring Vaccines

A

Depends on mode of transmission will determine the radius.

86
Q

Endemic disease control

A

Thinking about biosecurity (your own), is it contagious, is it a toxins, vaccination- do you vaccinate the herd that is affected or the ones next to affected? The ones next to the affected.

87
Q

Legislative basis for EAD (Emergency Animal Disease)?

A

Chief Veterinary Officer (CVO unit)- sampling and testing, control and vaccination, eradication (+/- slaughter)

*Nat’l coordination > 1 state

* animal health committee

*National management group

* Consultative Committee on EAD

AUSVET PLAN

88
Q

AUSVET plan for equine influenza?

A

Containment and control based on: movement control, +/- vaccination, +/- regionalisation/ zoning

89
Q

Rabies pathogenesis

A

Transmission usually occurs through bites (scratching and licking can too).

Virus replicates in muscle cells, very little virus replication in nerve cells, variable incubation period (weeks- years), bites around face, deep bites = shorter incubation period

90
Q

Where is rabies maintained?

A
  1. Sylvatic (wildlife) rabies
  2. urban (dog) rabies

* maintained when dog pop density > 4.5 dogs/km^2

* >55,000 human deaths per annum

*>95% of all human cases related to dog bites

*>99% of human cases occur in Asia and Africa

* Majority of cases in children <15 yo

91
Q

Sylvatic rabies UDA 2010

A

* 92% of reported cases were sylvatic rabies

*37% raccoons- of all animal cases

*23.5% skunks

*foxes- 7%

Other wild animals- 1.8%

92
Q

Foot and Mouth Disease

A

* Important disease of farmed livestock

*Highly contagious

*Very high morbidity/low mortality

* Dramatic reduction in production (especially cattle)

* Disease of economic important (direct and trade)

93
Q

FMD

A

* Multiple host species, multiple modes of transmission, multiple serotypes, small infective dose, rapid replication, virus shedding before clinical signs, highly contagious, carrier state

94
Q

FMD Vaccination vs. slaughter

A
  1. Excrete FMDV up to 4 days prior to clinical signs
  2. Incubation period <14 days- index (2-3 day outbreak)
  3. HIghly contagious- spready by aerosol, oral, fomites
  4. Pigs are amplifier hosts
  5. Cattle and sheep can be persistently infected
  6. Sheep are often inapparent carriers
  7. Vaccinated animals can be carriers, serologically indistinguishable from exposed animals
95
Q

What type of metabolism do all pathogenic bacteria have in common?

A

Chemoheterotrophs- an organism deriving energy by ingesting intermediates or building blocks that is incapable of creating its own (most chemoheterotrophs obtain energy by ingesting organic molecules like glucose)

96
Q

Compare and contrast viruses and bacteria

A
97
Q

What grows in a cell culture? What does it need?

A

Some bacteria, and all viruses will grow in animal cells. Often contain serum as an additive. Such as fetal bovine serum- the blood fraction remaining after natural coagulaton of the blood followed by centrifugation to remove any remaining blood cell lines. Why this one? It has a very low level of antibodies and contains more growth factors, allowing for versatility in many different cell culture applications.

98
Q

Why do you sometimes want to detect microbial proteins or carbohydrates?

A

Some microbial diseases are a result of the action of toxins. The organism itself may be located at a distant site of the body, or may not even have entered the animal’s body. In these cases, diagnosis is often based on detecting the pathogen’s protein (sometimes even if the pathogen detection is possible, it might be easier to detect the pathogen’s proteins- say, if it is hard to culture).

99
Q

What is a Toxin-Antitoxin test?

A

An animal is inoculated with tissue, serum, or gut contents from a clinical case and a second animal is inoculated with the sample from the animal and a specific antitoxin. If the animal inoculated with the sample alone is affected and the animal administered antitoxin is not, the test is positive.

100
Q

What is an antitoxin?

A

An antibody with the ability to neutralize a specific toxin. Antitoxins are produced by animals, plants, and bacteria. Examples are antitoxins to diphtheria and tetanus

101
Q

What are labelled antibody tests?

A

Antibodies specific for proteins of the pathogen are labelled and applied directly to prepared tissue samples or smears. The antibody is generally conjugated to flourescein or horseradish peroxidase. AND you must add some sort of treatment to detect the label (incubation with a substrate for the enzyme). Whole organisms, or cells in which they have replicated, are specifically labelled.

102
Q

What is EHV4 v. EHV1

Why is it important to ID horses infected with these viruses?

A

Major cause of respiratory disease in young horses vs. EHV1 is the most important cause of viral abortion in mares, but it can also cause respiratory disease in foals. As they are herpes viruses, both EHV4 and EHV1 become latent in their host and can cause recurrent disease as a result of reactivation.

* Important to use ELISA to detect infected horses because of contagious potential for EHV1 storms.

103
Q

What are two uses of ELISA regarding EHV1 and EHV4?

A

Determine which horses are latently infected carriers of EHV1 so it can be effectively managed on stud farms

AND

To determine the cause of abortion where no foetal tissue samples can be obtained. Paired serum is obtained from the mare and taken about 10-14 days apart. A rise in antibody levels is indicative of recent infection with EHV1, or alternatively the absence of antibody means EHV1 was not the cause of the abortion.

104
Q

What is the analytical sensitivity of ELISA tests?

A

Very high meaning they detect very small amounts of antibody in serum, possibly less than the amount required to neutralise the virus in an accompanying test.

105
Q

What was soy milk used for in the ELISA practical?

A

All sites in the wells that are not coated with specific antigen need to be covered to prevent non-specific binding of other antigens which give false results. So you used a highly concentrated protein solution that is unrelated to the antigens in our test serums. You remove the blocking solution before the test of course.

106
Q

What do you calculate in an ELISA?

What does seroconversion mean?

What do you use to read the plates?

Example of what is a negative, indeterminate, and positive

A

the mean optical density.

Seroconversion- Antibody becomes detectable in the blood (antibody in the blood > the amount of antigen)

* use a spectrometer at a wavelength of 410nm

O.D. < 1: Negative

O.D. between 0.1-0.2: Indeterminate (repeat test)

O.D. > 0.2 Positive

107
Q

Indirect vs. direct ELISA?

A

Direct ELISA- only one antibody is used and fewer steps required- used to test a specific antibody-to-antigen reaction and helps eliminate the cross reactivity between other antibodies (i.e. EHV4 and EHV1 could be misdiagnosed because they are so similar). Problem: The method of antigen immobilization is not specific: when serum is used as the source of test antigen, all proteins in the sample may stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding the well surface.

Indirect ELISA- In EHV4 and EHV1 example after you add the primary antibody, you add a secondary antibody directed against primary antibody (anti-horse, for example, goat antibody). Then you can add the color, the second antibody takes it up. This solves the problem with the Direct ELISA- the sandwhich ELISA- uses a “capture” antibody specific for the test antigen to pull it out of the serum’s molecular mixture.

108
Q

What are neutralisation or inhibition assays?

A

The growth of some bacterial species and all viruses can be blocked by specific antibodies directed against their surface proteins. Treatment of these pathogens with such antibodies before inoculation into culture can thus be used to identify them.

109
Q

What are haemagglutination tests?

A

Some viruses can agglutinate red blood cells from particular animal species. The clumping of red blood cells by large numbers of virions can help identification.

e.g. Neonatal Isoerythrolysis is a problem in foals that results from the passive transfer through colostrum of maternal antibodies capable of binding the foal’s erythrocytes

110
Q
A
111
Q

What is a specific immune response detection? 2 ways to do so?

A

You may measure antibody concentrations, cell mediated responses, or cytokine production. One test can only enable one to say that the animal has, at some time in its life, been infected. If you do two tests, one at the time of illness and several weeks later at the convalescent phase, an increase in response over this period indicates the infection has occurred recently. OR detection of recent immune response characteristics e.g. IgM

Antibody concentrations & Cell mediated responses are the two ways

112
Q

Cell Mediated Responses to ID an infection

A

Inoculation of a small amount of protein from the organism, usually into the dermis, induces a migration of T cells and macrophages into the region. The swelling and redness can be detected a few days later. Cultures of lymphocytes in the presence of protein from an organism can also be used. If the animal has previously encountered the organism the memory response of the lymphocytes to the protein will stimulate their growth. You can also use an ELISA to detect the production of IL-2 by the stimulated lymphocytes.

113
Q

Saprophytic- general example and how it infects

A

Living freely in the environment- most bacteria and fungi

e.g. Soil saprophyte- loss of epithelial integrity, immunocompromise, broad spectrum antimicrobial

114
Q

Normal Flora

A

Varies greatly between individuals and species with age, environment, nutrition, and location. Herbivores major part of digestion, other species it provides vitamins such as B & K. It can also breakdown potentially toxic products in food. Also by occupying a particular niche, competition for nutrients, inhibits potential pathogens.

Places: mouth and nasopharynx, stomach (low numbers due to low pH), duodenum, jejunum and ileum, large intestine (high numbers), trachea, bronchi, lungs, vulva and prepuce, bagina, skin, mammary gland

115
Q

Bacteriostatic agents vs. Bacteriocidal

A

Bacteriostatic agents prevent further multiplication but allow the organism to remain viable. While Bacteriocidal agents damage the cell irreversibly, so that further multiplication is not possible (such as nucleic acid damage or rupture of the virion or cell membrane). (Disinfectants must be bacteriocidal, chemotherapeutic agents can be either).

116
Q

What temperature are most agents inactivated? Spores?

A

50C-70C for a few minutes. Most spores at 100C.

e.g. Milk Pasteurisation- liquid held at 63C for 30 minutes or 72C for 15 seconds in flash pasteurisation. Removes 97-99% of bacteria thus slowing spoilage.

117
Q

What is moist heat?

A

Heat sterilisation involves protein denaturation and melting of membrane lipids. Such disruption is aided by the presence of water to create hydrogen bonds with the separated molecules. Thus lower temperatures are necessary to sterilise wet material than dry material. Boiling is commonly used but some pathogens, such as Clostridium tetani (Tetanus) can survive several hours of boiling

118
Q

What is dry heating?

A

Heating at 160C for 1 to 2 hours.

119
Q

Preservation of Microorganisms

A

Conventional refrigerators are not adequate for preservation of bacteria but liquid nitrogen, solid CO2, or low temperature refrigerators can be used. Lyophilised (freeze dried) microorganisms can be stored at room temperature (many live vaccins are supplied lyophilised and are then reconstituted by adding water).

120
Q

What is Peracetic Acid?

A

Strong oxidizing agent, toxic, used for sterilising gnotobiotic animal chambers

121
Q

What is Hydrogen Peroxide?

A

Oxidizing agent, not effective against many species due to possession of catalase (enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen)

122
Q

Cell culture viral infection diagnosis vs. PCR viral infection diagnosis

A

Cell culture: Time consuming, labour intensive, technically difficult, infectious virus, Cytopathic effect (CPE)

PCR: Rapid diagnosis, specialized equipment, may be quantitative, doesn’t need infectious virus (lab transportation considerations are less important- key because so many possible ways to kill a pathogen before analysis)

123
Q

Nosocomial Infection

A

Organism remains infectious within the hostpial environment e.g. Parvovirus (environment), MRSA (staff and patients)

124
Q

What is Viral Cytopathic Effect?

A

Refers to structural changes in the host cells that are caused by viral invasion. The infecting virus causes lysis of the host cell or when the cell dies without lysis due to an inability to reproduce. Common examples include rounding of the infected cell, fusion with adjacent cells to form synctyia (multi nucleated cell resulting from fusions of multiple uninuclear cells), and the appearance of nuclear or cytoplasmic inclusion bodies.

125
Q

Titre

A

A way of expressing concentration. Titer testing employs serial dilution to obtain approximate quantitative information from a procedure that only evaluates as positive or negative. The TITRE corresponds to the HIGHEST dilution factor that still yield a POSITIVE READING. E.g. a Viral titre- which is the lowest concentration of virus that still infects cells. to determie the titre, several dilutions are prepared such as 10^-1, 10^-2, etc.

126
Q

Iatrogenic Transmission

A

Transmission due to medical procedures such as injection or transplantation of infected material. e.g. Creutzfeldt Jakob Disease by injection of contaminated human growth hormone, or MRSA- stay in hospital

127
Q

Virus entering the respiratory tract through droplets or saliva- what size does it have to be to get to different locations?

A

> 5 microns- filtered by the turbinates

< 5 microns- lower respiratory tract

128
Q

When pathogens gain entry via the gastrointestinal tract, where can they go?

A

Local infections- confied to cells lining intestinal lumen

OR

Systemic infections- viruses cross intestinal mucosa and invade underlying tissues and spread within the body

e.g. Rotaviruses and Coronaviruses- secretory and osmotic diarrhoea

129
Q

What kind of test is this? What is happening with the antibody and the pathogen in each?

A

Virus neutralization test. In the uninfected, the antibody has bound the pathogen and neutralized it. So the antibody is present. IN the infected, the pathogen has destroyed the cells- you are seeing a plaque.

130
Q

In a virus neutralization test, why do we use three controls?

A

First we look at cell controls- if the cells are growing okay and cells are healthy- then we move to look at the serum control- ensure it is not toxic (some serum is toxic to the cells, so our assay wouldn’t read properly). Then we look at virus titration- work out what the concentration of the virus is- and if we have the right amount.

131
Q

PCR

A

Essentially you are amplifying a single copy of a piece of DNA across several orders of magnitude, generating millions of copies of a particular DNA sequence. You can look at the genetic sequences of an agent to determine if it is a pathogenic strain or non-pathogenic, for example. You can also quantify the viral load in a patient.

Require a thermal cycler.

Melting: denaturing of the DNA duplex at a high temperature to yield a single stranded DNA.

Annealing: primers anneal to the single stranded target DNA sequence.

Elongation: DNA polymerase extends the primers by adding dNTPs to the phosphate backbone. (dNTPS are molecules containing nucleotide bound to two phosphates. Nucleotides are the building blocks of nucleic acids. NTP provides the energy and phosphate group for phosphorylations)

132
Q

Non-replicating vs. Replicating vaccines

A

The goal with any vaccine is to develop one that is capable of inducing potent, persistent cellular immunity, and broadly reactive neutralizing antibody responses. Replicating recombinants can be better at eliciting specific cellular immune responses and better at priming humoral immunity compared to non-replicating recombinants carrying the same gene insert. Live viral vectors, such as adenovirus, as vaccine vehicles present one option for inducing more potent immunity.

Non-replicating: inactivated viral vaccines (whole), bacterins (whole), toxoids (sub-unit), recombinant proteins, direct DNA

Replicating vaccines: attenuated viral vaccines (vaccine created by reducing the virulence of a pathogen but still keeping it live), recombinant vector vaccines.

Microbiology (6 decks)

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