Week 8 + 9 Flashcards
What is a vaccine?
- a suspension of antigens that is administered to induce immunity
- currently majority of vaccines derive from microbial pathogens for controlling infectious diseases
What does a vaccine contain?
- antigens
- preservatives + stabilizers - preserving Ag
- specific antibiotics - inhibiting bacterial and fungal growth
- adjuvant - enhancing the immune response to Ag
What is the purpose of adjuvant in vaccines?
- delay release of antigen from the site of injection
- induce secretion of chemokines by leukocytes
- ex: aluminum hydroxide, saponin, etc.
What are the different types of adjuvants?
What are features of an ideal vaccine?
- inexpensive
- consistent in formation, minimal variation
- stable
- proper type of immune response
- range of immunological epitopes
- long-lived immunity
- immunological memory
- no adverse effects
What are the kinds of infectious vaccines?
- live attenuated
- recombinant organism
- marker
What are features of live attenuated vaccines?
- intact, viable organism
- low-level infection
- no clinical disease/pathology
- pros:
- rapid immunity
- single dose
- cons:
- can become virulent
- less stable in storage
What are features of recombinant organism vaccines?
- use carrier organisms
- cannot become virulent
- adjuvant not required
What are features of marker vaccines?
- differentiate between infected + vaccinated immune response/animals
- ex: IBR w/ deletion
What are the types of non-infectious vaccines?
- killed whole organism
- subunit
- naked dna
- mRNA
What are features of killed whole organism vaccines?
- antigenically intact
- unable to replicate or induce disease
- in chemical killing-formalin, alcohol, or alkylating agents
- pros:
- safe
- stable
- no interference w/ other vaccines
- cons:
- slow immunity
- boosters
- less protection
What are features of subunit vaccines?
- contain immunological proteins/metabolites from a organism
- ex: purified proteins, synthetic peptides,recombinant proteins
What are features of naked DNA vaccines?
- gene is cloned from plasmid
- plasmids transfer the APC’s; pathogen gene is expressed and processed in APC for antigen presentation
What are features of mRNA vaccines?
- mRNA of gene of pathogen
- mRNA is expressed and processed by APC for antigen presentation
- ex: covid-19 vaccine (spike protein)
What are the kinds of immunization?
- active + passive
- immunization: aka vaccination, artificial induction of immunity to protect from infectious diseases
What are features of passive immunization?
- performed (changed) antibodies administered
- particular antigen
- inhibit endogenous antibody response
- sensitize recipient for hypersensitive reaction
- immediate protection
- fast, TEMPORARY, no memory
- ex:
- artificial: tetanus antitoxin, antivenoms, mAb to SARS-CoV-2
- natural: colostrum
What are features of active immunization?
- antigen administered
- induced immune response (humoral/cell-mediated)
- memory
- protection proportional to infection severity (no infection to no protection)
- ex:
- artificial: vaccine
- natural: antibody from infection
What are methods of vaccine delivery?
- injection
- intranasal
- needle-free
What are examples of adverse effects to vaccines?
- type i hypersensitivity: facial or periorbital edema, pruritus
- FISS (feline injection site sarcoma): surgical removal challenging, poor prognosis
Why are diagnostic tests important?
- confirm diagnosis
- determine treatment
- epidemiological surveillance
- prevention, control, and eradication strategies
- identification of new pathogens
What are the phases of diagnostic testing?
- pre-analytical: test selection, sampling, storage, transportation
- analytical: handling and analysis of specimen
- post-analytical: results and interpretation
What is used to determine test selection?
- pathogen type
- sample type
- test characteristics
- disease phase
- availability
- cost
What is the difference between sensitivity and selectivity in test selection?
- sensitivity: capacity of a test to correctly identify positive individuals (true positives)
- specificity: capacity of a test to correctly identify the negative individuals (true negatives)
T/F: more selective tests can be used to confirm a diagnosis
- false; specific
What are the test predictive values?
- positive predictive value: probability of a positive test being a TP
- negative predictive value: probability of a negative test being a TN
- prevalence is very important for predictive values
What are different immunodiagnostic techniques?
- serology: direct, indirect, titers
- enzyme linked immunosorbant assay (ELISA): direct, indirect, sandwich
- flow assays: lateral, bidirectional
- agglutination
- immunoprecipitation assays: solution based, gel based
- immunofluorescence
- immunohstochemistry
What are features of serology?
- in-vitro study of antigen-antibody interactions
- most commonly used diagnostic tool
- basis of several techniques
- usuall used with serum samples
- test infectious diseases, hormones, cancer, autoimmunity
What are the 3 main kinds of serology?
- direct: detects presence of ANTIGEN in sample, positive result = INFECTION
- indirect: detects presence of ANTIBODIES in sample, positive result = EXPOSURE
- titers: express concentration of antibodies/antigens, correlates with highest dilution at which they are still detectable via serial dilutions.
What are features of elisa?
- enzyme-linked immunosorbant assay
- uses enzyme labeled antibodies or antigens, with addition of a substrate, to detect and measure the concentration of analyte
- direct (+ sandwich) and indirect techniques
What ELISA technique is this?
- direct
What ELISA technique is this?
- indirect
What ELISA technique is this?
- direct, sandwich
What are features of flow assays?
- lateral or bidirectional
- ELISA-based
- qualitative or semi-quantitative
- point-of-care testing
- fast results
- no lab
What are features of agglutination?
- reaction between:
- antibody + particulate antigen
- antibody + soluble antigen coated latex beads
What are features of immunopreciptation assays?
- precipitation of antigen-antibody complexes
- exploits the concentrations of antibody and antigen (zone of equivalence) to measure relative quantities (titers)
- 2 methods:
- solution based
- gel based
What kind of immunoprecipitation is this?
- solution based
What kind of immunoprecipitation is this?
- line of precipitation on the gel indicates the zone of equivalence
What are features of immunofluorescence?
- fluorescent-labeled antibodies: tissue or cells, on slides
- requires a fluorescent microscope
- 2 methods: direct + indirect
What are features of immunohistochemistry?
- used on frozen or formalin-fixed tissues
- in situ results
- can detect tissue proteins or pathogen antigens
What are diagnostic techniques?
- direct examination
- culture
- immunologic methods
- molecular analysis
What are kinds of molecular diagnosis?
- electrophoresis
- restriction fragment length polymorphism (RFLP)
- hybridization
- nuclear acid amplification - target
- protein detection
What are features of electrophoresis?
- separated in a electrophoretic field
- negatively charged particles —> positive end
- size: smaller is faster
- structure: supercoiled (fastest) > linear > nicked circles (DNA)
What are features of restriction fragment length polymorphism (RFLP?)
- analyzing differences among homologous DNA sequences by restriction enzymes
- restriction enzymes: cut DNA at the specific recognition nucleotide sequences (SPECIFIC sequence)
- EcoRI: sticky end
- SmaI - blunt end - use in paternity tests, forensics
What are features of hybridization? Of probes?
- denatured, single-stranded DNA, ssDNA, (probe) binds to a complementary single-stranded sequences
- ex: dot, in situ, Southern, Northern, microarray
- fragment of nucleic acids
- labeled via radioisotope,enzyme, or chemiluminescence
- detect complementary sequences in samples
- high specificity
- variable size
What are features of nucleic acid ampification?
- target amplification: enzyme mediated process to synthesize copies of targeted nucleic acid
- polymerase chain reaction (PCR): sensitive!
- isothermal amplification (ex: LAMP)
- true/q PCR
- polymerase chain reaction (PCR): sensitive!
What are PCR primers?
- single stranded DNA fragments complementary to sequences flanking the region to be amplified
- distance between primer and binding sites determines size of PCR product
- determine specificity
What are features of primers?
- types: random or specific
- primers: product size, annealing temperature, specificity
- nucleotide composition
What are PCR variations?
- reverse-transcriptase PCR
- nested PCR
- multiplex PCR
- quantitative or real-time PCR
- etc.
What are features of real-time or quantitative PCR?
- probe or dye to generate a fluorescent signal from the product
- signal in real time allows quantification of starting material
- signal: exponential curve with lag phase, log phase, and stationary phase
- lag phase: inversely proportional to amount of starting material
What are features of isothermal activation/LAMP?
- pros:
- no thermal cycles needed, but need room temp
- quick - 1H
- sensitivity > PCR
- visible results
- cons: complicated design of primer plates
What are phenotypic characteristics (observable characteristics) of microorganisms?
- morphology: size, shape, structure
- biochemical reactions: staining, cell inclusions
What are advantages and disadvantages to microscopic identification?
What are features of concentration techniques?
- work to increase concentration of pathogens in the sample
- common in parasite diagnosis
- ex:
- flotation techniques
- sedimentation techniques
- baermann test for larval identification
What are features of fecal flotation?
- gross examination
- fresh feces: detect parasites in GI tract, liver, bile ducts
- fecal flotation: based on differences in specific gravity between the parasites eggs, larvae, cysts, or oocysts, and the majority of fecal debris
- determine presence of parasite, egg types, general level of infection (eggs per field view)
- McMaster egg counting slide
What are features of the baermann technique?
- method for extraction of nematode larva from fresh feces (ex: lung worm larvae in feces, larvae from fecal cultures, pastures,soil,tissues, etc.)
- warm water stimulates live larvae in the sample to move out and gravity pulls them to the bottom of the container
What are the two main categories of staining techniques?
- simple stain (only 1 dye)
- identification of morphology and cellular arrangement
- drawback: may not stain all components, difficult interpretation
- differential stain (more than 1 dye)
- identification of morphology and cellular arrangement
- distinguish between different cell types + structures
- drawback: multistep method requires more time, reagents, expertise
What are features of hematoxylin and eosin stain (H&E)?
- common tissue stain used to identify a range of normal/abnormal cells + tissues, can reveal viral, bacterial, fungal, and parasitic infections
- different structures have different dye affinities
- hematoxylin (basic stain): stains acidic (basophilic) or - charged particles (ex: nuclei, chromatin) PURPLE
- eosin (acidic stain): stains basic (acidophilic) or + charged components (ex: cytoplasmic components + granules) and extracellular components (ex: muscle, RBC) PINK
- detection of VIRAL INCLUSION BODIES: aggregates made mostly of proteins, represent site of viral replication
- virus cause virus-specific changes in cell structure, organization, and morphology
What are features of Diff-Quick?
- commercial romanowsky stain
- used in histological rapid staining
- fixative (fast green in methanol) > stain solution 1 (eosin G) > stain solution 2 (methylene blue)
- air dried sample prior
- modified wright-gives a stain: differential stain for adherence of pathogenic bacteria to cells (ex:blood cells, stain chromosomes)
What are features of Gram-stain?
- distinguish betweeen Gram + and Gram - bacteria based on CELL WALLS (peptidoglycan)
- G+ : PURPLE
- G - : PINK
What are some stains for specific bacterial structures?
- acid fast stain: stains organisms with impenetrable cell wall (ex: mycobacterium, cryptosporidium)
- capsule stain: negative sting technique; contrast a translucent, darker colored background with stained cells but unstained capsule (ex: bacillus anthracis, klebsiella pneumoniae, clostridium sp.)
- endospore stain: spores are dyed by heating malachite green dye
- flagella stain: flagella are thickened with mordant
What are some fungal and parasite stains?
- lactophenol cotton blue stain: stain chitin in cell wall of fungi
- gomeri methenamine silver (GMS): dark brown staining of fungal cell wall, surrounding tissue green
- periodic acid schiff (PAS): mucin stain
- Wheatley’s trichrome stain: detection of Protozoa
What do cultures allow observation of? How?
- mostly bacteria + fungi
- viruses = cell cultures
- different environmental and nutritional requirements
- ensure pathogen growth of desired specimen
- enrich number of pathogens for more study
- determine pathogen identity
What are pros and cons of culturing?
- pros:
- identification
- assessment of AMR
- ability of microbe to cause disease
- study characteristic and genetics
- cons:
- time consuming (2-10 days)
- expensive
- un-culturable bacteria, no ID
What are fastidious bacteria?
- bacteria that require specific nutrients and culture conditions
What are some kinds of fastidious bacteria?
- aerobes, anaerobes, temperature dependency
What is the great-plate culture anomaly?
- ~1% of bacteria is culturally
- unculturable: used to describe bacteria not grown on artificial media to date (not enough info)
What are kinds of culture media?
- agar (solid)
- nutrient media: general growth
- selective media: growth of suspected agent
- differential media: most are selective and aid in ID
- broth (liquid)
- nutrient broth
- enrichment broth: increase number of specific bacteria and limit others
What are features of agar plate growth?
- bacteria grow as colonies
- bacterial colony: visible mass of bacteria originating from a single mother cell aka clones
- streak plate method to obtain pure cultures
- oxygen requirements
What are kinds of non-selective nutrient media?
- basic nutrient media
- trypticase soy agar (TSA)
- luria bertani (LB) agar
- mueller-hinton (MH) agar
- enriched nutrient media
- blood agar
- brain heart infusion (BHI) sugar
- chocolate agar
- lysed-blood agar
What are kinds of selective nutrient media?
- selective for gram-positive organisms
- phenylethyl alcohol agar (PEA)
- selective for fungi
- sabouraud dextrose agar (SDA)
- selective for gram-negative organisms
- eosin methylene blue agar (EMB)
What will occur if you add antibiotics to agar?
- only antibiotic resistant bacteria will grow
What are kinds of differential media?
- blood agar
- hemolysis
- MacConkey agar
- selective for G-
- lactose fermentation (pink colonies)
- mannitol salt agar
- G+
- mannitol fermentation (yellow colonies)
- CLED agar: urine bacteriology
- cysteine-lactose-electrolyte deficient
- supports growth of common urinary pathogens (ex: proteus, klebsiella)
- lactose fermentation
What are biochemical tests for bacterial identification?
- based on metabolic characteristics
- enzyme production
- catalase: breaks down H2O2
- coagulase: causes fibrin in blood to clot
- urease: hydrolysis urea
- tryptophanase: ability to convert tryptophane to indole
- carbon source utilization
- carbohydrate fermentation: lactose, sucrose, glucose, xylose
What are features of UTI culture paddles?
- semi-quantitative culture count
- presumptive ID of common uropathogens
- EMB agar: selective for gram - bacteria
- CLED agar: non-selective
- interpretation of results
- colony density: degree of infection
- growth on EMB: gram -
- color on CLED: lactose fermentation
What are features of Flexicult Vet urinary test?
- semi-quantitative colony count
- presumptive ID of uropathogens
- antibiotic susceptibility information
- test within 30 mins of mid-stream urine collection, incubate at 37 C for 18-24 hours
- > 10^3 pathogen/mL = infection
- no growth = antibiotic susceptible
What is the cycle of diagnostic process?
What are differences between on-site analysis and diagnostic lab?
What are features of a good diagnostic lab?
