Why do we need to detect pathogens

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Why do we need to detect pathogens?
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Who does pathogen detection and diagnosis?
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Disease diagnosis is often a process of
elimination
Is it a pathogen, pest, or an abiotic
problem? -experience -symptom and sign type and
distribution -crop history If it is a
pathogen -fungal? -bacterial? -nematode? -vir
us? -viroid?
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Disease diagnosis is often a process of
elimination
Example Bacterial Blight of Leek Outbreak,
1996-97
Is it a pathogen, pest, or an abiotic
problem? -experience have seen disease
before -symptom type and distribution symptoms
across entire field -crop history disease on
several farms, same seed source
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Experience is Critical! Resources
Growers Plant disease clinics and plant
pathologists Reference books, websites and
disease compendia APS website (apsnet.org)
common names and causal agents of plant diseases
listed by host
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First Step Isolation of causal agent
Example Bacterial Blight of Leek Outbreak,
1996-97
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Differential and selective media

• pathogen known or suspected to be bacterial or
  fungal, but identity unclear
• differential/selective media for known pathogens
  of specific host
• differential media target organism has
  identifiable colony morphology
• selective media growth of target organism is
  enhanced

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Differential Medium
Example Bacterial Blight of Leek Outbreak,
1996-97
Fluorescent on Kings B media suggesting causal
agent is Pseudomonas syringae
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Biochemical characterization LOPAT Test
(Lelliott et al., 1966)
Levan production of raised, mucoid colonies on
hypersucrose medium
Oxidase production of the enzyme cytochrome
oxidase
Potato Rot
Arginine dihydrolase conversion of I-argininine
to putrescine
Tobacco hypersensitive reaction
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Differential carbon utilization

• carbon utilization tests - BIOLOG

• different carbon sources
• cell respiration (carbon utilization) detected by
  reduction of tetrazolium dye ? color change
• pattern of utilization identifies organism (even
  to species or strain) metabolic fingerprint

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Differential hosts/indicator plants

• useful to identify viruses, virus like
  pathogens, sometimes bacteria

• Indicator plants
• certain plant species or cultivars show
  characteristic symptoms of infection diagnostic
  of pathogen
• infection may be natural (eg detect infected
  insect vectors), mechanical or induced by
  grafting

• Differential hosts
• host range can be tested and compared to known
  pathogens for identification

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Light Microscopy

• Inclusion Bodies
• Amorphous Inclusion
• Crystal Inclusion

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Electron Microscopy
Spherical / Icosahedral
Rigid rod
Bacilliform
Geminate
Flexuous rod
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Detection and Diagnosis Methods
Growth Media (differential, carbon utilization
profiles) Biochemical characterization Indica
tor Plants Microscopy (Light, Electron)
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Serology
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Serological diagnosis

• Use of antibodies to detect and identify
  pathogens
• Rapid, specific, sensitive
• Antibodies detect antigens (usually proteins) on
  surface of pathogen
• Antibody produced by animal immune system primed
  with pathogen antigen
• Antibody binds antigen with high specificity
• Commonly used for virus identification

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Antibody-antigen binding(Ab-Ag)

• Lock and key
• key (antigen) fits into
• lock (antibody)
• Antigen binds in variable region of heavy and
  light chains (end of the arms of the Y)
• High specificity of binding

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Specificity

• High degree of specificity in Ag-Ab reactions
• Antibody binds to only one antigenic determinant
  (epitope)
• Antibodies can distinguish differences in the
• primary structure of an antigen
• isomeric forms of an antigen
• secondary and tertiary structure of an antigen

primary
secondary
tertiary
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If specificity breaks down cross-reactivity

• Antibody reacts with more than one antigenic
  determinant
• Population of antibodies react with more than one
  antigen
• Cross reacting antigen shares an epitope in
  common with the immunizing antigen, or has an
  epitope that is structurally similar to one on
  the immunizing antigen

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Polyclonal vs. monoclonal antibodies

• Polyclonal antibodies mixture of antibodies
  produced in response to a single antigen, which
  recognize different epitopes on that antigen
• Monoclonal antibody single species of antibody
  which recognizes a single epitope on an antigen

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Enyme-linked immunosorbent assay - ELISA

• sensitive and specific immunoassay for the
  detection and quantitation of antigens
• assay results are based on the binding of
  antibody and antigen

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Direct ELISA Double Antibody Sandwich
ELISA (DAS-ELISA)
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DAS-ELISA
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Indirect ELISA
2. Primary Ab, specific to antigen, is added and
binds antigen. e.g. primary Ab produced in mouse
1. Antigen and other proteins in diseased plant
sample are adsorbed to tube walls.
3. Secondary Ab conjugate (e.g. rabbit Ab to
mouse IgG) is added, and binds primary Ab.
4. Substrate is added and cleaved by conjugated
enzyme, leading to color development.
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ImmunoStrips
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Immunostrip similar to ELISA

• Antibody 1
• monoclonal
• specific to antigen (one epitope)
• tagged with colored dye

• Antibody 2
• monoclonal
• specific to antigen (second epitope)
• fixed to membrane

• Antibody 3
• specific to antibody 1
• fixed to membrane

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Detection and Diagnosis Methods
Growth Media (differential, carbon utilization
profiles) Biochemical characterization Indica
tor Plants Microscopy (Light, Electron) Serology
- ELISA
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Nucleic Acids
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Detecting pathogens using nucleic acids (DNA and
RNA)

• Overcomes many of the problems associated with
  serological methods, such as
• Some pathogens (viroids) do not have proteins
• Difficult to make antibodies for some pathogens
• Low titer of antigen can be hard to detect
• Cross-reaction of antibodies with heterologous
  antigens
• Requires experimental animals for antibody
  production
• change in antigen production by pathogen due to
  developmental or environmental conditions
• Nucleic acid sequence is not affected by growth
  conditions

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Nucleic acids for pathogen detection

• Common methods
• PCR (reverse transcription PCR for RNA)
• nucleic acid hybridization
• sequencing
• Benefits
• reagents are more stable than serological
  reagents
• high sensitivity (can detect low levels of
  pathogen)
• can develop very specific or more general tests
  depending on need
• usually less expensive to develop than serology
  (BUT sample preparation is often more
  time-consuming)

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PCR
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PCR Assays
Gel electrophoresis (fingerprinting) DNA
sequencing RT-PCR for viruses and viroids
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DNA fingerprinting
Example Bacterial Blight of Leek Outbreak,
1996-97
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DNA fingerprinting
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DNA sequencing
Example Bacterial Blight of Leek Outbreak,
1996-97
16S
23S
ITS
ITS
ITS
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DNA sequencing
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(No Transcript)
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Nucleic Acid Spot Hybridization (NASH)
infected potato leaf
vector carrying PVX viral coat protein gene
RNA isolation
PCR
RNA spotted on a membrane, immobilized under UV
light
Probe labeling
PVS PVX PVY C
DNA CONTROL
Labeled probe
Hybridization with a labeled probe
Wash off the probe that did not bind
PVS PVX PVY H
Detection of label (often X-ray film)
DNA CONTROL
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Sensitivity of assay

• Virus detection in mixed extracts of infected
  plants and virus free plants
• 11 combine equal amounts of tissue from
  infected and virus free plants for RNA extraction
• 140 combine 1 part infected and 40 parts virus
  free plant tissue for RNA extraction
• Negative control (III)

• Signal still clearly present in infected tissue
  diluted 140 with virus free tissue

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PVS, PVX and PVY multiplex detection

• RNA from one infected potato plant tested
  simultaneously with three probes for coat protein
  genes from three separate potato viruses
• PVS
• PVX
• PVY
• I DNA controls viral coat protein gene PCR
  products
• II RNA extracted from potato plant infected with
  all three viruses
• SAVES time, labor, costs
• suitable approach when the recommended control
  will be the same for all pathogens, as in the
  case of these potato viruses

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Macroarrays for pathogen diagnostics

• DNA hybridization method to distinguish pathogen
  groups that have genome sequence variations in
  otherwise conserved sequences
• oligonucleotides (40-50 nt) from conserved
  sequences, but specific to each pathogen of
  interest, are spotted onto nylon membrane (
  target)
• oligonucleotides can be designed to distinguish
  at genera, species, sub-species levels
• sensitivity comparable with PCR
• DNA from pathogen isolate or diseased plant is
  PCR-amplified using primers that flank the
  target PCR products are labeled and used to
  probe the macroarray membrane

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Macroarray with oligonucleotides from 23S
ribosomal DNA for 7 bacterial pathogens of plants

• hybridized with probe made from Clavibacter
  michiganensis DNA
• Primers used to make probe are based on highly
  conserved sequence (all species have similar
  priming sites)
• DNA between priming sites varies between species
• eg probe made from C. michigenensis DNA is
  specific to C. michigenensis

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Detection and Diagnosis Methods
Growth Media gold standard, slow, inexpensive,
only works for some pathogens Indicator Plants
slow, inexpensive, only works for some
pathogens Microscopy only works for some
pathogens Serology (ELISA) inexpensive once
developed, robust, does not work for viroids,
pathogen mutations problematic Nucleic Acid
PCR (fingerprinting, sequencing), NASH,
Macroarray
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Detection and Diagnosis Methods
Nucleic Acid PCR inexpensive to develop,
often more expensive to apply than ELISA, very
sensitive (contamination!), important to have
multiple targets per pathogen, many variations of
PCR assays Fingerprinting more sensitive than
single target PCR Sequencing more sensitive
than fingerprinting most expensive of the PCR
methods NASH and macroarrays inexpensive, less
sensitive than PCR, easy to multiplex
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PCR plant pathogens

• False positive

• False negative

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PCR human pathogens on plants

• False positive

• False negative

RECALL
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PCR

• Dead or Alive????