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									                              Bordetella
                           Dave Warshauer, Ph.D
                        Wisconsin State Laboratory of Hygiene
                               Phone: 608-265-9115
                           david.warshauer@slh.wisc.edu

                                   January 11, 2012

       WISCONSIN STATE
       LABORATORY OF HYGIENE
WISCONSIN STATE
LABORATORY OF HYGIENE
Bordetella species
   •     B. pertussis--------------human
   •     B. parapertussis--------human/sheep
   •     B. bronchiseptica------animals/human
   •     B. holmesii--------------human
   •     B. avium-----------------birds
   •     B. hinzii------------------poultry
   •     B. trematum------------animals
   •     B. petrii------------------environmental

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Bordetella
     • Small gram-negative coccobacillus
     • Strictly aerobic (except B. petrii)
     • Oxidize amino acids
            – None ferment carbohydrates
     • Catalase positive
     • Optimal growth at 35°C, ambient air with
       humidity
     • B. pertussis most fastidious of the
       Bordetella
            – Inhibited by constituents in media
                   •    Fatty acids
                   •    Metal ions
                   •    Sulfides
                   •    Peroxides
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B. Pertussis Gram Stain




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Some virulence factors of B. pertussis
   • Non-invasive bacterium-does not normally
     invade cells
   • Attachment
          – Filamentous hemagglutinin (FHA) and pertactin
          – Pertussis toxin
   • Toxins
          – Tracheal cytotoxin – cell death
          – Endotoxin – cytokines, fever
          – Adenylate cyclase/hemolysin – inhibits lymphocyte
            function
          – Lipooligosaccharide (endotoxin)
          – Pertussis toxin (PT)* - inhibits immune cells
            chemotaxis
              and acts as an adhesin

          * = difference between pertussis and parapertussis
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Whooping Cough
     • Bordetella pertussis
     • Classical whooping cough
           – Cold-like illness (Catarrhal phase)
                  • Runny nose, sneezing, low-grade fever, tired
           – Dry non-productive cough for >2 weeks
           – “Whooping” cough (Paroxysmal phase)
                  • Severe cough with vomiting
           – Severe disease in infants
           – 7-10 day incubation (range 5-21)
           – Epidemic disease every 2-5 years
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Parapertussis
• Symptoms are similar to pertussis but
  typically milder.
       – cough that occurs in sudden, uncontrollable bursts
              • Shorter duration than pertussis (Avg 14 days)
       – high-pitched whooping sounds when breathing in
         after a coughing episode
       – posttussive vomiting
• Incubation & Transmission Periods similar to
  pertussis
• All age groups can be infected.
       – Young infants (e.g., <6 months of age) may have
         a more severe course
• Rarely, death can occur in infants with
  underlying health problems or those that are
  co-infected with B. pertussis.
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Pertussis CSTE Case Definition
    • Clinical case definition
           – Cough > 2 weeks and at least one
             symptom: paroxysms, whoop, posttussive
             vomiting
    • Case classification
           – Confirmed cases
                 • Culture positive
                 • Clinical case and PCR positive
                 • Clinical case and epi-linked to confirmed case
           – Probable case
                 • Only meets the clinical case definition
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Transmission
     • Respiratory droplets
     • Highly infectious
            – 80-90% of household contacts




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Bordetella pertussis
    • 20-40 million cases/year worldwide
    • 200,000-400,000 deaths
           – Primarily among children
    • Cases/yr in the US
           –   2002----------9,771
           –   2003----------11,647
           –   2004----------25,827
           –   2005----------25,616
           –   2006----------15,632
           –   2007----------10,454
           –   2008----------13,278
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Number of Specimens Positive for
Whooping Cough (6/1/03 – 10/22/05)
                  75



                  60
Number Positive




                  45



                  30



                  15



                   0
                       6/7/03
                                7/5/03
                                         8/2/03
                                                  8/30/03
                                                            9/27/03
                                                                      10/25/03
                                                                                 11/22/03
                                                                                            12/20/03
                                                                                                       1/17/04
                                                                                                                 2/14/04
                                                                                                                           3/13/04
                                                                                                                                     4/10/04
                                                                                                                                               5/8/04
                                                                                                                                                        6/5/04
                                                                                                                                                                 7/3/04
                                                                                                                                                                          7/31/04
                                                                                                                                                                                    8/28/04
                                                                                                                                                                                              9/25/04
                                                                                                                                                                                                        10/23/04
                                                                                                                                                                                                                   11/20/04
                                                                                                                                                                                                                              12/18/04
                                                                                                                                                                                                                                         1/15/05
                                                                                                                                                                                                                                                   2/12/05
                                                                                                                                                                                                                                                             3/12/05
                                                                                                                                                                                                                                                                       4/9/05
                                                                                                                                                                                                                                                                                5/7/05
                                                                                                                                                                                                                                                                                         6/4/05
                                                                                                                                                                                                                                                                                                  7/2/05
                                                                                                                                                                                                                                                                                                           7/23/05
                                                                                                                                                                                                                                                                                                                     8/27/05
                                                                                                                                                                                                                                                                                                                               9/24/05
                                                                                                                                                                                                                                                                                                                                         10/22/05
                                    Bordetella parapertussis                                                                                                                                  Bordetella pertussis
                                    Bordetella pertussis/Bordetella bronchiseptica                                                                                                            Bordetella pertussis/parapertussis


                       WISCONSIN STATE
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Pertussis Vaccine
• Included in childhood vaccines
       – DTaP at 2,4,6, and 15-18 months, 4-6
         years
       – Recent addition-------Tdap at 11-12 years
       – “Catch up” for 13-18 yr olds
• Issue of “waning immunity”



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   Treatment and Prophylaxis
     • Macrolides
           – Azithromycin
           – Erythromycin
           – Clarithromycin
     • Second choice =
       sulfa drugs (SXT)
                  • Not for infants <2mo

NOTE: Treatment should be administered during the catarrhal stage to
          lessen illness. If cough develops, does not affect illness course
          but may lessen transmission possibility.
    WISCONSIN STATE
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Specimen Collection
     • Nasopharyngeal specimen
           – Want ciliated epithelial cells
           – Timing critical
     • NP aspirates, washes, or swabs
     • Dacron or rayon swabs
           – NO cotton or calcium alginate swaps
     • NO throat, sputum, or mouth
       specimens

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Nasopharyngeal Specimen




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Transport

 • B. pertussis extremely labile
 • Direct culture optimal, not feasible
 • Transport media
        – If <2hr----0.5-1.0% Casamino Acid Sol’n at room
          temp
        – 2hr – 24hr----Amies with charcoal, room temp
        – >24hr----Regan-Lowe or Jones-Kendrick at 4°C




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Laboratory Diagnosis
   • Culture

   • DFA

   • Real-time
     Polymerase
     Chain Reaction
          – More rapid than
            culture
          – More sensitive
            than culture
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Laboratory Diagnosis


                        CULTURE
                          DFA
                            PCR


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Culture
      • “Gold Standard”
            – Essential for public health labs
      • 100% specific, but low sensitivity
            – (12-60%)
      • Highest yield
            – Young children
            – Unvaccinated
            – Early in cough illness prior to antibiotics

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Culture Media
    • Bordet-Gengou
           – Potato infusion, glycerol, sheep/horse blood
    • Regan-Lowe
           – Charcoal agar, 10% horse blood
    • Jones-Kendrick
           – Charcoal agar, yeast extract, starch
    • Stainer-Scholte synthetic medium
    • Legionella buffered charcoal yeast-
      extract
    • Incorporate antibiotics to suppress normal
      flora----cephalexin or methicillin
    • Incubate 35-36C with high humidity for a
      minimum of 7 days
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B. pertussis on Bordet-Gengou
7 days incub. Commercial Medium




                         “Pearl-like”



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Other Bordetella species
    • B. parapertussis
           – Colonies within 2-3 days
           – On Regan-Lowe appear grey
           – On BG have brown pigmentation
    • B. holmesii
           – Colonies similar to B. pertussis
           – Inhibited by cephalexin
    • B. bronchiseptica
           – Colonies within 1 day
           – On Regan-Lowe, large, slight brown

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Culture Sensitivity
     • Considered no more than 60% sensitive
     • Factors effecting sensitivity
            – Type and quality of specimen
            – Time specimen obtained in the course
              of illness
            – Age of the patient
            – Appropriate transport
            – Choice of culture media
            – Length of time cultures incubate

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Culture for Public Health
    • Important if an outbreak is suspected
    • Isolation of the organism confirms
      pertussis
           – Other agents can cause pertussis-like
             illness
           – Co-infection with other pathogens occurs
           – Culture helps identify other Bordetella
             spp.
    • Necessary for AST and subtyping

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Identification
• B. Pertussis              • B. parapertussis
       –   Catalase     +     –   Catalase     +
       –   Oxidase      +     –   Oxidase      -
       –   Urease       -     –   Urease       +
       –   Motility     -     –   Motility     -
       –   Nitrate      -     –   Nitrate      -
       –   Blood Agar   -     –   Blood Agar   +
       –   MacConkey    -     –   MacConkey    V
       –   Fluor. Ab          –   Fluor. Ab


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Direct Fluorescent Antibody
     • In use since 1960
     • Direct detection and ID of isolates




                        Courtesy of Mike Saubolle, PhD

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DFA
• Problems
      – Poor sensitivity (18-78%)
      – Requires large numbers of organisms (>104 /ml)
      – Best when test early in course of illness
      – Requires skilled and experienced microscopist
      – Antibiotic therapy can affect binding of DFA
        reagent to cell wall
      – Poor specificity (7-44% false positives)
• Advantage
      – More rapid than culture
• No longer recommended

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Pertussis PCR
   • Optimal diagnostic test

          – 70 – 90% Sensitivity

   • No prolonged asymptomatic “carrier
     state”
          – If positive, considered diagnostic

   • Recognized by CSTE as official
     laboratory confirmation of pertussis in
     addition to culture
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Pertussis PCR (cont.)
• Advantages
     – Rapid
     – Extremely sensitive
           • <=1 CFU (5ul sample)
     – Does not require viable organism
           • Transport delays and antibiotics do not prevent
             laboratory diagnosis
     – Positive longer than culture
     – Specific
           • Except for detection of B. holmesii if using IS481



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Pertussis PCR (cont.)
     • Disadvantages
            – Stringent requirements to perform PCR
            – Not presently standardized
            – More expensive than culture or DFA
            – PCR inhibitors
            – Cross reaction with B. holmseii




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WSLH Pertussis Data
July 2003-July 2004                         %Pos of Total % of Positives

3330 Tested by Culture and PCR


 450 Pos by Cult and/or PCR                    13.5%           100%


 167 Pos by BOTH Cult and PCR                   5.0%          37.1%


  27 Pos by Culture ONLY                        0.8%           6.0%

 256 Pos by PCR ONLY                            7.7%          56.9%

 20 culture positive for B. parapertussis
 WISCONSIN STATE
 LABORATORY OF HYGIENE
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                                                         PCR Crossing Points vs Culture
                                                                June 1, 2003- September 10,2004

                                             73.6
                       80                                      68.4

                       70
% Culture Positive




                       60
                                                                                 46.5
                       50


                       40
                                                                                                   * No. Specimens
                                                                                                   21.2
                       30                    53*           95*
                                                                          127*
                       20                                                                                          10

                                                                                         226*
                       10
                                                                                                          10*
                         0
                                 13-24.99           25-29.99          30-34.99          35-39.99           40-45


                                                       Crossing Point Range

                     WISCONSIN STATE
                     LABORATORY OF HYGIENE
                                                                                                                        37
Number of Specimens Tested for Bordetella
by PCR and/or Culture (6/1/03 – 1/29/05)




                                                                                                              38




                                                                                    1/29/05
                                                                                    1/15/05
                                                                                    1/1/05
                                                                                    12/18/04
                                                                                    12/4/04
                                                                                    11/20/04
                                                                                    11/6/04
                                                                                    10/23/04
                                                                                    10/9/04
                                                                                    9/25/04
                                                                                    9/11/04
                                                                                    8/28/04
                                                                                    8/14/04
                                                                                    7/31/04
                                                                                    7/17/04
                                                                                    7/3/04
                                                                                    6/19/04
                                                                                    6/5/04
                                                                                    5/22/04




                                                                                               Total Tested
                                                                                    5/8/04
                                                                                    4/24/04
                                                                                    4/10/04
                                                                                    3/27/04
                                                                                    3/13/04
                                                                                    2/28/04
                                                                                    2/14/04
                                                                                    1/31/04
                                                                                    1/17/04
                                                                                    1/3/04
                                                                                    12/20/03
                                                                                    12/6/03
                                                                                    11/22/03
                                                                                    11/8/03
                                                                                    10/25/03
                                                                                    10/11/03
                                                                                    9/27/03
                                                                                    9/13/03




                                                                                                              LABORATORY OF HYGIENE
                                                                                    8/30/03
                                                                                    8/16/03
                                                                                    8/2/03




                                                                                                              WISCONSIN STATE
                                                                                    7/19/03
                                                                                    7/5/03
                                                                                    6/21/03
                                                                                    6/7/03




                                                                                0
                                            2000



                                                   1600



                                                            1200



                                                                   800



                                                                          400
                                                          Number Tested
Bordetella spp. PCR at WSLH
   • Multi-target RT-PCR
          – B. pertussis
          – B. parapertussis
          – B. holmesii
   • Targets
          – IS481
          – plS1001
          – hIS1001
          – ptxS1
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Targets
• IS481
     – B. pertussis (80-200 copies/cell)
• hIS1001
     – B. holmesii (3-5 copies/cell)
• pIS1001
     – B. parapertussis
• RNP
     – Inhibition detection
• ptxS1---Not part of multi-plex
     – Single copy in B. pertussis and B. parapertussis
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Species Identification

Species                 ptxS1   IS481   hIS1001   pIS1001
B. pertussis
                         +       +         -        -
B. parapertussis
                         +       -         -        +
B. pertussis and
B. parapertussis
                         +       +         -        +
B. holmesii
                          -      +        +          -




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Pertussis Serology
     • Require acute and convalescent specimens
     • Infection in vaccinated individuals
       confounds interpretation
     • Not included in CSTE case definition
     • Can have epidemiologic value
     • No standardized assays
     • No FDA approved assays
     • New CDC developed EIA may provide a
       useful serologic tool

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CDC IgG Anti-PT ELISA

     • Microwell ELISA format
     • Allows for diagnosis of recent
       infections with a single specimen
     • Useful in later stages of the disease
       (>2 weeks from onset
     • Post-vaccination antibody levels do not
       interfere with diagnosis
     • Can be qualitative
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Bordetella holmesii
     • First identified in 1995
     • Associated with pertussis-like
       respiratory disease and pneumonia
            – 0.6% of patients
            – Role in respiratory disease still unclear
     • Bacteremia in asplenic children and
       sickle-cell patients
            – Endocarditis

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Bordetella holmesii (cont.)




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Bordetella holmesii (cont.)

     • Gram negative coccobacillus
     • Grows well on SBA in 5% CO2 after
       24hr
            – Inhibited by cephalexin
     • Diffusible brown pigment
            – Can be mistaken for alpha hemolysis
     • Poor or no growth on Mac

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Bordetella holmesii (cont.)

     • Negative for catalase (variable),
       oxidase, NO3 reductase, urease,
       indole, motility
     • Misidentified as Acinetobacter lwoffii
       on Vitek 2




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Bordetella bronchiseptica




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Bordetella bronchiseptica (cont.)
    • Rarely isolated from respiratory
      specimens
    • Pertussis-like disease and other
      respiratory symptoms
           – Infectious bronchitis
    • Most frequently in imunocompromised
    • Rare cases of bacteremia and
      septicemia

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Bordetella bronchiseptica (cont.)

     • Has genes for pertussis toxin
            – Not expressed
     • Positive for catalase, oxidase, nitrate,
       urease, motility
     • Grows on Mac, SBA
            – On SBA, small to medium gray colonies
              with beta hemolysis under the colonies


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Bordetella bronchiseptica (cont.)




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Bordetella bronchiseptica (cont.)

     • Susceptibility pattern
            – Beta-lactamase producer
                   • R to many penicillins and cephalosporins
                   • Anti-pseudomonal penicillins usually sensitive
            – Mostly resistant to SXT
            – Most S to amoxicillin-clavulanic acid,
              tetracycline, gentamicin, and quinolones



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                        Thank You




                        Questions????
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LABORATORY OF HYGIENE
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