A Non-Human Primate Model of Acute Group A Streptococcus Pharyngitis - Bacterial Pathogenesis: Methods and Protocols - page 247

1. Anesthetize animals with an intramuscular injection of ketamine (10 mg/kg) and
bring them individually into the examination room.
2. Obtain rectal temperature and weight of the animal.
3. Take a blood sample ( see Subheading 3.4.1. ) and place the animal onto sheets
of disposable absorbent bench covers.
4. Using a laryngoscope with disposable blades, score pharyngeal erythema and
tonsil size (performed by a veterinarian) ( see Subheading 3.4.2. ). Replace bench
covers and laryngoscope blades after examination of each animal.
5. Following clinical scoring, obtain five throat swabs ( see Subheading 3.4.3. ),
collect approximately 1 mL of saliva ( see Subheading 3.4.4. ), and perform a
nasal wash using PBS ( see Subheading 3.4.5. ).
6. Return animals to holding room.
3.4.1. Serum is Used to Measure Host Immune Responses to Infection
The host response to infection is monitored by measurement of serum C-
reactive protein levels and antibody development against various GAS antigens
(21,22,23,24) . C-reactive protein concentrations are determined by ELISA
with commercially available anti-C-reactive protein antibodies. Antibody
production is assayed by ELISA using purified streptococcal proteins (e.g.,
SLO; Subheading 3.2.2. ).
1. Take 3-mL blood samples from each of the anesthetized animals by venipuncture
in the cephalic vein.
2. Draw blood using a Vacutainer™ system with a red-capped collection tube and
incubate on ice until all macaque manipulations are completed.
3. Incubate blood samples at 4 °C for1htoallow the blood to clot before centrifuging
at 500 × g for 20 min at 4 °C.
4. Remove aqueous (serum) phase from each sample and place into individual red-
capped 2-mL tubes.
5. Freeze serum samples at -80 °C until required.
3.4.2. Clinical Scoring of Disease
Development of disease is monitored by determination of tonsillitis and
pharyngeal erythema severity scores prior to throat swabbing. Briefly, score
pharyngeal erythema on a 4-point scale as follows: no erythema (0) , mild
erythema with hyperemic blood vessels (+1), more intense erythema and palatal
petechiae (+2), and intense erythema with palatal petechiae and exudative
tonsillitis (+3). Score macaque tonsil size on a 5-point scale using the same
criteria as established by Feinstein and Levitt (27) for scoring tonsil enlargement
during human GAS pharyngitis ( see Fig. 1 ).