A Non-Human Primate Model of Acute Group A Streptococcus Pharyngitis - Bacterial Pathogenesis: Methods and Protocols - page 246

7. Add 100 μL of goat-anti-monkey IgG-HRP (diluted 1:30,000 with PBS) to each
well and incubate for1hatroom temperature on a rocking platform.
8. Wash wells with PBST.
9. Wash wells with PBS.
10. Add 100 μL of TMB solution to each well and incubate for 26 min at room
temperature on a rocking platform.
11. Add 100 μL of stop solution (0.18 M sulfuric acid) to each well.
12. Read absorbance at 450 nm.
13. Calculate mean (±standard deviation) values for each serum sample.

3.2.3. Additional Considerations for Animal Grouping
To ensure that the macaques do not have current carriage of GAS, the animals
should be cultured for the presence of GAS in the upper respiratory tract on
day minus-seven. Culturing for GAS is performed by swabbing the posterior
pharynx and plating to look for GAS colonies as described in Subheading 3.4.3 .
A final consideration for animal grouping should be the pre-infection scoring
of pharyngeal erythema and tonsil size. Scoring is performed as described in
Subheading 3.4.2 .

3.3. GAS Non-Human Primate Infection
Non-human primates are infected on day 0 of the experiment through instil-
lation of 1 mL PBS containing GAS into the nares of each animal. Several
parameters of infection are monitored during the 5-week protocol, with samples
isolated as listed in Table 1 . All animals should be housed individually during
the infection phase of the protocol to prevent cross-contamination.

1. Thaw 1 h prior to animal infection one tube each of the wild-type and mutant
strains on ice and dilute to1×10 7 CFUs/mL using PBS.
2. Fill four 1-mL syringes with GAS-PBS solution for each of the two GAS strains.
3. Perform day-0 sample isolations and clinical scorings as outlined in Table 1 and
Subheading 4.4 .
4. Infect each animal by slowly dribbling 1 mL of GAS-PBS solution into their
nares ( 0.5 mL per nostril).

5. Titer dilute GAS-PBS solutions to confirm that correct infectious dose was given
to the animals for each of the two GAS strains. Use THY agar or TSA blood agar
plates for plating serial dilutions for titer determination.

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3.4. Sample Isolations and Clinical Scoring of Disease
Samples and clinical scoring occurs on the days outlined in Table 1 . The
order of events is as follows.