Nonhuman primate model of pertussis

Abstract

Pertussis is a highly contagious, acute respiratory illness caused by the bacterial pathogen Bordetella pertussis. Despite nearly universal vaccine coverage, pertussis rates in the United States have been rising steadily over the last 20 years. Our failure to comprehend and counteract this important public health concern is due in large part to gaps in our knowledge of the disease and the mechanisms of vaccine-mediated protection. Important questions about pertussis pathogenesis and mechanisms of vaccine effectiveness remain unanswered due to the lack of an animal model that replicates the full spectrum of human disease. Because current animal models do not meet these needs, we set out to develop a nonhuman primate model of pertussis. We inoculated rhesus macaques and olive baboons with wild-type B. pertussis strains and evaluated animals for clinical disease. We found that only 25% of rhesus macaques developed pertussis. In contrast, 100% of inoculated baboons developed clinical pertussis. A strong anamnestic response was observed when convalescent baboons were infected 6 months following recovery from a primary infection. Our results demonstrate that the baboon provides an excellent model of clinical pertussis that will allow researchers to investigate pertussis pathogenesis and disease progression, evaluate currently licensed vaccines, and develop improved vaccines and therapeutics.

Fig 1

Leukocytosis and colonization of pertussis-infected rhesus macaques. Four weanling rhesus macaques were inoculated with B. pertussis strain D420 as described in Materials and Methods. Once prior to inoculation and on the indicated days postinoculation, blood was drawn from each rhesus macaque, and the number of circulating white blood cells per microliter of peripheral blood was determined as described in Materials and Methods and shown in the graph for each animal. Nasopharyngeal washes were performed as described in Materials and Methods, and the recovered washes were plated on two Regan-Lowe plates (50 μl per plate). The number of CFU per plate is indicated for each day by the shading of the bar over the WBC graph: white, 0 colonies; light gray, 1 to 100 colonies; dark gray, 101 to 1,000 colonies; and black, >1,000 colonies.

Fig 2

Characteristics of B. pertussis grown at 39°C. (A) Growth curve of B. pertussis grown in Stainer-Scholte medium at 37°C and 39°C (n = 3). (B) Hemolysis of B. pertussis strain D420 grown on BG agar plates at 37°C and 39°C. (C) Following growth of strain D420 on BG agar plates at 37°C and 39°C, the expression of the genes encoding pertussis toxin (ptxA), filamentous hemagglutinin (fhaB), and adenylate cyclase toxin (cyaA) were analyzed by real-time PCR as described in Materials and Methods. Data are presented as threshold cycle (C_T) values (for 37°C, n = 4; for 39°C, n = 3). (D) Protein expression of adenylate cyclase toxin, filamentous hemagglutinin, and pertussis toxin was analyzed by Western blotting as described in Materials and Methods following growth of strain D420 on BG agar plates at 37°C and 39°C. Blots shown are representative of 4 separate experiments. The numbers on the right indicate the molecular size markers.

Fig 3

Leukocytosis, cough illness, and fever in pertussis-infected baboons. (A) Once prior to inoculation and on the indicated days postinoculation, blood was drawn from each weanling baboon, and the number of circulating white blood cells per microliter of peripheral blood was determined (n = 9). (B) A subset of infected baboons (n = 6) were observed four times per day as described in Materials and Methods. The number of coughs per hour was determined for each animal at each observation period, and the overall mean was reported for each day postinoculation. (C) The core temperature of each baboon was measured three times prior to inoculation and at each examination postinoculation. One data point is presented for each baboon (n = 9) at each time point.

Fig 4

Serological response to pertussis toxin in B. pertussis-infected baboons. (A) Once prior to and on the indicated range of days after inoculation, sera were collected and anti-PT IgG was measured by ELISA as described in Materials and Methods (n = 7 for all time points except for days 117 to 131, for which n = 4). Data are presented as relative units (percent anti-PT IgG compared to a standard). (B) The complete anti-PT IgG kinetics for two of the seven baboons are shown. **, P < 0.01; ***, P < 0.001 versus preinfection.

Fig 5

Reinfected convalescent baboons fail to develop leukocytosis and mount an anamnestic serological response. Convalescent baboons that had recovered from a previous pertussis infection (n = 4) and age-matched naïve baboons (n = 2) were inoculated with B. pertussis. The number of circulating white blood cells per microliter of peripheral blood was determined for the naïve baboons (A) and the convalescent baboons (B) once prior to inoculation and on the indicated days postinoculation. (C) Anti-PT IgG was measured by ELISA, as described in Materials and Methods, for naïve (gray bars) and convalescent (black bars) baboons. Data are presented as relative units on a logarithmic scale. **, P < 0.01; ***, P < 0.001 for convalescent versus naïve baboons.