Pertussis is a highly contagious respiratory illness caused by the bacterial pathogen Bordetella pertussis. Pertussis rates in the United States have been rising and reached a 50-y high of 42,000 cases in 2012. Although pertussis resurgence is not completely understood, we hypothesize that current acellular pertussis (aP) vaccines fail to prevent colonization and transmission. To test our hypothesis, infant baboons were vaccinated at 2, 4, and 6 mo of age with aP or whole-cell pertussis (wP) vaccines and challenged with B. pertussis at 7 mo. Infection was followed by quantifying colonization in nasopharyngeal washes and monitoring leukocytosis and symptoms. Baboons vaccinated with aP were protected from severe pertussis-associated symptoms but not from colonization, did not clear the infection faster than naïve animals, and readily transmitted B. pertussis to unvaccinated contacts. Vaccination with wP induced a more rapid clearance compared with naïve and aP-vaccinated animals. By comparison, previously infected animals were not colonized upon secondary infection. Although all vaccinated and previously infected animals had robust serum antibody responses, we found key differences in T-cell immunity. Previously infected animals and wP-vaccinated animals possess strong B. pertussis-specific T helper 17 (Th17) memory and Th1 memory, whereas aP vaccination induced a Th1/Th2 response instead. The observation that aP, which induces an immune response mismatched to that induced by natural infection, fails to prevent colonization or transmission provides a plausible explanation for the resurgence of pertussis and suggests that optimal control of pertussis will require the development of improved vaccines.whooping cough | T-cell memory | animal models | adaptive immunity |

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-inanswer-out results in <30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 l of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.full integration ͉ micro total analysis system ͉ microdevice ͉ pumping ͉ valving T he next revolution in personalized medicine, forensic science, and biowarfare defense will be impelled by analysis systems that provide a quantum leap in terms of functionality, time to result, and cost effectiveness. These systems need to meet several requirements, including a design conducive with low-cost manufacturing, turn-key operation with fast analysis times, and the ability to manipulate small volumes from crude samples. One example is the micrototal analysis system (-TAS) described conceptually more than a decade ago by Manz et al. (1). Prophetically, they stated that, ''. . . the detector or sensor in a TAS does not need high selectivity, because the sample pretreatment serves to eliminate most of the interfering chemical compounds.'' There are multiple examples in the literature of steps taken toward the advancement of integrated microfluidic genetic analysis (MGA) systems (refs. 2-4; also see ref. 5 for a comprehensive review); however, after a decade and a half, no bona fide microfluidic device has been presented that is capable of nanoliter flow control and integration of an electrophoretic separation with comprehensive sample pretreatment (DNA purification and PCR amplification).The MGA system described in this report brings together many advances in microfluidics over the last decade, exploiting differential channel flow resistances (6), elastomeric valves (7, 8), laminar flow (9), and electrophoretic mobility within the device, in concert with external fluid flow control from a syringe pump for sample and reagent delivery. Nucleic acid purification through solid-phase e...

bPertussis 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. Whooping cough is a highly contagious, acute respiratory illness caused by the bacterial pathogen Bordetella pertussis (for a review, see references 10 and 17). The introduction of pertussis vaccines in the 1940s and nationwide coverage in excess of 95% led to a dramatic decrease in the incidence of the disease. However, for unexplained reasons, pertussis rates in the United States have been rising steadily over the last 20 years for infants, children, and adolescents (1). With more than 21,000 reported cases in the United States in 2010, the highest number since the 1950s, pertussis is the most common of the vaccine-preventable diseases (2). This resurgence is mirrored throughout the industrial world, despite similar high rates of vaccination (12,20,31). Several hypotheses have been suggested for the increase in cases, but there is no consensus within the scientific community (9). 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. In order to fill these gaps, a good animal model of pertussis is required.In humans, infection with B. pertussis results in a wide spectrum of clinical manifestations that depends on the age and immune status of the host and ranges from mild respiratory symptoms to a severe cough illness which may be accompanied by the hallmark inspiratory whoop and posttussive emesis (4). Clinical signs include high leukocytosis, hypoglycemia, and reduced pulmonary capacity. Because of the acute nature of pertussis infections and because B. pertussis is a strict human pathogen with...

Despite near universal vaccine coverage, the bacterial pathogen Bordetella pertussis has re-emerged as a major public health concern. We recently developed a baboon (Papio anubis) model of pertussis that provides an excellent model of human pertussis. Using this model, the immune response to pertussis was characterized by measuring cytokines in the nasopharyngeal mucosa of infected baboons. Notably, we observed mucosal expression of interleukin-17 (IL-17) as well as IL-6, IL-23, and several cytokines and chemokines that are orchestrated by IL-17 immune responses. We also found substantial populations of circulating B. pertussis-specific Th17 and Th1 cells in convalescent animals >2 years post-infection consistent with a role in immunological memory to pertussis. Collectively, these data shed important light on the innate and adaptive immune responses to pertussis in a primate infection model and suggest that Th17 and Th1 immune responses contribute to the immunity conferred by natural pertussis infection.

Pertussis is a contagious, acute respiratory illness caused by the bacterial pathogen Bordetella pertussis. Although it is widely believed that transmission of B. pertussis occurs via aerosolized respiratory droplets, no controlled study has ever documented airborne transmission of pertussis. We set out to determine if airborne transmission occurs between infected and naive animals, utilizing the baboon model of pertussis. Our results showed that 100% of exposed naive animals became infected even when physical contact was prevented, demonstrating that pertussis transmission occurs via aerosolized respiratory droplets.

The complement system, especially the alternative pathway (AP), plays essential roles in the induction of injury in collagen antibody-induced arthritis (CAIA) in mice. The goal of the current study was to directly compare the roles of receptors for C3a and C5a, as well as the membrane attack complex (MAC), as effector mechanisms in the pathogenesis of CAIA. Clinical disease activity (CDA) in C3aR−/−, C5aR−/−, and C6 deficient (C6-def) mice was decreased by 52%, 94%, and 65%, respectively, as compared with WT mice. Decreases in histopathologic injury as well as in IgG and C3 deposition paralleled the CDA. A decrease in the percentage of synovial neutrophils was observed in C3aR−/−, C5aR−/−, and C6-def mice, and a decrease in macrophages was observed in C3aR−/− and C5aR−/−, but not in C6-def, mice. Synovial mRNA obtained by laser capture microdissection exhibited a decrease in TNF-α in C5aR−/− mice and in IL-1β in both C5aR−/− and C6-def mice, while C3aR−/− mice demonstrated no change in either cytokine. Our findings show that absent C3aR-, C5aR- or MAC-initiated effector mechanisms each decreases susceptibility to CAIA, with clinical effects most pronounced in C5aR deficient mice. Although the absence of C3aR, C5aR, or C6 led to differential deficiencies in effector mechanisms, decreased proximal joint IgG and C3 deposition was common to all three genotypes in comparison to WT mice. These data suggest the existence of positive feedback amplification pathways downstream of all three effectors that promote additional IgG deposition and C3 activation in the joint.

Anthrax toxins significantly contribute to anthrax disease pathogenesis, and mechanisms by which the toxins affect host cellular responses have been identified with purified toxins. However, the contribution of anthrax toxin proteins to dissemination, disease progression, and subsequent immunity after aerosol infection with spores has not been clearly elucidated. To better understand the role of anthrax toxins in pathogenesis in vivo and to investigate the contribution of antibody to toxin proteins in protection, we completed a series of in vivo experiments using a murine aerosol challenge model and a collection of in-frame deletion mutants lacking toxin components. Our data show that after aerosol exposure to Bacillus anthracis spores, anthrax lethal toxin was required for outgrowth of bacilli in the draining lymph nodes and subsequent progression of infection beyond the lymph nodes to establish disseminated disease. After pulmonary exposure to anthrax spores, toxin expression was required for the development of protective immunity to a subsequent lethal challenge. However, immunoglobulin (immunoglobulin G) titers to toxin proteins, prior to secondary challenge, did not correlate with the protection observed upon secondary challenge with wild-type spores. A correlation was observed between survival after secondary challenge and rapid anamnestic responses directed against toxin proteins. Taken together, these studies indicate that anthrax toxins are required for dissemination of bacteria beyond the draining lymphoid tissue, leading to full virulence in the mouse aerosol challenge model, and that primary and anamnestic immune responses to toxin proteins provide protection against subsequent lethal challenge. These results provide support for the utility of the mouse aerosol challenge model for the study of inhalational anthrax.

In vivo bioluminescent imaging permits the visualization of bacteria in live animals, allowing researchers to monitor, both temporally and spatially, the progression of infection in each animal. We sought to engineer stably luminescent clinical strains of Staphylococcus aureus, with the goal of using such strains in mouse models. The gram-positive shuttle vector pMAD was used as the backbone for an integration plasmid. A chloramphenicol resistance gene, a modified lux operon from Photorhabdus luminescens, and approximately 650 bp of homology to the chromosome of the USA300 S. aureus strain NRS384 were added, generating plasmid pRP1195. Electroporation into strain RN4220 followed by temperature shift led to integration of pRP1195 into the chromosome. The integrated plasmid was transferred to clinical strains by phage transduction. Luminescent strains displayed no in vitro growth defects. Moreover, luminescence was stable in vitro after three rounds of subculture over 48 hours of growth in the absence of antibiotics. Mice were infected with a luminescent strain of NRS384 in skin and intravenous models. In a mouse skin model, luminescent bacteria were present in lesions that formed and cleared over the course of several days, and in an intravenous model, bacteria inoculated in the mouse tail vein were observed spreading to multiple tissues. No statistically significant difference in virulence was observed between NRS384 and the luminescent strain in either infection model. These preliminary data suggest that this luminescent USA300 strain is suitable for use in mouse models. Similar strains were engineered using other sequenced clinical strains. Because these strains are stably luminescent, they should prove useful in animal models of infection.