PREVALENCE, ISOLATION, AND GENETIC CHARACTERIZATION OF TOXOPLASMA GONDII IN CHICKEN

Toxoplasma gondii (T. gondii) is one of the most successful parasites in the world because of its capability of infecting all warm-blooded animals. It has been reported that up to one third of the world population is infected with this parasite. Chickens are recognized as good indicators of the environmental T. gondii oocysts contamination because they obtain food from the ground. Thus, the prevalence of T. gondii in chicken provides more insight related to public health concern from T. gondii. Previous studies have shown a high isolation rate from free-range chickens raised in the United States. The objectives of this study were to evaluate the microbial safety and infection of T. gondii in free-range chickens available at the grocery stores and farms for the consumers to purchase and genotype T. gondii isolates. Chicken hearts were obtained from the local markets and also from the farms raising free- range chickens. Heart juice was obtained from cavities of each heart. Modified agglutination test (MAT) for detection of IgG antibodies was conducted with those heart juice samples with titer of 1:5, 1:25, and 1: 100. Each seropositive heart was pepsin digested and bioassayed into a group of two mice. Six weeks post inoculation (p.i.) mice were bled and euthanized to examine the infection of T. gondii. In addition, multiplex multilocus nested PCR-RFLP was performed to genetically characterize T. gondii isolates with eleven PCR-RFLP markers including SAG1, SAG2, altSAT2, SAG3, BTUB, GRA6, c22-8, c29-a, L358, PK1, and Apico. One hundred fifty from a total of 997 samples (15.0%) were found seropositive for T. gondii. No viable T. gondii was isolated from chicken hearts that were sampled. A total of four genotypes were identified, including one new genotype and three previously identified genotypes. The results suggest that T. gondii oocysts could present in the environment and infect the food animals. T. gondii prevalence in chicken hearts could reflect the environmental contamination of T. gondii and prevalence information can be used to manage T. gondii infection risk.

ROLE OF ICAM-1-MEDIATED ENDOCYTOSIS IN ENDOTHELIAL FUNCTION AND IMPLICATIONS FOR CARRIER-ASSISTED DRUG DELIVERY

Intercellular adhesion molecule 1 (ICAM-1) is a transmembrane protein found on the surface of vascular endothelial cells (ECs). Its expression is upregulated at inflammatory sites, allowing for targeted delivery of therapeutics using ICAM-1-binding drug carriers. Engagement of multiple copies of ICAM-1 by these drug carriers induces cell adhesion molecule (CAM)-mediated endocytosis, which results in trafficking of carriers to lysosomes and across ECs. Knowledge about the regulation behind CAM-mediated endocytosis can help improve drug delivery, but questions remain about these regulatory mechanisms. Furthermore, little is known about the natural function of this endocytic pathway. To address these gaps in knowledge, we focused on two natural binding partners of ICAM-1 that potentially elicit CAM-mediated endocytosis: leukocytes (which bind ICAM-1 via β₂ integrins) and fibrin polymers (a main component of blood clots which binds ICAM-1 via the γ3 sequence). First, inspired by properties of these natural binding partners, we varied the size and targeting moiety of model drug carriers to determine how these parameters affect CAM-mediated endocytosis. Increasing ICAM-1-targeted carrier size slowed carrier uptake kinetics, reduced carrier trafficking to lysosomes, and increased carrier transport across ECs. Changing targeting moieties from antibodies to peptides decreased particle binding and uptake, lowered trafficking to lysosomes, and increased transport across ECs. Second, using cell culture models of leukocyte/EC interactions, inhibiting regulatory elements of the CAM-mediated pathway disrupted leukocyte sampling, a process crucial to leukocyte crossing of endothelial layers (transmigration). This inhibition also decreased leukocyte transmigration across ECs, specifically through the transcellular route, which occurs through a single EC without disassembly of cell-cell junctions. Third, fibrin meshes, which mimic blood clot fragments/remnants, bound to ECs at ICAM-1-enriched sites and were internalized by the endothelium. Inhibiting the CAM-mediated pathway disrupted this uptake. Following endocytosis, fibrin meshes trafficked to lysosomes where they were degraded. In mouse models, CAM-mediated endocytosis of fibrin meshes appeared to remove fibrin remnants at the endothelial surface, preventing re-initiation of the coagulation cascade. Overall, these results support a link between CAM-mediated endocytosis and leukocyte transmigration as well as uptake of fibrin materials by ECs. Furthermore, these results will guide the future design of ICAM-1-targeted carrier-assisted therapies.