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The goal of this study was to develop a passive, low-cost, low-technological, rapid surveillance method for detecting schistosome cercariae in schistosomiasis endemic regions, compared to other available methods. Schistosomiasis is a parasitic disease that is linked to abdominal pain, enlarged liver, and multiple organ failure, yet it is highly preventable as individuals are infected through dermal contact with contaminated waters. In areas where transmission mitigation efforts have been successful, a combination of control initiatives were employed, such as mass drug treatment, hygiene improvement, and monitoring of transmission foci. The environmental detection methods currently in use are labor- and time- intensive or cost-prohibitive in rural developing areas with high transmission rates and very limited resources. Three aims were formulated in order to develop and evaluate the device. The first aim was to examine the chemotactic response of Schistosoma mansoni and S. japonicum cercariae to media containing oleic acid (OA) and explore how cercarial age plays a role in that response. The second aim was to apply the findings from the first aim to the development of an environmental surveillance device for schistosome cercariae (ESDSC) and to laboratory optimize the device to maximize cercarial capture. After examining the performance of the device, the third aim was to compare the ESDSC to other environmental surveillance methods that are currently available for the detection of schistosome cercariae through a review of the literature. While both S. mansoni and S. japonicum cercariae exhibited highest attachment to media when freshly shed, they preferentially attached to different media types: S. mansoni to beeswax with 0.3 g/mL OA and S. japonicum to plain beeswax. An ESDSC was designed from inexpensive, easily sourced materials (acrylic and aluminum sheets) and a S. mansoni-specific ESDSC was laboratory tested by utilizing beeswax with 0.3 g/mL of OA to attract

In recent decades, transmission of Trypanosoma cruzi, the causative agent of Chagas disease, by Triatoma infestans and other vector insects has expanded from historically rural areas to urban centers across Latin America. The urbanization of the T. cruzi transmission cycle necessitates new understanding of Chagas disease ecology and epidemiology, as well as new approaches to the surveillance, control, and prevention of vector infestation and parasite transmission. In rural La Joya, Peru, analyses highlight how the complexities of human migration and intermittent intervention influence the prevalence and incidence of Chagas disease. Substantial prevalence of T. cruzi infection was found in the adult population as a result of relatively higher incidence of infection among long-term inhabitants and relatively lower incidence of infection among short-term in-migrants. While an insecticide intervention in 1995 effectively eliminated incidence of infection among children, T. infestans and T. cruzi were rapidly reemerging in the absence of continuing vector control. In Arequipa, Peru, T. infestans had extensively and intensively infested an urban and peri-urban landscape prior to vector control. Environmental and social factors, which may directly or indirectly influence insect biology and behavior, were associated with infestation. Large clusters of infestation and spatial dependence among infested households at short and long distances suggest that T. infestans can disperse by crawling or flying in an urban environment, which may challenge ongoing vector surveillance and control. Reemergence of vector insects, including T. infestans, complicates continuing control of Chagas disease. While relatively rare, reemergence of T. infestans is a present and possibly persistent problem in urban and peri-urban Arequipa. The probability of a reemergence event varied spatially. Events were both clustered and non-clustered, and were spatially dependent at distances up to 1,600 meters

Infection with Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis, causing an estimated 2.1 million cases annually. Although infections with C. jejuni resolve naturally, over 13,000 hospitalizations and 100 deaths are attributed to this organism. Despite these alarming numbers, relatively little is known about C. jejuni pathogenesis, when compared to other enteric pathogens. This dissertation outlines the identification and characterization of a novel virulence factor in C. jejuni, the protein expressed by the Cj1534c gene. Using microarray and RT real time PCR, Cj1534c was found to be greater than 10 fold over expressed in both swine and poultry. Employing immunoelectron microscopy, we determined that at least a subset of the protein is surface localized. Based on the surface localization and up regulation in poultry, colonization studies were performed. Results demonstrate a significant reduction in colonization by a Cj1534c deficient mutant as compared to wild type. In vitro binding assays using both biotic and abiotic surfaces indicate this protein is involved with attachment to surfaces, as well as the invasion of cultured epithelial cells. In vitro findings were confirmed in vivo using swine, with the Cj1534c mutant being highly attenuated as compared to wild type strains. Additionally, the Cj1534c protein was tested for its potential as a vaccine in poultry. Studies demonstrated that Cj1534c recombinantly expressed in a Salmonella expression vector partially protected chickens, reducing cecal colonization three logs as compared to wild type. Taken together, this data demonstrates a major role of Cj1534c in both chicken cecal colonization and infection of swine.

Giardia lamblia is a eukaryotic parasite that causes diarrhea in humans worldwide. Diarrheal diseases cause stunting and mental retardation in children in developing nations, therefore it is important to understand the molecular epidemiology of G. lamblia. Compounding this, it is not clear if companion animals such as dogs contribute to infections in humans through zoonotic transmission. The genotypes of G. lamblia that have been found in humans are A1, A2 and B, while those in dogs have been on rare occasions all three human genotypes, but largely C and D, which have only been reported in dogs and appear to be species-specific. The molecular epidemiology of G. lamblia in humans and dogs was assessed in an endemic region of Lima, Peru. With one exception, dogs were found to harbor the C and D dog genotypes of G. lamblia. A single family dog was found to harbor a human genotype of G. lamblia. A2 and B genotypes of G. lamblia, but not A1, were found in humans in the endemic region. Previous literature reported that A2 and B typing within genotype tools were available, however the A2 samples from the endemic region could not be distinguished from one another through nucleotide polymorphism sequence analysis. A molecular typing technique was developed to type A2 samples. The extensive sequence analysis performed on two chromosomes of G. lamblia, yielded different phylogenetic tree groupings for the same samples. This lead to algorithmic analysis, which demonstrated a significantly high probability that meiotic recombination is occurring in the A2 samples of G. lamblia. As G. lamblia is largely believed to be asexual, the conclusion of doctoral research performed in this study yielded controversial, yet significant evidence that sex in G. lamblia A2 genotype samples is indeed occurring.

Tests were conducted inhouse to determine the microbiological treatment efficacy of the Pre-Mac Model FWP hand-held water purifiers. Test waters represented various physical/chemical challenge conditions. Microbial challenges consisted of enteric bacteria (Klebsiella terrigena), enteroviruses (Echovirus 1 and Poliovirus 1), protozoan oocysts (Crytosporidiu parvum), and cyst simulant (latex beads). Additional tests were conducted at the University of Arizona (UA) to determine the viability of Cryptosporidium parvum oocysts after exposure to the Pre-Mac iodine iodine, and to evaluate Pre-Mac water purifiers fitted with second generation 1.0 micron fine mesh filters for physical removals of cysts from product water. Studies used the U.S. Environmental Protection Agency's interim Guide Standard and Protocol for Testing Microbiological Water Purifiers for guidance. Results indicated that bacteria and viruses were readily removed by the Pre-Mac filter units. However, test results from inhouse studies and UA studies indicated that the Pre-Mac water purifiers were not adequate to physically remove or reduce infectious levels of protozoan cysts to meet the 3-log reduction criteria, Prepared in collaboration with Arizona Univ., Tucson. Dept. of Veterinary Science.

Outbreaks of cryptosporidiosis have been traced to Cryptosporidium oocysts in finished drinking water. Indeed, water contaminated with oocysts may be judged perfectly safe by conventional coliform tests. Although oocysts can be specifically identified using immunofluorescence, it is not yet possible to determine their viability. The lack of a viability test means that each oocyst detected in finished water must be regarded as potentially infective even though water treatment may have killed them. The goal of this research was to develop a test for oocyst viability. In vitro excystation, oocyst morphology, vital dyes, and a monoclonal antibody were tested. In vitro excystation expressed as percent of theoretical sporozoite yield correlated best with neonatal mouse infectivity. Although not directly applicable to testing water samples, excystation provided a basis for screening other testing methods. None of the eight vital dyes tested showed any relationship between oocyst staining and viability. This was presumably due to inability of the dyes to penetrate the oocyst wall. Pretreatment strategies designed to increase oocyst wall permeability were either ineffective or damaged the oocysts in ways that rendered them nonviable. Initially, microscopic appearance appeared to be related to oocyst infectivity. However, regression analysis showed that phase contrast microscopic appearance had marginal utility for use as a viability test. Indeed, microscopic identification of internal structures of intact oocysts is not a reliable viability indicator because DAPI staining showed intact sporozoite nuclei within obviously dead oocysts that would not excyst. A monoclonal antibody (MAb OW64) was found which binds to internal sites along the oocyst suture. There was positive correlation between binding of this MAb and decreasing oocyst infectivity indicating that MAb OW64 bound preferentially to nonviable oocysts. Regression analysis showed that OW64 binding overestimated oocyst

Tests were conducted to determine the efficacy of CHLOR-FLOC water purification tablets for treating microbiological contaminants in drinking water. The test waters represented various physical/chemical challenge conditions. Microbial challenges consisted of enteric bacteria (Klebseilla terrigena) enterovirus (Echovirus 1), protozoan oocysts (Cryptosporidium parvum) , and cyst simulant (latex beads). Studies used the U.S. Environmental Protection Agency's interim Guide Standard and Protocol for Testing Microbiological Water Purifiers for guidance. Results indicated that the CHLOR- FLOC system effectively reduced both bacterial and viral challenge components of the study to the required levels under all conditions, but failed to physically remove the oocysts and cyst simulant to the required levels under all conditions. Viability studies also indicated that the chemical disinfection component of the CHLOR-FLOC tablets had minimal capabilities to kill the encysted organisms over the 20-minute contact time. The overall study results revealed that the CHLOR-FLOC system was not adequate to physically remove, or to provide adequate chemical disinfection of, Cryptosporidium oocysts to the required level of 99.9 percent reduction. Water, Purification, CHLOR-FLOC tablets, Micro-organisms, Cryptosporidium, Klebseilla, Echovirus, Latex beads, Protozoan cysts, Bacteria, Disinfection, Coagulation.

A HEp-2 cell culture model was used to investigate the invasive properties of Campylobacter species. Two of twenty-five Campylobacter isolates did not invade HEp-2 cells, and one of these isolates did not adhere to the epithelial cells. Penetration of HEp-2 epithelial cells by C. jejuni was significantly (P < 0.05) inhibited with C. jejuni lysates and a MAb (1B4) in competitive inhibition studies. Immunogold electron microscopic studies revealed that the 1B4 MAb bound to the flagella and cell surface of low passage (invasive) C. jejuni M 96, whereas only the flagella of high passage (non-invasive) C. jejuni were labelled. Western blot analysis revealed that the 1B4 MAb identified an epitope on antigens ranging in size from 66 to 44 kDa in invasive and non-invasive organisms. Antigens were also recognized in lysates prepared only from invasive strains from 42 to 38 kDa. Sodium meta-periodate chemical treatment of C. jejuni lysates significantly (P < 0.05) affected its inhibitory capacity. Additionally, proteinase K and sodium meta-periodate treatment of lysates changed the mobility of antigens recognized by the 1B4 MAb. This suggests that the antigens required for epithelial cell penetration by C. jejuni may be glycoprotein in nature and that the functional binding site is dependent upon an intact carbohydrate moiety. Co-infection of HEp-2 epithelial cells with coxsackievirus B3, echovirus 7, polio virus (LSc type 1), porcine enterovirus and Campylobacter isolates was performed to determine if a synergistic effect could be obtained. The invasiveness of C. jejuni was significantly increased for HEp-2 cells pre-infected with echovirus 7, coxsackievirus B3, and UV-inactivated (non-infectious) coxsackievirus B3 particles. Polio and porcine enterovirus had no effect on C. jejuni adherence and invasiveness. C. hyointestinalis and C. mucosalis, two non-invasive isolates, did not invade virus-infected HEp-2 cells. The increase of invasiveness of C. jejuni appears to be the r