Kathleen S. Logan

Isolation and characterization of an avian isolate of Encephalitozoon hellem

Members of the phylum Microspora are a group of unusual, obligate intracellular eukaryotic parasites that infect a wide range of hosts. However, there are a limited number of microsporidial infections reported in avian hosts, and no parasite species has been defined as an avian pathogen. A microsporidian organism was recovered from the droppings of a clinically normal peach-faced lovebird (Agapornis roseicollis) and established in in vitro culture. Intermittent parasite spore shedding was documented over a 2-month period using calcofluor M2R staining of cloacal swabs. The organism was identified as Encephalitozoon hellem based on protein and antigenic profiles and molecular sequencing of the small subunit and internal transcribed spacer regions of the ribosomal RNA gene.

Molecular Identification of Encephalitozoon hellem in an Ostrich

Microsporidia are obligate, intracellular, eukaryotic parasites found in a wide variety of vertebrate and invertebrate hosts. Scientific literature contains a small number of reports of these parasites in psittacine hosts, and recently microsporidiosis was reported in the first nonpsittacine host, an ostrich. DNA was extracted from formalin-fixed, paraffin-embedded ostrich tissues, and a portion of the small subunit ribosomal RNA gene was sequenced to identify the microsporidian species. The organisms were identified as Encephalitozoon hellem, a parasite species that was first described in immunocompromised humans and recently reported in three psittacine species.

Identification of Bloodmeal Sources and Trypanosoma cruzi Infection in Triatomine Bugs (Hemiptera: Reduviidae) from Residential Settings in Texas, the United States

ABSTRACT The host-vector-parasite interactions in Chagas disease peridomestic transmission cycles in the United States are not yet well understood. Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) infection prevalence and bloodmeal sources were determined for adult and immature triatomine (Hemiptera: Reduviidae) specimens collected from residential settings in central Texas. Sequenced cytochrome b DNA segments obtained from triatomine digestive tract identified nine vertebrate hosts and one invertebrate host in four triatomine species (Triatoma gerstaeckeri, Triatoma indictiva, Triatoma protracta, and Triatoma sanguisuga). The broad range of wild and domestic host species detected in triatomine specimens collected from residential sites indicates high host diversity and potential movement between the sylvatic and peridomestic settings. Domestic dogs appear to be key in the maintenance of the peridomestic transmission cycle as both a blood host for the triatomine vectors and a potential reservoir for the parasite. The high rate of T. cruzi infection among triatomine specimens that were collected from inside houses, outside houses, and dog kennels (69, 81, and 82%, respectively) suggests a current risk for Chagas disease vector-borne transmission for humans and domestic animals in residential settings in Texas because of overlap with the sylvatic cycle.

Identification of candidate vaccine antigens of bovine hemoparasites Theileria parva and Babesia bovis by use of helper T cell clones

Current vaccines for bovine hemoparasites utilize live attenuated organisms or virulent organisms administered concurrently with antiparasitic drugs. Although such vaccines can be effective, for most hemoparasites the mechanisms of acquired resistance to challenge infection with heterologous parasite isolates have not been clearly defined. Selection of potentially protective antigens has traditionally made use of antibodies to identify immunodominant proteins. However, numerous studies have indicated that induction of high antibody titers neither predicts the ability of an antigen to confer protective immunity nor correlates with protection. Because successful parasites have evolved antibody evasion tactics, alternative strategies to identify protective immunogens should be used. Through the elaboration of cytokines, T helper 1-(Th1)-like T cells and macrophages mediate protective immunity against many intracellular parasites, and therefore most likely play an important role in protective immunity against bovine hemoparasites. CD4+ T cell clones specific for soluble or membrane antigens of either Theileria parva schizonts or Babesia bovis merozoites were therefore employed to identify parasite antigens that elicit strong Th cell responses in vitro. Soluble cytosolic parasite antigen was fractionated by gel filtration, anion exchange chromatography or hydroxylapatite chromatography, or a combination thereof, and fractions were tested for the ability to induce proliferation of Th cell clones. This procedure enabled the identification of stimulatory fractions containing T. parva proteins of approximately 10 and 24 kDa. Antisera raised against the purified 24 kDa band reacted with a native schizont protein of approximately 30 kDa. Babesia bovis-specific Th cell clones tested against fractionated soluble Babesia bovis merozoite antigen revealed the presence of at least five distinct antigenic epitopes. Proteins separated by gel filtration revealed four patterns of reactivity, and proteins separated by anion exchange revealed two patterns of reactivity when selected T cell clones were assayed for stimulation by antigenic fractions. Studies using a continuous-flow electrophoresis apparatus have indicated the feasibility of identifying T cell-stimulatory proteins from parasite membranes as well as from the cytosolic fraction of B. bovis merozoites. The Th cell clones reactive with these different hemoparasites expressed either unrestricted or Th1 cytokine profiles, and were generally characterized by the production of high levels of IFN-gamma. A comprehensive study of T cell and macrophage responses to defined parasite antigens will help elucidate the reasons for vaccine failure or success, and provide clues to the mechanisms of acquired immunity that are needed for vaccine development.

Simple, Filter-based PCR Detection of Thelohania solenopsae (Microspora) in Fire Ants (Solenopsis invicta)

Abstract Thelohania solenopsae is a microsporidian parasite that may serve as a biological control agent for the red imported fire ant, Solenopsis invicta. A rapid, filter-based PCR amplification method detecting a portion of the small-subunit ribosomal RNA gene was developed to facilitate field studies detecting the parasite in fire ants. Processing ant homogenates with a commercially available membrane-based system, FTA Classic Card technology, compared favorably with traditional DNA extraction and PCR amplification methods. As few as 100 spores were detected. The FTA membrane system is a simple, extraction-free method for detecting T. solenopsae in fire ants, and allows for easy archival storage of DNA samples.