Maria Toivio-Kinnucan

EXAMINATION OF TISSUE CYST FORMATION BY TOXOPLASMA GONDII IN CELL CULTURES USING BRADYZOITES, TACHYZOITES, AND SPOROZOITES

Tissue cyst formation by a goat isolate (GT-1) of Toxoplasma gondii was examined in bovine monocyte, human fetal lung, and Madin-Darby bovine kidney cell cultures. Transmission electron microscopy (TEM) and cat feeding studies indicated that tissue cysts were present in all 3 cell lines examined. Tissue cysts were first seen 3 days postinoculation (PI) using TEM. Standard cell culture procedures were used and no additional condition was needed to induce tissue cyst formation. Cats fed cell cultures excreted T. gondii oocysts in their feces 5-7 days PI. These oocysts caused lethal infections in mice. Tissue cysts were produced in cell cultures regardless if the initiating inoculum consisted of bradyzoites, sporozoites, or a mixture of bradyzoites and tachyzoites. Tissue cyst formation has been followed through 40 subpassages of infected cells. By TEM tissue cysts still were present after 40 passages, but when 40th-passaged cultures were fed to cats, oocytsts were not excreted. This indicates that the parasite had become oocystless after repeated passage in vitro.

Mutation in β1-Tubulin Correlates with Macrothrombocytopenia in Cavalier King Charles Spaniels

BACKGROUND Cavalier King Charles Spaniels (CKCS) have a high prevalence of inherited macrothrombocytopenia. The purpose of this study was to determine if a mutation in beta1-tubulin correlated with presumptive inherited macrothrombocytopenia. HYPOTHESIS A mutation in beta1-tubulin results in synthesis of an altered beta1-tubulin monomer. alpha-beta tubulin dimers within microtubule protofilaments are unstable, resulting in altered megakaryocyte proplatelet formation. ANIMALS Blood samples were obtained from CKCS and non-CKCS dogs. METHODS DNA was used in polymerase chain reaction (PCR) assays to evaluate beta1-tubulin. Platelet numbers and mean platelet volume (MPV) were evaluated for a correlation with the presence or absence of a mutation identified in beta1-tubulin. Platelets obtained from homozygous, heterozygous, and clear CKCS were further evaluated using electron microscopy and immunofluorescence. RESULTS A mutation in the gene encoding beta1-tubulin correlated with macrothrombocytopenia in CKCS. Electron microscopy and immunofluorescence studies suggest that platelet microtubules are present but most likely are unstable and decreased in number. CONCLUSIONS AND CLINICAL IMPORTANCE The macrothrombocytopenia of CKCS correlated with a mutation in beta1-tubulin. alpha-beta tubulin dimers within protofilaments most likely are unstable, leading to altered proplatelet formation by megakaryocytes. This information will aid in distinguishing inherited from acquired thrombocytopenia. It also provides insight into the mechanism of platelet production by megakaryocytes, and also may prove useful in understanding heart-related changes in macrothrombocytopenic CKCS with concurrent mitral valve regurgitation.

Type I Glanzmann's thrombasthenia in a Great Pyrenees dog.

An 8-month-old female Great Pyrenees dog with chronic epistaxis and a history of gingival bleeding during shedding of deciduous teeth was evaluated for platelet function. Platelet morphology was normal at both the light and electron microscopic level. Platelet number and mean platelet volume were also normal. Platelet aggregation responses to adenosine diphosphate, collagen, platelet activating factor, and thrombin were markedly reduced, although shape change responses were normal. Clot retraction was markedly impaired. Monoclonal antibody (MoAb) Y2/51, a murine anti-human platelet β3 antibody that cross-reacts with canine platelet β3 , and MoAb 5G11, a murine anti-dog platelet αIIbβ3 antibody, bound minimally to affected dog platelets, as demonstrated by flow cytometry. Binding of MoAb Y2/51 was not detectable by immunoblot. MoAb CAP1, a murine anti-dog fibrinogen receptor-induced binding site antibody, failed to bind to affected dog platelets, as demonstrated by flow cytometry. A reduction in glycoproteins αIIb and β3 was demonstrated by two-dimensional protein electrophoresis. This is the first reported case of type I Glanzmanns thrombasthenia in the dog that closely resembles the clinical syndrome and the platelet morphology described in type I Glanzmanns thrombasthenia of human beings.

Ultrastructural determination of cystogenesis by various Toxoplasma gondii isolates in cell culture.

The tissue cyst stage of Toxoplasma gondii is important in relapsing disease seen in toxoplasmic encephalitis and retinochoroiditis. An in vitro culture system to examine the developmental biology of the tissue cyst stage would greatly aid in our understanding of this stage of the parasites life cycle. We used transmission electron microscopy (TEM) and acid-pepsin digestion of infected cell cultures to determine the capability of 21 isolates of T. gondii to produce tissue cysts in cell cultures. All 21 of the isolates had acid-pepsin-resistant stages present, and tissue cysts could be demonstrated in 19 using TEM. The present study demonstrates that tissue cyst formation in vitro is a common phenomenon for T. gondii isolates.

Gastrointestinal Pathogenicity of Adenoviruses and Reoviruses Isolated from Broiler Chickens in Alabama

Adenoviruses and reoviruses isolated from commercial broiler chickens were evaluated for gastrointestinal pathogenicity in specific-pathogen-free Leghorn chickens. The viruses were originally isolated from either the proventriculus or a gastrointestinal pool of tissues of broiler chickens with proventriculitis or enteritis. Isolates were cloned by terminal dilution. Day-old chickens were inoculated by oral and ocular routes with undiluted tissue culture fluids (titers of 102-104 TCID50/ml) and then examined at necropsy on days 5, 10, and 15 postinoculation. Chickens in all virus groups (but not the control group) developed wet, unformed fecal droppings that persisted for the duration of the study. Mild lesions occurred in reovirus-inoculated chickens and included hyperplasia of lymphocyte aggregates in various organs and mild gizzard erosions. Chickens inoculated with adenovirus isolates developed marked gizzard erosions and necrotizing pancreatitis as well as mild proventriculitis. Intranuclear viral inclusion bodies occurred in gizzard epithelium and pancreatic acinar cells at the sites of lesions. Lymphocytic atrophy occurred in the bursa of Fabricius. Respective viruses were reisolated from proventriculus and duodenum collected from chickens of each group; no viruses were isolated from controls. Under the conditions of this study, adenovirus isolates were more pathogenic than the reovirus isolates in the digestive system.

Novel Metal Clusters Isolated from Blood Are Lethal to Cancer Cells

Unfolding and subsequent aggregation of proteins is a common phenomenon that is linked to many human disorders. Misfolded hemoglobin is generally manifested in various autoimmune, infectious and inherited diseases. We isolated micrometer and submicrometer particles, termed proteons, from human and animal blood. Proteons lack nucleic acids but contain two major polypeptide populations with homology to the hemoglobin α-chain. Proteons form by reversible seeded aggregation of proteins around proteon nucleating centers (PNCs). PNCs are comprised of 1- to 2-nm metallic nanoclusters containing 40–300 atoms. Each milliliter of human blood contained approximately 7 × 1013 PNCs and approximately 3 × 108 proteons. Exposure of isolated blood plasma to elevated temperatures increased the number of proteons. When an aliquot of this heated plasma was introduced into untreated plasma that was subsequently heated, the number of proteons further increased, reaching a maximum after a total of three such iterations. Small concentrations of PNCs were lethal to cultured cancer cells, whereas noncancerous cells were much less affected.

Examination of extraintestinal tissue cysts of Isospora belli

Relapse is common in immunocompetent and immunosuppressed humans infected with Isospora belli and is believed to be associated with the presence of extraintestinal stages. In the present study, we examined this important stage in an AIDS patient using histological, immunohistological, histochemical, and ultrastructural methods to better understand the development and structure of this stage and to develop better means of detecting infections. Antisera made in rabbits to Isospora suis, Toxoplasma gondii, Hammondia hammondi, Sarcocystis neurona, Neospora caninum, and Caryospora bigenetica were tested against I. belli tissue cysts in the avidin-biotin peroxidase complex (ABC) immunohistological test. Most antisera reacted positively in the ABC test at dilutions of 1:100 but not at dilutions of 1:250. Some antisera to N. caninum and H. hammondi reacted positively at dilutions of 1:1,000 in the ABC test. Most reactive antisera stained the tissue cyst wall and not the enclosed zoite. Eight histochemical tests were examined and most were nonreactive with I. belli zoites or tissue cysts. Transmission electron microscopy revealed that the tissue cyst wall was composed of granular material and was directly beneath the parasitophorous vacuole membrane. Zoites were in the center of the tissue cysts and were surrounded by fibrillar material that appeared to originate from the zoite surface. Tubulelike structures were present in the granular tissue cyst wall and in the fibrillar material that surrounded the zoite. Zoites contained a crystalloid body. New findings in the present study consisted of identifying what are probably early tissue cysts that lack a developed tissue cyst wall, demonstrating that more than 1 tissue cyst can occupy a host cell, describing the distribution of micronemes and the shedding of zoite membranes, and identifying tubular structures in the inner tissue cyst wall and inner compartment.

Sarcocystis kirkpatricki n.sp. (Apicomplexa: Sarcocystidae) in muscles of raccoons (Procyon lotor) from Illinois.

Sarcocysts of Sarcocystis kirkpatricki n. sp. (Apicomplexa: Sarcocystidae) are described from the skeletal and heart musculature of 66 (66%) of 100 raccoons (Procyon lotor) from Illinois. Histologic examination of muscle tissues from tongue, diaphragm, esophagus, and heart revealed that 61%, 47%, 32%, and 2%, respectively, contained sarcocysts of this species. Juvenile raccoons (less than 1 yr old) were more likely (P less than 0.01) to have sarcocysts in the tissues examined (52/60 or 87%) than were adults (14/40 or 35%). Histologically, sarcocysts in the 4 tissues were similar: the cyst wall was 2-3 microns thick, PAS negative, and had fine hairlike surface projections; interior septa were indistinct. Ultrastructurally, sarcocyst walls had short (mean = 2.8 microns), straight to sloping, villuslike projections. Longitudinal tubular filaments inside these projections extended from the tips to the base, where they terminated in a granular electron-dense layer of the primary cyst wall. Thin septa were within the sarcocysts. Feeding experiments utilizing dogs and cats as potential definitive hosts were negative.

Cryptosporidium sp. infection in the proventriculus of an Australian diamond firetail finch (Staganoplura bella: Passeriformes, Estrildidae).

An Australian diamond firetail finch died following the acute onset and development of severe diarrhea. The bird was purchased from a wholesaler and was housed in a pet store aviary with 12 other birds. Necropsy, histologic evaluation, and electron microscopic evaluation revealed organisms in the proventriculus (surface, ductal, and glandular epithelium) compatible in site of development, size, and morphology with Cryptosporidium spp. Lesions in the proventriculus were focal cuboidal metaplasia of glandular epithelial cells and deposition of amyloid in the perivascular interstitial tissues at the base of the glands. Amyloid also was present in the duodenum, liver, spleen, pancreas, and kidney. Inability to recover other organisms suggested that Cryptosporidium was the primary cause of diarrhea and death. The affected bird likely suffered dehydration as a result of acute gastrointestinal disturbance, concomitant with renal amyloidosis and urate nephrosis.

Ultrastructural effects of diclazuril against Toxoplasma gondii and investigation of a diclazuril-resistant mutant

Diclazuril is an anticoccidial that inhibits tachyzoite production of the RH strain of Toxoplasma gondii by > 97% at a concentration of 0.005 micrograms/ml. The effects of 1.0 microgram/ml diclazuril on the development of 3 strains (RH and 2 tissue cyst formers, GT-1, and WTD-3) of T. gondii in vitro was examined using transmission electron microscopy. The effects of diclazuril were not noted until 2 days after treatment. Treatment with diclazuril interfered with endodyogeny and resulted in the production of multinucleate (> 2 nuclei) meront stages. Up to 12 nuclei were observed in some meronts. Tachyzoites attempted to bud from the surfaces of these stages but division was apparently blocked by the action of diclazuril. The parasitophorous vacuole (PV) enclosing developing stages became hypertrophic. As the meront stages degenerated the PV became filled with membranous material and the cytoplasmic contents of lysed stages. Formation of tissue cysts by the GT-1 and WTD-3 strains of T. gondii was not prevented by treatment with diclazuril. A mutant of the RH strain that was resistant to 1.0 microgram/ml diclazuril was selected by progressive culture in permissive levels of the agent. This DicR-1 mutant produced significantly fewer tachyzoites (P < 0.05) in vitro than its parent strain. Resistance to diclazuril appears to be stable because reversion to sensitivity to the agent did not occur after 50 passages in the absence of drug pressure. The DicR-1 mutant was somewhat less pathogenic for mice (70% mortality) than its parent RH strain (100% mortality) and persisted as tissue cysts in the brains of mice.(ABSTRACT TRUNCATED AT 250 WORDS)