Browsing by Author "Lindsay, David S."

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    Age and Sex Related Behavioral Changes in Mice Congenitally Infected with Toxoplasma gondii: Role of dopamine and other neurotransmitters in the genesis of behavioral changes due to congenital infection and attempted amelioration with interferon gamma
    Goodwin, David G. (Virginia Tech, 2011-07-12)
    Evidence suggests that the neurotropic parasite Toxoplasma gondii may play a role in the development of cognitive impairments. My hypothesis was that congenital exposure to T. gondii would lead to detectable age and sex related differences in behavior and neurotransmitter levels in mice. The neurotransmitter dopamine and commonly used anti-schizophrenic agents were evaluated against T. gondii in human fibroblast cells. Dopamine caused a significant increase in tachyzoite numbers at 250 nM but not 100 nM and the drugs valproic acid, fluphenazine, thioridazine and trifluoperazine inhibited T. gondii development. The effects T. gondii infection had on behavior were examined using a congenital mouse model. Previous work demonstrated maternal immune stimulation (MIS) with interferon gamma (INF-g) resulted in decreased fetal mortality from congenital T. gondii infections; therefore I examined the effects of INF- g treatment of mothers to determine if protection from the behavioral effects of T. gondii occurred in their offspring. No differences in concentrations of neurotransmitters in the brains of congenitally infected mice were observed. I found that mice infected with T. gondii developed adult onset behavior impairments with decreased rate of learning, increased activity and decreased memory, indicating cognitive impairment for male mice and not female mice. My findings support the evidence T. gondii is a factor in the development of cognitive impairments. My results for T. gondii exposed male mice are consistent with the convention that males have more cognitive impairments in the prodromal stage of schizophrenia. MIS with IFN-g had a minimal effect on behavior post sexual maturity but had a greater effect on pre sexual maturity female mice which exhibited difficulties with spatial memory, coordination and the ability to process stimuli. The results indicate the behavior alterations from IFN- g are transient. When MIS is given prior to congenital infection with T. gondii, we detected no behavior deficits in any group of mice, including male mice post sexual maturity. Based on the results of my study, I must reject the hypothesis that neurotransmitter levels are influenced by congenital toxoplasmosis and accept the hypothesis that congenital T. gondii infection caused cognitive impairments in male mice post sexual maturity.
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    Ancient, globally distributed lineage of Sarcocystis from sporocysts of the Eastern rat snake (Pantherophis alleghaniensis) and its relation to neurological sequalae in intermediate hosts
    Verma, Shiv K.; Lindsay, David S.; Rosenthal, Benjamin M.; Dubey, Jitender P. (2016-07)
    There is an emerging concern that snakes are definitive hosts of certain species of Sarcocystis that cause muscular sarcocystosis in human and non-human primates. Other species of Sarcocystis are known to cycle among snakes and rodents, but have been poorly characterized in the USA and elsewhere. Although neurological sequalae are known for certain species of Sarcocystis, no such neurological symptoms are known to typify parasites that naturally cycle in rodents. Here, sporocysts of a species of Sarcocystis were found in the intestinal contents of a rat snake (Pantherophis alleghaniensis) from Maryland, USA. The sporocysts were orally infective for interferon gamma gene knockout (KO) mice, but not to Swiss Webster outbred mice. The KO mice developed neurological signs, and were necropsied between 33 and 52 days post-inoculation. Only schizonts/merozoites were found, and they were confined to the brain. The predominant lesion was meningoencephalitis characterized by perivascular cuffs, granulomas, and necrosis of the neuropil. The schizonts and merozoites were located in neuropil, and apparently extravascular. Brain homogenates from infected KO mice were infective to KO mice and CV-1 cell line. DNA extracted from the infected mouse brain, and infected cell cultures revealed the highest identity with Sarcocystis species that employ snakes as definitive hosts. This is the first report of Sarcocystis infection in the endangered rat snake (P. alleghaniensis) and the first report of neurological sarcocystosis in mice induced by feeding sporocysts from a snake. These data underscore the likelihood that parasites in this genus that employ snakes as their definitive hosts constitute an ancient, globally distributed monophyletic group. These data also raise the possibility that neurological sequalae may be more common in intermediate hosts of Sarcocystis spp. than has previously been appreciated.
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    The anthelmintic effect of Bacillus thuringiensis Cry5B on Haemonchus contortus in sheep
    Sanders, John Patrick (Virginia Tech, 2020-06-30)
    Widespread anthelmintic resistance in trichostrongyle nematodes of ruminants has created an urgent need for alternatives to commercial anthelmintics. The bacterium Bacillus thuringiensis (Bt) can produce crystal proteins during sporulation, which can be lethal to insects in multiple orders when ingested. One protein, Cry5B, has demonstrated effectiveness against multiple parasitic nematodes. We hypothesized that Cry5B would be effective against Haemonchus contortus, a highly pathogenic parasite, in sheep. Two experiments tested efficacy of Cry5B in sheep experimentally infected with H. contortus. In the first, a live genetically modified, asporogenous strain of B. thuringiensis expressing cytosolic Cry5B protein (BaCC) was administered orally daily for four days (~40mg/kg Cry5B/day). The mean fecal egg count (FEC) of treated animals was reduced by 94% three days after treatment, and at necropsy the female worm burden was significantly reduced by 98%. In the second experiment inactivated, asporogenous Bt expressing cytosolic Cry5B (IBaCC) was used. Treated animals received 60mg/kg Cry5B, administered daily for three days. By 72 hours after the first treatment FEC was reduced by 91%. Mean total worm burden of treated sheep at necropsy was significantly reduced, with female worms reduced by 95%. A third study tested the effect of BaCC and IBaCC on development of eggs to infective larvae in feces under laboratory and outdoor environmental conditions. Cry5B (15mg) added to feces (10g) reduced numbers of infective larvae by 99% in both environments within 12 days. Cry5B appears to have potential for controlling H. contortus in sheep. All protocols approved by VT IACUC and IBC.
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    Approaches towards vaccine development against Neospora caninum
    Ramamoorthy, Sheela (Virginia Tech, 2006-06-05)
    Neospora caninum is an apicomplexan parasite that causes neuromuscular paralysis in dogs and abortions in cattle. N. caninum is responsible for losses of several million dollars to the dairy and beef industries in several parts of the world. The key players in the host immune response to N. caninum include CD4+ T cells, the Th1 cytokines IL-12, Interferon gamma and IgG2a isotype antibodies. There are currently no chemotherapeutic agents that are effective against adult cattle neosporosis. A commercially available, inactivated vaccine induces the undesirable Th2 type of immunity against N. caninum. Therefore, two approaches towards vaccine development against N. caninum that were designed to induce potent cell mediated immunity have been explored in this dissertation. The first approach consisted of the development of a bivalent recombinant vaccine for both brucellosis and neosporosis, while the second approach involved gamma irradiation of N. caninum tachyzoites for use as an attenuated vaccine against N. caninum. Since N. caninum research has been conducted with several strains of mice and the different strains of mice vary in their susceptibility to infection with N. caninum, there is a need to develop a standard lab animal model for N. caninum. A gerbil and a C57BL/6 mouse model for N. caninum vaccine testing have been developed. It was found that the LD50 of N. caninum tachyzoites in gerbils was 9.3 x105 tachyzoites per gerbil delivered intra-peritoneally, (i.p) while for C57BL/6 mice the LD50 was 1.5 x107 tachyzoites per mouse delivered i.p. Vertical transmission rates in C57BL/6 mice infected with N. caninum tachyzoites during mid-gestation were determined and found to be in the range of 96-100%. Putative protective antigens of N. caninum that included MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in B. abortus strain RB51 to create recombinant vaccine strains. C57BL/6 mice were vaccinated with either the recombinant strains or the irradiated tachyzoites. Antigen specific IgG2a and IgG1 responses and high levels of interferon gamma and IL-10 were induced by vaccination. Mice vaccinated with irradiated tachyzoites, RB51-MIC1 and RB51-GRA6 were completely protected against lethal challenge, while the mice vaccinated with RB51-SRS2, RB51-GRA2 and RB51-MIC3 were partially protected. To determine the efficacy of the vaccines in preventing vertical transmission of N. caninum, mice were vaccinated and bred after administration of a booster dose four weeks after the primary vaccination. Antigen specific IgG1 and IgG2a and significant levels of IFN-ã and IL-10 were detected in vaccinated, pregnant mice. Pregnant mice were challenged with 5 x 106 N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups three weeks after birth and examined for the presence of N. caninum by a semi-nested PCR. Protection against vertical transmission elicited by the RB51-GRA6, RB51-MIC3, irradiated tachyzoite, RB51-GRA2, RB51-MIC1 and RB51-SRS2 vaccinated groups were 43%, 38%, 34%, 34%, 18%, and 7% respectively. Since not all the antigens that were highly protective against acute disease were not very effective in preventing vertical transmission, the role of the selected antigens in preventing acute disease and vertical transmission appear to differ. Only GRA6 was found to be effective in protecting against an acute lethal challenge as well as preventing vertical transmission 43% of the time. In summary, two animal models for the testing of N. caninum vaccines were developed. N. caninum protective antigens were successfully expressed in B. abortus strain RB51. The irradiated tachyzoite and recombinant RB51-Neospora vaccines were highly effective in protecting against acute neosporosis and partially protective against vertical transmission. Therefore, both these approaches show great promise as practical and effective means to achieve the goal of successful prophylaxis against N. caninum induced abortions and reduce the chances of vertical transmission.
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    Besnoitia oryctofelisi n. sp (Protozoa : Apicomplexa) from domestic rabbits
    Dubey, Jitender P.; Sreekumar, C.; Lindsay, David S.; Hill, D.; Rosenthal, B. M.; Venturini, L.; Venturini, M. C.; Greiner, E. C. (Cambridge University Press, 2003-06)
    A species of Besnoitia from naturally infected rabbits from Argentina was propagated experimentally in mice, gerbils, rabbits, cats, and cell cultures. Cats fed tissue cysts from rabbits shed oocysts with a prepatent period of nine to 13 days. Sporulated oocysts were infective to gerbils, rabbits, outbred Swiss Webster and interferon gamma gene knockout mice. Bradyzoites were infective orally to gerbils and cats. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey kidney cells. Schizonts were seen in the lamina propria of the small intestine of cats fed tissue cysts; the largest ones measured 52 x 45 mum. Schizonts were also present in mesenteric lymph nodes, livers, and other extra-intestinal organs of cats fed tissue cysts. Oocysts were 10-14 x 10-13 mum in size. This rabbit-derived species of Besnoitia resembled B. darlingi of the North American opossum, Didelphis virginiana with an opossum-cat cycle, but it was not transmissible to D. virginiana, and B. darlingi of opossums was not transmissible to rabbits. Based on biological, serological, antigenic, and molecular differences between the rabbit and the opossum Besnoitia, a new name, B. oryctofelisi is proposed for the parasite from domestic rabbits from Argentina.
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    Bovine Coccidiosis: Dynamics of infection in grazing cattle and the potential role of stress and immunity
    Lucas, Aaron Scott (Virginia Tech, 2011-08-02)
    Eimerian parasites infect cattle worldwide. Information on the infection dynamics of these parasites is lacking in the central Appalachian region of the United States. Studies aimed at characterizing the seasonal dynamics of eimerian parasites in this region were carried out in order to assess the impact of these organisms in grazing systems. In these studies the prevalence of Eimeria spp. infection was highest in calves less than one year of age and subsequently decreased to stable levels in older animals. Although E. bovis was the most common species identified in calves, heifers and cows, mixed species infections dominated. Additional studies were carried out to investigate the effect of stress on Eimeria spp. infection in beef calves. Lower stress, two-stage, weaning methods had no effect on Eimeria spp. infection dynamics in beef calves. These findings must be interpreted in light of the fact that calves used in this study were not managed in a way typical of many calves in the U.S.A. The fact that they were only transported short distances, never commingled, or exposed to a livestock market may explain why a rise in post weaning FOC was not observed. A model of stress- induced coccidiosis was developed using dexamethasone and E. bovis challenge. In this model, an oral challenge of at least 500,000 sporulated E. bovis oocysts in addition to dexamethasone injection at 7 days post challenge increased subsequent FOC. Further investigation of the immune response to E. bovis challenge during times of stress indicates that stress-induced suppression of cell mediated immunity and E. bovis challenge are required to increase subsequent oocyst shedding. These findings may represent the mechanism associated with stress-induced outbreaks of coccidiosis reported to occur in beef cattle in the United States.
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    Can levamisole upregulate the equine cell mediated immune response in vitro?
    Santonastaso, Amy Marie (Virginia Tech, 2016-07-19)
    Equine Protozoal Myeloencephalitis (EPM) is arguably the most common and costly equine neurologic diseases nationwide. The national seroprevalence is >50%, but only 0.5-1% of all horses develops disease during their lifetimes. Some EPM affected horses have decreased immune response. A cell-mediated immune response has been shown to be protective for development of EPM after infection with Sarcocystis neurona in mouse models. Levamisole has been proposed as an adjunctive therapy for EPM to upregulate the cell-mediated immune response based on positive results in other species, but there are very limited studies in equids. We hypothesized that levamisole will upregulate the equine cell-mediated macrophage (M1) dendritic cell (DC1) CD4 T-helper 1 (Th1) CD8 Tc1 immune response in vitro. The first aim was to determine optimal conditions and effects of levamisole on cellular proliferation. Equine PBMCs were harvested from ten horses seronegative for S. neurona. The cells were cultured alone, or with one of the mitogens: concanavalin A (ConA) or phorbol 12-myristate 13-acetate and ionomycin (PMA/I), or with a combination of the above mitogens and levamisole at several conditions. Cellular proliferation was assessed using a colorimetric bromodeoxyuridine ELISA assay. The second aim was to determine the ability of levamisole, under optimized conditions, to upregulate the M1 DC1 CD4Th1 CD8 Tc1 response in vitro based on activation and function. PBMCs from the same 10 horses were cultured with each of the following: no stimulation, conA, and levamisole with and without ConA. To determine proliferation of each specific subset, cells were labeled with a fluorescent dye, CellTrace. Proliferation was determined based on dye dilution using flow cytometry. To determine the effects of levamisole on the specific immune response, cell subsets were labeled with fluorescent antibodies for cell surface markers (CD4, CD8, CD21, CD172a, CD14) and dendritic and macrophage activations markers (MHC Class II, CD86). Induction of T-regs was based on FoxP3 expression. Immune phenotypes were determined based on intracellular cytokine expression (IFNɣ, IL4, IL10). Study results indicate that levamisole alone did not significantly alter PBMC proliferation compared to the response of unstimulated cells. Cells cultured with either ConA or PMA/I resulted in a statistically significant increase (P<0.05) in proliferation compared to unstimulated cells. Cells cultured with ConA and levamisole at 1µg/mL resulted in a significant decrease (P<0.05) in proliferation compared with cells cultured with ConA alone. Flow cytometry data failed to elucidate the specific immune phenotype that is affected by levamisole. Subjectively, there appeared to be a trend for inceased IFNɣ production by CD14 and CD172a positive cells (macrophages and dendritic cells) and a decrease in IFNɣ production by CD4 and CD8 positive cells (T-lymphocytes). These results demonstrate that levamisole downregulates ConA stimulated PBMC proliferation. Based on these in vitro results, further studies to determine the effectiveness of levamisole on modulating the equine immune system in vivo and to more specifically evaluate the immune cell subets affected by levamisole are warranted.
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    Can levamisole upregulate the equine cell-mediated macrophage (M1) dendritic cell (DC1) T-helper 1 (CD4 Th1) T-cytotoxic (CD8) immune response in vitro?
    Witonsky, Sharon G.; Buechner-Maxwell, Virginia A.; Santonastasto, Amy; Pleasant, R. Scott; Werre, Stephen R.; Wagner, Bettina; Ellison, Siobhan; Lindsay, David S. (Wiley, 2019-03-01)
    Background: Equine protozoal myeloencephalitis (EPM) is a common and devastating neurologic disease of horses in the United States. Because some EPM-affected horses have decreased immune responses, immunomodulators such as levamisole have been proposed as supplemental treatments. However, little is known about levamisole's effects or its mechanism of action in horses. Objective: Levamisole in combination with another mitogen will stimulate a macrophage 1 (M1), dendritic cell 1 (DC1), T-helper 1 (CD4 Th1), and T-cytotoxic (CD8) immune response in equine peripheral blood mononuclear cells (PBMCs) in vitro as compared to mitogen alone. Animals: Ten neurologically normal adult horses serologically negative for Sarcocystis neurona. Methods: Prospective study. Optimal conditions for levamisole were determined based on cellular proliferation. Peripheral blood mononuclear cells were then cultured using optimal conditions of mitogen and levamisole to identify the immune phenotype, based on subset-specific activation markers, intracellular cytokine production, and cytokine concentrations in cell supernatants. Subset-specific proliferation was determined using a vital stain. Results: Concanavalin A (conA) with levamisole, but not levamisole alone, resulted in a significant decrease (P <.05) in PBMC proliferation compared to conA alone. Levamisole alone did not elicit a specific immune phenotype different than that induced by conA. Conclusion and Clinical Importance: Levamisole co-cultured with conA significantly attenuated the PBMC proliferative response as compared with conA. If the mechanisms by which levamisole modulates the immune phenotype can be further defined, levamisole may have potential use in the treatment of inflammatory diseases.
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    Case Report Chronic Microsporidial Enteritis in a Missionary from Mozambique
    Palmieri, James R.; Elswaifi, Shaadi F.; Lindsay, David S.; Junko, Gretchen; Callahan, Cathy (American Society for Tropical Medicine & Hygiene, 2010-11-01)
    Microsporidiosis often occurs in immunocompromised persons but may also occur in those who are immunocompetent. Infection by Microsporidia involves a variety of organs and systems, most notably, intestine, lung, kidney, brain, sinuses, muscle, and eyes. Enterocytozoon bieneusi and Encephalitozoon intestinalis are associated with gastroenteritis, and Enterocytozoon hellem and Encephalitozoon cuniculi are associated with keratoconjunctivitis. We report a case of chronic microsporidiosis in a 28-year-old woman missionary from Mozambique who came to our diagnostic laboratory with nausea, lower abdominal pain, and frequent bowel movements. Over two years, the patient was clinically assessed and treated for malaria and giardiasis without laboratory diagnosis while in Mozambique. Identification of the causative agent of her condition was not attempted during the course of her illness in Mozambique. Furthermore, adverse effects of malaria and giardiasis medications may have exacerbated the chronic illness in this patient and mimicked chronic microsporidiosis.
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    Caspase-11 Modulates Inflammation and Attenuates Toxoplasma gondii Pathogenesis
    Coutermarsh-Ott, Sheryl; Doran, John T.; Campbell, Caroline; Williams, Tere M.; Lindsay, David S.; Allen, Irving C. (Hindawi, 2016-06-09)
    Toxoplasma gondii is an obligate intracellular parasite that is the etiologic agent responsible for toxoplasmosis. Infection with T. gondii results in activation of nucleotide binding domain and leucine rich repeat containing receptors (NLRs). NLR activation leads to inflammasome formation, the activation of caspase-1, and the subsequent cleavage of IL-1β and IL-18. Recently, a noncanonical inflammasome has been characterized which functions through caspase-11 and appears to augment many biological functions previously considered to be dependent upon the canonical inflammasome. To better elucidate the function of this noncanonical inflammasome in toxoplasmosis, we utilized Asc−/− and Casp11−/− mice and infected these animals with T. gondii. Our data indicates that caspase-11 modulates the innate immune response to T. gondii through a mechanism which is distinct from that currently described for the canonical inflammasome. Asc−/− mice demonstrated increased disease pathogenesis during the acute phase of T. gondii infection, whereas Casp11−/− mice demonstrated significantly attenuated disease pathogenesis and reduced inflammation. This attenuated host response was associated with reduced local and systemic cytokine production, including diminished IL-1β. During the chronic phase of infection, caspase-11 deficiency resulted in increased neuroinflammation and tissue cyst burden in the brain. Together, our data suggest that caspase-11 functions to protect the host by enhancing inflammation during the early phase of infection in an effort to minimize disease pathogenesis during later stages of toxoplasmosis.
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    Characterization of Canine Leishmaniasis in the United States: Pathogenesis, Immunological Responses, and Transmission of an American Isolate of Leishmania infantum
    Rosypal, Alexa C. (Virginia Tech, 2005-04-06)
    Leishmania infantum, an etiologic agent of zoonotic visceral leishmaniasis, has recently emerged in the foxhound population in the United States and parts of Canada. Leishmania infections are usually spread to mammals by infected sand flies, however epidemiological data do not support a role for sand fly transmission in North America. The purpose of this work was to isolate and characterize L. infantum from a naturally infected foxhound from Virginia (LIVT-1 isolate). A mouse model of North American leishmaniasis was developed using immunocompetent and genetically immunodeficient mouse strains infected with LIVT-1 promastigotes by different inoculation routes. The intravenous route of infection was superior to the subcutaneous route for inducing consistent experimental infections and mice lacking interferon gamma, inducible nitric oxide synthase, or B-cells were resistant to clinical disease. Experimental infections in dogs were performed to examine the infectivity, immune responses, and pathogenicity of LIVT-1. Experimentally infected dogs developed parasitologically proven infections and a range of clinical manifestations that were similar to those observed in naturally occurring disease. Diagnostic tests including culture and cytologic evaluation of bone marrow and lymph node aspirates, polymerase chain reaction, and serology by indirect fluorescent antibody test, and recombinant K39 (rK39) immunoassay were evaluated. Kappa statistics revealed that PCR had the highest level of agreement with culture and cytology results although the rK39 dipstick assay consistently identified more experimentally infected dogs. Flow cytometry revealed no significant differences (p>0.05) in CD4+ or CD8+ expression on peripheral blood lymphocytes. Alternate transmission mechanisms in experimentally inoculated mice and dogs were investigated. PCR revealed a low level of vertical and direct transmission of LIVT-1 in inoculated BALB/c mice. Leishmania DNA was detectable by PCR in tissues from puppies from a LIVT-1 infected beagle. Although the strain of L. infantum infecting foxhounds in North America appears to predominantly use a non-vector transmission mode, the disease it produces is similar to canine leishmaniasis in other parts of the world. Non-sand fly transmission may be responsible for maintaining infections in the foxhound population. Results from this work will lead to improvement in diagnosis, clinical management, and control of canine leishmaniasis in North America.
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    Characterization of temperature-sensitive strains of Neospora caninum in mice
    Lindsay, David S.; Lenz, S. D.; Blagburn, B. L.; Brake, D. A. (American Society of Parasitology, 1999-02)
    Temperature-sensitive (ts) strains of the Neospora caninum tachyzoites were selected by chemical mutagenesis and selection for growth at 32 C. Three ts strains and the parental, N. caninum wild-type strain, NC-1, were examined in the present study for their ability to cause disease in inbred BALB/c mice, outbred ICR mice, and chemically immunosuppressed ICR mice. In BALB/c mice, all 3 strains failed to induce clinical disease, whereas infection with the NC-1 strain caused central nervous system disease and death in some mice. No disease was observed in ICR mice inoculated with the 3 ts strains or the NC-I strain. All immunosuppressed ICR mice inoculated with the NC-1 strain died, whereas no immunosuppressed mice inoculated with the NCts-4 strain and only 1 of 5 mice inoculated with the NCts-8 and NCts-12 strains died. The NCts-4 and NCts-12 strains reverted to a wild-type phenotype when grown at 37 C. Vaccination of BALB/c mice with live, but not frozen NCts-8 strain tachyzoites induced significant (P < 0.05) protection following NC-1 strain challenge.
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    Characterizing Cystoisospora canis as a Model of Apicomplexan Tissue Cyst Formation and Reactivation
    Houk-Miles, Alice Elizabeth (Virginia Tech, 2015-07-01)
    Cystoisospora canis is an Apicomplexan parasite of the small intestine of dogs. C. canis produces monozoic tissue cysts (MZT) that are similar to the polyzoic tissue cysts (PZT) of Toxoplasma gondii, a parasite of medical and veterinary importance, which can reactivate and cause toxoplasmic encephalitis. We hypothesized that C. canis is similar biologically and genetically enough to T. gondii to be a novel model for studying tissue cyst biology. We examined the pathogenesis of C. canis in beagles and quantified the oocysts shed. We found this isolate had similar infection patterns to other C. canis isolates studied. We were able to superinfect beagles that came with natural infections of Cystoisospora ohioensis-like oocysts indicating that little protection against C. canis infection occurred in these beagles. The C. canis oocysts collected were purified and used for future studies. We demonstrated in vitro that C. canis could infect 8 mammalian cell lines and produce MZT. The MZT were able to persist in cell culture for at least 60 days. We were able to induce reactivation of MZT treated with bile-trypsin solution. In molecular studies, we characterized C. canis genetically using ITS1 and CO1 to build phylogenetic trees and found C. canis was most similar to C. ohioensis-like with ITS1 and more similar to T. gondii than any other coccidia using ITS1 and CO1. We identified genes and proteins involved with virulence, cyst wall structure, and immune evasion of T. gondii and examined the DNA of C. canis for orthologs. C. canis had orthologs with 8 of 20 T. gondii genes examined. Monoclonal and polyclonal antibody and lectin studies demonstrated similar tissue cyst wall proteins on C. canis MZT and T. gondii PZT. Our findings in vitro and using genetic characterization of C. canis indicated the presence of similar genes and proteins, and its close phylogenetic location with T. gondii demonstrate that C. canis may serve as a model to examine tissue cyst biology. The system we described provides a simple model to produce tissue cysts and to study host factors that cause reactivation of tissue cysts.
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    Chronic Toxoplasma gondii in Nurr1-Null Heterozygous Mice Exacerbates Elevated Open Field Activity
    Eells, J. B.; Varela-Stokes, A.; Guo-Ross, S. X.; Kummari, E.; Smith, H. M.; Cox, E.; Lindsay, David S. (PLOS, 2015-04-09)
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    Comparable growth of Tritrichomonas mobilensis in two commercially available culture media
    Boggild, A. K.; Sundermann, C. A.; Estridge, B. H.; Lindsay, David S. (American Society of Parasitology, 2002-10)
    The investigation reported herein was undertaken to determine which medium is more practical for the axenic laboratory culture of trichomonads. The growth of Tritrichomonas mobilensis was monitored in 2 different types of commercially available growth media. Although Roswell Park Memorial Institute (RPMI) 1640 medium is typically used as a mammalian cell culture medium, it was found to support the growth of trichomonads as well as the American Type Culture Collection (ATCC) medium 745 under similar conditions. Environmental variables, such as temperature and pH, known to affect the success of trichomonad cultures were controlled. The mean generation times (MGTs) of T. mobilensis in the log phase of growth were 5.1 and 4.9 hr for RPMI 1640 and ATCC medium 745, respectively. A stationary phase of zero growth was reached more quickly in the ATCC medium 745 cultures, and in both media a phase of rapid attrition followed this period of static growth. In assessing the practicality of the media, total cell amplification, as well as factors such as cost, case of preparation, and storage capacity, were considered.
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    Comparison of diagnostic techniques for detection of Giardia duodenalis in dogs and cats
    Saleh, Meriam N.; Heptinstall, Jack R.; Johnson, Eileen M.; Ballweber, Lora R.; Lindsay, David S.; Werre, Stephen R.; Herbein, Joel F.; Zajac, Anne M. (American College of Veterinary Internal Medicine, 2019-05)
    Background An evaluation of currently available in-clinic diagnostic tests for Giardia duodenalis infection of dogs and cats has not been performed. In addition, there is discordance among published diagnostic comparisons. The absence of a true gold standard for detecting Giardia duodenalis also complicates diagnostic evaluations. Objectives To evaluate diagnostic tests commercially available in the United States for detecting Giardia duodenalis in dogs and cats, in comparison to a widely used reference test, the direct immunofluorescent assay (IFA), and also to compare the results of 2 methods of analysis: comparison of diagnostic tests to a reference test (IFA) and Bayesian analysis. Animals Fecal samples from a convenience sample of 388 cats and dogs located in Colorado, Oklahoma, and Virginia. Methods Fecal samples were tested for Giardia duodenalis by zinc sulfate centrifugal fecal flotation and 4 different commercial diagnostic immunoassays. Results were analyzed via Bayesian analysis and by comparison to the IFA as the reference test. Results Sensitivity and specificity by comparison to IFA was >= 82% and >= 90%, respectively, for all diagnostic tests in dogs and cats. When analyzed via Bayesian analysis, sensitivity and specificity were >= 83% and >= 95%, respectively. When ZnSO4 centrifugal fecal flotation results were combined with immunoassay results, there was no longer a significant difference between the sensitivities of the commercial in-clinic immunoassays. Conclusion and Clinical Relevance The Bayesian analysis validates using IFA as the reference test. Differences in commercial in-clinic immunoassay sensitivities can be mitigated when the results are combined with ZnSO4 centrifugal fecal flotation results.
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    Comparison of Neospora seroprevalence in Virginia dairy herds with high and low abortion rates
    Murphy, Julia M. (Virginia Tech, 1998-06-30)
    Neospora has become a commonly recognized infectious cause of abortion in dairy cattle. The organism is associated with mid to late term abortion outbreaks with rates exceeding 30% in some herds. Cows infected with this organism exhibit no other clinical signs. While this disease has been reported in other parts of the country, no assessment of Neospora seroprevalence has been undertaken in the Southeast. This study sampled commercial dairy herds to assess Neospora seroprevalence in dairy cows and investigate its significance as an abortifacient agent in Virginia. Twenty four herds participated in the study. Twelve herds had DHIA reported annual abortion rates of 6% or greater (high abortion rate herds) and twelve herds had abortion rates of 2% or less (low abortion rate herds). High abortion rate herds were each paired to a low abortion rate herd (control) herd within the same county. A single blood sample was collected from all cows confirmed to be 90 to 240 days pregnant, with a maximum of thirty samples per herd. A random sample of cows was selected in herds with more than 30 pregnant cows between 90 and 240 days gestation. Neospora antibody titers were determined using a serum ELISA test at the California Veterinary Diagnostic Laboratory. Both mean and median seroprevalence of high and low abortion rate herds were compared using the Mann-Whitney Rank Sum test and the Median test, respectively. No significant difference was found in either case (p=0.56, p=0.41). These findings suggest that Neospora does not contribute significantly to the average abortion rate in Virginia's dairy cattle.
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    Complete development of the porcine coccidium Isospora suis Biester, 1934 in cell cultures
    Lindsay, David S.; Quick, D. P.; Steger, A. M.; Toivio-Kinnucan, M. A.; Blagburn, B. L. (American Society of Parasitology, 1998-06)
    Development from inoculated sporozoites to unsporulated oocysts of Isospora suis Biester, 1934 is described in a swine testicular (ST) cell line. Sporozoites penetrated ST cells within 1 hr postinoculation (PI). Development was initially by endodyogeny to produce binucleate type I meronts and type I merozoites. Division by endodyogeny continued during the 13-day observation period and type I merozoites were the developmental stages most abundant at observation periods >3 days PI. Mutinucleate type II meronts and type II merozoites were first observed 7 days PI. Gamonts and oocysts were present 12 days PI. Oocysts did not sporulate in vitro. The ultrastructural features of stages were similar to those that occur in the pig host.
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    Confirmation of Sarcocystis jamaicensis Sarcocysts in IFN-γ Gene Knockout Mice Orally Inoculated With Sporocysts From a Red-Tailed Hawk (Buteo jamaicensis)
    Dubey, Jitender P.; Cerqueira-Cezar, Camila K.; Murata, Fernando H. A.; Mowery, J. D.; Scott, D.; von Dohlen, Alexa Rosypal; Lindsay, David S. (2019-02-22)
    Here, we report confirmation of sarcocysts of Sarcocystis jamaicensis in an experimental intermediate host, IFN-gamma gene knockout (KO) mice orally inoculated sporocysts from its natural definitive host, a red-tailed hawk (Buteo jamaicensis) (RTH). A RTH submitted to the Carolina Raptor Center, Huntersville, North Carolina, was euthanized because it could not be rehabilitated and released. Fully sporulated sporocysts from intestinal scrapings of the RTH were orally fed to 2 laboratory-reared outbred Swiss Webster mice (SW; Mus musculus) and to 2 KO mice. The sporocysts were infective for KO mice but not to SW mice. Both SW mice remained asymptomatic, and neither schizonts nor sarcocysts were found in their tissues when euthanized on day 54 post-inoculation (PI). The KO mice developed neurological signs and were necropsied 38-54 days PI. Schizonts/merozoites were found in both KO mice euthanized and they were confined to the brain. The predominant lesion was meningoencephalitis. Microscopic sarcocysts were found in muscles of both KO mice. When viewed with light microscopy, the sarcocyst wall appeared thin (<1 mu m thick) and smooth. Ultrastructural details of sarcocysts are described.
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    Congenital infection of mice with toxoplasma gondii induces minimal change in behavior and no change in neurotransmitter concentrations
    Goodwin, David G.; Hrubec, Terry C.; Klein, Bradley G.; Strobl, Jeannine S.; Werre, Stephen R.; Han, Qian; Zajac, Anne M.; Lindsay, David S. (American Society of Parasitology, 2012-08-01)
    We examined the effect of maternal Toxoplasma gondii infection on behavior and the neurotransmitter concentrations of congenitally infected CD-I mice at 4 and 8 wk of age when latent tissue cysts would be present in their brains. Because of sex-associated behavioral changes that develop during aging, infected female mice were compared with control females and infected male mice were compared with control males. Only the short memory behavior (distance between goal box and first hole investigated) of male mice congenitally infected with T. gondii was significantly different (P < 0.05) from that of uninfected control males at both 4 and 8 wk by using the Barnes maze test. The other parameters examined in the latter test, i.e., functional observational battery tests, virtual cliff, visual placement, and activity tests, were not significantly different (P > 0.05) at 4 and 8 wk. Concentrations of neurotransmitters and their metabolites (dopamine; 3,4-dihydroxyphenylacetic acid; homovanillic acid; norepinephrine; epinephrine; 3-methoxy-4-hydroxyphenylglycol; serotonin; and 5-hydroxyindoleacetic acid) in the frontal cortex and striatum were not different (P > 0.05) between infected and control mice at 8 wk of age. The exact mechanism for the observed effect on short-term memory in male mice is not known, and further investigation may help elucidate the molecular mechanisms associated with the proposed link between behavioral changes and T gondil infection in animals. We were not able, however, to confirm the widely held belief that changes in neurotransmitters result from chronic T. gondii infection of the brain.