Browsing by Author "Zajac, Anne M."

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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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    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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    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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    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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    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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    Characterizing physiological and genetic differences in the early immune response to Haemonchus contortus in resistant and susceptible sheep
    Bowdridge, Scott Alexander (Virginia Tech, 2009-10-12)
    This dissertation compares immune responses of resistant and susceptible sheep to infection with Haemonchus contortus during the peri-parturient period and larval stage of infection. Identification of immunological events resulting in parasite resistance in St. Croix hair sheep may provide better targets for differential gene expression analysis and eventual discovery of selectable markers for parasite resistance. Antibody levels of hair ewes and composite Dorset x Finnsheep-Rambouillet wool ewes were measured during breeding and again after parturition. Results demonstrated that hair ewes had higher levels immunoglobulin-A after infection and maintained a higher level of circulating antigen-specific antibody when compared to wool ewes. To characterize immune responses to the larval stage of infection, hair and wool lambs were sacrificed at 0, 3, 5, and 7 d after infection with H. contortus. Neutrophil migration to abomasal mucosa and lymph node development were higher in hair sheep than in wool sheep. Gene expression analysis indicated no difference in the abomasal lymph node as both breeds expressed a general T-helper cell type 2 (TH2) response. However, profound differences in TH2 responses were observed in the abomasal mucosa, where hair sheep expressed more IL-4, -13 and -33 than wool sheep. These data thus document the presence of immunological differences between the breeds. Immune responses to larval parasite infection in wool sheep are generally suppressed and may increase the magnitude and duration of infection whereas immune responses to larval infection in hair sheep was more robust and more strongly polarized towards a TH2
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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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    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.
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    Control of Internal Parasites in Sheep
    Whittier, W. Dee; Zajac, Anne M.; Umberger, Steven H. (Virginia Cooperative Extension, 2009)
    Internal parasites are a threat to sheep health and productivity. Increased understanding of the role and actions of internal parasites provides the basis for more effective control programs.
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    Cystoisospora canis Nemeseri, 1959 (Syn. Isospora canis), infections in dogs: Clinical signs, pathogenesis, and reproducible clinical disease in beagle dogs fed oocysts
    Mitchell, S. M.; Zajac, Anne M.; Charles, S.; Duncan, R. B.; Lindsay, David S. (American Society of Parasitology, 2007-04)
    Canine intestinal coccidiosis is a cause of diarrhea in young dogs and dogs that are immunocompromised. Reports in the literature indicate that experimental reproduction of clinical coccidiosis with Cystoisospora canis (syn. Isospora canis) is difficult, and few studies have been done with C canis. Experimental oral infections were attempted in 22, 6- to 8-wk-old female beagles with 5 x 10(4) (n = 2) or 1 x 10(5) (n = 20) sporulated C. canis oocysts. Diarrhea was observed in all inoculated dogs. Diarrhea began 2-3 days before oocyst excretion. Five of the 22 dogs were given an anticoccidial (sulfadimethoxine) because of their clinical signs. The mean prepatent period was 9.8 days (range, 9-11 days, n = 22 dogs), and the patent period was 8.9 days (range, 7-18 days, n = 20 dogs). Two dogs exhibiting clinical coccidiosis were examined at necropsy 10 days after infection. Developmental stages of C canis were present in cells in the lamina propria throughout the entire small intestine in both dogs. Microscopic lesions observed in both of these dogs were villous atrophy, dilation of lacteals, and hyperplasia of lymph nodes in Peyer's patches. Results of bacterial and viral examinations of these 2 dogs were negative, indicating that intestinal coccidiosis was the cause of the diarrhea. Our study indicates that C. canis can be a primary cause of diarrhea in young dogs.
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    Detecting Giardia: Clinical and Molecular Identification
    Saleh, Meriam Naim (Virginia Tech, 2017-11-13)
    The protozoan parasite Giardia duodenalis (syn. G. lamblia, G. intestinalis) can cause diarrhea in humans, cats, dogs and other animals. Giardia duodenalis consists of eight assemblages (A-H) that are morphologically identical but genetically distinct. Assemblages C-H are generally species-specific, while A and B infect people and animals and are considered potentially zoonotic. Most canine and feline isolates belong to their respective species-specific assemblages, but isolates of assemblages A and B (predominantly found in humans) have also been recovered from dogs and cats. Diagnosis of infection has historically been by morphologic techniques (observing trophozoites on direct fecal smears or cysts on centrifugal zinc sulfate fecal flotations), and it is currently recommended to use morphologic techniques in conjunction with a sensitive and specific antigen test. Diagnosis is important for management of clinical giardiasis in cats and dogs and also to identify the assemblage present to determine its zoonotic potential. In my dissertation research I evaluated diagnostic techniques in use for companion animals, including centrifugal zinc sulfate fecal flotation, antigen tests optimized for use in dogs and cats, direct immunofluorescent assay (IFA), and Polymerase Chain Reaction (PCR). I showed that when compared to the reference IFA the veterinary optimized antigen tests performed similarly and had no statistically significant differences in sensitivity or specificity when combined with a centrifugal zinc sulfate fecal flotation test. Sensitivity and specificity by comparison to IFA was ≥ 82% and ≥ 90%, respectively, for all diagnostic tests evaluated in dogs and cats. When analyzed via Bayesian analysis sensitivity and specificity for all diagnostic tests was ≥83% and ≥95%, respectively. The Bayesian analysis also showed that using the direct immunofluorescent assay (IFA) as the reference test was supported. I also evaluated PCR as a molecular diagnostic technique to detect Giardia infections in dogs with soft stool or diarrhea (mimicking clinical signs of infection). I utilized both conventional and real time PCR assays and compared the results to the recommended method of diagnosis, the zinc sulfate fecal flotation combined with an immunoassay test. I found that agreement between PCR and microscopy combined with an immunoassay was poor to fair and varied depending on the molecular parameters and size of the DNA target underscoring the complexity of test evaluation and molecular diagnostics for Giardia. I also evaluated cats from a varied population (owned, shelter, feral) in Virginia to determine to what extent (if any) they were infected with potentially zoonotic assemblages of Giardia. The species-specific assemblage F was detected in 57% of the samples and assemblage A, which is considered potentially zoonotic, was recovered from 32% of the sampleI. In 11% both assemblages F and A were detected. We showed for the first time that cats in Virginia are infected with potentially zoonotic assemblages of Giardia.
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    Development and ultrastructure of cystoisospora canis nemeseri, 1959 (syn. isospora canis) monozoic cysts in two noncanine cell lines
    Mitchell, S. M.; Zajac, Anne M.; Lindsay, David S. (American Society of Parasitology, 2009-08)
    Cystoisospora canis is a coccidial parasite of the intestinal tract that can cause severe disease in dogs. Clinical Signs include watery diarrhea, vomiting, fever, and weight loss. Extraintestinal stages of Cystoisospora spp. have been demonstrated in the mesenteric lymph nodes of paratenic hosts. information on the biology of extraintestinal stages of canine Cystoisospora species is limited. The Current study examined the development of C canis in 2 noncanine cell lines and the ultrastructure of the monozoic cysts that formed. Monolayers of bovine turbinate cells and African green monkey kidney cells were grown on coverslips and inoculated with excysted C canis sporozoites. Coverslips were collected oil various days and fixed and stained for light microscopy (LM) or transmission electron microscopy (TEM). A single, centrally located, slightly crescent-shaped sporozoite Surrounded by a thick cyst wall within a parasitophorous vacuole was observed with the use of LM and TEM. No division and no multinucleated stages were observed with either LM or TEM. With TEM, typical organelles of sporozoites were observed, such as rhoptries, dense granules, a crystalloid body, polysaccharide granules, and a conoid. The structure and ultrastructure of C. canis monozoic cysts produced in vitro are similar to extraintestinal cysts of other Cystoisospora species in experimentally infected animals and those of Cystoisospora belli observed in immunocompromised humans. This is the first Study that fully demonstrates in vitro the development of what structurally resemble extraintestinal cysts or a Cystoisospora spp.
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    Development of a Canine Coccidiosis Model and the Anticoccidial Effects of a New Chemotherapeutic Agent
    Mitchell, Sheila (Virginia Tech, 2008-04-11)
    Coccidia are obligate intracellular parasites belonging to the phylum Apicomplexa. Many coccidia are of medical and veterinary importance such as Cystoisospora species and Toxoplasma gondii. The need to discover new anticoccidial therapies has increased due to development of resistance by the parasite or toxicity issues in the patient. The goals of this work were to develop a model for canine coccidiosis while proving that Cystoisospora canis is a true primary pathogen in dogs and to determine the efficacy of a new anticoccidial agent. A canine coccidiosis model would be useful in evaluating new anticoccidial treatments. Oral infections with 5 X 104 (n=2) and 1 X 105 (n=20) sporulated C. canis oocysts were attempted in 22 purpose bred beagle puppies. Clinical signs associated with disease were observed in all dogs. Bacterial and viral pathogens were ruled out by transmission electron microscopy (TEM) and bacterial growth assays. Development of C. canis in cell culture was also evaluated. The efficacy of ponazuril, a new anticoccidial drug, was examined in T. gondii. In vitro studies were conducted to determine the activity of ponazuril on tachyzoites and how this agent affects development of apicomplexcan parasites. The tachyzoite production assay was conducted. Ponazuril at a dose of 1.0 µg/ml had a significant affect on tachyzoite reproduction. Comparisons were made on how ponazuril affects T. gondii and Neospora caninum. Inhibition of T. gondii tachyzoites occurred after the second round of replication and with N. caninum tachyzoites after 4 rounds of replication. Results of TEM revealed ponazuril affects replication of T. gondii and N. caninum differently. The efficacy of ponazuril to prevent and treat acute and chronic toxoplasmosis was investigated. Mice treated prophylactically with ponazuril were completely protected from developing an acute T. gondii infection. Fatal toxoplasmosis was prevented in mice starting treatment 3 and 6 days post infection at a dose of 20 mg/kg. Immunohistochemistry was used to evaluate ponazuril's effect on chronic toxoplasmosis. Sections of brain were scored according to the number of tissue cysts present. Ponazuril also proved to be highly active against toxoplasmic encephalitis in an interferon-gamma knockout mouse model.
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    Development of an antigen-specific ELISPOT to detect intestinal antibody responses to the swine whipworm, Trichuris suis
    Kellman, Maxine Franchestcê (Virginia Tech, 1997-06-05)
    The swine whipworm, Trichuris suis, is a parasite present throughout the United States and is of concern to the swine industry worldwide because it is very pathogenic to growing pigs. The economic threat posed by T. suis and other intestinal parasite infections has created a strong interest in the development of parasite vaccines for the swine industry. Use of a vaccine either alone or with anthelmintics should reduce the economic losses. However, before effective parasite vaccines can be created, the swine gastrointestinal immune response to parasite antigens must be understood. In this study, an enzyme-linked immunospot (ELISPOT) assay was developed to measure total and antigen-specific IgG and IgA antibody secreting cells (ASC) from gut-associated lymphoid tissues (GALT) [mesenteric lymph node explants from jejunal region of small intestine (SI-MLN) and cecum in large intestine (C-MLN); and ileocecal Peyer's patches (IC-PP)] and lamina propria from the proximal colon removed from T. suis infected pigs. Tbe local antibody responses were compared to peripheral antibody responses found in the spleen and submandibular lymph nodes. The hypotheses to be tested was that parasite antigen-specific antibody secreting cells would be greatest in lymphoid tissue draining the site of infection compared to peripheral lymphoid tissues and that 19A ASC would predominate over IgG ASC in the lamina propria of T. suis infected pigs. The total IgG and IgA ASC frequencies for the spleen, SI-MLN, and ICPP did not significantly change (P> 0.05) over time. For C-MLN, there was a significant increase (p< 0.05) of total IgG ASC during a primary infection with T. suis. Antigen-specific IgG ASC were greatest at the GALT site closest to the infection, CMLN, whereas, antigen-specific IgA ASC predominated in the proximal colonic: lamina propria. Host protection to T. suis develops after anthelmintic: treatment of a primary exposure to parasite. The ELISPOT assay provided valuable information on the localization and compartmentalization of the swine gastrointestinal immune response to T. suis which resides in the cecum and proximal colon. In the future, this technique may be useful for monitoring gastrointestinal immune parameters of pigs exposed to a T. sllis vaccine.
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    Diet and Feeding Ecology of the Coyotes, Black Bears, and Bobcats in Western Virginia and Preliminary Assessment of Coyote Parasites
    Montague, David Miles (Virginia Tech, 2014-10-22)
    Although deer abundance is high throughout most areas of Virginia, parts of western Virginia, especially on public lands, have comparatively low density deer populations. Concerns voiced by sportsmen regarding declining deer numbers in this region prompted interest in research to investigate the role of predation on deer populations. The coyote (Canis latrans) is a relative newcomer to Virginia, and relatively little is known about the role coyotes play in Virginia ecosystem dynamics, including their interactions with other sympatric predators. Research studies in other areas suggest that predation from coyotes, bobcats (Lynx rufus), and black bears (Ursus americanus) may be a significant source of deer (Odocoileus virginianus) mortality, especially for fawns, and the cumulative effects of predation from these sources may also increase the potential for additive mortality in deer populations. An important consideration when comparing feeding strategies of carnivores is the relative availability of food items across the landscape. I estimated the relative seasonal availability of several potential food items in a mosaic landscape that included some areas of prescribed burning and variable timber harvest in the mountains of western Virginia between June 2011 and May 2013. I focused on 4 broad categories of food items: white-tailed deer, mid-sized mammals (raccoons, Procyon lotor, opossums, Didelphis virginiana, cottontails, Sylvilagus spp., and squirrels, Sciurus spp.), small mammals, and soft mast. I used distance sampling to estimate deer density, camera trapping techniques to estimate mid-sized mammal trapping rates and occupancy, mark-recapture techniques to estimate small mammal abundance, and vegetation sampling to estimate % cover, which I used as measures of food availability for predation. To estimate carnivore diet, I analyzed scats of coyotes (n = 334), bobcats (n = 258), and black bears (n = 107) collected monthly from June 2011 and May 2013. Additionally, I compared estimates of % occurrence to estimates of seasonal availability of deer, mid-sized mammals, small mammals, and soft mast by ranking availability of food items from 0 (unavailable) to 4 (highly available) from my monthly and seasonal food item abundance and density estimates. I then ranked % occurrence in scat on a 0 - 4 scale and compared diet rankings to food item availability where changes in rank indicate differences from the generalist diet. Deer densities were substantially higher in Bath County (4.75 - 16.06 deer/km²) than in Rockingham County (0.17 - 3.55 deer/km²). I estimated availability of other food items only in Bath County. For mid-sized mammals, I estimated low activity as shown by constant, but low, trapping rates (#photo events/trap nights*100) of opossums, and relatively higher, constant trapping rates of cottontails. Raccoon and squirrel trapping rates were highly variable across seasons and raccoon occupancy (proportion of sites occupied) was higher in summer and fall (0.51 - 0.59) whereas squirrel occupancy was highest in October-November (0.51 - 0.53). Cottontail and opossum occupancies were higher in burned areas (0.40 - 0.57, 0.24 - 0.46, respectively), whereas raccoon and gray squirrel occupancies were higher in unburned areas (0.23 - 0.78, 0.12 - 0.58, respectively). Using program MARK I estimated high abundance and density of Peromyscus spp. in all seasons relative to other small mammal species. Densities of meadow voles and jumping mice were high seasonally in open field habitat. Densities of eastern chipmunks, red-backed voles, and Peromyscus spp. were significantly higher in burned areas relative to unburned sites. Among soft mast species, blueberries comprised the largest % occurrence. I expect a generalist predator to consume deer at a constant rate and have lower % deer occurrence in Rockingham County where deer densities are much lower. I expect seasonally constant, but low, occurrence of opossum and relatively higher, but similarly constant, occurrence of cottontail. I expect raccoon occurrence to be relatively high in summer and fall, and lower in winter. Squirrel occurrence should peak in October and November, followed by a steady decline until June. Based on availability, Peromyscus spp. should be the most commonly occurring small mammal in the diet of a generalist predator, rivaled by meadow voles (Clethrionomys gapperi) in fall and perhaps summer. I expect chipmunks (Tamias striatus) and red-backed voles to occur at constant but low rates in predator diets. I predict that various species of soft mast may occur during their respective fruiting seasons. Deer were the most frequently occurring food item of coyotes in all months in both counties, with % biomass consumed highest in June (Bath: 60.0 - 90.9 %; Rockingham: 55.6 - 92.3%). Deer occurrence in bobcat scat peaked in June (77.54 %). In black bear scat, deer occurrence was highest in February and March (100 %), however sample sizes in these months were extremely low (n = 2, 3 respectively). All 3 predators consumed deer more than expected in June and July. In most months, coyotes consumed deer and soft mast more often than expected (1 rank higher) and they consumed deer much higher (2-3 ranks higher) than expected in January and February. While deer was a major food item in the diets of all 3 predators, it is unclear whether this observed result was caused by predation or scavenging. It is unlikely that coyotes, bears, or bobcats are preying on adult deer with high frequency. I did not estimate the availability of deer carcasses for scavenging in this study. If deer carcasses are highly available in the study area, this may explain the high occurrence of deer in carnivore scat and the lack of seasonality in coyote samples. Mice (Peromyscus spp.) were consumed by predators less than expected based on availability, whereas squirrels and chipmunks were consumed by bobcats more than expected. Meadow and red-backed voles were consumed more than expected by both bobcats and coyotes. Mid-sized mammals occurred with expected frequency or lower in predator scats. Coyotes had the lowest diversity and variation in diet, whereas bobcat and bear diets were more diverse including seasonal use of many species. Coyotes exhibited more selectivity (based on changes, and magnitude of changes, in rankings for food item occurrence versus availability) and appear more specialized on deer in all months than bobcats. I analyzed 203 randomly-selected coyote fecal samples using a modified Wisconsin fecal flotation technique. I identified 13 parasite taxa, 9 of which occurred with adequate frequency to permit statistical analysis. No landscape patterns or statistically significant seasonal differences were found in prevalence of parasite taxa, and only A. caninum and Eimeria spp. were significantly higher, and Cystoisospora spp. was somewhat significantly higher, in Bath versus Rockingham Counties. By comparing my data on spatio-temporal patterns of food availability to patterns in the scats of predators, we have better understanding of predator foraging strategies and can identify opportunities for targeted management to balance predator-prey dynamics with human needs and values. More research is needed on the interactions of sympatric predators in specific areas to gain further inferences regarding population-level impacts of predation on deer in western Virginia.
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    Differential gene expression and immune regulatory mechanisms in parasite-resistant hair and susceptible wool sheep infected with the parasitic nematode, Haemonchus contortus
    MacKinnon, Kathryn Michelle (Virginia Tech, 2007-07-23)
    Among sheep producers, the parasitic nematode Haemonchus contortus is a major animal health concern. Caribbean hair sheep are more resistant than conventional wool breeds to this blood-feeding, abomasal parasite. Our objective was to determine differences in the immune response associated with parasite-resistant hair and susceptible wool lambs infected with 10,000 H. contortus and in uninfected controls. Animals were sacrificed and abomasum and lymph node tissues were collected at 3 or 27 days post-infection (PI), and for controls on day 17, 27, or 38 relative to d 0 of infected animals. Blood and fecal samples were collected throughout the study. Lower fecal egg counts, higher packed cell volumes, and heavier lymph nodes of infected hair compared to wool lambs, suggests hair lambs have increased parasite resistance. Greater tissue infiltration of eosinophils (P < 0.05) was observed in hair compared to wool sheep by 3 days PI, with no breed differences in globule leukocytes. Total serum IgA and IgE were greater in control hair versus wool sheep (P < 0.05). After 3, 5, and 21 of infection, total serum IgA (P< 0.05), total lymph node IgE (P < 0.01), but not total serum IgE were greater in hair sheep compared to wool sheep. Gene expression was measured between hair and wool lambs for abomasal and lymph node tissues using bovine cDNA microarrays and real-time RT-PCR. Microarray analysis revealed cell survival, endosome function, gut motility, and anti-coagulation pathways are important in abomasal and lymph node tissues during H. contortus infection. Immune genes, including IL-4, IL-4 Ra, IL-12 Rb1, and IL-12 Rb2, are also highly represented in abomasal or lymph node tissue of infected animals. Eleven genes were evaluated using real-time RT-PCR and included TH1 and TH2 cytokines, cytokine receptors, and IgE. Parasite infection leads to increased expression of IL-13 and IgE in both tissues and breeds when compared to control animals. Breed comparison of gene expression shows resistant hair sheep produce a stronger modified TH2-type immune response during infection. Differential cell infiltration, antibody production, and regulation of TH2 cytokines between breeds may be partially responsible for differences in parasite resistance.
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    Effect of hydrogen peroxide and other protease inhibitors on Cryptosporidium parvum excystation and in vitro development
    Kniel, Kalmia E.; Sumner, Susan S.; Pierson, Merle D.; Zajac, Anne M.; Hackney, Cameron Raj; Fayer, Ronald; Lindsay, David S. (American Society of Parasitology, 2004-08)
    This study was undertaken to observe the effects of hydrogen peroxide on Cryptosporidium parvum oocysts with respect to protease activity in comparison to known protease inhibitors. In assessing the possible mechanisms of action of hydrogen peroxide, treatment effectiveness was analyzed using 3 assays and the potential roles of proteases and cations were considered. Treatment of C. parvum oocysts with hydrogen peroxide inhibited protease activity up to 50% compared with untreated controls. Treatment of oocysts with chemicals that affect sulfhydryls, including N-ethylmaleimide and dithiolthreitol, inhibited protease activity by > 90%. Treatment of oocysts with these chemicals, along with the protease inhibitors, phenylmethylsulfonyl fluoride (PMSF), ethylenediamine-tetraacetic acid, and cystatin, inhibited protease activity as well as in vitro excystation and infection in a cell culture assay. Several mechanisms may result in the successful inhibition of infection and excystation by hydrogen peroxide treatment, including: oxidation of oocyst wall proteins or lipids, chelating of cations necessary for infection, or hydroxyl radical-induced DNA damage to sporozoites, or both.
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    Effects of high-pressure processing on in vitro infectivity of Encephalitozoon cuniculi
    Jordan, C. N.; Zajac, Anne M.; Holliman, D.; Flick, George J. Jr.; Lindsay, David S. (American Society of Parasitology, 2005-12)
    High-pressure processing (HPP) has been shown to be an effective means of eliminating bacteria and destructive enzymes from a variety of food products. HPP extends the shelf life of products while maintaining the sensory features of food and beverages. In this study, we examined the effects of HPP on the infectivity of Encephalitozoon cuniculi spores in vitro. Spores were exposed to between 140 and 550 MPa for 1 min in a commercial HPP unit. Following treatment, the spores were loaded onto cell culture flasks or were kept for examination by transmission electron microscopy. No effect was observed on the infectivity of spores treated with 140 MPa. Spores treated with between 200 and 275 MPa showed reduction in infectivity. Following treatment of 345 MPa or more, spores were unable to infect host cells. No morphologic changes were observed in pressure-treated spores with transmission electron microscopy.
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    The Effects of Naturally Occurring Plant Products on Experimental Haemonchus contortus Infection in Gerbils and Sheep
    Squires, Jill Marie (Virginia Tech, 2009-05-01)
    Haemonchus contortus is a blood-sucking abomasal helminth of small ruminants responsible for major economic losses to producers worldwide. Widespread resistance to commercial anthelmintics has created a need for alternative methods of parasite control. One method involves using plant products with natural anthelmintic properties. This thesis assessed the efficacy of several plant products against experimental Haemonchus contortus infection in gerbils and sheep. In gerbil assays, animals were orally infected with 600 third-stage Haemonchus larvae and treated once or daily for 5 days with artemisinin, Artemisia annua aqueous or ethanolic extract, an orange oil emulsion, or Asimina triloba ethanolic extract. Nine days post-infection, gerbils were euthanized, their stomachs removed, and the worms counted. Significant anthelmintic activity was not found for artemisinin, A. annua extracts, or A. triloba extract. The orange oil product caused significant parasite reductions up to 87.8% when administered for 5 days. The orange oil emulsion was tested in sheep to evaluate the product against Haemonchus in its natural host. Sheep were orally inoculated with 10,000 Haemonchus larvae and, one month later, dosed with the emulsion once or daily for 3 days. Fecal egg counts were monitored daily starting on the first day of dosing and continuing to 14 days post-dosing. Results showed that a single dose of the product caused highly significant fecal egg count reduction (97.4%) compared to control sheep and that there is no advantage to treating for 3 days. Thus, the orange oil emulsion shows promise as an alternative to commercial dewormers.