Browsing by Author "Zimmerman, Kurt L."

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    The ability of TLR agonists to upregulate Brucella abortus strain RB51 mediated protection in a murine respiratory model
    Walker, Michelle Kay (Virginia Tech, 2014-01-23)
    Brucella abortus is amongst the top 5 zoonotic diseases worldwide. The overall goal of this research is to generate a safe and effective vaccine for humans. Brucella abortus strain RB51, approved for use in cattle, provides protection by initiating a strong T-helper 1 (Th1) type response is a candidate vaccine. Based on a model for aerosol exposure mice were vaccinated intranasally (IN) with strain RB51 and challenged IN with B. abortus strain 2308, strain RB51 did not protect. Protection against Brucella is mediated through TLRs 2, 4 and 9. The addition of TLR 2 or TLR 4 and a trend with TLR9 agonists with intranasal RB51 vaccination significantly increased bacterial clearance in the lung after strain 2308 challenge. Therefore, we hypothesized that combining TLR agonists 2, 4, and 9 with strain RB51 IN would upregulate protection and clearance in the lung against strain 2308 challenge (IN), by upregulating the DC1 and CD4 Th1 and CD8 immune response. This study showed that protection is not upregulated by combining all TLR agonists. Overall the addition of TLR 2 and 4 vs. TLR 2, 4 and 9 agonists affects the immune response and impacts the level of clearance. Our data support the development of a DC1 Th1 CD8 response, based on serology, and both DC and T-cell activation and function by the group which received the TLR 2 and 4 agonists and to a lesser degree the group receiving TLR 2, 4, and 9 agonists. Additional studies are warranted to further define the differential mechanisms and endpoints of protection.
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    Adrenocortical Challenge Response and Genomic Analyses in Scottish Terriers With Increased Alkaline Phosphate Activity
    Zimmerman, Kurt L.; Panciera, David L.; Hoeschele, Ina; Monroe, William E.; Todd, S. Michelle; Werre, Stephen R.; LeRoith, Tanya; Fecteau, Kellie; Lake, Bathilda B. (Frontiers, 2018-10-09)
    Scottish terriers (ST) frequently have increased serum alkaline phosphatase (ALP) of the steroid isoform. Many of these also have high serum concentrations of adrenal sex steroids. The study’s objective was to determine the cause of increased sex steroids in ST with increased ALP. Adrenal gland suppression and stimulation were compared by low dose dexamethasone (LDDS), human chorionic gonadotropin (HCG) and adrenocorticotropic hormone (ACTH) response tests. Resting plasma pituitary hormones were measured. Steroidogenesis-related mRNA expression was evaluated in six ST with increased ALP, eight dogs of other breeds with pituitary-dependent hyperadrenocorticism (HAC), and seven normal dogs. The genome-wide association of single nucleotide polymorphisms (SNP) with ALP activity was evaluated in 168 ST. ALP (reference interval 8–70 U/L) was high in all ST (1,054 U/L) and HAC (985 U/L) dogs. All HAC dogs and 2/8 ST had increased cortisol post-ACTH administration. All ST and 2/7 Normal dogs had increased sex steroids post-ACTH. ST and Normal dogs had similar post-challenge adrenal steroid profiles following LDDS and HCG. Surprisingly, mRNA of hydroxysteroid 17-beta dehydrogenase 2 (HSD17B2) was lower in ST and Normal dogs than HAC. HSD17B2 facilities metabolism of sex steroids. A SNP region was identified on chromosome 5 in proximity to HSD17B2 that correlated with increased serum ALP. ST in this study with increased ALP had a normal pituitary-adrenal axis in relationship to glucocorticoids and luteinizing hormone.We speculate the identified SNP and HSD17B2 gene may have a role in the pathogenesis of elevated sex steroids and ALP in ST.
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    Cardiac Biomarkers in Hyperthyroid Cats
    Sangster, Jodi Kirsten (Virginia Tech, 2013-04-03)
    Background: Hyperthyroidism has substantial effects on the circulatory system. The cardiac biomarkers NT-proBNP and troponin I (cTNI) have proven useful in identifying cats with myocardial disease but have not been as extensively investigated in hyperthyroidism. Hypothesis: Plasma NT-proBNP and cTNI concentrations are higher in cats with primary cardiac disease than in cats with hyperthyroidism and higher in cats with hyperthyroidism than in healthy control cats. Animals: Twenty-three hyperthyroid cats, 19 cats with HCM without congestive heart failure, and 19 euthyroid, normotensive healthy cats eight years of age or older. Fourteen of the hyperthyroid cats were re-evaluated three months after administration of 131I. Methods: A complete history, physical examination, complete blood count, serum biochemistries, urinalysis, blood pressure measurement, serum T4 concentration, plasma concentrations of NT-proBNP and cTNI, and echocardiogram was prospectively obtained from each cat. Results: Hyperthyroid and HCM cats had plasma NT-proBNP and cTNI concentrations that were significantly greater than healthy older cats, but there was no significant difference between hyperthyroid and HCM cats with respect to concentration of either biomarker. In hyperthyroid cats that were re-evaluated three months after 131I treatment, plasma NT-proBNP and cTNI concentrations as well as ventricular wall thickness decreased. Conclusions and Clinical Relevance: Although there may be a role for NT-proBNP in monitoring the cardiac response to treatment of hyperthyroidism, neither NT-proBNP nor cTNI can be used to distinguish hyperthyroid cats from cats with HCM. Therefore, the thyroid status of older cats should be ascertained prior to interpreting results of cardiac biomarker testing.
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    Comparative efficacy of three common treatments for equine recurrent airway obstruction
    Lee, Laura Caryn (Virginia Tech, 2009-04-17)
    Objective - evaluate horses with acute airway obstruction using three treatment regimens: tapering doses of dexamethasone (DEX), environmental modification (ENV), and a combination of both treatments (DEX + ENV) by analyzing clinical parameters, pulmonary function testing, bronchoalveolar lavage fluid (BALF) cytology and BALF cell expression of the cytokines IFN-? and IL-4 Animals - 6 horses with recurrent airway obstruction (RAO) Procedures - Clinical examination, pulmonary function test, and collection of BALF prior to treatment and during 22 day treatment period Hypothesis - Alterations in clinical parameters, pulmonary function and airway inflammation in acute equine RAO will return to remission values by treating with DEX, ENV or DEX + ENV Results - All horses demonstrated clinical disease, reduced pulmonary dynamic compliance (Cdyn) and an increased maximum change in pleural pressures (?Pplmax) when in a challenge environment. All treatments improved clinical parameters, ?Pplmax and Cdyn. BALF cytology during an RAO crisis demonstrated neutrophilic inflammation. ENV or DEX + ENV resulted in a significant decrease in airway neutrophilia that was maintained throughout the treatment period. In contrast, treatment with DEX caused a reduction in airway neutrophilia initially followed by a rebound neutrophilia as the period between administrations of dexamethasone (0.05mg/kg) was increased to 72 hours. The rebound neutrophilia was not accompanied by equivalent deterioration in clinical parameters or pulmonary function. Conclusions - Environmental modification is important in the management of RAO horses. Treatment of clinical RAO with a decreasing dosage protocol of corticosteroids in the absence of environmental modification results in the persistence of airway inflammation without recrudescence of clinical disease.
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    A Comparison of Liver Biopsy Techniques in Dogs
    Kemp, Stephanie D. (Virginia Tech, 2013-05-15)
    Background: The liver biopsy technique in dogs that consistently provides samples adequate for accurate histopathologic interpretation and the variability of histopathology among lobes is unknown. Hypothesis:  Liver biopsy specimens obtained via punch, cup biopsy, and 14 gauge needle biopsy would result in similar histopathologic diagnoses to those found on deeply sectioned samples of liver obtained at necropsy and that discordant results would not differ between lobes.   Animals:  Seventy dogs undergoing necropsy. Methods: Liver specimens were obtained from the left lateral liver lobe with an 8 mm punch, a cup, and a 14 gauge needle. Two larger tissue samples were then collected near the center of the left lateral lobe and used as a histologic standard for comparison.  Samples were also obtained from all remaining lobes. Histopathologic features and numbers of portal triads in each sample were compared. Results: The mean number of portal triads were 2.9 in needle biopsies, 3.4 in cup biopsies, 12 in punch biopsies, and 30.7 in the necropsy samples. Sixty-six percent of needle biopsies,  60% of laparoscopic cup samples, and 69% of punch samples were in agreement with the necropsy samples, differences that were not significantly different. The corresponding kappa coefficient were 0.59 for needle biopsies, 0.52 for cup biopsies, and 0.62 for punch biopsies.  Discordant results did not differ between the liver lobes. Conclusions and Clinical Relevance:  A single biopsy using any of the tested techniques is insufficient for reliable diagnosis of liver disease in the dog.  Multiple biopsies from 2 lobes is recommended.
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    A Comparison of Liver Sampling Techniques in Dogs
    Kemp, Stephanie D.; Zimmerman, Kurt L.; Panciera, David L.; Monroe, William E.; Leib, Michael S.; Lanz, Otto I. (American College of Veterinary Internal Medicine, 2015-01)
    Background: The liver sampling technique in dogs that consistently provides samples adequate for accurate histopathologic interpretation is not known. Hypothesis/ Objectives: To compare histopathologic results of liver samples obtained by punch, cup, and 14 gauge needle to large wedge samples collected at necropsy. Animals: Seventy dogs undergoing necropsy. Methods: Prospective study. Liver specimens were obtained from the left lateral liver lobe with an 8 mm punch, a 5 mm cup, and a 14 gauge needle. After sample acquisition, two larger tissue samples were collected near the center of the left lateral lobe to be used as a histologic standard for comparison. Histopathologic features and numbers of portal triads in each sample were recorded. Results: The mean number of portal triads obtained by each sampling method were 2.9 in needle samples, 3.4 in cup samples, 12 in punch samples, and 30.7 in the necropsy samples. The diagnoses in 66% of needle samples, 60% of cup samples, and 69% of punch samples were in agreement with the necropsy samples, and these proportions were not significantly different from each other. The corresponding kappa coefficients were 0.59 for needle biopsies, 0.52 for cup biopsies, and 0.62 for punch biopsies. Conclusion and Clinical Importance: The histopathologic interpretation of a liver sample in the dog is unlikely to vary if the liver biopsy specimen contains at least 3- 12 portal triads. However, in comparison large necropsy samples, the accuracy of all tested methods was relatively low.
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    Data Standardization and Machine Learning Models for Histopathology
    Awaysheh, Abdullah Mamdouh (Virginia Tech, 2017-03-27)
    Machine learning can provide insight and support for a variety of decisions. In some areas of medicine, decision-support models are capable of assisting healthcare practitioners in making accurate diagnoses. In this work we explored the application of these techniques to distinguish between two diseases in veterinary medicine; inflammatory bowel disease (IBD) and alimentary lymphoma (ALA). Both disorders are common gastrointestinal (GI) diseases in humans and animals that share very similar clinical and pathological outcomes. Because of these similarities, distinguishing between these two diseases can sometimes be challenging. In order to identify patterns that may help with this differentiation, we retrospectively mined medical records from dogs and cats with histopathologically diagnosed GI diseases. Since the pathology report is the key conveyer of this information in the medical records, our first study focused on its information structure. Other groups have had a similar interest. In 2008, to help insure consistent reporting, the World Small Animal Veterinary Association (WSAVA) GI International Standardization Group proposed standards for recording histopathological findings (HF) from GI biopsy samples. In our work, we extend WSAVA efforts and propose an information model (composed of information structure and terminology mapped to the Systematized Nomenclature of Medicine - Clinical Terms) to be used when recording histopathological diagnoses (HDX, one or more HF from one or more tissues). Next, our aim was to identify free-text HF not currently expressed in the WSAVA format that may provide evidence for distinguishing between IBD and ALA in cats. As part of this work, we hypothesized that WSAVA-based structured reports would have higher classification accuracy of GI disorders in comparison to use of unstructured free-text format. We trained machine learning models in 60 structured, and independently, 60 unstructured reports. Results show that unstructured information-based models using two machine learning algorithms achieved higher accuracy in predicting the diagnosis when compared to the structured information-based models, and some novel free-text features were identified for possible inclusion in the WSAVA-reports. In our third study, we tested the use of machine learning algorithms to differentiate between IBD and ALA using complete blood count and serum chemistry data. Three models (using naïve Bayes, neural networks, and C4.5 decision trees) were trained and tested on laboratory results for 40 Normal, 40 IBD, and 40 ALA cats. Diagnostic models achieved classification sensitivity ranging between 63% and 71% with naïve Bayes and neural networks being superior. These models can provide another non-invasive diagnostic tool to assist with differentiating between IBD and ALA, and between diseased and non-diseased cats. We believe that relying on our information model for histopathological reporting can lead to a more complete, consistent, and computable knowledgebase in which machine learning algorithms can more efficiently identify these and other disease patterns.
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    Development of an ontology of animals in context within the OBO Foundry framework from a SNOMED-CT extension and subset
    Santamaria, Suzanne Lamar (Virginia Tech, 2012-04-25)
    Animal classification needs vary by use and application. The Linnaean taxonomy is an important animal classification scheme but does not portray key animal identifying information like sex, age group, physiologic stage, living environment and role in production systems such as farms. Ontologies are created and used for defining, organizing and classifying information in a domain to enable learning and sharing of information. This work develops an ontology of animal classes that form the basis for communication of animal identifying information among animal managers, medical professionals caring for animals and biomedical researchers involved in disciplines as diverse as wildlife ecology and dairy science. The Animals in Context Ontology (ACO) was created from an extension and subset of the Systematized Nomenclature of Medicine — Clinical Terms (SNOMED-CT). The principles of the Open Biological and Biomedical Ontologies (OBO) Foundry were followed and freely available tools were used. ACO includes normal development and physiologic animal classes as well animal classes where humans have assigned the animal's role. ACO is interoperable with and includes classes from other OBO Foundry ontologies such as the Gene Ontology (GO). Meeting many of the OBO Foundry principles was straightforward but difficulties were encountered with missing and problematic content in some of the OBO ontologies. Additions and corrections were submitted to four ontologies. Some information in ACO could not be represented formally because of inconsistency in husbandry practices. ACO classes are of interest to science, medicine and agriculture, and can connect information between animal and human systems to enable knowledge discovery.
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    Diagnostic accuracy of stereotactic brain biopsy for intracranial neoplasia in dogs: Comparison of biopsy, surgical resection, and necropsy specimens
    Kani, Yukitaka; Cecere, Thomas E.; Lahmers, Kevin K.; LeRoith, Tanya; Zimmerman, Kurt L.; Isom, Scott; Hsu, Fang-Chi; Debinski, Waldemar; Robertson, John L.; Rossmeisl, John H. Jr. (American College of Veterinary Internal Medicine, 2019-05)
    Background Stereotactic brain biopsy (SBB) is a technique that allows for definitive diagnosis of brain lesions. Little information is available regarding the diagnostic utility of SBB in dogs with intracranial diseases. Objective To investigate the diagnostic accuracy (DA) of SBB in dogs with brain tumors. Animals Thirty-one client-owned dogs that underwent SBB followed by surgical resection or necropsy examinations. Methods Retrospective observational study. Two pathologists blinded to SBB and reference standard diagnoses reviewed histologic specimens and typed and graded tumors according to World Health Organization and revised canine glioma classification criteria. Agreement between tumor type and grade from SBB were compared to reference standards and assessed using kappa statistics. Patient and technical factors associated with agreement also were examined. Results Stereotactic brain biopsy specimens were obtained from 24 dogs with gliomas and 7 with meningiomas. Tumor type agreement between SBB and the reference standard was observed in 30/31 cases (kappa = 0.95). Diagnostic concordance was perfect for meningiomas. Grade agreement among gliomas was observed in 18/23 cases (kappa = 0.47). Stereotactic brain biopsy underrepresented the reference standard glioma grade in cases with disagreement. The DA of SBB was 81%, with agreement noted in 56/69 biopsy samples. Smaller tumors and fewer SBB specimens obtained were significantly associated with diagnostic discordance. Conclusions and Clinical Importance The DA of SBB readily allows for the diagnosis of common brain tumors in dogs. Although glioma grade discordance was frequent, diagnoses obtained from SBB are sufficient to currently inform therapeutic decisions. Multiple SBB specimens should be collected to maximize DA.
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    Evaluation of cerebrospinal fluid biomarkers of endothelial damage and basement membrane degradation as indirect indicators of blood-brain barrier dysfunction in chronic canine hypothyroidism
    Pancotto, Theresa E. (Virginia Tech, 2011-02-24)
    A variety of neurologic illnesses including peripheral and cranial neuropathies, central vestibular disease, seizures and coma have been associated with hypothyroidism in dogs. Repeated studies have shown that there is loss of blood brain barrier (BBB) integrity in these animals. Current research has also shown the development cerebrospinal fluid abnormalities in neurologically normal hypothyroid dogs; a finding that is related to BBB degradation. This derangement may be secondary to atherosclerosis and vascular accidents. One possible mediator of vasospasm and ischemic brain injury is endothelin-1 (ET-1). Another group of mediators of vascular dysfunction that has been found in CSF of dogs with various other CNS diseases is matrix metalloproteinases (MMP). The purpose of this study was to assay molecular markers that may contribute to disruption in the blood brain barrier in chronically hypothyroid canines. We hypothesized that BBB disruption in hypothyroidism is mediated by ET-1 and MMPs, as evidenced by increased concentrations of these proteins in CSF compared to controls. Cerebrospinal fluid (CSF) previously collected from 9 control and 9 experimentally induced hypothyroid dogs was used. Administration of I-131 was used to create the experimental model. CSF from time points 0, 6, 12, and 18 months post-induction were evaluated using an ELISA kit for endothelin-1. CSF from each time point was also evaluated using gelatinase zymography to detect MMP-2,9, and 14. The endothelin assay was able to detect ET-1 in CSF as determined by a spike and recovery method. However, ET-1 was undetectable in CSF of control and hypothyroid dogs at all time points. Constitutively expressed MMP-2 was detectable in all dogs at all time points. No other MMPs were detectable in CSF. ET-1 and gelatinase MMP,-9, and -14 are not primary mediators of BBB damage in chronically hypothyroid dogs. They could be involved secondarily and may be better evaluated with different assays or in temporal association with the development of clinical signs of neurologic dysfunction. Additional research is needed to confirm this finding and to evaluate biomarkers of non-vascular components of the BBB.
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    Evaluation of Sex Differences in the Hippocampus and Pituitary of Egr1 conditional knockout mice mediated by Nestin-Cre
    Swilley, Cody Lynn (Virginia Tech, 2023-08-29)
    Early growth response 1 (Egr1) is a transcription factor critical for learning and memory in the hippocampus and pituitary cell differentiation. Egr1 has been shown to extend continuation of the long-term potentiation in the hippocampus and is credited for forming long-term memories. The somatotrophs in the pituitary produce growth hormone and are found to be decreased in Egr1KO mice. These animals are also found to be sterile due to a decrease in LHB, which blocks ovulation. All previous studies have evaluated these physiological processes with complete Egr1KO research strains or antisense oligonucleotides, up until now, no data specific to individual type of cells has been generated. In an attempt to focus on the understanding of the functions of Egr1 gene in neural cell lineage, we are using an Egr1cKO Nestin-Cre model. Nestin allows for targeting neuronal lineage specific cells. In Chapter 1, we provide a systemic view of Egr1 gene and Nestin-Cre as a system for generating conditional knockout mouse strains. The Chapter begins with the identification of Egr1 gene and its protein structure, then proceeds to grasp its link to memory with behavior testing. The critical role of Egr1 in the pituitary and what cell populations are affected is also described. The same goes for Nestin-Cre, along with its limitations and understanding how to account for them in a study. The Egr1cKO Nestin-Cre system is the best form to understand neurological cell populations with Egr1 removal. In Chapter 2 and Chapter 3, we employ the Egr1cKO Nestin-Cre mouse model to understand cell-specific knockout of Egr1 in the nervous system by evaluating the hippocampus and pituitary. We explore learning and memory through behavioral tests and ribonucleic acid sequencing (RNA-seq) analysis to understand gene expression changes with Egr1 removal. Females showed higher activity during behavior tests, with more movement in the elevated plus maze and lower freezing times during the contextual fear conditioning. RNA-seq had higher changes in females than males but was not affected by the Nestin-Cre system overall. The same RNA-seq changes in the pituitary gland were present, with females having higher genomic differentiation. Females had growth-specific pathways altered by Nestin-Cre.
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    Extending Snomed to Include Explanatory Reasoning
    Zimmerman, Kurt L. (Virginia Tech, 2003-12-01)
    The field of medical informatics comprises many subdisciplines, united by a common interest in the establishment of standards to facilitate the sharing, reuse, and understanding of information. This work depends in large part on the ability of controlled medical terminologies to represent relevant concepts. This work augments a controlled terminology to provide not only standardized content, but also standardized explanatory knowledge for use in expert systems. This experiment consisted of four phases centered on the use of the controlled terminology-- Systemized Nomenclature of Medicine (SNOMED). The first phase evaluated SNOMED's ability to express explanatory knowledge for clinical pathology. The second developed the Normalized Medical Explanation (NORMEX) syntax for expressing and storing pathways of causal reasoning in the domain of clinical pathology. The third segment examined SNOMED's capacity to represent concepts used in the NORMEX model of clinical pathology. The final phase incorporated NORMEX-based pathways of influence in a Bayesian network to assess ability to predict causal mechanisms as implied by serum analyte results. Findings from this work suggest that SNOMED's capacity to represent explanatory information parallels its coverage of clinical pathology findings. However, SNOMED currently lacks much of the content necessary for both of these purposes. Additional explanatory content was created with an ontology-modeling tool. The NORMEX syntax was defined by SNOMED hierarchy names. Complex sequences of explanations were created using the NORMEX syntax. In addition, medical explanatory knowledge represented in the NORMEX format could be stored in an architectural framework consistent with that used by a controlled terminology such as SNOMED. Once stored, such knowledge could be retrieved from storage without loss of meaning or introduction of errors. Lastly, a Bayesian network constructed from the retrieved NORMEX knowledge produced a network whose prediction performance equaled or exceeded that of a network produced by more traditional means.
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    Frame-Based Stereotactic Biopsy of Canine Brain Masses: Technique and Clinical Results in 26 Cases
    Rossmeisl, John H. Jr.; Andriani, Rudy T.; Cecere, Thomas E.; Lahmers, Kevin K.; LeRoith, Tanya; Zimmerman, Kurt L.; Gibo, Denise M.; Debinski, Waldemar (2015)
    This report describes the methodology, diagnostic yield, and adverse events (AE) associated with frame-based stereotactic brain biopsies (FBSB) obtained from 26 dogs with solitary forebrain lesions. Medical records were reviewed from dogs that underwent FBSB using two stereotactic headframes designed for use in small animals and compatible with computed tomographic (CT) and magnetic resonance (MR) imaging. Stereotactic plans were generated from MR and CT images using commercial software, and FBSB performed both with (14/26) and without intraoperative image guidance. Records were reviewed for diagnostic yield, defined as the proportion of biopsies producing a specific neuropathological diagnosis, AE associated with FBSB, and risk factors for the development of AE. Postprocedural AE were evaluated in 19/26 dogs that did not proceed to a therapeutic intervention immediately following biopsy. Biopsy targets included intra-axial telencephalic masses (24/26), one intra-axial diencephalic mass, and one extra-axial parasellar mass. The median target volume was 1.99 cm(3). No differences in patient, lesion, or outcome variables were observed between the two headframe systems used or between FBSB performed with or without intraoperative CT guidance. The diagnostic yield of FBSB was 94.6%. Needle placement error was a significant risk factor associated with procurement of non-diagnostic biopsy specimens. Gliomas were diagnosed in 24/26 dogs, and meningioma and granulomatous meningoencephalitis in 1 dog each. AE directly related to FBSB were observed in a total of 7/26 (27%) of dogs. Biopsy-associated clinical morbidity, manifesting as seizures and transient neurological deterioration, occurred in 3/19 (16%) of dogs. The case fatality rate was 5.2% (1/19 dogs), with death attributable to intracranial hemorrhage. FBSB using the described apparatus was relatively safe and effective at providing neuropathological diagnoses in dogs with focal forebrain lesions.
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    Histopathologic Variation between Liver Lobes in Dogs
    Kemp, Stephanie D.; Zimmerman, Kurt L.; Panciera, David L.; Monroe, William E.; Leib, Michael S. (American College of Veterinary Internal Medicine, 2015-01)
    Background: Biopsy of the liver evaluates a small portion of tissue, with inferences made to the entire organ. The method and number of biopsies obtained are tempered by consideration of the risks and benefits. Recommendations often include biopsy of more than one liver lobe, although the consistency of histopathology among lobes in dogs is unknown. Hypothesis/Objectives: To describe the distribution of histopathologic abnormalities between liver lobes. We hypothesized that discordant results would be evenly distributed among all liver lobes. Animals: Seventy dogs undergoing necropsy. Methods: Prospective study. Liver samples were obtained from all lobes. A primary diagnosis was assigned to each liver sample based on the predominant histopathologic abnormality. Results: In this population of dogs, biopsy of at least 2 liver lobes identified the predominant histologic abnormality in 98.6% of the cases. Ten ( 14%) of the dogs had = 3 lobes in agreement and could not be assigned a predominant diagnosis. The same diagnosis was present in 6/ 6 lobes in 39 ( 56.5%) dogs, 5/ 6 lobes in 10 ( 14.5%) dogs, 4/ 6 lobes in 10 ( 14.5%) dogs, 3/ 6 lobes in 7 ( 10.1%) dogs, and 2/ 6 in 3 ( 4.3%) dogs. The number of discordant results did not differ between the liver lobes. Conclusion and Clinical Importance: The likelihood of obtaining a sample that is reflective of the predominant histologic abnormality in the liver is increased when multiple liver lobes are biopsied.
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    Improving the Knowledge of EPM
    Helber, Lauren Anne (Virginia Tech, 2024-06-05)
    Equine protozoal Myeloencephalitis (EPM) is a neurologic disease in horses predominantly caused by the protozoa Sarcocystis neurona. Carried by the North American opossum, Didelphis virginiana, horses are exposed to S. neurona when eating or drinking food or water contaminated with opossum feces. While exposure to the protozoa is high, only around 1% of horses develop clinical disease. While the mechanism by which S. neurona causes central nervous system damage is still unknown, this dissertation examines the histopathologic damage and potential persistence of S. neurona after anti-protozoal treatment between groups of horses with acute and chronic disease. This dissertation also examines the ability of two diagnostic techniques, immunohistochemistry (IHC) and polymerase chain reaction (PCR), to detect S. neurona. Horses were grouped based on duration of clinical signs; acutely affected horses exhibited clinical signs <6 months, while chronically affected horses exhibited clinical signs >6 months, including those previously treated for EPM. A comparison of necropsy reports revealed that chronically affected horses with EPM had more degenerative changes compared to acutely affected horses with EPM. However, when histologic changes were quantified, acutely affected horses had similar degenerative changes compared to chronically affected horses. When IHC and PCR were compared, IHC detected S. neurona presence (9/9 horses with EPM) significantly more often than PCR 4/9 horses with EPM). Our studies also show that S. neurona can be persistent in horses, as evidenced by the detection of S. neurona in the CNS of horses previously treated for EPM. Four horses had previously been treated for EPM, and all four had S. neurona present in their CNS even after anti-protozoal treatment, suggesting the ability for S. neurona to persist. In addition, this dissertation examines the possibility of using soluble CD14 (sCD14) as a supplemental assay for differentiating neurologic diseases such as EPM and cervical vertebral stenotic myelopathy (CVSM). When sCD14 levels were assessed in control EPM, CVSM, and EPM+CVSM horses, sCD14 concentrations in the cerebral spinal fluid (CSF) were significantly different between control and EPM horses and EPM horses and CVSM horses. With this information, clinicians and researchers may use sCD14 as a supplemental assay for differentiating between healthy, EPM, and CVSM horses. Finally, future directions include preliminary data that may lead to a potential for a peptide vaccine protecting horses from EPM clinical disease. Further insight into the persistence of S. neurona after anti-protozoal treatment is needed, the classification of acutely and chronically affected horses and the ability of sCD14 detection as a supplemental assay will be required; this this dissertation allows for the continuation of knowledge in combating the elusive protozoa, S. neurona.
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     NF-kB Inducing Kinase (NIK) Influences Eosinophil Development, Survival, and Plasticity
    Trusiano, Briana Lynn (Virginia Tech, 2024-04-22)
    Hypereosinophilic (HES) syndrome is an umbrella term encompassing several disease subsets that affects humans and veterinary species, ultimately resulting in >1,500 eosinophils/uL circulating in the blood documented over six-months. This eventually culminates in end-organ infiltration and increased patient morbidity and mortality. In mice where the gene Map3k14 encoding NF -kB inducing kinase (NIK) is knocked out, a HES-like syndrome develops that is dependent on Th2 cells and cytokines. NIK is the upstream regulator of the noncanonical NF-kB pathway and is involved in lymphoid organ development, B cell lymphopoiesis, and myelopoiesis. In addition to regulating the noncanonical NF-kB pathway, NIK is also involved in regulation of kB dimers of the canonical NF-kB pathway and can function independent of NF-kB signaling by regulating lipid and glucose metabolism, mitochondrial, and RIP1 binding to influence cell survival and death. Despite previous studies performed in the Nik-/- model, the mechanisms underlying eosinophil development, plasticity, and fitness in conjunction with the bone marrow and splenic microenvironments have not been fully elucidated. In the present work, we reviewed current data exploring the influence of the noncanonical NF-kB pathway and NIK specifically on the development of acute myeloid leukemias (AMLs) and Myelodysplastic Syndrome (MDS) with a focus on how these mechanisms might induce subvariants of HES. We next examined the effect of NIK loss on eosinophilopoiesis within hematopoietic tissues in vivo and in various cell culture environments in vitro via cytology, histology, flow cytometry, FACS, positive cell selection, MTT assay, BrDU assay, and protein microarray analysis. Overall, our findings suggest that NIK influences eosinophil maturation, proliferation, metabolism, survival, and potentially plasticity in vivo and in vitro under different environmental conditions and Th2 cytokine influence. NIK loss was also associated with altered free and bound TNFR1 levels on day 13 in vitro. TNFR1 acts upstream of RIP1 and suggests that these differences may be due to NF-kB independent functions of NIK. Overall, these results provide further insight into the potential mechanisms underlying eosinophilopoiesis in the Nik-/- murine model. This information may prove useful in discovering new treatment options underlying subvariants of HES in both human and veterinary patients.
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    Optic nerve astrocytoma in a dog
    Rozov, Orr; Piñyero, Pablo E.; Zimmerman, Kurt L.; Herring, Ian P.; Matusow, Rachel; Rossmeisl, John H. Jr.; Jortner, Bernard S.; Dreyfus, Jennifer (Wiley, 2016)
    Intraocular neoplasms in dogs are uncommon in comparison with other anatomic locations [1–4]. Over 75% of these cases are attributed to melanocytic neoplasia [1]. A majority of the remaining cases (~22%) are comprised of lymphoma, metastatic neoplasia, iridociliary epithelial tumors, optic nerve meningiomas, and histiocytic sarcomas in decreasing order of frequency [1, 2]. Rarely, optic nerve astrocytomas have been reported in humans, dogs, and horses [5–7]. In humans, astrocytomas involving the optic nerve are uncommon accounting for only about 1% of neoplasms at this site in comparison with 25% of neoplasms occurring in the brain [5, 8–14]. Most of these neoplasms occur unilaterally, are benign, and arise in children under 10 years of age; those involving the cerebellum have a more favorable prognosis [8, 14]. These younger patients commonly have an underlying familiar disorder such as neurofibromatosis. In older patients in their fourth and fifth decade of life, astrocytomas more commonly involve the cerebrum with a less favorable prognosis and can be associated with familiar disorders such as Li–Fraumeni syndrome [13, 15–17]. Most canine ocular astrocytoma cases are sporadic and not associated with any familial disorder [1, 13, 18–21]. However, Thomas et al. demonstrated a genomic risk factor associated with frequency of chromosome copy number aberrations within canine brain astrocytomas and tumor grade [22]. Similar to humans, canine astrocytomas account for less than 1% of ocular and optic nerve neoplasms and 10–36% of primary intracranial neoplasms [1, 4, 16–18, 20, 21, 23]. There appears to be a breed predisposition for development of intracranial astrocytomas in English Toy Spaniels, Boston Terriers, French Bulldogs, Boxers, and English Bulldog with a peak prevalence at 7– 8 years and 1.5 odds ratio in favor of larger versus smaller breeds [13, 23]. These risk factors have not been shown in association with canine ocular forms of this neoplasm [1, 16, 17]. Gender as a risk factor has not been described for either anatomic location [1, 13, 16–18, 23]. Case reports related to ocular astrocytomas are still rare in veterinary literature. The purpose of this report is to add to this sparse body of information. This report presents the clinical, histological, and immunohistochemical features of an optic nerve astrocytoma in a dog.
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    Probable paraneoplastic leukocytosis in a dog with a gastrointestinal stromal tumor
    Gidcumb, Emily M.; Bolton, Timothy A.; Trusiano, Brie; Zimmerman, Kurt L.; Oakes, Vanessa J. (Wiley, 2022-05-09)
    A 9-year-old female spayed Boston Terrier presented for diagnostic investigation of lethargy, poor appetite, weight loss, and a marked leukocytosis. Significant muscle wasting and a palpable abdominal mass were present on physical examination. Abdominal imaging revealed the mass to be of small intestinal origin; consequently, an intestinal resection and anastomosis were performed without complication. The histopathologic diagnosis was a gastrointestinal stromal tumor, verified by immunohistochemical positivity to CD117 (KIT). Two weeks after discharge, the leukocytosis had resolved. Though the exact molecular mediator of the severe leukocytosis was undetermined, resolution following tumor removal suggests a paraneoplastic cause. To the authors' knowledge, this is the first reported case of probable paraneoplastic leukocytosis secondary to a gastrointestinal stromal tumor in the dog. Gastrointestinal tract imaging should be performed when this uncommon hematologic abnormality is present.
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    Properties of Nanoscale Biomaterials for Cancer Detection and Other Applications
    Geist, Brian Lee (Virginia Tech, 2009-04-24)
    The first thermal cycling experiments of ionic self-assembled multilayer (ISAM) films have been reported examining their survivability through repeated thermal cycles from -20° C to 120° C in ambient atmospheric conditions. The films were constructed from alternating layers of Nile Blue A and gold nanoparticles which provided a strong absorbance in the optical wavelength range. No degradation of the optical characteristics of the ISAM films was observed [1]. Techniques for measuring the capacitance and resistivity of various ISAM films have also been developed allowing for a more complete electrical characterization of ISAM films. Capacitance measurements enabled a calculation of the dielectric function and breakdown field strength of the ISAM films. The capacitance measurement technique was verified by measuring the dielectric function of a spin-coated thin film PMMA, which has a well characterized dielectric function [2]. Surface-enhanced Raman spectroscopy (SERS) has been studied as a possible detection method for malignant melanoma revealing spectral differences in blood sera from healthy horses and horses with malignant melanoma. A SERS microscope system was constructed with the capability of resolving the Raman signal from biologically important molecules such as beta-carotene and blood sera. The resulting Raman signals from sera collected from horses with malignant melanoma were found to have additional peaks not found in the Raman signals obtained from sera collected from healthy horses. A systematic analysis of the combination of absorbance and fluorescence signals of blood sera collected from populations of healthy dogs and dogs with cancer has resulted in a rapid and cost-effective method for monitoring protein concentrations that could possibly be used as part of a cancer screening process. This method was developed using the absorbance and fluorescence signals from known serum proteins, the combinations of which were used to match the absorbance and fluorescence signals of blood sera allowing for an accurate determination of protein concentrations in blood sera [3]. Finally, a novel method for measuring the melting point of DNA in solution using capacitance measurements is presented. This method allows for the determination of the melting temperature as well as the melting entropy and melting enthalpy of DNA strands. Two different short strands of DNA, 5'-CAAAATAGACGCTTACGCAACGAAAAC-3' along with its complement and 5'-GGAAGAGACGGAGGA-3' along with its complement were used to validate the technique as the characteristics of these strands could be modeled using theoretical methods. This experimental technique allows for the precise determination of the melting characteristics of DNA strands and can be used to evaluate the usefulness of theoretical models in calculating the melting point for particular strands of DNA. Additionally, a micro-fluidic device has been proposed that will allow for a rapid and cost-effective determination of the melting characteristics of DNA [4].
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    Real Time PCR-Based Infectivity Assay and Characterization of Cell Surface Receptors for Turkey Hemorrhagic Enteritis Virus
    Mahsoub, Hassan Mostafa Mohammed (Virginia Tech, 2016-01-19)
    Turkey hemorrhagic enteritis virus (THEV) is responsible for the hemorrhagic enteritis (HE) disease in commercial turkeys through infections by its virulent strains. HE is an acute condition characterized by depression, immunosuppression, bloody droppings, intestinal hemorrhage, and death. THEV (also known as turkey adenovirus 3) is an official member of the family Adenoviridae, genus Siadenovirus, species Turkey siadenovirus A. Two main types of live vaccines are currently used for the protection of turkeys against HE; a crude splenic vaccine propagated in live turkeys, and a cell culture-based vaccine generated in RP19 cells. The only laboratory-adapted tests for assessing the titers of these vaccines are agar gel immunodiffusion test and cell culture endpoint dilution, respectively. The assays suffer from low sensitivity, inaccuracy, and time consumption. A SYBR Green-based real time PCR assay for determining the genomic titer of THEV through the quantification of its hexon gene was developed. The assay was applied as a quality control for the titration of splenic vaccines and was found useful in distinguishing the differences in virus titer among many vaccine batches. Additionally, using the qPCR assay along with a cell culture system, a novel infectivity assay was developed for the titration of THEV, as an alternative for the endpoint dilution assay. Applying the assay on nine batches of commercial HE cell culture vaccines, high variations in infectious virus titers were detected. The new method is rapid, sensitive, and very accurate. A strong correlation was found between the genomic titer and qPCR infectious titer in HE cell culture vaccines. Moreover, the qPCR infectivity assay proved as an instrumental research tool. It was used to measure the effect of several treatments of RP19 cells on virus infection. The main target cell type for THEV infection and replication is B-lymphocytes, which are represented in vitro by the B lymphoblastoid, RP19 cells. The cellular surface components used by the virus to gain entry into cells are unknown. As an adenovirus, we hypothesized that THEV uses two different molecules on RP19 cells for the attachment and internalization. A recent study has shown that the synthesized THEV fiber knob domain binds to sialyllactose, based on a glycan array analysis. In our studies, the treatment of RP19 cells with neuraminidases and lectins resulted in high reduction of virus entry, which provide a strong evidence of the utilization of cell surface sialic acids as attachment receptor for THEV. Destruction of surface carbohydrates and proteins on RP19 cells also reduced virus entry, indicating that these components are part of the THEV receptor. Using virus overlay protein blot assay, THEV was found to specifically bind to two RP19 surface membrane proteins, most likely, representing primary and secondary receptors for virus entry. Further studies are required to identify these proteins and verify their role in THEV endocytosis in host cells.