Diagnostic Medicine/Pathobiology Faculty Research and Publications
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Item Open Access Genome Characterization of a Pathogenic Porcine Rotavirus B Strain Identified in Buryat Republic, Russia in 2015(2018-04-20) Alekseev, Konstantin P.; Penin, Aleksey A.; Mukhin, Alexey N.; Khametova, Kizkhalum M.; Grebennikova, Tatyana V.; Yuzhakov, Anton G.; Moskvina, Anna S.; Musienko, Maria I.; Raev, Sergey A.; Mishin, Alexandr M.; Kotelnikov, Alexandr P.; Verkhovsky, Oleg A.; Aliper, Taras I.; Nepoklonov, Eugeny A.; Herrera-Ibata, Diana M.; Shepherd, Frances K.; Marthaler, Douglas G.An outbreak of enteric disease of unknown etiology with 60% morbidity and 8% mortality in weaning piglets occurred in November 2015 on a farm in Buryat Republic, Russia. Metagenomic sequencing revealed the presence of rotavirus B in feces from diseased piglets while no other pathogens were identified. Clinical disease was reproduced in experimentally infected piglets, yielding the 11 RVB gene segments for strain Buryat15, with an RVB genotype constellation of G12-P[4]-I13-R4-C4-M4-A8-N10-T4-E4-H7. This genotype constellation has also been identified in the United States. While the Buryat15 VP7 protein lacked unique amino acid differences in the predicted neutralizing epitopes compared to the previously published swine RVB G12 strains, this report of RVB in Russian swine increases our epidemiological knowledge on the global prevalence and genetic diversity of RVB.Item Open Access The Convergence of High-Consequence Livestock and Human Pathogen Research and Development: A Paradox of Zoonotic Disease(2018-05-30) Michelotti, Julia M.; Yeh, Kenneth B.; Beckham, Tammy R.; Colby, Michelle M.; Dasgupta, Debanjana; Zuelke, Kurt A.; Olinger, Gene G.The World Health Organization (WHO) estimates that zoonotic diseases transmitted from animals to humans account for 75 percent of new and emerging infectious diseases. Globally, high-consequence pathogens that impact livestock and have the potential for human transmission create research paradoxes and operational challenges for the high-containment laboratories that conduct work with them. These specialized facilities are required for conducting all phases of research on high-consequence pathogens (basic, applied, and translational) with an emphasis on both the generation of fundamental knowledge and product development. To achieve this research mission, a highly-trained workforce is required and flexible operational methods are needed. In addition, working with certain pathogens requires compliance with regulations such as the Centers for Disease Control (CDC) and the U.S. Department of Agriculture (USDA) Select Agent regulations, which adds to the operational burden. The vast experience from the existing studies at Plum Island Animal Disease Center, other U.S. laboratories, and those in Europe and Australia with biosafety level 4 (BSL-4) facilities designed for large animals, clearly demonstrates the valuable contribution this capability brings to the efforts to detect, prepare, prevent and respond to livestock and potential zoonotic threats. To raise awareness of these challenges, which include biosafety and biosecurity issues, we held a workshop at the 2018 American Society for Microbiology (ASM) Biothreats conference to further discuss the topic with invited experts and audience participants. The workshop covered the subjects of research funding and metrics, economic sustainment of drug and vaccine development pipelines, workforce turnover, and the challenges of maintaining operational readiness of high containment laboratories.Item Open Access Longitudinal Surveillance of Porcine Rotavirus B Strains from the United States and Canada and In Silico Identification of Antigenically Important Sites(2017-12-03) Shepherd, Frances K.; Murtaugh, Michael P.; Chen, Fangzhou; Culhane, Marie R.; Marthaler, Douglas G.Rotavirus B (RVB) is an important swine pathogen, but control and prevention strategies are limited without an available vaccine. To develop a subunit RVB vaccine with maximal effect, we characterized the amino acid sequence variability and predicted antigenicity of RVB viral protein 7 (VP7), a major neutralizing antibody target, from clinically infected pigs in the United States and Canada. We identified genotype-specific antigenic sites that may be antibody neutralization targets. While some antigenic sites had high amino acid functional group diversity, nine antigenic sites were completely conserved. Analysis of nucleotide substitution rates at amino acid sites (dN/dS) suggested that negative selection appeared to be playing a larger role in the evolution of the identified antigenic sites when compared to positive selection, and was identified in six of the nine conserved antigenic sites. These results identified important characteristics of RVB VP7 variability and evolution and suggest antigenic residues on RVB VP7 that are negatively selected and highly conserved may be good candidate regions to include in a subunit vaccine design due to their tendency to remain stable.Item Open Access Zearalenone Promotes Cell Proliferation or Causes Cell Death?(2018-05-02) Zheng, Wanglong; Wang, Bingjie; Li, Xi; Wang, Tao; Zou, Hui; Gu, Jianhong; Yuan, Yan; Liu, Xuezhong; Bai, Jianfa; Bian, Jianchun; Liu, ZongpingZearalenone (ZEA), one of the mycotoxins, exerts different mechanisms of toxicity in different cell types at different doses. It can not only stimulate cell proliferation but also inhibit cell viability, induce cell apoptosis, and cause cell death. Thus, the objective of this review is to summarize the available mechanisms and current evidence of what is known about the cell proliferation or cell death induced by ZEA. An increasing number of studies have suggested that ZEA promoted cell proliferation attributing to its estrogen-like effects and carcinogenic properties. What’s more, many studies have indicated that ZEA caused cell death via affecting the distribution of the cell cycle, stimulating oxidative stress and inducing apoptosis. In addition, several studies have revealed that autophagy and some antioxidants can reverse the damage or cell death induced by ZEA. This review thoroughly summarized the metabolic process of ZEA and the molecular mechanisms of ZEA stimulating cell proliferation and cell death. It concluded that a low dose of ZEA can exert estrogen-like effects and carcinogenic properties, which can stimulate the proliferation of cells. While, in addition, a high dose of ZEA can cause cell death through inducing cell cycle arrest, oxidative stress, DNA damage, mitochondrial damage, and apoptosis.Item Open Access Current Status of Rift Valley Fever Vaccine Development(2017-09-19) Faburay, Bonto; LaBeaud, Angelle Desiree; McVey, D. Scott; Wilson, William C.; Richt, Juergen A.Rift Valley Fever (RVF) is a mosquito-borne zoonotic disease that presents a substantial threat to human and public health. It is caused by Rift Valley fever phlebovirus (RVFV), which belongs to the genus Phlebovirus and the family Phenuiviridae within the order Bunyavirales. The wide distribution of competent vectors in non-endemic areas coupled with global climate change poses a significant threat of the transboundary spread of RVFV. In the last decade, an improved understanding of the molecular biology of RVFV has facilitated significant progress in the development of novel vaccines, including DIVA (differentiating infected from vaccinated animals) vaccines. Despite these advances, there is no fully licensed vaccine for veterinary or human use available in non-endemic countries, whereas in endemic countries, there is no clear policy or practice of routine/strategic livestock vaccinations as a preventive or mitigating strategy against potential RVF disease outbreaks. The purpose of this review was to provide an update on the status of RVF vaccine development and provide perspectives on the best strategies for disease control. Herein, we argue that the routine or strategic vaccination of livestock could be the best control approach for preventing the outbreak and spread of future disease.Item Open Access Biological Control Strategies for Mosquito Vectors of Arboviruses(2017-02-10) Huang, Yan-Jang S.; Higgs, Stephen; Vanlandingham, Dana L.Historically, biological control utilizes predatory species and pathogenic microorganisms to reduce the population of mosquitoes as disease vectors. This is particularly important for the control of mosquito-borne arboviruses, which normally do not have specific antiviral therapies available. Although development of resistance is likely, the advantages of biological control are that the resources used are typically biodegradable and ecologically friendly. Over the past decade, the advancement of molecular biology has enabled optimization by the manipulation of genetic materials associated with biological control agents. Two significant advancements are the discovery of cytoplasmic incompatibility induced by Wolbachia bacteria, which has enhanced replacement programs, and the introduction of dominant lethal genes into local mosquito populations through the release of genetically modified mosquitoes. As various arboviruses continue to be significant public health threats, biological control strategies have evolved to be more diverse and become critical tools to reduce the disease burden of arboviruses.Item Open Access Harnessing Invariant NKT Cells to Improve Influenza Vaccines: A Pig Perspective(2017-12-27) Yang, Guan; Richt, Jürgen A.; Driver, John P.Invariant natural killer T (iNKT) cells are an “innate-like” T cell lineage that recognize glycolipid rather than peptide antigens by their semi-invariant T cell receptors. Because iNKT cells can stimulate an extensive array of immune responses, there is considerable interest in targeting these cells to enhance human vaccines against a wide range of microbial pathogens. However, long overlooked is the potential to harness iNKT cell antigens as vaccine adjuvants for domestic animal species that express the iNKT cell–CD1d system. In this review, we discuss the prospect of targeting porcine iNKT cells as a strategy to enhance the efficiency of swine influenza vaccines. In addition, we compare the phenotype and tissue distribution of porcine iNKT cells. Finally, we discuss the challenges that must be overcome before iNKT cell agonists can be contemplated for veterinary use in livestock.Item Open Access Influence of Intestinal Microbiota Transplantation and NleH Expression on Citrobacter rodentium Colonization of Mice(2018-03-30) Wang, Gaochan; Feuerbacher, Leigh Ann; Hardwidge, Philip R.The intestinal microbiota plays an important role in regulating host resistance to enteric pathogens. The relative abundance of the microbiota is dependent upon both genetic and environmental factors. The attaching and effacing pathogens enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium cause diarrheal disease and translocate type III secretion system effector proteins into host cells to inhibit pro-inflammatory host responses. Here we determined the influence of both the intestinal microbiota and the expression of the C. rodentium NleH effector on C. rodentium colonization in different mouse models. We performed fecal transplantation experiments between C57BL/6J and C57BL/10ScNJ mice and found that such microbiota transfers altered both the host resistance to C. rodentium infection as well as the benefit or detriment of expressing NleH to C. rodentium intestinal colonization.Item Open Access Evaluation of the Zoonotic Potential of Transmissible Mink Encephalopathy(2013-07-30) Comoy, Emmanuel E.; Mikol, Jacqueline; Ruchoux, Marie-Madeleine; Durand, Valérie; Luccantoni-Freire, Sophie; Dehen, Capucine; Correia, Evelyne; Casalone, Cristina; Richt, Juergen A.; Greenlee, Justin J.; Torres, Juan Maria; Brown, Paul; Deslys, Jean-PhilippeSuccessful transmission of Transmissible Mink Encephalopathy (TME) to cattle supports the bovine hypothesis for the still controversial origin of TME outbreaks. Human and primate susceptibility to classical Bovine Spongiform Encephalopathy (c-BSE) and the transmissibility of L-type BSE to macaques indicate a low cattle-to-primate species barrier. We therefore evaluated the zoonotic potential of cattle-adapted TME. In less than two years, this strain induced in cynomolgus macaques a neurological disease similar to L-BSE but distinct from c-BSE. TME derived from another donor species (raccoon) induced a similar disease with even shorter incubation periods. L-BSE and cattle-adapted TME were also transmissible to transgenic mice expressing human prion protein (PrP). Secondary transmissions to transgenic mice expressing bovine PrP maintained the features of the three tested bovine strains (cattle TME, c-BSE and L-BSE) regardless of intermediate host. Thus, TME is the third animal prion strain transmissible to both macaques and humanized transgenic mice, suggesting zoonotic potentials that should be considered in the risk analysis of animal prion diseases for human health. Moreover, the similarities between TME and L-BSE are highly suggestive of a link between these strains, and therefore the possible presence of L-BSE for many decades prior to its identification in USA and Europe.Item Open Access Enterotoxigenic Escherichia coli Flagellin Inhibits TNF-Induced NF-κB Activation in Intestinal Epithelial Cells(2017-05-17) Wang, Gaochan; Geisbrecht, Brian V.; Rueter, Christian; Hardwidge, Philip R.Enterotoxigenic Escherichia coli (ETEC) causes childhood diarrhea in developing countries. ETEC strains produce the heat-labile enterotoxin (LT) and/or heat-stable enterotoxins (ST) and encode a diverse set of colonization factors used for adherence to intestinal epithelial cells. We previously found that ETEC secretes a heat-stable protein we designated as ETEC Secreted Factor (ESF) that inhibits the extent of NF-κB activation normally induced by tumor necrosis factor alpha (TNF). Here we fractionated ETEC supernatants using fast protein liquid chromatography (FPLC) and determined that ETEC flagellin was necessary and sufficient to protect IκBα from degradation in response to TNF stimulation. These data suggest a potentially novel mechanism by which ETEC may evade the host innate immune response by down-regulating NF-κB-dependent host responses.Item Open Access Modified Heat-Stable Toxins (hSTa) of Enterotoxigenic Escherichia coli Lose Toxicity but Display Antigenicity after Being Genetically Fused to Heat-Labile Toxoid LT(R192G)(2011-09-15) Liu, Mei; Zhang, Chengxian; Mateo, Kristy; Nataro, James P.; Robertson, Donald C.; Zhang, WeipingEnterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrhea in humans and animals. Heat-stable (STa) and heat-labile (LT) enterotoxins produced by ETEC disrupt fluid homeostasis in host small intestinal epithelial cells and cause fluid and electrolyte hyper-secretion that leads to diarrhea. ETEC strains producing STa or LT are sufficiently virulent to cause diarrhea, therefore STa and LT antigens must be included in ETEC vaccines. However, potent toxicity and poor immunogenicity (of STa) prevent them from being directly applied as vaccine components. While LT toxoids, especially LT(R192G), being used in vaccine development, STa toxoids have not been included. A recent study (IAI, 78:316-325) demonstrated porcine-type STa toxoids [pSTa(P12F) and pSTa(A13Q)] elicited protective anti-STa antibodies after being fused to a porcine-type LT toxoid [pLT(R192G)]. In this study, we substituted the 8th, 9th, 16th, or the 17th amino acid of a human-type STa (hSTa) and generated 28 modified STa peptides. We tested each STa peptide for toxicity and structure integrity, and found nearly all modified STa proteins showed structure alteration and toxicity reduction. Based on structure similarity and toxic activity, three modified STa peptides: STa(E8A), STa(T16Q) and STa(G17S), were selected to construct LT192-STa-toxoid fusions. Constructed fusions were used to immunize mice, and immunized mice developed anti-STa antibodies. Results from this study provide useful information in developing toxoid vaccines against ETEC diarrhea.Item Open Access Flavivirus-Mosquito Interactions(2014-11-24) Huang, Yan-Jang S.; Higgs, Stephen; Horne, Kate McElroy; Vanlandingham, Dana L.The Flavivirus genus is in the family Flaviviridae and is comprised of more than 70 viruses. These viruses have a broad geographic range, circulating on every continent except Antarctica. Mosquito-borne flaviviruses, such as yellow fever virus, dengue virus serotypes 1–4, Japanese encephalitis virus, and West Nile virus are responsible for significant human morbidity and mortality in affected regions. This review focuses on what is known about flavivirus-mosquito interactions and presents key data collected from the field and laboratory-based molecular and ultrastructural evaluations.Item Open Access Evolution of Diagnostic Tests for Chronic Wasting Disease, a Naturally Occurring Prion Disease of Cervids(2017-08-05) Haley, Nicholas J.; Richt, Jürgen A.Since chronic wasting disease (CWD) was first identified nearly 50 years ago in a captive mule deer herd in the Rocky Mountains of the United States, it has slowly spread across North America through the natural and anthropogenic movement of cervids and their carcasses. As the endemic areas have expanded, so has the need for rapid, sensitive, and cost effective diagnostic tests—especially those which take advantage of samples collected antemortem. Over the past two decades, strategies have evolved from the recognition of microscopic spongiform pathology and associated immunohistochemical staining of the misfolded prion protein to enzyme-linked immunoassays capable of detecting the abnormal prion conformer in postmortem samples. In a history that parallels the diagnosis of more conventional infectious agents, both qualitative and real-time amplification assays have recently been developed to detect minute quantities of misfolded prions in a range of biological and environmental samples. With these more sensitive and semi-quantitative approaches has come a greater understanding of the pathogenesis and epidemiology of this disease in the native host. Because the molecular pathogenesis of prion protein misfolding is broadly analogous to the misfolding of other pathogenic proteins, including Aβ and α-synuclein, efforts are currently underway to apply these in vitro amplification techniques towards the diagnosis of Alzheimer’s disease, Parkinson’s disease, and other proteinopathies. Chronic wasting disease—once a rare disease of Colorado mule deer—now represents one of the most prevalent prion diseases, and should serve as a model for the continued development and implementation of novel diagnostic strategies for protein misfolding disorders in the natural host.Item Open Access Bunyavirus-Vector Interactions(2014-11-13) Horne, Kate McElroy; Vanlandingham, Dana L.The Bunyaviridae family is comprised of more than 350 viruses, of which many within the Hantavirus, Orthobunyavirus, Nairovirus, Tospovirus, and Phlebovirus genera are significant human or agricultural pathogens. The viruses within the Orthobunyavirus, Nairovirus, and Phlebovirus genera are transmitted by hematophagous arthropods, such as mosquitoes, midges, flies, and ticks, and their associated arthropods not only serve as vectors but also as virus reservoirs in many cases. This review presents an overview of several important emerging or re-emerging bunyaviruses and describes what is known about bunyavirus-vector interactions based on epidemiological, ultrastructural, and genetic studies of members of this virus family.Item Open Access Association of the Host Immune Response with Protection Using a Live Attenuated African Swine Fever Virus Model(2016-10-22) Carlson, Jolene; O’Donnell, Vivian; Alfano, Marialexia; Velazquez Salinas, Lauro; Holinka, Lauren G.; Krug, Peter W.; Gladue, Douglas P.; Higgs, Stephen; Borca, Manuel V.African swine fever (ASF) is a lethal hemorrhagic disease of swine caused by a double-stranded DNA virus, ASF virus (ASFV). There is no vaccine to prevent the disease and current control measures are limited to culling and restricting animal movement. Swine infected with attenuated strains are protected against challenge with a homologous virulent virus, but there is limited knowledge of the host immune mechanisms generating that protection. Swine infected with Pretoriuskop/96/4 (Pret4) virus develop a fatal severe disease, while a derivative strain lacking virulence-associated gene 9GL (Pret4Δ9GL virus) is completely attenuated. Swine infected with Pret4Δ9GL virus and challenged with the virulent parental virus at 7, 10, 14, 21, and 28 days post infection (dpi) showed a progressive acquisition of protection (from 40% at 7 dpi to 80% at 21 and 28 dpi). This animal model was used to associate the presence of host immune response (ASFV-specific antibody and interferon (IFN)-γ responses, or specific cytokine profiles) and protection against challenge. With the exception of ASFV-specific antibodies in survivors challenged at 21 and 28 dpi, no association between the parameters assessed and protection could be established. These results, encompassing data from 65 immunized swine, underscore the complexity of the system under study, suggesting that protection relies on the concurrence of different host immune mechanisms.Item Open Access Impact of Three Different Mutations in Ehrlichia chaffeensis in Altering the Global Gene Expression Patterns(2018-04-18) Kondethimmanahalli, Chandramouli; Ganta, Roman R.The rickettsial pathogen Ehrlichia chaffeensis causes a tick-borne disease, human monocytic ehrlichiosis. Mutations within certain genomic locations of the pathogen aid in understanding the pathogenesis and in developing attenuated vaccines. Our previous studies demonstrated that mutations in different genomic sites in E. chaffeensis caused variable impacts on their growth and attenuation in vertebrate and tick hosts. Here, we assessed the effect of three mutations on transcriptional changes using RNA deep-sequencing technology. RNA sequencing aided in detecting 66–80% of the transcripts of wildtype and mutant E. chaffeensis. Mutation in an antiporter gene (ECH_0379) causing attenuated growth in vertebrate hosts resulted in the down regulation of many transcribed genes. Similarly, a mutation downstream to the ECH_0490 coding sequence resulted in minimal impact on the pathogen’s in vivo growth, but caused major changes in its transcriptome. This mutation caused enhanced expression of several host stress response genes. Even though the ECH_0660 gene mutation caused the pathogen’s rapid clearance in vertebrate hosts and aids in generating a protective response, there was minimal impact on the transcriptome. The transcriptomic data offer novel insights about the impact of mutations on global gene expression and how they may contribute to the pathogen’s resistance and/or clearance from the host.Item Open Access A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis(2017-11-17) Wang, Ying; Wei, Lanjing; Liu, Huitao; Cheng, Chuanmin; Ganta, Roman R.Obligate intracellular bacteria (obligates) belonging to Rickettsiales and Chlamydiales cause diseases in hundreds of millions of people worldwide and in many animal species. Lack of an efficient system for targeted mutagenesis in obligates remains a major impediment in understanding microbial pathogenesis. Challenges in creating targeted mutations may be attributed to essential nature of majority of the genes and intracellular replication dependence. Despite success in generating random mutations, a method that works well in creating mutations in specific genes of interest followed by complementation remains problematic for obligates and is a highly sought-after goal. We describe protocols to generate stable targeted mutations by allelic exchange in Ehrlichia chaffeensis, an obligate intracellular tick-borne bacterium responsible for human monocytic ehrlichiosis. Targeted mutations in E. chaffeensis were created to disrupt two genes, and also to restore one gene by another allelic exchange mutation leading to the restoration of transcription and protein expression from the inactivated gene and the recovered organisms also express mCherry, which distinguishes from the wild type. We expect that the methods developed are broadly applicable to other obligates, particularly to rickettsial pathogens, to routinely perform targeted mutations to enable studies focused on protein structure-function analyses, host-pathogen interactions and in developing vaccines.Item Open Access Transcription of Ehrlichia chaffeensis genes is accomplished by RNA polymerase holoenzyme containing either sigma 32 or sigma 70(2013-11-21) Liu, Huitao; Ohlen, Tonia Von; Cheng, Chuanmin; Faburay, Bonto; Ganta, Roman R.Bacterial gene transcription is initiated by RNA polymerase containing a sigma factor. To understand gene regulation in Ehrlichia chaffeensis, an important tick-transmitted rickettsiae responsible for human monocytic ehrlichiosis, we initiated studies evaluating the transcriptional machinery of several genes of this organism. We mapped the transcription start sites of 10 genes and evaluated promoters of five genes (groE, dnaK, hup, p28-Omp14 and p28-Omp19 genes). We report here that the RNA polymerase binding elements of E. chaffeensis gene promoters are highly homologous for its only two transcription regulators, sigma 32 and sigma 70, and that gene expression is accomplished by either of the transcription regulators. RNA analysis revealed that although transcripts for both sigma 32 and sigma 70 are upregulated during the early replicative stage, their expression patterns remained similar for the entire replication cycle. We further present evidence demonstrating that the organism’s -35 motifs are essential to transcription initiations. The data suggest that E. chaffeensis gene regulation has evolved to support the organism’s growth, possibly to facilitate its intraphagosomal growth. Considering the limited availability of genetic tools, this study offers a novel alternative in defining gene regulation in E. chaffeensis and other related intracellular pathogens.Item Open Access Ehrlichia chaffeensis Infection in the Reservoir Host (White-Tailed Deer) and in an Incidental Host (Dog) Is Impacted by Its Prior Growth in Macrophage and Tick Cell Environments(2014-10-10) Nair, Arathy D. S.; Cheng, Chuanmin; Jaworski, Deborah C.; Willard, Lloyd H.; Sanderson, Michael W.; Ganta, Roman R.Ehrlichia chaffeensis, transmitted from Amblyomma americanum ticks, causes human monocytic ehrlichiosis. It also infects white-tailed deer, dogs and several other vertebrates. Deer are its reservoir hosts, while humans and dogs are incidental hosts. E. chaffeensis protein expression is influenced by its growth in macrophages and tick cells. We report here infection progression in deer or dogs infected intravenously with macrophage- or tick cell-grown E. chaffeensis or by tick transmission in deer. Deer and dogs developed mild fever and persistent rickettsemia; the infection was detected more frequently in the blood of infected animals with macrophage inoculum compared to tick cell inoculum or tick transmission. Tick cell inoculum and tick transmission caused a drop in tick infection acquisition rates compared to infection rates in ticks fed on deer receiving macrophage inoculum. Independent of deer or dogs, IgG antibody response was higher in animals receiving macrophage inoculum against macrophage-derived Ehrlichia antigens, while it was significantly lower in the same animals against tick cell-derived Ehrlichia antigens. Deer infected with tick cell inoculum and tick transmission caused a higher antibody response to tick cell cultured bacterial antigens compared to the antibody response for macrophage cultured antigens for the same animals. The data demonstrate that the host cell-specific E. chaffeensis protein expression influences rickettsemia in a host and its acquisition by ticks. The data also reveal that tick cell-derived inoculum is similar to tick transmission with reduced rickettsemia, IgG response and tick acquisition of E. chaffeensis.Item Open Access Evaluation of Culture Methods To Identify Bovine Feces with High Concentrations of Escherichia coli O157(2007-08-15) Fox, J. Trent; Renter, David G.; Sanderson, Michael W.; Thomson, Daniel U.; Lechtenberg, Kelly F.; Nagaraja, T. G.Our objective was to evaluate methods for identifying cattle with high concentrations of Escherichia coli O157 in their feces. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid (Nal)-resistant E. coli O157, and direct plating of diluted feces on sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) containing Nal was considered the gold standard (GS) method. In experiment 1, methods evaluated were preenrichment direct streak, immunomagnetic separation with most probable number (MPN), and postenrichment direct streak with MPN, all using CT-SMAC. The mean concentration of Nal-resistant E. coli O157 in samples (n = 59) by use of the GS was 3.6 log10 CFU/g. The preenrichment streak detected >3.0 log10 CFU/g samples with a 74.4% sensitivity and 68.8% specificity. Postenrichment direct streak-MPN and immunomagnetic separation-MPN concentrations were correlated significantly with GS concentrations (r = 0.53 and r = 0.39, respectively). In experiment 2 (480 samples), pre- and postenrichment direct streaking performed in triplicate and spiral plating on CT-SMAC were evaluated. For preenrichment streaks, sensitivity was 79.7% and specificity was 96.7% for detecting >3.0 log10 CFU/g when the criterion was positive cultures on at least two plates. For spiral plating at that concentration, sensitivity and specificity were 83.9% and 56.3%, respectively. Postenrichment streaking performed relatively poorly. Triplicate preenrichment streaks of 1:10-diluted feces on CT-SMAC may be useful for identifying cattle shedding high concentrations of E. coli O157. Estimates of sensitivity and specificity enable appropriate application of methods and interpretation of results and may enhance applied research, surveillance, and risk assessments.
