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ABSTRACT: Escherichia coli O157:H7 is an enteric pathogen of animals and humans that can result in deadly sequelae. Cattle are asymptomatic carriers and shedders of the bacteria and serve as an important reservoir of human infection. E. coli O157:H7 colonizes the gastrointestinal tract, most frequently at the rectoanal junction mucosa in cattle. Vaccination is a potentially highly effective means of decreasing cattle colonization and shedding and thereby decreasing human infections. Currently available vaccines are administered subcutaneously or intramuscularly and immune responses have been evaluated solely by systemic immunoglobulin responses. This study evaluated local and systemic lymphoproliferative responses in addition to immunoglobulin responses following subcutaneous or mucosal (rectal) immunization with E. coli O157:H7 outer membrane protein intimin over three trials. In all three trials, significant local and systemic lymphoproliferative responses (P<0.05) occurred following immunization in the majority of animals, as well as significant immunoglobulin responses (P<0.001) in all animals. Surprisingly, local responses in the mesorectal lymph nodes were very similar between the subcutaneous and mucosal immunization groups. Moreover, the responses in mesorectal lymph nodes appeared targeted rather than generalized, as minimal or no significant responses were observed in the associated prescapular lymph nodes of subcutaneously immunized animals. The results indicate that both subcutaneous and mucosal immunizations are effective methods of inducing immune responses against E. coli O157:H7 in cattle.
Clinical and vaccine immunology: CVI 02/2013; · 2.37 Impact Factor
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ABSTRACT: Staphylococcus aureus is a commensal bacterium associated with the skin and mucosal surfaces of humans and animals that can also cause chronic infection. The emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA) and strains causing chronic intramammary infections (IMI) in cows results in severe human and livestock infections. Conventional approaches to vaccine development have yielded only a few noneffective vaccines against MRSA or IMI strains, so there is a need for improved vaccine development. CD4 T lymphocytes are required for promoting gamma interferon (IFN-γ) mediated immunoglobulin isotype switching in B lymphocytes to produce high-affinity IgG antibodies and IFN-γ-mediated phagocyte activation for an effective resolution of bacterial infection. However, the lack of known CD4 T cell antigens from S. aureus has made it difficult to design effective vaccines. The goal of this study was to identify S. aureus proteins recognized by immune CD4 T cells. Using a reverse genetics approach, 43 antigens were selected from the S. aureus Newman strain. These included lipoproteins, proteases, transcription regulators, an alkaline shock protein, conserved-domain proteins, hemolysins, fibrinogen-binding protein, staphylokinase, exotoxin, enterotoxin, sortase, and protein A. Screening of expressed proteins for recall T cell responses in outbred, immune calves identified 13 proteins that share over 80% sequence identity among MRSA or IMI strains. These may be useful for inclusion in a broadly protective multiantigen vaccine against MRSA or IMI.
Clinical and vaccine immunology: CVI 02/2012; 19(4):477-89. · 2.37 Impact Factor
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ABSTRACT: Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) is an important cause of food and waterborne illness in the developed countries. Cattle are a reservoir host of EHEC O157 and a major source of human exposure through contaminated meat products. Shiga toxins (Stxs) are an important pathogenicity trait of EHEC O157. The insertion sites of the Stx-encoding bacteriophages differentiate EHEC O157 isolates into genogroups commonly isolated from cattle but rarely from sick humans (bovine-biased genotypes [BBG]) and those commonly isolated from both cattle and human patients (clinical genotypes [CG]). Since BBG and CG share the cardinal virulence factors of EHEC O157 and are carried by cattle at similar prevalences, the infrequent occurrence of BBG among human disease isolates suggests that they may be less virulent than CG. We compared the virulence potentials of human and bovine isolates of CG and BBG in newborn conventional pig and weaned Dutch Belted rabbit models. CG-challenged piglets experienced severe disease accompanied by early and high mortality compared to BBG-challenged piglets. Similarly, CG-challenged rabbits were likely to develop lesions in kidney and intestine compared with the BBG-challenged rabbits. The CG strains used in this study carried stx2 and produced significantly higher amounts of Stx, whereas the BBG strains carried the stx2c gene variant only. These results suggest that BBG are less virulent than CG and that this difference in virulence potential is associated with the Stx2 subtype(s) carried and/or the amount of Stx produced.
Infection and immunity 01/2012; 80(1):369-80. · 4.21 Impact Factor
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ABSTRACT: Mycobacterium avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease (JD) in ruminants, has been implicated in the pathogenesis of Crohn's disease (CD) in humans. We developed a bovine ileal cannulation model to facilitate comparison of the immune response to Map and the mechanisms of pathogenesis in cattle and humans. Initial studies showed a T cannula could be maintained for up to a year in calves without inducing inflammation or adversely affecting intestinal function. Map introduced through the cannula established a persistent low level of infection without inflammation. Infection elicited an immune response to Map antigens detectable by flow cytometry. Further studies now show the cannulation model can be used with cows during the later stage of infection, affording access to the target tissue at all stages of infection. The studies also revealed no difference in infectivity or immunogenicity of isolates of Map obtained from cattle or humans with CD. Comparison of the immune response to Map during the early and late stages of infection using PCR, flow cytometry and QRT-PCR, showed the immune response early in the disease process is dominated by CD4 T cells. A CD8 response is delayed but comparable at later stages of infection. Genes for pro-inflammatory cytokines IFN-γ and the recently identified genes encoding IL-17 and IL-22 are up regulated in infected animals. These findings reveal that both human and bovine isolates of Map can establish infection and induce similar immune responses in a bovine model. They also reveal the cytokine responses elicited in cattle are similar to those implicated in CD pathogenesis.
Veterinary Immunology and Immunopathology 06/2011; 141(3-4):258-66. · 2.08 Impact Factor
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ABSTRACT: A 10-year-old Akita mix became acutely paraplegic. Upon magnetic resonance imaging, multiple, slightly T2-hyperintense, T1-isointense extradural masses, relative to spinal cord were found in the vertebral canal. The retroperitoneal masses had mixed T2-signal intensity. The contrast enhancement pattern for the spinal masses was both homogenous and heterogenous. The diagnosis was metastatic pheochromocytoma. Signal intensity of the tumors in this dog was similar to reports of pheochromocytoma in human beings.
Veterinary Radiology & Ultrasound 05/2011; 52(5):534-7. · 1.08 Impact Factor
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ABSTRACT: The ABCB gene subfamily of ABC (ATP-binding cassette) transporters is responsible for transporting a wide spectrum of molecules including peptides, iron, bile salts, drugs, and phospholipids. In humans, ABCB4 appears to be exclusively expressed on the apical membrane of hepatocytes where it translocates phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane. Functional alterations in the ABCB4 transporter are associated with a number of cholestatic syndromes in humans. Because of its role in biliary lipid homeostasis in humans, investigation of the ABCB4 gene in dogs is warranted. Thus, the full cDNA sequence of canine ABCB4 was elucidated and its mRNA and protein expression levels in tissues were determined. Canine ABCB4 consists of 3804 nucleotides spanning 26 exons and is 89% identical to human ABCB4. Expression of ABCB4 in canine liver supports a potential role for the protein in normal biliary function similar to that in humans. The function of ABCB4 expressed in brain tissue has yet to be determined.
Research in Veterinary Science 02/2010; 89(1):65-71. · 1.65 Impact Factor
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Christine G Elsik,
Ross L Tellam,
Kim C Worley,
Richard A Gibbs,
Donna M Muzny,
George M Weinstock,
David L Adelson,
Evan E Eichler,
Laura Elnitski,
Roderic Guigó, [......],
Ashley J Waardenberg,
Zhiquan Wang,
Robert Ward,
Rosemarie Weikard,
Thomas H Welsh,
Stephen N White,
Laurens G Wilming,
Kris R Wunderlich,
Jianqi Yang,
Feng-Qi Zhao
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ABSTRACT: To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production.
Science 05/2009; 324(5926):522-8. · 31.20 Impact Factor
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ABSTRACT: To report and compare the clinical diagnosis, surgical treatment, histopathologic changes, and outcomes of dogs with mineralized and nonmineralized supraspinatus tendinopathy (ST).
Case series.
Dogs (n=24) with ST.
Medical records (1995-2006) of dogs with ST that had surgical treatment were reviewed. Results of clinical examination, diagnostic imaging, surgery, histopathology of resected tendon tissue, and outcome were compared between dogs with mineralized and nonmineralized ST.
There were 15 dogs with mineralized ST and 9 with nonmineralized ST. Chronic, unilateral, intermittent or waxing-waning lameness, and pain elicited on palpation of the cranial aspect of the shoulder were the most consistent findings. On ultrasonographic or magnetic resonance imaging (MRI) of 35 shoulders, enlargement of the supraspinatus tendon (54%), increased fluid content (63%), and medial displacement of the biceps tendon (60%) were observed. Eleven of 12 dogs with bilateral abnormalities only had unilateral lameness. Surgery was performed in 30 shoulders. Resected tendon specimens had myxomatous degeneration and/or cartilaginous metaplasia in 11 of 13 dogs in the mineralized group and all 9 dogs in the nonmineralized group. Functional outcome after surgery was poor in 3 dogs and good-to-excellent in 16.
Mineralized and nonmineralized ST have many similarities. Although lameness is usually unilateral, the supraspinatus tendon may be affected bilaterally.
Ultrasonography and MRI are good imaging techniques for detection of ST especially the nonmineralized form. Surgical treatment results in good recovery of limb function. Nonmineralized ST is a recently described disorder in dogs and evaluation of more cases is necessary to determine outcome after surgical or medical treatment.
Veterinary Surgery 05/2009; 38(3):380-7. · 1.26 Impact Factor
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ABSTRACT: An ileal cannulation model was developed in conjunction with a flow cytometric assay to gain a better understanding of the mechanisms of immunopathogenesis of Johne's disease caused by Mycobacterium avium subsp. paratuberculosis. Initial studies with calves showed that M. avium subsp. paratuberculosis DNA is detectable by PCR in ileal biopsies during the first months following experimental infection. Inflammatory lesions were not detected on endoscopic evaluation up to 8 months postexperimental infection. M. avium subsp. paratuberculosis DNA was detected in multiple tissues at necropsy 8 months postinfection. Examination of the activation status of epithelial lymphocytes from the jejunum and ileum from infected and control animals at necropsy revealed that none of the major subsets of lymphocytes (NK, CD2(+), and CD2(-) gammadelta T lymphocytes, or CD4 and CD8 alphabeta T lymphocytes) expressed activation molecules CD25, CD26, CD71, ACT1, or ACT16. Subsets of CD4 and CD8 T lymphocytes from control and infected animals expressed CD26. The majority of CD4 and CD8 T lymphocytes expressed CD45R0, the memory T-lymphocyte marker. An immune response to M. avium subsp. paratuberculosis was detected by 3 months postinfection, dominated by a strong proliferative response of CD4 memory T lymphocytes. The findings indicate an immune response develops following initial exposure to M. avium subsp. paratuberculosis that controls but does not eliminate the pathogen. This persistence of M. avium subsp. paratuberculosis possibly leads to erosion and dysregulation of protective immunity at later time points postinfection. Continuous access to the ileum offers an opportunity to elucidate the cellular and molecular events leading to immune dysregulation and development of chronic inflammatory ileitis.
Clinical and vaccine immunology: CVI 03/2009; 16(4):453-63. · 2.37 Impact Factor
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ABSTRACT: Acquired T cell immunity is central for protection against infection. However, the immunological consequences of exposing memory T cells to high Ag loads during acute and persistent infection with systemic pathogens are poorly understood. We investigated this by using infection with Anaplasma marginale, a ruminant pathogen that replicates to levels of 10(9) bacteria per ml of blood during acute infection and maintains mean bacteremia levels of 10(6) per ml during long-term persistent infection. We established that immunization-induced Ag-specific peripheral blood CD4(+) T cell responses were rapidly and permanently lost following infection. To determine whether these T cells were anergic, sequestered in the spleen, or physically deleted from peripheral blood, CD4(+) T lymphocytes from the peripheral blood specific for the major surface protein (MSP) 1a T cell epitope were enumerated by DRB3*1101 tetramer staining and FACS analysis throughout the course of immunization and challenge. Immunization induced significant epitope-specific T lymphocyte responses that rapidly declined near peak bacteremia to background levels. Concomitantly, the mean frequency of tetramer(+)CD4(+) cells decreased rapidly from 0.025% before challenge to a preimmunization level of 0.0003% of CD4(+) T cells. Low frequencies of tetramer(+)CD4(+) T cells in spleen, liver, and inguinal lymph nodes sampled 9-12 wk postchallenge were consistent with undetectable or unsustainable Ag-specific responses and the lack of T cell sequestration. Thus, infection of cattle with A. marginale leads to the rapid loss of Ag-specific T cells and immunologic memory, which may be a strategy for this pathogen to modulate the immune response and persist.
The Journal of Immunology 01/2009; 181(11):7759-69. · 5.79 Impact Factor
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ABSTRACT: The ability to rapidly screen a complex pathogen proteome for proteins that elicit recall T-lymphocyte responses from immune individuals would accelerate vaccine development. An outer membrane fraction of the rickettsial pathogen Anaplasma marginale induces protective immunity against infection and disease in cattle. We have used this immunization model to evaluate high-throughput screening of proteins expressed by in vitro transcription and translation (IVTT) for recognition by memory CD4(+) T-lymphocytes. Fifty selected vaccine candidate antigens identified from the A. marginale genome were expressed from transcriptionally active PCR products using an Escherichia coli-based IVTT system, and bead-affinity purified using antibodies to His and FLAG epitope tags. IVTT-expressed bead-bound antigens were processed and presented by antigen presenting cells to T-lymphocytes from outer membrane immunized animals and evaluated for immunogenicity in proliferation assays. Antigens that consistently stimulated responses were known T-cell antigens major surface protein (MSP)2, MSP3, VirB9, and VirB10 and newly identified T-cell antigens outer membrane protein (OMP)4, OMP9, elongation factor-Tu, Ana29, and OMA87. Specific T-cell stimulation was achieved even at low antigen concentration, and was highly sensitive when compared with unbound IVTT reaction products. This method allows rapid expression and identification of T-lymphocyte antigens for any pathogen for which the genome sequence is available.
Journal of Immunological Methods 04/2008; 332(1-2):129-41. · 2.20 Impact Factor
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ABSTRACT: 6 healthy dogs given human albumin solution as part of a study were examined following development of an immediate hypersensitivity reaction (1 dog) and signs suggestive of a type III hypersensitivity reaction (all 6 dogs).
All 6 dogs were healthy prior to administration of human albumin solution. One dog developed signs of an immediate hypersensitivity reaction, characterized by vomiting and facial edema, during administration of human albumin solution. All 6 dogs developed signs of a delayed adverse reaction 5 to 13 days after administration of human albumin solution. Initial clinical signs included lethargy, lameness, edema, cutaneous lesions indicative of vasculitis, vomiting, and inappetance.
In the dog with signs of immediate hypersensitivity, signs resolved after administration of human albumin solution was discontinued and diphenhydramine was administered. Supportive treatment was provided after dogs developed signs of a delayed adverse reaction. Four dogs recovered, but 2 dogs died despite treatment. All 6 dogs were found to have antihuman albumin antibodies. There was no evidence of contamination of the human albumin solution.
Findings suggest that administration of human albumin solution in healthy dogs with normal serum albumin concentrations may result in signs of a type III hypersensitivity reaction.
Journal of the American Veterinary Medical Association 04/2007; 230(6):873-9. · 1.79 Impact Factor
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ABSTRACT: The functions of gammadelta T cells are enigmatic, and these cells are often considered as evolutionary remnants of well-characterized alphabeta T cells. However, their conservation throughout evolution suggests that gammadelta T cells are biologically unique. In ruminants, gammadelta T cells expressing the workshop cluster 1 (WC1) scavenger receptor comprise a large proportion of circulating lymphocytes, suggesting these cells are biologically relevant and functionally different from alphabeta T cells. In fact, bovine WC1(+) gammadelta T cells can act as APC for alphabeta T cells, indicating they may express genes encoding proteins associated with innate immunity. The present study was designed to compare immune function gene expression profiles of clonal populations of WC1(+) gammadelta and CD4(+) alphabeta T cells derived from the same animal, which respond to major surface protein 2 (MSP2) of the intraerythrocytic rickettsial pathogen of cattle, Anaplasma marginale. Gene expression profiles of activated T cell clones were compared using a microarray format, and differential gene expression was confirmed by real-time RT-PCR and protein analyses. We demonstrate that although MSP2-specific alphabeta and gammadelta T cell clones express many of the same genes, gammadelta T cell clones express high levels of genes associated with myeloid cells, including chemokines CCL2, CXCL1, CXCL2, CXCL6, and surface receptors CD68, CD11b, macrophage scavenger receptor 1, macrophage mannose receptor, and galectin-3. It is important that many of these genes were also expressed at higher levels in polyclonal WC1(+) gammadelta T cells when compared with CD4(+) alphabeta T cells selected from peripheral blood.
Journal of Leukocyte Biology 11/2006; 80(4):939-52. · 4.99 Impact Factor
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ABSTRACT: WC1 molecules are implicated in augmenting cellular activation as well as inducing cell cycle arrest of gammadelta T cells. Since WC1 is a large multigene family differences in outcome could result from modulation of different WC1 molecules. To further investigate this family of molecules, peripheral blood WC1(+) gammadelta T cell subpopulations were evaluated by 2-D Western blotting and RT-PCR. We found 13 cDNA intracytoplasmic tail sequences with differences in signaling motifs among them and at least 20 biochemically distinguishable WC1 spots associated with cell membranes, with some in lipid rafts. An understanding of the diversity of 2-D spots could not be resolved by evaluating T cell clones, removing sialyated carbohydrates or blotting with anti-WC1.1 or anti-WC1.2-specific antibodies. Nevertheless, while the major gammadelta T cell subpopulations in blood (WC1.1(+)/WC1.2(-) and WC1.2(+)/WC1.1(-)) both had complex 2-D patterns, virtually all spots associated with WC1.2(+)/WC1.1(-) cells bore the WC1.2 epitope, distinguishing them from the WC1.1(+) cells.
Cellular Immunology 03/2006; 239(2):151-61. · 1.97 Impact Factor
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ABSTRACT: A 4-year-old spayed female Australian Cattle Dog (Blue Heeler) was evaluated because of right forelimb lameness of 5 months' duration. Orthopedic evaluation revealed signs of pain localized to the cranial aspects of both shoulder joints. Via magnetic resonance imaging, the mass of the supraspinatus tendon insertion in both shoulder joints was increased, compared with findings in cadavers of clinically normal dogs; additional imaging procedures revealed that, compared with clinically normal tendons, the tendon had increased signal intensity that was consistent with increased fluid content. The increased supraspinatus tendon mass in each shoulder joint was associated with medial displacement of the biceps brachii tendon, which was more severe in the right limb. Arthroscopic evaluations of both shoulder joints revealed no abnormalities. The dog underwent surgery, and the abnormal parts of the tendons were resected. The most prominent finding on histologic examination of excised tissues was severe myxomatous degeneration. The lameness resolved, and at 22 months after surgery, the dog was reported to have had no recurrence of lameness. The clinical signs and histologic appearance of the tendons in this dog strongly resemble findings associated with tendinosis in humans. Decompression of the biceps brachii tendon may have contributed to the successful outcome after surgery in this dog. Supraspinatus tendinosis should be considered among the differential diagnoses in dogs with uni- or bilateral forelimb lameness.
Journal of the American Veterinary Medical Association 12/2005; 227(9):1429-33, 1416. · 1.79 Impact Factor
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ABSTRACT: Major surface protein 2 (MSP2) of the bovine rickettsial pathogen Anaplasma marginale is an abundant, serologically immunodominant outer membrane protein. Immunodominance partially results from numerous CD4+ T cell epitopes in highly conserved amino and carboxy regions and the central hypervariable region of MSP2. However, in long-term cultures of lymphocytes stimulated with A. marginale, workshop cluster 1 (WC1)+ gammadelta T cells and CD4+ alphabeta T cells proliferated, leading to a predominance of gammadelta T cells. As gammadelta T cells proliferate in A. marginale-stimulated lymphocyte cultures, this study hypothesized that gammadelta T cells respond to the abundant, immunodominant MSP2. To test this hypothesis, gammadelta T cell clones were isolated from MSP2 vaccinates and assessed for antigen-specific proliferation and interferon-gamma secretion. Seven WC1+ gammadelta T cell clones responded to A. marginale and MSP2, and three of these proliferated to overlapping peptides from the conserved carboxy region. The gammadelta T cell response was not major histocompatibility complex-restricted, although it required antigen-presenting cells and was blocked by addition of antibody specific for the T cell receptor (TCR). Sequence analysis of TCR-gamma and -delta chains of peripheral blood lymphocytes identified two novel TCR-gamma chain constant (Cgamma) regions. It is important that all seven MSP2-specific gammadelta T cell clones used the same one of these novel Cgamma regions. The TCR complementarity-determining region 3 was less conserved than those of MSP2-specific CD4+ alphabeta T cell clones. Together, these data indicate that WC1+ gammadelta T cells recognize A. marginale MSP2 through the TCR and contribute to the immunodominant response to this protein.
Journal of Leukocyte Biology 03/2005; 77(2):199-208. · 4.99 Impact Factor
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ABSTRACT: WC1 molecules are implicated in augmenting cellular activation as well as inducing cell cycle arrest of γδ T cells. Since WC1 is a large multigene family differences in outcome could result from modulation of different WC1 molecules. To further investigate this family of molecules, peripheral blood WC1+ γδ T cell subpopulations were evaluated by 2-D Western blotting and RT-PCR. We found 13 cDNA intracytoplasmic tail sequences with differences in signaling motifs among them and at least 20 biochemically distinguishable WC1 spots associated with cell membranes, with some in lipid rafts. An understanding of the diversity of 2-D spots could not be resolved by evaluating T cell clones, removing sialyated carbohydrates or blotting with anti-WC1.1 or anti-WC1.2-specific antibodies. Nevertheless, while the major γδ T cell subpopulations in blood (WC1.1+/WC1.2− and WC1.2+/WC1.1−) both had complex 2-D patterns, virtually all spots associated with WC1.2+/WC1.1− cells bore the WC1.2 epitope, distinguishing them from the WC1.1+ cells.
Cellular Immunology.
