@article{meichner_stokol_tarigo_avery_burkhard_comazzi_fogle_stowe_rütgen_seelig_et al._2020, title={Multicenter flow cytometry proficiency testing of canine blood and lymph node samples}, volume={49}, ISSN={0275-6382}, url={http://dx.doi.org/10.1111/vcp.12843}, DOI={10.1111/vcp.12843}, abstractNote={Abstract}, number={2}, journal={Veterinary Clinical Pathology}, publisher={Wiley}, author={Meichner, Kristina and Stokol, Tracy and Tarigo, Jaime and Avery, Anne and Burkhard, Mary J. and Comazzi, Stefano and Fogle, Jonathan and Stowe, Devorah Marks and Rütgen, Barbara and Seelig, Davis and et al.}, year={2020}, month={Apr}, pages={249–257} }
 @article{howard_burkhard_2007, title={FIV infection induces unique changes in phenotype and cellularity in the medial iliac lymph node and intestinal IEL}, volume={23}, ISSN={["1931-8405"]}, DOI={10.1089/aid.2006.0025}, abstractNote={Studies of human immunodeficiency virus-1 (HIV-1)-infected patients and simian immunodeficiency virus (SIV)-infected macaques have identified profound depletion of CD4(+) T cells and expansion of CD8(+) T cells in the gastrointestinal lamina propria. Less attention has been given to CD8(+) intraepithelial lymphocytes (IEL), and no studies have concurrently examined inductive sites such as draining lymph nodes. Our preliminary data in the feline immunodeficiency virus (FIV) animal model suggested additional changes in IEL, and marked differences in the responses of lymph nodes draining different mucosal sites. To address this, we quantified the absolute leukocyte yield and examined the phenotype of cells from small intestinal IEL, mesenteric lymph node (MLN), and medial iliac lymph node (ILN) from chronically FIV-infected cats. The cellularity of the ILN was increased 530% in FIV-infected animals with an expansion of CD62L(+) cells, suggesting an increased population of naive T cells. The number of CD4(+), as well as CD8(+), T cells was increased in the ILN, resulting in a CD4:CD8 ratio greater than 1:1. In contrast, reduced cellularity, specific loss of CD4(+) T cells, and inversion of the CD4:CD8 ratio was observed in the MLN, which drains the intestine. In IEL, loss of CD8alpha, CD8beta, and CD4-expressing T cells was found in FIV-infected cats. Furthermore, expression intensity of CD8alpha and CD5, markers known to be important in T cell function, was markedly decreased on IEL. These findings expand the array of immune alterations induced by lentiviral infection and indicate that characterization of multiple mucosal sites will be necessary to fully understand the pathogenesis of HIV-1 infection.}, number={5}, journal={AIDS RESEARCH AND HUMAN RETROVIRUSES}, author={Howard, Kristina E. and Burkhard, Mary Jo}, year={2007}, month={May}, pages={720–728} }
 @article{howard_fisher_dean_burkhard_2005, title={Methodology for isolation and phenotypic characterization of feline small intestinal leukocytes}, volume={302}, ISSN={["1872-7905"]}, DOI={10.1016/j.jim.2005.04.019}, abstractNote={Critical assessment of intestinal immune responses requires the ability to characterize leukocytes from different anatomic locations as leukocytes from inductive sites such as Peyer's patches and lymphoid follicles vary significantly from their effector counterparts, intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL). This study describes (1) methods developed to isolate specific intestinal leukocyte populations with high yield and purity, (2) difficulties encountered in establishing a panel of monoclonal antibodies to assess phenotype, and (3) the phenotypic characterization of effector and inductive sites in the feline small intestine. We found that the phenotypic distribution of feline intestinal leukocytes was similar to that found in other species such as humans, macaques and mice. The majority of IEL were CD5(+) T-cells with less than 7% B-cells. CD8(+) T-cells comprised approximately 60% of the IEL with roughly half displaying CD8alphaalpha homodimers. Approximately 10% of IEL were CD4(+) T-cells. In the LPL, CD4(+) T-cells predominated at 42%, with 33% CD8(+) T-cells and 10% B-cells. As would be expected, B-cells predominated in Peyer's patches with 40% B-cells, 28% CD4(+) T-cells and 20% CD8(+) T-cells. Increased MHCII expression was found in the Peyer's patches as compared to the IEL and LPL. B7.1 expression was significantly higher in mucosal leukocyte populations as compared to organized lymphoid tissue in the periphery with expression detected on 65% of IEL and 53% of LPL. Plasma cells were found in all regions of small intestine examined with greater numbers in lamina propria and Peyer's patches. Lymphoblasts were only identified in inductive tissue. In general, no differences were found between the phenotype of mucosal leukocyte populations from specific pathogen free or random source cats. However, the percentage of CD4(+) CD25(+) T-cells was significantly greater in both IEL and LPL from random source animals. This study provides techniques and a baseline from which future studies of the feline intestinal immune system can be conducted.}, number={1-2}, journal={JOURNAL OF IMMUNOLOGICAL METHODS}, author={Howard, KE and Fisher, IL and Dean, GA and Burkhard, MJ}, year={2005}, month={Jul}, pages={36–53} }
 @article{sirriyah_dean_lavoy_burkhard_2004, title={Assessment of CD4+ and CD8+ IFN-gamma producing cells by ELISPOT in naive and FIV-infected cats}, volume={102}, ISSN={["1873-2534"]}, DOI={10.1016/j.vetimm.2004.06.011}, abstractNote={IFN-gamma is critical for the development of antiviral cell-mediated immunity in HIV infected humans and FIV infected cats. The ELISPOT has proven to be a technically straightforward assay to quantify the number of IFN-gamma producing cells and offers a reasonable alternative for the quantitative measurement of T-cell function in cats. We used a feline-specific ELISPOT to identify constitutive as well as Con A stimulated IFN-gamma production in T-cell subsets and determine if there were differences between purified (positively sorted) and negatively depleted populations from naïve and FIV infected cats. We found no difference in the total number of PBMC constitutively producing IFN-gamma in naïve and FIV+ cats. Con A exposure was associated with increased numbers of IFN-gamma producing PBMC in naïve, but not FIV+, cats. Equivalent numbers of CD4+ and CD8+ T cells constitutively expressed IFN-gamma in naïve cats. However, in FIV+ cats, the number of IFN-gamma producing CD8+ T-cells was approximately two-fold over that seen for CD4+ T-cells. We found minimal differences between purified (e.g. CD4+ or CD8+) and corresponding depleted (e.g. CD8- or CD4-) populations in samples from FIV+ cats. In contrast, depleted populations from naïve cats showed greater response to Con A than did purified populations. Thus, while determination of the number of IFN-gamma producing cells by feline-specific ELISPOT is a useful tool for the evaluation of the feline immune response, determination of the initial sample population and T-cell subset is critical for optimal interpretation of the IFN-gamma ELISPOT.}, number={1-2}, journal={VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY}, author={Sirriyah, J and Dean, GA and LaVoy, A and Burkhard, MJ}, year={2004}, month={Nov}, pages={77–84} }
 @article{stevens_howard_nordone_burkhard_dean_2004, title={Oral immunization with recombinant Listeria monocytogenes controls virus load after vaginal challenge with feline immunodeficiency virus}, volume={78}, ISSN={["1098-5514"]}, DOI={10.1128/JVI.78.15.8210-8218.2004}, abstractNote={ABSTRACT}, number={15}, journal={JOURNAL OF VIROLOGY}, author={Stevens, R and Howard, KE and Nordone, S and Burkhard, M and Dean, GA}, year={2004}, month={Aug}, pages={8210–8218} }
 @article{dean_lavoy_burkhard_2004, title={Peptide mapping of feline immunodeficiency virus by IFN-gamma elispot}, volume={100}, ISSN={["1873-2534"]}, DOI={10.1016/j.vetimm.2004.03.001}, abstractNote={Interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISPOT) has become an important tool in studying antigen-specific T lymphocyte responses. Soluble peptides can be used to map T-cell epitopes, providing information that is useful in the design and evaluation of vaccines as well as studies of immunopathogenesis. To date, this assay has not been widely utilized in feline immunodeficiency virus (FIV) research. We have developed a feline IFN-γ ELISPOT assay and used it to determine FIV-specific T-cell epitopes recognized by infected cats. A panel of 331 peptides, 15 amino acids in length and overlapping by 10 residues was synthesized. The peptide library spanned the FIV structural (Gag), envelope (Env), reverse transcriptase (RT), and open-reading-frame A (OrfA) proteins. Initially, 34 pools, containing 7–10 peptides each were screened by IFN-γ ELISPOT against peripheral blood mononuclear cells (PBMC) from eight cats chronically infected with the NCSU1 molecular clone of FIV and four uninfected control cats. Individual peptides from pools recognized by FIV+ cats were then evaluated and optimal peptides were combined into pools representing Gag, Env, RT, and OrfA. A higher percentage of FIV infected cats were identified as responders against the peptide pools when using fresh PBMC as compared to cryopreserved PBMC. In vitro restimulation of cryopreserved PBMC with the peptide pools improved the sensitivity of the assay to similar levels as observed from fresh samples. Individual peptides used in the pools were generally found to stimulate CD8+ T-cells more efficiently than CD4+ T-cells. Comparison of the peptide sequences to representative FIV sequences from clades A–D showed conservation was high among Gag and RT peptides, variable among Env peptides and low for OrfA peptides. The IFN-γ ELISPOT assay and FIV-specific peptide pools we describe here will be useful in assessing cell-mediated responses to experimental FIV vaccines.}, number={1-2}, journal={VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY}, author={Dean, GA and LaVoy, A and Burkhard, MJ}, year={2004}, month={Jul}, pages={49–59} }
 @misc{burkhard_dean_2003, title={Transmission and immunopathogenesis of FIV in cats as a model for HIV}, volume={1}, ISSN={["1873-4251"]}, DOI={10.2174/1570162033352101}, abstractNote={The feline immunodeficiency virus (FIV) model provides a system to study lentivirus transmission, virus kinetics, pathogenesis, host responses, and immune dysfunction in a natural, out-bred host, under controlled conditions with specific-pathogen-free animals. The diversity of primary FIV strains can be exploited to mirror the range of disease manifestations associated with HIV infection. FIV is infectious via intravenous, intraperitoneal, intradermal, or subcutaneous injection as well as by atraumatic instillation onto the oral, vaginal, or rectal mucosa. Together, these features allow investigators to model specific aspects of HIV infection in a highly relevant and relatively inexpensive animal model. Well-developed areas of the FIV model include: (1) transmission of cell-associated as well as cell-free virus; (2) mucosal infectivity and immunopathogenesis; (3) vertical transmission; (4) acquired immunodeficiency including defects of the innate immune system; (5) thymic dysfunction; (6) neurotropism and neuropathogenesis; (7) host-virus interactions and the role of specific gene products; (8) efficacy of antiviral therapy; and (9) efficacy and immune correlates of experimental vaccines. This review will encompass areas specific to transmission and immunopathogenesis.}, number={1}, journal={CURRENT HIV RESEARCH}, author={Burkhard, MJ and Dean, GA}, year={2003}, month={Jan}, pages={15–29} }
 @inproceedings{de voe_flammer_degernes_burkhard_2002, title={Blood lactate: potential applications in avian medicine}, booktitle={Proceedings Annual Conference of the Association of Avian Veterinarians}, author={De Voe, R. and Flammer, K. and Degernes, L. and Burkhard, M. J.}, year={2002}, pages={19–22} }
 @article{burkhard_valenski_leavell_dean_tompkins_2002, title={Evaluation of FIV protein-expressing VEE-replicon vaccine vectors in cats}, volume={21}, ISSN={["0264-410X"]}, DOI={10.1016/S0264-410X(02)00455-3}, abstractNote={Venezuelan equine encephalitis (VEE) virus-replicon particles (VRP) were used to generate feline immunodeficiency virus (FIV) Gag- and ENV-expressing vaccine vectors. Serum and mucosal FIV-specific antibody was detected in cats immunized subcutaneously, once monthly for 5 months, with FIV-expressing VRP. Expansion of the CD8+ L-selectin negative phenotype and transient CD8+ noncytolytic suppressor activity were seen in cats immunized with FIV-expressing or control VRP. Despite induction of FIV-specific immune responses and nonspecific suppressor responses, all cats became infected following vaginal challenge with high dose, pathogenic cell-associated FIV-NCSU(1) although relative early maintenance of CD4+ cells was seen in FIV-immunized cats.}, number={3-4}, journal={VACCINE}, author={Burkhard, MJ and Valenski, L and Leavell, S and Dean, GA and Tompkins, WAF}, year={2002}, month={Dec}, pages={258–268} }
 @article{burkhard_mathiason_k o'halloran_hoover_2002, title={Kinetics of early FIV infection in cats exposed via the vaginal versus intravenous route}, volume={18}, ISSN={["1931-8405"]}, DOI={10.1089/08892220252781284}, abstractNote={To determine the influence of route of virus exposure on early pathogenesis of feline immunodeficiency virus (FIV) infection, cats were exposed to either of two FIV isolates (FIV-B-2542 or FIV-A-PPR) by vaginal or intravenous (IV) inoculation. Exposure to either virus clade by either route of inoculation resulted in vaginal and systemic infection. Peak plasma viremia and tissue proviral burden were 1-3 log(10) greater in cats infected with FIV-B-2542 vs. FIV-A-PPR, irrespective of inoculation route. Plasma RNA levels paralleled provirus titers in FIV-B-2542-infected cats and were highest in those exposed IV. In contrast, plasma RNA titers were higher in cats infected vaginally with FIV-A-PPR than in those infected IV. Despite early differences, PBMC provirus titers were similar in all groups by 9 weeks postinfection. In cats infected IV, but not vaginally, CD4(+) lymphocyte counts declined significantly independent of the magnitude of viremia. Mitogen-induced lymphoproliferation was decreased in all infected cats regardless of CD4(+) cell counts; this decline correlated with the magnitude of peak plasma viremia in FIV-B-2542, but not FIV-A-PPR, infected cats. These results establish that the kinetics of early FIV infection differ with route of exposure as well as virus isolate and that properties extrapolated from one virus isolate may not be universal.}, number={3}, journal={AIDS RESEARCH AND HUMAN RETROVIRUSES}, author={Burkhard, MJ and Mathiason, CK and K O'Halloran and Hoover, EA}, year={2002}, month={Feb}, pages={217–226} }
 @article{burkhard_brown_mcgrath_meador_mayle_keaton_hoffman_zimmermann_abbott_sun_2001, title={Evaluation of the erythroid regenerative response in two different models of experimentally induced iron deficiency anemia}, volume={30}, ISSN={["1939-165X"]}, DOI={10.1111/j.1939-165X.2001.tb00262.x}, abstractNote={Abstract:Anemia was induced in weanling Sprague Dawley rats either by feeding an iron‐deficient diet or by chronic phlebotomy. The erythroid regenerative response was then evaluated before and after a hemolytic event, and results were compared with those of a third group of control nonphlebotomized rats fed an iron‐replete diet. Diet and phlebotomy groups developed a similar degree of anemia (mean hemoglobin concentration 7.9 g/dL and 7.8 g/dL, respectively; controls, 13.9 g/dL) and hypoferremia (mean serum iron concentration 25.4 μg/dL and 34.9 μg/dL, respectively; controls, 222.0 μg/dL). However, the anemia in diet rats was nonregen‐erative (reticulocyte count, 83.1×103cells/μL) and associated with bone marrow erythroid hypoplasia; whereas the anemia in phlebotomy rats was regenerative (reticulocyte count, 169.6×103cells/μL) and associated with bone marrow erythroid hyperplasia. Thrombocytosis was seen in diet rats (l,580×103cells/μL) but not phlebotomy rats (901×103cells/μL) when compared with controls (809×103cells/μL). To further evaluate the regenerative capability, phenylhydrazine (PHZ) was administered to induce hemolysis. Erythrocyte mass declined approximately 25% in all groups, including controls. The reticulocytosis (265.3×103cells/μL) seen in phlebotomy rats was earlier and significantly greater than that seen in either diet or control rats. Hemoglobin concentration returned to pre‐PHZ concentrations (7.9 g/dL) in phlebotomy rats within 4 days posthemolysis. In diet rats, the maximal regenerative response (176.3×103cells/μL) was not seen until 8 days posthemolysis, and hemoglobin (7.5 g/dL) did not return to pre‐PHZ concentrations during the 8‐day study. In many aspects, the anemia seen following diet‐or phlebotomy‐induced iron deficiency was similar. However, the erythroid regenerative capability varied depending on the mechanism by which anemia was induced and furthermore altered the efficiency of hemoglobin production following a hemolytic event. These results suggest that the availability of iron in the diet may modulate the pathogenesis of iron deficiency anemia.}, number={2}, journal={VETERINARY CLINICAL PATHOLOGY}, author={Burkhard, MJ and Brown, DE and McGrath, JP and Meador, VP and Mayle, DA and Keaton, MJ and Hoffman, WP and Zimmermann, JL and Abbott, DL and Sun, SC}, year={2001}, pages={76–85} }
 @article{burkhard_mathiason_bowdre_hoover_2001, title={Feline immunodeficiency virus Gag- and Env-specific immune responses after vaginal versus intravenous infection}, volume={17}, ISSN={["1931-8405"]}, DOI={10.1089/08892220152741469}, abstractNote={To better understand the correlation of mucosal and systemic immune responses with lentiviral containment, we contrasted the early mucosal and systemic immune responses induced by vaginal versus intravenous exposure of cats to feline immunodeficiency virus (FIV) isolates of differing pathogenicity and clade (i.e., FIV-B-2542 and FIV-A-PPR). We found that despite divergence in viral genotype, the mucosal and systemic immune responses induced differed more with route of exposure than virus isolate. In intravenously exposed cats, Gag-specific antibody (both IgG and IgA isotype) predominated in the serum, saliva, and vaginal wash fluid irrespective of infecting virus isolate. While Env-specific responses were more variable, they were more often detected in vaginally infected cats. Both IgG and IgA directed against Gag and Env were consistently present in vaginal wash fluids independent of route of infection or virus isolate. FIV Gag- and Env-specific cytotoxic lymphocytes (CTLs) were detected in blood and tissue lymphocytes of cats infected with either virus strain but were greatest in intravenously infected animals. Likewise, FIV-specific CTLs were detected in CD8(+) vaginal lymphocytes of animals infected by either route but were also more frequent in intravenously inoculated animals. In summary, we found qualitative differences in the immune responses following vaginal infection but no evidence (1) that mucosal immune responses were enhanced in vaginally exposed cats, (2) that local mucosal infection led to measurably greater immune responses in either compartment; or (3) that more prominent immune responses correlated with lower viral burden.}, number={18}, journal={AIDS RESEARCH AND HUMAN RETROVIRUSES}, author={Burkhard, MJ and Mathiason, CK and Bowdre, T and Hoover, EA}, year={2001}, month={Dec}, pages={1767–1778} }
 @article{rooney_burkhard_greiner_zeng_johnson_2001, title={Intestinal and blood parasites in Amazon parrots destined for relocation in Guatemala}, volume={32}, number={1}, journal={Journal of Zoo and Wildlife Medicine}, author={Rooney, M. B. and Burkhard, M. J. and Greiner, E. and Zeng, Q. Y. and Johnson, J.}, year={2001}, pages={71–73} }
 @article{burkhard_hoover_1999, title={Feline immunodeficiency virus (FIV): Clinical manifestations and management part 2}, volume={27}, number={1}, journal={Feline Practice}, author={Burkhard, M. J. and Hoover, E. A.}, year={1999}, pages={10–14} }
 @article{burkhard_hoover_1998, title={Feline Immunodeficiency Virus (FIV): Immunopathogenesis}, volume={26}, number={6}, journal={Feline Practice}, author={Burkhard, M. J. and Hoover, E. A.}, year={1998}, pages={10–13} }