Phenotypic characterization of equine monocyte-derived dendritic cells generated ex vivo utilizing commercially available serum-free medium.

The impact of culture conditions on equine monocyte-derived dendritic cells (MoDC) generation has not been fully characterized. We hypothesized that 1) MoDC could be cultured in a commercially available serum-free medium (AIM-V); and 2) that differential culture conditions would influence MoDC viability, yield and phenotype. MoDC generated from adult horses were cultured under variable conditions in a series of experiments. Viability was assessed using trypan blue and propidium iodide staining. Yield was determined by manual hemocytometer counting. Phenotype was assessed by flow cytometric analysis of surface markers (MHC class-II, CD86 and CD14). Data were analyzed using paired t-tests and repeated measures ANOVA. Two MoDC populations that differed in size and phenotype were identified: larger MoDC (LgMoDC) and smaller MoDC (SmMoDC). Medium type, plate chemistry, or length of monocyte adhesion time did not impact MoDC viability or yield. LgMoDC generated in serum-free medium expressed more MHC class-II and CD86 (P ≤ 0.03). A prolonged duration in culture reduced MoDC yield (P ≤ 0.04). MoDC can be consistently and reliably generated using AIM-V serum-free medium in standard tissue culture plates with a recommended culture duration of 3-4 days.

Rapid clinical progression of B-cell chronic lymphocytic leukemia in a horse.

CASE DESCRIPTION: A 17-year-old Friesian gelding was examined at a referral hospital because of a 1-month history of mild exercise intolerance and marked lymphocytosis. CLINICAL FINDINGS: Physical examination revealed no peripheral lymphadenopathy or other abnormalities. Results of an abdominal palpation examination per rectum and thoracic and abdominal ultrasonographic examinations were unremarkable. B-cell chronic lymphocytic leukemia (CLL) was diagnosed on the basis of severe lymphocytosis and positive expression of the B-cell marker CD20 by lymphocytes in the bone marrow and peripheral blood. TREATMENT AND OUTCOME: Treatment with prednisolone (2 mg/kg [0.9 mg/lb], PO, every other day) and chlorambucil (20 mg/m2, PO, every 3 weeks for 2 doses, then every 2 weeks) was initially associated with improvement in clinical signs and a decrease in the lymphocyte count. However, 3 weeks after administration of the first dose of chlorambucil, the lymphocyte count began to increase. One week later, the horse developed episodes of recurrent fever and the lymphocyte count continued to increase. Despite continued administration of the prednisolone-chlorambucil protocol, the horse's clinical condition deteriorated rapidly, and it was euthanized 6 weeks after initial examination at the referral hospital because of a poor prognosis. A necropsy was not performed. CLINICAL RELEVANCE: B-cell CLL has been infrequently described in horses. This report was the first to describe the use of chemotherapy, albeit unsuccessful, for the treatment of B-cell CLL in a horse. This information should be useful for guiding expectations for prognosis and management of other horses affected with the disease.

Successful management of hydrallantois in a Standardbred mare at term resulting in the birth of a live foal.

A 6-year-old Standardbred mare was presented at 339 days of gestation for investigation of abnormal abdominal distension and ventral edema. Transrectal palpation and ultrasound examination revealed the uterus to be enlarged with an excessive volume of fetal fluid, characteristic of hydrops. Gradual transcervical drainage of 55 L of allantoic fluid over 45 minutes, with concurrent intravenous fluid therapy followed by assisted vaginal delivery, resulted in the birth of a live foal with long-term survival. The birth and long-term survival of a foal from a mare with hydrallantois at term has not been previously reported in horses. However, this report demonstrates that successful outcome for both mare and foal may be achieved in a mare at term with hydrallantois.

Gestion réussie de l'hydrallantois chez une jument Standardbred à terme donnant lieu à la naissance d'un poulain vivant. Une jument Standardbred âgée de 6 ans a été présentée à 339 jours de gestation pour investiguer une distension abdominale anormale et un oedème ventral. La palpation transrectale et l'échographie ont révélé que l'utérus était enflé en raison d'un volume excessif de liquide foetal, ce qui est caractéristique de l'hydrops fetalis. Un drainage transcervical graduel de 55 L de liquide allantoïdien pendant plus de 45 minutes et une fluidothérapie par intraveineuse suivis d'une mise bas vaginale assistée ont donné lieu à la naissance d'un poulain vivant avec survie à long terme. La naissance et la survie à long terme d'un poulain provenant d'une jument atteinte de l'hydrallantois à terme n'avaient pas été précédemment signalées chez les chevaux. Cependant, des résultats fructueux pour la jument et le poulain peuvent être obtenus chez une jument atteinte d'hydrallantois à terme.(Traduit par Isabelle Vallières).

The effect of age on foal monocyte-derived dendritic cell (MoDC) maturation and function after exposure to killed bacteria.

Neonatal foals are uniquely susceptible to certain infections early in life. Dendritic cells (DC) are vital in the transition between the innate and adaptive immune response to infection, but DC biology in foals is not fully characterized. Monocyte-derived DC represent a suitable in vitro model similar to DC that differentiate from monocytes recruited from circulation. We hypothesized that foal monocyte-derived DC (MoDC) would exhibit age-dependent phenotypic and functional differences compared to adult horse MoDC. MoDC generated from 9 horses (collected once) and from 8 foals (collected at 1, 7, and 30 days-of-age) were exposed to killed whole cell Escherichia coli or Staphylococcus aureus bacteria. MoDC expression of MHC class II (MHC class-II), CD86, and CD14 were measured by flow cytometry, and supernatant cytokine concentrations of IL-4, IL-17, IFN-γ, and IL-10 were quantified with a validated immunoassay. The percentage of MoDC expressing MHC class-II and CD86 was lower and CD14 was higher for cells generated from 1-day-old foals compared to cells generated from adult horses (P < 0.0001). Bacterial exposure increased the percentage of cells expressing CD86 at all ages (P < 0.0001). Bacteria-exposed MoDC from 1-day-old foals produced significantly less IL-4, IL-17, and IFN-γ than adult MoDC produced in response to bacterial exposure (P ≤ 0.04). Following bacterial exposure, foal MoDC phenotype and cytokine secretion were different than those of mature horses. These differences could reduce the ability of foals to generate a protective immune response against bacterial infection.

Neuroborreliosis in a horse with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a rare condition in adult horses characterized by hypogammaglobulinemia and increased susceptibility to parasitic and bacterial infections, including recurrent respiratory diseases, septicemia, and meningitis. Lyme disease is often included as a differential diagnosis in CVID horses with signs of meningitis; however, the Borrelia burgdorferi organism has not been demonstrated previously within central nervous system tissues of CVID horses with neurologic disease, to our knowledge. We report herein a case of neuroborreliosis in a CVID horse, confirmed by combined immunologic testing, histopathology, real-time PCR assay, fluorescent in situ hybridization, and immunohistochemical staining. Implications of these findings include heightened monitoring of CVID horses for Lyme disease in endemic areas and appropriate therapy in the case of neurologic disease.

Multispectral fluorescence-activated cell sorting of B and T cell subpopulations from equine peripheral blood.

Immune phenotyping of equine peripheral blood mononuclear cells (PBMC) is commonly described by single or double marker labeling, which limits complex phenotypic descriptions and subpopulation identification. Our objective was to develop a new multispectral flow cytometry protocol to identify and sort equine lymphocyte subpopulations using commercially available, pre-conjugated monoclonal antibodies to cell surface markers. Two clones of anti-equine CD8 (CVS8 and CVS21) were compared in combination with CD3. Clone CVS21 bound non-T CD3- cells in addition to CD8+ T cells. Further analysis using co-labeling with CD3 and multiple B cell antibodies revealed that most of these CVS21+CD3- cells were B cells. Clone CVS8 and anti-equine CD4 clone CVS4 only labeled CD3+ cells and were mutually exclusive. To identify equine B-cell subpopulations, anti-equine Pan-Ig (clone CVS36), IgM (clone 1-22), and IgG1 (clone CVS45), as well as anti-human CD21 (clone B-ly4) were tested. Anti-equine Pan-Ig antibody labeled 88 ±â€¯7.6% of CD3- lymphocytes. Anti-equine IgM often produced a continuum of stain intensity of PBMC from several adult horses. Anti- IgG1 and -CD21 labeled overlapping subsets of CD3- cells. Based on these results, combined with conjugate availability, a final panel of anti-CD4 (CVS4), -CD8 (CVS8), -IgG1 (CVS45), and -CD21 (B-ly4) antibodies was used to sort CD4+ T cells, CD8+ T cells, and CD21+ and/or IgG1+ B cells simultaneously. The identity of the sorted populations was confirmed by qRT-PCR for cell lineage markers. The described analysis emphasizes the need for increased availability of reagents and use of multispectral flow for equine immunophenotypic analysis. This new multispectral flow cytometry protocol will facilitate studies on equine lymphocyte responses and will have broad application across studies of infectious and immune-mediated disease.

Effect of a Histone Demethylase Inhibitor on Equine Herpesvirus-1 Activity In Vitro.

Equine herpesvirus type 1 (EHV-1) is a ubiquitous and highly contagious pathogen that causes a range of disease severities with outbreaks of notable economic impact. Given the limitations in immune protection of current vaccines and the limited effectiveness of antiviral drugs on EHV-1 infections in vivo, improved treatment measures are needed to control disease. The use of drugs that alter the epigenetic state of herpes simplex virus genome has been shown to limit viral primary infection and reactivation both in vitro and in vivo. Therefore, we tested the hypothesis that maintaining a repressive epigenetic state on the EHV-1 genome in the host equine cell would decrease viral load during lytic infection. Equine fetal kidney cells (EFKCs) or isolated peripheral blood leukocytes were treated in vitro with (a) the nucleoside analog ganciclovir; (b) the histone demethylase inhibitor OG-L002; (c) both ganciclovir and OG-L002; or (d) dimethyl sulfoxide (DMSO, vehicle control); and then infected with a clinical EHV-1 isolate. Treatment of EFKCs with ganciclovir (mean 22.3 DNA copies per cell, p = 0.0005), OG-L002 (mean 25.6, p = 0.005) or both ganciclovir and OG-L002 (mean 7.1, p = 0.0001) resulted in decreased EHV-1 viral load at 24 h post-infection (hpi) in comparison with DMSO (mean 42.0), with greater impact using the combined treatment. Further, EHV-1 gene expression at 3 hpi decreased when EFKCs were infected in the presence of ganciclovir (p = 0.04) and combined treatment of ganciclovir and OG-L002 (p = 0.0003). In contrast, under similar conditions, neither ganciclovir nor OG-L002 suppressed EHV-1 infection in leukocytes. Differences between cell types, drug penetrance, or drug turnover, may have contributed to the distinct effects observed in this study.

Sequence analysis of feline immunoglobulin mRNAs and the development of a felinized monoclonal antibody specific to feline panleukopenia virus.

In response to immunization, B-cells generate a repertoire of antigen-specific antibodies. Antibody-based immunotherapies hold great promise for treating a variety of diseases in humans. Application of antibody-based immunotherapy in cats is limited by the lack of species-specific complete sequences for mRNAs encoding rearranged heavy and light chain immunoglobulins in B cells. To address this barrier, we isolated mRNAs from feline peripheral blood mononuclear cells (PBMCs), and used available immunoglobulin sequences and 5' and 3' RACE to clone and sequence heavy and light chain immunoglobulin mRNAs. We recovered mRNA from PBMCs from two cats, cloned and sequenced the variable and constant domains of the feline heavy chains of IgG1a (IGHG1a), IgG2 (IGHG2), and IgA (IGHA), and the light chains (lambda and kappa). Using these sequences, we prepared two bicistronic vectors for mammalian expression of a representative feline heavy (IGHG1a) together with a light (lambda or kappa) chain. Here we report novel feline Ig sequences, a technique to express antigen-specific felinized monoclonal antibodies, and the initial characterization of a functional felinized monoclonal antibody against feline panleukopenia virus.

Effects of autologous stromal cells and cytokines on differentiation of equine bone marrow-derived progenitor cells.

OBJECTIVE To develop an in vitro system for differentiation of equine B cells from bone marrow hematopoietic progenitor cells on the basis of protocols for other species. SAMPLE Bone marrow aspirates aseptically obtained from 12 research horses. PROCEDURES Equine bone marrow CD34+ cells were sorted by use of magnetic beads and cultured in medium supplemented with cytokines (recombinant human interleukin-7, equine interleukin-7, stem cell factor, and Fms-like tyrosine kinase-3), murine OP9 stromal cell preconditioned medium, and equine fetal bone marrow mesenchymal stromal cell preconditioned medium. Cells in culture were characterized by use of flow cytometry, immunocytofluorescence microscopy, and quantitative reverse-transcriptase PCR assay. RESULTS For these culture conditions, bone marrow-derived equine CD34+ cells differentiated into CD19+IgM+ B cells that expressed the signature transcription factors early B-cell factor and transcription factor 3. These conditions also supported the concomitant development of autologous stromal cells, and their presence was supportive of B-cell development. CONCLUSIONS AND CLINICAL RELEVANCE Equine B cells were generated from bone marrow aspirates by use of supportive culture conditions. In vitro generation of equine autologous B cells should be of use in studies on regulation of cell differentiation and therapeutic transplantation.

Acute leukemia in six horses (1990-2012).

Acute leukemia is rare in horses. Herein we describe historical, clinicopathologic, and postmortem findings in 6 horses with acute leukemia. Medical records of horses with >20% bone marrow blasts and cytochemical or immunophenotyping results were reviewed. Affected horses were 2-8 y of age and of different breeds and sex. Horses were presented acutely with nonspecific signs (e.g., fever, lethargy). Characteristic hemogram findings were bi- or pancytopenia with low blast numbers. Histologic examination revealed extramedullary infiltrates, especially in lymph nodes, spleen, kidney, liver, and lungs. Leukemias were classified as B-cell ( n = 3) and acute myeloid leukemia (AML) ( n = 3). Tumors in 4 cases expressed multiple lineage markers, which complicated classification. Acute leukemia should be suspected in horses with moderate-to-severe bi- or pancytopenia. Blood smears should be reviewed for neoplastic cells, and bone marrow examination is required for diagnosis. Leukemia classification is best achieved using combined morphologic, cytochemical, and immunophenotyping results.

Clinical and immunological assessment of therapeutic immunization with a subunit vaccine for recurrent ocular canine herpesvirus-1 infection in dogs.

Latent canine herpesvirus-1 (CHV-1) infections are common in domestic dogs and reactivation of latent virus may be associated with recurrent ocular disease. The objectives of the present study were to evaluate the ability of a subunit CHV-1 vaccine to stimulate peripheral CHV-1 specific immunity and prevent recurrent CHV-1 ocular disease and viral shedding. Mature dogs with experimentally-induced latent CHV-1 infection received a 2-dose CHV-1 vaccine series. Recurrent ocular CHV-1 infection was induced by corticosteroid administration in the prevaccinal, short-term postvaccinal (2 weeks post-vaccination), and long-term postvacccinal (34 weeks post-vaccination) periods. Immunological, virological, and clinical parameters were evaluated during each study period. Quantitative assessment of peripheral immunity included lymphocyte immunophenotyping, proliferation response, and interferon-γ production; and CHV-1 virus neutralizing antibody production. In the present study, vaccination did not prevent development of ocular disease and viral shedding; however, there was a significant decrease in clinical ocular disease scores in the short-term postvaccinal period. Significant alterations in peripheral immunity detected in the dogs during the short-term and long-term postvaccinal periods included increased T and B lymphocyte subpopulation percentage distributions, increased lymphocyte expression of major histocompatibility complex class I and II, increased CHV-1 virus neutralizing antibody titers, decreased lymphocyte proliferation, and decreased interferon-γ production. Vaccination of latently infected mature dogs with the selected subunit CHV-1 vaccine was not effective in preventing recurrent ocular CHV-1 infection and viral shedding induced by corticosteroid administration. The vaccine did induce long-term CHV-1 specific immunity and may decrease the severity of clinical ocular disease in the immediate postvaccinal period.

Applied Protein and Molecular Techniques for Characterization of B Cell Neoplasms in Horses.

Mature B cell neoplasms cover a spectrum of diseases involving lymphoid tissues (lymphoma) or blood (leukemia), with an overlap between these two presentations. Previous studies describing equine lymphoid neoplasias have not included analyses of clonality using molecular techniques. The objective of this study was to use molecular techniques to advance the classification of B cell lymphoproliferative diseases in five adult equine patients with a rare condition of monoclonal gammopathy, B cell leukemia, and concurrent lymphadenopathy (lymphoma/leukemia). The B cell neoplasms were phenotypically characterized by gene and cell surface molecule expression, secreted immunoglobulin (Ig) isotype concentrations, Ig heavy-chain variable (IGHV) region domain sequencing, and spectratyping. All five patients had hyperglobulinemia due to IgG1 or IgG4/7 monoclonal gammopathy. Peripheral blood leukocyte immunophenotyping revealed high proportions of IgG1- or IgG4/7-positive cells and relative T cell lymphopenia. Most leukemic cells lacked the surface B cell markers CD19 and CD21. IGHG1 or IGHG4/7 gene expression was consistent with surface protein expression, and secreted isotype and Ig spectratyping revealed one dominant monoclonal peak. The mRNA expression of the B cell-associated developmental genes EBF1, PAX5, and CD19 was high compared to that of the plasma cell-associated marker CD38. Sequence analysis of the IGHV domain of leukemic cells revealed mutated Igs. In conclusion, the protein and molecular techniques used in this study identified neoplastic cells compatible with a developmental transition between B cell and plasma cell stages, and they can be used for the classification of equine B cell lymphoproliferative disease.

Bone marrow transcriptome and epigenome profiles of equine common variable immunodeficiency patients unveil block of B lymphocyte differentiation.

Common variable immunodeficiency (CVID) is a late-onset humoral deficiency characterized by B lymphocyte dysfunction or loss, decreased immunoglobulin production, and recurrent bacterial infections. CVID is the most frequent human primary immunodeficiency but still presents challenges in the understanding of its etiology and treatment. CVID in equine patients manifests with a natural impairment of B lymphocyte differentiation, and is a unique model to identify genetic and epigenetic mechanisms of disease. Bone marrow transcriptome analyses revealed decreased expression of genes indicative of the pro-B cell differentiation stage, importantly PAX5 (p≤0.023). We hypothesized that aberrant epigenetic regulation caused PAX5 gene silencing, resulting in the late-onset and non-familial manifestation of CVID. A significant increase in PAX5 enhancer region methylation was identified in equine CVID patients by genome-wide reduced-representation bisulfite sequencing and bisulfite PCR sequencing (p=0.000). Thus, we demonstrate that integrating transcriptomics and epigenetics in CVID enlightens potential mechanisms of dysfunctional B lymphopoiesis or function.

Intramuscular administration of a synthetic CpG-oligodeoxynucleotide modulates functional responses of neutrophils of neonatal foals.

Neutrophils play an important role in protecting against infection. Foals have age-dependent deficiencies in neutrophil function that may contribute to their predisposition to infection. Thus, we investigated the ability of a CpG-ODN formulated with Emulsigen to modulate functional responses of neutrophils in neonatal foals. Eighteen foals were randomly assigned to receive either a CpG-ODN with Emulsigen (N = 9) or saline intramuscularly at ages 1 and 7 days. At ages 1, 3, 9, 14, and 28, blood was collected and neutrophils were isolated from each foal. Neutrophils were assessed for basal and Rhodococcus equi-stimulated mRNA expression of the cytokines interferon-γ (IFN-γ), interleukin (IL)-4, IL-6, and IL-8 using real-time PCR, degranulation by quantifying the amount of ß-D glucuronidase activity, and reactive oxygen species (ROS) generation using flow cytometry. In vivo administration of the CpG-ODN formulation on days 1 and 7 resulted in significantly (P<0.05) increased IFN-γ mRNA expression by foal neutrophils on days 3, 9, and 14. Degranulation was significantly (P<0.05) lower for foals in the CpG-ODN-treated group than the control group at days 3 and 14, but not at other days. No effect of treatment on ROS generation was detected. These results indicate that CpG-ODN administration to foals might improve innate and adaptive immune responses that could protect foals against infectious diseases and possibly improve responses to vaccination.

Diversity of immunoglobulin lambda light chain gene usage over developmental stages in the horse.

To further studies of neonatal immune responses to pathogens and vaccination, we investigated the dynamics of B lymphocyte development and immunoglobulin (Ig) gene diversity. Previously we demonstrated that equine fetal Ig VDJ sequences exhibit combinatorial and junctional diversity levels comparable to those of adult Ig VDJ sequences. Herein, RACE clones from fetal, neonatal, foal, and adult lymphoid tissue were assessed for Ig lambda light chain combinatorial, junctional, and sequence diversity. Remarkably, more lambda variable genes (IGLV) were used during fetal life than later stages and IGLV gene usage differed significantly with time, in contrast to the Ig heavy chain. Junctional diversity measured by CDR3L length was constant over time. Comparison of Ig lambda transcripts to germline revealed significant increases in nucleotide diversity over time, even during fetal life. These results suggest that the Ig lambda light chain provides an additional dimension of diversity to the equine Ig repertoire.

Developmental progression of equine immunoglobulin heavy chain variable region diversity.

Humoral immunity is a critical component of the immune system that is established during fetal life and expands upon exposure to pathogens. The extensive humoral immune response repertoire is generated in large part via immunoglobulin (Ig) heavy chain variable region diversity. The horse is a useful model to study the development of humoral diversity because the placenta does not transfer maternal antibodies; therefore, Igs detected in the fetus and pre-suckle neonate were generated in utero. The goal of this study was to compare the equine fetal Ig VDJ repertoire to that of neonatal, foal, and adult horse stages of life. We found similar profiles of IGHV, IGHD, and IGHJ gene usage throughout life, including predominant usage of IGHV2S3, IGHD18S1, and IGHJ1S5. CDR3H lengths were also comparable throughout life. Unexpectedly, Ig sequence diversity significantly increased between the fetal and neonatal age, and, as expected, between the foal and adult age.

Fell Pony syndrome: characterization of developmental hematopoiesis failure and associated gene expression profiles.

Fell Pony syndrome (FPS) is a fatal immunodeficiency that occurs in foals of the Fell Pony breed. Affected foals present with severe anemia, B cell lymphopenia, and opportunistic infections. Our objective was to conduct a prospective study of potential FPS-affected Fell Pony foals to establish clinical, immunological, and molecular parameters at birth and in the first few weeks of life. Complete blood counts, peripheral blood lymphocyte phenotyping, and serum immunoglobulin concentrations were determined for 3 FPS-affected foals, 49 unaffected foals, and 6 adult horses. In addition, cytology of bone marrow aspirates was performed sequentially in a subset of foals. At birth, the FPS-affected foals were not noticeably ill and had hematocrit and circulating B cell counts comparable to those of unaffected foals; however, over 6 weeks, values for both parameters steadily declined. A bone marrow aspirate from a 3-week-old FPS-affected foal revealed erythroid hyperplasia and concurrent erythroid and myeloid dysplasia, which progressed to a severe erythroid hypoplasia at 5 weeks of life. Immunohistochemical staining confirmed the paucity of B cells in primary and secondary lymphoid tissues. The mRNA expression of genes involved in B cell development, signaling, and maturation was investigated using qualitative and quantitative reverse transcriptase PCR (RT-PCR). Several genes, including CREB1, EP300, MYB, PAX5, and SPI1/PU.1, were sequenced from FPS-affected and unaffected foals. Our study presents evidence of fetal erythrocyte and B cell hematopoiesis with rapid postnatal development of anemia and B lymphopenia in FPS-affected foals. The transition between fetal/neonatal and adult-like hematopoiesis may be an important aspect of the pathogenesis of FPS.

Investigation of an outbreak of besnoitiosis in donkeys in northeastern Pennsylvania.

OBJECTIVE: To describe the clinical, endoscopic, and serologic features of an outbreak of besnoitiosis in 2 donkey operations in northeastern Pennsylvania and to report the outcome of attempted treatment of 1 naturally infected individual. DESIGN: Observational study. ANIMALS: 29 donkeys (Equus asinus) in northeastern Pennsylvania. PROCEDURES: Donkeys were examined for lesions suggestive of besnoitiosis in an outbreak investigation. Information was collected regarding the history and signalment of animals on each premises. Rhinolaryngoscopy was performed to identify nasopharyngeal and laryngeal lesions. Serum samples were collected for immunofluorescent antibody testing and immunoblotting for Besnoitia spp. Skin biopsy samples were obtained from 8 animals with lesions suggestive of besnoitiosis for histologic examination. Quantitative real-time PCR assay for Besnoitia spp was performed on tissue samples from 5 animals. RESULTS: Besnoitiosis was confirmed in 6 of the 8 suspected cases. The most common lesion site was the nares, followed by the skin and sclera. Donkeys with clinical signs of disease had higher serum antibody titers and tested positive for a greater number of immunoblot bands than did donkeys without clinical signs of disease. All animals evaluated by PCR assay tested positive. Putative risk factors for disease included age and sex. Ponazuril was not effective at treating besnoitiosis in a naturally infected donkey. CONCLUSIONS AND CLINICAL RELEVANCE: Knowledge of clinical and serologic features of besnoitiosis in donkeys will assist clinicians in the diagnosis and prevention of this disease in donkey populations. Besnoitiosis may be an emerging disease of donkeys in the United States.