Danielle D. Carrade

Canine granulocytic anaplasmosis: a review.

Anaplasma phagocytophilum is an emerging pathogen of humans, horses, and dogs worldwide that is transmitted by Ixodid ticks and maintained in a variety of small wild mammal species. Recent studies suggest that multiple strains of A. phagocytophilum may be circulating in wild and domestic animal populations, and these strains may have differential host tropisms and pathogenicity. The organism infects and survives within neutrophils by disabling key neutrophil functions, including neutrophil motility, phagocytosis, the oxidative burst mechanism, and neutrophil-endothelial cell interactions, as well as interfering with neutrophil apoptosis. Coinfections with other tick-borne pathogens may occur, especially Borrelia burgdorferi. A. phagocytophilum causes an acute febrile illness in dogs with lethargy and inappetence. Less frequent signs include lameness, coughing, polydipsia, intermittent vomiting, and hemorrhages. Diagnosis is based on finding morulae within granulocytes in the peripheral blood, the combination of acute and convalescent serology using immunofluorescent antibody techniques, and detection of the DNA of A. phagocytophilum using specific polymerase chain reaction assays. Whether persistent infection or reinfection with A. phagocytophilum occurs after natural infection requires additional study, with most reports suggesting that anaplasmosis is a self-limiting disease in dogs that responds well to a 2-week course of doxycycline therapy.

Clinicopathologic findings following intra-articular injection of autologous and allogeneic placentally derived equine mesenchymal stem cells in horses.

BACKGROUND AIMS The development of an allogeneic mesenchymal stem cell (MSC) product to treat equine disorders would be useful; however, there are limited in vivo safety data for horses. We hypothesized that the injection of self (autologous) and non-self (related allogeneic or allogeneic) MSC would not elicit significant alterations in physical examination, gait or synovial fluid parameters when injected into the joints of healthy horses. METHODS Sixteen healthy horses were used in this study. Group 1 consisted of foals (n = 6), group 2 consisted of their dams (n = 5) and group 3 consisted of half-siblings (n = 5) to group 1 foals. Prior to injection, MSC were phenotyped. Placentally derived MSC were injected into contralateral joints and MSC diluent was injected into a separate joint (control). An examination, including lameness evaluation and synovial fluid analysis, was performed at 0, 24, 48 and 72 h post-injection. RESULTS MSC were major histocompatibility complex (MHC) I positive, MHC II negative and CD86 negative. Injection of allogeneic MSC did not elicit a systemic response. Local responses such as joint swelling or lameness were minimal and variable. Intra-articular MSC injection elicited marked inflammation within the synovial fluid (as measured by nucleated cell count, neutrophil number and total protein concentration). However, there were no significant differences between the degree and type of inflammation elicited by self and non-self-MSC. CONCLUSIONS The healthy equine joint responds similarly to a single intra-articular injection of autologous and allogeneic MSC. This pre-clinical safety study is an important first step in the development of equine allogeneic stem cell therapies.

Comparative Analysis of the Immunomodulatory Properties of Equine Adult-Derived Mesenchymal Stem Cells().

Mesenchymal stem cells (MSCs) derived from bone marrow (BM), adipose tissue (AT), umbilical cord blood (CB), and umbilical cord tissue (CT) are increasingly being used to treat equine inflammatory and degenerative lesions. MSCs modulate the immune system in part through mediator secretion. Animal species and MSC tissue of origin are both important determinants of MSC function. In spite of widespread clinical use, how equine MSCs function to heal tissues is fully unknown. In this study, MSCs derived from BM, AT, CB, and CT were compared for their ability to inhibit lymphocyte proliferation and secrete mediators in response to activation. Five MSC lines from each tissue were isolated. Lymphocyte proliferation was assessed in a mixed leukocyte reaction, and mediator secretion was determined by ELISA. Regardless of tissue of origin, quiescent MSCs did not alter lymphocyte proliferation or secrete mediators, except for transforming growth factor-β (TGF-β1). When stimulated, MSCs of all tissue types decreased lymphocyte proliferation, increased prostaglandin (PGE(2)) and interleukin-6 (IL-6) secretion, and decreased production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). BM-MSCs and CB-MSCs also produced nitric oxide (NO), while AT-MSCs and CT-MSCs did not. Equine MSCs did not produce indoleamine 2,3-dioxygenase (IDO). These data suggest that activated equine MSCs derived from BM, AT, CT, and CB secrete high concentration of mediators and are similar to MSCs from rodents and humans in their immunomodulatory profiles. These findings have implication for the treatment of inflammatory lesions dominated by activated lymphocytes and TNF-α and IFN-γ in vivo.

Intradermal injections of equine allogeneic umbilical cord-derived mesenchymal stem cells are well tolerated and do not elicit immediate or delayed hypersensitivity reactions

BACKGROUND AIMS. The use of allogeneic mesenchymal stem cells (MSC) to treat acute equine lesions would greatly expand equine cellular therapy options; however, the safety and antigenicity of these cells have not been well-studied. We hypothesized that equine allogeneic umbilical cord tissue (UCT)-derived MSC would not elicit acute graft rejection or a delayed-type hypersensitivity response when injected intradermally. METHODS. Six Quarterhorse yearlings received 12 intradermal injections (autologous MSC, allogeneic MSC, positive control and negative control, in triplicate) followed by the same series of 12 injections, 3-4 weeks later, at another site. Wheals were measured and palpated at 0.25, 4, 24, 48, 72 h and 7 days post-injection. Biopsies were obtained at 48 and 72 h and 7 days post-injection. Mixed leukocyte reactions were performed 1 week prior to the first injections and 3 weeks after the second injections. RESULTS. There were no adverse local or systemic responses to two intradermal injections of allogeneic MSC. MSC injection resulted in minor wheal formation, characterized by mild dermatitis, dermal edema and endothelial hyperplasia, that fully resolved by 48-72 h. No differences were noted between allogeneic and autologous MSC. The second injection of MSC did not elicit more significant physical or histomorphologic alterations compared with the first MSC injection. Neither allogeneic nor autologous UCT-derived MSC stimulated or suppressed baseline T-cell proliferation in vitro prior to or after two MSC administrations. CONCLUSIONS. Equine allogeneic UCT MSC may be safely administered intradermally on multiple occasions without eliciting a measurable cellular immune response.

Identification of variables that optimize isolation and culture of multipotent mesenchymal stem cells from equine umbilical-cord blood

OBJECTIVE-To optimize the isolation and culture of mesenchymal stem cells (MSCs) from umbilical-cord blood (UCB), identify variables that predicted successful MSC isolation, and determine whether shipping, processing, and cryopreservation altered MSC viability, recovery rates, and expansion kinetics. SAMPLE POPULATION-UCB samples from 79 Thoroughbred and Quarter Horse mares. PROCEDURES-UCB samples were processed to reduce volume and remove RBCs. Nucleated cells (NCs) were cryopreserved or grown in various culture conditions to optimize MSC monolayer expansion and proliferation. Donor and UCB-sample factors were analyzed to determine their influence on the success of MSC isolation and monolayer expansion. RESULTS-MSCs capable of multilineage in vitro differentiation were expanded from > 80% of UCB samples. Automated UCB processing and temperature-controlled shipping facilitated sterile and standardized RBC reduction and NC enrichment from UCB samples. The number of NCs after UCB samples were processed was the sole variable that predicted successful MSC expansion. The UCB-derived MSCs and NCs were successfully cryopreserved and thawed with no decrease in cell recovery, viability, or MSC proliferation. The use of fibronectin-coated culture plates and reduction of incubator oxygen tension from 20% to 5% improved the MSC isolation rate. Some UCB-derived MSC clones proliferated for > 20 passages before senescence. Onset of senescence was associated with specific immunocytochemical changes. CONCLUSIONS AND CLINICAL RELEVANCE-Equine UCB samples appeared to be a rich source of readily obtainable, highly proliferative MSCs that could be banked for therapeutic use.

Collection of equine cord blood and placental tissues in 40 Thoroughbred mares

REASONS FOR PERFORMING STUDY Stem cells derived from umbilical cord tissue (UCT) and umbilical cord blood (UCB) in human subjects and horses can be obtained in a minimally invasive fashion with successful propagation of mesenchymal stem cells (MSCs). Currently there are no detailed protocols documenting a procedure to harvest UCB and UCT safely for equine stem cell propagation. HYPOTHESIS UCB and UCT could be collected without harm to mare or foal. OBJECTIVES To develop a standard and safe method for UCB and UCT collection, and prospectively to compare foal and mare health between groups of animals where tissue was and was not collected. METHODS This study was conducted at a Thoroughbred breeding facility in central California in 2008. UCB and UCT were collected from 40 mare and foal pairs. Clinical parameters including time for foal to stand and nurse, time for mare to pass the placenta, and foal haematology data at age 24 h were documented and compared to a control group, consisting of the succeeding 40 mare and foal pairs. RESULTS UCB was obtained successfully from 36 of 40 (90%) mares and UCT from 38 of 40 (95%) mares. Bacterial contamination was documented in 6 out of 36 (16.6%) UCB samples. There were no significant differences in time to stand or nurse for foals or time to pass the placenta for mares, between the experimental and control groups. There were no clinically relevant differences identified in haematological data obtained from foals with and without UCB collection. CONCLUSIONS UCB and UCT can be harvested safely without harm to mares or foals. POTENTIAL RELEVANCE UCB and UCT samples collected in an inherently contaminated environment can be successfully disinfected and transported with minimal bacterial overgrowth for use in cell culture to isolate MSCs.

Safety and immunomodulatory effects of allogeneic canine adipose-derived mesenchymal stromal cells transplanted into the region of the lacrimal gland, the gland of the third eyelid and the knee joint.

BACKGROUND AIMS Mesenchymal stromal cells (MSCs) have been extensively studied as a cellular therapeutic for various pathologic conditions. However, there remains a paucity of data regarding regional and systemic safety of MSC transplantations, particularly with multiple deliveries of allogeneic cells. The purpose of this study was to investigate the safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs into the region of the lacrimal gland, the gland of the third eyelid and the knee joint in dogs. METHODS Allogeneic adipose tissue-derived canine MSCs were delivered to the regions of the lacrimal gland and the third eyelid gland as well as in the knee joints of six healthy laboratory beagles as follows: six times with 1-week intervals for delivery to the lacrimal gland and the third eyelid gland regions and three to four times with 1- to 2-week intervals for intra-articular transplantations. Dogs were sequentially evaluated by clinical examination. At the conclusion of the study, dogs were humanely euthanized, and a complete gross and histopathologic examination of all organ systems was performed. Mixed leukocyte reactions were also performed before the first transplantation and after the final transplantation. RESULTS Clinical and pathologic examinations found no severe consequences after repeated MSC transplantations. Results of mixed leukocyte reactions demonstrated suppression of T-cell proliferation after MSC transplantations. CONCLUSIONS This is the first study to demonstrate regional and systemic safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs in vivo.

Spatial distribution of seroprevalence for Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia canis, and Dirofilaria immitis in dogs in Washington, Oregon, and California

BACKGROUND In the US little spatially defined information regarding exposure to most vector-borne pathogens in dogs is available for the states of California (CA), Oregon (OR), and Washington (WA). OBJECTIVES The purpose of the present study was to evaluate the spatial distribution of seroprevalence for 4 vector-borne pathogens, Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia canis, and Dirofilaria immitis, across the 3 western coastal states of the contiguous United States that extend from the northern Mexican to the southern Canadian border. METHODS A convenience sample, targeting blood from 20 pet dogs per county across CA, OR, and WA, was evaluated using a canine point-of-care ELISA kit. Geographic coordinates of home zip code were displayed using a geographic information system. A total of 2431 dogs from CA, OR, and WA were tested. RESULTS The overall seroprevalence was highest for A. phagocytophilum (2.4%), followed by B. burgdorferi (1.2%), and E. canis (0.7%). The prevalence of infection with D. immitis was 0.7%. At the individual dog level, there was a significant association between seropositivity to B. burgdorferi and A. phagocytophilum (odds ratio=18.7, 95% confidence interval=6.8-47.1). For most positive results, prevalence tended to decrease with increasing latitude; thus, the highest rates of seropositivity occurred in CA, followed by OR, and then WA; one exception was seropositivity for B. burgdorferi, which was higher in WA (0.38%) than in OR (0.15%), but considerably lower than in CA (2.00%). In WA, dogs that tested positive for A. phagocytophilum, E. canis, and B. burgdorferi were in the southern Puget Sound area. For D. immitis, none of the dogs in WA was positive. CONCLUSIONS Seropositivity for vector-borne pathogens is broadly but patchily distributed in dogs in CA, OR, and WA.

Use of an in vitro biotinylation technique for determination of posttransfusion survival of fresh and stored autologous red blood cells in Thoroughbreds

OBJECTIVE To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days. ANIMALS 13 healthy adult Thoroughbreds. PROCEDURES Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfused or stored at 4 degrees C for 1, 14, or 28 days prior to labeling with NHS-biotin and reinfusion. Posttransfusion blood samples were collected 15 minutes and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after reinfusion. Biotin-labeled RBCs were detected via flow cytometry by use of streptavidin-PE. Posttransfusion lifespan of RBCs and RBC half-life were determined. RESULTS Optimal biotin concentration was 0.04 pg of biotin/RBC, and the optimal streptavidin-PE ratio was 1.2 microg of streptavidin-PE/1 x 10(6) RBCs. Posttransfusion lifespan of autologous RBCs was 99, 89, 66, and 59 days after storage for 0, 1, 14, and 28 days, respectively. Storage did not result in significant alterations in RBC lifespan. Mean posttransfusion RBC half-life was 50, 45, 33, and 29 days for 0, 1, 14, and 28 days of storage, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Biotin can be used to label equine RBCs for RBC survival studies. Posttransfusion survival of equine autologous RBCs was greater than previously reported.

The effects of therapeutic concentrations of gentamicin, amikacin and hyaluronic acid on cultured bone marrow-derived equine mesenchymal stem cells

REASONS FOR PERFORMING STUDY Joint inflammation and septic arthritis are both potential complications of intra-articular injections of bone marrow-derived mesenchymal stem cells (BM-MSCs). Clinicians may prophylactically co-inject BM-MSCs admixed with either antimicrobials or hyaluronic acid; however, the effect of these agents on cultured BM-MSCs is unknown. OBJECTIVE To determine the effects of therapeutic levels of gentamicin, amikacin and hyaluronic acid on cultured equine BM-MSCs in vitro. STUDY DESIGN In vitro experimental study. METHODS Equine BM-MSCs from 4 healthy mature horses were isolated. Cultured BM-MSCs from each donor were incubated with gentamicin (150 mg), amikacin (250 mg), hyaluronic acid (22 mg) or 1% penicillin/streptomycin (control) under sterile conditions. Mesenchymal stem cells viability, proliferation, mediator secretion and culture media pH were measured. RESULTS Incubation of BM-MSCs with gentamicin resulted in >95% MSC death after 45 min, and incubation of BM-MSCs with amikacin resulted in >95% MSC death after 2 h. Incubation of BM-MSCs with hyaluronic acid or penicillin/streptomycin (control) for up to 6 h resulted in sustained BM-MSC viability of 80% and >93%, respectively. All additives resulted in decreased media pH in the first minute; however, the pH then remained constant over the 6 h incubation period. No significant differences in BM-MSC proliferation or mediator secretion between the penicillin/streptomycin (control) and cells treated with hyaluronic acid were observed. CONCLUSION Therapeutic concentrations of aminoglycoside antimicrobials are toxic to cultured equine BM-MSCs. The effects of hyaluronic acid on cultured MSC viability, proliferation and mediator secretion are minimal. POTENTIAL RELEVANCE Based on these findings, the mixing of aminoglycoside antimicrobials and cultured equine BM-MSCs prior to therapeutic use is not recommended.