Gregory L. Ferraro

Comparison of the osteogenic potential of equine mesenchymal stem cells from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue

OBJECTIVE To determine the optimal osteogenic source of equine mesenchymal stem cells (eMSCs) and optimize collection of and expansion conditions for those cells. ANIMALS 10 adult Quarter Horses and 8 newborn Thoroughbred foals. PROCEDURES eMSCs were isolated from bone marrow (BM), adipose tissue, and umbilical cord blood and tissue, and the osteogenic potential of each type was assessed. Effects of anatomic site, aspiration volume, and serum type on eMSC yield from BM were investigated. RESULTS BM-eMSCs had the highest overall expression of the osteogenic genes Cbfa1, Osx, and Omd and staining for ALP activity and calcium deposition. There was no significant difference in BM-eMSC yield from the tuber coxae or sternum, but yield was significantly greater from the first 60-mL aspirate than from subsequent aspirates. The BM-eMSC expansion rate was significantly higher when cells were cultured in fetal bovine serum instead of autologous serum (AS). CONCLUSIONS AND CLINICAL RELEVANCE eMSCs from BM possessed the highest in vitro osteogenic potential; eMSCs from adipose tissue also had robust osteogenic potential. The tuber coxae and the sternum were viable sources of BM-eMSCs in yearlings, and 60 mL of BM aspirate was sufficient for culture and expansion. Expanding BM-eMSCs in AS to avoid potential immunologic reactions decreased the total yield because BM-eMSCs grew significantly slower in AS than in fetal bovine serum. Additional studies are needed to determine optimal ex vivo eMSC culture and expansion conditions, including the timing and use of growth factor—supplemented AS.

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.

Equine herpesvirus-1 myeloencephalopathy: a review of recent developments.

Equine herpes myeloencephalopathy (EHM), although a relatively uncommon manifestation of equine herpesvirus-1 (EHV-1) infection, can cause devastating losses on individual farms or boarding stables. Although outbreaks of EHM have been recognized for centuries in domestic horse populations, many aspects of this disease remained poorly characterized. In recent years, an improved understanding of EHM has emerged from experimental studies and from data collected during field outbreaks at riding schools, racetracks and veterinary hospitals throughout North America and Europe. These outbreaks have highlighted the contagious nature of EHV-1 and have prompted a re-evaluation of diagnostic procedures, treatment modalities, preventative measures and biosecurity protocols for the disease. This review concentrates on these and other selected, clinically relevant aspects of EHM.

Equine herpesvirus-4 kinetics in peripheral blood leukocytes and nasopharyngeal secretions in foals using quantitative real-time TaqMan PCR

Based on the hypothesis that the viral load of cells infected with EHV-4 will likely change during the course of disease, TaqMan PCR was used to investigate and characterize the kinetics of EHV-4 viral DNA load (glycoprotein B gene) and transcriptional activity (glycoprotein B and latency-associated transcripts) in peripheral blood leukocytes (PBLs) and nasopharyngeal secretions (NSs) collected from 11 foals during a field outbreak of respiratory disease. The EHV-4 DNA load in PBLs was low and of short duration after onset of clinical signs. In contrast, the EHV-4 load in NSs remained high for the majority of the foals over a period of 4 weeks. Viral replication determined by detection of mRNA expression of the structural glycoprotein B was detected only in NSs during the first 7 days after onset of clinical signs for most foals. The majority of foals expressed latency-associated transcripts in NS sonly during the first 7 days after onset of clinical signs. Persistence of the expression of latency-associated transcripts in NS, as a reflection of a latent viral state, was not documented during the 28-day study period. Based on these results, it was concluded that lytic infection with EHV-4 can be diagnosed either by high EHV-4 DNA load of glycoprotein B gene or by detection of transcriptional activity of glycoprotein B.

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.

Cytokine gene signatures in neural tissue of horses with equine protozoal myeloencephalitis or equine herpes type 1 myeloencephalopathy

This study was designed to determine the relative levels of gene transcription of selected pathogens and cytokines in the brain and spinal cord of 12 horses with equine protozoal myeloencephalitis (epm), 11 with equine herpesvirus type 1 (ehv-1) myeloencephalopathy, and 12 healthy control horses by applying a real time pcr to the formalin-fixed and paraffin-embedded tissues. Total rna was extracted from each tissue, transcribed to complementary dna (cdna) and assayed for Sarcocystis neurona, Neospora hughesi, ehv-1, equine gapdh (housekeeping gene), tumour necrosis factor (tnf)-α, interferon (ifn)-γ, interleukin (il)-1β, il-2, il-4, il-6, il-8, il-10 and il-12 p40. S neurona cdna was detected in the neural tissue from all 12 horses with epm, and two of them also had amplifiable cdna of N hughesi. The relative levels of transcription of protozoal cdna ranged from 1 to 461 times baseline (mean 123). All the horses with ehv-1 myeloencephalopathy had positive viral signals by pcr with relative levels of transcription ranging from 1 to 1618 times baseline (mean 275). All the control horses tested negative for S neurona, N hughesi and ehv-1 cdna. The cytokine profiles of each disease indicated a balance between pro- and anti-inflammatory markers. In the horses with epm the pro-inflammatory Th1 cytokines (il-8, tnf-α and ifn-γ) were commonly expressed but the anti-inflammatory Th2 cytokines (il-4, il-6 and il-10) were absent or rare. In the horses with ehv-1 the proinflammatory cytokine il-8 was commonly expressed, but il-10 and ifn-γ were not, and tnf-α was rare. Tissue from the control horses expressed only the gene gapdh.

QUALITATIVE EVALUATION OF SELECTIVE TESTS FOR DETECTION OF NEOSPORA HUGHESI ANTIBODIES IN SERUM AND CEREBROSPINAL FLUID OF EXPERIMENTALLY INFECTED HORSES

Neospora hughesi is a newly recognized protozoan pathogen in horses that causes a myeloencephalitis similar to Sarcocystis neurona. There are no validated serologic tests using the gold standard sera that are currently available to detect specific N. hughesi antibodies and, thus, no tests available to detect antemortem exposure or estimate seroprevalence in the horse. The objectives of the present study were to establish a bank of gold standard equine sera through experimental infections with N. hughesi and to assess several serologic tests for the detection of related protozoan antibodies. Seven horses were inoculated with N. hughesi tachyzoites, and 7 horses received uninfected cell culture material. The horses were monitored, and blood and cerebrospinal fluid were collected repeatedly over a 4-mo period. With the sera, 4 different serologic techniques were evaluated, including a whole-parasite lysate enzyme-linked immunosorbent assay (ELISA), a recombinant protein ELISA, a modified direct agglutination test, and an indirect fluorescent antibody test. Qualitative and quantitative evaluation of the results showed that the N. hughesi indirect fluorescent antibody test (IFAT) consistently discriminated between experimentally infected and noninfected horses, using a cutoff of 1:640. Sera from 3 naturally infected horses had titers >1:640. Cerebrospinal fluid in all but 1 infected horse had very low N. hughesi IFAT titers (<1:160), starting at postinoculation day 30.

Detection of Antibodies to West Nile Virus in Equine Sera Using Microsphere Immunoassay

One hundred and ninety-one sera from horses that recently were exposed to West Nile virus (WNV) by either vaccination or natural infection or that were not vaccinated and remained free of infection were used to evaluate fluorescent microsphere immunoassays (MIAs) incorporating recombinant WNV envelope protein (rE) and recombinant nonstructural proteins (rNS1, rNS3, and rNS5) for detection of equine antibodies to WNV. The rE MIA had a diagnostic sensitivity and specificity, respectively, of 99.3% and 97.4% for detection of WNV antibodies in the serum of horses that were recently vaccinated or naturally infected with WNV, as compared to the plaque reduction neutralization test (PRNT). The positive rE MIA results were assumed to be WNV-specific because of the close agreement between this assay and the PRNT and the fact that unvaccinated control horses included in this study were confirmed to be free of exposure to the related St Louis encephalitis virus. The NS protein–based MIA were all less sensitive than either the rE MIA or PRNT (sensitivity 0–48.0), although the rNS1 MIA distinguished horses vaccinated with the recombinant WNV vaccine from those that were immunized with the inactivated WNV vaccine (P < 0.0001) or naturally infected with WNV (P < 0.0001). The rE MIA would appear to provide a rapid, convenient, inexpensive, and accurate test for the screening of equine sera for the presence of antibodies to WNV.

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.

Progressive Swelling, Hyperkeratosis, and Fibrosis of Distal Limbs in Clydesdales, Shires, and Belgian Draft Horses, Suggestive of Primary Lymphedema

BACKGROUND A condition characterized by progressive swelling, hyperkeratosis, and fibrosis of the distal limbs has been recognized in Shire, Clydesdale, and Belgian draft horses. This chronic progressive disease starts at an early age, progresses throughout the life of the horse, and often ends in disfigurement and disability of the limbs that inevitably leads to the horses premature death. This study was undertaken to better characterize this disease. METHODS AND RESULTS Six affected horses were donated for diagnostic workup. A detailed clinical, radiologic, gross, and histologic description is given in this report. The lesions in the limb consisted of progressive development of thick-walled lymphatics, associated with chronic dermal edema, inflammation, fibrosis, neovascularization, and elastin degeneration. In the end stages, arteriosclerosis and fibrosed veins were also present. The clinical signs and pathologic changes in this disease closely resemble the human condition of elephantiasis nostras verrucosa, a state in which chronic lymphedema plays a pivotal pathogenic role.