Johanna L. Watson

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.

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.

Virulent and avirulent strains of equine arteritis virus induce different quantities of TNF-α and other proinflammatory cytokines in alveolar and blood-derived equine macrophages

Equine arteritis virus (EAV) infects endothelial cells (ECs) and macrophages in horses, and many of the clinical manifestations of equine viral arteritis (EVA) reflect vascular injury. To further evaluate the potential role of EAV-induced, macrophage-derived cytokines in the pathogenesis of EVA, we infected cultured equine alveolar macrophages (AMphi), blood monocyte-derived macrophages (BMphi), and pulmonary artery ECs with either a virulent (KY84) or an avirulent (CA95) strain of EAV. EAV infection of equine AMphi, BMphi, and ECs resulted in their activation with increased transcription of genes encoding proinflammatory mediators, including interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha. Furthermore, the virulent KY84 strain of EAV induced significantly higher levels of mRNA encoding proinflammatory cytokines in infected AMphi and BMphi than did the avirulent CA95 strain. Treatment of equine ECs with the culture supernatants of EAV-infected AMphi and BMphi also resulted in EC activation with cell surface expression of E-selectin, whereas infection of ECs with purified EAV alone caused only minimal expression of E-selectin. The presence of TNF-alpha in the culture supernatants of EAV-infected equine AMphi, BMphi, and ECs was confirmed by bioassay, and the virulent KY84 strain of EAV induced significantly more TNF-alpha in all cell types than did the avirulent CA95 strain. Thus, the data indicate that EAV-induced, macrophage-derived cytokines may contribute to the pathogenesis of EVA in horses, and that the magnitude of the cytokine response of equine AMphi, BMphi, and ECs to EAV infection reflects the virulence of the infecting virus strain.

Incidence, management, and outcome of complications of castration in equids: 324 cases (1998–2008)

OBJECTIVE To determine the incidence of complications and identify risk factors associated with development of complications following routine castration of equids. DESIGN Retrospective case series. Animals-311 horses, 10 mules, and 3 donkeys. PROCEDURES Medical records of equids undergoing routine castration were reviewed. Age, breed, surgical techniques (closed vs semiclosed castration and use of ligatures), anesthesia method (general IV anesthesia vs standing sedation with local anesthesia) and repeated administration of IV anesthetic agents, administration of antimicrobials and anti-inflammatory drugs, and details regarding development, management, and outcome of complications were recorded. Odds ratios and 95% confidence intervals were determined. Associations between additional doses of anesthetic agents during surgery and development of complications were analyzed with a Jonckheere-Terpstra test. RESULTS 33 of 324 (10.2%) equids developed a complication after surgery; 32 recovered and 1 was euthanized because of eventration. Equids that underwent semiclosed castration had significantly higher odds of developing a complication (OR, 4.69; 95% confidence interval, 2.09 to 10.6) than did those that underwent closed castration. Equids that received additional doses of anesthetic agents to maintain adequate general anesthesia developed complications more frequently than those that did not require this treatment. CONCLUSIONS AND CLINICAL RELEVANCE Incidence of complications was low, and most evaluated variables were not significantly associated with development of complications following castration in equids. However, findings suggested that the choice of surgical technique (closed vs semiclosed) is an important factor in this regard. Future studies should investigate whether duration of surgery is associated with complications following castration in equids.

Molecular cloning and sequencing of the low-affinity IgE receptor (CD23) for horse and cattle

Expression of the low-affinity IgE receptor (CD23) on the surface of mononuclear cells is a critical event in the development of IgE-mediated immunologic responses. Using PCR and cDNA library screening, a 2.7kb cDNA encoding equine CD23 and a 627bp PCR fragment of cattle CD23 were sequenced and characterized. The equine CD23 sequence encodes a complete and continuous open reading frame of 327 amino acids, while the shorter cattle fragment encodes 209 amino acids corresponding to nucleotides 325-1098 of the equine CD23 transcript. In addition to high identities in their nucleotides and translated amino acids with CD23 sequences published for other species, the translated equine CD23 protein also shares many of the structural features of this molecule described for human and rodents. Interestingly, an additional repetitive element of possible functional significance consisting of 18 amino acids, located between the transmembrane region and the carbohydrate-binding lectin domain of horse CD23, was also identified. Based on these sequences, molecular assays will be developed to measure CD23 mRNA in tissues and expression of recombinant proteins will be essential to the production of species-specific antibody reagents. These assays and reagents will be useful in future studies of allergic and lymphoproliferative diseases in horses and cattle.

Characterization of horse (Equus caballus) T-cell receptor beta chain genes

Genes encoding the horse (Equus caballus) T-cell receptor beta chain (TCRB) were cloned and characterized. Of 33 cDNA clones isolated from the mesenteric lymph node, 30 had functionally rearranged gene segments, and three contained germline sequences. Sixteen unique variable segments (TCRBV), 14 joining genes (TCRBJ), and two constant region genes (TCRBC) were identified. Horse TCRBV were grouped into nine families based on similarity to human sequences. TCRBV2 and TCRBV12 were the most commonly represented horse families. Analysis of predicted protein structure revealed the presence of conserved regions similar to those seen in TCRB of other species. A decanucleotide promoter sequence homologous to those found in humans and mice was located in the 5′ untranslated region of one horse gene. Germline sequences included the 5′ region of the TCRBD2 gene with flanking heptamer/nonamer recombination signals and portions of the TCRBJ-C2 intron. Southern blot hybridizations demonstrated restriction fragment length polymorphisms at the TCRBC locus among different horse breeds.

Development of a live, attenuated, potential vaccine strain of R. equi expressing vapA and the virR operon, and virulence assessment in the mouse.

Pneumonia caused by Rhodococcus equi remains a significant problem in foals. The objective of this study was to develop a safe and efficacious attenuated strain of R. equi for eventual use in oral immunization of foals. The approach involved expression of vapA in a live, virulence plasmid-negative, strain of R. equi (strain 103-). PCR-amplified fragments of the vapA gene, with and without the upstream genes virR, orf5, vapH, orf7 and orf8 (orf4-8), were cloned into a shuttle vector pNBV1. These plasmids, named pAW48A and pAWVapA respectively, were electroporated into strain 103-. The presence of the recombinant vectors in the attenuated strain (103-) and the integrity of the inserted genes were confirmed, and both constructs expressed VapA. The virulence of the two strains was compared to that of wild type R. equi 103+ and negative controls by their intravenous inoculation into mice, followed by examination of liver clearance 4 days later. Mice inoculated with R. equi 103-, 103-/pAWVapA and 103-/pNBV1 completely cleared infection, whereas strain 103-/pAW48A persisted in 47% of mice.

Characterization of Brucella abortus infection of bovine monocyte-derived dendritic cells.

Brucella abortus is a Gram negative facultative intracellular pathogen of cattle, and an important zoonosis in humans worldwide. Previous studies have shown that dendritic cells (DC) from humans and mice are highly permissive for Brucella survival and proliferation. Impairment of DC activation and maturation by Brucella infection has also been reported in these two species. The aim of this study was to characterize infection of bovine DC with B. abortus. Monocyte-derived DC (mdDC) were cultured from bovine peripheral blood mononuclear cells (PBMC) using the recombinant bovine cytokines IL-4 and GM-CSF. The resulting mdDC were DEC205(+), MHC class II(hi). Approximately 70% of the cultured cells were DEC205(+), MHC II(+). MdDC were infected with B. abortus strain 2308 at an MOI of 1 and 100. Parallel infection experiments were performed in monocyte derived macrophages (mdM) isolated from the same subjects. Bacteria were successfully killed by mdDC by 24 hours post infection (PI) with high and low dose of B. abortus, bacteria persisted in mdM infected with a high dose. Expression of IL-1b, IL-6, IL-10, IL-12p40, IFNγ, iNOS and TNFα in B. abortus infected and LPS stimulated mdDC at 6 and 24 hours PI were evaluated using RT-qPCR. At 6 hours PI all transcripts were increased over control cells and significantly less IL-10, IL-12p40, and IFNγ were expressed in mdDC infected with B. abortus compared to LPS stimulation. Evaluation of mdDC cultures by flow cytometry was performed. Flow cytometric analysis of infected and LPS stimulated mdDC 24 hours PI showed expression of CD80 and CD86 was impaired in two of the three animals analyzed. MHC class II expression was equivocal between the groups. From these results we conclude that cultured bovine mdDC are not permissive for intracellular proliferation of B. abortus, and infected mdDC exhibit some signs of maturational and activational impairment.

A potential role for indoleamine 2,3-dioxygenase (IDO) in Rhodococcus equi infection.

Rhodococcus equi is a facultative intracellular bacterial pathogen of foals and immunocompromised humans that infects and proliferates within host macrophages and dendritic cells (DC). Indoleamine 2,3-dioxygenase (IDO), the initial enzyme in the tryptophan catabolism pathway, is upregulated in R. equi infected equine monocyte-derived DC and alveolar macrophages. Tryptophan requirement of R. equi for extracellular and intracellular growth was assessed. Growth of R. equi in minimal media did not require tryptophan and pharmacologic inhibition of IDO had no effect on intracellular proliferation of R. equi in equine alveolar macrophages. To investigate an immune-regulatory role for INDO in R. equi infection, IDO(-/-) (B6.129-(Indotm1Alm)/J) (n=22) and strain matched control (C57BL/6J) (n=20) mice were infected with R. equi by intraperitoneal injection, for 3 and 6 days. There was no difference in bacterial counts in liver or spleen between the two groups. Histological sections of liver and spleen were assigned inflammation scores and RT-PCR for interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα), IL-4, IL-6, IL-10, IL-12, IL-23, forkhead box P3 (FoxP3), and transforming growth factor-beta (TGFβ) was performed on liver and spleen. Liver tissue of IDO(-/-) had higher inflammation scores at 6 days post-infection (PI) (P=0.05) and had decreased expression of TGFβ at 3 days PI (P=0.01), and FOXP3 at 3 days (P=0.02) and 6 days (P=0.03) compared to control mice. Immunostaining for FOXP3 showed lower numbers of FOXP3+ regulatory T cells in liver of IDO(-/-) mice 6 days PI. Prolonged inflammation in the liver tissue of IDO(-/-) mice corresponded with lower expression of FOXP3 and TGFβ in that tissue, and also with lower numbers of FOXP3+ regulatory T cells. We conclude that IDO expression by activated macrophages and DC plays a role in dampening the inflammatory response to R. equi infection in mice.

Identification of immunologically relevant genes in mare and foal dendritic cells responding to infection by Rhodococcus equi.

Rhodococcus equi is a facultative intracellular bacterial pathogen of horses; infected foals develop pyogranulomatous pneumonia, however adult horses are largely unaffected. R. equi infects and proliferates within host macrophages and dendritic cells (DCs). DCs initiate the appropriate adaptive immune response, thereby playing a critical role in determining the outcome of infection. Our aim was to identify genes that are differentially expressed in R. equi infected monocyte-derived DCs (mdDCs). Peripheral blood monocytes from mares and foals were used to derive mdDCs by culturing with recombinant equine IL-4 and recombinant human GM-CSF. RNA harvested 24h after infection with R. equi (ATCC 33701+) was used to perform suppression subtractive hybridization (SSH) experiments. Approximately 38 unique sequences were obtained from these experiments. Differential expression of 19 immunologically relevant genes was validated by PCR. These genes are characterized by the following functions: cell adhesion, chemotaxis/migration, immune/inflammatory response, ion transport, signal transduction, T-cell regulation, and vesicular transport. In summary, we identified several novel genes that are differentially expressed in foal and adult mdDCs in response to R. equi infection. These genes provide promising targets for further research into the host response to R. equi, and the susceptibility of foals to this disease.