An update on equine post‐operative ileus: Definitions,pathophysiology and management

Post‐operative ileus (POI) is a serious condition which any horse undergoing abdominal surgery is at risk of developing, leading to increased hospitalisation time and resulting costs. Advances in the understanding of the development of equine POI are mainly based on human and rodent literature, where manipulation‐induced inflammation has been identified as a trigger, with activation of resident muscularis externa macrophages playing a crucial role in the pathophysiology. Despite many pharmacological trials in all species, there is no single completely successful treatment for POI, highlighting that the condition is multifactorial in cause and requires a multimodal approach to minimise its incidence.     

Generation and characterisation of an equine macrophage cell line (e-CAS cells) derived from equine bone marrow cells

Macrophages play a pivotal role in the pathophysiology of many diseases by mediating the host immune response to infections and intoxications. The species-specific activation of macrophages and the differential response in cytokine production impedes the extrapolation of results between species. Therefore, the aim of this study was to isolate and immortalise macrophages from equine bone marrow (BM) cells in order to study equine-specific signalling pathways. The isolated BM-derived macrophages (referred to as e-CAS cells) showed proliferation kinetics similar to that of standardised cell lines and were maintained in culture for >76 passages. To characterise the cells, a number of typical parameters of macrophages were tested. Morphological evaluation (May-Grünwald Giemsa staining) and non-specific esterase activity indicated the e-CAS cells to be macrophages. The presence of CD14 and their ability to phagocytose Escherichia coli bioparticles further confirmed their identity, as did their ability to produce cytokines, reactive oxygen and nitrogen intermediates in response to LPS. These data show that the established cell line (e-CAS) shows the characteristics of equine macrophages and may, therefore, prove to be a unique in vitro model for studying the cellular biology of equine inflammation.     

Eosinophils of the horse: Part I: Development,distribution, structure and biochemical mediators

Eosinophils are becoming the target of increasing research interest as recent studies suggest that their role in immune homoeostasis and the immune response to disease is far more complex than previously understood. Historically, the horse eosinophil has been used to study basic eosinophil biology because of the considerable volume of blood required to obtain enough viable cells for reliable, repeatable experiments. This resulted in a large but disseminated body of literature pertaining to the structure and function of the horse eosinophil. More recently, equine clinicians have produced case reports and clinical studies in an effort to define the role of the eosinophil in diseases of the horse. A thorough review of the equine eosinophil incorporating both bench research and clinical reports does not exist. The objective of this two-part review is to fill this need by integrating the basic science and clinical research into a comprehensive body of work on what is known specifically about the horse eosinophil, and its role in equine health and disease. Part I summarises the development and tissue distribution of eosinophils in the normal horse, and presents what is known about the cell structure, migration and biochemical mediators of the horse eosinophil. Part II reviews the role of the eosinophil in diseases of the horse, and concludes with a summary of knowledge gaps and open research questions to benefit both those who wish to use the equine eosinophil as a model for basic science research, and those whose primary interest lies with diseases of horse.     

Concepts for the clinical use of stem cells in equine medicine

Stem cells from various tissues hold great promise for their therapeutic use in horses, but so far efficacy or proof-of-principle has not been established. The basic characteristics and properties of various equine stem cells remain largely unknown, despite their increasingly widespread experimental and empirical commercial use. A better understanding of equine stem cell biology and concepts is needed in order to develop and evaluate rational clinical applications in the horse. Controlled, well-designed studies of the basic biologic characteristics and properties of these cells are needed to move this new equine research field forward. Stem cell research in the horse has exciting equine specific and comparative perspectives that will most likely benefit the health of horses and, potentially, humans.     

Molecular cloning and characterization of markers and cytokines for equid myeloid cells

The myeloid cell system comprises of monocytes, macrophages (MPhi), dendritic cells (DC), Kupffer cells, osteoclasts or microglia and is also known as the mononuclear phagocytic system (MPS). Essential cytokines to differentiate or activate these cells include GM-CSF or IL-4. Important markers for characterization include CD1, CD14, CD68, CD163 and CD206. All these markers, however, were not cloned or further characterized in equids by use of monoclonal antibodies earlier. To overcome this problem with the present study, two approaches were used. First, we cloned equine cytokines and markers, and second we analyzed cross-reactivity of human homologues or anti-human monoclonal antibodies. For cloning of equine cytokines and markers, we used degenerate primers delineated from other species, or equine-specific primers based on previous information in Genbank. Flow cytometry was used to determine the expression of markers on myeloid cells. Cross-reactivity could be shown for anti-human CD14, CD163 and mannose receptor (CD206) mAbs. Surface markers such as CD1 and CD68 that distinguish MPhi and DC were cloned and sequenced. According to blast homology, equine CD1a and CD1b could be identified and distinguished. With the resulting information, dendritic cells and macrophages of horses may be characterized.     

Deciphering the Role of Bronchial Hyper-Responsiveness in Equine Pasture Asthma

Bronchial hyper-responsiveness (BHR) describes a lung abnormality in which airways are easily triggered to constrict in response to normally harmless inhaled stimuli, and is a key element of human asthma pathophysiology. BHR contributes to equine respiratory diseases including inflammatory airway disease and recurrent airway obstruction. Collectively these diseases account for over 80% of poor performance in equine athletes, and at least 10% of veterinary admissions. BHR is also a contributing factor in ‘exercise induced pulmonary hemorrhage’. Increased sensitivity to airway constriction that characterizes BHR is a documented sequel to viral respiratory infections in several species, including horses and humans. Five respiratory viruses known to circulate extensively in equine populations place the horse at risk for BHR. Despite adverse effects of BHR on equine health, there remains a gap in our fundamental understanding of how gene products coordinate in the lung to cause BHR. Leveraging the equine genome sequence, we employ systems biology including proteomics and RNA sequencing to model the complex biology of BHR in the lungs of horses with pasture asthma. Using a self controlled experimental design, gene products that segregate with seasonal asthma exacerbation in diseased horses are being identified and their relevant physiology identified to address the need for better recognition and management of BHR in equine disease.     

Equine post operative ileus: A review of current thinking on pathophysiology and management

Equine post operative ileus (POI) is a serious post surgical complication in the horse, with a significant fatality rate. Despite the ongoing debate with regard to both the clinical definition of equine POI and the optimal management of this condition, there is increasing awareness and acceptance, supported by scientific research, that inflammation plays a key role in its pathophysiology. This review aims to outline the current thinking on the pathophysiology and management of this condition, with reference to the published literature on equine, rodent and human POI. Although studies conducted in other species are likely to provide an abundant source of information with potentially useful translational applications for the equine condition, such an approach needs to be cognisant of potential interspecies differences both in the pathogenesis of the condition and in basic gastrointestinal physiology.     

Equine multinodular pulmonary fibrosis: a newly recognized herpesvirus-associated fibrotic lung disease

Pulmonary fibrosis and interstitial lung disease are poorly understood in horses; the causes of such conditions are rarely identified. Equine herpesvirus 5 (EHV-5) is a gamma-herpesvirus of horses that has not been associated with disease in horses. Pathologic and virologic findings from 24 horses with progressive nodular fibrotic lung disease associated with EHV-5 infection are described and compared with 23 age-matched control animals. Gross lesions consisted of multiple nodules of fibrosis throughout the lungs. Histologically, there was marked interstitial fibrosis, often with preservation of an "alveolar-like" architecture, lined by cuboidal epithelial cells. The airways contained primarily neutrophils and macrophages. Rare macrophages contained large eosinophilic intranuclear viral inclusion bodies; similar inclusion bodies were also found cytologically. The inclusions were identified as herpesviral-like particles by transmission electron microscopy in a single horse. In situ hybridization was used to detect EHV-5 nucleic acids within occasional macrophage nuclei. With polymerase chain reaction (PCR), the herpesviral DNA polymerase gene was detected in 19/24 (79.2%) of affected horses and 2/23 (8.7%) of the control horses. Virus genera-specific PCR was used to detect EHV-5 in all of the affected horses and none of the control horses. EHV-2 was detected in 8/24 (33.3%) of affected horses and 1/9 (11.1%) of the control horses. This disease has not been reported before, and the authors propose that based upon the characteristic gross and histologic findings, the disease be known as equine multinodular pulmonary fibrosis. Further, we propose that this newly described disease develops in association with infection by the equine gamma-herpesvirus, EHV-5.     

In vitro model of equine muscle regeneration

Equine satellite cells are responsible for muscle healing and regeneration in the mature horse. We describe the in vitro cell culture conditions required for clonal populations of equine satellite cells to undergo both proliferation and differentiation. Our hypothesis is that these in vitro conditions model regeneration of muscle and can be used to evaluate potential therapeutics. In this study, 2 areas of satellite cell response were tested: proliferation of clones induced by growth factors, and fusion induced by culture conditions. Equine satellite cell clones showed differences in their response to growth factors as well as accumulation of cellular protein concentrations. Equine satellite cells proliferate in response to both human and bovine FGF. IGF-1, a powerful mitogen of other satellite cell culture systems, was not as effective for inducing equine satellite cell proliferation. Protein concentrations were also measured in satellite cell cultures. Clones differed in cellular protein produced depending on growth conditions. Conditions inducing differentiation into myotubes was also determined for a 96 well assay and can be used to study the final stage of functioning muscle production. This in vitro model is the first step in identifying potential therapeutics to speed wound healing and promote muscle regeneration in horses.     

The role of alveolar macrophages in the pathogenesis of recurrent airway obstruction in horses

When challenged with allergens and pro-inflammatory agents, such as Aspergillus fumigatus (AF), hay dust solution (HDS) and lipopolysaccharide (LPS), the innate immune response will not only activate the immune system but also increase the amount of pro-inflammatory cytokines in the bronchoalveolar space. The aim of this study was to assess the response of equine alveolar macrophages to different aerosolized challenges and to investigate the differences in this response between horses susceptible or nonsusceptible to recurrent airway obstruction (RAO). Seven susceptible and 5 nonsusceptible horses were challenged with saline, LPS, HDS, or AF, and bronchoalveolar lavage (BAL) cytology, total cell counts, and lung function were assessed. In addition, alveolar macrophages were isolated 6 and 24 hours after challenge, and macrophage mRNA expression of tumor necrosis factor (TNF)-alpha and interleukins (IL) IL-1beta, IL-6, IL-8, and IL-10 were measured by means of real-time (RT) polymerase chain reaction (PCR). There was a significant difference in lung function, neutrophil ratios, and total cell counts in the bronchoalveolar lavage fluid between RAO-susceptible and nonsusceptible horses. In addition, the expression of TNF-alpha, IL-1beta, and IL-8 by alveolar macrophages after challenges were higher in susceptible horses, than in nonsusceptible horses. In contrast, I1-6, considered an anti-inflammatory cytokine, showed a higher expression in nonsusceptible horses 6 hours after inhalation challenge with allergens and pro-inflammatory antigens. These data suggest that the differences between susceptible and nonsusceptible horses to RAO are not only dependent on adaptive immunity but also start with an innate immune response.     

The cellular Toll-like receptor 4 antagonist E5531 can act as an agonist in horse whole blood

Sepsis and endotoxaemia are important causes of morbidity and mortality in humans. Research on sepsis focuses on rodent models most of which are poorly responsive to lipopolysaccharide (LPS), and thus do not mimic very well the high sensitivity of humans. Therefore, there is a need to develop more clinically relevant models. Horses suffer from a similar endotoxaemic syndrome to humans with high morbidity and mortality. LPS analogues that act as antagonists at Toll-like receptor 4 (TLR4) are being developed as novel treatments for endotoxaemia. Due to differences in recognition of ligands by TLR4 from different mammalian species, individual LPS molecules may act as agonists in some species and antagonists in others. The synthetic lipid A analogue E5531 is an antagonist at TLR4 in humans and mice, but its effects at TLR4 from other species are unknown. In the studies reported here, Escherichia coli LPS is a full agonist on equine bone marrow macrophage-like cells and its effects are antagonised by E5531. Similarly, E. coli LPS is an agonist and E5531 an antagonist on monocytes isolated from peripheral blood of healthy horses and human embryonic kidney (HEK) cells, transiently transfected to express horse TLR4 and its associated cell surface proteins MD2 and CD14. In contrast, both E. coli LPS and E5531 behave as agonists in horse whole blood by inducing production of equivalent amounts of the inflammatory mediator prostaglandin. This finding suggests that modification of E5531 may occur in whole blood, for example, deacylation, which alters its activity. This comparative study has revealed a novel pharmacological action of E5531 and emphasises the importance of extending studies of this nature beyond the normal rodent models.     

Science alone is not always enough: The importance of ethical assessment for a more comprehensive view of equine welfare

Researchers in equitation science have worked diligently to use objective measures in horse behavior and welfare studies, ones that colleagues will appreciate. This is appropriate. However, because equine welfare is not purely an empirical matter, we must recognize that science alone may not be enough when assessing equine treatment. The science community must realize, as does the public already, that different value assumptions will lead to different welfare emphases. Thus, there is an intimate interplay between science and values where the quality of life of both human and nonhuman animals are concerned. If certain training practices or aesthetic modifications are shown to the public and a majority finds them objectionable, the practices should be further scrutinized. There are still many that believe if a horse is healthy and performing competitively then its welfare is good. On closer scrutiny, equine welfare is conceptually more nuanced than this and includes an ethical/values-based component. Welfare is not only concerned with biological functioning, but also with “affective states” such as emotions, pain, suffering, and frustration when opportunities to express species-characteristic behaviors are thwarted. These emphases, informed by empirical insights, reflect value frameworks that influence the scientific study of equine welfare. Here, we consider the complementary values-based side of equine welfare and suggest how ethical assessments have the potential to enhance equine welfare. Three different ethical accounting methods are presented in this article and an example from the horse industry is used to illustrate each. Each subdiscipline of the horse industry contains at least a few ethically questionable practices. Stakeholders are challenged to examine their area of the horse industry and evaluate questionable practices, possibly using the ethical accounting processes discussed in this article.     

Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes.

In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes.

The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response.

In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.     

Toxin production by and adhesive properties of Clostridium difficile isolated from humans and horses with antibiotic-associated diarrhea

Clostridium difficile is a common nosocomial pathogen in humans and animals that causes diarrhea and colitis following antibiotic therapy. Isolates of C. difficile obtained from faecal material from 20 human patients and 6 equine subjects with antibiotic-associated diarrhea were investigated regarding production of toxins A and B, their capacity to adhere to the human intestinal Caco-2 cell line and equine intestinal cells, and for the presence of fimbriae. The results showed that most (17/20) of the human clinical isolates produced both toxins A and B. One of the human isolates proved toxin A-negative/toxin B-positive. All (6/6) horse isolates were positive for both toxins A and B. Both the human and horse isolates possessed the capacity to adhere, to varying degree, to human and equine intestinal cells. It appeared that human isolates produced greater amounts of toxin B, and that there was a host-species dependency on ability to attach to intestinal epithelial cells. No fimbriae were found in any of the investigated isolates.     

Proadifen-induced production of prostacyclin by equine peritoneal macrophages

A study was performed to determine the effect of proadifen hydrochloride on prostacyclin (prostaglandin I2 [PGI2]) and thromboxane A2 (TxA2) synthesis by equine peritoneal macrophages and the effect of proadifen on endotoxin-induced synthesis of PGI2 and TxA2 by equine macrophages. Peritoneal macrophages (2.5 x 10(6)/ml) were incubated for 6 hours in tissue culture media containing 1) nothing (nontreated control), 2) proadifen hydrochloride (20, 100, 250, and 500 mumol/L, 3) endotoxin (5 ng/ml), or 4) the calcium ionophore A23187 (0.95 mumol/L). In a second series of experiments, peritoneal macrophages were incubated with endotoxin (5 ng/ml) and proadifen (250 umol/L), for 6 hours. Concentrations of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2, the stable metabolites of PGI2 and TxA2, were determined in the incubation media by radioimmunoassay. Proadifen caused increased synthesis of PGI2 by equine macrophages, without affecting TxA2 production. The increased PGI2 production was similar to that induced by endotoxin and calcium ionophore; however, the latter 2 agents significantly stimulated TxA2 production as well (P less than 0.05). There were no significant differences among mean concentrations of 6-keto-PGF1 alpha in media from macrophages treated with 100, 250, or 500 mumol/L proadifen, but there was a significant curvilinear regression between their concentrations. The ratio of thromboxane B2 to 6-keto-PGF1 alpha was significantly lower than baseline in incubation media from macrophages exposed to proadifen, endotoxin, and calcium ionophore. Proadifen hydrochloride did not significantly change equine peritoneal macrophage production of PGI2 or TxA2 in response to endotoxin.     

Endotoxin-induced production of interleukin 6 by equine peritoneal macrophages in vitro.

A study was performed to determine whether equine peritoneal macrophages produce interleukin 6 (IL-6) in vitro in response to endotoxin. Peritoneal fluid was collected from 14 clinically normal adult horses and was used as the source of peritoneal macrophages. Macrophages from each horse were isolated and cultured separately in vitro in the absence or presence of various concentrations (0.5, 5, 500 ng/ml) of endotoxin (lipopolysaccharide from Escherichia coli 055:B5). Culture medium supernatants were collected after 3, 6, 12, and 24 hours' incubation and were frozen at -70 C until assayed for IL-6 activity. Supernatant IL-6 activity was determined by use of a modified colorimetric assay and the murine hybridoma cell line B 13.29 clone B.9, which is dependent on IL-6 for survival. Results indicated that equine peritoneal macrophages produce IL-6 in vitro and that supernatant medium IL-6 activity was significantly (P less than 0.05) increased by exposure to endotoxin. Significant (P less than 0.05) time and treatment effects on macrophage IL-6 production were apparent. The IL-6 activity peaked at 6 or 12 hours' incubation, then remained high through 24 hours' incubation, regardless of endotoxin exposure. Medium IL-6 activity during 3 and 6 hours' incubation was significantly (P less than 0.05) greater in macrophages exposed to 5 or 500 ng of endotoxin/ml than in those exposed to 0.5 ng of endotoxin/ml; however peak IL-6 activity was similar among all endotoxin concentrations. Endotoxin concentration did not have an effect on medium IL-6 activity from macrophages exposed to endotoxin for 12 or 24 hours.     

Kinetics of equine neutrophil elastase release and superoxide anion generation following secretagogue activation: a potential mechanism for antiproteinase inactivation

Man and horses both suffer from neutrophil mediated pulmonary diseases however there are striking species differences in the underlying pathology. In particular while pulmonary emphysema is a common pathological sequel to human respiratory disease it is not a major feature of the common equine neutrophil mediated condition, chronic obstructive pulmonary disease (COPD). The proposed reason for this difference is that equine neutrophils contain less elastase than equivalent human cells and therefore there is a reduced risk of excess and/or uninhibited elastase activity, which is considered the major cause of pulmonary emphysema in man, in the horse lung. In previous studies equine neutrophil elastase (ENE) has been assayed by measuring elastinolytic activity whereas human neutrophil elastase content has been determined using immunological techniques. Neutrophils contain several intracellular protease inhibitors therefore measurement of elastase activity may underestimate the total NE content. The aim of the current study was to develop immunological techniques to allow investigation of the cellular content, distribution and release of ENE from purified equine neutrophils. Equine neutrophil elastase 2A (ENE 2A), the most abundant elastase in equine neutrophils, and equine alpha-1-proteinase inhibitor (API), the main inhibitor of elastase were found to be present at 0.813 pg +/- 0.179 and 0.021 pg +/- 0.003 (mean +/- SEM, n = 11 individual horses) per neutrophil, respectively. This represents twice as much elastase as previously found in the equine neutrophil and a comparable amount to that reported in human neutrophils. Immunolocalisation demonstrated that ENE 2A has a granular distribution within the cytosol of neutrophils, whereas API exhibits a uniform non-granular cytoplasmic appearance. In addition the kinetics of simultaneous generation and release of superoxide anions (SOA) and release of ENE 2A from equine neutrophils, stimulated in vitro by zymosan-activated serum (ZAS) in the presence and absence of the cation chelator ethylene glycol-N,N,N',N'-tetraacetic acid (EGTA), showed a close relationship between total SOA generation and total ENE 2A release during the initial 90 min post-ZAS stimulation and the dependence of both events on extracellular cations. In conclusion these studies have shown that horse and human neutrophil elastase content and mediator release functions are more closely matched than was previously thought. This suggests that the species differences in pathology resulting from neutrophil-mediated respiratory disease are determined by other factors such as differences in the abundance and function of intra- and extra-cellular protease inhibitors.     

Phagocytotic activation of muscularis resident macrophages inhibits smooth muscle contraction in rat ileum

Intestinal muscularis resident macrophages distributed in myenteric region may play an important role in the immunological host defense against infection. In this study, we investigated the phagocytic stimulation of resident macrophages on cyclooxygenase-2 (COX-2) expression and smooth muscle contraction in the small intestine of rat. After the injection of FITC-dextran to rat, phagocytosed macrophages could be detected in the myenteric plexus. FITC-positive macrophages were also immunostained with COX-2 antibody. The number of COX-2 immunopositive cells increased in a time-dependent manner reaching its maximum at 4 hr after the injection, which then decreased gradually but considerable number of cells were still remained on 7 days. The injection of FITC-dextran, however, did not change the population of ED2-positive resident macrophages even on 7 days. Production of PGE2 was significantly higher in the dextran treated tissue as compared to control tissue. In the smooth muscle tissue phagocytosed dextran, carbachol-induced contraction was significantly decreased. The suppression of the carbachol-induced contraction was completely restored by COX inhibitor, indomethacin. Finally we demonstrated that, in freshly isolated macrophage cells, addition of dextran induced a slow and sustained increase in intracellular Ca2+ concentration. These results indicate that phagocytotic activation of muscularis resident macrophages induces COX-2 gene expression and then results in production of PGE2 to suppress the smooth muscle contractile activity.