John F. Marshall

The effect of nonsteroidal anti-inflammatory drugs on the equine intestine

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in the management of pain and endotoxaemia associated with colic in the horse. While NSAIDs effectively treat the symptoms of colic, there is evidence to suggest that their administration is associated with adverse gastrointestinal effects including right dorsal colitis and inhibition of mucosal barrier healing. Several studies have examined the pathophysiology of NSAID associated effects on the large and small intestine in an effort to avoid these complications and identify effective alternative medications. Differences in the response of the large and small intestines to injury and NSAID treatment have been identified. Flunixin meglumine has been shown in the small intestine to inhibit barrier function recovery and increase permeability to lipopolysaccharide (LPS). A range of NSAIDs has been examined in the small intestine and experimental evidence suggests that those NSAIDs with cyclooxygenase independent anti-inflammatory effects or a COX-2 selective mode of action may offer significant advantages over traditional NSAIDs.

An inertial sensor-based system can objectively assess diagnostic anaesthesia of the equine foot

REASONS FOR PERFORMING STUDY In order for changes in lameness to be accurately and repeatably detected and recorded during diagnostic investigations, an objective measure of lameness is required. OBJECTIVES To ascertain whether an inertial sensor-based system can distinguish between a positive and negative response to diagnostic anaesthesia of the foot and objectively assess the effect of a positive response on the trot. STUDY DESIGN Restrospective clinical study. METHODS Data obtained during lameness investigations undertaken between August 2011 and December 2012 in which either a palmar digital or abaxial sesamoid nerve block was performed were retrospectively reviewed. Response to diagnostic anaesthesia was categorised as positive (n = 14) or negative (n = 9) by one of 2 evaluators before analysis of kinematic data (i.e. blinded). Changes in maximum and minimum head difference (ΔHDMax and ΔHDMin) and change in head movement asymmetry/change in pelvic movement asymmetry (ΔHMA/PMA, measure of asymmetry) allocated to each limb were calculated. A Kruskal-Wallis one way analysis of variance on ranks was performed. Receiver operating characteristic (ROC) curves were generated for ΔHDMax, ΔHDMin and ΔHMA for the blocked limb to identify cut-off values to distinguish positive and negative responses to the block. RESULTS Median ΔHDMax and ΔHDMin were significantly greater after a positive response to diagnostic anaesthesia (P<0.01). Change in head movement asymmetry allocated to the blocked limb and contralateral forelimb and ΔPMA allocated to the contralateral hindlimb were significantly greater in the positive response group (P<0.05). Change in head movement asymmetry allocated to the blocked limb and ΔHDMax and ΔHDMin are useful diagnostic tests for identifying positive response to anaesthesia (area under the curve = 0.98, 0.83 and 0.96 respectively). CONCLUSIONS An inertial sensor-based system can identify a positive response to diagnostic anaesthesia of the foot. Symmetry of movement allocated to the blocked limb, contralateral forelimb and contralateral hindlimb significantly improve, and head movement significantly decreases in horses with a positive response to the block. Cut-off values for a positive response have been identified with good sensitivity and specificity. Forelimb lameness significantly affects contralateral hindlimb movement, which has implications for the investigation of multi-limb lameness.

Infection and pathogenesis of canine, equine and human influenza viruses in canine tracheas.

ABSTRACT Influenza A viruses (IAVs) can jump species barriers and occasionally cause epidemics, epizootics, pandemics, and panzootics. Characterizing the infection dynamics at the target tissues of natural hosts is central to understanding the mechanisms that control host range, tropism, and virulence. Canine influenza virus (CIV; H3N8) originated after the transfer of an equine influenza virus (EIV) into dogs. Thus, comparing CIV and EIV isolates provides an opportunity to study the determinants of influenza virus emergence. Here we characterize the replication of canine, equine, and human IAVs in the trachea of the dog, a species to which humans are heavily exposed. We define a phenotype of infection for CIV, which is characterized by high levels of virus replication and extensive tissue damage. CIV was compared to evolutionarily distinct EIVs, and the early EIV isolates showed an impaired ability to infect dog tracheas, while EIVs that circulated near the time of CIV emergence exhibited a CIV-like infection phenotype. Inoculating dog tracheas with various human IAVs (hIAVs) showed that they infected the tracheal epithelium with various efficiencies depending on the virus tested. Finally, we show that reassortant viruses carrying gene segments of CIV and hIAV are viable and that addition of the hemagglutinin (HA) and neuraminidase (NA) of CIV to the 2009 human pandemic virus results in a virus that replicates at high levels and causes significant lesions. This provides important insights into the role of evolution on viral emergence and on the role of HA and NA as determinants of pathogenicity. IMPORTANCE Influenza A viruses (IAVs) have entered new host species in recent history, sometimes with devastating consequences. Canine influenza virus (CIV) H3N8 originated from a direct transfer of an equine influenza virus (EIV) in the early 2000s. We studied the infection patterns of IAVs that circulate in dogs or to which dogs are commonly exposed and showed that CIV emergence was likely caused by an adaptive driver, as evolutionarily distinct EIVs display distinct infection phenotypes. We also showed that many human viruses can infect dog tracheas and that reassortment with CIV results in viable viruses. Finally, we showed that the hemagglutinin and neuraminidase of CIV act as virulence factors. Our findings have significant implications because they show that dogs might act as “mixing vessels” in which novel viruses with pandemic potential could emerge and also provide experimental evidence supporting the role of viral evolution in influenza virus emergence.

Naturally-occurring forelimb lameness in the horse results in significant compensatory load redistribution during trotting

This study aimed to quantify the compensatory response to naturally-occurring forelimb lameness on load redistribution. Data from lameness investigations using an inertial sensor based system to monitor the response to forelimb diagnostic anaesthesia were reviewed. Horses with primary forelimb lameness were grouped for analysis as (1) all horses combined (n= 28), (2) forelimb-only lameness (n= 8/28), (3) forelimb-contralateral hindlimb lameness (n= 14/28), (4) forelimb-ipsilateral hindlimb lameness (n= 6/28). The effect of diagnostic anaesthesia on measures of head and pelvic movement asymmetry was determined using the Wilcoxon signed rank test. Spearmans correlation analysis was performed between forelimb and hindlimb variables. Statistical significance was set at P< 0.05. Forelimb diagnostic anaesthesia resulted in a decrease in pelvic movement asymmetry among all horses and the forelimb-only and forelimb-contralateral hindlimb lameness groups. Pelvic movement asymmetry associated with the contralateral hindlimb decreased by a median of 38% (interquartile range [IQR] 10-65%), 43% (IQR 28-60%) and 28% (IQR 12-67%) in all horses, forelimb-only and forelimb-contralateral hindlimb groups respectively (P< 0.05). Maximum pelvic height difference (PDMax) significantly decreased in all horses combined and the forelimb-contralateral hindlimb lameness group by a median of 66% (IQR 24-100%) and 78% (IQR 27-100%, P< 0.01), respectively. Change in head movement asymmetry and vector sum was significantly positively correlated with PDMax in all horses combined and the forelimb-contralateral hindlimb group (P< 0.05). Forelimb lameness had a significant effect on hindlimb and pelvic movement in horses with clinical lameness resulting in compensatory load redistribution and decreased push-off from the contralateral hindlimb.

Evaluation of the cyclooxygenase selectivity of robenacoxib and its effect on recovery of ischemia-injured jejunal mucosa in horses

OBJECTIVE To determine the cyclooxygenase (COX) selectivity of robenacoxib and its effect on recovery of jejunal mucosa following ischemic injury in horses. ANIMALS 12 healthy horses. PROCEDURES Half the maximal inhibition (EC₅₀) of robenacoxib for COX-1 and COX-2 activity was established in bloods samples from 6 horses via measurement of thromboxane B₂ (TXB₂) and prostaglandin E₂ concentrations, respectively; COX selectivity was subsequently calculated. Six other horses were anesthetized, and ischemia was induced in the jejunum for 2 hours. Control and ischemia-injured mucosa were collected and incubated with Ringers solution (control treatment), flunixin meglumine (2.7 × 10⁻⁵M), or robenacoxib (2.7 × 10⁻⁵M). Transepithelial electrical resistance and mannitol flux were measured over a 4-hour recovery period. Bathing solution TXB₂ and prostaglandin E metabolite concentrations were measured to assess COX-1 and COX-2 function, respectively. RESULTS The mean ± SD EC₅₀ value of robenacoxib for COX-1 and COX-2 was 11.46 ± 4.46 μM and 0.19 ± 0.07 μM, respectively, resulting in a COX selectivity ratio of 61.01. The transepithelial electrical resistance of ischemia-injured jejunum treated with flunixin meglumine was significantly lower than that of control and robenacoxib-treated tissues. A significant increase in concentrations of prostaglandin E metabolites and TXB₂ was detected in control and robenacoxib-treated tissues but not flunixin meglumine-treated tissues. CONCLUSIONS AND CLINICAL RELEVANCE Robenacoxib selectively inhibited COX-2 and allowed recovery of barrier function in ischemia-injured equine jejunal tissue in vitro.

Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1.

ABSTRACT Influenza A viruses (IAVs) are common pathogens of birds that occasionally establish endemic infections in mammals. The processes and mechanisms that result in IAV mammalian adaptation are poorly understood. The viral nonstructural 1 (NS1) protein counteracts the interferon (IFN) response, a central component of the host species barrier. We characterized the NS1 proteins of equine influenza virus (EIV), a mammalian IAV lineage of avian origin. We showed that evolutionarily distinct NS1 proteins counteract the IFN response using different and mutually exclusive mechanisms: while the NS1 proteins of early EIVs block general gene expression by binding to cellular polyadenylation-specific factor 30 (CPSF30), NS1 proteins from more evolved EIVs specifically block the induction of IFN-stimulated genes by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially and were rapidly selected for during EIV evolution, highlighting the importance of evolutionary processes in immune evasion mechanisms during IAV adaptation. IMPORTANCE Influenza A viruses (IAVs) infect certain avian reservoir species and occasionally transfer to and cause epidemics of infections in some mammalian hosts. However, the processes by which IAVs gain the ability to efficiently infect and transmit in mammals remain unclear. H3N8 equine influenza virus (EIV) is an avian-origin virus that successfully established a new lineage in horses in the early 1960s and is currently circulating worldwide in the equine population. Here, we analyzed the molecular evolution of the virulence factor nonstructural protein 1 (NS1) and show that NS1 proteins from different time periods after EIV emergence counteract the host innate immune response using contrasting strategies, which are associated with two mutations that appeared sequentially during EIV evolution. The results shown here indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.

Effect of flunixin meglumine and firocoxib on ex vivo cyclooxygenase activity in horses undergoing elective surgery

OBJECTIVE To evaluate ex vivo cyclooxygenase (COX) inhibition and compare in vitro and ex vivo COX-1 inhibition by flunixin meglumine and firocoxib in horses. ANIMALS 4 healthy horses for in vitro experiments and 12 healthy horses (6 males and 6 females; 5 Thoroughbreds, 5 Warmbloods, and 2 ponies) undergoing elective surgery for ex vivo experiments. PROCEDURES 12 horses received flunixin meglumine (1.1 mg/kg, IV, q 12 h) or firocoxib (0.09 mg/kg, IV, q 24 h). Blood samples were collected before (baseline) and 2 and 24 hours after NSAID administration. Prostanoids (thromboxane B2, prostaglandin E2, and prostaglandin E metabolites) served as indicators of COX activity, and serum drug concentrations were measured by use of high-performance liquid chromatography. An in vitro coagulation-induced thromboxane B2 assay was used to calculate drug concentration-COX-1 inhibition curves. Effect of time and treatment on COX activity was determined. Agreement between in vitro and ex vivo measurement of COX activity was assessed with Bland-Altman analysis. RESULTS At 2 and 24 hours after NSAID administration, COX-1 activity was reduced, compared with baseline activity, for the flunixin meglumine group only and relative COX-1 activity was significantly greater for the firocoxib group, compared with that for the flunixin meglumine group. There was no significant change in COX-2 activity after surgery for either group. Bland-Altman analysis revealed poor agreement between in vitro and ex vivo measurement of COX-1 activity. CONCLUSIONS AND CLINICAL RELEVANCE Compared with flunixin meglumine, firocoxib had COX-1-sparing effects ex vivo in equine patients that underwent elective surgery.

Disease and pharmacologic risk factors for first and subsequent episodes of equine laminitis: A cohort study of free-text electronic medical records

Electronic medical records from first opinion equine veterinary practice may represent a unique resource for epidemiologic research. The appropriateness of this resource for risk factor analyses was explored as part of an investigation into clinical and pharmacologic risk factors for laminitis. Amalgamated medical records from seven UK practices were subjected to text mining to identify laminitis episodes, systemic or intra-synovial corticosteroid prescription, diseases known to affect laminitis risk and clinical signs or syndromes likely to lead to corticosteroid use. Cox proportional hazard models and Prentice, Williams, Peterson models for repeated events were used to estimate associations with time to first, or subsequent laminitis episodes, respectively. Over seventy percent of horses that were diagnosed with laminitis suffered at least one recurrence. Risk factors for first and subsequent laminitis episodes were found to vary. Corticosteroid use (prednisolone only) was only significantly associated with subsequent, and not initial laminitis episodes. Electronic medical record use for such analyses is plausible and offers important advantages over more traditional data sources. It does, however, pose challenges and limitations that must be taken into account, and requires a conceptual change to disease diagnosis which should be considered carefully.

Validation of an Improved Computer-Assisted Technique for Mining Free-Text Electronic Medical Records

Background The use of electronic medical records (EMRs) offers opportunity for clinical epidemiological research. With large EMR databases, automated analysis processes are necessary but require thorough validation before they can be routinely used. Objective The aim of this study was to validate a computer-assisted technique using commercially available content analysis software (SimStat-WordStat v.6 (SS/WS), Provalis Research) for mining free-text EMRs. Methods The dataset used for the validation process included life-long EMRs from 335 patients (17,563 rows of data), selected at random from a larger dataset (141,543 patients, ~2.6 million rows of data) and obtained from 10 equine veterinary practices in the United Kingdom. The ability of the computer-assisted technique to detect rows of data (cases) of colic, renal failure, right dorsal colitis, and non-steroidal anti-inflammatory drug (NSAID) use in the population was compared with manual classification. The first step of the computer-assisted analysis process was the definition of inclusion dictionaries to identify cases, including terms identifying a condition of interest. Words in inclusion dictionaries were selected from the list of all words in the dataset obtained in SS/WS. The second step consisted of defining an exclusion dictionary, including combinations of words to remove cases erroneously classified by the inclusion dictionary alone. The third step was the definition of a reinclusion dictionary to reinclude cases that had been erroneously classified by the exclusion dictionary. Finally, cases obtained by the exclusion dictionary were removed from cases obtained by the inclusion dictionary, and cases from the reinclusion dictionary were subsequently reincluded using Rv3.0.2 (R Foundation for Statistical Computing, Vienna, Austria). Manual analysis was performed as a separate process by a single experienced clinician reading through the dataset once and classifying each row of data based on the interpretation of the free-text notes. Validation was performed by comparison of the computer-assisted method with manual analysis, which was used as the gold standard. Sensitivity, specificity, negative predictive values (NPVs), positive predictive values (PPVs), and F values of the computer-assisted process were calculated by comparing them with the manual classification. Results Lowest sensitivity, specificity, PPVs, NPVs, and F values were 99.82% (1128/1130), 99.88% (16410/16429), 94.6% (223/239), 100.00% (16410/16412), and 99.0% (100×2×0.983×0.998/[0.983+0.998]), respectively. The computer-assisted process required few seconds to run, although an estimated 30 h were required for dictionary creation. Manual classification required approximately 80 man-hours. Conclusions The critical step in this work is the creation of accurate and inclusive dictionaries to ensure that no potential cases are missed. It is significantly easier to remove false positive terms from a SS/WS selected subset of a large database than search that original database for potential false negatives. The benefits of using this method are proportional to the size of the dataset to be analyzed.

Prevalence, survival analysis and multimorbidity of chronic diseases in the general veterinarian-attended horse population of the UK

The average age of the global human population is increasing, leading to increased interest in the effects of chronic disease and multimorbidity on health resources and patient welfare. It has been posited that the average age of the general veterinarian-attended horse population of the UK is also increasing, and therefore it could be assumed that chronic diseases and multimorbidity would pose an increasing risk here also. However, evidence for this trend in ageing is very limited, and the current prevalence of many chronic diseases, and of multimorbidity, is unknown. Using text mining of first-opinion electronic medical records from seven veterinary practices around the UK, Kaplan-Meier and Cox proportional hazard modelling, we were able to estimate the apparent prevalence among veterinarian-attended horses of nine chronic diseases, and to assess their relative effects on median life expectancy following diagnosis. With these methods we found evidence of increasing population age. Multimorbidity affected 1.2% of the study population, and had a significant effect upon survival times, with co-occurrence of two diseases, and three or more diseases, leading to 6.6 and 21.3 times the hazard ratio compared to no chronic disease, respectively. Laminitis was involved in 74% of cases of multimorbidity. The population of horses attended by UK veterinarians appears to be aging, and chronic diseases and their co-occurrence are common features, and as such warrant further investigation.