Dynamic changes in circulating leukocytes during the induction of equine laminitis with black walnut extract

Administration of black walnut heartwood extract (BWHE) via nasogastric tube induces acute laminitis in horses. However, the processes responsible for the development of laminitis, including laminitis induced with BWHE, remain unclear. The results of recent studies indicate that administration of BWHE initiates an inflammatory response in the laminar tissues and that this response may be due to extravasation of activated leukocytes from the circulation. This study examines the effects of BWHE administration on the dynamics of circulating neutrophils and monocytes, and the capacity of blood leukocytes to produce radical oxygen species (ROS) over the time period from administration of BWHE to the development of lameness consistent with Obel grade I laminitis.

Individual horses, free of pre-existing musculoskeletal disease, were administered either 6 l of BWHE or an equal volume of water at time 0 (T = 0). Blood samples were collected prior to dosing and at 1, 2, 3, 4, 6, 8, 10 and 12 h after dosing, or until the onset of Obel grade I laminitis. For each sample, total leukocyte counts were determined followed by collection of buffy coats and removal of erythrocytes by hypotonic lysis. Leukocytes were either fixed for flow cytometric assessment of differential counts or maintained in culture to measure endogenous and phorbol ester-induced production of ROS. At each sample time, the number of cells recovered and the flow cytometric differential counts were compared with corresponding total leukocyte counts determined by the Clinical Pathology laboratory.

Horses administered BWHE had a significant reduction in circulating leukocytes at 3–4 h relative to values for horses administered the same volume of water. Horses that developed Obel grade I laminitis had a significant reduction in circulating leukocytes when compared to values for horses administered BWHE that did not become lame. Flow cytometric analysis revealed a consistent decrease in the total number of monocytes obtained from horses that developed laminitis. In these same horses, the endogenous level of ROS production was significantly higher at T = 0 than for horses that did not become lame. Furthermore, production of ROS by leukocytes from horses that developed laminitis increased significantly and coincided with the decrease in circulating leukocytes.

Collectively, these findings support a role for systemic activation of leukocytes and induction of inflammation by BWHE as a factor in the early pathogenesis of acute laminitis. Because laminitis often develops as a sequel to diseases characterized by systemic inflammatory events, activation and emigration of neutrophils and monocytes may be important factors in the early pathogenesis of laminitis in clinical cases.     

Recent developments in equine dentistry

ABSTRACT

Equine dentistry is a rapidly developing clinical specialty. It has benefitted from key advances in anatomical and physiological research, development of equipment and instrumentation, utilisation of standing sedation and anaesthesia protocols, a change towards minimally invasive surgical techniques, and the introduction of restorative and endodontic techniques translated from techniques used in human and canine dentistry. Anatomical research has provided further insight into the endodontic system of incisors and cheek teeth and how it changes throughout development with age. Studies of the periodontium have demonstrated a rich vascular supply and repair capacity. Routine dental visits are increasingly being performed utilising sedation and clinical instruments for routine examinations. Equipment has become more efficient, battery-operated and miniaturised giving benefits to both equine dental maintenance work and advanced techniques, assisting the transition to minimally invasive techniques, and the development of endodontic and restorative dentistry. Diagnosis has also benefitted from advances in equipment such as patient-side digital radiography systems, high definition oroscopy, and small diameter flexible fibrescopes that are capable of visualising inside a pulp canal. Dental units combining endodontic high- and low-speed drills, suction and air or water flush are becoming increasingly used and adapted for equine use. Sedative combinations and standing anaesthesia protocols have meant that revisions of traditional techniques, as well as novel techniques, can be performed with almost no requirement for general anaesthesia. Equine dentistry can only continue to advance in this way if there is early identification of dental disease through clinical oral examinations, leading to a system based on prophylaxis, as in human dentistry. This necessitates a change in attitude of the public and industry in general to a proactive approach, with early intervention based on examination and diagnostic findings of practitioners and not necessarily based on the clinical signs displayed by the patient.     

Emerging outbreaks associated with equine coronavirus in adult horses

The purpose of this study was to describe clinical, hematological and fecal PCR results from 161 horses involved in outbreaks associated with ECoV. The outbreaks happened at four separate boarding facilities between November 2011 and April 2012 in the States of CA, TX, WI and MA. Following the molecular detection of ECoV in the feces from the initial index cases, the remaining herdmates were closely observed for the development of clinical signs. Fecal samples were collected from sick and healthy horses for the PCR detection of ECoV. All four outbreaks involved primarily adult horses. Fifty-nine horses developed clinical signs with 12–16 sick horses per outbreak. The main clinical signs reported were anorexia, lethargy and fever. Four horses from 3 different outbreaks were euthanized or died due to rapid progression of clinical signs. The cause of death could not be determined with necropsy evaluation in 2 horses, while septicemia secondary to gastrointestinal translocation was suspected in 2 horses. Blood work was available from 10 horses with clinical disease and common hematological abnormalities were leucopenia due to neutropenia and/or lymphopenia. Feces were available for ECoV testing by real-time PCR from 44 and 96 sick and healthy horses, respectively. 38/44 (86%) horses with abnormal clinical signs tested PCR positive for ECoV, while 89/96 (93%) healthy horses tested PCR negative for ECoV. The overall agreement between clinical status and PCR detection of ECoV was 91%. The study results suggest that ECoV is associated with self-limiting clinical and hematological abnormalities in adult horses.     

Current practice relating to equine castration in the UK

This study aimed to characterise current practice relating to equine castration in the UK. A questionnaire was posted to all 655 veterinary practices specified to provide veterinary care for horses, or classified as specialist equine practices. Respondents were asked to cite the number of equine castrations performed annually by the practice, describe techniques used for castration, outline anaesthetic/sedative/analgesic drug protocols used and provide details of post-operative medication. There was a 43% response rate to the questionnaire. Considerable variation in techniques and analgesia provision was identified, with the majority of respondents using a number of sedation/anaesthetic protocols rather than a single technique. This characterisation of current practice provides a useful platform from which subsequent investigations into welfare implications of current equine castration techniques can be directed.     

A case-control study of risk factors for equine influenza spread onto horse premises during the 2007 epidemic in Australia

The 2007 epidemic of equine influenza in Australia provided an opportunity to investigate the effectiveness of on-farm biosecurity measures in preventing the spread of a novel pathogen in a largely naive population. We conducted a case-control study of 200 horse premises from highly affected regions of the state of New South Wales (NSW), to investigate risk factors for the spread of equine influenza onto horse premises, specifically, non-compliance with biosecurity measures recommended to horse owners by the relevant animal health authority, the NSW Department of Primary Industries. The study was restricted to cases occurring during the first seven weeks of the epidemic, a period prior to vaccination and the relaxation of some movement restrictions. Case and control premises were selected from a laboratory testing dataset and interviews were conducted with horse owners and managers on premises between July and November 2009. The proximity of premises to the nearest infected premises was the factor most strongly associated with case status. Case premises were more likely than control premises to be within 5 km and beyond 10 km of an infected premises. Having a footbath in place on the premises before any horses were infected was associated with a nearly four-fold reduction in odds of infection (odds ratio=0.27; 95% confidence interval: 0.09, 0.83). This protective association may have reflected overall premises biosecurity standards related to the fomite transmission of equine influenza. Compliance with certain on-farm biosecurity practices seemingly prevented horses on premises in high risk areas being infected with equine influenza during the 2007 outbreak in Australia. In future outbreaks, in addition to broader disease control measures, on-farm biosecurity practices should be adopted by horse owners and managers to prevent equine influenza spread.     

Molecular cloning and characterization of equine Toll-like receptor 9

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4(+) and CD8(+) T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.     

Bovine papillomavirus infection in equine sarcoids and in bovine bladder cancers

Bovine papillomavirus (BPV) type 2 is involved in carcinogenesis of the urinary bladder in cattle, while BPV-1 is commonly associated with equine sarcoid tumours. In both cases the early viral proteins are expressed, but virion is not produced. Given the similarities in BPV biology between the tumours in cattle and horses, bovine bladder cancers and equine sarcoids were compared with respect to physical status, load of viral DNA and variability of the E5 open reading frame (ORF). Rolling circle amplification demonstrated that BPV-1 and BPV-2 genomes exist as double stranded, episomal, circular forms in the two tumours. Realtime quantitative PCR revealed that equine sarcoids contained higher viral DNA loads compared to bovine bladder cancers. The BPV-1 E5 ORF showed sequence variation but BPV-2 ORF did not. The presence of BPV-1 E5 variations or their absence in the BPV-2 E5 ORF does not appear to have an effect on viral DNA load in either tumour type.     

Kinematic Characterization of the Menorca Horse at the Walk and the Trot: Influence of Hind Limb Pastern Angle

This paper describes the handled walking and trotting kinematics (linear, temporal, and angular traits) of 35 Menorca Purebred (MEN) stallions, and the relationships among these variables is presented for the first time, along with a discussion of the influence of the hind limb pastern angle on kinematic variables at both gaits. For data collection, all animals, aged between 3 and 10 years old and belonging to 28 different studs, were recorded under the same experimental and environmental conditions, using a three-dimensional (3D) semiautomatic movement analysis system. A total of 24 kinematic variables (temporal, linear, and angular) at the walk and the trot and a morphometric variable measured at the mid stance position of walking (hind pastern angle) were included in this analysis. Angle-time diagrams of the hind pastern angle while walking and trotting normalized to stride duration were also obtained. Generally the MEN stallions' forelimb movements closely resembled the movement characteristics of other European dressage performance breeds, while the hind limb locomotion showed a greater likeness to Iberian dressage Purebreds. Despite this, their ability in collection and propulsion at the walk and the trot was relatively low. The hind limb pastern conformation was partially connected to the hind limb movements for both gaits, with an apparently negative effect of excessively upright pasterns on the amplitude at the trot, which indirectly reduced collection ability.     

Identification and quantification of amines in the equine caecum

Acute laminitis has been associated with the release of compounds, as yet unidentified, produced by hindgut fermentation which affect blood flow to the digit. The objectives of this study were to identify amine compounds in equine caecal and colonic contents, some of which are known to have vasoactive properties. In addition, the concentrations of amines in caecal contents of horses fed either grass or hay diets were compared. Fifteen amines were identified in equine hindgut contents in concentrations greater than 1 microM. The caecal concentrations of phenylethylamine, isoamylamine, cadaverine, diaminoheptane and spermidine were significantly higher in horses on spring/summer grass compared with those on winter grass or hay. These data show that many amines are present in the equine hindgut, some of which may have the potential to cause peripheral vasoconstriction if released into the circulation from the gastrointestinal tract.     

Platelets in equine recurrent airway obstruction

Platelets contribute to the pathogenesis of human allergic airway disease. The aim of this study was to compare platelet activating factor (PAF)-induced platelet aggregation and thromboxane (Tx) production, plasma Tx and 5-hydroxytryptamine (5-HT) in ponies with recurrent airway obstruction (RAO), an hypersensitivity to inhaled antigens, and normal ponies, before and after antigen exposure. Plasma 5-HT was significantly higher in ponies with RAO but was not further increased by antigen challenge. There was no difference between PAF-induced platelet aggregation or Tx production, or in plasma Tx before or after challenge. These data suggest there may be a difference between platelet 5-HT uptake in RAO and normal ponies but do not provide evidence of platelet activation following antigen exposure.     

Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0 = day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17β (E₂) were detected in day 25 conceptuses. Concentrations of E₂ were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10 ng/mL E₂ and 10 ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1α and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E₂ and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.     

The Michigan equine monitoring system. I. Design, implementation and population estimates

The Michigan equine monitoring system (MEMS) was designed and implemented in the State of Michigan, starting in 1991. The program was designed systematically to track the State's equine population, its health, and its economic implications to the equine industry. The MEMS was designed as a two-phase program. Phase I (the population and economic survey; the subject of this paper) was designed to provide new and statistically valid information describing the size, composition, location and economic characteristics of the Michigan equine industry. A standardized questionnaire was used to collect data via mail, telephone and personal interviews.

Of the 3000 randomly selected list-frame samples, 2800 (93%) participated. However, 650 of these had no equids. There were 129932 equids reported compared with 160000 in 1984. The American Quarter Horse, Standardbred and Arabian breeds were the most numerous. Detailed results, including the size of equine operations/herds, uses, geographical distribution and the financial structure of the industry, are presented. A detailed account of the strategies used in designing and implementing the system is provided.     

Cloning and nucleotide sequence of the equine and elk pituitary pre-prolactin cDNA

We report the equine (Equs equs) and elk (Cervus elaphus) pituitary pre-prolactin (PRL) cDNA cloning, and their nucleotide and deduced amino acid sequences. Pre-PRL cDNA was obtained by RNA ligation mediated-rapid amplification of cDNA ends (RLM-RACE) and polymerase chain reaction (PCR). The elk pre-PRL cDNA exhibits two polymorphisms at positions 96 and 672, which are silent since they encode for the same amino acids, proline and isoleucine, respectively. We found no polymorphisms in the equine pre-PRL cDNA. The deduced amino acid sequence of the equine pre-PRL is 99% identical to the previously reported protein sequence. Pre-PRL mRNA is <1 kb in length and is highly expressed in the anterior pituitary gland, as demonstrated by Northern hybridization analysis. In summary, we cloned and sequenced the equine and elk pre-PRL cDNAs. The deduced amino acid sequence of elk and equine pre-PRL appears to be moderately conserved among other mammalian species. The polymorphic sites found in the elk cDNA could potentially be used in parentage testing and gene mapping.     

The first reported outbreak of equine herpesvirus myeloencephalopathy in New Zealand

CASE HISTORY AND CLINICAL FINDINGS: On 9 January 2014 (Day 0) a mare from a stud farm in the Waikato region presented with urinary incontinence without pyrexia. Over the following 33 days 15 mares were clinically affected with neurological signs. All but one mare had a foal at foot. The most commonly observed clinical signs were hind limb paresis and ataxia. In some cases recumbency occurred very early in the course of disease and seven mares were subject to euthanasia for humane reasons.

LABORATORY FINDINGS: Equid herpesvirus (EHV) type 1 was detected using PCR in various tissues collected post mortem from two mares with neurological signs. DNA sequencing data from the DNA polymerase gene of the virus showed a nucleotide transition at position 2254, a mutation encoding amino acid D₇₅₂ that is highly associated with the neuropathogenic genotype of EHV-1. In total 12/15 mares were confirmed positive for EHV-1 on PCR. Results from a virus neutralisation test and ELISA on paired serum samples, and PCR on whole blood and nasal swabs, indicated that of four paddocks in a high-risk area where a cluster of cases had occurred, 20/21 (95%) horses were likely to have been exposed or were confirmed infected with EHV-1. Subsequent to the outbreak two mares aborted, one at 9 months and one at 10 months of gestation. The cause of abortion was confirmed as EHV-1 with the same genotype as that involved in the outbreak.

DIAGNOSIS: Equine herpesvirus myeloencephalopathy.

CLINICAL RELEVANCE: The outbreak described shows the considerable impact that can occur in outbreaks of equine herpesvirus myeloencephalopathy in New Zealand. Early biosecurity controls not only reduced the effect on the farm but mitigated the potential for the virus to spread to other horse enterprises.     

A pre- and post-intervention study of infection control in equine hospitals in Sweden

Background

Detection of nosocomial methicillin-resistant Staphylococcus aureus infections in horses in Sweden has increased attention on infection control (IC) in equine hospitals. This study established baseline data on IC programmes within such settings, evaluated compliance with some IC procedures before and after an education intervention, and examined barriers to compliance.The study was carried out between 2008 and 2011 in four Swedish equine hospitals. Data on current IC of each hospital, purchase data on hand sanitisers and disposable gloves per patient, and direct observations of compliance with procedures were monitored pre- and post-intervention. The intervention comprised a lecture on common IC and a review of each hospital’s current procedures. For comparison, retrospective purchase data were reviewed. A questionnaire on individual compliance, experiences and opinions of IC was issued to employees.

Results

Three hospitals completed the study, while the fourth reported its IC procedures and completed the questionnaire. Actual numbers of procedures, content and level of documentation differed among the hospitals. Similarities were poor or absent IC implementation strategy, lack of active surveillance of compliance with procedures and no monitoring of such as nosocomial infections. Among the hospitals which completed the study, two reported pre-intervention observation of compliance, while all three reported post-intervention observations. The purchase data showed trends for changes over time, although not uniformly related to the intervention. One hospital demonstrated a significant post-intervention increase in compliance with glove procedures, accompanied by a non-significant post-intervention increase in purchases figures. Compliance with dress code and personal appearance was high in all three hospitals (92-100%), while compliance with hand hygiene procedures was generally poorer. Barriers to compliance cited in the questionnaire (data from four hospitals) included insufficient supplies of hygiene products, lack of readily accessible places for cleaning, insufficient knowledge and high workload.

Conclusions

Potential for easily attainable improvements in IC, such as traceability of documents, implementation strategies and surveillance of efficacy, was revealed. Attention to hand hygiene implementation and improvement of logistics appeared important. Data on purchases per patient were readily available and therefore applicable for intra-hospital surveillance of IC trends over time.     

云南地区马流感病毒检测及其HA基因序列分析

马流感能引起马属动物的高度接触性呼吸道传染病。该病毒已知仅存在H7N7(马流感病毒1型)、H3N8(马流感病毒2型)两个亚型,目前仅H3N8亚型毒株在全球范围内引起马流感流行和暴发。采用A型及H3亚型特异性RT-PCR技术,检测云南疑似马流感病例鼻腔棉拭子样品;HA基因PCR扩增产物经纯化后,克隆至pMD18-T载体进行测序,并与已知代表性毒株对应序列进行比对及系统发育分析。研究发现云南马流感病毒与已知代表毒株血凝素基因核苷酸及氨基酸序列同源性分别介于91.2%~99.2%和89.5%~98.4%,属于美洲谱系(American lineage)佛罗里达亚谱系(Florida sub-lineage)毒株。     

Evaluation of the long-term oral consequences of equine exodontia in 50 horses

The aims of this study were to objectively evaluate and quantify the process of post-extraction cheek teeth (CT) dental drift in horses, and to report on associated disorders of CT wear and long-term periodontal health. Fifty horses that had CT oral extraction because of apical infection were prospectively re-examined and a full oral examination, including measurements of some dental parameters, was performed.Narrowing of the extraction space was noted in all cases with complete closure occurring in 18% of horses. The rate of dental drift was calculated as 15.7% of extraction space/year (range 4–50%) and was not associated with the age at extraction (P = 0.78) or frequency of dental care since extraction (P = 0.48). There was a significant negative relationship between the rate of dental drift and the duration of time since extraction (P = 0.008). Overgrowths were present on the opposite CT row in 98% of horses, including opposite the extracted CT and on the Triadan 06s and 11s. No significant difference was noted in either the number of diastemata (P = 0.9) or periodontal disease score (P = 0.8) between the extraction and the contralateral cheek tooth rows.     

Repeatability of equine health and management information obtained via a telephone questionnaire

The repeatability of equine health and management data obtained via a telephone survey of 60 horse owners and trainers was evaluated by using a test-retest method. One month following completion of the initial telephone questionnaire, the owners and trainers were asked to complete a retest questionnaire which contained a subset of 31 questions from the original survey. The second (retest) questionnaire collected information concerning the same time period as the first interview, i.e. the 4 week period prior to the original admission date of the horse. Measures of agreement were calculated using Spearman's rank correlation coefficient for continuous variables and the Kappa statistic for categorical variables.

Overall, measures of agreement were very favorable, ranging from good to excellent for nearly all questions examined. The results of this study indicate that repeatable information concerning equine health and management data can be obtained via a telephone interview and that a telephone questionnaire is a feasible method for obtaining information from horse owners and trainers.     

Prevalence of serum neutralising antibody to equine rhinitis A virus (ERAV), equine rhinitis B virus 1 (ERBV1) and ERBV2

The objective of this study was to determine the incidence of serum neutralising (SN) antibody to ERAV, ERBV1 and ERBV2 in a population of horses from birth to 22 years of age. The prevalences of ERAV, ERBV1 and ERBV2 SN antibodies in 381 sera obtained from 291 horses were 37%, 83% and 66%, respectively. ERAV, ERBV1 and ERBV2 maternal antibody was present in foals 12 h postsuckling but by 10-12 months, ERAV SN antibody was not detected in any of the horses, while ERBV1 and ERBV2 SN antibodies were common (83% and 100%, respectively). Sera were obtained from 44 Thoroughbred horses when they were newly introduced into a training centre when their average age was 23 months and a second sample was obtained approximately 7 months later. ERAV SN antibody was present in 8 (18%) when first bled and in 27 (61%) when tested 7 months later. Accordingly 19 of the 44 horses (43%) seroconverted to ERAV within 7 months of entering the training stable. Among all the horses the average ERAV SN antibody titre was relatively high (3796) and in contrast, ERBV1 and ERBV2 titres were relatively low (average 84 and 45, respectively) and often fell to below detectable levels over time and at a rate comparable to new seroconversions in the same group of horses.