INFLUENCE OF THE CHEMICAL SHIFT ARTIFACT ON MEASUREMENTS OF COMPACT BONE THICKNESS IN EQUINE DISTAL LIMB MR IMAGES

The effect of the chemical shift artifact, resulting from misregistration or phase cancellation at the interface between compact and trabecular bone, on apparent bone thickness was quantified in six isolated equine limbs. Sagittal T1‐weighted spin echo (SE) and in‐phase three‐dimensional spoiled gradient echo (SPGR) images were acquired twice with a 1.5 T magnetic resonance (MR) unit, switching the frequency encoding direction between acquisitions. Out‐of‐phase SPGR images were also obtained. MR images with different frequency encoding directions were compared with each other and to radiographs made from corresponding 3‐mm‐bone sections. Compact bone thickness was significantly different when comparing images acquired with different frequency encoding directions for both SE and SPGR sequences. Significant differences were identified in the frequency but not the phase encoding direction when measurements of compact bone in MR images were compared with measurements obtained from thin section radiographs for the majority of surfaces studied (P<0.05). Correction of MR measurements with the calculated chemical shift abolished these differences (P>0.05). Measurements of compact bone from out‐of‐phase SPGR sequences were significantly different than from in‐phase sequences (P<0.001) with out‐of‐phase measurements greater than in‐phase measurements by an average of 0.38 mm. These results indicate that the chemical shift artifact results in errors in MR evaluation of compact bone thickness when measurements are performed in the frequency encoding direction or in out‐of‐phase images. For better accuracy, measurements should be performed parallel to the phase encoding direction and avoiding out‐of‐phase gradient echo sequences.     

Isolation and cultivation of equine corneal keratocytes, fibroblasts and myofibroblasts

Objective  To establish an in vitro model for the investigation of equine corneal wound healing. To accomplish this goal, a protocol to isolate and culture equine corneal keratocytes, fibroblasts and myofibroblasts was developed.
Animal material  Equine corneal buttons were aseptically harvested from healthy research horses undergoing humane euthanasia for reasons unrelated to this study. Slit-lamp biomicroscopy was performed prior to euthanasia by a board-certified veterinary ophthalmologist to ensure that all samples were harvested from horses free of anterior segment disease.
Procedure  Equine corneal stroma was isolated using mechanical techniques and stromal sub-sections were then cultured. Customized media at different culture conditions was used to promote growth and differentiation of corneal stromal cells into keratocytes, fibroblasts and myofibroblasts.
Results  Cell culture techniques were successfully used to establish a method for the isolation and culture of equine corneal keratocytes, fibroblasts and myofibroblasts. Immunohistochemical staining for alpha-smooth muscle and F-actin was used to definitively differentiate the three cell types.
Conclusion  Equine corneal stromal keratocytes, fibroblasts and myofibroblasts can be predictably isolated and cultured in vitro using this protocol.     

Report of the Second Havemeyer EHV‐1 Workshop,Steamboat Springs,Colorado, USA,September 2008

This report summarises the findings of the Second Havemeyer EHV‐1 Workshop, which was held in Steamboat Springs, Colorado, USA in September 2008. A total of 38 delegates, consisting of veterinary clinicians and scientists from academia and industry participated in a series of sessions that focused on equine herpesvirus myeloencephalopathy (EHM). Each session consisted of a review, followed by short presentations on current research topics. The sessions included EHM epidemiology, in vivo and in vitro models for studying EHM, EHV‐1 virulence determinants, real‐time PCR diagnostics, antiviral medications and new vaccination technologies. The report summarises the key advances identified during and since the meeting. Citations are restricted to selected reviews and papers published since the workshop.     

Effect of dietary restriction on body condition,composition and welfare of overweight and obese pony mares

Reasons for performing study: Increased prevalence of obesity among UK horses and ponies demands evidence‐based advice to promote weight loss. Hypothesis: Restriction of dry matter intake (DMI) to 1% of body mass (BM, 6% of predicted maintenance digestible energy [DE] requirements) would promote weight loss without compromise to health. Methods: Five mature (mean ± s.e. 10 ± 2 years), overweight/obese pony mares (BM, 257 ± 20 kg: body condition score [BCS] 6.8/9 ± 0.5) were studied over 12 weeks. Animals were individually housed. Daily provision of a chaff‐based, complete diet (measured DE, 8.5 MJ/kg DM) was restricted to 1% of actual BM as DMI daily. BCS, girth measurements and ultrasound‐derived measures of subcutaneous fat depth overlying the gluteal region and 12th intercostal space (rib‐eye) were recorded weekly. Body fat content was estimated at the beginning and end of the study by deuterium oxide dilution methods. Clinical biochemistry was monitored weekly. Behaviour was observed (24 h, 3/5 ponies) on 3 occasions. Results: BM decreased by 4.3 ± 1.1% during the first week and thereafter by 0.7 ± 0.1% of BM at end of Week 1 each week. BCS remained constant. Heart and belly girths, rump width and subcutaneous fat depth at rib‐eye decreased significantly with time and BM. Fat comprised 45 ± 19% of BM loss. Fatter animals lost relatively more fat. With decreased feeding activity, time spent in ‘play’ and rest increased by 36 ± 11% and 438 ± 95%, respectively. Conclusions: This plane of nutrition resulted in an overall rate of weight loss of 1% of outset BM weekly. BCS was not a useful index of early weight loss but heart and belly girths and subcutaneous rib‐eye fat were identified as alternative markers. Potential relevance: This study provides an evidence‐base for the management of weight loss in obese animals, especially those for which exercise may be contra‐indicated.     

In vitro effects of lidocaine on the contractility of equine jejunal smooth muscle challenged by ischaemia‐reperfusion injury

Reasons for performing study: Post operative ileus (POI) in horses is a severe complication after colic surgery. A commonly used prokinetic drug is lidocaine, which has been shown to have stimulatory effects on intestinal motility. The cellular mechanisms through which lidocaine affects smooth muscle activity are not yet known. Objectives: To examine the effects of lidocaine on smooth muscle in vitro and identify mechanisms by which it may affect the contractility of intestinal smooth muscle. Hypothesis: Ischaemia and reperfusion associated with intestinal strangulation can cause smooth muscle injury. Consequently, muscle cell functionality and contractile performance is decreased. Lidocaine can improve basic cell functions and thereby muscle cell contractility especially in ischaemia‐reperfusion‐challenged smooth muscle. Methods: To examine the effects of lidocaine on smooth muscle function directly, isometric force performance was measured in vitro in noninjured and in vivo ischaemia‐reperfusion injured smooth muscle tissues. Dose‐dependent response of lidocaine was measured in both samples. To assess membrane permeability as a marker of basic cell function, release of creatine kinase (CK) was measured by in vitro incubations. Results: Lidocaine‐stimulated contractility of ischaemia‐reperfusion injured smooth muscle was more pronounced than that of noninjured smooth muscle. A 3‐phasic dose‐dependency was observed with an initial recovery of contractility especially in ischaemia‐reperfusion injured smooth muscle followed by a plateau phase where contractility was maintained over a broad concentration range. CK release was decreased by lidocaine. Conclusion: Lidocaine may improve smooth muscle contractility and basic cell function by cellular repair mechanisms which are still unknown. Improving contractility of smooth muscle after ischaemia‐reperfusion injury is essential in recovery of propulsive intestinal motility. Potential relevance: Characterisation of the cellular mechanisms of effects of lidocaine, especially on ischaemia‐reperfusion injured smooth muscle, may lead to improved treatment strategies for horses with POI.     

Dysautonomia in a six‐year‐old mule in the United States

Equine dysautonomia, also known as equine grass sickness (EGS), is a well documented disease in several countries. To the authors’ knowledge, EGS has not been reported previously in North America. This report describes EGS in a 6‐year‐old female mule in the USA. Failure initially to consider EGS resulted in a delayed diagnosis. EGS should be considered as a differential diagnosis and appropriate diagnostic tests performed in similar cases in North America.     

马疱疹病毒gD蛋白主要抗原域的表达及初步应用

根据GenBank马疱疹病毒1型(EHV-1)和4型(EHV-4)gD基因进行分析,选取同源性较高且抗原性强的C端序列设计一对引物进行扩增。将扩增的基因插入pET-30a的BamHⅠ和SalⅠ之间构建原核表达载体pET-gD。然后将pET-gD质粒转化至BL21(DE3)宿主菌,对培养和表达条件进行优化,实现EHV gD蛋白主要抗原区域的高效表达。将重组pET-gD蛋白进行纯化并接种长白兔制备高免血清。经免疫印迹试验鉴定,证实所得纯化表达产物具有良好的抗原性。     

Comparison of two modern vaccines and previous influenza infection against challenge with an equine influenza virus from the Australian 2007 outbreak

During 2007, large outbreaks of equine influenza (EI) caused by Florida sublineage Clade 1 viruses affected horse populations in Japan and Australia. The likely protection that would be provided by two modern vaccines commercially available in the European Union (an ISCOM-based and a canarypox-based vaccine) at the time of the outbreaks was determined. Vaccinated ponies were challenged with a representative outbreak isolate (A/eq/Sydney/2888-8/07) and levels of protection were compared. A group of ponies infected 18 months previously with a phylogenetically-related isolate from 2003 (A/eq/South Africa/4/03) was also challenged with the 2007 outbreak virus. After experimental infection with A/eq/Sydney/2888-8/07, unvaccinated control ponies all showed clinical signs of infection together with virus shedding. Protection achieved by both vaccination or long-term immunity induced by previous exposure to equine influenza virus (EIV) was characterised by minor signs of disease and reduced virus shedding when compared with unvaccinated control ponies. The three different methods of virus titration in embryonated hens’ eggs, EIV NP-ELISA and quantitative RT-PCR were used to monitor EIV shedding and results were compared. Though the majority of previously infected ponies had low antibody levels at the time of challenge, they demonstrated good clinical protection and limited virus shedding. In summary, we demonstrate that vaccination with current EIV vaccines would partially protect against infection with A/eq/Sydney/2888-8/07-like strains and would help to limit the spread of disease in our vaccinated horse population.     

Evolutionary Patterns of the Proviral gp90 V3 to V5 Regions of Equine Infectious Anemia Virus Associated with Immune Selection in Progressors and Nonprogressors

The aim of this study was to determine the genomic evolutionary pattern of virulent equine infectious anemia virus (EIAV)during persistent infection.The evolutionary dynamics of proviral genomes were examined by challenging an EIAV seronegative equine (pony 1) and three EIAV vaccinated equines (ponies 4,7,and 8) with the Chinese virulent strain EIAVL.Ponies I and 7 succumbed to disease and were called progressors,while ponies 4 and 8 lacked clinical symptoms and were considered nonprogressors.Sequences spanning the V3,V4,and V5 hyper-variable regions of the EIAV-L envelope gp90 gene were sequenced from each pony as evolutionary markers of the provirus.The proviral genome of the EIAV-Linoculum evolved during persistent infection and displayed different patterns between EIA progressors and nonprogressors.Inoculum-like variants were isolated from nonprogressors during persistent infection,but only from progressors during acute infection.Variant mutations from nonprogressors were dispersed throughout the sequenced region,while those from progressors were predominantly localized to V3.Humoral immunity and virus variant population selection analyses indicated that immune selection was positive in chronically infected progressors and weak in nonprogressors.In-frame stop codons were frequently localized to a defect "hot spot".The high number of defective variants in nonprogressors may promote disease survival.     

Assessment of hygienic quality of haylage fed to healthy horses

Wrapped forages such as haylage and silage are increasing in popularity in equine feed rations, but knowledge of their microbial composition, especially of haylage, is scarce. Haylage may be more at risk of mould growth compared to silage, and methods for the assessment of hygienic quality of haylage are needed. Varying culturing conditions, including aerobic/anaerobic incubation, four substrates and three incubation temperatures were therefore evaluated for analysis of fungi in haylage samples from eighteen horse farms, taken on two occasions (autumn and the following spring). Average mould counts in forage samples were low, but fungi with the potential for producing mycotoxins, such as Aspergillus fumigatus and Penicillium spp., were present. The microbial composition and content of fermentation products were similar in haylage samples taken in autumn and in spring. The type of substrate used for cultivation did not influence mould or yeast counts, but not all mould species were present on all substrates. Incubation temperature influenced fungal counts and the presence of mould species. By using at least two substrates (malt extract agar and dichloran 18% glycerol agar) and two incubation temperatures (25 and 37°C), all mould species/genera that were identified in this study could be detected.