Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses: Implications for laminitis

Equine laminitis, a disease of the lamellar structure of the horse's hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Toll-like receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-α and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-α, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease.     

慢病毒介导的水牛CD14基因shRNA序列的筛选

研究慢病毒载体介导的RNA干扰对水牛CD14基因表达的影响,设计并筛选具有抑制作用的靶序列。将真核表达载体pDsRed-N1-buffaloCD14转染293细胞株,建立稳定表达水牛CD14基因细胞系。同时设计5条水牛CD14 shRNA(319/421/755/970/1 041)以及1条阴性对照序列(NC-1864),构建慢病毒重组质粒pSicoR-GFP-shRNA。采用磷酸钙沉淀法将三质粒共转染293T细胞包装慢病毒,并进行滴度测定。最后,通过对慢病毒感染的pDsRed1-N1-buffalo CD14-293细胞进行QRT-PCR检测,筛选具有抑制效果的shRNA。结果显示:在各感染细胞组中,外源水牛CD14基因mRNA的表达均受到不同程度的抑制,其中,CD14 shRNA-970靶点的干扰效率最高,达到79.3%(P<0.01)。成功筛选出具有较高抑制效果的水牛CD14 shRNA靶序列,为研究CD14基因在布氏杆菌所致炎症反应中的作用,建立家畜布氏杆菌病防治新方法奠定了基础。     

Morphological data indicate a stress response at the oral border of strangulated small intestine in horses

Strangulation colic often leads to surgery. We aimed to document the molecular response in the non-resected intestine in these horses using quantitative Western blot analysis, and immunohistochemistry. The expression of hypoxia-inducible factor 1-alpha (HIF1α) was investigated together with two molecular pathways initiated after protein destruction: proteasome degradation via ubiquitin chain formation and protein restoration via molecular chaperones such as inducible heat shock protein 70 (HSP70). In addition, the expression of c-fos and c-jun could indicate an early proinflammatory response. Ubiquitin, HSP70, c-jun and c-fos protein levels did not differ between the control and colic samples nor were they related to the clinical outcome in case of strangulation colic. However, the immunohistochemical distribution of several of these proteins (ubiquitin, HSP70 and c-jun) differed significantly between colic and control samples. The elevated presence of ubiquitin in the enterocytes’ nucleus, of HSP70 in the smooth muscle cells’ nucleus and of c-jun in enteric neurons suggest protective and degenerative pathways are activated in the apparently healthy non-resected tissue in case of strangulation obstruction, perhaps providing a molecular and morphological basis for the development of complications like post-operative ileus.     

Concentrations of ionized and total magnesium and calcium in healthy horses: effects of age, pregnancy, lactation, pH and sample type

Magnesium (Mg) and calcium (Ca) are the main divalent cations in the body. The free ionized fraction is the physiologically active form of both Mg and Ca. As ion-selective-electrode (ISE) analyzers are becoming widely used in veterinary practice it will be useful to establish reference intervals for horses. In the present study, reference intervals were established for ionized, total and the ratio of the ionized to total Mg and Ca concentrations in four horse groups: adults, neonatal foals, and pregnant and lactating mares, as well as in three sample types, whole blood, plasma and serum, using an ISE-analyzer. Sample pH effect was assessed.Concentration differences in both total and ionized cations were recorded for some measures between horse groups and different sample types. The most significant differences were recorded between neonatal foals and other groups. These results demonstrate the need to use specific reference intervals for horses of different physiologic status and in specific sample types.     

Clinical Outcome of 14 Obese,Laminitic Horses Managed with the Same Rehabilitation Protocol

A specific method of rehabilitation was used to manage obese horses with laminitis, and clinical outcome was evaluated after 5 to 20 months. Clinical data from 14 similar laminitis cases were statistically analyzed to evaluate response to rehabilitation. Data were analyzed using repeated measures or logistic regression methodologies. Each horse presented as obese and laminitic with no history of a systemic inflammatory disease. The rehabilitation method emphasized a mineral-balanced, low nonstructural carbohydrate diet; daily exercise; hoof trimming that minimized hoof wall loading; and sole protection in the form of rubber hoof boots and/or hoof casts. Distal phalanx alignment within the hoof capsule was significantly improved, and hoof wall thickness was significantly decreased (P < .0001) following treatment. Solar depth was significantly increased (P < .0015). Reduction of palmar angle measurements was detected in acutely and chronically affected horses. This treatment effect was statistically greater for horses with chronic laminitis than for horses with acute laminitis (P interaction < .0001). Horses were 5.5 times more likely to be sound post-treatment than before treatment. Daily exercise, dietary modification, and removal of ground reaction force from the hoof wall were foci of the rehabilitation program. Hoof care and husbandry as applied to these horses may be an effective method of rehabilitation of horses from obesity-associated laminitis.     

Faecal shedding and serological cross‐sectional study of Lawsonia intracellularis in horses in the state of Minas Gerais,Brazil

Reason for performing the study: Proliferative enteropathy, caused by the intracellular bacterium Lawsonia intracellularis, has been described in horses in Australia, the USA, Canada and European countries but has not been reported in Latin America. The prevalence of the disease in horses worldwide is unknown. Objective: To evaluate the presence of subclinical L. intracellularis infection in horses in the state of Minas Gerais, Brazil. Methods: A longitudinal study using serology and PCR for detecting antibodies (IgG) and shedding of L. intracellularis in faecal samples, respectively, was conducted using a total of 223 horses from 14 different horse farms in Minas Gerais, and from the Veterinary School of UFMG equine herds in Minas Gerais. The immunoperoxidase technique in glass slides was used as the serological test. Results: Twenty‐one horse sera had immunoglobulin G titres of 1:60 and were considered positive. The PCR technique in faeces for L. intracellularis DNA identified 7 horses as faecal shedders. Horses shedding the organism appeared healthy, indicating that subclinical infection of L. intracellularis occurred in the horses. Conclusion: Seropositivity and detection of faecal shedding of L. intracellularis indicates the presence of the agent in the equine population in Minas Gerais. Potential relevance: Results of this study should alert clinicians in countries where proliferative enteropthy in horses has not been reported to consider this disease as a possible cause of enteric disease.     

Maintenance of arytenoid abduction following carbon dioxide laser debridement of the articular cartilage and joint capsule of the cricoarytenoid joint combined with prosthetic laryngoplasty in horses: An in vivo and in vitro study

The objective was to evaluate CO₂ laser debridement of the cricoarytenoid joint (CAJ) combined with prosthetic laryngoplasty to prevent post-operative loss of arytenoid abduction in seven horses. Horses were assigned to either laser debridement of the left CAJ and laryngoplasty (laser treated, n = 5) or control laryngoplasty (sham, n = 2), and were evaluated with endoscopic examinations and measurement of right to left angle quotients (RLQ) to assess maintenance of arytenoid abduction. The animals were euthanased at intervals after surgery and larynges were harvested for post-mortem testing, including determination of translaryngeal flow, pressure, impedance and RLQ. Measurements were obtained under increasing vacuum-generated negative pressure with laryngoplasty sutures intact and with the knot/crimp of the laryngoplasty sutures removed. Following post-mortem testing the cricoarytenoid joints were examined histologically.Post-operative endoscopic examinations revealed no significant differences between RLQ measurements calculated for day 1 following surgery to the termination date of the study for the seven horses. Post-mortem RLQ at airflows of 10 and 60 L/s was significantly higher in sham than in laser treated horses both before and after knot/crimp removal. Translaryngeal impedance at 10 and 60 L/s was not statistically different between groups. Histopathology revealed necrosis and loss of articular cartilage in the laser treated horses. The lymphoid cell infiltration subsided but joint capsule and periarticular fibrosis increased over the course of the study. Post-operative loss of arytenoid abduction after laryngoplasty can be minimized with CO₂ laser debridement of the CAJ joint.     

Estimation of genetic parameters for racing speed at different distances in young and adult Spanish Trotter horses using the random regression model

A total of 71 522 records (from 3154 horses) with the times per kilometre (TPK), recorded in Spanish Trotter horses (individual races) from racing performances held from 1991 to 2007, were available for this study. The TPK values for the different age groups (young and adult horses) and different distances (1600–2700 m) were considered as different traits, and a bi character random regression model (RRM) was applied to estimate the (co)variance components throughout the trajectory of age groups and distances. The following effects were considered as fixed: the combination of hippodrome‐date of race (404 levels); sex of the animals (3 levels); type of start (2 levels) and a fixed regression of Legendre polynomials (order 2). Those considered as random effects were the random regression Legendre polynomial (order 1) for animals (9201 animals in the pedigree); the individual environment permanent (3154 animals with data) and the driver (n = 957 levels). The residual variance was considered as heterogeneous with two classes (ages). The heritability estimated by distance ranged from 0.12 to 0.34, with a different trajectory for the two age groups. Within each age group, the genetic correlations between adjacent distances were high (>0.90), but decreased when the differences between them were over 400 metres for both age groups. The genetic correlations for the same distance across the age groups ranged from 0.47 to 0.78. Accordingly, the analysed trait (TPK) can be considered as positive genetic correlated but as different traits along the trajectory of distance and age. Therefore, some re‐ranking should be expected in the breeding value of the horses at different characteristics of the racing. The use of RRM is recommended because it allows us to estimate the breeding value along the whole trajectory of race competition.     

Qualitative and quantitative characteristics of the electroencephalogram in normal horses during spontaneous drowsiness and sleep

BACKGROUND: The influence of sleep on the equine electroencephalogram (EEG) has not been well documented. HYPOTHESIS: The objectives were to develop a noninvasive method of electrode placement for recording the EEG in horses and to establish normal EEG parameters for the various states of vigilance. Findings are compared with previously published reports on equine sleep based on electrocorticography (ECoG). ANIMALS: Five neurologically normal horses. METHODS: Overnight EEGs were recorded digitally in association with simultaneous videotaping of the horses' behavior. Data were analyzed by visual inspection, states of vigilance were identified, and representative segments were quantitatively processed. Transient EEG events were examined. RESULTS: Slow wave sleep (SWS) was significantly different (P < .05) in frequency and power from drowsiness and rapid eye movement (REM) sleep. Second-degree heart block was associated with SWS as were transient events commonly recognized in EEGs of humans. Drowsiness and REM sleep were similar. In both, background activity was low-amplitude beta activity admixed with prominent activity of approximately 4 Hz. Standing REM sleep was associated with numerous partial collapses in 1 horse. CONCLUSIONS AND CLINICAL IMPORTANCE: Normative data for several states were described and probable benign variants identified. This information will serve as control data for sedative and anesthetic studies in this species. The sleep patterns observed during this study are those of horses removed from their usual surroundings, and thus may represent those encountered in a clinical environment.     

荧光定量RT-PCR检测鸡CD4、CD8基因表达水平

白细胞分化抗原CD4和CD8在机体免疫应答及其信号传递过程中发挥着重要作用。本试验建立了定量检测鸡CD4和CD8 mRNA表达水平的SYBR Green I实时荧光定量RT-PCR（RRT-PCR）方法,并采用该方法对26-50日龄商品鸡外周血淋巴细胞（PBL）中CD4和CD8 mRNA表达水平进行了检测。结果显示：所建立的RRT-PCR对CD4和CD8 mRNA的扩增效率分别为93%和91%;线性范围分别在10^-4-10^-9和10^-3-10^-9;相关系数分别为0.998 0和0.999 9;最低分别能检测105和120拷贝;熔解曲线分别在78.2和86.7℃附近出现1个单特异峰;组内变异系数分别在1.13%-2.15%和1.17%-3.68%,组间变异系数分别在1.16%-3.25%和1.66%-2.86%。26-50日龄鸡PBL CD4和CD8的mRNA表达水平有小幅波动,与采用流式细胞术检测结果的报道一致。本试验建立的RRT-PCR方法敏感性高、稳定性和再现性好,为检测鸡CD4、CD8基因表达水平提供了精确定量的新方法。     

Detection of bovine papillomavirus DNA on the normal skin and in the habitual surroundings of horses with and without equine sarcoids

The purpose of the present study was to examine whether bovine papillomavirus (BPV) DNA can be detected on the normal skin and in the habitual surroundings of horses with and without equine sarcoids by means of superficially taken swabs. In affected horses, no significant difference in presence of BPV-DNA could be observed between samples obtained from the equine sarcoid surface, from normal skin close to the tumour and from a normal skin site in direct contact with the tumour. From the group of healthy horses living in contact with affected horses, 44% were BPV-DNA positive. The surroundings of affected and non-affected horses are probably not a major source of BPV-DNA contamination. It can be concluded that BPV-DNA is present on the normal skin of horses affected by equine sarcoid and to a lesser degree, on the normal skin of horses living in contact with affected horses.     

Comparative efficacy of diminazene diaceturate and diminazene aceturate for the treatment of babesiosis in horses

The present work was conducted to study the comparative efficacy of two anti-protozoan (babesicidal) drugs on the recovery and health of horses. A total of 80 horses, showing typical clinical symptoms of the disease, were selected for this study; the presence of babesiosis was confirmed through blood smear examination. These animals were divided into two groups i.e. A and B. Horses of Group A, were treated with diminazene diaceturate, while horses of Group B were treated with diminazene aceturate. Efficacy of the drugs was determined by the reversal of clinical signs and a negative blood smear examination. The efficacy of diminazene diaceturate was demonstrated to be 80% while diminazene aceturate was found to have 90% efficacy against babesiosis.     

高温下清凉冲剂对鸡免疫器官中CD4+、CD8+ T细胞数量的影响

采用免疫组化方法检测鸡免疫器官中CD4^＋、CD8^＋T细胞数量，探讨高温下清凉冲剂对机体细胞免疫机能的影响及其药理作用。108只农大3号35目龄公雏，随机分为3组，每纽36只鸡，即常温对照组、高温对照组和高温用药组。用药组饲喂中药煎荆（浓度为1g／ml），用药量与饲料量比为1：1000。在试验的第1、4、8、10天各组捕杀5只。分别采取胸腺、脾脏、法氏囊检测。结果高温下。用药组免疫器官中CD4^＋、CD8^＋T细胞数量均高于高温对照组。第8、10天差异极显著（P〈0．01）。表明高温下清凉冲剂可以增加鸡免疫器官中CD4^＋、CD8^＋T细胞数量，有效地提高鸡细胞免疫水平。     

The role of CD44 adhesion factor in canine mammary carcinomas

CD44 is an adhesion molecule implicated in the progression of human breast cancer. The purpose of this study was to describe CD44 antigen expression in canine mammary carcinomas and to evaluate its prognostic significance in relation to other clinico-pathological variables.A reduction in CD44 expression was significantly related to variables such as tumour size and adherence to underlying tissues but was not related to tumour location or to ulceration of the overlying skin. Complex (grade I) and anaplastic (grade III) carcinomas exhibited more intense expression of this antigen than did some tubulopapillary and most solid carcinomas (grade II). Although reduced CD44 expression was associated with infiltrative growth and vascular invasion in solid carcinomas, intense expression was also observed in anaplastic tumours. Although overall these findings suggest a role for this adhesion factor in canine mammary tumour development and progression, the complexity and apparently paradoxical nature of some of the findings currently limit the use of this immunohistochemical marker as a prognostic indicator.     

Purification and analyses of the specificity of two putative diagnostic antigens for larval cyathostomin infection in horses

Cyathostomins are important equine gastrointestinal parasites. Mass emergence of mucosal stage larvae causes a potentially fatal colitis. Mucosal stages are undetectable non-invasively. An assay that would estimate mucosal larval stage infection would greatly assist in treatment, control and prognosis. Previously, we identified two putative diagnostic antigens (20 and 25 kDa) in somatic larval preparations. Here, we describe their purification and antigen-specific IgG(T) responses to them. Western blots confirmed the purity of the antigens and showed that epitopes in the 20 kDa complex were specific to larval cyathostomins. No cross-reactive antigens appeared to be present in Parascaris equorum or Strongyloides westeri species. Low levels of cross-reactivity were observed in Strongylus edentatus and Strongylus vulgaris species. Use of purified antigens greatly reduced background binding in equine sera. These results indicate that both antigen complexes may be of use in a diagnostic assay.     

Seroprevalence of Fasciolosis in Equines of the Black Sea Region,Turkey

This study determined the seroprevalence of fasciolosis in equines (n = 140) by enzyme-linked immunosorbent assay and Western blot. Blood samples were collected from different locations of the Black Sea Region of Turkey. The overall seroprevalence of fasciolosis in equines was 19.3%: 18% of horses (15), 22.6% of donkeys (7), and 19.2% of mules (5). The seroprevalence rates across provinces were as follows: 25% (5/20) in Samsun, 21.9% (7/32) in Tokat, 31.4% (11/35) in Amasya, 7.4% (2/27) in Ordu, and 7.7% (2/26) in Sinop. Three protein bands with molecular weights of 13, 30-40, and 40-65 kDa were determined in antigen clusters in sera from Fasciola spp-infected equine animals.