Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.     

Septicaemia secondary to infection by Corynebacterium macginleyi in an Indian python (Python molurus)

A seven-year-old female Indian python (Python molurus) weighing about 35kg was euthanased after several clinical episodes of stomatitis, pneumonia, ophthalmitis and dystocia over a period of four years. The animal had been maintained in a terrarium in a circus truck at an adequate temperature. During shows, however, the snake was considered to be exposed to stressful conditions for several hours at a time at low temperatures and with noise and bright lights. A post-mortem examination indicated ulcerative stomatitis, osteomyelitis, severe pneumonia and numerous granulomata and multifocal necrosis in stomach and spleen. Corynebacterium macginleyi was isolated in pure culture from the ulcerative stomatitis, and mixed with Stenotrophomonas maltophilia from the lungs and spleen. The findings indicated that the snake had died from a septicaemic process caused by C. macginleyi, probably originating from the stomatitis. The role of S. maltophilia as a secondary agent is discussed. The stress of the circus show and poor husbandry may have predisposed the animal to infection and septicaemia. This is the first report of C. macginleyi causing disease in a snake.     

Non-invasive assessment of growth, gender and time of day related changes of respiratory pattern in healthy cats by use of barometric whole body plethysmography

The objective of this study was to establish a reference base for respiratory variables (respiratory rate [R(R)], inspiratory and expiratory time [T(i) and T(e)], peak inspiratory and expiratory pseudoflow [PIF and PEF], tidal volume [V(T)], minute ventilation [V(E)] and enhanced pause [Penh]) of healthy cats by use of barometric whole body plethysmography (BWBP). Eighteen healthy European cats (10 male, 8 female) were studied from the age of 3 to 13 months in order to assess growth- and gender-related changes of BWBP variables. Chest radiographs and bronchoalveolar lavage cytology were performed to confirm pulmonary health status. Diurnal changes were investigated every 2 h over a period of 24 h when the cats were adult. V(T), V(E), PIF and PEF significantly increased during somatic growth and were higher in males than in females, whereas R(R), T(i), T(e), T(e)/T(i) ratio, PEF/PIF ratio and Penh remained unchanged and were not affected by gender. When measured over 24 h, Penh, T(e) and T(i) were significantly increased in the early morning hours (04:00 h), whereas R(R), PIF and PEF were decreased at that time. This study provides reference values of BWBP variables for healthy male and female cats and indicates when circadian changes might be observed.     

Comparison of biopsy samples obtained using standard endoscopic instruments and the harmonic scalpel during laparoscopic and laparoscopic-assisted surgery in normal dogs

Objectives— To compare tissue specimens of canine abdominal organs collected by standard techniques (ST) and harmonic scalpel (HS) and to determine the effect of each technique on wound healing.
Study Design— Experimental.
Animals— Twelve dogs.
Methods— Paired biopsy samples (ST, HS) were collected from liver, spleen, adrenal gland, pancreas, stomach, jejunum, and bladder using laparoscopic or laparoscopic-assisted methods. Hemorrhage at biopsy sites was assessed (present or absent). Specimens were evaluated for diagnostic quality and histologic changes. Dogs were necropsied at 4 or 14 days postoperatively.
Results— HS incision caused less frequent hemorrhage of all organs except spleen. HS specimens had more coagulation necrosis than ST specimens, but both techniques yielded specimens that were sized similarly and were of diagnostic quality. At necropsy, HS biopsy sites of the stomach, jejunum, bladder, adrenal gland, and pancreas were more hyperemic than ST sites. There were more adhesions at jejunal and pancreatic ST biopsy sites. HS biopsy sites had more coagulation necrosis at days 4 and 14 postoperatively. Differences in fibrin deposition, inflammation, and fibrosis were present at biopsy sites of some organs at days 4 or 14 and in comparisons between days 4 and 14.
Conclusions— Diagnostic quality biopsy specimens were obtained with HS and ST. Although HS-induced gross and histologic changes during the first 2 postoperative weeks, no clinical complications were observed.
Clinical Relevance— Both HS and ST can yield specimens with minimal hemorrhage and HS resulted in no apparent postoperative problems in normal dogs. Although HS caused more inflammation and adhesions at biopsy sites of the pancreas, adrenal gland, and jejunum, no clinical complications occurred.     

Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background

The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells.

Methods

A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses.

Results

Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles.

Conclusion

We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.     

新疆北部绢蒿荒漠水源圈土壤养分的梯度变化

由于干旱缺水,新疆北部绢蒿（Seriphidium）荒漠形成以饮水点为中心的定居放牧,导致水源圈大量出现。2014年5月底,以新疆木垒县天山北坡绢蒿荒漠一个典型的牧场水源点为中心,采集了东南西北4个方向和6个采样距离（50 m,100 m,200 m,400 m,800 m和1200 m）的土样,分析了土壤含水率、电导率、pH值以及土壤有机碳、土壤全量和速效养分变化特征。结果表明,随着离水源点距离的增加,土壤含水量、全钾和速效钾没有明显变化,土壤电导率、pH值、土壤有机碳、全氮、全磷、速效氮和速效磷的含量随着离水源点距离的增加而增加,说明放牧扰动强度的增加降低了土壤有机碳和土壤养分含量。结果说明,在新疆北部涓蒿荒漠,过度放牧导致水源点附近土壤养分明显减少,甚至枯竭。     

Microbiological identification and analysis of swine tonsils collected from carcasses at slaughter

The primary objective of this 7-month study was to determine the prevalence of porcine pathogens of the tonsil of the soft palate of swine at slaughter. Additional objectives were to determine if sampling the carcasses of normal or abnormal hogs provided different microbiological profiles and if the slaughter plant provides a feasible sampling frame and environment for detecting and monitoring important pathogens in tonsils that have health implications for both swine and humans. A total of 395 samples were collected from 264 farms. Of these, 180 tonsils were collected from normal carcasses and 215 tonsils were collected from carcasses that were diverted to the hold rail. Laboratory testing included bacteriological culture and identification as well as real time-polymerase chain reaction (PCR) testing for porcine reproductive and respiratory syndrome virus (PPRSV) and immunohistochemistry (IHC) for porcine circovirus-2 (PCV-2). The most commonly isolated bacteria included: Streptococcus suis (53.7%), Arcanobacterium pyogenes (29.9%), Pasteurella multocida (27.3%), and Streptococcus porcinus (19.5%). Virus screening revealed evidence of PRRSV and PCV-2 in 22.0% and 11.9% of the samples, respectively. Salmonella Typhimurium and Yersinia enterocolitica were isolated in 0.5% and 1.8% of the samples, respectively. Tonsils collected from the hold rail were more likely to be positive for Staphylococcus hyicus [odds ratio (OR) = 7.51, confidence interval (CI) = 2.89 to 19.54], Streptococcus porcinus (OR = 9.93, CI = 4.27 to 23.10), and Streptococcus suis (OR = 2.16, CI = 1.45 to 3.24). Tonsils collected from abnormal carcasses were less likely to be positive for Staphylococcus aureus (OR = 0.05, CI = 0.005 to 0.482).     

Type I immune-mediated polyarthritis in dogs: 39 cases (1997-2002)

OBJECTIVE: To determine clinical signs, laboratory findings, relationship to vaccination, and response to treatment for type I immune-mediated polyarthritis (IMPA) in dogs. DESIGN: Retrospective study. ANIMALS: 39 dogs PROCEDURE: Clinical records and radiographic reports from 3 university referral hospitals were reviewed. Clinical signs, laboratory and investigative findings, relationship to vaccination, and response to treatment were evaluated. RESULTS: Clinical signs and initial laboratory and clinical investigative findings were frequently abnormal but were nonspecific and not associated with likelihood of recovery. Time of vaccination was not associated with onset of disease. Chemotherapeutic immunosuppression resulted in complete cure in 56% of dogs. Continuous medication was required in 18% (7/39) of dogs, relapses were treated successfully in 13% (5/39) of dogs, and 15% (6/39) of dogs died or were euthanatized as a result of disease. CONCLUSIONS AND CLINICAL RELEVANCE: The possible involvement of vaccination in type I IMPA was not made clear from this study because of the small population size. Signalment, clinical signs, and results of diagnostic tests other than multiple synovial fluid analyses were generally nonspecific. Most dogs with type I IMPA responded to initial immunosuppressive treatment, but 31% (12/39) of dogs relapsed, required further treatment, or both.     

Surgical management of urinary incontinence.

Urethral sphincter mechanism incompetence and ureteral ectopia are the two most common causes of urinary incontinence in dogs and cats. Surgical treatments for both disorders have been described.Once a diagnosis is made, surgical intervention may lead to improved outcomes with resolution of incontinence in many patients. Proper case selection and surgical technique are critical in achieving clinical success when managing these difficult cases.