Secretion of multi-protein migratory complex induced by Toxoplasma gondii infection in macrophages involves the uPA/uPAR activation system

Toxoplasmosis is a world wide spread zoonosis caused by Toxoplasma gondii, an obligate intracellular parasite that is able to disseminate into deep tissues and cross biological barriers, reaching immunoprivileged sites such as the brain and retina. The parasite is able to infect macrophages and dendritic cells for dispersal throughout the body. However, the molecular mechanisms or outcomes of the subversion of the host cell are largely unknown. Recently our group established that metalloproteinases are involved in migration of infected macrophages. Herein, we evaluated the recruitment of host invasive machinery components in T. gondii infected murine macrophages. We showed by immunoprecipitation assays that MMP-9, CD44 TIMP-1 and uPAR were secreted as a multi-protein complex by infected macrophages. Zymographic analysis revealed that MMP-9 was present in its pro- and active form. Moreover, inhibition of uPA/uPAR pathway by PAI-1 decreased secretion of MMP-9 active forms, as well those associated to uPAR and TIMP-1, but not to CD44. Data presented here suggest that MMP-9 is secreted as a multiprotein complex by T. gondii infected macrophages, similar to that observed in metastatic cells. We further speculate that uPA/uPAR system is involved in the expression/secretion of complexes containing active MMP-9 forms.     

Effects of Short-term Hyper- and Hypoprolactinaemia on Hormones of the Pituitary, Gonad and -Thyroid Axis and on Semen Quality in Male Beagles

Effects of a short-term hyper- and hypoprolactinaemia on serum concentrations of LH, testosterone and semen quality in six male Beagles were investigated. Blood samples were collected at 3-day intervals for 12 weeks. The time span was divided into five 3-week periods: pre-treatment, metoclopramide (MCP) treatment (0.2 mg/kg orally three times daily), cabergoline (CAB) treatment (5 μg/kg orally once daily), post-treatment 1 and post-treatment 2. In the latter, only semen characteristics were evaluated. Semen parameters were analyzed once per week during the whole 15-week investigation time. At the end of each period, the effects of a single intravenous injection of thyrotropin-releasing hormone (TRH; 10 μg/kg) on the secretion of prolactin (PRL), LH, testosterone, thyroid-stimulating hormone and thyroxine (T4) were investigated. Pre-treatment serum PRL concentration increased under MCP (p < 0.05), followed by a decrease under CAB administration (p < 0.05). Luteinizing hormone and testosterone concentrations were not affected. Except for straight-line sperm velocity, semen quality did not differ between collection periods. A single iv TRH injection induced a significant PRL increase at 20 min in all experimental periods except during CAB treatment. Luteinizing hormone and testosterone did not show clear TRH-related changes. Basic T4 levels were significantly reduced after CAB treatment (p < 0.05). The results of the present study demonstrate that MCP-induced short-term hyperprolactinaemia in male beagles does not seriously affect the hypothalamo-pituitary axis and semen quality.     

Determination of the minimum alveolar concentration of isoflurane in Shetland ponies using constant current or constant voltage electrical stimulation

Objective To determine the minimum alveolar concentration (MAC) of isoflurane in Shetland ponies using a sequence of three different supramaximal noxious stimulations at each tested concentration of isoflurane rather than a single stimulation. Study design Prospective, experimental trial. Animals Seven 4‐year‐old, gelding Shetland ponies. Methods The MAC of isoflurane was determined for each pony. Three different modes of electrical stimulation were applied consecutively (2 minute intervals): two using constant voltage (90 V) on the gingiva via needle‐ (CVneedle) or surface‐electrodes (CVsurface) and one using constant current (CC; 40 mA) via surface electrodes applied to the skin over the digital nerve. The ability to clearly interpret the responses as positive, the latency of the evoked responses and the inter‐electrode resistance were recorded for each stimulus. Results Individual isoflurane MAC (%) values ranged from 0.60 to 1.17 with a mean (±SD) of 0.97 (±0.17). The responses were more clearly interpreted with CC, but did not reach statistical significance. The CVsurface mode produced responses with a longer delay. The CVneedle mode was accompanied by variable inter‐electrode resistances resulting in uncontrolled stimulus intensity. At 0.9 MAC, the third stimulation induced more positive responses than the first stimulation, independent of the mode of stimulation used. Conclusions The MAC of isoflurane in the Shetland ponies was lower than expected with considerable variability among individuals. Constant current surface electrode stimulations were the most repeatable. A summation over the sequence of three supramaximal stimulations was observed around 0.9 MAC. Clinical relevance The possibility that Shetland ponies require less isoflurane than horses needs further investigation. Constant current surface‐electrode stimulations were the most repeatable. Repetitive supramaximal stimuli may have evoked movements at isoflurane concentrations that provide immobility when single supramaximal stimulation was applied.     

A study of cardiovascular function under controlled and spontaneous ventilation in isoflurane–medetomidine anaesthetized horses

Objective To determine, in mildly hypercapnic horses under isoflurane–medetomidine balanced anaesthesia, whether there is a difference in cardiovascular function between spontaneous ventilation (SV) and intermittent positive pressure ventilation (IPPV). Study design Prospective randomized clinical study. Animals Sixty horses, undergoing elective surgical procedures under general anaesthesia: ASA classification I or II. Methods Horses were sedated with medetomidine and anaesthesia was induced with ketamine and diazepam. Anaesthesia was maintained with isoflurane and a constant rate infusion of medetomidine. Horses were assigned to either SV or IPPV for the duration of anaesthesia. Horses in group IPPV were maintained mildly hypercapnic (arterial partial pressure of carbon dioxide (PaCO₂) 50–60 mmHg, 6.7–8 kPa). Mean arterial blood pressure (MAP) was maintained above 70 mmHg by an infusion of dobutamine administered to effect. Heart rate (HR), respiratory rate (f_R), arterial blood pressure and inspiratory and expiratory gases were monitored continuously. A bolus of ketamine was administered when horses showed nystagmus. Cardiac output was measured using lithium dilution. Arterial blood‐gas analysis was performed regularly. Recovery time was noted and recovery quality scored. Results There were no differences between groups concerning age, weight, body position during anaesthesia and anaesthetic duration. Respiratory rate was significantly higher in group IPPV. Significantly more horses in group IPPV received supplemental ketamine. There were no other significant differences between groups. All horses recovered from anaesthesia without complications. Conclusions There was no difference in cardiovascular function in horses undergoing elective surgery during isoflurane–medetomidine anaesthesia with SV in comparison with IPPV, provided the horses are maintained slightly hypercapnic. Clinical relevance In horses with health status ASA I and II, cardiovascular function under general anaesthesia is equal with or without IPPV if the PaCO₂ is maintained at 50–60 mmHg.     

In vitro sensitivity of Hungarian Actinobaculum suis strains to selected antimicrobials

In vitro antimicrobial sensitivity of 12 Hungarian isolates and the type strain ATCC 33144 of Actinobaculum suis to different antimicrobial compounds was determined both by the agar dilution and by the disc diffusion method. By agar dilution, MIC50 values in the range of 0.05-3.125 micrograms/ml were determined for penicillin, ampicillin, ceftiofur, doxycycline, tylosin, pleuromutilins, chloramphenicol, florfenicol, enrofloxacin and lincomycin. The MIC50 value of oxytetracycline and spectinomycin was 6.25 and 12.5 micrograms/ml, respectively. For ofloxacin, flumequine, neomycin, streptomycin, gentamicin, nalidixic acid, nitrofurantoin and sulphamethoxazole + trimethoprim MIC50 values were in the range of 25-100 micrograms/ml. With the disc diffusion method, all strains were sensitive to penicillin, cephalosporins examined, chloramphenicol and florfenicol, tetracyclines examined, pleuromutilins, lincomycin and tylosin. Variable sensitivity was observed for fluoroquinolones (flumequine, enrofloxacin, ofloxacin), most of the strains were susceptible to marbofloxacin. Almost all strains were resistant to aminoglycosides but most of them were sensitive to spectinomycin. A strong correlation was determined for disc diffusion and MIC results (Spearman's rho 0.789, p < 0001). MIC values of the type strain and MIC50 values of other tested strains did not differ significantly. Few strains showed a partially distinct resistance pattern for erythromycin, lincomycin and ampicillin in both methods.     

Quantitative Evaluation of Equine Deep Digital Flexor Tendon Morphology using Magnetic Resonance Imaging

Injury of the distal aspect of the deep digital flexor tendon (DDFT) is a recognized cause of lameness, but diagnosis is difficult. This study aimed to improve understanding of DDFT morphology and pathology using retrospective evaluation of magnetic resonance (MR) images. We hypothesized that: (1) The distal aspect of the DDFT in normal horses would have a repeatable proximal/distal pattern and symmetry between limbs and between lobes; (2) DDFT dimensions would be related to bodyweight, navicular bone dimensions and hoof size; (3) this symmetry and pattern would be lost in DDFT injury; and (4) DDFT size would increase with injury. MR images of 64 live horse limbs, 26 with no identified DDFT lesion and 38 with identified DDFT abnormalities, and 19 normal cadaver limbs were analyzed. Using standardized transverse images, measurements of DDFT cross-sectional area, medial-lateral (ML) width and dorsal-palmar depth were obtained at six preselected sites. A uniform distal to proximal shape pattern was identified in all horses. The flattened crescent shape at the insertion changed to a deeper bilobed shape more proximally, with the mid-navicular area having the greatest cross-sectional area. Strong ML (P < 0.0006) and left/right symmetry (P < 0.02) were observed. In addition, there was a strong association between DDFT cross-sectional area and horse weight (P = 0.005) and between DDFT and navicular bone ML width (P = 0.004). Symmetry between sides or between lobes was lost at sites with a unilateral lesion and correlation between horse weight and DDFT cross-sectional area was lost in the presence of lesions. DDFTs with core lesions had a consistent increase in cross-sectional area overall, but other lesion types had no significant increase in size. The shape and symmetry seen in normal tendons could be related to the mechanical demands placed upon individual lobes. The limited increase in cross-sectional area with injury may be explained by the restrictive structures of the hoof, possibly explaining the ongoing pain seen in such lesions.     

Computed tomography-guided fine-needle aspiration and tissue-core biopsy of bone lesions in small animals

In humans, free-hand computed tomography (CT)-guided biopsy is an accurate method to obtain a tissue sample. There are only a few reports of this technique in veterinary medicine. In the present study, 21 dogs and two cats underwent a free-hand CT-guided tissue-core biopsy (17 animals) or fine-needle aspiration (six animals) of a bone lesion. Two out of 17 tissue-core samples were also cultured. All 17 tissue-core biopsy samples were diagnostic (accuracy of 100%). Five out of six aspirates were diagnostic (accuracy of 83.3%). The overall accuracy was 95.7%. In one aspirate, cytologic quality was insufficient containing only blood. No major complications were encountered. Fourteen neoplastic, two infectious and six benign lesions were diagnosed. CT examination after intravenous contrast medium added useful information to avoid large vessels and to biopsy-viable tissue. Free-hand CT-guided tissue-core biopsy and aspiration appears to be a safe and very accurate procedure for use in the diagnosis of bone-associated diseases in small animals.