Evaluation of various compounds to inhibit activity of matrix metalloproteinases in the tear film of horses with ulcerative keratitis

OBJECTIVE: To examine in vitro effects of various antiproteolytic compounds on activity of matrix metalloproteinase (MMP)-2 and -9 in the tear film of horses with active corneal ulcers. SAMPLE POPULATION: Samples of tear film obtained from the eyes of 34 horses with active ulcerative keratitis. PROCEDURE: Horses were sedated, and tear samples were collected from the lower fornix of 34 ulcerated eyes by use of capillary tubes. The protease inhibitors 0.2% EDTA, 0.1% doxycycline, 10% N-acetylcysteine (NAC), 0.1% solution of a modified dipeptide that contains hydroxamic acid (ie, ilomostat), 0.1% alpha1-proteinase inhibitor (PI), 0.5% alpha1-PI, and 100% fresh equine serum (ES) were used to treat pooled samples. Amount of latent and active MMP-2 and -9 was measured by optical density scanning of gelatin zymograms of treated and untreated tear samples. RESULTS: Pooled tear samples obtained from ulcerated eyes contained the latent and active forms of MMP-2 and -9. Compared with MMP activity in untreated samples, total MMP activity (sum of all bands detected) observed on the gelatin zymogram gels was reduced by 99.4% by EDTA, 96.3% by doxycycline, 98.8% by NAC, 98.9% by ilomostat, 52.4% by 0.1% alpha1-PI, 93.6% by 0.5% alpha1-PI, and 90.0% by ES. CONCLUSIONS AND CLINICAL RELEVANCE: We documented that EDTA, doxycycline, NAC, ilomostat, alpha1PI, and ES inhibited MMP activity in vitro. Because these compounds use different mechanisms to inhibit various families of proteases in the tear film of horses, a combination of these protease inhibitors may be beneficial for treatment of corneal ulcers in horses.     

Pharmacokinetics of topically applied ciprofloxacin in equine tears

OBJECTIVE: To evaluate the pharmacokinetics of topically applied ciprofloxacin 0.3% ophthalmic solution in tears of healthy horses. ANIMAL STUDIED: Twenty healthy, adult, mixed-breed horses. PROCEDURES: Twenty study horses were confirmed free of ophthalmic disease by complete ophthalmic examination. Seventy microliters of 0.3% ciprofloxacin (Ciloxan) was placed in the ventral cul-de-sac of each eye using a microliter syringe and 19-g cannula. Population kinetics were carried out by sampling the tear film from the lower cul-de-sac of each eye with tear test strips at 5, 10, 15 and 30 min and 1, 2, 4 and 6 h post administration for a total of five samples at each time-point. Sample collection time was 15 s. Concentrations of ciprofloxacin were determined using high performance liquid chromatography. RESULTS: Mean (+/-SD) of the Schirmer tear test results from all eyes was 23.4 +/- 4.8 mm wetting in 1 min. Mean concentration of ciprofloxacin in the tears at 5 min post administration was 498.4 +/- 266.8 microg/g. Mean concentration rapidly declined and began to plateau at 30 min. The mean tear concentrations of ciprofloxacin at 30 min and at 1, 2, 4 and 6 h were 66.6 +/- 56.0, 60.25 +/- 55.7, 42.25 +/- 30.9, 36.25 +/- 32.0, and 45.5 +/- 46.5 microg/g, respectively. CONCLUSIONS: The pharmacokinetics of ciprofloxacin in normal horses are similar to those in rabbits and humans. Topical application of ciprofloxacin resulted in a mean tear concentration of ciprofloxacin that remained above the MIC(90) levels for most pathogenic bacteria for 6 h post administration.     

Pharmacokinetics of topically applied ciprofloxacin in tears of mesocephalic and brachycephalic dogs

OBJECTIVE: To evaluate the pharmacokinetics of ophthalmic ciprofloxacin in the tear film of normal mesocephalic and brachycephalic dogs. ANIMALS STUDIED: Twenty mesocephalic dogs and 15 brachycephalic dogs. PROCEDURES: Thirty-five microliters of ciprofloxacin were placed on the cornea of both eyes of each dog. Five brachycephalic dogs were used twice. A tear-test strip placed in the ventral cul de sac for 30 s was used to obtain samples. The tear film of each eye was sampled once at eight time-points post administration, resulting in five samples at each time-point. Samples were evaluated using high performance liquid chromatography. Data from the two skull types were compared using the unpaired two-tailed t-test. RESULTS: The mean concentration of ciprofloxacin in the tears of mesocephalic dogs was 192.8 +/- 269.97, 140.6 +/- 91.06, 56.60 +/- 28.47, 13.6 +/- 6.3, 43.25 +/- 59.71, 16.6 +/- 10.62, 15.6 +/- 13.16 and 6.25 +/- 9.84 microg/g at 5, 10, 15, 30 min and 1, 2, 4 and 6 h, respectively. The mean concentration of ciprofloxacin in the tears of brachycephalic dogs was 272.6 +/- 106.21, 144.4 +/- 142.32, 131.2 +/- 147.07, 75 +/- 80.07, 40.8 +/- 30.35, 35 +/- 21.98, 52.75 +/- 51.87 and 8.6 +/- 12.10 microg/g at 5, 10, 15, 30 min and 1, 2, 4 and 6 h, respectively. There was no statistical difference in tear concentration at any time-point between skull types. CONCLUSIONS: Topical application of ciprofloxacin resulted in a mean tear concentration of ciprofloxacin that remained above the MIC(90) levels for most pathogenic bacteria for 6 h in normal mesocephalic and brachycephalic dogs.     

Doxycycline levels in preocular tear film of horses following oral administration

Objective To determine the concentration of doxycycline in preocular tear film following oral administration in horses as a possible therapeutic modality for infectious and keratomalacic equine keratitis. Procedure Eight broodmares without ocular disease from a Thoroughbred breeding facility were included in this study. Each mare received 20 mg/kg of doxycycline by mouth once daily in the morning for five consecutive days. Tears were collected 1 h after doxycycline administration starting on day one of administration and continuing for 10 consecutive days. Doxycycline levels in the tears were measured using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). Results Doxycycline was present in the tears of each mare at low µg/mL levels with the highest concentration appearing on the third to fifth days (8.21–9.83 µg/mL). Doxycycline levels had fallen below quantifiable ranges by day 10. No systemic side-effects were noted in any of the horses included in this study. Conclusions Oral doxycycline is present in preocular tear film of normal horses with noninflamed eyes and may be useful as treatment in equine ulcerative keratomalacia. The oral dose listed was tolerated well by the horses in this study. The drug levels attained at 20 mg/kg once daily orally of doxycycline may aid in the treatment of corneal ulceration in horses, but further study is warranted.     

The effect of drugs commonly used in the treatment of equine articular disorders on the activity of equine matrix metalloproteinase-2 and 9

Loss of articular cartilage, which is the most important pathological lesion occurring in osteoarthritis, has been shown to be enzymatically mediated. The matrix metalloproteinases (MMPs) are a group of enzymes which have been implicated in this degradation of articular cartilage matrix. The use of pharmacological agents to inhibit this catabolic process in the joint is a potential route for therapeutic intervention.
  The gelatinase MMPs, MMPs-2 and 9, were purified by affinity chromatography from equine cell cultures. The ability of phenylbutazone, flunixin, betamethasone, dexamethasone, methylprednisolone acetate (MPA), hyaluronan, pentosan polysulphate and polysulphated glycosaminoglycan (PSGAG) to inhibit equine MMPs-2 and 9 were assessed by two degradation assays. Whilst some agents did have direct effects on MMP activity, these effects were only obtained at concentrations which were unlikely to be achieved for any length of time in vivo . It is improbable that any pharmacological agent, currently used in the horse, has a significant effect on gelatinase MMP activity.     

Biomicroscopy of the tear film: aqueous and lipid tear substitutes in the normal and abnormal eye

Polarised light biomicroscopy was used to assess the behaviour and interactions of two polymer-containing preparations of artificial tears, and two lipid-containing ointments, with the normal and abnormal pre-corneal tear film. The preparations were used topically in six normal dogs, and three dogs with keratoconjunctivitis sicca. A transient morphological alteration of the surface lipid layer of the tear film was commonly observed after the use of polymer solutions. The ointments spread as a continuous or semi-continuous layer over the normal, or polymer-supplemented, aqueous tears. Further applications of artificial tears produced only a temporary disruption of this layer. In dogs with keratoconjunctivitis sicca, thickening of the lipid layer of the tear film was observed for over 20 hours after the administration of ointment. These observations are discussed in relation to the prospects for the improved medical treatment of keratoconjunctivitis sicca by the use of polymer-containing solutions and ointments in combination.     

A preliminary study of changes in tear film proteins in the feline eye following nictitating membrane removal

Objective To investigate the influence of nictitating membrane (third eyelid) removal on selected proteins in feline tears. Animal studied Domestic short‐haired cats (7–17 months; 2.6–5.2 kg) were used. Procedures Eye‐flush tears were collected periodically for up to 18 weeks from both eyes of animals with nictitating membranes removed, but nictitating gland left intact, (n = 4) or with nictitating membranes intact (n = 4). Tear comparisons were based on total protein content (TPC) using micro bicinchoninic acid assay, immunoglobulin A (IgA), and matrix‐metalloproteinase (MMP)‐9 measurements using sandwich enzyme‐linked immunosorbent assay (ELISA) and tear gelatinase activity using gelatin zymography. Expression of MMP‐2 and ‐9 in nictitating membranes removed at baseline (week 0) and eyes collected at 18 weeks were also investigated in histological sections using immunoperoxidase for visualization. Results Nictitating membrane removal did not significantly change TPC and MMP‐9 in tears within the first 4 weeks. MMP‐9 was not detected by ELISA in tears from eyes without nictitating membranes from week 5 onwards. IgA (%IgA of TPC) data varied between animals. Gelatin zymography showed increased MMP‐2 and ‐9 activity in tears from eyes without nictitating membranes at week 1 and a decrease following week 2 post‐surgery. MMP‐2 and ‐9 were immunolocalised to conjunctival goblet cells of removed nictitating membranes and to the conjunctival epithelium, respectively. After 18 weeks, the distribution of MMPs in tissue was comparable between eyes with and without nictitating membranes. Conclusions Based on this preliminary study, nictitating membrane removal appeared to cause long‐term changes in expression of tear proteins, including reduced MMP‐9 expression.     

Metalloproteinases in canine experimental traumatic keratoconjunctivitis

Matrix metalloproteinases (MMPs) are thought to be involved in eye disease and may be present in tears. MMP activity was measured by gelatine zymography. Active gelatinase levels were determined by a gelatine degradation ELISA. Potential MMP-9 (gelatinase-B) monomer enzyme activity was elevated (P < 0.001) in keratoconjunctivitis in comparison to normal tears. MMP-9, dimer form, enzyme activity was elevated (P < 0.001) in fluids from keratoconjunctivitis cases in comparison to fluids from normal eyes. Significant increases in gelatinase bioactivities were seen in tears from keratoconjunctivitis disease cases (P < 0.01). Enzymes in dogs with eye disease were biologically active indicating that both the active forms of the enzyme were present. Hence, they could be an important indicator in deterioration of the cornea during eye disease.     

Effects of polysulfated glycosaminoglycan and hyaluronan on prostaglandin E2 production by cultured equine synoviocytes

OBJECTIVE: To investigate effects of the anti-arthritic agents hyaluronan and polysulfated glycosaminoglycan (PSGAG) on inflammatory metabolism in cultured equine synoviocytes. SAMPLE POPULATION: Synoviocytes cultured from samples obtained from the metacarpophalangeal joints of 4 horses. PROCEDURE: Equine synoviocytes were grown in monolayer culture. Synoviocytes were stimulated with lipopolysaccharide (LPS) and simultaneously treated with various concentrations of hyaluronan or PSGAG for 48 hours. Three hyaluronan preparations were compared. Prostaglandin E2 (PGE2) concentrations in culture medium were measured, using radioimmunoassay. RESULTS: The highest concentrations of hyaluronan and PSGAG tested inhibited PGE2 production. CONCLUSIONS AND CLINICAL RELEVANCE: Clinically achievable concentrations of hyaluronan and PSGAG inhibited PGE2 synthesis by cultured equine synoviocytes. This anti-inflammatory action may be a mechanism through which these agents exert anti-arthritic effects. The effect was obtained at concentrations that can be achieved by use of intra-articular, but not systemic, administration of hyaluronan or PSGAG.     

Proteinases of the cornea and preocular tear film

Maintenance and repair of corneal stromal extracellular matrix (ECM) requires a tightly coordinated balance of ECM synthesis, degradation and remodeling in which proteolytic enzymes (proteinases) perform important functions. There are natural proteinase inhibitors present in preocular tear film (PTF) and cornea simultaneously with proteinases that prevent excessive degradation of normal healthy tissue. Disorders occur when there is an imbalance between proteinases and proteinase inhibitors in favor of the proteinases, causing pathologic degradation of stromal collagen and proteoglycans in the cornea. Two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are of major importance in terms of remodeling and degradation of the corneal stromal collagen. Immunohistochemical studies have shown different origins of MMP-2 and -9. MMP-2 is synthesized by corneal keratocytes and performs a surveillance function in the normal cornea, becoming locally activated to degrade collagen molecules that occasionally become damaged. Alternatively, MMP-9 may be produced by epithelial cells and polymorphonuclear neutrophils following corneal wounding. Because the cornea is in close contact with the preocular tear film (PTF), proteinases have been evaluated in the PTF. In damaged corneas, total proteolytic activity in the tear fluid was found to be significantly increased compared to normal eyes and contralateral eyes. Studies analyzing the proteolytic activity in serial PTF samples during corneal healing led to the following conclusions: ulcerative keratitis in animals is associated with initially high levels of tear film proteolytic activity, which decrease as ulcers heal; proteinase levels in melting ulcers remain elevated leading to rapid progression of the ulcers. The success of medical and surgical treatment of the corneal ulcers is reflected by the proteolytic activity in tears. In animals, successful treatment leads to a rapid reduction in tear film proteolytic activity that corresponds with the improvement in the clinical signs of corneal ulceration. The in vitro effects of various compounds on proteolytic activity in the tear fluid of animals with ulcerative keratitis have been evaluated and their important inhibitory effects have been confirmed. Because these various compounds utilize different mechanisms to inhibit various families of proteinases, a combination of these proteinase inhibitors may be beneficial.     

马波沙星对猪临床分离病原菌的体外抑菌活性研究

考察马波沙星对猪临床分离病原菌的体外抑菌活性,以期为临床应用提供依据。采用琼脂二倍稀释法测定马波沙星对猪临床分离大肠杆菌、金黄色葡萄球菌和链球菌的最小抑菌浓度(MIC),并以土霉素、恩诺沙星、诺氟沙星、环丙沙星、庆大霉素和多西环素作为对照药物。结果表明:马波沙星对猪临床分离病原菌大肠杆菌、金黄色葡萄球菌和链球菌均有较低的最小抑菌浓度,对临床分离大肠杆菌、金黄色葡萄球菌和链球菌的抑菌活性明显高于诺氟沙星、环丙沙星、庆大霉素和强力霉素,对临床分离大肠杆菌的抑菌活性高于恩诺沙星。马波沙星对引起猪乳房炎-子宫炎-无乳综合征(MMA)的主要病原菌的抑菌活性优于常用抗菌药。     

Preliminary results of antibiotic resistance monitoring in the Netherlands

Qualitative tests are used to monitor antimicrobial resistance in bacteria of animal origin in the Netherlands. Quantitative information on trends in resistance is thus not obtained. Moreover, in general a limited panel of antibiotics is tested. The present study describes resistance in zoonotic food-borne pathogens Salmonella, Campylobacter, and Escherichia coli O157 isolated from human clinical cases and from faeces of healthy food animals in 1998 and 1999, as determined with quantitative susceptibility tests. The resistance of the indicator organisms E. coli and Enterococcus faecium isolated from faecal samples of broilers and pigs randomly sampled at slaughterhouses was also determined. For this end, faecal samples from veal calves were sampled in 1996 and 1997 at the three main Dutch veal calf slaughterhouses. In 1998 only a limited number of faecal samples of veal calves were taken at farms. For E. coli and Salmonella the following antibiotics were tested: amoxicillin, amoxicillin-clavulanic acid, piperacillin, cefotaxime, ceftazidime, imipenem, gentamicin, doxycycline, trimethoprim, trimethoprim/sulphamethoxazole, ciprofloxacin, chloramphenicol, florfenicol, carbadox, and flumequine. For E. faecium the following antibiotics were tested: amoxicillin, amoxicillin-clavulanic acid, chloramphenicol, doxycycline, erythromycin, vancomycin, teicoplanin, streptomycin ('high level' > 2000 mg/ml), gentamicin ('high level' > 500 mg/ml), ciprofloxacin, bacitracin, flavofosfolipol, salinomycin, quinupristin-dalfopristin, virginiamycin, tilmicosin, avilamycin, and everninomycin. For Campylobacter the following antibiotics were tested: erythromycin, doxycycline, gentamicin, carbadox, flavofosfolipol, ciprofloxacin, trimethoprim/sulphamethoxazole, amoxicillin, and metronidazole.     

Analysis of tear uptake by the Schirmer tear test strip in the canine eye

OBJECTIVE: To analyze the uptake of tears in a Schirmer tear test (STT) in vitro and in vitro. MATERIALS AND METHODS: Uptake of fluid by Schirmer tear test strips was studied in vitro by examining fluid uptake over time from an unlimited fluid supply as well as with specific fluid volumes applied to the test strip. Uptake of fluid by Schirmer tear test strips was evaluated in a population of 100 ophthalmologically normal dogs together with a group of 40 dogs with tear film abnormalities such as keratoconjunctivitis sicca (KCS) or epiphora. Each animal was given a full ophthalmic examination followed by a standard Schirmer tear test extended over between 3 and 5 min with the STT reading recorded every 5 s and plotted over time. To determine the effect of ocular irritation by the test strip, uptake of tears by test strips was determined before and after topical anesthesia in 20 dogs. RESULTS: In vitro examination of fluid uptake by the STT strips showed an initial rapid uptake followed by a gradual reduction in rate of uptake. Temporal evaluation of STT in vivo showed a similar rapid initial uptake of tear fluid, followed in the majority of cases by a sudden change to a steady state uptake of fluid. The initial gradient was 29.3 +/- 16.9 mm/min followed by a steady state uptake of 5.2 +/- 2.3 mm/min in normal dogs and 1.9 +/- 1.3 mm/min in dogs with KCS. This corresponds to a steady state tear turnover of 7.8 +/- 3.4 microL/min in normal dogs and 2.8 +/- 1.9 microL/min in animals with KCS. Dogs with nasolacrimal blockage and resultant epiphora showed a high initial gradient but final gradients were not statistically different from those of normal dogs. Discussion and conclusions Temporal evaluation of tear uptake by the STT shows substantial differences in rate of tear uptake at different time-points during the period of the test. RESULTS: of this study suggest that the initial rapid rise in STT value represents uptake from the tear lake followed by a slower tear uptake of tears from steady state tear production. Temporal examination of the Schirmer tear test allows a more precise evaluation of tear production than the standard STT measuring tear uptake in 1 min, together with estimation of the contribution to the test strip tear uptake of tears from the residual tear lake volume and those from continual tear production.     

Effect of polysulfated glycosaminoglycan on DNA content and proteoglycan metabolism in normal and osteoarthritic canine articular cartilage explants

OBJECTIVE: To study the effect of polysulfated glycosaminoglycan (PSGAG) on proteoglycan metabolism and DNA content of control and osteoarthritic (OA) cartilage. STUDY DESIGN: An in vitro study comparing the effects of PSGAG on articular cartilage explants from canine stifle joints with and without chronic OA after transection of the left cranial cruciate ligament. SAMPLE POPULATION: Five large cross-breed dogs. METHODS: Cartilage explants (6 to 13 per treatment group) from the medial side of the femoral trochlea and medial femoral condyle from both stifles of each dog were incubated in a defined medium containing 0, 0.05, 0.5, or 5 mg/mL of PSGAG. After 72 hours in culture, explants were pulsed for 6 hours with sodium [35S]sulfate. Aminophenylmercuric acetate (APMA) was used to activate endogenous neutral matrix metalloproteinases (MMPs) and induce proteoglycan degradation in the radiolabeled explants. DNA content and radioactivity were measured in papain-digested explants, and radioactivity was measured in the medium by liquid scintillation counting. Proteoglycan synthesis and degradation were calculated. Cartilage was examined histologically for signs of OA. A mixed model analysis of variance and linear contrasts were used to test for significant (P < .05) effects of OA and treatment with PSGAG. RESULTS: Transection of the cranial cruciate ligament produced OA in operated joints. DNA content and proteoglycan synthesis of OA cartilage were significantly lower than in cartilage from control joints. For both DNA content and proteoglycan synthesis, significant interactions occurred between the concentration of PSGAG and whether the articular cartilage was from OA or control joints. The two lower concentrations of PSGAG (0.05 and 0.5 mg/mL) predominantly increased DNA content in OA cartilage (7 and 18%, respectively, compared with 0 mg/mL PSGAG) while the highest concentration (5 mg/mL) predominantly increased DNA content in control cartilage (30% compared with 0 mg/mL PSGAG). PSGAG at .05 mg/mL predominantly decreased proteoglycan synthesis in OA cartilage (19% reduction compared with 0 mg/mL PSGAG) while PSGAG at .5 and 5 mg/mL predominantly decreased proteoglycan synthesis in control cartilage (17 and 55% reduction, respectively, compared with 0 mg/mL PSGAG). Following activation of MMPs, PSGAG caused a dose-dependent decrease in degradation of radiolabeled proteoglycan in both OA and control cartilage. CONCLUSIONS: OA cartilage was responsive to treatment with PSGAG at 100-fold lower concentration than control cartilage. When treated with PSGAG, articular cartilage explants maintained or increased DNA content at the expense of proteoglycan synthesis. Following MMP activation, proteoglycan degradation was inhibited in OA and control explants in a dose-dependent manner. CLINICAL RELEVANCE: If the results of this study extend to in vivo use, treatment with PSGAG may modify the progression of OA in articular cartilage by maintaining chondrocyte viability or stimulating chondrocyte division as well as protecting against extracellular matrix degradation.     

Protein microanalysis of animal tears

Sub-microlitre volumes of normal koala, mouse, dog, rat and cat tears were fractionated using size exclusion-high performance liquid chromatography (SE - HPLC), giving reproducible profiles which were different for each species. Microlitre volumes of tears were also fractionated using sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS - PAGE), resulting in good separation of individual tear proteins with a species specific distribution. Tears from koalas with conjunctivitis and mice with keratitis were similarly examined and showed mostly quantitative changes. These simple, rapid techniques gave reproducible results and, in contrast to conventional separation techniques, used easily obtainable volumes (as little as 0.75 microl) of tears. Their expansion could allow isola tion, identification and quantitation of individual tear components, enabling effective investigation of changes occurring in disease.     

Polysulfated glycosaminoglycan accelerates net synthesis of collagen and glycosaminoglycans by arthritic equine cartilage tissues and chondrocytes

Low molecular weight polysulfated glycosaminoglycan (PSGAG) stimulated net collagen and glycosaminoglycan synthesis by normal and arthritic equine fetlock cartilage tissues in organ culture. Arthritic tissues were more sensitive to PSGAG stimulation. The rates of cartilage-specific type-II collagen and chondroitin sulfate-rich glycosaminoglycan synthesis by confluent chondrocyte cell cultures obtained from normal and arthritic equine cartilage tissues were increased by 25 and 50 mg of PSGAG/ml. Cells from arthritic cartilage were also more sensitive to the presence of PSGAG. In addition, concentrations of PSGAG (25 and 50 mg/ml) approximate to those in synovial fluid after intra-articular injection of 250 mg of PSGAG inhibited the rate of collagen and glycosaminoglycan degradation in cell culture. These findings suggest that PSGAG may have a role in the healing of mild cartilage degeneration by encouraging the production of replacement hyaline matrix materials, while delaying their subsequent degradation. In contrast, growth of cell cultures was inhibited by PSGAG, suggesting that these compounds may fail to stimulate chondrocyte replication, a prerequisite for tissue regeneration. Nonetheless, these observations provide direct evidence of a truly chondroprotective role for low molecular weight PSGAG in the treatment of equine degenerative joint disease.     

禽源结肠弯曲杆菌的耐药性分析与MLST分型

为了解禽肉结肠弯曲杆菌的耐药表型和分子型,采用琼脂平板稀释法和多位点序列分型(Multilocus Se-quence Typing,MLST)技术分别对54株禽肉结肠弯曲杆菌进行耐药性及分子分型研究。耐药性试验结果得到单重耐药菌株有41株(75.9%),分别是对环丙沙星耐药有9株(16.7%)、对强力霉素耐药有23株(42.6%)和对红霉素耐药有9株(16.7%);多重耐药菌株有10株(18.5%),其中4株(7.4%)对环丙沙星和强力霉素耐药,1株(1.9%)对环丙沙星和红霉素耐药,4株(7.4%)对红霉素和强力霉素耐药,1株(1.9%)对环丙沙星、红霉素和强力霉素耐药;所有菌株对硫酸庆大霉素敏感。MLST得到了38个(含1个新的)等位基因(allele);26个(含8个新的)序列型(Sequence type,ST);2个已知序列型克隆系(ST clonal complexes),ST-828克隆系(45株,83.3%)和ST-1150克隆系(3株,5.6%),以及5个(6株,11.1%)没有序列型克隆系。耐药性与序列型和序列克隆系相关性比较,相关性不大。结果提示,禽肉结肠弯曲杆菌出现了对环丙沙星、红霉素和强力霉素的单重及多重耐药菌株;禽肉中结肠弯曲杆菌主要流行ST-828克隆系;耐药性与序列型及序列克隆系相关性差,揭示耐药菌株来源广泛。     

Estimation of lacrimal level and testing methods on normal beagles

Five methods of testing tears including the Schirmer tear test (STT-1), phenol red thread tear test (PRT) and modified Schirmer tear test (STT-2) were conducted on 44 eyes of 22 normal Beagles, and the tear film break up time (BUT) and lacrimal pH were tested in 32 eyes of 16 normal Beagles. The coefficient of variation of PRT demonstrated a low value (C.V. 14%) compared to the value of STT-1 (C.V. 12%). The lacrimal pH showed a more constant value (C.V. 3%). Measurements of STT-2 (C.V. 48%) and BUT (C.V. 34%) varied significantly. The results show the clinical usefulness of the PRT. Mean values (mean ± SD) of tests were: PRT, 29.3 ± 3.45 mm/15 s; STT-1, 18.89 ± 2.62 mm/60 s; STT-2, 9.52 ± 4.55 mm/60 s; pH, 7.29 ± 0.22 and BUT, 21.53 ± 7.42 s.     

In vitro killing of Escherichia coli, Staphylococcus pseudintermedius and Pseudomonas aeruginosa by enrofloxacin in combination with its active metabolite ciprofloxacin using clinically relevant drug concentrations in the dog and cat

Enrofloxacin is a fluoroquinolone antibacterial agent used to treat infections in companion animals. Enrofloxacin's antimicrobial spectrum includes Gram positive and Gram-negative bacteria and demonstrates concentration-dependent bacteriocidal activity. In dogs and cats, enrofloxacin is partially metabolized to ciprofloxacin and both active agents circulate simultaneously in treated animals at ratios of approximately 60-70% enrofloxacin to 30-40% ciprofloxacin. We were interested in determining the killing of companion animal isolates of Escherichia coli, Staphylococcus pseudintermedius and Pseudomonas aeruginosa by enrofloxacin and ciprofloxacin combined using clinically relevant drug concentrations and ratios. For E. coli isolates exposed to 2.1 and 4.1μg/ml of enrofloxacin/ciprofloxacin at 50:50, 60:40 and 70:30 ratios, a 1.7-2.5log(10) reduction (94-99% kill) was seen following 20min of drug exposure; 0.89-1.7log(10) (92-99% kill) of S. pseudintermedius following 180min of drug exposure; 0.85-3.4log(10) (98-99% kill) of P. aeruginosa following 15min of drug exposure. Killing of S. pseudintermedius was enhanced in the presence of enrofloxacin whereas killing of P. aeruginosa was enhanced in the presence of ciprofloxacin. Antagonism was not seen when enrofloxacin and ciprofloxacin were used in kill assays. The unique feature of partial metabolism of enrofloxacin to ciprofloxacin expands the spectrum of enhanced killing of common companion animal pathogens.     

Use of tears for diagnosis of feline leukemia virus infection

A comparison was made of the use of serum, tears, and saliva for the detection of feline leukemia virus (FeLV) infection in cats. Cotton swabs were used to collect saliva, and tear-test strips were used to collect tears. Specimens were analyzed by a commercially available ELISA. Using a 10- to 15-minute specimen incubation period, FeLV was detected in 70% of the saliva specimens and in 73% of the tear specimens from viremic (serum-positive) cats. Feline leukemia virus antigen was not detected in saliva and tear specimens from serum-negative cats. The sensitivity of the tear assay was improved by increasing the incubation time to 24 hours. Tear strips could be air-dried and stored at room temperature for up to 7 days without any appreciable loss of activity. Client-owned and experimentally infected laboratory cats were tested for FeLV, using air-dried tear-test strips and a 24-hour incubation period. Tears were positive (contained FeLV antigen) in 65 of 72 (90%) serum-positive cats and did not contain antigen in 46 of 46 (100%) serum-negative cats. Results of ELISA obtained from serum and tears also were compared with results obtained from indirect fluorescent antibody testing of blood smears. Results of indirect fluorescent antibody and ELISA compared favorably with each other and with the results of tear testing.