In vitro study of neutrophil apoptosis in dogs

In the present study, to investigate the apoptosis of the polymorphonuclear neutrophil (PMN) from healthy dogs, we carried out TUNEL assay and DNA analysis by electrophoresis on dog PMNs. The TUNEL assay indicated that apoptotic PMNs in dogs were 0.15+/-5% before incubation, 0.3+/-5% at 4h incubation, 1+/-6% at 8h, 9+/-4% at 12h and 28+/-5% at 24h, respectively. The ladder formation was much more clearly observed in DNA from PMNs after 24h incubation at 37 degrees C than that before incubation. The results in this study indicated that healthy dog PMNs undergo apoptosis spontaneously within hours to days, and that the apoptosis of PMNs might be related to the high turnover of these circulating cells in dogs.     

Thyroid and immunologic status of dogs with perianal fistula

Lymphocyte-proliferation responses, absolute lymphocyte counts, and thyrotropin-stimulation responses were determined in 33 dogs with perianal fistula; serum immunoglobulin values also were determined in 15 of the 33 dogs. Lymphocytes were stimulated with concanavalin A, pokeweed mitogen, and phytohemagglutinin and were cultured with medium containing normal pooled canine serum or fresh patient's autologous serum. Initially, lymphocytes from 9 dogs (27.3%) had depressed stimulation responses to greater than or equal to 1 phytomitogen, and 4 of the 9 dogs had absolute lymphopenia. One month after recovery in these 9 dogs, lymphocytes from 4 dogs (66.7%) had normal proliferation responses. Of immunoglobulin determinations in 15 dogs, serum IgA values were 32 to 185 mg/dl (mean, 69 +/- 10 mg/dl) and were low in 2 dogs (13%), and serum IgM values were 48 to 610 mg/dl (mean, 263 to 46 mg/dl) and were high in 8 dogs (53%). Serum IgG values were 1,050 to 3,220 mg/dl (mean, 2,339 +/- 165 mg/dl) and were high in 10 dogs (71%). After thyrotropin stimulation, 1 dog was considered hypothyroid. Neither pathogenesis nor prognosis of canine perianal fistula was clarified via immunoglobulin concentrations or absolute lymphocyte counts. Based on lymphocyte-proliferation assays, suppression of cell-mediated immunity was probably a result of perianal fistula, rather than a cause of the fistula.     

Vaginal resection and anastomosis for treatment of vestibulovaginal stenosis in 4 dogs with recurrent urinary tract infections

CASE DESCRIPTION-4 dogs were evaluated because of recurrent urinary tract infections. CLINICAL FINDINGS-All dogs had recurrent urinary tract infections and similar clinical signs; 3 dogs had urinary incontinence. Digital vaginal examination revealed vestibulovaginal stenosis in all dogs, which was confirmed by results of contrast vaginourethrography. From image measurements, the vestibulovaginal ratio (ratio of the height of the vestibulovaginal junction to the maximum height of the vagina on a lateral vaginourethrogram) was calculated for each dog. Three dogs had severe stenosis (vestibulovaginal ratio, < 0.20; severe stenosis is defined as a vestibulovaginal ratio < 0.20), whereas the fourth dog had moderate stenosis (vestibulovaginal ratio, 0.24; ratio range for moderate stenosis is 0.20 to 0.25). TREATMENT AND OUTCOME-All dogs were anesthetized for surgical correction of the vestibulovaginal stenosis. Vaginal resection and anastomosis of the stenosis was performed in all 4 dogs, with 1 dog also undergoing episioplasty. Complete resolution of clinical signs was apparent in 3 dogs; 1 dog had postoperative complications including pollakiuria and stranguria, which resulted in rectal and vaginal prolapse. This dog underwent ovariohysterectomy, after which clinical signs resolved. All dogs had resolution of urinary tract infections at the time of follow-up (6 to 8 months after surgery). CLINICAL RELEVANCE-Resection and anastomosis may resolve recurrent urinary tract infections in dogs with severe or moderate vestibulovaginal stenosis. Episiotomy was not necessary for success of surgical treatment, and overall, that procedure increased morbidity, the severity of intraoperative hemorrhage, and duration of surgery.     

In vitro antifungal susceptibility of Malassezia pachydermatis from dogs with and without skin lesions

Canine Malassezia dermatitis is frequently treated with systemic ketoconazole (KTZ) and itraconazole (ITZ). However, no information is available on the antifungal susceptibility to azoles and allilamine of Malassezia pachydermatis isolates from dogs with or without skin lesions. The present study was designed to evaluate the in vitro antifungal susceptibility of M. pachydermatis strains from dogs with or without skin lesions to KTZ, ITZ, miconazole (MICO), fluconazole (FLZ), posaconazole (POS), voriconazole (VOR) and terbinafine (TER) using the Clinical and Laboratory Standards Institute reference Broth Microdilution Method (CLSI M27-A2). The association between the susceptibility to antifungal compounds and the origin of M. pachydermatis, from skin with or without lesions has been also assessed. A total of 62 M. pachydermatis strains from healthy dogs (i.e., Group A=30) or with skin lesions (i.e., Group B=32) were tested. ITZ, KTZ and POS showed the highest activity against M. pachydermatis strains, whereas MICO TER and FLZ the lowest. A higher number of Malassezia resistant strains were registered among isolates from Group B than those from Group A. This study indicates that M. pachydermatis strains were susceptible to ITZ, KTZ, and POS. However, dogs with lesions may harbour strains with low susceptibility to antifungal agents and displaying cross-resistance phenomena to azole. The antifungal therapy in Malassezia infections requires careful appraisal of choice of drugs especially in cases of unresponsiveness to antifungal treatment or recurrent infections.     

Lymphoscintigraphy in healthy dogs and dogs with experimentally created thoracic duct abnormalities.

Lymphoscintigraphic evaluation of the thoracic duct (TD) was performed in 10 healthy and 12 dogs with experimentally created TD abnormalities (6 dogs with TD lacerations and 6 dogs with cranial vena ligations). Complete imaging took 4 hours and caused no adverse effects or complications. Lymphoscintigraphy of healthy dogs failed to image the TD; however, background activity in the abdomen and thorax, and radioactivity in the kidneys, bladder, liver, and heart were noticed. Lacerations and transections of the TD were experimentally created in 6 dogs to ascertain whether TD rupture could be detected with lymphoscintigraphy. Lymphoscintigraphy was performed within 48 hours of creating the TD defect. There was no significant difference in the scintigraphic pattern of healthy dogs and those with experimentally created TD defects. Ligation of the cranial vena cava was performed in 6 dogs; 3 dogs developed chylothorax. In those 3 dogs, diffuse radioactivity was imaged in the thorax and was compatible with thoracic lymphangiectasia. In one of these dogs, linear activity consistent with the TD and localized regions of radioactivity cranial to the heart (compatible with the mediastinal lymph nodes) were noticed. Lymphoscintigraphic findings in these dogs correlated with lymphangiographic findings.     

In vitro characteristics of normal and dystrophic skeletal muscle from dogs

Explants were prepared from skeletal muscle tissue from 5 nondystrophic pups and from 5 pups with X-linked muscular dystrophy; pups were 2 to 17 weeks old. A serial reexplant method resulted in optimal cell density with minimal fibroblast growth. Cultures were examined daily by use of phase-contrast microscopy; differentiated (post-fusion) cultures were examined by electron microscopy. Moderate nuclear pleomorphism and cell clustering were observed in cultures of normal and dystrophic muscle cells. Cultures were maintained to 27 days after plating. Minimal myofilament synthesis was observed in multinucleate cells from nondystrophic and dystrophic pups, but spontaneous contraction of myotubes was not observed during this period. Differences in growth, fusion, or differentiation of myogenic cells into multinucleate cells and myotubes were not found between dystrophic and normal muscle.     

Leukoencephalitis and vasculitis with perivascular demyelination in a Weimaraner dog

Aseptic and noninfectious diseases of the central nervous system are being recognized with increasing frequency. After multiple episodes of neurologic illness, this 7-year-old Weimaraner dog was euthanatized and submitted for postmortem examination. Lesions in the central nervous system were found mainly in the white matter of the cerebral cortex and cervical spinal cord and represented acute and more chronic injury. Necrotizing vasculitis with fibrinoid change and a marked neutrophilic infiltrate dominated the acute lesions. More chronic changes consisted of perivascular demyelination and accumulation of foamy macrophages with positive staining for myelin. An immune-complex (Arthus-type) vasculitis is suspected.     

Assessment of neutrophil function in dogs with primary ciliary dyskinesia

Compared with neutrophils from healthy dogs, neutrophils from 2 dogs with primary ciliary dyskinesia had increased distance of random migration, but fewer of the neutrophils migrated. The affected dogs had an increase in the numbers of Staphylococcus aureus phagocytized. Lymphocyte blastogenesis in the affected dogs in response to standard mitogens was considered to be normal.     

Thyroid hormone abnormalities and outcome in dogs with non-thyroidal illness

OBJECTIVES: To document thyroid hormone abnormalities in dogs with non-thyroidal illness and identify markers of prognostic value. METHODS: Circulating total and free thyroxine, total triiodothyronine and thyrotropin concentrations were measured in 196 dogs with non-thyroidal illness. Clinical signs, previous medications and outcome were recorded in each case. Data were analysed to determine endocrine prognostic factors, and to document the prevalence of thyroid hormone abnormalities. RESULTS: Total triiodothyronine, and total and free thyroxine concentrations were decreased in 75.9, 34.7 and 4.5 per cent of cases, respectively. Dogs which were euthanased had significantly decreased total triiodothyronine, and total and free thyroxine concentrations compared with those which made a full recovery. Total triiodothyronine concentrations were significantly lower in dogs that were euthanased compared with those which made a partial recovery. CLINICAL SIGNIFICANCE: Thyroid hormone concentrations may be used as prognostic indicators in dogs with non-thyroidal illness. Low triiodothyronine syndrome may be more common in dogs than previously recognised.     

In vitro evaluation of a novel prosthesis for laryngoplasty of horses with recurrent laryngeal neuropathy

A prosthesis, composed of a steel cable and stress-reducing washers, was developed to prevent failure of laryngoplasty, a common treatment for horses affected by recurrent laryngeal neuropathy. Laryngoplasties were performed on 15 cadaveric larynges using a polyester suture on one side and the cable prosthesis on the other. Each prosthesis was distracted at a displacement rate of 20 mm/s using a servohydraulic materials testing machine until laryngoplasty failed. Distraction force and actuator displacement were recorded and analysed. All 15 laryngoplasties performed with a suture failed at the muscular process at a mean +/- s.d. force of 55.8 +/- 13.1 N. Six laryngoplasties performed with the cable prosthesis failed at the muscular process at mean force 219.6 +/- 125.0 N. In the other 9, the arytenoid cartilage was avulsed from the larynx at mean force 206.4 +/- 75.3 N, and the cable then tore through the muscular process at mean force 357.0 +/- 32.0 N. The difference in force required to cause failure of laryngoplasty was significant (P<0.0001). Although the prosthesis resisted substantially higher forces than did the suture, the effects of the prosthesis in vivo must be evaluated.     

In vitro allergy tests compared to intradermal testing in horses with recurrent airway obstruction

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterised by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of IgE-mediated reactions in RAO remains unclear.The aim of the study was to investigate with a serological IgE ELISA test (Allercept?), an in vitro sulfidoleukotriene (sLT) release assay (CAST^®) and with intradermal testing (IDT) whether serum IgE and IgE-mediated reactions against various mould, mite and pollen extracts are associated with RAO. IDT reactions were evaluated at different times in order to detect IgE-mediated immediate type reactions (type I hypersensitivity reactions, 0.5–1 h), immune complex-mediated late type reactions (type III reactions, 4–10 h) and cell-mediated delayed type reactions (type IV hypersensitivity reactions 24–48 h).In the serological test, overall the control horses displayed more positive reactions than the RAO-affected horses but the difference was not significant. Comparison of the measured IgE levels showed that the RAO-affected horses had slightly higher IgE levels against Aspergillus fumigatus than controls (35 and 16 AU, respectively, p < 0.05), but all values were below the cut off (150 AU) of the test. In the sLT release assay, seven positive reactions were observed in the RAO-affected horses and four in the controls but this difference was not significant.A significantly higher proportion of late type IDT reactions was observed in RAO-affected horses compared to controls (25 of 238 possible reactions versus 12 of 238 possible reactions, respectively, p < 0.05). Interestingly, four RAO-affected but none of the control horses reacted with the recombinant mould allergen A. fumigatus 8 (rAsp f 8, p < 0.05), but only late phase and delayed type reactions were observed.In all three tests the majority of the positive reactions was observed with the mite extracts (64%, 74% and 88% of all positive reactions, respectively) but none of the tests showed a significant difference between RAO-affected and control animals. Our findings do not support that IgE-mediated reactions are important in the pathogenesis of RAO. Further studies are needed to investigate whether sensitisation to mite allergens is of clinical relevance in the horse and to understand the role of immune reactions against rAsp f 8.     

In vitro effect of recombinant human tumor necrosis factor-alpha on canine neutrophil apoptosis

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-alpha (TNF-alpha) is suggested to play an important role. Since an effect of TNF-alpha on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-alpha in the present study to investigate the effect of TNF-alpha on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-alpha at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-alpha combined with cycloheximide. Therefore, it is indicated that TNF-alpha is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-alpha requires protein synthesis in the PMNs.     

Hemostatic abnormalities in dogs with primary immune-mediated hemolytic anemia

Hemostatic parameters were prospectively measured in 20 dogs with primary immune-mediated hemolytic anemia. Eight of 20 dogs had received prior treatment with prednisone. Activated partial thromboplastin time was increased in nine dogs; one-stage prothrombin time was increased in two dogs; fibrinogen concentration was increased in 17 dogs; and antithrombin activity was decreased in 10 dogs. Fibrin(ogen) degradation products concentration was increased in 12 dogs, and D-dimer concentration was increased in 16 dogs. Four or more laboratory criteria of disseminated intravascular coagulation (DIC) were present in nine dogs, and three criteria of DIC were found in four additional dogs. Thromboembolism was the most common finding in the dogs that died. In this study population, mortality was not significantly associated with any clinical finding or laboratory variable.     

In vivo effects of chloramphenicol, tetracycline, and gentamicin on bovine neutrophil function and morphologic features

Antibiotics that have been shown in vitro to have a detrimental effect on bovine polymorphonuclear leukocytes (PMNL) were injected into the mammary gland. Chloramphenicol, tetracycline, gentamicin, or phosphate-buffered saline solution (PBSS) were administered to uninfected mammary quarters of four cows at recommended doses. Each cow received each of the 4 treatments. Total milk somatic cell count and N-acetyl-beta-D-glucosaminidase activity in milk in response to drug, changes in ultrastructure of PMNL, and effects on in vitro percentage phagocytosis, reduction of nitroblue tetrazolium, and chemiluminescence were studied. Chloramphenicol and tetracycline caused a significant (P less than 0.01) increase in somatic cell count, compared with baseline values. During the first 12 hours, no effect on NAGase activity was observed. All 3 antibiotics caused a significant (P less than 0.05) alteration of PMNL morphologic features. More abnormal PMNL (63%) were found in tetracycline-injected quarters. Gentamicin-injected quarters contained 33% abnormal PMNL, compared with only 5% for PBSS-injected quarters. A significant (P less than 0.01) decrease in percentage phagocytosis was observed for tetracycline and gentamicin. Tetracycline inhibited all chemiluminescence activity, whereas no effect was observed for the other 2 drugs. Nitroblue tetrazolium reduction was nonsignificantly (P greater than or equal to 0.05) decreased for the 3 drugs, compared with that for PBSS controls. On the basis of our findings, we concluded that some antibiotics may be inhibitory to phagocyte function and, thereby, impair host defense mechanisms against invading microbes.     

Ascarid infections of cats and dogs

The ascarids Toxocara canis, Toxocara cati, and Toxascaris leonina are probably the most common gastrointestinal helminths encountered in small animal practice. Both T. canis and T. cati can cause serious disease in kittens and puppies; T. leonina is generally less pathogenic. Prenatal transmission assures that virtually all puppies are born infected with T. canis. Transmammary transmission is probably the major route of infection for kittens with T. cati. In addition, all three species of worm produce resistant eggs and use paratenic hosts to facilitate transmission. Much is now known about the complex biology and life history of T. canis. However, many questions, such as those concerning the mechanisms of larval survival within host tissues and of larval reactivation and migration during pregnancy, await further study. The mechanism of resistance to ascarid infections in cats and dogs has not been clearly defined. Ascariasis is traditionally thought to be a disease of young animals, with older animals being considered immune. However, at least in the case of T. canis, adult dogs can be repeatedly infected. A wide range of anthelmintics is available with extremely high efficacy against patent ascarid infections. The problem of prenatal infection with T. canis may be overcome by strategic use of the newer benzimidazole-carbamates, and the production of ascarid-free puppies now seems possible. However, complete larvicidal activity against somatic stages has not been convincingly demonstrated. Visceral larva migrans-like syndromes are now being recognized in dogs and cats. In addition, visceral larva migrans in children due to T. canis continues to be a significant zoonotic disease in North America and underscores the need for the veterinary profession to control ascarid infections in cats and dogs at every opportunity.     

In vitro responses of bronchial spirals and parenchymal strips from healthy and heartworm-infected dogs

Bronchial spirals and pulmonary parenchymal strips from control (noninfected) and Dirofilaria immitis-infected dogs were tested for isometric contractile response to histamine and carbamylcholine. Responses of bronchial spirals from control dogs and from D immitis-positive groups did not differ in responses to histamine or carbamylcholine. Parenchymal strips from D immitis-positive dogs showed greater response to histamine than did those from controls; responses to carbamylcholine did not differ.     

In vitro and in vivo comparison of applanation tonometry and rebound tonometry in dogs

Intraocular pressure (IOP) evaluated by applanation tonometry via TONO-PEN XL (TP), and rebound tonometry via TonoVet (TV) were compared in enucleated canine eyes with varied pressure of the anterior chamber (AC) and in clinical cases. TV measured IOP values were lower than IOP measurements of TP in the enucleated eyes with 5-10 mmHg of AC (P<0.0001), though there was no significant difference in IOP values obtained with TP and TV on the pressure ranges of 15-20 mmHg. However, TP detected IOP values were lower than IOP measurements of TV in the eyes with over 25 mmHg of AC (P<0.0001). The results of clinical cases were similar to the enucleated eye model. There was no significant difference in IOP values obtained from TP and TV in dogs with normotensive eyes. IOP measurements of TP were lower than those of TV in glaucomatous eyes (P<0.0001). TV was a reliable tonometer for measurement of IOP in hypertensive eyes, whereas it was less accurate than TP in hypotensive eyes. The characteristics of TP and TV should be considered in the evaluation of IOP in practice.     

Coagulation abnormalities associated with acute Angiostrongylus vasorum infection in dogs

Ten dogs were experimentally infected with 150 3rd-stage larvae of the lungworm Angiostrongylus vasorum (Baillet, 1866). Blood samples obtained once a week for 5 weeks after infection was induced were examined for coagulation alterations. Thrombocytopenia developed in infected dogs before and after parasitic patency, which occurred 6 weeks after infection was induced. Prolongation of activated partial thromboplastin time and thrombocytopenia initially were associated with periods of larval migration and, later, at patency, with pulmonary egg embolism and hatching of 1st-stage larvae in the lung. Decreased factor-V activity, significantly shortened values for the prothrombin time, and increased factor-VIII activity were evident during postpatent periods of parasitic infection. Significant changes were not found in factors-XII and -IX activities throughout the course of infection. Coagulation abnormalities were attributed to excessive intravascular coagulation. Immature adults and first stages of the parasite seemed to have initiated the coagulopathy in the prepatent phase of infection, with subsequent coagulation abnormalities occurring at the time of patency.     

Correlation of haemostatic abnormalities with tumour stage and characteristics in dogs with mammary carcinoma

Sixty female dogs with untreated mammary carcinoma, comprising equal numbers of dogs in tumour stages I to IV, were evaluated for haemostatic abnormalities using the following tests: platelet count, prothrombin time, activated partial thromboplastin time, thrombin time, plasma activity of factor V, VIII and X, plasma concentration of fibrinogen, fibrin monomers and fibrinogen degradation products, and plasma antithrombin III activity. Two-thirds of all dogs had one or more haemostatic test abnormality of which the likelihood and frequency was increased in those with stage III and IV neoplasia. Haemostatic abnormalities were more frequently observed in dogs which had mammary tumours with distant metastases, extended tumour necrosis, inflammatory carcinomas, tumours fixed to underlying structures, or tumours in which there was penetration of the tumour capsule by tumour cells. As in humans with mammary carcinoma, these haemostatic abnormalities might be used as prognostic indicators, but their clinical importance remains unknown.