Ozone therapy by rectal insufflation in dogs: safety and oxidative stress - a randomized cross-over study.

Ozone therapy acts in the body inducing controlled oxidative stress, thereby improving the antioxidant, immune and circulatory responses. However, very little is known about how this therapy affects oxidative stress indicators in dogs. We aimed to assess the clinical, hematological, biochemical and oxidative stress parameters of healthy dogs subjected to ozone therapy and oxygen therapy by rectal insufflation. Ten healthy dogs were allocated into three experimental groups in a cross-over design: control, without intervention; ozone, which received 100 µg of O3/kg through rectal insufflation; and oxygen, which received an ozone-equivalent volume of medicinal O2 through rectal insufflation. Dogs received four applications weekly and were followed up until the seventh week. Ozone therapy significantly increased the weight, mean corpuscular volume and mean platelet volume and decreased total cholesterol of treated dogs. Regarding oxidative stress, ozone therapy reduced total antioxidant capacity by ferric reduction (TAC-FRAP) in D7 compared with baseline and the control, significantly increased total antioxidant capacity by cupric reduction (TAC-CUPRAC) in D42 and D49 compared with the control group, caused an increase in uric acid compared with the oxygen group and decreased lipid peroxidation on D21 compared with the control group. In conclusion, ozone therapy through rectal insufflation causes transient oxidative stress followed by an antioxidant response and discreetly interferes with a few clinical, hematological and biochemical variables in healthy dogs, although variables still remained within the reference ranges for the species, thus proving the safety of the therapy. Furthermore, oxygen therapy causes oxidative stress without inducing a subsequent antioxidant response.

Amphotericin B-loaded natural latex dressing for treating Candida albicans wound infections using Galleria mellonella model.

Amphotericin B (AmB) is the gold standard for antifungal drugs. However, AmB systemic administration is restricted because of its side effects. Here, we report AmB loaded in natural rubber latex (NRL), a sustained delivery system with low toxicity, which stimulates angiogenesis, cell adhesion and accelerates wound healing. Physicochemical characterizations showed that AmB did not bind chemically to the polymeric matrix. Electronic and topographical images showed small crystalline aggregates from AmB crystals on the polymer surface. About 56.6% of AmB was released by the NRL in 120 h. However, 33.6% of this antifungal was delivered in the first 24 h due to the presence of AmB on the polymer surface. The biomaterial's excellent hemo- and cytocompatibility with erythrocytes and human dermal fibroblasts (HDF) confirmed its safety for dermal wound application. Antifungal assay against Candida albicans showed that AmB-NRL presented a dose-dependent behavior with an inhibition halo of 30.0 ± 1.0 mm. Galleria mellonella was employed as an in vivo model for C. albicans infection. Survival rates of 60% were observed following the injection of AmB (0.5 mg.mL-1) in G. mellonella larvae infected by C. albicans. Likewise, AmB-NRL (0.5 mg.mL-1) presented survival rates of 40%, inferring antifungal activity against fungus. Thus, NRL adequately acts as an AmB-sustained release matrix, which is an exciting approach, since this antifungal is toxic at high concentrations. Our findings suggest that AmB-NRL is an efficient, safe, and reasonably priced ($0.15) dressing for the treatment of cutaneous fungal infections.

Effect of acupuncture on hematologic, muscular biomarkers, fibrinogen and serum lactate parameters in training rodeo bulls.

The study aimed to evaluated the effects of acupuncture in rodeo bulls in training, by determining hematological variables, creatine kinase (CK), aspartate aminotransferase (AST), fibrinogen, and plasma lactate. Thirty adult healthy bulls, crossbred, were included in the study and randomly allocated into two groups of 15 animals, according to the use of acupuncture treatment for six months (GA) or not (GB). The variables were measured 30 min before (TP0) and 10 min (TP10min), 12 (TP12h), 24 (TP24h), 48 (TP48h), and 72 h (TP72h) after a single episode of jumping emulating rodeo exercise. The GB group showed variations in hemoglobin between TP0 and TP10min (p = 0.002) and TP0 and TP12h (p = 0.004), and the GA presented an increase in eosinophil values between TP0 and TP12h (p = 0.013) and TP0 and TP24h (p = 0.034). Leukopenia was observed in GB between TP10min and TP72h ((p = 0.008). The CK values were high (↑ 300 UI/l) after exercise until the TP24h, and decreased in TP48h, in both groups. The plasma lactate elevation was lower in the GA at TP10min (p = 0.011), TP12h (p = 0.008), TP72h (p < 0.001). The rodeo bulls submitted to acupuncture treatment showed smaller variations in hemogram, elevated eosinophils levels, and lower plasma lactate levels after exercise.

Feline obesity causes hematological and biochemical changes and oxidative stress - a pilot study.

Obesity, an extremely important factor in feline clinical practice, is estimated to affect up to one third of the feline population. Moreover, it can trigger chronic inflammation, which could predispose to oxidative stress by increasing reactive oxygen species, thereby generating potentially irreversible cellular damage. This study analyzed hematological, biochemical and oxidative stress profiles at various degrees of feline obesity. Forty-five cats were selected and divided into three groups: control (n = 17), overweight (n = 13) and obese (n = 15), after clinical and laboratory evaluation and body condition score. Biochemical and oxidative stress analyses were performed using a photocolorimeter and hematological analyses were performed in a veterinary cell counter. Obese cats showed increased mean corpuscular volume (MCV), red cell distribution width (RDW), HDL cholesterol and triglycerides and decreased activity of gamma-glutamyl transferase (GGT) than control cats, although within the reference ranges for the species. As for oxidative stress, obese cats showed higher total antioxidant capacity (TAC), by the inhibition of 2,2'-Azino-Bis-3-Ethylbenzthiazoline-6-Sulfonic Acid (ABTS), inhibition of ABTS associated with horseradish peroxidase (ABTS + HRP), cupric ion reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) methods, while overweight cats had a higher TAC-ABTS + HRP and TAC-FRAP than control cats. We conclude that the conditions of natural obesity and overweight in the feline species alter its hematological, biochemical and oxidative stress parameters.

Does postprandial lipemia interfere with blood gas analysis and assessment of acid-base status in dogs?

To evaluate the interference of postprandial lipemia on blood gas parameters and to assess the acid-base status by the quantitative approach of the strong ion model blood samples of 15 healthy dogs were collected during fasting (0 h) and at one (1 h), three (3 h) and five (5 h) hours after the induction of lipemia with a hypercaloric diet. Total cholesterol (TC) and triglyceride (TG) levels were used to assess lipemia and these were correlated with the parameters evaluated accordingly. Anion gap decreased at 5 h without correlation with TC and TG, whereas other parameters measured by the blood gasometer did not change. In the evaluation of the acid base state, the apparent strong ion difference (SIDa) and the strong ion gap (SIG) showed a decrease at 5 h without correlation with lipemia. Lipid levels correlated with the effective strong ion difference (SIDe), the concentration of total non-volatile weak acids (Atot), albumin, phosphate, and magnesium. The SIDe increased at 1 h and at 3 h; the Atot at 1 h, 3 h, and 5 h; albumin increased at 1 h and 3 h; phosphate increased at 1 h, 3 h and 5 h; and magnesium decreased at 5 h. Though postprandial lipemia does not interfere with blood gas analysis, it can cause errors in the variables used to assess the acid-base status, which are dependent on biochemical analytes. Therefore, caution is required when interpreting electrolyte disturbances that result from the postprandial state.

Neospora caninum infection and reproductive problems in dairy cows from Brazil: A case-control study.

Neosporosis, an infectious disease caused by the protozoan Neospora caninum, has been associated with economic losses in cattle rearing worldwide. However, previous studies have not presented any evidence regarding the association between serological status of neosporosis and alteration of the reproductive parameters. Thus, this study aimed to determine whether N. caninum is associated with reproductive disorders and to evaluate the possible risk factors of the infection. Blood samples from 202 dairy cows, 51 with a history of reproductive disorders (case group) and 151 without (control group), were collected from different farms in Brazil. Epidemiological questionnaires were conducted with all the farmers. Serum samples were subjected to an indirect fluorescent antibody test to detect antibodies against the parasite. In total, 28.22% (57/202) of the cows were seropositive: 47.06% (24/51) from the case group and 21.85% (33/151) from the control group. By logistic regression, cows aged ≥48 months and cows with history of abortion were 4.8 (95% confidence interval [CI] = 1.91-12.05; p = 0.001) and 2.3 (95% CI = 1.06-5.1; p = 0.034) times more likely to be seropositive, respectively. Furthermore, our results show an association between N. caninum seropositivity and abortion in dairy cows from Brazil with poor management conditions and N. caninum seropositivity risk factors for reproductive disorders.

Evaluation of hematological, biochemical and oxidative stress profile in calves under propofol anesthesia.

Propofol is a widely used drug in veterinary medicine to induce anesthesia; as well as the chosen compound for protocols of intravenous anesthesia. The present study aimed to describe the hematological, biochemical and oxidative stress alterations in calves kept under anesthesia by propofol in different dosages. In order to achieve this, eight Holstein calves were induced using propofol in a 5 mg/kg dosage and maintained under continuous propofol infusion for 60 min, having being administered 0.6 mg/kg/h or 0.8 mg/kg/h in crossover design with seven days interval. Blood samples were collected immediately before the anesthesia induction (baseline), and 30 min, 1, 2, 3, 4 and 5 h after the procedure started. Statistically relevant propofol influence was observed both in blood and biochemical parameters, with differences between dosages according to the time of infusion. The drug action over oxidative stress was also observed, causing a raise of the total antioxidant capacity (TAC) with an uric acid increase. Additionally, the increase of triglycerides, induced by the anesthesia maintenance with propofol, caused lipemia in the samples, which was capable of interfering directly in the measurements made by refractometry and spectrophotometry. It was concluded that, in spite of propofol induced alterations in blood and biochemical parameters, such alterations are subtle. In addition to that, the drug presented an antioxidative effect, which reinstates the safety of anesthesia maintenance with propofol in calves.

Vertical transmission of Neospora caninum in bovine fetuses from a slaughterhouse in Brazil.

Neospora caninum is considered as one of the main causes of reproductive failure in cattle. Vertical transmission is the main route of infection in the bovine host and plays an important role in maintaining the parasite in the herd. Molecular detection of N. caninum is important to determine the occurrence of the disease and to evaluate the genetic diversity of the parasite. The present study aimed at assessing the vertical transmission of N. caninum using molecular techniques to detect the parasite in tissue samples from bovine fetuses collected in a slaughterhouse in the state of São Paulo, Brazil. Seventy fetuses and 70 blood samples from pregnant cows were collected in a slaughtering line. Fresh samples of heart and brain tissue from fetuses were analyzed using molecular assays. Serum samples from fetuses and cows were subjected to an indirect fluorescent antibody test (IFAT) to detect antibodies against N. caninum. Nested PCR targeting the internal transcriber 1 (ITS1) region of the protozoan organism was used in the molecular testing. From the total of fetuses examined, 71.42% were positive for N. caninum by PCR. A higher number of heart samples (47.1%) were positive for the parasite using this technique. Antibodies against the protozoa were detected in 12.9% of serum samples of cows; 2.8% of fetuses were seropositive for this pathogen. Our results show that vertical transmission of N. caninum occurs in cattle from this region of Brazil, and that the use of different diagnostic techniques contributes to successful diagnosis of congenital transmission of the parasite in cattle.

PD-1 function in apoptosis of T lymphocytes in canine visceral leishmaniasis.

Dogs infected with Leishmania infantum have a reduced number of T lymphocytes. PD-1 (Programmed cell death 1) a new member of the B7-CD28 family that is expressed by immune cells, and its binding to PD-L1 (CD274) or PD-L2 (CD273) induces the deactivation or apoptosis of T cells. This study aimed to evaluate the expression of PD-1 and its ligands, as well as blocking in the induction of apoptosis in T lymphocytes, TNF-α, IL-4 and nitric oxide production by leucokocytes from PBMC and spleen and the parasite load in dogs with visceral leishmaniasis (VL). Our results showed that the expression of PD1 and its ligands was increased in CD3(+) T cells and CD21(+) B lymphocytes within the peripheral blood and splenic mononuclear cells of dogs with VL. In peripheral blood monocytes, only PD-1 ligands exhibited increased expression; however, in spleen macrophages, increased expression of both PD-1 and its ligands was observed. Levels of apoptosis in peripheral blood and splenic T lymphocytes were higher in dogs with VL compared to healthy dogs. Blocking monoclonal antibodies to PD-1 and its ligands in the culture of mononuclear cells from the peripheral blood and spleen decreased the amount of CD3(+) T lymphocyte apoptosis. The concentration of nitric oxide, TNF-α and IL-4 increased in the culture supernatants of peripheral blood mononuclear cells treated with a blocking monoclonal antibody against PD-1. The TNF-α concentration increased in the culture supernatants of splenic cells following all treatments with antibodies blocking PD-1 and its ligands; however, the amount of IL-4 increased only in the presence of a PD-1 blocking agent. Treatment with a PD-1 blocking monoclonal antibody in the mononuclear peripheral blood of dogs with VL reduced the parasite burden while increased TNF-α. We conclude that in canine visceral leishmaniasis, PD-1 and its ligands are involved in the induction of T lymphocyte apoptosis and in regulating the production of nitric oxide, TNF-α, and IL-4, as well as the parasitic load.

Hypertension and its correlation with renal lesions in dogs with leishmaniosis.

To evaluate the prevalence of hypertension and its correlation with the severity of renal injury and proteinuria in dogs with leishmaniosis, sixty-six dogs were divided into two groups. Group 1 (G1) was composed of 54 dogs included in stage 1 of chronic kidney disease (CKD), and group 2 (G2) of twelve dogs in stages 2 and 3 of CKD. Prevalence of hypertension was 28.8%, comprising 22.2% of the dogs from G1 and 58.3% from G2 (P=0.011). The mean arterial blood pressure (BP) of dogs from G1 (135.7 ± 20.5) was lower than from G2 (170.0 ± 26.3) (P <0.001). Urine protein-creatinine ratio (UP/C) revealed values above 0.5 in 75.7% of the dogs, with 34% presenting hypertension. All dogs with hypertension had histopathological and laboratory evidence of glomerular disease. Although there was no statistically significant correlation between elevated BP and the severity of glomerular lesions (P=0.408), there was a statistically significant correlation between elevated BP and increased UP/C in the studied population (P=0.002). Thus, dogs with leishmaniosis and renal disease must be screened for the presence of hypertension so that treatment may be instituted as early as possible, in countries where treatment is allowed, to prevent the progression of renal damage.

Alteration of sFAS and sFAS ligand expression during canine visceral leishmaniosis.

Visceral leishmaniosis (VL) is caused by intracellular parasites of the genus Leishmania that affect humans and several animal species. Dogs are one of the main urban reservoirs of Leishmania infantum and play a central role in the transmission cycle to humans via sandflies. CD3+ cells apoptosis is involved in the immune response in VL. Dysregulation of apoptosis has been implicated in various disease states. An important regulator of apoptosis is the FAS-FAS-associated death domain protein (cluster of differentiation 95 - CD95) and FASL-FAS ligand protein (cluster of differentiation 178 - CD178) system involved in the down-regulation of immune reactions and in T cell-mediated cytotoxicity. FAS is a member of the tumor necrosis factor (TNF) receptor super family, which can be expressed in transmembrane or soluble forms. The soluble levels of FAS (sFAS), FASL (sFASL) and active Caspase-3, this last related to apoptotic cascade, were investigated in the spleen of 19 symptomatic dogs presenting moderate VL and 6 healthy dogs, determined by ELISA assay. The splenic parasite load was determined by real-time PCR monitoring of amplification of the intergenic internal transcribed spacer (ITS1) gene of parasite rRNA. sFAS levels were lower (p<0.05). sFASL and active Caspase-3 levels were higher (p<0.05) in dogs with VL compared with controls. Negative correlation was observed between parasite burden and sFASL levels. The increase in sFASL could be related to the mechanism involved in the elimination of the parasite.

Oxidative stress, superoxide production, and apoptosis of neutrophils in dogs with chronic kidney disease.

Oxidative stress is a key component in the immunosuppression of chronic kidney disease (CKD), and neutrophil function may be impaired by oxidative stress. To test the hypothesis that in uremic dogs with CKD, oxidative stress is increased and neutrophils become less viable and functional, 18 adult dogs with CKD were compared with 15 healthy adult dogs. Blood count and urinalysis were done, and the serum biochemical profile and plasma lipid peroxidation (measurement of thiobarbituric acid reactive substances) were determined with the use of commercial reagents. Plasma total antioxidant capacity (TAC) was measured with a spectrophotometer and commercial reagents, superoxide production with a hydroethidine probe, and the viability and apoptosis of neutrophils with capillary flow cytometry and the annexin V-PE system. The plasma concentrations of cholesterol (P = 0.0415), creatinine (P < 0.0001), and urea (P < 0.0001) were significantly greater in the uremic dogs than in the control dogs. The hematocrit (P = 0.0004), urine specific gravity (P = 0.015), and plasma lipid peroxidation (P < 0.0001) were significantly lower in the dogs that were in late stages of CKD than in the control group. Compared with those isolated from the control group, neutrophils isolated from the CKD group showed a higher rate of spontaneous (0.10 ± 0.05 versus 0.49 ± 0.09; P = 0.0033; median ± standard error of mean) and camptothecin-induced (18.53 ± 4.06 versus 44.67 ± 4.85; P = 0.0066) apoptosis and lower levels of superoxide production in the presence (1278.8 ± 372.8 versus 75.65 ± 86.6; P = 0.0022) and absence (135.29 ± 51.74 versus 41.29 ± 8.38; P = 0.0138) of phorbol-12-myristate-13-acetate stimulation. Thus, oxidative stress and acceleration of apoptosis occurs in dogs with CKD, the apoptosis diminishing the number of viable neutrophils and neutrophil superoxide production.

Le stress oxydatif est un élément clé dans l'immunosuppression de maladie rénale chronique (MRC), et la fonction des neutrophiles peut être affectée par le stress oxydatif. Afin de vérifier l'hypothèse que chez les chiens urémiques avec MRC le stress oxydatif est augmenté et les neutrophiles deviennent moins viables et fonctionnels, 18 chiens adultes avec MRC ont été comparés à 15 chiens adultes en santé. Des analyses sanguines et urinaires ont été effectuées, de même que le profil biochimique sérique et la peroxydation des lipides plasmatiques (mesures des substances réactives à l'acide thiobarbiturique) ont été déterminés au moyen de réactifs commerciaux. La capacité antioxydante plasmatique totale (CAT) a été mesurée à l'aide d'un spectrophotomètre et de réactifs commerciaux, la production de superoxyde avec une sonde hydroéthidine, et la viabilité et l'apoptose des neutrophiles avec un cytomètre à flux capillaire et le système d'annexine V-PE. Les concentrations plasmatiques de cholestérol (P = 0,0415), de créatinine (P < 0,0001) et d'urée (P < 0,0001) étaient significativement plus élevées chez les chiens urémiques comparativement aux chiens témoins. L'hématocrite (P = 0,0004), la gravité spécifique de l'urine (P = 0,015), et la peroxydation des lipides plasmatiques (P < 0,0001) étaient significativement plus faibles chez les chiens dans les stades avancés de MCR que chez les chiens témoins. Comparativement aux neutrophiles provenant des chiens témoins, ceux provenant des chiens avec MRC montraient un taux plus élevé d'apoptose spontanée (0,10 ± 0,05 versus 0,49 ± 0,09; P = 0,0033; médiane ± écart-type) et d'apoptose induite par la camptothécine (18,53 ± 4,06 versus 44,67 ± 4,85; P = 0,0066) et des niveaux plus faibles de production de superoxyde en présence (1278,8 ± 372,8 versus 75,65 ± 86,6; P = 0,0022) et en absence (135,29 ± 51,74 versus 41,29 ± 8,38; P = 0,0138) de stimulation par l'acétate de phorbol-12-myristate-13. Ainsi, le stress oxydatif et l'accélération de l'apoptose surviennent chez des chiens avec MRC, l'apoptose diminuant le nombre de neutrophiles viables et la production de superoxyde par les neutrophiles.(Traduit par Docteur Serge Messier).