Effects of etamsylate on equine platelets: in vitro and in vivo studies

The aim of this study was to investigate whether etamsylate produces equine platelet activation. In vitro and in vivo studies were designed in which seven and eight adult healthy horses were included, respectively. In the in vitro study, citrated blood was incubated with different concentrations of etamsylate, and P-selectin expression and annexin V binding were determined by flow cytometry. In the in vivo study, blood was collected before and 1 and 2h after IV administration of etamsylate, and P-selectin expression was evaluated. In the in vitro study, a significant increase in P-selectin expression, leukocyte-platelet aggregate formation and annexin V binding were observed. In the in vivo study, a marked increase in P-selectin expression and heterotypic aggregate formation was seen in two and five horses, respectively, although no significant differences were detected when analyzing results from all the animals together. The results of the in vitro study indicate that etamsylate produces a pre-activation state in equine platelets, but this fact could be confirmed by the in vivo study.     

Antimicrobial activity of gallium against virulent Rhodococcus equiin vitro and in vivo

Rhodococcus equi, a facultative intracellular bacterium, causes severe pneumonia in foals. Evidence suggests that most foals become infected very early in life, when they have immature or ineffective innate immune responses. This study evaluated the antimicrobial activity of gallium against R. equi, as a potential chemoprophylactic and therapeutic agent. Rhodococcus equi was grown in media with various concentrations of gallium nitrate (GN), with and without excess iron. GN significantly inhibited growth and killed R. equi, and these effects were abolished with excess iron. Antimicrobial effects of Ga appear to be related to its interference with iron metabolism. Mice were treated orally with gallium maltolate (GaM), 10 or 50 mg/kg BW, or distilled H2O prior to and after experimental infection with R. equi. Six days post-infection, organs were harvested and R. equi concentrations assessed, and serum gallium concentrations determined. GaM was absorbed in a dose-dependent manner, and R. equi tissue burdens were greater in control mice than in all GaM-treated mice. GaM may aid in the control of disease by preventing development of overwhelming R. equi tissue burdens prior to the establishment of requisite innate and adaptive immune responses.     

Effect of feeding selected carbohydrates on the in vivo attachment of Salmonella typhimurium in chick ceca.

Two-day-old chicks were orally inoculated with 1 ml of Salmonella typhimurium (10(5) colony-forming units/ml) and divided into four groups. Three groups were fed 2.5% carbohydrates starting on day 1 (arabinose, galactose, and lactose), while the fourth group served as the control. Ceca were obtained from each group at 7, 14, and 21 days. At the end of 14 days, all three carbohydrates statistically reduced Salmonella recovery. However, lactose failed to reduce recovery between day 14 and day 21. Arabinose and galactose continued to show significant reductions of recovery through 21 days. No statistical difference was found between Salmonella recovery from whole ceca (with cecal material) and inverted ceca (washed free of cecal material).     

Effect of growth hormone on estrogen receptor binding properties in the chick oviduct in vivo

The equilibrium dissociation constant (K_d) and the maximum binding capacity (B_max) of estrogen receptor in cytosolic and nuclear fractions in oviduct of pullets following various daily injections (1–10 times) of growth hormone (GH: 50 µg/chick) were examined by Scathchard analysis of specific [³H]estradiol‐17β (E₂) binding. The K_d values of receptor in both fractions decreased after three times of GH‐injection. In the B_max values, three times of the injection caused a marked decrease in that value in the cytosolic fraction with a concomitant increase in that in the nuclear fraction, whereas the total B_max (sum of B_max in the cytosolic and nuclear fractions) did not change. A similar relationship between the K_d values in the binding of the two fractions was also observed in 4–10 times of GH‐injection. However, in 4–10 times of GH‐injection the B_max of the both fractions and total B_max was greater than that in vehicle‐injection (control). When the chicks were injected with 6–10 times of GH‐injection, the weight of oviduct was increased. No change in the plasma concentrations of E₂ was found following GH‐ and vehicle‐injections into the chicks. The results suggest that growth hormone stimulates the growth of the chick oviduct by increasing the binding affinity and capacity of estrogen receptor.     

Effect of feeding selected short-chain fatty acids on the in vivo attachment of Salmonella typhimurium in chick ceca.

Two groups of 20 chicks each were fed 1% fatty acid continuously starting at 1 day of age, while a control group of 20 chicks received unsupplemented feed. At 2 days of age, chicks were inoculated orally with 1 ml of Salmonella typhimurium (1 x 10(6) colony-forming units/ml). Ceca were obtained from six chicks of each group at 7, 14, and 21 days of age. At 14 days of age, formic and propionic acids had statistically reduced Salmonella recovery by 2.56 logs and 3.09 logs, respectively, compared with controls. At 21 days of age, both test groups showed significant reductions of approximately 3.6 logs compared with controls. There were no statistical differences in body weights among the groups at 21 days of age.     

Interaction of heparin with canine coagulation proteins: in vivo and in vitro studies.

The effects of heparin on the coagulation profile and on specific factor activity in canine plasma have been examined both in vivo and in vitro. The results show that the prolongation of the partial thromboplastin time of plasma produced by heparin is, at least in part, the result of the interaction of heparin with the intrinsic Factors VIII, IX and XI and the inhibition of their procoagulant activity by heparin. A significant correlation was found between the partial thromboplastin time assay and the circulating heparin activity following intravenous administration of heparin to dogs. The results confirm the suitability of the partial thromboplastin time assay for monitoring heparin therapy in the dog.     

Endocytosis of erythrocytes in vivo and particulate substances in vitro by feline neoplastic mast cells.

Clinical evidence for the phagocytic capability of neoplastic feline mast cells was provided by recognition of endocytosed erythrocytes in seven of 12 cytological smears of mast cell neoplasms, particularly in those cells collected from splenic tumors. The capability of these neoplastic mast cells to endocytose particulate substances was also studied in vitro. Evidence is presented that under cultural conditions, feline neoplastic mast cells are capable of endocytosing a variety of substances including polystyrene latex microspheres, zymosan particles, horse spleen ferritin, salmon sperm nuclei, horseradish peroxidase, and carbon particles.     

In vitro and ex vivo pharmacodynamics of selected non-steroidal anti-inflammatory drugs in equine whole blood

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenases (COX), and the inhibition of COX-2 rather than COX-1 can limit the onset of NSAID-related adverse effects. The pharmacodynamic properties of eltenac, naproxen, tepoxalin, SC-560 and NS 398 in healthy horses were investigated using an in vitro whole blood assay. To predict COX selectivity in clinical use, eltenac and naproxen were also studied ex vivo after intravenous administration. SC-560 acted as a selective COX-1 inhibitor, tepoxalin as a dual inhibitor with potent activity against COX-1, and NS 398 as a preferential COX-2 inhibitor. Eltenac was a preferential COX-2 inhibitor in vitro but un-selective in the ex vivo study. Naproxen maintained its non-selectivity both in vitro and ex vivo. These findings have demonstrated that in vitro studies may not accurately predict in vivo NSAID selectivity for COX and should be confirmed using an ex vivo whole blood assay.     

In vitro and in vivo bioactivity of recombinant canine hepatocyte growth factor

Hepatocyte growth factor (HGF) is crucial for the development and regeneration of the liver and offers a possible new therapeutic strategy for the treatment of canine liver disease. In this study, the in vitro and in vivo bioactivity of recombinant canine HGF (rcHGF) produced with a baculoviral expression system in insect cells was measured. In vitro rcHGF induced mitogenesis, motogenesis, and phosphorylated the HGF receptor c-MET and its downstream mediators PKB and ERK1/2 in two canine epithelial cell lines. After a partial hepatectomy (phx) in dogs, rcHGF increased phosphorylation of c-MET, PKB and ERK1/2. A moderate increase was seen with the cell cycle protein PCNA in rcHGF treated livers, but no HGF-induced increase in liver weight was seen 7 days after phx. Furthermore, rcHGF treated livers showed lower levels of the key mediator of apoptosis, caspase-3, at 7days after phx. It is concluded that rcHGF is a biologically active protein in vitro and in vivo and the baculoviral expression system supplies sufficient amounts of rcHGF for future clinical studies in dogs.     

Further studies on in vitro and in vivo assays of hemorrhagic enteritis virus (HEV)

Isolation of hemorrhagic enteritis virus (HEV) from spleens of infected turkeys in the MDTC-RP19 lymphoblastoid cell line was compared with detection of HEV antigen in the same spleens using the agar gel precipitation (AGP) test. A concordance of 80% was found between the two assays. Virus isolation had a sensitivity of 84% and a specificity of 88% compared with the AGP test. RP19 cells were also susceptible to infection with several other avian adenoviruses, but such infection was easily differentiated from that of HEV by a fluorescent-antibody (FA) test. Turkeys required 10(2) tissue-culture-infectious doses (TCID) to develop HE-specific lesions and 10(5) TCID to be killed. On the other hand, as little as 10 TCID of apathogenic HEV protected the poults against challenge with virulent HEV. The enzyme-linked immunosorbent assay (ELISA) for detection of HEV antibody was improved by using virus-infected RP19 cells as antigen. The ELISA appears to be more sensitive than the serum-neutralization test.     

Comparative study on the in vitro and in vivo properties of two bovine herpesvirus-5 reference strains

Background

Bovine herpesvirus 5 (BoHV-5) is an alphaherpesvirus responsible for meningoencephalitis in young cattle and it is antigenically and genetically related to bovine herpesvirus 1. BoHV-5 outbreaks are sporadic and restricted in their geographical distribution, being mostly detected in the Southern hemisphere. The N569 and A663 strains are prototypes of the "a" and "b" subtypes of BoHV-5, however, scarce information about their in vitro and in vivo properties is currently available.

Methods

For the in vitro comparison between BoHV-5 A663 and N569 strains, viral growth kinetics, lysis and infection plaque size assays were performed. Additionally, an experimental infection of cattle with BoHV-5 A663 and N569 strains was carried out. Viral excretion, development of neurological signs, presence of specific antibodies in serum and nasal swabs and presence of latent BoHV-5 DNA in trigeminal ganglion, were analyzed. Histopathological examination of samples belonging to inoculated animals was also performed.

Results

The lytic capacity and the cell-to-cell spread was lower for the A663 strain compared to the N569 strain, however, the production of total infectious viral particles was similar between both strains. Concerning the in vivo properties, the A663 and N569 strains are able to induce similar degrees of pathogenicity in cattle.

Conclusions

Our results show that the A663 strain used in this study is less adapted to in vitro replication in MDBK cells than the N569 strain and, although slight differences were observed, both strains are able to induce a similar degree of virulence in the natural host.     

Comparative behaviour studies in horses infested with flying insects treated with insecticide or repellent substances

The aim of this study was to estimate the effects of flying insects (Order Diptera) on the behaviour of grazing horses in relation to the use of insecticide and repellent substances. The investigations were done between June and August in 2008 in 3 periods of 7 days each. As insecticide and repellent two substances were used: "Well-care emulsion" (Co. Essex Tierarznei, München, GER) containing Permethrin and "Bremsen-Frei-Plus" (Co. Dr. Schaette AG, Bad Waldsee, GER) based on etheric oils. Both groups were compared with a non treated control group in a crossover-design. Each group (n = 3-5) was used alternately as control and treatment group. Several climate parameters were taken during the study. Furthermore, the number of insects per animal was estimated at certain times. Once per observation period insects were caught using Malaise traps and differentiated by species. The proportion of horse relevant species of the genera Diptera, especially Culex pipiens and Musca autumnalis, caught was 9% on an average. There was no correlation between the number of Tabanidae caught in the Malaise traps and the number observed near by the horses. Behaviour parameters like tailswishing, headshaking, stamping, skintwitching, snapping at the body, and moving were observed more frequently with increasing insect infestation. When horses were infested with a high number of flying insects, feeding activity was significant lower, whereas locomotion activity was significant higher. Both substances had positive effects for about 50 hours after application with no apparent difference between the substances. However, a lower frequency of headshaking and tailswishing could be observed in the Permethrin treated horses.