In vitro generation of mature neutrophils from canine Lin- bone marrow cells

The major goal of this work was to describe the in vitro generation of mature functional neutrophils derived from a canine enriched haematopoietic progenitor cell population. We have utilised lineage depletion by immunomagnetic selection to isolate a canine haematopoietic progenitor cell population. The physical, immunological, metabolical and morphological methodologies employed in this study have permitted us to isolate and define a cell population enriched in Rh-123low and CD34+ cells. Irradiated pre-established long-term bone marrow cultures (LTBMC) were utilised to determine the self-renewal ability of lineage negative (Lin-) cells, as well as their capacity to differentiate into mature functional neutrophils. The authors demonstrate for the first time that canine neutrophils derived from Lin- cells are able to produce oxyradicals, express a specific neutrophil surface antigen, and contain gelatinase granules. These characteristics enable them to migrate through basement membranes to act as a first line defence mechanism. The fact that these cells are able to differentiate into functional mature cells, and give rise to long-term culture-initiating cells (LTC-IC) after 35 days of culture, allows the authors to assure that the isolated canine enriched haematopoietic cell population exhibit functional characteristics, associated with primitive haematopoietic cells.     

Use of ear tags and injectable transponders for the identification and traceability of pigs from birth to the end of the slaughter line

A total of 557 newborn piglets were used to compare eight identification devices, including one plastic ear tag as a control (C, n = 348) and two types of electronic ear tags (E1, n = 106; and E2, n = 103), and five types of injectable transponders (n = 557): small 12-mm (D12, n = 116; and S12, n = 110), medium 23-mm (T23, n = 108), and large (32-mm, T32, n = 115; and 34-mm, S34, n = 108). Injections were made s.c. in the auricle base (n = 248) and intraperitoneally (n = 309) using a new technique. All piglets were identified with two devices, but using electronic ear tags in conjunction with injection in the auricle was avoided on the same pig. Readability of devices was checked during fattening (until 110 kg BW) and slaughtering. On-farm losses were lower for control than for electronic ear tags (C = 1.1%; E1 = 8.8%; and E2 = 44.9%; P < 0.01); the latter also suffered electronic failures (E1 = 5.5%; and E2 = 55.1%; P < 0.001). On-farm losses of transponders injected in the auricle base were greater in large (S34 = 72.5%; and T32 = 46.3%; P < 0.05) than in small transponders (S12 = 19.4%; and D12 = 17.1%), but T23 (29.8%) only differed from S34. Transponder size did not affect on-farm losses for intraperitoneal injections in which only one loss was recorded (0.4%). All ear tags had similar losses during transportation to the slaughterhouse (1.2%), but no losses were observed in injectables. Slaughtering losses did not differ between ear tags (C = 11.2%; and E1 = 6.4%), but apart from losses, 12.8% of E1 failed electronically. Injection site affected losses and breakages during slaughtering (auricle base = 6.4%; and intraperitoneal = 0%), but recovery time did not significantly differ (auricle base = 28.6 s; and intraperitoneal = 18.9 s). Transponders in the auricle base were recovered by sight (30.2%), palpation (27.4%), or by cutting (42.5%). Intraperitoneal transponders were mainly recovered loose in the abdominal cavity (81.4%), whereas 18.6% fell on the floor. As a result, traceability varied significantly (P < 0.05) between control (86.7%) and electronic ear tags (0 to 68.1%) and injectable transponders, with the auricle base (17.8 to 75.0%) having lower values than intraperitoneal (98 to 100%). Intraperitoneal injection was a very effective tool for piglet identification and traceability, ensuring the transfer of information from farm to slaughterhouse. To warrant the use of this technique in practice, transponder recovery requires further investigation.     

Granulocyte and granulocyte macrophage colony-stimulating factors as therapy in human and veterinary medicine

Granulocyte colony-stimulating factors (G-CSFs) and granulocyte macrophage colony-stimulating factors (GM-CSFs) are endogenous cytokines that regulate granulocyte colonies and play a major role in the stimulation of granulopoiesis (neutrophils, basophils and eosinophils) and in the regulation of microbicidal functions. There are numerous pathological conditions in which neutrophils are decreased, the most common being neutropenia associated with cancer chemotherapy, which increases the risk of serious microbial infections developing with the potential for high morbidity and mortality. New methods in molecular biology have led to the identification and cloning of CSF genes and biopharmaceutical production. Since then, CSFs have been widely used for the prevention and treatment of neutropenia associated with cancer chemotherapy, for mobilising haematopoietic cell precursors, and for other neutropenia-related pathologies. This review focuses on the use of CSFs within both human and veterinary medicine. Clinical applications, pharmacology, tolerability and the potential role of these factors in veterinary medicine are considered.     

Analytical and Clinical Validation of a Time-resolved Immunofluorometric Assay (TR-IFMA) for Canine C-reactive Protein in Serum

A time-resolved immunofluorometric assay (TR-IFMA) was developed for the determination of C-reactive protein (CRP) in canine serum. CRP was isolated from canine acute-phase serum by affinity chromatography on agarose coupled with phosphorylethanolamine. This isolated dog CRP was used as standard to calibrate the assay. Intra-assay and inter-assay coefficients of variation were in the ranges 5.3–7.1% and 4.8–13.3%, respectively. Accuracy, evaluated by adding 2 and 10 μg/ml of CRP to serum samples, provided recoveries of 99.9% and 106.8%. High correlation was found between CRP measurements by TR-IFMA and a by commercial enzyme-linked immunosorbent assay (R² = 0.98). The limit of detection for the TR-IFMA method was 0.000067 μg/ml and the measurement of CRP in serial dilutions of acute-phase dog sera generated curves with the same slope as the one constructed with purified CRP. The TR-IFMA provides a precise, accurate and highly sensitive assay for CRP determination in dog samples. CRP levels in dogs with different diseases ranged between 10.2 and 210.7 μg/ml and were significantly higher than those observed in healthy dogs (< 7.1 μmg/ml).     

Metabolic Differences Between Draft-cross and Mustang Horses Detected by Metabonomic Analyses

Nuclear magnetic resonance (NMR) spectroscopy-based metabonomics is a powerful multivariate tool that can be used to characterize the unique metabolic profiles of living creatures. To test the hypothesis that NMR-based metabonomic analyses of serum would reveal metabolic differences between two types of horses, NMR spectra of serum samples drawn on 3 separate days from 2-year-old Mustangs (MU, n = 4) and 2 year-old Draft-cross (DC, n = 4) horses were compared. Metabonomic multivariate statistical analysis of the NMR spectra revealed clear and distinct clustering of each group. The metabolic separation between the Mustangs and Draft-crosses was due mainly to differences in urea, certain amino acids, acetate, lipoproteins, and glucose that probably reflected differences in growth rates and efficiency of protein utilization. NMR-based metabonomic analyses may be useful in detection and evaluation of metabolic differences between different types and physiologic states of horses.     

Effects of Garlic and Ginger Oils on Hematological and Biochemical Variables of Sea Bass Dicentrarchus labrax

Abstract

This study was conducted to investigate the effects of garlic and ginger oils on hematological and biochemical health characteristics of sea bass Dicentrarchus labrax. Fish were exposed to garlic oil (0.01 or 0.02 mL/L), ginger oil (0.01 or 0.02 mL/L), or a combination of the two oils (each oil at a concentration of 0.005 or 0.01 mL/L) for 96 h via bath immersion. Results showed that the red blood cell count, hematocrit (%), hemoglobin (Hb) concentration (g/dL), mean corpuscular volume (μm³), mean corpuscular Hb (pg), and mean corpuscular Hb concentration (%) were not significantly affected by herb oil exposure. However, some changes in biochemical variables were observed. Sea bass exposed to the 0.005-mL/L garlic oil–ginger oil mixture exhibited a significant increase in serum glucose. Serum total protein and albumin levels decreased in sea bass that were exposed to a garlic oil–ginger oil mixture (0.005 or 0.01 mL/L) or to garlic oil at 0.02 mL/L. Serum globulin levels decreased and triglyceride levels increased in sea bass exposed to 0.02-mL/L garlic oil or to the 0.01-mL/L mixture. The serum lipase level decreased and the cholesterol level increased in fish that were exposed to 0.02-mL/L garlic oil. In summary, ginger oil at 0.01–0.02 mL/L can be used without negative effects, while the garlic oil or garlic oil–ginger oil mixture should be applied at a concentration below 0.005 mL/L for bath immersion of sea bass. This is the first study to examine how garlic oil and ginger oil exposure via bath immersion affects the hematological and biochemical status of sea bass.

Received March 19, 2012; accepted July 2, 2012     

In vivo and ex vivo assessment of the interaction between ivermectin and danofloxacin in sheep

The impact of an efflux pump-related interaction between ivermectin and danofloxacin on their intestinal transport (ex vivo) and disposition kinetics (in vivo) was assessed. Eighteen male Corriedale sheep were randomly assigned to one of three groups. Animals in Group A received 0.2mg/kg ivermectin by SC injection, those in Group B were given 6 mg/kg danofloxacin SC on two occasions 48 h apart and those in Group C were treated with both compounds at the same rates. Plasma concentrations of ivermectin and danofloxacin were measured by HPLC using fluorescence detection. Ex vivo intestinal drug transport activity was measured by the use of the Ussing chamber technique. Plasma concentrations of ivermectin in the first 6 days after injection tended to be higher in Group C than Group A. Contemporaneous treatment with ivermectin significantly increased systemic exposure to danofloxacin (AUC values were 32-35% higher) and prolonged the elimination half-life of danofloxacin (40-52% longer). Ex vivo, incubation with ivermectin significantly decreased the efflux transport of rhodamine 123, a P-glycoprotein substrate, in sheep intestine, but no significant effect of danofloxacin on transport activity was observed. Evaluation of the interaction of danofloxacin with the breast cancer resistance protein (BCRP) showed that pantoprazole and ivermectin significantly decreased danofloxacin secretion in the rat intestine. Thus, the ivermectin-induced reduction of danofloxacin efflux transport observed in this study may involve BCRP activity but the involvement of P-glycoprotein cannot be ruled out.     

Subolesin/akirin orthologs from Ornithodoros spp. soft ticks: cloning, RNAi gene silencing and protective effect of the recombinant proteins

Subolesin/akirin is a well characterized protective antigen highly conserved across vector species and thus potentially useful for the development of a broad-spectrum vaccine for the control of arthropod infestations including hard ticks, mosquitoes, sand flies and the poultry red mite Dermanyssus gallinae. Soft ticks could be also targeted by this vaccine if proved that the soft tick subolesin orthologs are conserved and induce protective immune responses too. However, to date no soft tick subolesin orthologs have been fully characterized nor tested as recombinant antigens in vaccination trials. The objectives of the present work were to clone and characterize the subolesin orthologs from two important vector species of soft ticks as Ornithodoros erraticus and O. moubata, to evaluate the effect of subolesin gene silencing by RNAi, and to test the protective value of the recombinant antigens in vaccination trials. The obtained results demonstrate that both soft tick subolesins are highly conserved showing more than 69% and 74% identity with those of hard ticks in their nucleotide and amino acid sequences, respectively. Additionally, we demonstrate that both soft ticks possess fully operative RNAi machinery, and that subolesin gene silencing by dsRNA injection inhibits oviposition indicating the involvement of subolesin in tick reproduction. Finally, vaccination with the recombinant soft tick subolesins induced a partial protective effect resulting in the reduction of the oviposition rate. These preliminary results encourage further studies on the use of recombinant subolesins as vaccines for the control of soft tick infestations, either alone or in combination with other specific molecules.     

In vivo anthelmintic activity of Phytolacca icosandra against Haemonchus contortus in goats

Two separate controlled and blinded studies were conducted to confirm the dose and non-interference of spinosad and milbemycin oxime (MO) administered orally in combination or alone to dogs for the treatment and control of experimentally induced flea infestations (Ctenocephalides felis) and adult hookworm infections (Ancylostoma caninum). For each study, dogs were allocated randomly based on pre-treatment adult flea and hookworm egg counts to one of four treatment groups of 10 animals each. In each study, spinosad and MO in combination, using the lower half (30-45 mg/kg spinosad; 0.5-0.75 mg/kg MO) of the US commercial dose band (30-60 mg/kg spinosad; 0.5-1.0mg/kg MO) of each active ingredient, or individually alone using the full dose range, were given orally to dogs on Day 0 using a tablet formulation. A placebo control was treated similarly. In one study, on Days -1, 5, 12, 19, 28 and 35 each dog was infested with approximately 100 unfed adult C. felis obtained from the investigator's established flea colony. All dogs were infested via the same method. Forty-eight hour post-infestation flea comb counts were conducted on Days 1, 7, 14, 21, 30 and 37 and were used to determine the knockdown and residual flea activity. In the second study, on Day -27 each of 48 dogs were experimentally inoculated with 100 third-stage infective larvae of the hookworm, A. caninum. Dogs were treated on Day 0 and necropsied on Day 7 or Day 8. All nematodes in the intestinal tract were collected on Day 7 or Day 8, identified and counted by species and stage. Post-treatment, the geometric mean live flea counts were significantly different (p-value<0.0001) between the spinosad/MO combination and the spinosad only treatment groups as compared to the vehicle control group. The flea counts in the MO only group and the control group were not statistically different. The spinosad and MO combination group and the spinosad only treatment group demonstrated significantly different knockdown (100%) and post-treatment residual flea efficacy at Day 30 was 100% for both groups as compared to the vehicle control. The presence of MO in combination with spinosad did not interfere with the flea efficacy of spinosad as compared to the spinosad only group. MO alone did not demonstrate any flea efficacy. Post-treatment, the geometric mean A. caninum worm counts were significantly different (p-value<0.0001) between the spinosad and MO combination group as compared to the vehicle control group. The worm counts in the MO only group and the combination group were not statistically different. The spinosad and MO combination group (99.8% reduction) and the MO only treatment group (99.5% reduction) both demonstrated significantly different hookworm efficacy as compared to the vehicle control group. The presence of spinosad in combination with MO did not interfere with the hookworm efficacy of MO as compared to the MO only group. Spinosad alone did not demonstrate any hookworm efficacy. In summary, flavored spinosad and MO combination tablets administered orally to dogs at the lower end (30-45 mg/kg spinosad; 0.5-0.75 mg/kg MO) of the US commercial tablet unit dose range (30-60 mg/kg spinosad; 0.5-1.0mg/kg MO) were both safe and highly efficacious delivering 100% knockdown and 30 days of residual adult flea control on experimentally infested dogs as well as >99% adult hookworm efficacy evaluated under laboratory conditions. Interference between either drugs was not demonstrated for both of these dose limiting parasites.     

Trichinella: differing effects of antigens from encapsulated and non-encapsulated species on in vitro nitric oxide production

Trichinellosis is a cosmopolitan zoonotic disease affecting a wide variety of animals, including man. Non-encapsulated and encapsulated species diverge with respect to their developmental strategies. Little is known at the molecular level about parasite-derived mediators responsible for host muscle cell transformation occurring during trichinellosis. In this context, host-parasite relationships in Trichinella-infected animals could be related to different host-immune and cell mediators, e.g. nitric oxide (NO). Here, we investigate the stimulatory/inhibitory role of L1 antigens from four encapsulated (T. spiralis, T. britovi, T. nelsoni and T. nativa) and one non-encapsulated (T. pseudospiralis) Trichinella species on NO production from rat macrophages in vitro. Our results demonstrate that encapsulated and non-encapsulated Trichinella species differ in their capacity to stimulate the secretion of NO from host macrophages. Biological significance of these differences should be further assessed in the available experimental models.