Porcine hepatocyte–Kupffer cell co‐culture as an in vitro model for testing the efficacy of anti‐inflammatory substances

As Kupffer cells are highly involved in the regulation of hepatic inflammatory response, the main goal of this study was to improve and to characterize a hepatocyte–Kupffer cell co‐culture of pig origin for modelling endotoxin‐induced hepatic inflammation and for testing the efficacy of potential anti‐inflammatory substances. This monolayer co‐culture was prepared from primary isolated swine hepatocytes and Kupffer cells in the ratio of 6:1 and 2:1, mimicking different states of liver inflammation. The prepared cell cultures were characterized by immunohistochemical CD‐68 detection. Lipopolysaccharide (LPS) challenge of both co‐cultures resulted in elevated interleukin‐8 (IL‐8) and that of 6:1 co‐cultures in increased IL‐6 production with a higher extent than on hepatocyte monocultures, justifying the key role of Kupffer cells in pro‐inflammatory cytokine production. LPS‐induced IL‐8 production was successfully attenuated by concomitant application of both sodium butyrate and terpinen‐4‐ol on hepatocyte monocultures, but not on co‐cultures, demonstrating the importance of the presence of Kupffer cells in cell cultures as inflammatory models. Based on these initial data, the applied porcine primary hepatocyte–Kupffer cell co‐culture is suggested to be a proper tool for in vitro investigations on liver physiology and hepatic inflammation in pigs and can be used as a useful model mimicking in vivo conditions in veterinary research.     

Failure of viral protein 3 of infectious bursal disease virus produced in prokaryotic and eukaryotic expression systems to protect chickens against the disease

In recent years, infectious bursal disease virus (IBDV) has become a serious economic problem as a result of the emergence of new and very virulent strains. Most of the antibodies produced against IBDV are for the structural proteins viral protein (VP) 2 (VP2) and VP3. The purpose of this study was to test the potential of recombinant VP3 to induce protective antibodies. The gene for VP3 was isolated from a virulent strain of the virus and cloned into prokaryotic (Escherichia coli) and eukaryotic (baculovirus) expression systems. The protein expressed by both systems was of the expected size (32 kD) and was detected by anti-IBDV antibodies. Following partial purification, the polypeptides were injected into intact birds and induced the production of high levels of anti-IBDV antibodies, as detected by immunoblot and enzyme-linked immunosorbent assay tests. These antibodies did not prevent changes in the bursa and mortality when birds were challenged with a virulent IBDV strain after vaccination with the recombinant VP3. The results show that VP3 polypeptide cannot be used as a subunit vaccine against IBDV and raise questions concerning the nature of the neutralizing epitope on this structural protein.     

Assessment of diclofenac LC50 reference values in juvenile and embryonic stages of the zebrafish (Danio rerio)

The aim of the study was to compare the acute toxicity of diclofenac to juvenile and embryonic stages of the zebrafish (Danio rerio). Acute toxicity tests were performed on the aquarium fish Danio rerio, which is one of the model organisms most commonly used in toxicity testing. The tests were performed using a semi-static method according to OECD guideline No. 203 (Fish, acute toxicity test). Embryo toxicity tests were performed in zebrafish embryos (Danio rerio) in compliance with OECD No. 212 methodology (Fish, short-term toxicity test on embryo and sac-fry stages). The results were subjected to a probit analysis using the EKO-TOX 5.2 programme to determine 96hLC50 and 144hLC50 (median lethal concentration, 50% mortality after a 96 h or 144 h interval, respectively) values of diclofenac. The statistical significance of the difference between LC50 values in juvenile and embryonic stages of Danio rerio was tested using the Mann-Whitney non-parametric test implemented in the Unistat 5.1 programme. The LC50 mean value of diclofenac was 166.6 +/- 9.8 mg/L in juvenile Danio rerio, and 6.11 +/- 2.48 mg/L in embryonic stages of Danio rerio. The study demonstrated a statistically higher sensitivity to diclofenac (P < 0.05) in embryonic stages compared to the juvenile fish.     

Real-time PCR detection of Mycoplasma felis in domestic cats suffering from chronic conjunctivitis (Poland)

Real-time PCR directed to intergenic spacer (IGS) noncoding region between 16S and 23S rRNA genes was used for species specific detection of Mycoplasma felis in conjunctival scrapings. Samples were collected from 57 cats suffering from chronic conjunctivitis in 2008-2010 (Wroc?aw, Poland). Samples from 36 cats (63.2%) were shown to be positive for Mycoplasma felis. Our research gives a first insight in the occurrence of Mycoplasma felis among domestic cats in Poland suggesting that this pathogen may constitute an underestimated cause of chronic conjunctivitis.     

Association of polymorphisms for nuclear receptor coactivator 1 gene with egg production traits in the maternal line of Shaobo hens

1. The objectives of the study were to find polymorphic sites and elucidate the association between SNPs in the nuclear receptor coactivator 1 (NCOA1) gene and reproductive traits. 2. SNPs were detected by PCR-SSCP and DNA sequencing. Four SNPs were detected, including T10155007A, T10125838C, G10118492A and G10109315T. Three polymorphisms were associated with total egg production at the age of 300 d and the G10109315T polymorphism was associated with age at first egg. 3. In conclusion, the NCOA1 gene can be used as a molecular marker for reproductive traits in hens.     

Substitution of wheat dried distillers grains with solubles for barley grain or barley silage in feedlot cattle diets: intake, digestibility, and ruminal fermentation

The objective of this study was to evaluate the effects of substituting wheat dried distillers grains with solubles (DDGS) for barley grain and barley silage on intake, digestibility, and ruminal fermentation in feedlot beef cattle. Eight ruminally cannulated Angus heifers (initial BW 455 ± 10.8 kg) were assigned to a replicated 4 × 4 Latin square design with 4 treatments: control, low (25%), medium (30%), and high (35%) wheat DDGS (DM basis). The diets consisted of barley silage, barley concentrate, and wheat DDGS in ratios of 15:85:0 (CON), 10:65:25 (25DDGS), 5:65:30 (30DDGS), and 0:65:35 (35DDGS; DM basis), respectively. The diets were formulated such that wheat DDGS was substituted for both barley grain and barley silage to evaluate whether wheat DDGS can be fed as a source of both energy (grain) and fiber in feedlot finishing diets. Intakes (kg/d) of DM and OM were not different, whereas those of CP, NDF, ADF, and ether extract (EE) were greater (P < 0.01) and intake of starch was less (P < 0.01) for the 25DDGS compared with the CON diet. The digestibilities of CP, NDF, ADF, and EE in the total digestive tract were greater (P < 0.05) for 25DDGS vs. CON. Ruminal pH and total VFA concentrations were not different (P > 0.15) between 25DDGS and CON diets. Replacing barley silage with increasing amounts of wheat DDGS (i.e., from 25DDGS to 35DDGS) linearly reduced (P < 0.05) intakes of DM and other nutrients without altering (P=0.40) CP intake. In contrast, digestibilities of DM and other nutrients in the total digestive tract linearly increased (P < 0.05) with increasing wheat DDGS except for that of EE. Additionally, with increasing amounts of wheat DDGS, mean ruminal pH tended (P=0.10) to linearly decrease, and ruminal pH status decreased with longer (P=0.04) duration of pH <5.5 and <5.2, and greater (P=0.01) curve area under pH <5.8 and <5.5 without altering (P > 0.19) ruminal VFA and NH(3)-N concentrations. Results indicated that wheat DDGS can be effectively used to replace both barley grain and silage at a moderate amount to meet energy and fiber requirements of finishing cattle. However, when silage content of the diet is low (<10%), wheat DDGS is not an effective fiber source, so replacing forage fiber with wheat DDGS in finishing diets decreases overall ruminal pH status even though the rapidly fermentable starch content of the diet is considerably reduced.     

Effect of experimental endotoxemia on thrombelastography parameters, secondary and tertiary hemostasis in dogs

Background: Thrombelastography (TEG) and indicators of secondary and tertiary hemostasis might be altered in dogs with endotoxemia. Hypothesis: Endotoxemia influences measures of coagulation in dogs. Animals: Ten healthy cross‐bred dogs. Material and Methods: Prospective laboratory study between controls (n = 5) receiving 0.9% saline IV and the study group (n = 5) treated with low‐dose lipopolysaccharide (0.02 mg/kg IV). Physical examination and sampling for measurement of leukocytes, platelets, and coagulation variables were performed at time points 0, 1, 4, and 24 hours. Coagulation variables included kaolin‐activated TEG, 1‐stage prothrombin time (OSPT), activated partial thromboplastin time (aPTT), fibrinogen, factor VIII, antithrombin, protein C, protein S, activated protein C (APC)‐ratio calculated from aPTT with and without presence of APC), and D‐Dimers. Results: Endotoxemia‐induced clinical signs included lethargy (n = 5/5), diarrhea (n = 4/5), emesis (n = 4/5), and abdominal pain (2/5). After 1 hour there was severe leukopenia (2.5 ± 0.7 × 10⁹/L; mean ± SD, P < .0001) and a 2.2‐fold increase in D‐Dimers (0.81 ± 0.64 mg/L, P < .0001). After 4 hours there was hyperthermia (40.3 ± 0.4°C, P < .0001) and increases in OSPT (10.5 ± 2.7 seconds, P < .0001), aPTT (16.7±5.2 seconds, P= 0.002). A significant decrease in fibrinogen (1.5±1.0 g/L, P= 0.001), protein C (31 ± 33%, P <.0001), protein S (63 ± 47%, P < .0001), TEG α (58 ± 19, P= .007), and TEG maximal amplitude (50 ± 19 mm, P= .003) was seen compared with the controls. APC‐ratio rose significantly (2.5 ± 0.2, P < .0001) without exceeding the reference interval (n = 4/5). Conclusion and Clinical Importance: D‐Dimers are the earliest indicator for endotoxemia‐associated coagulation abnormalities followed by decreased protein C concentration. APC‐ratio and TEG were not good screening variables.     

Interindividual variability in plasma concentrations after systemic exposure of swine to dietary doxycycline supplied with and without paracetamol: a population pharmacokinetic approach

Anorexigenic substances released during infection may hinder the therapeutic efficacy of in-feed antibiotics. Paracetamol (acetaminophen; PARA) inhibits the anorexia of infection and seems to improve the clinical efficacy of doxycycline (DOX) against bacterial respiratory disease in swine herds. In order to verify whether PARA selectively stimulates intake of DOX-medicated feed in diseased pigs, we documented the pharmacokinetics (PK) of DOX when coadministered with PARA and examined the effect of in-feed PARA on the interindividual variability in plasma concentrations after systemic exposure to in-feed DOX in swine herds with respiratory disease. Systemic exposure to DOX was measured with the area under the curve (AUC) of its plasma concentrations over time. First, a rich-sampling PK study of in-feed and i.v. DOX (10 mg/kg of BW) and PARA (30 and 10 mg/kg of BW, respectively) was performed on 5 pigs. The PK profiles of in-feed DOX were used in mathematical simulations to determine 5 optimal sampling times for the farm-based population PK study. A randomized, blind, parallel PK study was performed in 2 herds with bacterial respiratory disease, where liquid feed was fortified with DOX alone (5 mg x kg of BW(-1) x meal(-1)) or combined with PARA (15 mg x kg of BW(-1) x meal(-1)). Medicated meals were given twice, 12 h apart, to group-housed growing pigs (n > 50 pigs x treatment(-1) x herd(-1), totaling 215 pigs). Plasma concentrations of DOX and PARA were measured with HPLC. At variance with our expectations, PARA decreased (P = 0.069) mean AUC of in-feed DOX and did not decrease its variability (P > 0.34). Mean AUC of DOX increased with feed intake and with initial exposure to DOX, and was greater in sick animals. Therefore, symptomatic PARA-induced improvement in bacterial respiratory disease control with DOX is more likely caused by its analgesic/antipyretic effects than by its orexigenic effect. Interindividual variation in the AUC of DOX was large in pigs given group medication, even when sufficient feeding space was allowed and the amount of feed offered was greater than their requirements. Therefore, future studies to improve the efficacy of group antibiotic therapy should focus on feeding behavior characteristics as well as biopharmaceutical properties of medicated feeds.     

Dosage assessment of valnemulin in pigs based on population pharmacokinetic and Monte Carlo simulation

To estimate the valnemulin pharmacokinetic profile in a swine population and to assess a dosage regimen for increasing the likelihood of optimization. This study was, respectively, performed in 22 sows culled by p.o. administration and in 80 growing‐finishing pigs by i.v. administration at a single dose of 10 mg/kg to develop a population pharmacokinetic model and Monte Carlo simulation. The relationships among the plasma concentration, dose, and time of valnemulin in pigs were illustrated as C_i,v = X₀(8.4191 × 10^‐4 × e^?0.2371t + 1.2788 × 10^?5 × e^?0.0069t) after i.v. and C_p.o = X₀(?8.4964 × 10^?4 × e^?0.5840t + 8.4195 × e^?0.2371t + 7.6869 × 10^?6 × e^?0.0069t) after p.o. Monte Carlo simulation showed that T_>MIC was more than 24 h when a single daily dosage at 13.5 mg/kg BW in pigs was administrated by p.o., and MIC was 0.031 mg/L. It was concluded that the current dosage regimen at 10–12 mg/kg BW led to valnemulin underexposure if the MIC was more than 0.031 mg/L and could increase the risk of treatment failure and/or drug resistance.