Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models

In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.     

Mycorrhizal Hyphal Length as a Function of Plant Community Richness and Composition in Restored Northern Tallgrass Prairies (USA)

We assessed the total length of external arbuscular mycorrhizal hyphae as a function of plant species and functional form richness in restored northern tallgrass prairies. Total hyphal length increased with species and functional form richness. Hyphal length also increased when plant communities were dominated by species with high root density, high root to shoot ratios, and high nitrogen use efficiency. Hyphal length was positively correlated with the biomass of late successional C₄ grasses (Andropogon gerardi Vitman, Panicum virgatum L., Schizachyrium scoparium [Michx.] Nash-Gould, and Sorghastrum nutans L.), which are obligately mycorrhizal and characterized by high root to shoot ratios, and high root surface area per unit of root biomass. We thus conclude that in order to recover extraradical arbuscular mycorrhizal hyphal length in restored northern tallgrass prairies, at least three factors need to be given priority: 1) achieving high levels of species and functional form richness; 2) making sure that late successional C₄ grasses are present; and 3) making sure that the seed mixture includes species that are characterized by high root to shoot ratio, high root density, and high nitrogen-use efficiency.     

Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test

An outbreak of human leishmaniosis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.     

The Neocortex of the Dog

This article describes the structural organization of the neocortex of the dog from purely morphological point of view. Series of 50 urn transverse sections were cut from the frozen right hemiencephala of adult greyhounds, stained by N issl's method, and drawn. His's method was then used to construct life-size lateral, dorsal and medial views of the hemiencephalon from 38 sections located at regular intervals. Once the cytoarchitectonic criteria to be used had been established on the basis of preliminary observations, all the sections of each hemiencephalon were examined microscopically and the cytoarchitectonic types observed in each mapped on the corresponding drawing. These maps were then projected onto lateral, dorsal and medial views of the whole hemiencephalon to produce a classical cytoarchitectonic map of the neocortex made up of 21 types, 7 of them agranular (with no inner granular layer), 2 dysgranular (with a granular layer with diffuse boundans) and the rest granular (with a well-defined layer IV). Various areas of cortex whose characteristics were intermediate between those of adjoinin areas were classified in a residual typus intermedius.     

Culture system and long-term storage of culture media in the in vitro production of bovine embryos

The optimum culture system for in vitro matured and fertilised oocytes still remains to be clarified. Culture media (CM) for mammalian embryos are routinely prepared fresh for use and preserved under refrigeration during one or two weeks. The purposes of this work were (1) to compare the efficiency of a synthetic oviduct fluid (SOF) with two different bovine serum albumin (BSA) concentrations (3 and 8 g/L) for the in vitro production of bovine blastocysts, (2) to test the effect of timing on adding fetal calf serum (FCS) to the SOF, and (3) to evaluate the effects on bovine embryo development of freezing and lyophilisation as procedures for preserving the SOF. Supplementation of SOF with 3 g/L BSA increased Day-7 blastocyst expansion rates (18.3 ± 1.6 vs. 14.4 ± 0.7; P < 0.05), although no differences in hatching rates were found. Addition of FCS to SOFaa (SOF with amino acids) medium supplemented with sodium citrate (SOFaaci) at 48 and at 72 h post-insemination (PI) allowed obtaining higher Day-6 embryo development rates than when FCS was added at 18 or 96 h PI (Day-6 morulae + blastocyst rate: 30.0 ± 1.1, 40.8 ± 1.1, 43.9 ± 2.3 and 39.3 ± 0.5 for FCS addition at 18, 48, 72 and 96 h, respectively). Hatching rates were significantly improved when serum was added at 72 h PI. Finally, both refrigeration and lyophilisation appeared as useful cryopreservation procedures for SOFaaci, although a significant loss of its ability to support embryo development, compared to the control fresh culture medium, was observed.     

Portal site metastasis of invasive mesothelioma after diagnostic thoracoscopy in a dog

CASE DESCRIPTION: A 10-year-old Golden Retriever evaluated because of recurrent pericardial and pleural effusion underwent thoracoscopy with biopsy of the pleura and mediastinum. CLINICAL FINDINGS: Before thoracoscopy, 5 L of serosanguinous fluid was removed from the pleural cavity via thoracocentesis. During thoracoscopic exploration, it was observed that the parietal pleura and mediastinum were covered by miliary white to tan nodules 1 to 3 mm in diameter. Biopsy specimens were obtained, and partial pericardiectomy was performed. Portal sites were closed routinely. Cytologic evaluation of the pleural fluid revealed high protein concentration and cellularity, with cellular changes consistent with an exfoliating carcinoma. Results of bacterial culture were negative. TREATMENT AND OUTCOME: Carboplatin was administered via intracavitary instillation, and prednisone was administered orally. Twenty-one days later, 1 firm, irregularly shaped 6.5 x 3-cm mass and 4 smaller masses were detected in the area of the left thoracic wall where the cannula had been inserted during thoracoscopy. Histologic analysis of tissue from the masses collected at necropsy confirmed that they were malignant tumors with similar appearance to the pleural mesothelioma and immunohistochemical staining properties identical to those of the primary tumor. CLINICAL RELEVANCE: Although thoracoscopy is associated with less postoperative pain, shorter hospitalization times, and faster patient recovery than sternotomy procedures, complications are also possible with minimally invasive endoscopic surgery. Portal site metastasis can develop from contamination of portal sites with cells on instruments or cannulas or via leakage of effusion fluid. Although rare, this potential complication should be discussed with owners prior to performing the procedure.     

Immunoproteomic analyses of outer membrane antigens of Actinobacillus pleuropneumoniae grown under iron-restricted conditions

Actinobacillus pleuropneumoniae, a bacterial pathogen of swine and agent of porcine pneumonia, causes a highly infectious disease of economic importance in the pig industry. Commercial vaccines for A. pleuropneumoniae include whole-cell bacterins and second generation subunit vaccines but they only confer partial protective immunity. Our search for new vaccine candidates identified antigens that are expressed during conditions that mimic infection; the outer membrane (OM) proteome of A. pleuropneumoniae serotype 5b was examined under iron restriction. Quantitative profiling by 2D-DiGE technology revealed that iron restriction induced expression of previously described transferrin binding proteins (TbpA, TbpB) plus four lipoproteins including spermidine/putrescine binding periplasmic protein 1 precursor (PotD2). Immunoproteomic analyses with antisera from na?ve animals and from infected pigs were able to differentiate antigens within the OM proteome that were specifically recognized during A. pleuropneumoniae infection. Immunoblots of iron-restricted profiles detected PotD2, heme-binding protein A (HbpA), and capsule polysaccharide export protein (CpxD) as well as surface antigens TbpA, TbpB, and OmlA. These data identify OM proteins that demonstrate immunogenicity and upregulation under conditions mimicking infection, providing emphasis on lipoproteins as an important class of antigens to exploit for vaccine development for A. pleuropneumoniae.     

PULSED WAVE-DOPPLER ULTRASONOGRAPHIC EVALUATION OF THE COMMON CAROTID ARTERY IN THE RESTING HORSE: PHYSIOLOGIC DATA

A pulsed wave-Doppler ultrasonographic evaluation of common carotid arterial blood flow was carried out on 63 healthy Italian Saddlebred horses. Vessel diameter and tracing morphology were evaluated were evaluated and blood flow parameters (systolic, diastolic and mean velocity, acceleration and deceleration of the systolic wave, cartid pulse volume) were calculated and correlated with class variables (sex, age and body weight). On the basis of the presence of an incisure in the ascending branch of the systolic curve, subjects were divided in two groups: one with a bifid systolic curve and the other with a monophasic aspect. Correlations between: 1) diameter of the vessel and body weight and 2) carotid pulse volume and flow velocity (systolic, diastolic and mean) were found. A greater systolic pulse volume was found in male subjects, in subjects with greater body weight and in those which had a monophasic systolic wave.