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.     

Type and frequency of contacts between Belgian pig herds

Knowledge of the frequency of direct and indirect contacts between pig herds is a requirement for understanding the potential between-herd transmission of pathogens. Our aim was to investigate the different contacts between Belgian pig herds. We obtained data by conducting a postal survey on 421 pig herds in August 2005 and by analysis of available information on livestock movements in the national identification-and-registration database (18-months period in 2004-2006). Direct contacts included transports of pigs by onto-farm, off-farm and between-farm movements. Indirect contacts included vehicles entering the herd and visitors entering the stables. The median number of direct contacts per herd made by onto-farm movements was 0.2/month (Q1: 0; Q3: 0.5). About 1.2% of herds had > or = 3 onto-farm movements/month. We used a zero-inflated negative-binomial regression model to describe differences in the number of onto-farm movements according to herd size and herd type. Piglet multipliers followed by finishing herds were predicted to have the most onto-farm movements. Farrow-to-finishing herds made less movements compared to breeding herds. A median of 3997 between-farm movements/month was made in Belgium; these mainly concerned piglets. The median number of origin herds during an 8-month period for between-farm movements was 4 (Q1: 2; Q3: 8). For a typical 1-month period, we constructed directed graphs of between-farm piglet and replacement stock movements, illustrating potential receivers and distributors of infection. Of these between-farm movements, many were made over a short distance (median straight-line distance 19 km (Q1: 8; Q3: 36)). The median number of vehicles entering a herd and visitors entering the stables was 8/month (Q1: 6; Q3: 13) and 3/month (Q1: 2; Q3: 6) respectively. The number of indirect contacts by vehicles and persons were associated with herd size (Spearman's r: 0.7 and 0.2), herd type and other factors. Skewness of both direct and indirect contacts, illustrated that there was a wide variety in contact structure between pig herds in Belgium. Infection control might benefit by accounting for this variation in contacts and by targeting 'high-risk' herds in case of animal-disease emergencies.     

Effect of Progesterone and Ionomycin on Domestic Cat Sperm Motility Patterns and Acrosome Reaction

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P₄). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25°C, were incubated for 30 min at 38°C in 5% CO₂ and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P₄ was added (10 μg/ml) to the P1 group. Groups P2 and P3 were supplemented with P₄ (10 and 20 μg/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 μ m , respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P₄ treatments led to a hyperactivation-like sperm motion and an increase (p < 0.05) in the percentage of AR. Although a higher (p < 0.05) percentage of AR was obtained in group I2 when compared with all P₄ groups, a decrease (p < 0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 μ m seems to be more suitable to trigger AR in domestic cat sperm.     

Beacon-like immunoreactivity in the hypothalamus of domestic chick

Beacon-immunoreactive (B-ir) fibres and neurons in the hypothalamus of the domestic chick (Gallus domesticus) were studied using an immunohistochemical technique in order to verify the presence and elucidate the pattern of distribution of this novel peptide in an avian brain. B-ir neurons were seen in the n. supraopticus, pars ventralis and pars externus; n. magnocellularis preopticus, pars dorsalis, medialis and ventralis; n. preopticus periventricularis; n. suprachiasmaticus, pars medialis; n. ventrolateralis thalami. Only few B-ir cells were scattered in the most anterior part of the lateral hypothalamic area. B-ir fibres, appearing as thin punctuate structures, were seen mainly along the walls of the third ventricle and in the ventromedial hypothalamus. Labelled fibres and terminals were located in the external and internal zones of the anterior and posterior median eminence. In particular, fibre terminals were seen close to the capillary loops of the hypothalamo-hypophysial portal system. The anatomical data of the present study regarding the distribution of B-ir in the chick hypothalamus suggest that beacon may play a key role in the regulation of the neuroendocrine system by acting as a neuromodulator and/or neurotransmitter.     

A survey of mutations in the Cochliomyia hominivorax (Diptera: Calliphoridae) esterase E3 gene associated with organophosphate resistance and the molecular identification of mutant alleles

Cochliomyia hominivorax (Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. In Brazil, C. hominivorax has been controlled mainly with organophosphate (OP) insecticides, although the inappropriate use of these chemicals can result in the selection of resistant flies. Changes in carboxylesterase activity have been associated with OP insecticides in some arthopodan species. In this work, we isolated and characterized part of the E3 gene in C. hominivorax (ChE7), which contained the same substitutions responsible for the acquisition of OP hydrolase activity in Lucilia cuprina (Calliphoridae). Digestion of the polymerase chain reaction products with a restriction enzyme that specifically recognized the mutation site unambiguously differentiated wild and mutated esterase alleles. The PCR-RFLP assay therefore provided a fast, reliable DNA-based method for identifying C. hominivorax individuals with a mutation in the esterase gene. Further bioassays to determine the association of this mutation with OP resistance in C. hominivorax should allow the development of more effective strategies for managing this species.     

Bluetongue in The Netherlands; description of the first clinical cases and differential diagnosis. Common symptoms just a little different and in too many herds

For the first time Bluetongue (BT) has been diagnosed in the Netherlands. The clinical symptoms of BT on five farms during the first outbreak ever in the Netherlands are described. Fever and swollen sensitive coronets leading to reluctance to stand and walk were sometimes the first symptoms. Later lesions in the mouth occurred with foamy salivation and respiratory problems. In other cases a swollen head with swollen lips and foamy salivation were the first clinical signs. Also sudden death occurred. In the first sixteen confirmed cases morbidity and mortality were lower than described in outbreaks in other countries. Good collaboration between practitioners, specialists of the Animal Health Service (GD-Deventer), and specialists of the Food and Consumer Product Safety Authority (VWA) and CIDC-Lelystad (Wageningen UR) led to a rapid notification and ultimately confirmation of the suspected diagnosis BT.     

The biochemical, morphological and virulence profiles of Bacillus anthracis isolated in the Kruger National Park

The biochemical, morphological and virulence profiles of 44 Bacillus anthracis isolates, obtained from various localities and carcass remains of wild animals in the Kruger National Park, South Africa, were examined. The morphological characteristics tested for included: the formation of capsules on bicarbonate agar, the motility of the vegetative organism, the presence of haemolysis on blood tryptose agar, the sensitivity of the vegetative organism to bacteriophage, the production of lecithinase on egg yolk agar, the liquefaction (hydrolysis) of gelatine and the capability of each isolate to produce mucoid colonies when grown on bicarbonate agar with horse serum in an atmosphere containing CO2. The API 50CHB and 20E systems were used to evaluate the biochemical activity of each isolate. The virulence of each isolate was determined by its LD50, using an inbred line of Balb/C mice. A clear pattern in the biochemical reactions emerged that appeared to be specific for each isolate. On the API 50CHB test strip, only 2 of the 44 isolates gave a 100% positive reaction to all 10 of the biochemical substances to which it was supposed to react, 9 gave positive results to 90%, 19 were positive to 80%, and 14 were positive to 70%. The reactions on the API 20E were completely different from what was expected, with only 1 of the biochemical activities (gelatinase production) showing a positive reaction to all the isolates. The virulence test indicated that 27/44 isolates could be regarded as highly virulent with a LD50 of less than 1,000 organisms, and the rest of the isolates as virulent with a LD 50 of 1,001-10,000 organisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Classical swine fever in The Netherlands 1997-1998: a description of organisation and measures to eradicate the disease

The central and regional organisation of the campaign to eradicate the CSF epidemic in the Netherlands in 1997/1998 is described. The main instruments used in the campaign were based on stamping-out and movement restrictions specified by the European Union. Additional instruments were used for the first time, namely, pre-emptive culling of contact and neighbouring farms, compartmentalisation of transport, monthly serological screening in established surveillance areas and supervised repopulation of all farms in the former surveillance zone. Two other measures, the killing of very young piglets and a breeding ban were introduced to reduce production in established surveillance zones. Several factors complicated the eradication campaign, for instance, the late detection of the first infection; artificial insemination as a source of infection; the organisation of pig farming in the Netherlands, with its highly concentrated production and dependence on the transport of stock from one unit to another; insufficient rendering capacity; decreasing sensitivity of clinical inspection; and extremely high costs.