Role of the “National Reference Centre for Genetically Modified Organisms (GMO) Detection” in the Official Control of Food and Feed

The National Reference Centre for Genetically Modified Organisms (GMO) detection was established in 2002 within the Istituto Zooprofilattico Sperimentale Lazio e Toscana, with the aim of providing scientific and technical support to the National Health System and to the Ministry of Health within the scope of the regulation of GMO use in food and feed.

The recently adopted EU legislation on GMOs (Regulation CE no. 1829/2003 and no. 1830/2003) introduced more rigorous procedures for the authorisation, labelling and analytical control of food and feed consisting, containing or derived from GMOs. The National Reference Centre, besides its institutional tasks as one of the laboratories of the Italian National Health System, collects and analyses data and results of the national official control of GMOs; carries out scientific research aimed at developing, improving, validating and harmonising detection and quantification methods, in cooperation with other scientific institutions, the Community Reference Laboratory and within the European Network of GMOs laboratories (ENGL); collaborates with the Ministry of Health in the definition of control programmes and promotes educational and training initiatives. Objectives defined for 2004–2006, activities in progress and goals already achieved are presented.

     

Virulence factors and antimicrobial susceptibility of Escherichia coli isolated from calves in Turkey

Escherichia coli isolates from calves were investigated by multiplex PCR assays for the presence of genes encoding K99, F41, F17-related fimbriae, heat-stabile enterotoxin a (STa), intimin (eae) and Shiga toxins (stx1 and stx2). A total of 120 E. coli isolates, 75 isolated from diarrhoeic or septicemic calves and 45 from clinically healthy calves aged between 1 day and 2 months were tested. Each isolate was obtained from different calves in different herds. Among the isolates from diseased animals, 12 (16%) isolates from 1- to 7-day-old diarrhoeic calves were detected as enterotoxigenic E. coli which possessed K99, F41 and STa in combination; F17-related fimbriae genes were detected in 33 (44%) isolates and they were found in combination with K99 + F41 + STa in two isolates. Of 120 isolates, 16 carried eae, eight stx1 and five stx2 genes alone or in combination. None of the eae- or stx-positive strains was identified as O157:H7. However, results indicate that calves may be carrier of Shiga toxin-producing E. coli which have potential as a human pathogen. Antimicrobial susceptibility of 75 isolates from diseased calves was determined by agar disk diffusion method for 14 antimicrobial agents. In 77.3% of the isolates, multiresistance was detected. Higher resistance rates were detected for cephalothin (72%), tetracycline (69.3%), kanamycin (69.3%), ampicillin (65.3%), nalidixic acid (53.3%), trimethoprim-sulphamethoxazole (52%) and enrofloxacin (41.3%), respectively. No resistance was found for ceftiofur and cefoxitin.     

Pharmacokinetics of meloxicam in adult goats: a comparative study of subcutaneous,oral and intravenous administration

AIMS: To determine the plasma disposition of meloxicam in goats following S/C, oral or I/V administration at a single dose of 0.5?mg/kg bodyweight.

METHODS: Five healthy Saanen goats, aged 12–14 months and weighing 35–40?kg, were used for a three phase cross-over design with a 10-day washout period, with meloxicam administered I/V, then orally and S/C. Heparinised blood samples (5?mL) were collected from all animals prior to drug administration (0 hours) and subsequently up to 96 hours. Concentrations of meloxicam in plasma were measured using high performance liquid chromatography. Concentration-time curves were fitted and pharmacokinetic parameters were estimated for each administration group.

RESULTS: Subcutaneous administration of meloxicam exhibited unique plasma distribution characteristics that differed from oral and I/V administration. Mean peak plasma concentrations were greater (1.91 (SD 0.39) vs. 0.71 (SD 0.17) µg/mL) and the time to reach them shorter (3.20 (SD 1.64) vs. 14.33 (SD 2.19) hours) following S/C compared with oral administration (p<0.05). The terminal half-life was longer (15.16 (SD 4.74) vs. 10.69 (SD 1.49) hours) and the MRT was shorter (15.67 (SD 2.37) vs. 24.33 (SD 3.12) hours) following S/C than oral administration (p<0.05), but bioavailability was similar (98.24 (SD 9.62) vs. 96.49 (SD 10.71)%).

CONCLUSION AND CLINICAL RELEVANCE: Subcutaneous administration of meloxicam resulted in long-term presence of drug at high concentration in goat plasma. This unique plasma disposition characteristic may offer an advantage in some clinical cases towards potentially improving the treatment efficacy in goats.     

Computed Tomography of the Normal Bovine Tarsus

The objective of this study was to provide a detailed multiplanar computed tomographic (CT) anatomic reference for the bovine tarsus. The tarsal regions from twelve healthy adult cow cadavers were scanned in both soft and bone windows via a 16‐slice multidetector CT scanner. Tarsi were frozen at ?20^o C and sectioned to 10‐mm‐thick slices in transverse, dorsal and sagittal planes respecting the imaging protocol. The frozen sections were cleaned and then photographed. Anatomic structures were identified, labelled and compared with the corresponding CT images. The sagittal plane was indispensable for evaluation of bone contours, the dorsal plane was valuable in examination of the collateral ligaments, and both were beneficial for assessment of the tarsal joint articulations. CT images allowed excellent delineation between the cortex and medulla of bones, and the trabecular structure was clearly depicted. The tarsal soft tissues showed variable shades of grey, and the synovial fluid was the lowest attenuated structure. This study provided full assessment of the clinically relevant anatomic structures of the bovine tarsal joint. This technique may be of value when results from other diagnostic imaging techniques are indecisive. Images presented in this study should serve as a basic CT reference and assist in the interpretation of various bovine tarsal pathology.     

A quantitative approach to analysing cortisol response in the horse

The cortisol response to glucocorticoid intervention has, in spite of several studies in horses, not been fully characterized with regard to the determinants of onset, intensity and duration of response. Therefore, dexamethasone and cortisol response data were collected in a study applying a constant rate infusion regimen of dexamethasone (0.17, 1.7 and 17 μg/kg) to six Standardbreds. Plasma was analysed for dexamethasone and cortisol concentrations using UHPLC‐MS/MS. Dexamethasone displayed linear kinetics within the concentration range studied. A turnover model of oscillatory behaviour accurately mimicked cortisol data. The mean baseline concentration range was 34–57 μg/L, the fractional turnover rate 0.47–1.5 1/h, the amplitude parameter 6.8–24 μg/L, the maximum inhibitory capacity 0.77–0.97, the drug potency 6–65 ng/L and the sigmoidicity factor 0.7–30. This analysis provided a better understanding of the time course of the cortisol response in horses. This includes baseline variability within and between horses and determinants of the equilibrium concentration–response relationship. The analysis also challenged a protocol for a dexamethasone suppression test design and indicated future improvement to increase the predictability of the test.     

Tetanus in a Camel (Camelus dromedarius) – A Case Report

Twenty days after an open castration, a 5-year-old dromedary was presented to the Dubai Camel Hospital with severe central nervous symptoms. The dromedary showed the following signs: off feed, stiff gait with extended neck, external swelling of the preputial sheath and groin region, and foamy saliva drooling from the mouth. The dromedary was unable to swallow. Three days after admission, the camel developed lockjaw, and on the fifth day it was unable to stand owing to paralysis of the hindquarters. Because of the severity of the disease and because it did not respond to treatment, the camel was euthanized 26 days after the operation and submitted to the Central Veterinary Research Laboratory for further investigation. Both castration wounds were closed and spermiducts were filled with necrotic masses from which Clostridium tetani was isolated. Two mice, which were injected with the filtrate of the thioglycolate broth, developed typical signs of tetanic spasm of the hind leg. Faecal samples from camel and horse paddocks that were only 50 metres apart were negative for C. tetani. However, C. tetani was isolated from two soil samples of the horse paddock. It is recommended that camels should be vaccinated against tetanus prior to castration.     

Antiapoptotic activity of bovine herpesvirus type-1 (BHV-1) UL14 protein

Viruses have evolved different strategies to interfere with apoptotic pathways in order to halt cellular responses to infection. One previous study showed that transient transfection of bovine herpesvirus type-1 (BHV-1) UL14 protein is efficient in protecting Madin Darby kidney (MDBK) and human chronic myelogenous leukemia (K562) cells from sorbitol-induced apoptosis. This protein corresponds to a putative protein of BHV-1, which shares aminoacid sequence with a part of the peptide-binding domain conserved in human heat shock protein (HSP70) family. The pBK-CMV-UL14 plasmid transfected MDBK cells treated with sorbitol did not show caspase-3 and caspase-9 activation with respect to non-transfected MDBK cells (UL14 negative). Furthermore, we report that the expression of the full length sequence of BHV-1 UL14 is evident after 7 h of infection of BHV-1 on MDBK cells which were then treated with sorbitol. These results indicate that UL14 gene product has important implications to enhance cell survival in response to apoptotic stimuli.     

Total and Differential Leucocyte Counts and Lymphocyte Subpopulations in Lymph, Afferent and Efferent to the Supramammary Lymph Node, During Endotoxin-Induced Bovine Mastitis

Leucocyte trafficking in afferent and efferent mammary lymph and the supramammary lymph node in cows was examined during 4 h after intramammary infusion of endotoxin from Escherichia coli. Total and differential leucocyte counts were measured in milk, blood and lymph. The proportions of CD4(+), CD8(+), major histocompatibility complex (MHC) class II(+) and IgM(+) lymphocytes were examined in the lymph and lymph node. At post-infusion hour (PIH) 4, the flow rates of both lymph fluids had increased approximately eightfold. Total leucocyte concentration increased in afferent lymph, but decreased in efferent lymph. Neutrophils increased in afferent lymph at PIH 2 and in efferent lymph and milk at PIH 4. The predominant cell type in afferent lymph shifted from lymphocyte to neutrophil while lymphocyte was still at PIH 4 the predominant type in efferent lymph. Among the lymphocytes, B cells were predominant in afferent lymph and lymph node at PIH 4 while T cells, mainly CD4(+) cells, were predominant in efferent lymph both at PIH 0 and PIH 4. The CD4 : CD8 ratio was higher in efferent lymph and the challenged lymph node than in afferent lymph and the control node, respectively. There was a significant difference in proportions of each lymphocyte subpopulation except for IgM(+) cells, between afferent and efferent lymph after infusion. According to the results, there was already during the first hours of the immune response, a non-random trafficking of neutrophils and lymphocyte subpopulations resulting in a changed distribution of cells in afferent and efferent lymph and a difference in lymphocyte reactivity between the two lymph fluids.     

Importance of the rearing period for laying hens in alternative systems

Feather pecking and cannibalism are still major problems in alternative systems for laying hens. Literature and practical experience indicate that unfavourable rearing conditions might be important risk factors for the occurrence of these behavioural disturbances during the laying period.Typical rearing conditions of laying hens from 50 rearing units in Germany and Austria are presented. Obvious risk factors during rearing for feather pecking and cannibalism during the laying period were found. Most flocks were kept under high stocking density (mean: 15 pullets per m' useable area) and some flocks had access to litter only after the second week of life or access to raised perches after the fourth week of life. Plumage condition of pullets and laying hens varied widely in non-beak-trimmed as well as in beak-trimmed flocks.The percentage of pullets with damaged plumage was higher in beak-trimmed than in non-beak-trimmed flocks (medians: 53 % versus 30 %, p = 0,022). In laying hens there was a higher percentage of hens with plumage damage in non-beak-trimmed flocks compared to beak-trimmed flocks (medians: 23 % versus 50 %, p = 0,007). Data analysis will be continued, especially with regard to particular risk factors.     

Comparison of Cryoprotective Effects of Lycopene and Cysteamine in Different Cryoprotectants on Bull Semen and Fertility Results

The objectives of this study were to compare glycerol and ethylene glycol at different concentrations as cryoprotectants and lycopene or cysteamine (with/without) as antioxidants in Tris extender for bull semen. Twenty‐four ejaculates were obtained from three bulls. Each ejaculate was split into four equal aliquots and diluted using both of the Tris extenders with glycerol (5% or 7%) or ethylene glycol (3% or 5%). After that, each extenders were split into three equal aliquots and added using both of the cysteamine 5 mm or lycopene 500 μg/ml, and control (without additives). The addition of 7% glycerol with cysteamine, 5% ethylene glycol with cysteamine and 3% ethylene glycol with cysteamine groups gave the lowest CASA motility than the other groups. However, 7% glycerol and 7% glycerol with lycopene resulted in a better rate of CASA progressive motility compared with that of other groups. Generally, all the lycopene groups signed better protective effects on acrosome and total morphology than the other groups. Glycerol 7% and 3% ethylene glycol with lycopene groups yielded to slight higher percentages of membrane integrity assessed by HOST than that of the other groups, but 7% glycerol with cysteamine and 3% ethylene glycol with cysteamine showed the worst percentages of membrane integrity. Glycerol 7% and 5% glycerol with lycopene gave rise to a higher value of VAP, VSL and VCL compared with that of the other groups. On the contrary, adding to 5% glycerol with cysteamine showed negative effect for VAP, VSL, VCL and ALH values. All cryoprotectant groups with lycopene decreased chromatin damage than the other groups. Ethylene glycol 3% led to lower non‐return rates of inseminated cows. However, this result was not considered to be statistically important.