The role of national and international veterinary laboratories.

In the field of diagnostic test validation, World Organisation for Animal Health (OIE) Reference Laboratories (RLs) have a pivotal role and provide the international community with impartial advice and support in the selection, development and validation of diagnostic tests, which can be applied to the specialist diseases for which they are designated. National RLs provide an invaluable function in supporting the introduction, ongoing validation and application of validated diagnostic tests in line with international standards. Experienced staff with extensive knowledge of such systems and access to specialist facilities for conducting work are available to monitor changes or advancements in technology. They consider their relevance and value to evolving diagnostic test requirements. Reference Laboratories often have a broad mandate of activity linking research or development programmes and surveillance activities to benefit the continual assessment and, if necessary, improvement of diagnostic tools. Reference Laboratories maintain or have access to unique biological archives (known positive and negative sample populations) and produce international reference standards, both of which are vital in establishing the necessary and detailed validation of any diagnostic test. Reference Laboratories act either singularly or in collaborative partnerships with other RLs or science institutes, but also, when required, and with impartiality, with the commercial sector, to ensure new tests are validated according to OIE standards. They promote and apply formal programmes of quality assurance (including proficiency testing programmes) for newly validated tests, ensuring ongoing monitoring and compliance with standards, or as required set out any limitations or uncertainties. Reference Laboratories publish information on test validation in the scientific literature and on relevant websites, as well as disseminating information at workshops and international conferences. Furthermore, they can offer training in the processes and systems underpinning test validation.

Dans le domaine de la validation des tests de diagnostic, les Laboratoires de référence de l'Organisation mondiale de la santé animale (OIE) jouent un rôle central et fournissent à la communauté internationale des conseils impartiaux ainsi qu'un soutien pour la sélection, la mise au point et la validation des tests de diagnostic utilisés pour la détection des maladies correspondant à leur domaine de spécialisation. Les Laboratoires de référence nationaux remplissent une fonction inestimable en facilitant l'introduction, la validation continue et l'application de tests de diagnostic validés conformément aux normes internationales. Ces laboratoires sont dotés de personnels expérimentés possédant une connaissance approfondie de ces systèmes et qui ont accès à des installations spécialisées pour mener à bien leurs opérations et suivre de près les changements ou les avancées technologiques. Ils peuvent ainsi examiner leur pertinence et intérêt au regard de l'évolution des exigences relatives aux tests de diagnostic. Le mandat des Laboratoires de référence recouvre souvent un large éventail d'activités reliant les programmes de recherche ou développement et les activités de surveillance, ce qui permet de réaliser une évaluation continue des outils diagnostiques et, si besoin, de procéder à leur amélioration. Les Laboratoires de référence entretiennent ou ont accès à des banques de matériels biologiques uniques (panels d'échantillons positifs et négatifs connus) et produisent des réactifs de référence internationale, deux catégories de matériels essentielles pour procéder à la validation point par point d'un test diagnostique suivant les critères requis. Les Laboratoires de référence interviennent individuellement ou en partenariat avec d'autres Laboratoires de référence ou instituts scientifiques, mais aussi, lorsque c'est nécessaire et dans le respect des règles d'impartialité, avec le secteur privé, afin de s'assurer que les nouveaux tests sont validés conformément aux normes de l'OIE. Ils soutiennent et appliquent des programmes officiels d'assurance de la qualité (y compris en participant à des programmes d'essais d'aptitude inter-laboratoires) pour les tests nouvellement validés et garantissent leur suivi continu ainsi que leur conformité avec les normes, ou, suivant les cas, définissent les limites ou le niveau d'incertitude à prendre en considération. Les Laboratoires de référence publient les données relatives à la validation des tests dans des journaux scientifique et sur les sites Web pertinents et diffusent également des informations sur le sujet lors d'ateliers et de conférences internationales. En outre, ils peuvent proposer des formations sur les procédures et les systèmes qui sous-tendent la validation des tests.

En el terreno de la validación de pruebas de diagnóstico, los Laboratorios de Referencia de la Organización Mundial de Sanidad Animal (OIE) cumplen una función central y proporcionan a la comunidad internacional servicios de apoyo y asesoramiento imparcial para la selección, el desarrollo y la validación de pruebas de diagnóstico, que pueden aplicarse a la enfermedad para la que cada laboratorio esté designado. Los laboratorios de referencia nacionales cumplen una inestimable función de apoyo a la implantación, la continua validación y la utilización de pruebas de diagnóstico validadas con arreglo a las normas internacionales. Disponen de personal experimentado y muy buen conocedor de estos sistemas y de acceso a instalaciones especializadas de trabajo, lo que les permite seguir de cerca los cambios o adelantos tecnológicos y estudiar su utilidad o interés en relación con la evolución de los requisitos de las pruebas de diagnóstico. Los Laboratorios de Referencia suelen tener un mandato amplio, que a los programas de investigación y desarrollo aúna actividades de vigilancia, en aras de la continua evaluación y, en caso necesario, mejora de las herramientas de diagnóstico. Estos laboratorios poseen (o tienen acceso a) archivos biológicos únicos (conjuntos de muestras probadamente positivas y negativas) y elaboran patrones de referencia internacional, elementos ambos indispensables para llevar a buen fin la necesaria validación detallada de toda prueba de diagnóstico. Los Laboratorios de Referencia pueden trabajar en solitario o en colaboración con otros Laboratorios de Referencia, con institutos científicos e incluso, cuando hace falta, y procediendo con imparcialidad, con entidades del sector privado, a fin de garantizar que toda nueva prueba sea validada con arreglo a las normas de la OIE. También promueven y llevan adelante programas oficiales de garantía de la calidad de pruebas recién validadas (incluidos programas de pruebas de competencia), lo que asegura un seguimiento continuo y el cumplimiento de la normativa en todo momento, o fijan, cuando es necesario, limitaciones o niveles de incertidumbre. Asimismo, estos laboratorios publican datos sobre la validación de pruebas en revistas científicas y sitios web conexos y difunden información al respecto en talleres y conferencias internacionales. Además, pueden impartir formación sobre los procesos y sistemas que fundamentan la validación de pruebas de diagnóstico.

Evaluation of ELISA and haemagglutination inhibition as screening tests in serosurveillance for H5/H7 avian influenza in commercial chicken flocks.

Avian influenza virus (AIV) subtypes H5 and H7 can infect poultry causing low pathogenicity (LP) AI, but these LPAIVs may mutate to highly pathogenic AIV in chickens or turkeys causing high mortality, hence H5/H7 subtypes demand statutory intervention. Serological surveillance in the European Union provides evidence of H5/H7 AIV exposure in apparently healthy poultry. To identify the most sensitive screening method as the first step in an algorithm to provide evidence of H5/H7 AIV infection, the standard approach of H5/H7 antibody testing by haemagglutination inhibition (HI) was compared with an ELISA, which detects antibodies to all subtypes. Sera (n = 1055) from 74 commercial chicken flocks were tested by both methods. A Bayesian approach served to estimate diagnostic test sensitivities and specificities, without assuming any 'gold standard'. Sensitivity and specificity of the ELISA was 97% and 99.8%, and for H5/H7 HI 43% and 99.8%, respectively, although H5/H7 HI sensitivity varied considerably between infected flocks. ELISA therefore provides superior sensitivity for the screening of chicken flocks as part of an algorithm, which subsequently utilises H5/H7 HI to identify infection by these two subtypes. With the calculated sensitivity and specificity, testing nine sera per flock is sufficient to detect a flock seroprevalence of 30% with 95% probability.

Match Running Performance and Success Across a Season in German Bundesliga Soccer Teams.

The aim of the study was to quantify the association between match running performance and success across a season in soccer teams competing within a European top league. We analyzed the match running performance data of all soccer teams from the German Bundesliga across the season 2012/13 (306 matches). The following match running performance data were used: total distance covered as well as number of running activities>18.0 km/h and > 22.7 km/h. Depending on the team's ball possession status, all match running performance data were also analyzed as those with and without ball possession. The success across the season was defined as the final competition points accumulated. The match running performance alone was not significantly correlated with the final points accumulated (best r=0.24; p=0.34). In contrast, positive-significant correlations were observed for the match running performance with ball possession (best r=0.77; p<0.01). However, of these latter correlations, only the total distance covered with ball possession was a significant predictor (p<0.01) and accounted for 60% of the variance (R(2)=0.60) in the final points accumulated. It is concluded that it is not the match running performance alone that is important for achieving success in German Bundesliga soccer teams, but rather its relation to technical/tactical skills with respect to ball possession.

Evaluation of the pooling of swabs for real-time PCR detection of low titre shedding of low pathogenicity avian influenza in turkeys.

The purpose of this study was to determine whether pooling avian influenza (AI)-positive swabs with negative swabs has a detrimental effect on the sensitivity of AI real-time reverse transcription-polymerase chain reactions (rRT-PCRs). Cloacal and buccal swabs were sampled daily from 12 turkeys infected with A/goose/England/07(H2N2). For half the turkeys, each swab was mixed with four swabs from known AI-negative turkeys, and for the other half the swabs were tested individually. Bayesian modelling was used to (i) determine whether pooling the positive swabs compromised the cycle threshold (C(t)) value obtained from the rRT-PCRs, and (ii) estimate the likelihood of detection of an H2N2 infected turkey flock via rRT-PCR for pooled and individually tested swabs (cloacal and buccal) vs. the number of days post-infection of the flock. Results indicated that there was no significant effect of compromising AI rRT-PCR sensitivity by pooling a weak positive swab with negative swabs on the Ct values which were obtained. Pooled sampling was able to widen the detection window compared to individual sampling, for the same number of rRT-PCR tests. This indicates that pooled sampling would be an effective method of reducing the number of tests to be performed to determine flock status during an AI outbreak and for surveillance.

Highly pathogenic avian influenza virus H5N6 (clade 2.3.4.4b) has a preferable host tropism for waterfowl reflected in its inefficient transmission to terrestrial poultry.

Highly-pathogenic avian influenza virus (HPAIV) H5N6 (clade 2.3.4.4b) incurred into Europe in late 2017 and was predominantly detected in wild birds, with very few terrestrial poultry cases. Pekin ducks directly-infected with a UK virus (H5N6-2017) were donors of infection to investigate contact transmission to three recipient species: Ducks, chickens and turkeys. H5N6-2017 transmission to ducks was 100% efficient, but transmission to in-contact galliforme species was infrequent and unpredictable, thereby reflecting the European 2017-2018 H5N6 epidemiology. Although only two of 28 (7%) infected ducks died, the six turkeys and one chicken which became infected all died and displayed systemic H5N6-2017 dissemination, while pathogenesis in ducks was generally milder. Analysis of H5N6-2017 progeny in the contacts revealed no emergent polymorphisms in an infected duck, but the galliforme species included changes in the polymerase (PB2 A199T, PA D347A), matrix (M1 T218A) and neuraminidase genes (T88I). H5N6-2017 environmental contamination was associated with duck shedding.

Role of melatonin in mucosal gastroprotection against aspirin-induced gastric lesions in humans.

Melatonin and its precursor, l-tryptophan, have been shown to exert gastroprotective effects in animals, but their influence on the gastric damage by aspirin (ASA) in humans has been sparingly investigated. In this study, we designed to determine the effects of melatonin and l-tryptophan on ASA-induced gastric mucosal damage, gastric microbleeding, mucosal generation of prostaglandin E(2), and plasma melatonin, and gastrin levels. Three groups of healthy male volunteers (n = 30) with intact gastric mucosa received daily for 11 days either ASA alone or that combined with melatonin or tryptophan. Gastric blood loss and mucosal damage were evaluated at 3rd, 7th, and 11th days of ASA administration by endoscopy using Lanza score. ASA alone caused a marked rise of gastric damage and gastric blood loss, mainly at day 3rd and 7th, but they were significantly reduced at 11th day. Pretreatment with melatonin or tryptophan remarkably reduced ASA induced gastric lesions and microbleeding. Gastric mucosal generation of PGE(2) was suppressed by about 90% in all subjects treated with ASA alone without or with addition of melatonin or tryptophan. Plasma melatonin was markedly increased after treatment with melatonin or tryptophan plus ASA, but it was also raised significantly after application of ASA alone. Plasma gastrin levels were raised in subjects given melatonin or tryptophan plus ASA, but not in those with ASA alone. We conclude that melatonin and its precursor tryptophan given orally significantly reduce gastric lesions induced by ASA possibly due to (a) direct gastroprotective action of exogenous melatonin or that generated from tryptophan and (b) gastrin released from the gastric mucosa by melatonin or tryptophan.

Role of real-time RT-PCR platform technology in the diagnosis and management of notifiable avian influenza outbreaks: experiences in Great Britain.

Diagnosis and management of avian influenza outbreaks now include the use of validated real-time reverse transcription PCR (RRT-PCR) methods in many countries, including all member states of the European Union. Two outbreaks in poultry of notifiable avian influenza (H5 and H7 subtypes) that occurred in Great Britain during 2007 will serve as examples in which RRT-PCR demonstrated its value in 1) rapid diagnosis and confirmation of disease by sensitive and specific laboratory testing of samples derived from the index cases and 2) high-volume, rapid testing of surveillance samples. The two poultry outbreaks followed the incursion of a H7N2 low-pathogenicity notifiable avian influenza (LPNAI) virus (May-June 2007) and a Eurasian lineage H5N1 highly pathogenic notifiable avian influenza (HPNAI) virus (November 2007). Coupled with the use of high-throughput, robotic RNA extraction methods, a total of approximately 9300 and 20,300 field samples were tested by appropriate, validated RRT-PCR assays during the 4- and 5-wk duration of the H7N2 LPNAI and H5N1 HPNAI outbreaks, respectively. Fundamental features of the validated RRT-PCR assays used included their high degree of sensitivity, specificity, and rapidity, attributes that were invaluable in providing timely and accurate information for notifiable AI outbreak management.

HRM screening of the UBC9 gene encoding the SUMO-E2-conjugating enzyme - case-control study in breast cancer.

AIM: UBC9 (E2) small ubiquitin-like modifier conjugating enzyme plays a key role in the post-translational modification of proteins named sumoylation. Defects in small ubiquitin-like modifier modification may contribute to breast carcinogenesis. In the present work, we examined UBC9 genetic variation. MATERIALS AND METHODS: UBC9 genetic variation was analyzed by using the high resolution melting (HRM) method. HRM study was conducted on 173-182 healthy women and 188-190 women with breast cancer. RESULTS: During HRM screening, we analysed three known single-nucleotide polymorphisms in introns: rs4984806, rs909916 and rs909917, and one known single nucleotide polymorphism rs8063 in exon 7, in a non-coding region. The genotype frequencies for all polymorphisms were in accordance with Hardy - Weinberg equilibrium among the control subjects and breast cancer patients. The linkage disequilibrium analysis displayed that there was one polymorphism block, which consisted of three single nucleotide polymorphisms: rs909916, rs909917 and rs4984806. We identified two common haplotypes CCG and TTC, but we did not find significant differences in the distribution of these haplotypes between cases and controls. CONCLUSION: Our study showed no differences in the occurrence of indicated polymorphisms in the UBC9 gene in a group of healthy women compared to women with breast cancer. These results suggest that the polymorphisms of the UBC9 gene - rs4984806, rs909916, rs909917 and rs8063 can be not associated with breast cancer risk.

Hospitalization due to community-acquired pneumonia in patients with chronic obstructive pulmonary disease: incidence, epidemiology and outcomes.

OBJECTIVES: Community-acquired pneumonia (CAP) is an important complication in patients with chronic obstructive pulmonary disease (COPD). This study aimed to define incidence, and outcomes of COPD patients hospitalized with pneumonia in the city of Louisville, and to estimate the burden of disease in the US population. METHODS: This was a secondary analysis of a prospective population-based cohort study of residents in Louisville, Kentucky, 40 years old and older, from 1 June 2014 to 31 May 2016. All adults hospitalized with CAP were enrolled. The annual incidence of pneumonia in COPD patients in Louisville was calculated and the total number of adults with COPD hospitalized in the United States was estimated. Clinical outcomes included time to clinical stability (TCS), length of hospital stay (LOS) and mortality. RESULTS: From a Louisville population of 18 246 patients with COPD, 3419 pneumonia hospitalizations were documented during the 2-year study. The annual incidence was 9369 patients with pneumonia per 100 000 COPD population, corresponding to an estimated 506 953 adults with COPD hospitalized due to pneumonia in the United States. The incidence of CAP in patients without COPD was 509 (95% CI 485-533) per 100 000. COPD patients had a median (interquartile range) TCS and LOS of 2 (1-4) and 5 (3-9) days respectively. The mortality of COPD patients during hospitalization, at 30 days, 6 months and 1 year was 193 of 3419 (5.6%), 400 of 3374 (11.9%), 816 of 3363 (24.3%) and 1104 of 3349 (33.0%), respectively. CONCLUSIONS: There was an annual incidence of 9369 cases of hospitalized CAP per 100 000 COPD patients in the city of Louisville. This was an approximately 18-fold greater incidence of CAP in COPD patients than in those without COPD.

Altered basal and postprandial plasma melatonin, gastrin, ghrelin, leptin and insulin in patients with liver cirrhosis and portal hypertension without and with oral administration of melatonin or tryptophan.

This investigation was designed to assess the effects of oral administration of melatonin (10 mg) and tryptophan (Trp) (500 mg) on fasting and postprandial plasma levels of melatonin, gastrin, ghrelin, leptin and insulin in 10 healthy controls and in age-matched patients with liver cirrhosis (LC) and portal hypertension. Fasting plasma melatonin levels in LC patients were about five times higher (102 +/- 15 pg/mL) than in healthy controls (22 +/- 3 pg/mL). These levels significantly increased postprandially in LC patients, but significantly less so in controls. Treatment with melatonin or L-Trp resulted in a further significant rise in plasma melatonin, both under fasting and postprandial conditions, particularly in LC patients. Moreover, plasma gastrin, ghrelin, leptin and insulin levels under fasting and postprandial conditions were significantly higher in LC subjects than in healthy controls and they further rose significantly after oral application of melatonin or Trp. This study shows that: (a) patients with LC and portal hypertension exhibit significantly higher fasting and postprandial plasma melatonin levels than healthy subjects; (b) plasma ghrelin, both in LC and healthy controls reach the highest values under fasting conditions, but decline postprandially, especially after oral application of melatonin or Trp; and (c) plasma melatonin, gastrin, ghrelin and insulin levels are altered significantly in LC patients with portal hypertension compared with that in healthy controls possibly due to their portal systemic shunting and decreased liver degradation.

The influence of bilberry fruit on memory and the expression of parvalbumin in the rat hippocampus.

Therefore, the aim of the present study was to evaluate the possible effect of bilberry fruit (Vaccinium myrtillus L.) supplement in a daily diet on the cognitive behaviour of the rats and the expression of paravalbumin (PV) in populations of hippocampal neurons. It has been postulated that the antioxidants present in bilberry fruit may act as neuroprotective factors playing also a significant role as memory enhancements. Forty Wistar rats with a similar average body weight (460 ± 0.4 g) were divided into four groups (n=10 per group). The control group received standard feed (210 g/week), whereas animals of experimental groups received standard feed supplemented with bilberry (per os) at consumed doses of 2 g (group I), 5 g (group II), and 10 g/kg b.w./ /day (group III). After three months of feeding with bilberry, the modified elevated plus-maze test (mEPM) was performed. After 32 weeks of feeding, brains were collected and PV-immunoreactive (ir) neurons were immunohistochemically visualized. In the modified elevated plus-maze test, transfer latency examined 2 h and 24 h after the acquisition session was significantly shorter (p⟨0.05) in the group II in comparison with the control group. In CA1 and CA2/CA3 hippocampal fields as well as dentate gyrus of all experimental groups, a significant (p⟨0.05) decrease in number of PV-ir neurons were found. In relation to the control group, the mean subpopulation of PV-ir neurons found in groups II and III were significantly reduced. The subpopulations of PV-ir neurons found in DG of all experimental groups were significantly reduced in comparison to the control. In conclusion the in the present paper we demonstrated a relationship between the diet rich in a bilberry fruit and process of memory as well as numbers of calcium- binding protein-expressing hippocampal neurons. Our results may be source of basic knowledge for further research aiming at neuroprotective role of the bilberry fruit.

Behavioral and Biochemical Impact of Chronic Unpredictable Mild Stress on the Acquisition of Nicotine Conditioned Place Preference in Rats.

Addiction is a chronic psychiatric disease which represents a global problem, and stress can increase drug addiction and relapse. Taking into account frequent concomitance of nicotine dependence and stress, the purpose of the present study was to assess behavioral and biochemical effects of chronic unpredictable mild stress (CUMS) exposure on nicotine reward in rats measured in the conditioned place preference (CPP) paradigm. Rats were submitted to the CUMS for 3 weeks and conditioned with nicotine (0.175 mg/kg) for 2 or 3 days. Our results revealed that only CUMS-exposed animals exhibited the CPP after 2 days of conditioning indicating that stressed rats were more sensitive to the rewarding properties of nicotine and that chronic stress exacerbates nicotine preference. Administration of metyrapone (50 mg/kg), a glucocorticosteroid antagonist, and imipramine (15 mg/kg), an antidepressant, abolished nicotine CPP in stressed rats after 2 days of conditioning. The biochemical experiments showed increased markers of oxidative stress after nicotine conditioning for 2 and 3 days, while the CUMS further potentiated pro-oxidative effects of nicotine. Moreover, metyrapone reversed oxidative changes caused by stress and nicotine, while imipramine was not able to overwhelm nicotine- and stress-induced oxidative damages; however, it could exert antioxidant effect if administered repeatedly. The results suggest that recent exposure to a stressor may augment the rewarding effects of nicotine through anhedonia- and stress-related mechanisms. Our study contributes to the understanding of behavioral and biochemical stress-induced modification of the rewarding effects of nicotine on the basis of the development of nicotine dependence.

Direct evidence of H7N7 avian influenza virus mutation from low to high virulence on a single poultry premises during an outbreak in free range chickens in the UK, 2008.

H5 and H7 subtypes of low pathogenicity avian influenza viruses (LPAIVs) have the potential to evolve into highly pathogenic avian influenza viruses (HPAIVs), causing high mortality in galliforme poultry with substantial economic losses for the poultry industry. This study provides direct evidence of H7N7 LPAIV mutation to HPAIV on a single poultry premises during an outbreak that occurred in June 2008 in free range laying hens in Oxfordshire, UK. We report the first detection of a rare di-basic cleavage site (CS) motif (PEIPKKRGLF), unique to galliformes, that has previously been associated with a LPAIV phenotype. Three distinct HPAIV CS sequences (PEIPKRKKRGLF, PEIPKKKKRGLF and PEIPKKKKKKRGLF) were identified in the infected sheds suggesting molecular evolution at the outbreak premises. Further evidence for H7N7 LPAIV preceding mutation to HPAIV was derived by examining clinical signs, epidemiological descriptions and analysing laboratory results on the timing and proportions of seroconversion and virus shedding at each infected shed on the premises. In addition to describing how the outbreak was diagnosed and managed via statutory laboratory testing, phylogenetic analysis revealed reassortant events during 2006-2008 that suggested likely incursion of a wild bird origin LPAIV precursor to the H7N7 HPAIV outbreak. Identifying a precursor LPAIV is important for understanding the molecular changes and mechanisms involved in the emergence of HPAIV. This information can lead to understanding how and why only some H7 LPAIVs appear to readily mutate to HPAIV.

Validated H5 Eurasian real-time reverse transcriptase-polymerase chain reaction and its application in H5N1 outbreaks in 2005-2006.

Real time reverse transcriptase (RRT)-polymerase chain reaction (PCR) for the detection of Eurasian H5 avian influenza virus (AIV) isolates was adapted from an existing protocol, optimized, and validated using a number of genetically diverse H5 isolates (n = 51). These included 34 "Asian lineage" H5N1 highly pathogenic avian influenza (HPAI) viruses (2004-2006), plus 12 other H5 isolates from poultry outbreaks and wild birds in the Eastern Hemisphere (1996-2005). All 51 were positive by H5 Eurasian RRT-PCR. Specificity was assessed by testing representative isolates from all other AL virus subtypes (n = 52), non-AI avian pathogens (n = 8), plus a negative population of clinical specimens derived from AI-uninfected wild birds and poultry (n = 604); all were negative by H5 Eurasian RRT-PCR. RNA was directly extracted from suspect HPAI H5N1 clinical specimens (Africa, Asia, and Europe; 2005-2006; n = 58) from dead poultry and wild birds, and 55 recorded as positive by H5 Eurasian RRT-PCR: Fifty-one of these 55 were in agreement with positive AIV isolation in embryonated chickens' eggs. H5 Eurasian RRT-PCR was invaluable in H5 outbreak diagnosis and management by virtue of its rapidity and high degree of sensitivity and specificity. This method provides a platform for automation that can be applied for large-scale intensive investigations, including surveillance.

Identification of sensitive and specific avian influenza polymerase chain reaction methods through blind ring trials organized in the European Union.

Many different polymerase chain reaction (PCR) protocols have been used for detection and characterization of avian influenza (AI) virus isolates, mainly in research settings. Blind ring trials were conducted to determine the most sensitive and specific AI PCR protocols from a group of six European Union (EU) laboratories. In part 1 of the ring trial the laboratories used their own methods to test a panel of 10 reconstituted anonymized clinical specimens, and the best methods were selected as recommended protocols for part 2, in which 16 RNA specimens were tested. Both panels contained H5, H7, other AI subtypes, and non-AI avian pathogens. Outcomes included verification of 1) generic AI identification by highly sensitive and specific M-gene real-time PCR, and 2) conventional PCRs that were effective for detection and identification of H5 and H7 viruses. The latter included virus pathotyping by amplicon sequencing. The use of recommended protocols resulted in improved results among all six laboratories in part 2, reflecting increased sensitivity and specificity. This included improved H5/H7 identification and pathotyping observed among all laboratories in part 2. Details of these PCR methods are provided. In summary, this study has contributed to the harmonization of AI PCR protocols in EU laboratories and influenced AI laboratory contingency planning following the first European reports of H5N1 highly pathogenic AI during autumn 2005.

Genetic polymorphism of alcohol dehydrogenase 3 in digestive tract alcohol damage.

BACKGROUND/AIMS: Genetic polymorphism of enzymes involved in alcohol metabolism plays a relevant role in etiopathogenesis of alcohol disease. The aim of the present study was to find in the Polish population the ADH3 genotypes, which are likely to be responsible for higher susceptibility to alcohol disease of the liver and chronic alcohol pancreatitis. METHODOLOGY: The ADH3 genotype and ADH3*1 and ADH3*2 alleles frequency were examined in 266 patients. Genotyping of the ADH3 was performed using polymerase chain reaction-restriction fragment length polymorphism methods on white cell DNA. RESULTS: The genotype ADH3*1/ADH3*1 was found to be significantly more frequent in alcohol abusers compared to non-drinkers. The examinations of the group of alcohol abusers showed that the genotype ADH3*2/ADH3*2 occurred statistically significantly less frequently in patients with chronic pancreatitis than those without alimentary lesions and patients with cirrhosis. Thus it is likely to be the protective factor of chronic pancreatitis. CONCLUSIONS: The alleles ADH3*1 and genotype ADH3*1/ADH3*1 were significantly more frequent in men than in women, while alleles ADH3*2 and genotype ADH3*2/ADH3*2 were more common in women. This may account for rarer alcohol addiction observed in Polish women.

Behavioral and Biochemical Interaction Between Nicotine and Chronic Unpredictable Mild Stress in Mice.

Nicotine, the main component of tobacco smoke, exerts influence on mood, and contributes to physical and psychological dependence. Taking into account frequent concomitance of nicotine abuse and stress, we aimed to research behavioral and biochemical effects associated with nicotine administration in combination with chronic unpredictable mild stress (CUMS). Mice were submitted to the procedure of CUMS for 4 weeks, 2 h per day. Our results revealed that CUMS-exposed animals exhibited behavioral alteration like anxiety disorders in the elevated plus maze (EPM) test, the disturbances in memory in the passive avoidance (PA) test and depressive effects in the forced swim test (FST). Moreover, nicotine (0.05-0.5 mg/kg), after an acute or subchronic administration decreased stress-induced depression- and anxiety-like effect as well as memory deficit. Administration of metyrapone (50 mg/kg), a glucocorticosteroid antagonist, alleviated the depressive effect induced by the CUMS. The biochemical experiments showed decreased values of the total antioxidant status (TAS), activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) with simultaneously increased in malondialdehyde (MDA) concentration in mice submitted to the CUMS. The same effects were observed after an acute and subchronic nicotine administration within all examined brain structures (i.e., hippocampus, cortex, and cerebellum) and in the whole brain in non-stressed and stressed mice confirming pro-oxidative effect of nicotine. Our study contributes to the understanding of behavioral and biochemical mechanisms involved in stress-induced disorders such as depression, anxiety and memory disturbances as well as dual nicotine-stress interactions on the basis of the development of nicotine dependence.