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.

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.

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.

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.

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.

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.

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.

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.

Effects of renin-angiotensin system inhibitors on fibrosis in patients with alcoholic chronic pancreatitis.

Chronic pancreatitis (CP) results in impairment of exocrine as well as endocrine functions and progressive fibrosis. Previous studies, have demonstrated the presence of renin-angiotensin system receptors within different pancreatic cells. The aim of the present study was to assess the effects of renin-angiotensin system (RAS) inhibitors on serum levels of fibrosis biomarkers (matrix metalloproteinase 2 and 9 (MMP-2, MMP-9), tissue inhibitor of MMP (TIMP- 1, TIMP-2), hyaluronic acid (HA)) and fasting glucose levels in patients with alcoholic CP. Seventy seven outpatients (mean age 43 years, 62 males) with diagnosed alcoholic CP were randomly enrolled into 5 study groups depending on the RAS inhibitors administered and their doses (2.5 or 5 mg and 12.5 or 25 mg for ramipril or losartan, respectively). Venous blood was sampled monthly for a period of one year to monitor serum drug levels. MMP-2, -9, TIMP-1, TIMP-2 and HA were measured with ELISA method on the onset and at the end of the study. Only forty five patients regularly participated in follow-up visits and completed the study. The fluctuations in serum HA levels observed among patients from the remaining groups also did not reach statistical significance. Serum MMP-2 levels (P = 0.06) and MMP-2/TIMP-1 ratio (P = 0.06) showed increasing tendency in the losartan 25 mg group. High doses of ramipril and losartan statistically significantly reduced fasting glucose levels. High doses of losartan can increase the MMP-2 activity in serum of alcoholic CP patients, which potentially is likely to affect turnover of extracellular matrix proteins within the pancreas. Moreover, high doses of both RAS inhibitors decrease the fasting glucose level.

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.

Effects of treatment with melatonin and tryptophan on liver enzymes, parameters of fat metabolism and plasma levels of cytokines in patients with non-alcoholic fatty liver disease--14 months follow up.

Non-alcoholic fatty liver disease (NAFLD), most common chronic hepatic pathology, that occurs in the developed countries is estimated at 1/3 of the population. Amongst the numerous pathogenetic factors, oxidative stress and apoptosis of hepatocytes initiate many inflammatory processes and are involved in the progression of disease, particularly in transformation of non-alcoholic steatohepatitis (NASH) to cirrhosis. The aim of our study was to determine the effects of tryptophan and melatonin on the selected biochemical parameters in patients with NAFLD, and additionally, to evaluate the effects of tryptophan and melatonin in improvement of liver tissue in selected NAFLD patients. Seventy four patients with NAFLD confirmed by histopathological examination of liver biopsy samples, were admitted to the study. They were randomly assigned to three groups. Group I received the preparation Essentiale forte in the dose of 3 x 1 tablet per day and tryptophan 2 x 500 mg/day over the period of 14 months, group II received Essentiale forte and melatonin 2 x 5 mg/day over 14 months and group III received only Essentiale over the period of 14 months. In nine patients of groups I, II, and III, the liver biopsy was performed after 14-months of treatment period. Out of nine patients whom biopsy was performed, three of them were from group I, four from group II and two of them were from group III, respectively. After the 14-month treatment period, gamma-glutamyl transferase (GGPT) activity and levels of triglycerides and LDL-cholesterol were found to be significantly reduced in group I and II. The level of melatonin after the therapy was significantly elevated in group I and II and did not change in group III. Statistically significantly lower levels of IL-1, IL-6 and TNF-α were observed in patients receiving melatonin and tryptophan, comparing with group III treated with Essentiale forte only. These study findings demonstrate that melatonin and tryptophan substantially reduce the levels of pro-inflammatory cytokines and improve some parameters of fat metabolism in patients with NAFLD. In few patients with NASH melatonin and tryptophan reduced the inflammation in liver. We conclude that melatonin is worth considering for the therapy of NAFLD, particularly in patients with impaired fat metabolism accompanied by hypertriglyceridemia and hyper-LDL cholesterolemia.

Comparison of anti-inflammatory properties of peroxisome proliferator-activated receptor gamma agonists rosiglitazone and troglitazone in prophylactic treatment of experimental colitis.

Non-specific inflammatory bowel disease (IBD), including ulcerative colitis and Crohn`s disease, is a chronic noninfectious inflammatory disease whose incidence is increasingly high, especially in the developed countries. Effective methods of its treatment and prevention of recurrences are still under investigation. Amongst the options to control effectively the inflammatory processes of the gastrointestinal tract are thiazolidinediones - peroxisome proliferator-activated receptors gamma (PPAR-γ) agonists, whose beneficial effects on macroscopic and histopathological features of colitis have been confirmed in numerous studies. In the present study, possible effects of PPAR-γ agonists rosiglitazone and troglitazone enhancing the resistance of colonic tissues to the damaging factor were examined and compared. Rats received the food with 0.01% rosiglitazone or troglitazone for 4 weeks; during the final 2 weeks, colitis-inducing 1.5% DSS (dextran sodium sulfate) was additionally administered in the drinking water. The large intestine specimens were microscopically evaluated and the levels of Th1- (IL-2, INF) and Th2-dependent (IL-4, IL-10) cytokines were determined in the serum and intestinal homogenates. Prophylactic treatment with rosiglitazone and troglitazone ameliorated colitis substantially down-regulating the microscopic inflammatory parameters. Rosiglitazone and troglitazone administered before the induction of colitis exerted comparable effects on colitis. Both substances significantly reduced the levels of pro-inflammatory cytokines and increased the levels of inflammation-limiting cytokines. We conclude that thiazolidinedione drugs are likely to be successfully used for therapy and prevention of non-specific bowel diseases.

Phylogenetic and molecular characteristics of Eurasian H9 avian influenza viruses and their detection by two different H9-specific RealTime reverse transcriptase polymerase chain reaction tests.

Avian influenza viruses (AIVs) of the H9 haemagglutinin subtype are endemic in many Asian and Middle-East countries, causing mortality and morbidity in poultry. Consequently there is a need for accurate and sensitive detection of Eurasian H9 subtype viruses. Two H9 RealTime reverse transcriptase polymerase chain reaction (RRT-PCR) tests, developed by Monne et al. (2008) and Ben Shabat et al. (2010), were originally validated with a limited number of H9 specimens. In the present study, the two tests have been assessed using 66 diverse H9 isolates and 139 clinical specimens from six H9 poultry outbreaks in four geographically disparate Eurasian countries. The Monne et al. (2008) test was modified and successfully detected all H9 viruses from all three Eurasian H9 lineages. Bayesian analysis of the clinical specimens' results revealed this test to be more sensitive (97%) than the Ben Shabat et al. (2010) test (31%). The latter test detected most H9 isolates of the G1 lineage, but no isolates from other H9 lineages. Mismatches in the primer/probe binding sequences accounted for sensitivity differences between the two H9 RRT-PCRs. Genetic analysis of 34 sequenced H9 haemagglutinin genes showed the South Asian and Middle-East H9 isolates to belong to the H9 G1 lineage, and possessed residues that appear to preferably bind alpha 2,6-linked sialic acid receptors which indicate a potential for human infection. European H9s clustered phylogenetically in a broader geographical group that includes recent North American H9 wild bird isolates and contemporary Asian viruses in the Y439 H9 lineage.

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.

First reported detection of a low pathogenicity avian influenza virus subtype H9 infection in domestic fowl in England.

In December 2010, infection with a H9N1 low pathogenicity avian influenza (LPAI) virus was detected in a broiler breeder flock in East Anglia. Disease suspicion was based on acute drops in egg production in two of four sheds on the premises, poor egg shell quality and evidence of diarrhoea. H9N1 LPAI virus infection was confirmed by real-time reverse transcription PCR. Sequencing revealed high nucleotide identity of 93.6 per cent and 97.9 per cent with contemporary North American H9 and Eurasian N1 genes, respectively. Attempted virus isolation in embryonated specific pathogen free (SPF) fowls' eggs was unsuccessful. Epidemiological investigations were conducted to identify the source of infection and any onward spread. These concluded that infection was restricted to the affected premises, and no contacts or movements of poultry, people or fomites could be attributed as the source of infection. However, the infection followed a period of extremely cold weather and snow which impacted on the biosecurity protocols on site, and also led to increased wild bird activity locally, including waterfowl and game birds around the farm buildings. Analysis of the N1 gene sequence suggested direct introduction from wild birds. Although H9 infection in poultry is not notifiable, H9N2 LPAI viruses have been associated with production and mortality episodes in poultry in many parts of Asia and the Middle East. In the present H9N1 outbreak, clinical signs were relatively mild in the poultry with no mortality, transient impact on egg production and no indication of zoonotic spread. However, this first reported detection of H9 LPAI virus in chickens in England was also the first H9 UK poultry case for 40 years, and vindicates the need for continued vigilance and surveillance of avian influenza viruses in poultry populations.