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.

DISCONTOOLS: Identifying gaps in controlling bovine spongiform encephalopathy.

This article summarizes the 2016 update of the DISCONTOOLS project gap analysis on bovine spongiform encephalopathy (BSE), which was based on a combination of literature review and expert knowledge. Uncertainty still exists in relation to the pathogenesis, immunology and epidemiology of BSE, but provided that infected material is prohibited from entering the animal feed chain, cases should continue to decline. BSE does not appear to spread between cattle, but if new strains with this ability appear then control would be considerably more difficult. Atypical types of BSE (L-BSE and H-BSE) have been identified, which have different molecular patterns and pathology, and do not display the same clinical signs as classical BSE. Laboratory transmission experiments indicate that the L-BSE agent has zoonotic potential. There is no satisfactory conclusion regarding the origin of the BSE epidemic. C-BSE case numbers declined rapidly following strict controls banning ruminant protein in animal feed, but occasional cases still occur. It is unclear whether these more recent cases indicate inadequate implementation of the bans, or the possibility that C-BSE might occur spontaneously, as has been postulated for H- and L-BSE. All of this will have implications once existing bans and levels of surveillance are both relaxed. Immunochemical tests can only be applied post-mortem. There is no immunological basis for diagnosis in the live animal. All aspects of disease control are expensive, particularly surveillance, specified risk material removal and feed controls. There is pressure to relax feed controls, and concurrent pressure from other sources to reduce surveillance. While the cost benefit argument can be applied successfully to either of these approaches, it would be necessary to maintain the ban on intraspecies recycling and some baseline surveillance. However, the potential risk is not limited to intraspecies recycling; recycling with cross-species transmission may be an ideal way to select or/and modify properties of transmissible spongiform encephalopathies agents in the future.

Intestinal carriage of verocytotoxigenic Escherichia coli O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica, in cattle, sheep and pigs at slaughter in Great Britain during 2003.

An abattoir survey was undertaken to determine the prevalence of foodborne zoonotic organisms colonizing cattle, sheep and pigs at slaughter in Great Britain. The study ran for 12 months from January 2003, involved 93 abattoirs and collected 7703 intestinal samples. The design was similar to two previous abattoir surveys undertaken in 1999-2000 allowing comparisons. Samples were examined for VTEC O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica. The prevalence of VTEC O157 faecal carriage was 4.7% in cattle, 0.7% in sheep and 0.3% in pigs. A significant decrease in sheep was detected from the previous survey (1.7%). Salmonella carriage was 1.4% in cattle, a significant increase from the previous survey of 0.2%. In sheep, faecal carriage was 1.1% a significant increase from the previous survey (0.1%). In pigs, carriage was 23.4%, consistent with the previous study. Thermophilic Campylobacter spp. were isolated from 54.6% of cattle, 43.8% of sheep and 69.3% of pigs. Y. enterocolitica was isolated from 4.5% of cattle, 8.0% of sheep and 10.2% of pigs.

Nocardia sepsis in a multigravida with systemic lupus erythematosus and autoimmune hepatitis.

This report describes a pregnant woman with systemic lupus erythematosus and autoimmune hepatitis who presented with threatened labour and acute renal failure. She developed respiratory distress, haematemesis and became coagulopathic. Intrauterine death occurred and she was admitted to the intensive care unit after caesarean section. She suffered sudden cardiovascular collapse and succumbed. At autopsy, Nocardia was cultured from multiple renal abscesses. The co-existence of Nocardia sepsis, systemic lupus erythematosus, autoimmune hepatitis and pregnancy are discussed. This case illustrates diagnostic challenges associated with Nocardia infection in the presence of co-existing disease.

Rapid detection of Escherichia coli virulence factor genes using multiplex real-time TaqMan PCR assays.

Three multiplex real-time TaqMan PCR assays were developed for the detection of Escherichia coli virulence factor genes in veterinary samples. Target virulence factors chosen were the fimbriae K88 (F4), K99 (F5), F41, F17, F18 and 987p (F6) and the toxins LT, STa and CDT IV. Detection of genes coding GAD were included in each assay as an internal control. These assays allow rapid identification of virulence factor genes using identical cycling conditions on an Mx3000Ptrade mark real-time PCR machine with the capacity to test up to 20 strains for 9 virulence genes in 1h.

Development and initial characterization of xenomitochondrial mice.

Xenomitochondrial mice harboring trans-species mitochondria on a Mus musculus domesticus (MD) nuclear background were produced. We created xenomitochondrial ES cell cybrids by fusing Mus spretus (MS), Mus caroli (MC), Mus dunni (Mdu), or Mus pahari (MP) mitochondrial donor cytoplasts and rhodamine 6-G treated CC9.3.1 or PC4 ES cells. The selected donor backgrounds reflected increasing evolutionary divergence from MD mice and the resultant mitochondrial-nuclear mismatch targeted a graded respiratory chain defect. Homoplasmic (MS, MC, Mdu, and MP) and heteroplasmic (MC) cell lines were injected into MD ova, and liveborn chimeric mice were obtained (MS/MD 18 of 87, MC/MD 6 of 46, Mdu/MD 31 of 140, and MP/MD l of 9 founder chimeras, respectively). Seven MS/MD, 1 MC/MD, and 11 Mdu/MD chimeric founder females were mated with wild-type MD males, and 18 of 19 (95%) were fertile. Of fertile females, only one chimeric MS/MD (1% coat color chimerism) and four chimeric Mdu/MD females (80-90% coat color chimerism) produced homoplasmic offspring with low efficiency (7 of 135; 5%). Four male and three female offspring were homoplasmic for the introduced mitochondrial backgrounds. Three male and one female offspring proved viable. Generation of mouse lines using additional female ES cell lineages is underway. We hypothesize that these mice, when crossbred with neurodegenerative-disease mouse models, will show accelerated age-related neuronal loss, because of their suboptimal capacity for oxidative phosphorylation and putatively increased oxidative stress.

Determinants of bone density in 30- to 65-year-old women: a co-twin study.

UNLABELLED: Reported effects of body composition and lifestyle on bone mineral density in pre-elderly adult women have been inconsistent. In a co-twin study, we measured bone mineral density, lean and fat mass, and lifestyle factors. Analyzing within pair differences, we found negative associations between bone mineral density and tobacco use (2.3-3.3% per 10 pack-years) and positive associations with sporting activity and lean and fat mass. INTRODUCTION: Reported effects of body composition and lifestyle of bone mineral density in pre-elderly adult women have been inconsistent. METHODS: In a co-twin study of 146 female twin pairs aged 30 to 65 years, DXA was used to measure bone mineral density at the lumbar spine, total hip, and forearm, total body bone mineral content, and lean and fat mass. Height and weight were measured. Menopausal status, dietary calcium intake, physical activity, current tobacco use, and alcohol consumption were determined by questionnaire. Within-pair differences in bone measures were regressed through the origin against within-pair differences in putative determinants. RESULTS: Lean mass and fat mass were associated with greater bone mass at all sites. A discordance of 10 pack-years smoking was related to a 2.3-3.3% (SE, 0.8-1.0) decrease in bone density at all sites except the forearm, with the effects more evident in postmenopausal women. In all women, a 0.8% (SE, 0.3) difference in hip bone mineral density was associated with each hour per week difference in sporting activity, with effects more evident in premenopausal women. Daily dietary calcium intake was related to total body bone mineral content and forearm bone mineral density (1.4 +/- 0.7% increase for every 1000 mg). Lifetime alcohol consumption and walking were not consistently related to bone mass. CONCLUSION: Several lifestyle and dietary factors, in particular tobacco use, were related to bone mineral density. Effect sizes varied by site. Characterization of determinants of bone mineral density in midlife and thereafter may lead to interventions that could minimize postmenopausal bone loss and reduce osteoporotic fracture risk.

A comparison of antimicrobial susceptibilities in nontyphoidal salmonellas from humans and food animals in England and Wales in 2000.

A joint study by the Public Health Laboratory Service and the Veterinary Laboratories Agency of resistance to antimicrobials in isolates of Salmonella enterica serotypes Enteritidis, Typhimurium, Hadar, and Virchow from humans and food-producing animals in England and Wales in 2000 has demonstrated that resistance was most common in Typhimurium, particularly in strains of definitive phage type (DT) 104. However resistance was also common in other phage types, particularly DTs 193 and 208 and phage type U302. Multiresistant strains of DT208 appeared to be predominantly associated with pigs; for the other phage types, the human/food-producing animal relationships of drug-resistant isolates were more complex. For Enteritidis, Virchow, and Hadar, there were substantial differences in the resistance spectra of isolates from humans and food-producing animals, suggesting that food-producing animals bred in England and Wales may not be the primary sources of drug-resistant strains of these serotypes causing infections in humans. Further phenotypic and molecular comparison of drug-resistant isolates of these serotypes may be required to ascertain the sources of strains responsible for infections in humans.

Investigation of the genetic diversity among isolates of Salmonella enterica serovar Dublin from animals and humans from England, Wales and Ireland.

AIMS: To assess the degree of genetic diversity among animal Salmonella Dublin UK isolates, and to compare it with the genetic diversity found among human isolates from the same time period. METHODS AND RESULTS: One hundred isolates (50 human and 50 animal) were typed using plasmid profiling, XbaI-pulsed field gel electrophoresis (PFGE) and PstI-SphI ribotyping. Antimicrobial resistance data to 16 antibiotics was presented, and the presence of class-I integrons was investigated by real-time PCR. Seven different plasmid profiles, 19 ribotypes and 21 PFGE types were detected. A combination of the three methods allowed clear differentiation of 43 clones or strains. Eighteen isolates were resistant to at least one antimicrobial; five of them were multi-resistant and of these, only three presented class I integrons. CONCLUSIONS: Ribotyping data suggest the existence of at least three very different clonal lines; the same distribution in well-defined groups was not evident from the PFGE data. The existence of a variety of clones in both animals and humans has been demonstrated. A few prevalent clones seem to be widely disseminated among different animal species and show a diverse geographical and temporal distribution. The same clones were found in animals and humans, which may infer that both farm and pet animals may act as potential vehicles of infection for humans. Some other clones seem to be less widely distributed. Clustering analysis of genomic fingerprints of Salmonella Dublin and Salm. Enteritidis isolates confirms the existence of a close phylogenetic relationship between both serotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper describes the utility of a multiple genetic typing approach for Salm. Dublin. It gives useful information on clonal diversity among human and animal isolates.

Gonadotrophin surge-induced upregulation of mRNA for plasminogen activator inhibitors 1 and 2 within bovine periovulatory follicular and luteal tissue.

The serine proteinases, tissue-type (tPA) and urokinase (uPA) plasminogen activator, are implicated in the ovulatory processes via their ability to convert plasminogen to its active form, plasmin. One mechanism for regulation of plasmin-directed ovarian extracellular matrix remodelling during follicle rupture and corpus luteum formation is through inhibition of plasminogen activation by the plasminogen activator inhibitors (PAI-1 and PAI-2). The effect of the preovulatory gonadotrophin surge on the temporal and spatial regulation of expression of PAI-1 and PAI-2 mRNA and PAI activity in preovulatory bovine follicles and new corpora lutea collected at 0, 6, 12, 18, 24 and 48 h after a GnRH-induced gonadotrophin surge was examined. Both PAI-1 and PAI-2 mRNAs were upregulated markedly after the gonadotrophin surge, with the highest expression observed in follicles collected at about the time of ovulation (24 h) and in corpora lutea (48 h). PAI-1 mRNA was localized primarily to the thecal layer of preovulatory follicles. In contrast, PAI-2 mRNA was localized specifically to the granulosa cell layer. Significant PAI activity was detected in follicle extracts, but temporal or spatial differences in PAI activity were not detected in response to the gonadotrophin surge. These results indicate that PAI-1 and PAI-2 mRNAs are upregulated in preovulatory bovine follicles after the gonadotrophin surge in a cell-specific way. Regulation of PAI-1 and PAI-2 may help to control plasminogen activator activity associated with ovulation and early corpus luteum formation.

Effect of the preovulatory LH surge on bovine follicular progesterone receptor mRNA expression.

A growing body of evidence indicates that intrafollicular progesterone receptor signaling pathways are obligatory for follicle rupture. However, the intrafollicular localization and regulation of progesterone receptor expression during the periovulatory period in cattle are not known. In this study, we determined the effect of the preovulatory gonadotropin surge on localization and expression of progesterone receptor mRNA in bovine periovulatory follicular and luteal tissue. Ovaries containing preovulatory follicles or new corpora lutea (CL) were collected at approximately 0, 6, 12, 18, 24 (preovulatory follicles) and 48 h (CL) after a GnRH-induced LH surge (n=5-8 per timepoint). Expression of progesterone receptor mRNA was detected in periovulatory follicular and luteal tissue at all timepoints examined. Relative levels of progesterone receptor mRNA were dramatically upregulated within 6h after the LH surge compared to all other time points (P<0.0001). In situ hybridization analysis revealed that the significant increase in progesterone receptor mRNA expression was localized to the granulosal layer of preovulatory follicles. Our results indicate that progesterone receptor mRNA expression is upregulated specifically in the granulosal layer of bovine preovulatory follicles following the LH surge. Progesterone receptor signaling pathways may help mediate the effects of the preovulatory LH surge on follicle rupture in cattle.

The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand.

On the western border of Thailand, Plasmodium falciparum has become resistant to almost all antimalarial agents. The molecular basis of resistance in these parasite populations has not been well characterized. This study assessed genetic polymorphisms in the pfmdr1 gene in 54 parasites collected from the western border of Thailand to determine the relationship of pfmdr1 copy number and codon mutations with parasite sensitivities to mefloquine, chloroquine, halofantrine, quinine, and artesunate assessed in vitro. A point mutation at codon 86 (resulting in a change of Asn to Tyr) was associated with a significantly lower 50% inhibitory concentration (IC(50)) of mefloquine (median, 9 ng/ml versus 52.4 ng/ml; P = 0.003). Overall 35% of the isolates (19 of 54) had an increase in pfmdr1 copy number, and all 19 carried the wild-type allele at codon 86. Increased pfmdr1 copy number was associated with higher IC(50)s of mefloquine (P = 0.04) and artesunate (P = 0.005), independent of polymorphism at codon 86. The relationship between pfmdr1 and resistance to structurally distinct antimalarial agents confirms the presence of a true multidrug-resistant phenotype.

Trisodium phosphate increases sensitivity of gram-negative bacteria to lysozyme and nisin.

Cell suspensions of Campylobacter jejuni, Escherichia coli, Pseudomonas fluorescens, and Salmonella enteritidis exposed to sublethal concentrations (0.5 to 5 mM) of trisodium phosphate (TSP) for 10 min showed greatly increased susceptibility to lysozyme (10 micrograms ml-1) and/or nisin (1 microM). Under optimal conditions at 37 degrees C, reductions in viable count after 30 min were up to six log cycles. At 4 degrees C, C. jejuni showed greater resistance than at 37 degrees C, and maximal cell kills (95%) were reduced by more than two log cycles. Cells dried on the surface of chicken skin were more resistant than suspended cells to TSP-lysozyme and TSP-nisin treatments; nevertheless, at 37 degrees C, kills varied from approximately 95% for S. enteritidis cells with nisin (30 microM) or lysozyme (100 micrograms ml-1) to > 99.9% for C. jejuni and E. coli cells with nisin. Under the experimental conditions used, nisin also reduced viable counts of skin-attached Staphylococcus aureus by > 99.9%. The results suggest that the high TSP concentrations (approximately 10% wt/vol, 0.25 M) needed for successful decontamination of gram-negative bacteria, on the surface of poultry and other foodstuffs, may be substantially reduced by following TSP treatment with exposure to low lysozyme or nisin concentrations.

An in situ method for determining bacterial survival on food preparation surfaces using a redox dye.

A simple method is described for the direct enumeration of viable bacteria dried on test surfaces. Inoculated surfaces were overlayed with agar and after incubation nitroblue tetrazolium solution (pale yellow) was used to stain colonies (purple) at the agar-test surface interface. Stained colonies could be readily detected and counted even against the opaque background of ceramic tile or stainless steel or when present within opaque films of milk or serum. Recovery of bacterial by this method was approximately fivefold greater than using a conventional swabbing procedure. The method was used to demonstrate the marked effect of the composition of the suspension fluid, in which bacteria were dried, and the length of surface exposure upon bacterial survival.