Validation, certification and registration of veterinary diagnostic test kits by the World Organisation for Animal Health Secretariat for Registration of Diagnostic Kits.

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.

Examples of proper reporting for evaluation (Stage 2 validation) of diagnostic tests for diseases listed by the World Organisation for Animal Health.

Reporting and design standards are key indicators of the quality of diagnostic accuracy (validation) studies but, with the exception of aquatic animal diseases and paratuberculosis in ruminants, there is limited guidance for designing these studies in animals. There is, therefore, a need for generic guidelines that are based on disease characteristics, such as mode of transmission, latent period and pathogenesis. Comprehensive, clear and transparent reporting of primary test accuracy studies for diseases listed by the World Organisation for Animal Health (OIE) has value for the end users of diagnostic tests and, ultimately, for decision-makers, who require systematic reviews and meta-analysis of multiple tests for specified diseases and testing purposes. The recent publication of reporting standards for Bayesian latent class models, to analyse test-accuracy data from naturally occurring disease events, fills an important gap as these methods are being increasingly used for OIE-listed diseases. Adherence to design and reporting standards, as well as to guidelines, helps to ensure that research funding for test validation studies is used appropriately and that the strengths and limitations of single tests or test combinations are made clear to test users. The authors provide a review of key points that are often overlooked or misinterpreted in test validation studies, as well as two concrete examples of good practice for use as a reference point for future studies.

Les normes de notification et de conception sont des indicateurs essentiels de la qualité des études de validation des tests destinées à déterminer leur exactitude diagnostique ; or, en dehors des maladies des animaux aquatiques et de la paratuberculose chez les ruminants, il n'existe guère de lignes directrices pour concevoir ce type d'études pour les tests utilisés en santé animale. À la connaissance des auteurs, il n'existe pas non plus de normes de conception applicables aux études de validation en santé humaine. Par conséquent, il conviendrait de disposer de lignes directrices génériques fondées sur les caractéristiques des maladies telles que leurs modalités de transmission, leur période de latence et leur pathogénie. Une notification complète, claire et transparente des études d'exactitude des tests primaires pour les maladies listées par l'Organisation mondiale de la santé animale (OIE) serait une aide précieuse pour les utilisateurs finaux des tests de diagnostic, mais aussi pour les responsables de l'élaboration des politiques, dont les décisions reposent sur des examens et des méta-analyses systématiques couvrant un grand nombre de tests pour certaines maladies ou pour certains usages d'un test. La publication récente des normes de notification applicables aux modèles bayésiens à classe latente pour analyser les données de performance d'un test à partir de foyers naturels de maladie comble une lacune importante dans la mesure où ces méthodes sont de plus en plus utilisées pour les maladies listées par l'OIE. L'adhésion à des normes de conception et de notification ainsi qu'à des lignes directrices en la matière permettra de garantir que les fonds alloués aux études de validation des tests sont bien utilisés et que les atouts et les limitations de certains tests individuels ou associations de tests sont clairement perçus par les utilisateurs. Les auteurs passent en revue certains points essentiels qui sont souvent ignorés ou mal interprétés lors des études de validation des tests et proposent deux exemples concrets de bonnes pratiques qui pourront servir de références pour les études à venir.

Las normas de comunicación y diseño son indicadores básicos de la calidad de los estudios encaminados a determinar la exactitud de diagnóstico (validación) pero, con la excepción de las enfermedades de los animales acuáticos y la paratuberculosis en rumiantes, hay escasas directrices que se apliquen al diseño de esos estudios en animales. Además, hasta donde saben los autores, en el ámbito de la salud humana no hay normas de diseño. De ahí la necesidad de directrices genéricas que estén basadas en las características de las enfermedades, como modo de transmisión, período de latencia o patogénesis. La comunicación exhaustiva, clara y transparente de estudios primarios sobre la exactitud de pruebas de diagnóstico de enfermedades incluidas en las listas de la Organización Mundial de Sanidad Animal (OIE) reviste utilidad no solo para los usuarios finales de la prueba, sino también, en última instancia, para los órganos decisorios, que necesitan metaanálisis y estudios sistemáticos de múltiples pruebas que se apliquen a una u otra enfermedad y sirvan para una u otra finalidad. La reciente publicación de normas de comunicación de modelos bayesianos de clases latentes para analizar los datos de exactitud de pruebas a partir de episodios de enfermedad de origen natural viene a colmar una importante laguna, en la medida en que estos métodos se aplican cada vez más al diagnóstico de enfermedades incluidas en las listas de la OIE. El cumplimiento de las normas de diseño y comunicación, y también de las directrices, ayuda a garantizar que los fondos de investigación destinados a estudios de validación de pruebas sean utilizados debidamente y que el usuario final de una prueba reciba información clara sobre los puntos fuertes y las limitaciones de una prueba o combinación de pruebas. Los autores pasan revista a los principales aspectos que se suelen pasar por alto o malinterpretar en los estudios de validación de pruebas y ofrecen dos ejemplos concretos de buenas prácticas que se pueden utilizar como referencia en futuros estudios.

Introduction - Validation of tests for OIE-listed diseases as fit-for-purpose in a world of evolving diagnostic technologies.

The World Organisation for Animal Health (OIE) has made leading contributions to the discipline of test validation science by providing standards and guidelines that inform the test validation process in terrestrial and aquatic animals. The OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, and the Manual of Diagnostic Tests for Aquatic Animals describe the test validation pathway in the context of fitness for purpose, elaborate on the importance of diagnostic sensitivity (DSe) and specificity (DSp) as measures of test accuracy, and designate additional factors (e.g. test cost, laboratory throughput capacity and rapidity of test results) that influence choices of a single test over others or the inclusion of a new test in a diagnostic process that includes multiple tests. This paper provides examples of each of the six main testing purposes listed in the Terrestrial Manual and describes additional metrics such as ruggedness and robustness that should be included in the validation of point-of-care tests. Challenges associated with new diagnostic technologies and platforms are described. Validated tests with estimates of DSe and DSp are needed to measure confidence in test results for OIE-listed diseases, to facilitate risk assessments related to animal movement, to estimate true prevalence, and for certification of disease freedom and use in epidemiological (risk factor) studies.

L'Organisation mondiale de la santé animale (OIE) a apporté d'importantes contributions dans le domaine de la validation des tests en élaborant des normes et des lignes directrices qui informent sur le processus de validation des tests chez les animaux terrestres et aquatiques. Le Manuel des tests de diagnostic et des vaccins pour les animaux terrestres et le Manuel des tests de diagnostic pour les animaux aquatiques de l'OIE décrivent le processus de validation des tests dans le contexte de leur aptitude à l'emploi, expliquent l'importance de la sensibilité (DSe) et de la spécificité (DSp) diagnostiques pour mesurer l'exactitude des tests, et désignent d'autres facteurs (ex. coût des tests, capacité de traitement des laboratoires et rapidité d'obtention des résultats des tests) qui influencent le choix d'un test par rapport à un autre ou l'inclusion d'un nouveau test dans un processus de diagnostic composé de multiples tests. Le présent article fournit des exemples pour chacun des six principaux objectifs définis pour les tests figurant dans le Manuel terrestre et décrit des mesures supplémentaires, telle la robustesse (aussi bien interne qu'externe), qu'il conviendrait d'inclure dans la validation des tests au point d'intervention. Il aborde également les défis soulevés par les nouvelles technologies et plateformes de diagnostic. Des tests validés accompagnés d'estimations de la DSe et de la DSp sont nécessaires pour mesurer la fiabilité des résultats des tests pour les maladies listées par l'OIE, faciliter les évaluations des risques associés aux mouvements des animaux, estimer le véritable taux de prévalence et certifier l'absence de maladies ; ils sont également indispensables pour les études (des facteurs de risque) épidémiologiques.

La Organización Mundial de Sanidad Animal (OIE), con su labor de elaboración de normas y directrices que fundamentan el proceso de validación de pruebas para enfermedades de los animales terrestres y acuáticos, ha hecho aportaciones punteras a la disciplina científica que se ocupa de la validación de pruebas. En su Manual de las Pruebas de Diagnóstico y de las Vacunas para los Animales Terrestres y su Manual de las Pruebas de Diagnóstico para los Animales Acuáticos, la OIE describe el procedimiento de validación de pruebas en clave de idoneidad para determinados propósitos, ahonda en la importancia de la sensibilidad y la especificidad diagnósticas (DSe y DSp) como medidas de la exactitud de una prueba y señala otros factores (como el costo de la prueba, la productividad del laboratorio o la rapidez de los resultados) que también influyen en la elección de una determinada prueba por delante de otras o en la inclusión de una nueva prueba en un proceso de diagnóstico que entraña el uso de varias. Los autores ofrecen ejemplos de cada uno de los seis principales propósitos con las que puede utilizarse una prueba, según vienen enunciados en el Manual Terrestre, y describen otros parámetros que es preciso tener en cuenta a la hora de validar pruebas practicadas en el punto de consulta, como la robustez o también la solidez (ruggedness en inglés; llamada a veces «robustez interlaboratorios¼). También describen las dificultades ligadas a nuevas tecnologías y plataformas de diagnóstico. Se necesitan pruebas validadas y acompañadas de un cálculo de la DSe y la DSp para fines tan diversos e importantes como medir la confianza que merecen los resultados de pruebas para enfermedades inscritas en las listas de la OIE, facilitar la evaluación del riesgo ligado al desplazamiento de animales, estimar la prevalencia real, certificar la ausencia de enfermedad o realizar estudios epidemiológicos (factores de riesgo).

Design standards for experimental and field studies to evaluate diagnostic accuracy of tests for infectious diseases in aquatic animals.

Design and reporting quality of diagnostic accuracy studies (DAS) are important metrics for assessing utility of tests used in animal and human health. Following standards for designing DAS will assist in appropriate test selection for specific testing purposes and minimize the risk of reporting biased sensitivity and specificity estimates. To examine the benefits of recommending standards, design information from published DAS literature was assessed for 10 finfish, seven mollusc, nine crustacean and two amphibian diseases listed in the 2017 OIE Manual of Diagnostic Tests for Aquatic Animals. Of the 56 DAS identified, 41 were based on field testing, eight on experimental challenge studies and seven on both. Also, we adapted human and terrestrial-animal standards and guidelines for DAS structure for use in aquatic animal diagnostic research. Through this process, we identified and addressed important metrics for consideration at the design phase: study purpose, targeted disease state, selection of appropriate samples and specimens, laboratory analytical methods, statistical methods and data interpretation. These recommended design standards for DAS are presented as a checklist including risk-of-failure points and actions to mitigate bias at each critical step. Adherence to standards when designing DAS will also facilitate future systematic review and meta-analyses of DAS research literature.

Sensitivity and specificity of real-time PCR and bacteriological culture for francisellosis in farm-raised Nile tilapia (Oreochromis niloticus L.).

Despite the worldwide occurrence of Francisella noatunensis subsp. orientalis (Fno) infection in farmed tilapia, sensitivity and specificity estimates of commonly used diagnostic tests have not been reported. This study aimed to estimate the sensitivity and specificity of bacteriological culture and qPCR to detect Fno infection. We tested 559 fish, sampled from four farms with different epidemiological scenarios: (i) healthy fish in a hatchery free of Fno; (ii) targeted sampling of diseased fish with suggestive external clinical signs of francisellosis during an outbreak; (iii) convenience sampling of diseased and clinically healthy fish during an outbreak; and (iv) sampling of healthy fish in a cage farm without a history of outbreaks, but with francisellosis reported in other farms in the same reservoir. The qPCR had higher median sensitivity (range, 48.8-99.5%) than culture (range, 1.6-74.4%). Culture had a substantially lower median sensitivity (1.6%) than qPCR (48.8%) to detect Fno in carrier tilapia (farm 4). Median specificity estimates for both tests were >99.2%. The qPCR is the superior test for use in surveillance and monitoring programmes for francisellosis in farmed Nile tilapia, but both tests have high sensitivity and specificity which make them fit for use in the diagnosis of Fno outbreaks.

Sudden death in racing Thoroughbred horses: an international multicentre study of post mortem findings.

REASONS FOR PERFORMING STUDY: To improve the understanding of exercise related sudden death in Thoroughbred racehorses. OBJECTIVES: To describe the post mortem findings in cases of sudden death associated with exercise in 268 Thoroughbred racehorses. METHODS: Gross and histological post mortem findings of 268 cases of sudden death were collated and reviewed. Cases originated from 6 racing jurisdictions around the world. Sudden death was defined as acute collapse and death in a closely observed and previously apparently healthy Thoroughbred racehorse, during, or within one hour after, exercise. Cause of death as determined by the attending pathologist was categorised as definitive, presumptive or unexplained and compared between the different populations. Cardiopulmonary lesions recorded at post mortem examination were compared between different populations. RESULTS: Pathologists recorded a definitive cause of death in 53% (143/268) of cases. Major definitive causes of sudden death included cardiac failure, apparent pulmonary failure, pulmonary haemorrhage, haemorrhage associated with pelvic fractures or with idiopathic blood vessel rupture, and spinal cord injury. A presumptive cause of death was made in 25% (67/268) of cases and death remained unexplained in 22% (58/268) of cases. There were several statistically significant inter-population differences in the cause of death and in reporting of cardiopulmonary lesions. CONCLUSIONS: Sudden death can be attributed to a variety of causes. Causes of sudden death and the lesions found in cases of exercise-related sudden death are similar in different racing jurisdictions. However, the lesions are often not specific for the cause of death and determination of the cause of death is therefore affected by interpretation by the individual pathologist.

Accumulation and metabolism of drugs and CYP probe substrates in zebrafish larvae.

This study examined the accumulation and metabolism of a number of drugs and commonly used probes for human cytochrome P450s (CYPs) in zebrafish larvae under conditions relevant to pharmacological and toxicological assays. Studies with cisapride, chlorpromazine, verapamil, testosterone, and dextromethorphan showed that the zebrafish larvae catalyze a range of phase 1 (oxidation, N-demethylation, O-de-ethylation, and N-dealkylation) and phase 2 (sulfation and glucuronidation) reactions. Both similarities and differences in the metabolic pathways were observed in zebrafish larvae when compared to mammals. Metabolism of phenacetin to paracetamol and dextromethorphan to dextrorphan (metabolic reactions catalyzed by CYP 1A2 and 2D6 in humans respectively) were observed in the zebrafish larvae. In addition the zebrafish larvae 7 days post fertilization (7 d.p.f.) hydroxylated diclofenac, bupropion, tacrine, and testosterone. Although metabolites of several compounds were detected in zebrafish larvae, in the instances where the metabolite amounts were quantified, the amount of any specific metabolite formed was low, accounting for only a small percentage of the amount of parent compound added. Furthermore, when the concentrations of metabolite present in the zebrafish larvae were compared with the measured level of parent compound, the metabolite concentrations were always much lower than that of parent compound. Overall, for the compounds used in the current study it is unlikely that the quantified metabolites would significantly contribute to the outcome of safety pharmacology or toxicology studies conducted in zebrafish larvae under the paradigms typically used for such investigations.

The REFLECT statement: reporting guidelines for randomized controlled trials in livestock and food safety: explanation and elaboration.

Concerns about the completeness and accuracy of reporting of randomized clinical trials (RCTs) and the impact of poor reporting on decision-making have been documented in the medical field over the past several decades. Experience from RCTs in human medicine would suggest that failure to report critical trial features can be associated with biased estimated effect measures, and there is evidence to suggest similar biases occur in RCTs conducted in livestock populations. In response to these concerns, standardized guidelines for reporting RCTs were developed and implemented in human medicine. The Consolidated Standards of Reporting Trials (CONSORT) statement was first published in 1996 with a revised edition published in 2001. The CONSORT statement consists of a 22-item checklist for reporting a RCT and a flow diagram to follow the number of participants at each stage of a trial. An explanation and elaboration document not only defines and discusses the importance of each of the items, but also provides examples of how this information could be supplied in a publication. Differences between human and livestock populations necessitate modifications to the CONSORT statement to maximize its usefulness for RCTs involving livestock. These have been addressed in an extension of the CONSORT statement titled the REFLECT statement: Methods and processes of creating reporting guidelines for randomized control trials for livestock and food safety. The modifications made for livestock trials specifically addressed the common use of group housing and group allocation to intervention in livestock studies, the use of a deliberate challenge model in some trials, and common use of non-clinical outcomes, such as contamination with a foodborne pathogen. In addition, the REFLECT statement for RCTs in livestock populations proposed specific terms or further clarified terms as they pertained to livestock studies.

The REFLECT statement: methods and processes of creating reporting guidelines for randomized controlled trials for livestock and food safety.

The conduct of randomized controlled trials in livestock with production, health, and food-safety outcomes presents unique challenges that may not be adequately reported in trial reports. The objective of this project was to modify the CONSORT (Consolidated Standards of Reporting Trials) statement to reflect the unique aspects of reporting these livestock trials. A two-day consensus meeting was held on November 18-19, 2008 in Chicago, Ill, United States of America, to achieve the objective. Prior to the meeting, a Web-based survey was conducted to identify issues for discussion. The 24 attendees were biostatisticians, epidemiologists, food-safety researchers, livestock production specialists, journal editors, assistant editors, and associate editors. Prior to the meeting, the attendees completed a Web-based survey indicating which CONSORT statement items may need to be modified to address unique issues for livestock trials. The consensus meeting resulted in the production of the REFLECT (Reporting Guidelines for Randomized Control Trials) statement for livestock and food safety (LFS) and 22-item checklist. Fourteen items were modified from the CONSORT checklist, and an additional sub-item was proposed to address challenge trials. The REFLECT statement proposes new terminology, more consistent with common usage in livestock production, to describe study subjects. Evidence was not always available to support modification to or inclusion of an item. The use of the REFLECT statement, which addresses issues unique to livestock trials, should improve the quality of reporting and design for trials reporting production, health, and food-safety outcomes.

Association between Mycobacterium avium subspecies paratuberculosis infection and milk production in two California dairies.

The association between Mycobacterium avium ssp. paratuberculosis (MAP) and milk production was estimated on 2 California dairies using longitudinal data from 5,926 cows. Both study herds had moderate MAP seroprevalence, housed cows in freestalls, and had Johne's disease control programs. Cow MAP status was determined using both serum ELISA and fecal culture results from cows tested at dry-off and from whole-herd tests. Potential confounders were evaluated based on a causal diagram. Mixed models with 2 functions (splines) for days in milk (DIM) representing milk production pre- and postpeak used in similar studies were further modified to use each cow's observed DIM at peak and lactation length. Cows that were seropositive produced 2.5kg less 4% fat-corrected milk (FCM) per day than their seronegative herdmates. In addition, cows that were fecal-culture positive by liquid culture and confirmed by PCR produced 2.2kg less 4% FCM per day than their fecal-culture negative herdmates. The decrease in milk production in MAP test-positive compared with test-negative cows started in the second lactation. A switch in MAP status in either ELISA or fecal culture results from positive to negative had no significant association with milk production. Modified DIM functions that used the observed DIM at peak had better model fit than another function that assumed a fixed peak at 60 DIM. Cows that tested positive for MAP on serum ELISA or fecal culture produced less milk than cows that tested negative, and the association between MAP and milk production was not confounded by mastitis, elevated somatic cell counts, or uterine or metabolic cow conditions.

The REFLECT statement: methods and processes of creating reporting guidelines for randomized controlled trials for livestock and food safety.

The conduct of randomized controlled trials in livestock with production, health, and food-safety outcomes presents unique challenges that might not be adequately reported in trial reports. The objective of this project was to modify the CONSORT (Consolidated Standards of Reporting Trials) statement to reflect the unique aspects of reporting these livestock trials. A 2-day consensus meeting was held on November 18-19, 2008 in Chicago, IL, to achieve the objective. Before the meeting, a Web-based survey was conducted to identify issues for discussion. The 24 attendees were biostatisticians, epidemiologists, food-safety researchers, livestock production specialists, journal editors, assistant editors, and associate editors. Before the meeting, the attendees completed a Web-based survey indicating which CONSORT statement items would need to be modified to address unique issues for livestock trials. The consensus meeting resulted in the production of the REFLECT (Reporting Guidelines for Randomized Control Trials) statement for livestock and food safety and 22-item checklist. Fourteen items were modified from the CONSORT checklist, and an additional subitem was proposed to address challenge trials. The REFLECT statement proposes new terminology, more consistent with common usage in livestock production, to describe study subjects. Evidence was not always available to support modification to or inclusion of an item. The use of the REFLECT statement, which addresses issues unique to livestock trials, should improve the quality of reporting and design for trials reporting production, health, and food-safety outcomes.

Species differences in the multiple-dose pharmacokinetics of the non-nucleoside reverse transcriptase inhibitor (NNRTI) UK-453,061 in animals and man: implications for safety considerations.

The requirements for safe testing of pharmaceuticals in humans places substantial emphasis on the translation of safety testing performed in animals to man. The comparison of systemic exposure in animals and man has taken on increasing importance in this assessment, with the underlying assumption that plasma concentrations will elicit the same response in different species. This assumption may be flawed for a number of different reasons, one of which is differences in drug disposition between species leading to high doses required in animal species to yield equivalent systemic exposure to humans and consequent higher exposure to organs such as the intestine and liver. Hepatic clearance can vary substantially, particularly between rodents and man, resulting in vast differences in the dose-exposure relationship. A specific example of a non-nucleoside reverse transcriptase inhibitor, which causes substantial auto-induction in rodents, is used to illustrate this situation and the impact this has on the interpretation of safety extrapolation from animals to man. In such circumstances, it is important to recognize the impact of species differences in drug clearance and disposition and consider broader input in the assessment of clinical safety.

Naturally occurring and melengestrol acetate-associated reproductive tract lesions in zoo canids.

As husbandry practices have improved, safe and effective contraception for captive wildlife management has become a necessity. Melengestrol acetate (MGA), a synthetic progestin, is highly effective and has been used in many zoo species. Long-term use of MGA has been associated with uterine lesions in zoo felids, but effects in zoo canids have not been evaluated. This retrospective study documented spontaneously occurring lesions and investigated the impact of MGA on the reproductive health of zoo canids. Reproductive tracts from adult females were submitted by US zoos to the Association of Zoos & Aquariums' Wildlife Contraception Center Health Surveillance Program. Reproductive tracts were sampled and processed for histopathologic examination following standard protocols. Microscopic evaluations were performed without prior knowledge of MGA treatment status. Prevalence of uterine lesions was evaluated and compared between MGA-treated animals (n = 20) and control (untreated) animals (n = 61). Common lesions within the study population as a whole included endometrial hyperplasia (predominantly cystic) (53%), hydrometra (33%), and adenomyosis (25%). Treatment with MGA was a risk factor for endometrial hyperplasia, hydrometra, fibrosis, and adenomyosis. Uterine mineralization occurred exclusively in MGA-treated animals. Results indicate that MGA contraception can lead to lesions that may permanently impair the fertility of females. Therefore, if long-term contraception of zoo canids is necessary, the use of alternate methods of reproductive control such as gonadotropin-releasing hormone (GnRH) analogs or GnRH vaccines that reduce gonadal hormone exposure should be pursued.

Reliability of environmental sampling to quantify Mycobacterium avium subspecies paratuberculosis on California free-stall dairies.

The reliability of environmental sampling to quantify Mycobacterium avium ssp. paratuberculosis (MAP) based on collector and time was evaluated. Fecal slurry samples were collected using a standardized protocol simultaneously by 2 collectors of different experience levels. Samples were collected from 30 cow pens on 4 dairies every other day on 3 occasions while cow movements between pens were minimal. The 4 study herds had moderate MAP seroprevalence and were housed in free-stall dairies in central California. Results of testing the environmental samples for MAP using PCR and culture were strongly correlated. The reliability of environmental sampling simultaneously by different collectors as estimated by the intraclass correlation coefficient was excellent (81%) for PCR and good (67%) for culture and may justify comparison of quantitative results of samples collected by different investigators. The reliability of environmental sampling over a 5-d period was good (67 and 64% for PCR and culture results, respectively), which justifies the utility of environmental sampling to identify pens with a high MAP bioburden between routine cow pen changes on a dairy. Environmental sampling of free-stall pens using the standardized sampling protocol yielded comparable PCR and culture results across collectors with different experience levels and at different times within a 5-d period.

VIVD: Virtual in vitro distribution model for the mechanistic prediction of intracellular concentrations of chemicals in in vitro toxicity assays.

In vitro toxicity testing routinely uses nominal treatment concentrations as the driver for measured toxicity endpoints. However, test compounds can bind to the plastic of culture vessels or interact with culture media components, such as lipids and albumin. Additionally, volatile compounds may partition into the air above culture media. These processes reduce the free concentrations of compound to which cells are exposed. Models predicting the freely dissolved concentrations by accounting for these interactions have been published. However, these have only been applied to neutral compounds or assume no differential ionisation of test compounds between the media and cell cytoplasm. Herein, we describe an in vitro distribution model, based on the Fick-Nernst Planck equation accounting for differential compound ionisation in culture medium and intracellular water. The model considers permeability of ionised and unionised species and accounts for membrane potential in the partitioning of ionised moieties. By accounting for lipid and protein binding in culture medium, binding to cell culture plastic, air-partitioning, and lipid binding in the cell, the model can predict chemical concentrations (free and total) in medium and cells. The model can improve in vitro in vivo extrapolation of toxicity endpoint by determining intracellular concentrations for translation to in vivo.

Strategies to prevent N-acetyltransferase-mediated metabolism in a series of piperazine-containing pyrazalopyrimidine compounds.

1. (1-Methyl-5-piperazine-1-yl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)-(5-methyl-pyridin-2-yl)-amine (UK-469,413) was identified as a lead compound in a new medicinal chemistry programme. UK-469,413 had good physicochemical properties and was slowly metabolized by cytochromes P450 in rat and human liver microsomes. 2. In the rat in vivo the compound was rapidly cleared. Subsequent studies showed that UK-469,413 was rapidly acetylated in rat liver cytosol to an N-acetylpiperazine metabolite that was the major circulating metabolite in rat plasma in vivo. 3. Analogues of UK-469,413 containing the unsubstituted piperazine moiety were rapidly acetylated in rat liver cytosol and had high plasma clearance in the rat in vivo. These compounds were also acetylated in human liver cytosol and the N-acetyl metabolite was a major metabolite formed in incubations with cryopreserved human hepatocytes. 4. Using specific inhibitors, correlation analysis and expressed human N-acetyltransferase (NAT) enzymes the compounds were shown to be substrates of the polymorphically expressed NAT-2 isozyme. 5. Further experiments showed that it was possible to make small structural changes to the piperazine group that retained potency but prevented metabolism by NAT.

Effects of oral tetrachlorvinphos fly control (Equitrol) administration in horses: physiological and behavioural findings.

Highly reactive horses may pose risks to humans involved in equestrian activities. Among the factors that may affect horses' reactivity to external stimuli are pesticides used for fly control in equine facilities. The organophosphorus (OP) insecticide tetrachlorvinphos (TCVP) is used as a feed-through larvicide to prevent completion of the fly larval life cycle in horse manure. TCVP exerts its effect by inhibiting the enzyme cholinesterase (ChE) leading to the accumulation of the neurotransmitter acetylcholine (AChE) in synapses of the central and peripheral nervous systems. The aim of the present study was to investigate alterations of whole-blood ChE levels associated with feeding a commercially available product (Equitrol, Farnam Companies, Inc.) to horses for fly control. A second aim was to report neurological, physiological and behavioural findings in addition to profiles of selected immune markers (IFN-gamma, IL-12p40 and COX-2) and serum thyroid hormones during and after a 30-day treatment period of TCVP feeding. The results indicated significant decreases in whole-blood ChE activity and concomitant behavioural alterations, manifested as increased reactivity and decreased controllability in treated horses. No changes were detected in physiological or neurological parameters, immune markers or thyroid hormones in treated (n=6) or control (n=4) horses during the course of the study.

Connectivity-based risk ranking of infectious salmon anaemia virus (ISAv) outbreaks for targeted surveillance planning in Canada and the USA.

Infectious salmon anaemia (ISA) can be a serious viral disease of farmed Atlantic salmon (Salmo salar). A tool to rank susceptible farms based on the risk of ISA virus (ISAv) infection spread from infectious farms after initial incursion or re-occurrence in an endemic area, can help guide monitoring and surveillance activities. Such a tool could also support the response strategy to contain virus spread, given available resources. We developed a tool to rank ISAv infection risks using seaway distance and hydrodynamic information separately and combined. The models were validated using 2002-2004 ISAv outbreak data for 30 farms (24 in New Brunswick, Canada and 6 in Maine, United States). Time sequence of infection spread was determined from the outbreak data that included monthly infection status of the cages on these farms. The first infected farm was considered as the index site for potential spread of ISAv to all other farms. To assess the risk of ISAv spreading to susceptible farms, the second and subsequent infected farms were identified using the farm status in the given time period and all infected farms from the previous time periods. Using the three models (hydrodynamic only, seaway-distance, and combined hydrodynamic-seaway-distance based models), we ranked susceptible farms within each time interval by adding the transmission risks from surrounding infected farms and sorting them from highest to lowest. To explore the potential efficiency of targeted sampling, we converted rankings to percentiles and assessed the model's predictive performance by comparing farms identified as high risk based on the rank with those that were infected during the next time interval as observed in the outbreak data. The overall predictive ability of the models was compared using area under the ROC curve (AUC). Farms that become infected in the next period were always within the top 65% of the rank predicted by our models. The overall predictive ability of the combined (hydrodynamic-seaway-distance based model) model (AUC = 0.833) was similar to the model that only used seaway distance (AUC = 0.827). Such models can aid in effective surveillance planning by balancing coverage (number of farms included in surveillance) against the desired level of confidence of including all farms that become infected in the next time period. Our results suggest that 100% of the farms that become infected in the next time period could be targeted in a surveillance program, although at a significant cost of including many false positives.