Reshaping surveillance for infectious diseases: less chasing of pathogens and more monitoring of drivers.

Animal health surveillance, despite its name, tends to focus on looking for disease. Often this involves searching for cases of infection with known pathogens (â€Ëœpathogen chasing'). Such an approach is both resource intensive and limited by the requirement for prior knowledge of disease likelihood. In this paper, the authors propose the gradual reshaping of surveillance towards the systems level, focusing on the processes (â€Ëœdrivers') that promote disease or health, rather than on the presence or absence of specific pathogens. Examples of relevant drivers include land-use change, increasing global interconnectedness, and finance and capital flows. Importantly, the authors suggest that surveillance should focus on detecting changes in patterns or quantities associated with such drivers. This would generate systems-level, risk-based surveillance information to identify areas where additional attention may be needed, and, over time, inform the implementation of prevention efforts. The collection, integration and analysis of data on drivers is likely to require investment in improving data infrastructures. A period of overlap would allow the two systems (traditional surveillance and driver monitoring) to be compared and calibrated. This would also lead to a better understanding of the drivers and their linkages, and thereby generate new knowledge that can improve surveillance and inform mitigation efforts. Since surveillance of drivers may give signals when changes are occurring, which could act as alerts and enable targeted mitigation, this might even enable disease to be prevented before it happens by directly intervening in the drivers themselves. Such surveillance focused on the drivers could be expected to bring additional benefits, since the same drivers promote multiple diseases. Further, focusing on drivers rather than pathogens should enable control of currently unknown diseases, making this approach particularly timely, given the increasing risk of emergence of new diseases.

La surveillance de la santé animale a tendance, malgré son nom, à se focaliser sur la recherche des maladies. Elle implique souvent de chercher les cas d'infection par des agents pathogènes connus (" chasse aux agents pathogènes "). Ce type d'approche exige non seulement beaucoup de ressources, mais elle est aussi limitée par la nécessité d'avoir une connaissance préalable de la probabilité de survenue de la maladie en question. Dans cet article, les auteurs proposent une refonte progressive de la surveillance pour la déplacer au niveau systémique, en se concentrant sur les processus (" facteurs ") influençant la maladie ou la santé plutôt que sur la présence ou non d'agents pathogènes spécifiques. Parmi les facteurs pertinents, on peut citer les changements dans l'utilisation des sols, l'interconnexion accrue au niveau mondial et les flux financiers et de capitaux. Les auteurs soulignent cet élément important : la surveillance devrait se focaliser sur la détection de changements au niveau des schémas ou des quantités associés à ces facteurs. Cela permettrait d'obtenir des informations de surveillance au niveau systémique et basées sur les risques, afin d'identifier les domaines auxquels il pourrait être nécessaire de porter une attention particulière - ce qui informerait, à terme, la mise en oeuvre des efforts de prévention. Il est probable qu'une amélioration des infrastructures de données soit nécessaire pour assurer la collecte, l'intégration et l'analyse des données sur les facteurs. Une période de chevauchement permettrait de comparer et de calibrer les deux systèmes (surveillance traditionnelle et surveillance des facteurs). Les facteurs et les liens entre eux seraient également mieux compris, ce qui générerait de nouvelles connaissances pouvant améliorer la surveillance et informer les efforts d'atténuation. Grâce à la surveillance des facteurs, des signaux pourraient être identifiés lorsque des changements se produisent, ce qui constituerait une alerte pour que des efforts d'atténuation ciblés soient mis en place afin d'intervenir directement sur les facteurs eux-mêmes et donc prévenir une maladie avant même qu'elle ne survienne. On peut s'attendre à ce que ce type de surveillance centrée sur les facteurs apporte des bénéfices supplémentaires, puisque les mêmes facteurs peuvent favoriser de multiples maladies. De surcroît, cette orientation axée sur les facteurs plutôt que sur les agents pathogènes devrait permettre de contrôler des maladies aujourd'hui inconnues, ce qui rend cette approche d'autant plus opportune, compte tenu du risque croissant d'émergence de nouvelles maladies.

La vigilancia zoosanitaria, pese a lo que su nombre indica, tiende a centrarse en la búsqueda de enfermedades, lo que a menudo pasa por tratar de localizar casos de infección por un patógeno conocido ("persecución de patógenos"). Semejante método no solo exige cuantiosos recursos, sino que además presenta la limitación de que obliga a conocer de antemano la probabilidad de aparición de una enfermedad. Los autores proponen una remodelación gradual de la vigilancia tendente a dotarla de carácter sistémico y a centrarla no tanto en la presencia o ausencia de determinados patógenos, sino en los procesos ("inductores" o "factores de inducción", drivers) que favorecen la enfermedad o la salud. Son ejemplo de tales procesos la evolución de los usos del suelo, el creciente nivel de interconexión mundial o los flujos financieros y de capitales. Un aspecto importante que apuntan los autores es que la vigilancia debería tener por objetivo la detección de cambios en las características o cantidades de esos factores de inducción. Ello generaría información de vigilancia basada en el riesgo de carácter sistémico, que serviría para determinar aquellas zonas a las que convendría prestar más atención y, con el tiempo, fundamentar la realización de actividades de prevención. Es probable que la obtención, integración y análisis de datos sobre los factores de inducción exijan inversiones para mejorar las infraestructuras de datos. Si hubiera una fase de solapamiento, sería posible comparar y valorar los resultados de ambos sistemas (vigilancia tradicional y seguimiento de los factores de inducción). Ello serviría para entender mejor los inductores y su vinculación recíproca, lo que generaría nuevos conocimientos con los que perfeccionar la vigilancia y en los que cimentar las actividades de mitigación. Dado que la vigilancia de los inductores puede generar una señal cuando se estén produciendo cambios, señal que a su vez activaría una alerta y propiciaría medidas selectivas de mitigación, podría ser que ello sirviera incluso para prevenir una enfermedad antes de que surgiera, actuando directamente sobre los propios factores de inducción. Cabría pensar que semejante tipo de vigilancia, centrarse en los inductores, puede tener otros efectos beneficiosos, en la medida en que un mismo inductor alimenta la aparición de varias enfermedades. Además, el hecho de centrarse en los factores de inducción, y no tanto en los patógenos, debería servir para controlar enfermedades actualmente desconocidas, por lo que este planteamiento, ante el creciente riesgo de aparición de nuevas enfermedades, resulta especialmente oportuno.

Surveillance and risk assessment for early detection of emerging infectious diseases in livestock.

Those who work in the area of surveillance and prevention of emerging infectious diseases (EIDs) face a challenge in accurately predicting where infection will occur and who (or what) it will affect. Establishing surveillance and control programmes for EIDs requires substantial and long-term commitment of resources that are limited in nature. This contrasts with the unquantifiable number of possible zoonotic and non-zoonotic infectious diseases that may emerge, even when the focus is restricted to diseases involving livestock. Such diseases may emerge from many combinations of, and changes in, host species, production systems, environments/habitats and pathogen types. Given these multiple elements, risk prioritisation frameworks should be used more widely to support decision-making and resource allocation for surveillance. In this paper, the authors use recent examples of EID events in livestock to review surveillance approaches for the early detection of EIDs, and highlight the need for surveillance programmes to be informed and prioritised by regularly updated risk assessment frameworks. They conclude by discussing some unmet needs in risk assessment practices for EIDs, and the need for improved coordination in global infectious disease surveillance.

Les personnes travaillant dans le domaine de la surveillance et de la prévention des maladies infectieuses émergentes (MIE) sont confrontées à la difficulté de prédire avec exactitude le lieu d'émergence d'une maladie, ainsi que l'espèce, le système ou le site affectés. La mise en place de programmes de surveillance et de lutte contre les MIE exige une mobilisation conséquente et durable de ressources nécessairement limitées. Par contraste, le nombre des maladies infectieuses zoonotiques et non zoonotiques pouvant se déclarer est impossible à quantifier, même si l'on s'en tient aux seules maladies affectant les animaux d'élevage. Ces maladies surviennent à la faveur des nombreuses et diverses configurations, associations ou modifications qui peuvent se produire parmi les espèces hôtes, les systèmes de production, les environnements ou habitats et les types d'agents pathogènes. Compte tenu de la multiplicité de ces éléments, il devrait être fait plus largement appel à des cadres de priorisation du risque afin de soutenir les processus de prise de décision et d'allocation des ressources en matière de surveillance. Les auteurs s'appuient sur des exemples récents d'événements liés à des MIE pour faire le point sur les méthodes de surveillance appliquées pour la détection précoce de ces maladies et soulignent l'importance de documenter et de prioriser les programmes de surveillance en procédant à des mises à jour régulières des cadres utilisés pour l'évaluation du risque. Ils concluent en évoquant certains aspects importants que les pratiques actuelles d'évaluation du risque ne permettent pas de couvrir lorsqu'il s'agit de MIE, ainsi que l'importance d'améliorer la coordination de la surveillance des maladies infectieuses au niveau mondial.

Cuantos trabajan en el ámbito de la vigilancia y la prevención de enfermedades infecciosas emergentes (EIE) tienen dificultades para predecir con precisión dónde va a surgir y a quién (o qué) afectará una infección. La instauración de programas de vigilancia y control de EIE exige una inversión sustancial y duradera de recursos que por definición son escasos, sobre todo teniendo en cuenta el número incalculable de enfermedades infecciosas zoonóticas y no zoonóticas que pueden aparecer, aun considerando solo aquellas que afectan al ganado. Este tipo de enfermedades pueden surgir como resultado de muchas combinaciones distintas de especie hospedadora, sistema productivo, medio/hábitat y tipo de patógeno o por efecto de cambios que se den en cualquiera de estos elementos. En vista de la multiplicidad de factores que concurren, convendría emplear de modo más generalizado un sistema de jerarquización de los riesgos en el cual fundamentar las decisiones de vigilancia y la distribución de los recursos destinados a ella. Los autores, valiéndose de ejemplos recientes de episodios infecciosos emergentes que afectaron al ganado, pasan revista a distintos métodos de vigilancia para la detección temprana de EIE y recalcan que los programas de vigilancia deben reposar en procedimientos de determinación del riesgo periódicamente actualizados y en las prioridades fijadas a partir de estos procedimientos. Por último, los autores se detienen en algunas necesidades desatendidas en la praxis de la determinación del riesgo de EIE y en la necesidad de una mejor coordinación de la vigilancia mundial de las enfermedades infecciosas.

Regulation of bone mineral density in the grey squirrel, Sciurus carolinensis: Bioavailability of calcium oxalate, and implications for bark stripping.

The damage caused when grey squirrels strip the outer bark off trees and ingest the underlying phloem can result in reduced timber quality or tree death. This is extremely costly to the UK forestry industry and can alter woodland composition, hampering conservation efforts. The calcium hypothesis proposes that grey squirrels ingest phloem to ameliorate a seasonal calcium deficiency. Calcium in the phloem predominantly takes the form of calcium oxalate (CaOx), however not all mammals can utilise CaOx as a source of calcium. Here, we present the results of a small-scale study to determine the extent to which grey squirrels can utilise CaOx. One of three custom-made diets containing calcium in varying forms and quantities (CaOx diet, Low-calcium carbonate (CaCO3 ) diet and Control diet) were fed to three treatment groups of six squirrels for 8 weeks. Bone densitometric properties were measured at the end of this time using peripheral quantitative computed tomography and micro-computed tomography. Pyridinoline-a serum marker of bone resorption-was measured regularly throughout the study. Bone mineral density and cortical mineralisation were lower in squirrels fed the CaOx diet compared to the Control group but similar to that of those on the Low-calcium diet, suggesting that calcium from calcium oxalate was not effectively utilised to maintain bone mineralisation. Whilst no differences were observed in serum pyridinoline levels between individuals on different diets, females had on average higher levels than males throughout the study. Future work should seek to determine if this apparent lack of ability to utilise CaOx is common to a large sample of grey squirrels and if so, whether it is consistent across all areas and seasons.

Quantitative interferon-gamma responses predict future disease progression in badgers naturally infected with Mycobacterium bovis.

The diagnosis and control of Mycobacterium bovis infection (bovine tuberculosis: TB) continues to present huge challenges to the British cattle industry. A clearer understanding of the magnitude and duration of immune response to M. bovis infection in the European badger (Meles meles) - a wildlife maintenance host - may assist with the future development of diagnostic tests, and vaccination and disease management strategies. Here, we analyse 5280 diagnostic test results from 550 live wild badgers from a naturally-infected population to investigate whether one diagnostic test (a gamma interferon release [IFNγ] assay, n = 550 tests) could be used to predict future positive results on two other tests for the same disease (a serological test [n = 2342 tests] and mycobacterial culture [n = 2388 tests]) and hence act as an indicator of likely bacterial excretion or disease progression. Badgers with the highest IFNγ optical density (OD) values were most likely to subsequently test positive on both serological and culture tests, and this effect was detectable for up to 24 months after the IFNγ test. Furthermore, the higher the original IFNγ OD value, the greater the chance that a badger would subsequently test positive using serology. Relationships between IFNγ titres and mycobacterial culture results from different types of clinical sample suggest that the route of infection may affect the magnitude of immune response in badgers. These findings identify further value in the IFNγ test as a useful research tool, as it may help us to target studies at animals and groups that are most likely to succumb to more progressive disease.

Diagnosis of tuberculosis in groups of badgers: an exploration of the impact of trapping efficiency, infection prevalence and the use of multiple tests.

Accurate detection of infection with Mycobacterium bovis in live badgers would enable targeted tuberculosis control. Practical challenges in sampling wild badger populations mean that diagnosis of infection at the group (rather than the individual) level is attractive. We modelled data spanning 7 years containing over 2000 sampling events from a population of wild badgers in southwest England to quantify the ability to correctly identify the infection status of badgers at the group level. We explored the effects of variations in: (1) trapping efficiency; (2) prevalence of M. bovis; (3) using three diagnostic tests singly and in combination with one another; and (4) the number of badgers required to test positive in order to classify groups as infected. No single test was able to reliably identify infected badger groups if 80% sensitive, at least 94% specific, and able to be performed rapidly in the field.

Evaluation of the usefulness at national level of the dairy cattle health and production recording systems in Great Britain.

The aim of this study was to formally evaluate, qualitatively, the ability of existing recording systems to generate accurate and reliable estimates of the frequency of selected health conditions in the dairy herd of Great Britain. Fifty-nine recording systems were identified, of which 36 had their key characteristics defined through a web-based questionnaire. Nineteen of them were further assessed following the SERVAL, a SuRveillance EVALuation framework against a set of 12 attributes: benefit, bias, communication, coverage, data collection, data management, data analysis, data completeness, flexibility, multiple utility, representativeness and stability/sustainability. The evaluated systems showed considerable differences in their coverage, implementation and objectives. There were overlaps in recorded conditions, with Johne's disease, bovine viral diarrhoea, mastitis and lameness being recorded by most of the systems. Selection bias, data ownership and lack of integration of data from different systems appeared to be a key limitation on the future use of existing systems for nationwide monitoring. The results showed that even though the individual systems can provide reliable estimates of dairy health for individual farmers, none of the systems alone could provide accurate and reliable estimates for any of the conditions of interest at national level.

Risk factors for visible lesions or positive laboratory tests in bovine tuberculosis reactor cattle in Northern Ireland.

An observational case-control study was conducted to investigate risk factors for confirmed bovine tuberculosis (bTB) infection in cattle reacting positively to the single intradermal comparative cervical test (SICCT) in Northern Ireland in the years 1998, 2002 and 2006. Macroscopic lesions were detected at slaughter (positive visible lesion (VL) status) in 43.0% of reactor cattle, whilst 45.3% of those sampled were confirmed as bTB positive due to the presence of lesions or positive histopathology/mycobacterial culture (positive bTB status). In 97.5% of the reactors, the VL status and bTB status were either both negative or both positive. Generalized linear mixed model analyses were conducted on data of 24,923 reactor cattle with the variables herd identifier, local veterinary office (DVO) and abattoir being used as random effects within all the models generated at univariable and multivariable level. The other variables within the dataset were used as fixed effects. Significant risk factors associated with VL status and bTB status at multivariable level (p<0.05) included age at death, breed, sex, test year, net increase in skin thickness at bovine tuberculin injection site, epidemiological status of skin test, total number of reactors at the disclosure test, mean herd size and prior response to the skin test. These risk factors are likely related to the time since infection, the strength of the challenge of infection and the susceptibility of the animal. These findings are important as the detection of visible lesions and the confirmation of bTB are an integral part of the overall bTB control programme in Northern Ireland and the veterinary meat inspection and hygiene programme. The visible lesion status and bTB status of an animal can affect the way in which bTB breakdowns are managed, since failure to detect visible lesions and recovery of Mycobacterium bovis can lead to a less stringent follow-up after other risk factors have been taken into account.

SERVAL: a new framework for the evaluation of animal health surveillance.

Animal health surveillance programmes may change in response to altering requirements or perceived weaknesses but are seldom subjected to any formal evaluation to ensure that they provide valuable information in an efficient manner. The literature on the evaluation of animal health surveillance systems is sparse, and those that are published may be unstructured and therefore incomplete. To address this gap, we have developed SERVAL, a SuRveillance EVALuation framework, which is novel and aims to be generic and therefore suitable for the evaluation of any animal health surveillance system. The inclusion of socio-economic criteria ensures that economic evaluation is an integral part of this framework. SERVAL was developed with input from a technical workshop of international experts followed by a consultation process involving providers and users of surveillance and evaluation data. It has been applied to a range of case studies encompassing different surveillance and evaluation objectives. Here, we describe the development, structure and application of the SERVAL framework. We discuss users' experiences in applying SERVAL to evaluate animal health surveillance systems in Great Britain.

Assessing the expenditure distribution of animal health surveillance: the case of Great Britain.

Animal health surveillance in Great Britain (GB) is conducted through public and private initiatives, yet there is no consolidated information on these activities and their outcomes. We developed an inventory of livestock health surveillance programmes in GB to identify gaps in resource use and potential synergies that could be exploited. The inventory contained details of 36 livestock surveillance activities active in 2011. Data were collected by questionnaire and interviews. Livestock health surveillance funding was found to be unevenly distributed between species: the vast majority (approximately 94 per cent) was spent on cattle diseases (tuberculosis surveillance accounted for most of this expenditure), with 2 per cent on pigs, 2 per cent on sheep/goats, 1 per cent on poultry, and 1 per cent on antimicrobial resistance surveillance across all species. Consequently, surveillance efforts in GB appears heavily skewed towards regions with high cattle densities, particularly high-prevalence tuberculosis areas such as the southwest. The contribution of private schemes to surveillance funding was hard to quantify due to limited access to data, but was estimated to be about 10 per cent. There is scope to better understand the benefits of surveillance, enhance data sharing, clarify costs and identify who pays and who gains. Health surveillance should be considered within the sharing of responsibilities for disease control.

Spatial and temporal interactions between livestock and wildlife in South Central Spain assessed by camera traps.

The diversification of livestock farms into hunting estates in South Central Spain (SCS) may impede the success of Mycobacterium bovis eradication programmes by facilitating transmission between wildlife and livestock. In this observational study we aimed to provide information of relevance about the nature and frequency of interactions (observed visits to study points) between livestock (cattle and domestic pigs) and wildlife (wild boar and red deer). The study was conducted in an extensive cattle farm in SCS where the land is also used for game hunting. During a period of one year, camera traps (n=16) were placed at a priori risk points for interspecies interactions: water (natural and artificial troughs), food placed on the ground for baiting wildlife, and pasture. To define indirect interspecies interactions, a critical time window for M. bovis to survive in the environment was selected based on the literature. Results suggest that wildlife frequented food and pasture points more often than water points, and that the number of visits increased through the dry season, peaking during the acorn season (October-January) and the deer breeding season (June-July). Direct interactions were rare (n=10), as opposed to indirect interactions (n=8992). Wildlife-followed-by-livestock interactions (n=7714) occurred much more often than livestock-followed-by-wildlife (n=1278) and were frequent at water points (66% water points, 17% food, 17% pasture). Results also suggest that water points are a hotspot for indirect interactions and might therefore be a source of infection at the wildlife-livestock interface in the territory covered, particularly for M. bovis, as it is around water where the bacteria seem to survive the longest. Preventing aggregation and therefore reducing contact rates between domestic and wild animals especially at water points may be valuable for disease control in South Central Spain.

Proposed terms and concepts for describing and evaluating animal-health surveillance systems.

The information provided by animal-health surveillance helps to reduce the impact of animal diseases. The widespread movement of animals and their products around the world results in an increasing risk that disease will spread. There is, therefore, a need for exchange between countries of comparable information about disease incidence; the exchange must be based on a common understanding of surveillance approaches and how surveillance systems are designed and implemented. Establishing agreed-upon definitions of surveillance terms would be a first step in achieving this standardisation, and will enhance transparency and confidence. To this end, a workshop was held with the aim of agreeing upon key terms and concepts for animal-health surveillance. In this paper, we describe the methods used at the workshop and summarise the discussions. A complete list of all the proposed definitions including lists of characteristics that can be used to describe surveillance activities and attributes for evaluation of surveillance is available in the workshop report (available at http://www.defra.gov.uk/ahvla-en/disease-control/surveillance/icahs-workshop/). Some important issues were highlighted during these discussions; of particular note was the importance of economic efficiency as an evaluation attribute. Some remaining inconsistencies in the proposed use of terms are highlighted (including the definition of 'risk-based surveillance' and the use of the term 'event-based surveillance').

Patterns of direct and indirect contact between cattle and badgers naturally infected with tuberculosis.

Tuberculosis (TB) due to infection with Mycobacterium bovis is transmitted between cattle and badgers (Meles meles) in the UK and Ireland but it is unclear where or when transmission occurs. We investigated direct and indirect interactions between badgers and cattle using automated proximity loggers on animals and at badger latrines located on pasture, in an area of south-west England with a high-density badger population. Direct contacts (interactions within 1.4 m) between badgers and cattle at pasture were very rare (four out of >500000 recorded animal-to-animal contacts) despite ample opportunity for interactions to occur. Indirect interactions (visits to badger latrines by badgers and cattle) were two orders of magnitude more frequent than direct contacts: 400 visits by badgers and 1700 visits by cattle were recorded. This suggests that indirect contacts might be more important than direct contacts in terms of disease transmission at pasture. The TB infection status of individual badgers (ascribed with 93% accuracy using three diagnostic tests) did not affect the frequency or duration of their visits to latrines located on pasture grazed by cattle. Nevertheless, there was wide variation in contact behaviour between individuals, which highlights the importance of understanding heterogeneity in contact patterns when developing strategies to control disease spread in wildlife and livestock.

Evaluation of animal and public health surveillance systems: a systematic review.

Disease surveillance programmes ought to be evaluated regularly to ensure they provide valuable information in an efficient manner. Evaluation of human and animal health surveillance programmes around the world is currently not standardized and therefore inconsistent. The aim of this systematic review was to review surveillance system attributes and the methods used for their assessment, together with the strengths and weaknesses of existing frameworks for evaluating surveillance in animal health, public health and allied disciplines. Information from 99 articles describing the evaluation of 101 surveillance systems was examined. A wide range of approaches for assessing 23 different system attributes was identified although most evaluations addressed only one or two attributes and comprehensive evaluations were uncommon. Surveillance objectives were often not stated in the articles reviewed and so the reasons for choosing certain attributes for assessment were not always apparent. This has the potential to introduce misleading results in surveillance evaluation. Due to the wide range of system attributes that may be assessed, methods should be explored which collapse these down into a small number of grouped characteristics by focusing on the relationships between attributes and their links to the objectives of the surveillance system and the evaluation. A generic and comprehensive evaluation framework could then be developed consisting of a limited number of common attributes together with several sets of secondary attributes which could be selected depending on the disease or range of diseases under surveillance and the purpose of the surveillance. Economic evaluation should be an integral part of the surveillance evaluation process. This would provide a significant benefit to decision-makers who often need to make choices based on limited or diminishing resources.

Pathology of Mycobacterium bovis infection in wild meerkats (Suricata suricatta).

Pathological lesions associated with Mycobacterium bovis infection (bovine tuberculosis; bTB) in free-living meerkats (Suricata suricatta) in the Kalahari Desert of South Africa are described. The pathology of bTB in meerkats was determined through detailed post-mortem examinations of 57 animals (52 meerkats showing clinical signs of bTB, and five not showing signs of disease). Lymph nodes and tissue lesions thought to be associated with bTB were cultured for mycobacteria. All 52 bTB-infected meerkats showed gross or microscopical granulomatous lesions, but M. bovis was cultured from only 42% (22/52) of these animals. The majority (96%, 50/52) of diseased meerkats had lesions in multiple sites, the pattern of which suggested haematogenous spread of M. bovis infection in this species. The histological characteristics of the tuberculous lesions, together with the gross pathology and the wide range of body systems affected, indicate that infection in meerkats is acquired principally via the respiratory and oral routes, whereas excretion is most likely via the respiratory tract and suppurating skin wounds. Urine and faeces appear to be unlikely sources of infection. The findings of this study provide information on the transmission, pathogenesis and epidemiology of bTB in meerkats that is likely to be relevant to the understanding of M. bovis infection in other social mammal species such as the European badger (Meles meles).

Pentylenetetrazole-induced inhibition of recombinant gamma-aminobutyric acid type A (GABA(A)) receptors: mechanism and site of action.

Pentylenetetrazole (PTZ) is a central nervous system convulsant that is thought, based on binding studies, to act at the picrotoxin (PTX) site of the gamma-aminobutyric acid type A (GABA(A)) receptor. In the present study, we have investigated the mechanism and site of action of PTZ in recombinant GABA(A) receptors. In rat alpha 1 beta 2 gamma 2 receptors, PTZ inhibited GABA-activated Cl(-) current in a concentration-dependent, voltage-independent manner, with an IC(50) of 0.62 +/- 0.13 mM. The mechanism of inhibition appeared competitive with respect to GABA in both rat and human alpha 1 beta 2 gamma 2 receptors. Varying subunit configuration (change or lack of alpha subunit isoform or lack of gamma 2 subunit) had modest effects on PTZ-induced inhibition, as evidenced by comparable IC(50) values (0.6-2.2 mM) in all receptor configurations tested. This contrasts with PTX and other PTX-site ligands, which have greater affinity in receptors lacking an alpha subunit. Using a one-site model for PTZ interaction with alpha 1 beta 2 gamma 2 receptors, the association rate (k(+1)) was found to be 1.14 x 10(3) M(-1) s(-1) and the dissociation rate (k(-1)) was 0.476 s(-1), producing a functional k(d) of 0.418 mM. PTZ could only gain access to its binding site extracellularly. Single-channel recordings demonstrated that PTZ decreased open probability by increasing the duration of closed states but had no effect on single-channel conductance or open state duration. alpha-Isopropyl-alpha-methyl-gamma-butyrolactone, a compound known to antagonize effects of PTX, also diminished the effects of PTZ. Taken together, our results indicate that pentylenetetrazole and picrotoxin interact with overlapping but distinct domains of the GABA(A) receptor.

Influence of subunit configuration on the interaction of picrotoxin-site ligands with recombinant GABA(A) receptors.

We have assessed the interaction of picrotoxin and a putative picrotoxin-site ligand [4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1, 5-a) quinoxaline] (U-93631) with varying configurations of recombinant GABA(A) receptors, using the whole-cell patch clamp technique. In alpha2beta2gamma2 GABA(A) receptors, coapplication of picrotoxin with GABA had minimal effects on initial GABA-activated Cl(-) current amplitude, and subsequently enhanced decay of GABA-activated Cl(-) currents. The half-maximal inhibitory concentration (IC(50)) for picrotoxin in alpha2beta2gamma2 receptors was 10.3+/-1.6 microM. The alpha subunit isoform did not affect picrotoxin-induced inhibition, as IC(50) values for alpha3beta2gamma2 (5.1+/-0.7 microM) and alpha6beta2gamma2 receptors (7.2+/-0.4 microM) were comparable to those obtained in alpha2beta2gamma2 receptors. Interestingly, in receptors lacking an alpha subunit (beta2gamma2 configuration), picrotoxin had a markedly lower IC(50) (0.5+/-0.05 microM) compared to alpha-containing receptors. The inhibitory profile was generally similar for the presumed picrotoxin-site ligand U-93631, i.e., IC(50) values were comparable in all alphabetagamma-containing receptors, but the IC(50) in beta2gamma2 receptors was greater than 10-fold lower. In addition, a modest but significant initial stimulation of GABA-activated current by U-93631 was observed in alpha2beta2gamma2 and beta2gamma2 receptors. A mutation in the second transmembrane domain, shown previously to abolish picrotoxin sensitivity, also greatly attenuated sensitivity to U-93631. Moreover, incubation of receptors with excess U-93631 hindered picrotoxin's ability to gain access to its binding site; both results indicate that U-93631 interacts at the picrotoxin site of the receptor. Our results indicate the presence of an alpha subunit hinders the ability of picrotoxin to block the GABA(A) receptor, and thus provides additional insight into the site of action of picrotoxin. In addition, we have shown that domains important for the actions of picrotoxin also affect U-93631. Thus, this compound should prove to be a useful ligand for analysis of the convulsant site of this receptor.

Substituted 3beta-phenylethynyl derivatives of 3alpha-hydroxy-5alpha-pregnan-20-one: remarkably potent neuroactive steroid modulators of gamma-aminobutyric acidA receptors.

Neuroactive steroids are positive allosteric modulators of gamma-aminobutyric acidA (GABAA) receptor complexes. Synthetic modification generally does not increase neuroactive steroid potency beyond that of the naturally occurring progesterone metabolite, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-P). Recently, it has been shown that introduction of appropriately para-substituted phenylethynyl groups at the 3beta-position of 5beta steroids increases receptor potency. The present report presents the synthesis and pharmacological profile of an analogous series of 5alpha steroids. The most striking feature of this series is the further enhancement of in vitro and in vivo potency obtained. In particular, 3beta-(p-acetylphenylethynyl)-3alpha-hydroxy-5alpha-pr egnan-20-one (Co 152791) was 11-, 16- and 49-fold more potent than 3alpha, 5alpha-P in modulating the binding of [35S]TBPS, [3H]flunitrazepam and [3H]muscimol, respectively, in rat brain membranes (Co 152791 IC50 or EC50 = 2-7.5 nM). Similarly, Co 152791 was 3- to 20-fold more potent than 3alpha,5alpha-P as an inhibitor of [35S]TBPS binding in human recombinant receptor combinations containing alpha1, alpha2, alpha3 or alpha5 and beta2gamma2L subunits (Co 152791 IC50 1.4-5.7 nM). Co 152791 displayed low efficacy and 3alpha,5alpha-P had low potency at alpha4/6beta3gamma2L GABAA receptor complexes. Interestingly, Co 152791 demonstrated remarkable potency as a potentiator of GABA-evoked currents in Xenopus oocytes expressing alpha1beta2gamma2L receptors (EC50 0.87 nM), being 184-fold more potent than 3alpha,5alpha-P. High in vitro potency was also reflected in enhanced in vivo activity in that Co 152791 exhibited exceptional anticonvulsant potency, protecting mice from pentylenetetrazol-induced seizures at a approximately 5-fold lower dose than 3alpha,5alpha-P after i.p. administration (Co 152791 ED50 0.6 mg/kg). Moreover, Co 152791 was orally active (ED50 1.1 mg/kg) and exhibited a therapeutic index of 7 relative to rotorod impairment. The remarkable potency of Co 152791 as a positive allosteric modulator of GABAA receptors may be explained by its interaction with an auxiliary binding pocket in the neuroactive steroid binding site. In addition, modification at the 3beta-position probably hinders metabolism of the 3alpha-hydroxy group contributing to the exceptional anticonvulsant potency of this compound relative to other neuroactive steroids.

Structure-activity relationships for a series of bis(phenylalkyl)amines: potent subtype-selective inhibitors of N-methyl-D-aspartate receptors.

A series of bis(phenylalkyl)amines, structural analogues of ifenprodil and nylidrin, were synthesized and tested for antagonism of N-methyl-D-aspartate (NMDA) receptors. Potency and subunit selectivity were assayed by electrical recordings in Xenopus oocytes expressing three binary combinations of cloned rat NMDA receptor subunits: NR1A expressed in combination with either NR2A, NR2B, or NR2C. The bis(phenylalkyl)amines were selective antagonists of NR1A/2B receptors. Assayed under steady-state conditions, the most potent of these, N-[2-(4-hydroxyphenyl)ethyl]-5-phenylpentylamine hydrochloride (20), has an IC50 value of 8 nM and >1000-fold selectivity with respect to NR1A/2A and NR1A/2C receptors. The structure-activity relationship of the bis(phenylalkyl)amine series indicates that the piperidine ring and alkyl chain substitutions common to NR2B-selective antagonists such as ifenprodil, CP 101,606, and Ro 25-6981 are not necessary to generate potent and selective ligands. The primary determinants of potency are the phenolic OH group, acting as a hydrogen bond donor, the distance between the two rings, and an electrostatic interaction between the receptor and the basic nitrogen atom. This study provides a framework for designing structurally novel NR2B-selective antagonists which may be useful for treatment of a variety of neurological disorders.

Pharmacology of the human gamma-aminobutyric acidA receptor alpha 4 subunit expressed in Xenopus laevis oocytes.

The human gamma-aminobutyric acidA (GABAA) receptor alpha 4 subunit was recently cloned and characterized pharmacologically using radioligand binding techniques. These studies suggested that alpha 4 subunits confer a novel diazepam-insensitive binding site. To further investigate the pharmacology of the alpha 4 subunit, we expressed human alpha 4 beta 2 gamma 2L subunit combinations in oocytes and compared the expression and pharmacology of these receptors with alpha 1 beta 2 gamma 2L, beta 2 gamma 2L, and other possible binary subunit combinations. Apparent GABA affinity was 2-3-fold higher for alpha 4 beta 2 gamma 2L than for alpha 1 beta 2 gamma 2L receptors. Functional modulation of receptors by benzodiazepine-site ligands and other classes of allosteric modulator were assayed over a broad concentration range (0.01-100 microM) on currents that were 10% of the maximum GABA response. Diazepam (0.01-1 microM) did not modulate GABA responses at alpha 4 beta 2 gamma 2L receptors, whereas it increased alpha 1 beta 2 gamma 2L responses by approximately 110%. Bretazenil (0.01-1 microM), a benzodiazepine partial agonist, induced higher efficacy modulation of alpha 4 beta 2 gamma 2L receptors (approximately 83%) than of alpha 1 beta 2 gamma 2L (approximately 25%). The benzodiazepine antagonist flumazenil (0.1-10 microM) unexpectedly potentiated alpha 4 beta 2 gamma 2L responses up to approximately 41%, and the benzodiazepine partial inverse agonist Ro15-4513 (1 microM) potentiated alpha 4 beta 2 gamma 2L responses by approximately 63%. Two other benzodiazepine-site ligands, CGS-9895 and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, had qualitatively similar effects at alpha 1 beta 2 gamma 2L and alpha 4 beta 2 gamma 2L. Modulators such as pentobarbital, 3 alpha-hydroxy-5 alpha-pregnan-20-one, mefenamic acid, and loreclezole also induced similar potentiation at both subtypes of receptor. The pharmacology conferred by the alpha 4 subunit was similar to that conferred by the alpha 6 subunit, to which it shows highest levels of homology, but the two subunits differ in sensitivity to the beta-carboline methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate. Properties of the alpha 4-containing receptors are consistent with diazepam-insensitive binding sites found in cerebral cortex and other forebrain structures. Characterization of these receptors should further our understanding of mechanisms underlying the behavioral effects of GABA modulators and help in the design of drugs with improved, or novel, therapeutic profiles.