Identifying potential candidate Culicoides spp. for the study of interactions with Candidatus Cardinium hertigii.

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors responsible for the transmission of several viruses of veterinary importance. Previous screens of Culicoides have described the presence of the endosymbiont Candidatus Cardinium hertigii (Bacteroidetes). However, any impacts of this microbe on vectorial capacity, akin to those conferred by Wolbachia in mosquitoes, are yet to be uncovered and await a suitable system to study Cardinium-midge interactions. To identify potential candidate species to investigate these interactions, accurate knowledge of the distribution of the endosymbiont within Culicoides populations is needed. We used conventional and nested PCR assays to screen Cardinium infection in 337 individuals of 25 Culicoides species from both Palearctic and Afrotropical regions. Infections were observed in several vector species including C. imicola and the Pulicaris complex (C. pulicaris, C. bysta, C. newsteadi and C. punctatus) with varying prevalence. Phylogenetic analysis based on the Gyrase B gene grouped all new isolates within 'group C' of the genus, a clade that has to date been exclusively described in Culicoides. Through a comparison of our results with previous screens, we suggest C. imicola and C. sonorensis represent good candidates for onward study of Cardinium-midge interactions.

The UK's suitability for Aedes albopictus in current and future climates.

The Asian tiger mosquito Aedes albopictus is able to transmit various pathogens to humans and animals and it has already caused minor outbreaks of dengue and chikungunya in southern Europe. Alarmingly, it is spreading northwards and its eggs have been found in the UK in 2016 and 2017. Climate-driven models can help to analyse whether this originally subtropical species could become established in northern Europe. But so far, these models have not considered the impact of the diurnal temperature range (DTR) experienced by mosquitoes in the field. Here, we describe a dynamical model for the life cycle of Ae. albopictus, taking into account the DTR, rainfall, photoperiod and human population density. We develop a new metric for habitat suitability and drive our model with different climate data sets to analyse the UK's suitability for this species. For now, most of the UK seems to be rather unsuitable, except for some densely populated and high importation risk areas in southeast England. But this picture changes in the next 50 years: future scenarios suggest that Ae. albopictus could become established over almost all of England and Wales, indicating the need for continued mosquito surveillance.

The effect of temperature, farm density and foot-and-mouth disease restrictions on the 2007 UK bluetongue outbreak.

In 2006, bluetongue (BT), a disease of ruminants, was introduced into northern Europe for the first time and more than two thousand farms across five countries were affected. In 2007, BT affected more than 35,000 farms in France and Germany alone. By contrast, the UK outbreak beginning in 2007 was relatively small, with only 135 farms in southeast England affected. We use a model to investigate the effects of three factors on the scale of BT outbreaks in the UK: (1) place of introduction; (2) temperature; and (3) animal movement restrictions. Our results suggest that the UK outbreak could have been much larger had the infection been introduced into the west of England either directly or as a result of the movement of infected animals from southeast England before the first case was detected. The fact that air temperatures in the UK in 2007 were marginally lower than average probably contributed to the UK outbreak being relatively small. Finally, our results indicate that BT movement restrictions are effective at controlling the spread of infection. However, foot-and-mouth disease restrictions in place before the detection and control of BT in 2007 almost certainly helped to limit BT spread prior to its detection.

The challenges posed by equine arboviruses.

Equine populations worldwide are at increasing risk of infection by viruses transmitted by biting arthropods, including mosquitoes, biting midges (Culicoides), sandflies and ticks. These include the flaviviruses (Japanese encephalitis, West Nile and Murray Valley encephalitis), alphaviruses (eastern, western and Venezuelan encephalitis) and the orbiviruses (African horse sickness and equine encephalosis). This review provides an overview of the challenges faced in the surveillance, prevention and control of the major equine arboviruses, particularly in the context of these viruses emerging in new regions of the world.

The role of climate change in a developing threat: the case of bluetongue in Europe.

There is a solid theoretical basis for expecting climate change to have a considerable effect on the infectious diseases of humans, animals and plants. Vector-borne diseases are the most likely to be affected. It is, however, rare to observe such impacts, as diseases are also influenced by many other drivers, some of which may have stronger effects over shorter time scales than climate change. Nevertheless, there is evidence that our warming climate has already influenced some animal diseases, of which bluetongue is considered a prime example. Bluetongue emerged dramatically in southern Europe after 1998 and in northern Europe from 2006. While the speed and scale of this emergence is a challenge to explain, there is evidence, principally from the development of climate-driven models, that recent climate change has played a significant role. Climate-driven models point to an increase in the risk of bluetongue transmission in Europe in recent decades, caused by an increased suitability of parts of southern Europe for the Afro-tropical biting midge, Culicoides imicola, as well as an increase in the vectorial capacity of indigenous Culicoides vectors in northern Europe. Farm-to-farm transmission models of bluetongue in England and Wales under predicted climatic conditions further suggest that, under high-emission scenarios, the scale of future outbreaks could far exceed those experienced to date. The role of climate change in the developing threat of animal disease is, therefore, likely to be economically and socially costly, unless lower emission targets can be set and followed.

Nombre d'arguments théoriques sérieux confirment l'ampleur des effets du changement climatique sur les maladies infectieuses affectant l'être humain, les animaux et les végétaux. Les maladies à transmission vectorielle sont probablement les plus sujettes à cette influence. Toutefois, il est très rare de pouvoir observer ces effets directement, dans la mesure où d'autres facteurs exercent également une influence sur les maladies, dont certains ont des effets plus marquants et plus rapides que le changement climatique. L'influence du réchauffement climatique sur certaines maladies animales a néanmoins été prouvée ; à ce titre, le cas de la fièvre catarrhale ovine est considéré comme exemplaire. La fièvre catarrhale ovine a fait son apparition en Europe méridionale après 1998, puis en Europe du Nord à partir de 2006. Si la rapidité et l'envergure de cette émergence sont difficiles à expliquer, plusieurs démonstrations, recourant pour la plupart à des modèles axés sur le climat font état du rôle important joué par le changement climatique. Les modèles axés sur le climat font ressortir un risque accru de transmission de la fièvre catarrhale ovine en Europe au cours des dernières décennies, associé, d'une part, à l'adéquation croissante de régions entières de l'Europe méridionale vis-à-vis du moucheron afro-tropical Culicoides imicola et, d'autre part, à l'accroissement de la capacité vectorielle des vecteurs Culicoides autochtones dans le nord de l'Europe. D'après les études basées sur des modèles de transmission de la fièvre catarrhale ovine entre exploitations en Angleterre et au Pays de Galles, dans les conditions climatiques prévisibles, il apparaît qu'en cas de fortes émissions, l'ordre de grandeur des foyers futurs serait considérablement plus élevé que dans les épisodes que nous avons connus jusqu'à présent. Par conséquent, le rôle du changement climatique dans les menaces évolutives de santé animale risque d'avoir un coût économique et social élevé, à moins que des objectifs de réduction de l'émission soient mis en place et fassent l'objet d'un suivi approprié.

Existen sólidas bases teóricas para prever que el cambio climático tendrá efectos considerables en las enfermedades infecciosas que afectan al hombre, los animales o las plantas. Las que más probablemente se verán afectadas son las enfermedades transmitidas por vectores. Sin embargo, rara vez pueden observarse tales efectos, pues hay otros muchos factores que influyen en las enfermedades, algunos de los cuales, a una escala temporal más breve, pueden tener una influencia más marcada que el cambio climático. Aun así, hay pruebas de que el clima, en pleno proceso de calentamiento, ya ha incidido en algunas enfermedades animales, de las que la lengua azul se considera un perfecto ejemplo. La lengua azul hizo una espectacular aparición en el sur de Europa a partir de 1998, y en la Europa septentrional a partir de 2006. Aunque resulta difícil explicar la velocidad y las proporciones de tal aparición, existen sólidos indicios, obtenidos principalmente de la elaboración de modelos regidos por variantes climáticas, de que el reciente cambio climático ha cumplido una función importante. Estos modelos apuntan a un incremento del riesgo de transmisión de la lengua azul en Europa en los últimos decenios, lo que se explica por las condiciones más propicias al jején afrotropical, Culicoides imicola, que ofrecen ciertas partes de Europa meridional y por un aumento de la capacidad vectorial de los Culicoides autóctonos del norte de Europa. Los modelos de transmisión de la lengua azul entre explotaciones agropecuarias de Inglaterra y Gales en las condiciones climáticas predichas indican además que, en la hipótesis de un elevado volumen de emisiones, los futuros brotes pueden revestir una escala muy superior a cuanto hemos conocido hasta ahora. Por consiguiente, a menos que se logre establecer y cumplir objetivos de emisiones menos cuantiosas, es probable que el cambio climático resulte económica y socialmente gravoso por su incidencia en la creciente amenaza que plantean las enfermedades animales.

Potential vectors of equine arboviruses in the UK.

There is growing concern about the increasing risk of disease outbreaks caused by arthropod-borne viruses (arboviruses) in both human beings and animals. There are several mosquito-borne viral diseases that cause varying levels of morbidity and mortality in horses and that can have substantial welfare and economic ramifications. While none has been recorded in the UK, vector species for some of these viruses are present, suggesting that UK equines may be at risk. The authors undertook, therefore, the first study of mosquito species on equine premises in the UK. Mosquito magnet traps and red-box traps were used to sample adults, and larvae were collected from water sources such as tyres, buckets, ditches and pools. Several species that are known to be capable of transmitting important equine infectious arboviruses were trapped. The most abundant, with a maximum catch of 173 in 72 hours, was Ochlerotatus detritus, a competent vector of some flaviviruses; the highest densities were found near saltmarsh habitats. The most widespread species, recorded at >75 per cent of sites, was Culiseta annulata. This study demonstrates that potential mosquito vectors of arboviruses, including those known to be capable of infecting horses, are present and may be abundant on equine premises in the UK.

African horse sickness: The potential for an outbreak in disease-free regions and current disease control and elimination techniques.

African horse sickness (AHS) is an arboviral disease of equids transmitted by Culicoides biting midges. The virus is endemic in parts of sub-Saharan Africa and official AHS disease-free status can be obtained from the World Organization for Animal Health on fulfilment of a number of criteria. AHS is associated with case fatality rates of up to 95%, making an outbreak among naïve horses both a welfare and economic disaster. The worldwide distributions of similar vector-borne diseases (particularly bluetongue disease of ruminants) are changing rapidly, probably due to a combination of globalisation and climate change. There is extensive evidence that the requisite conditions for an AHS epizootic currently exist in disease-free countries. In particular, although the stringent regulations enforced upon competition horses make them extremely unlikely to redistribute the virus, there are great concerns over the effects of illegal equid movement. An outbreak of AHS in a disease free region would have catastrophic effects on equine welfare and industry, particularly for international events such as the Olympic Games. While many regions have contingency plans in place to manage an outbreak of AHS, further research is urgently required if the equine industry is to avoid or effectively contain an AHS epizootic in disease-free regions. This review describes the key aspects of AHS as a global issue and discusses the evidence supporting concerns that an epizootic may occur in AHS free countries, the planned government responses, and the roles and responsibilities of equine veterinarians.

Morphometric discrimination of two sympatric sibling species in the Palaearctic region, Culicoides obsoletus Meigen and C. scoticus Downes & Kettle (Diptera: Ceratopogonidae), vectors of bluetongue and Schmallenberg viruses.

BACKGROUND: Some Palaearctic biting midge species (subgenus Avaritia) have been implicated as vectors of bluetongue virus in northern Europe. Separation of two species (C. obsoletus and C. scoticus) is considered difficult morphologically and, often, these female specimens are grouped in entomological studies. However, species-specific identification is desirable to understand their life history characteristics, assess their roles in disease transmission or measure their abundance during arboviral outbreaks. This study aims to investigate whether morphometric identification techniques can be applied to female C. obsoletus and C. scoticus individuals trapped at different geographical regions and time periods during the vector season. METHODS: C. obsoletus and C. scoticus were collected using light-suction traps from the UK, France and Spain, with two geographical locations sampled per country. A total of 759 C. obsoletus/C. scoticus individuals were identified using a molecular assay based on the cytochrome c oxidase subunit I gene. Fifteen morphometric measurements were taken from the head, wings and abdomen of slide-mounted specimens, and ratios calculated between these measurements. Multivariate analyses explored whether a combination of morphometric variables could lead to accurate species identification. Finally, Culicoides spp. collected in France at the start, middle and end of the adult vector season were compared, to determine whether seasonal variation exists in any of the morphometric measurements. RESULTS: The principal component analyses revealed that abdominal characteristics: length and width of the smaller and larger spermathecae, and the length of the chitinous plates and width between them, are the most reliable morphometric characteristics to differentiate between the species. Seasonal variation in the size of each species was observed for head and wing measurements, but not abdominal measurements. Geographical variation in the size of Culicoides spp. was also observed and is likely to be related to temperature at the trapping sites, with smaller individuals trapped at more southern latitudes. CONCLUSIONS: Our results suggest that female C. obsoletus and C. scoticus individuals can be separated under a stereomicroscope using abdominal measurements. Although we show the length and width of the spermathecae can be used to differentiate between the species, this can be time-consuming, so we recommend undertaking this using standardized subsampling of catches.

Comparison of the h-Index Scores Among Pathogens Identified as Emerging Hazards in North America.

Disease surveillance must assess the relative importance of pathogen hazards. Here, we use the Hirsch index (h-index) as a novel method to identify and rank infectious pathogens that are likely to be a hazard to human health in the North American region. This bibliometric index was developed to quantify an individual's scientific research output and was recently used as a proxy measure for pathogen impact. Analysis of more than 3000 infectious organisms indicated that 651 were human pathogen species that had been recorded in the North American region. The h-index of these pathogens ranged from 0 to 584. The h-index of emerging pathogens was greater than non-emerging pathogens as was the h-index of frequently pathogenic pathogens when compared to non-pathogenic pathogens. As expected, the h-index of pathogens varied over time between 1960 and 2011. We discuss how the h-index can contribute to pathogen prioritization and as an indicator of pathogen emergence.

Repellent effect of topical deltamethrin on blood feeding by Culicoides on horses.

African horse sickness (AHS) is a vectorborne disease spread by Culicoides biting midges. The UK's Department for Environment, Food and Rural Affairs currently suggests using topical deltamethrin for AHS control; however, no data are available regarding its efficacy in the horse. The aims of this study were to investigate the effect of topical deltamethrin on blood feeding by Culicoides on horses and to investigate which Culicoides species blood fed on horses. Three pairs of horses were placed in partially enclosed cages that allowed samples representing the Culicoides interacting with individual horses to be sampled. Four data collection sessions were completed before one horse from each pair was topically treated with 10 ml of 1 per cent deltamethrin solution and another four sessions were then carried out. Collected Culicoides were identified and each biting midge examined to see if it had blood fed. The most abundant species collected were C. chiopterus, C. dewulfi, C. obsoletus and C. scoticus (44.3 per cent) and either C. pulicaris or C. punctatus (34.7 per cent). These species were also more likely to have blood fed than other species, supporting their potential role as AHS vectors if the virus were to reach the UK. There was no significant effect of treatment on blood feeding by Culicoides. The results do not support the use of topical deltamethrin to prevent blood feeding by Culicoides on individual horses; however, the study does not investigate the effect that the widespread use of topical deltamethrin might have on vector numbers or disease transmission from viraemic individuals during an outbreak of AHS.

Evaluation of a temperate climate mosquito, Ochlerotatus detritus (=Aedes detritus), as a potential vector of Japanese encephalitis virus.

The U.K. has not yet experienced a confirmed outbreak of mosquito-borne virus transmission to people or livestock despite numerous autochthonous epizootic and human outbreaks of mosquito-borne diseases on the European mainland. Indeed, whether or not British mosquitoes are competent to transmit arboviruses has not been established. Therefore, the competence of a local (temperate) British mosquito species, Ochlerotatus detritus (=Aedes detritus) (Diptera: Culicidae) for transmission of a member of the genus Flavivirus, Japanese encephalitis virus (JEV) as a model for mosquito-borne virus transmission was assessed. The JEV competence in a laboratory strain of Culex quinquefasciatus (Diptera: Culicidae), a previously incriminated JEV vector, was also evaluated as a positive control. Ochlerotatus detritus adults were reared from field-collected juvenile stages. In oral infection bioassays, adult females developed disseminated infections and were able to transmit virus as determined by the isolation of virus in saliva secretions. When pooled at 7-21 days post-infection, 13% and 25% of O. detritus were able to transmit JEV when held at 23 °C and 28 °C, respectively. Similar results were obtained for C. quinquefasciatus. To our knowledge, this study is the first to demonstrate that a British mosquito species, O. detritus, is a potential vector of an exotic flavivirus.

First detection of endosymbiotic bacteria in biting midges Culicoides pulicaris and Culicoides punctatus, important Palaearctic vectors of bluetongue virus.

Heritable bacteria have been highlighted as important components of vector biology, acting as required symbionts with an anabolic role, altering competence for disease transmission, and affecting patterns of gene flow by altering cross compatibility. In this paper, we tested eight U.K. species of Culicoides (Diptera: Ceratopogonidae) midge for the presence of five genera of endosymbiotic bacteria: Cardinium (Bacteroidales: Bacteroidaceae); Wolbachia (Rickettsiales: Rickettsiaceae); Spiroplasma (Entomoplasmatales: Spiroplasmataceae); Arsenophonus (Enterobacteriales: Enterobacteriaceae), and Rickettsia (Rickettsiales: Rickettsiaceae). Cardinium spp. were detected in both sexes of Culicoides pulicaris and Culicoides punctatus, two known vectors of bluetongue virus. Cardinium spp. were not detected in any other species, including the Culicoides obsoletus group, the main vector of bluetongue and Schmallenberg viruses in northern Europe. The other endosymbionts were not detected in any Culicoides species. The Cardinium strain detected in U.K. Culicoides species is very closely related to the Candidatus Cardinium hertigii group C, previously identified in Culicoides species in Asia. Further, we infer that the symbiont is not a sex ratio distorter and shows geographic variation in prevalence within a species. Despite its detection in several species of Culicoides that vector arboviruses worldwide, the absence of Cardinium in the C. obsoletus group suggests that infections of these symbionts may not be necessary to the arboviral vector competence of biting midges.

The threat of midge-borne equine disease: investigation of Culicoides species on UK equine premises.

There are concerns that outbreaks of exotic or novel vector-borne viral diseases will increasingly occur within northern Europe and the UK in the future. African horse sickness (AHS) is a viral disease of equids that is transmitted by Culicoides and is associated with up to 95 per cent mortality. AHS has never occurred in the UK; however, it has been suggested that appropriate Culicoides species and climatic conditions are present in northern Europe to support an outbreak. No data are currently available regarding the Culicoides species present on UK equine properties. This study demonstrates the presence of potential AHS virus vector Culicoides species on both urban and rural equine properties within the south-east UK. PCR analysis revealed that engorged members of these species contained equine DNA, proving a direct vector-host interaction. It is therefore possible that an AHS outbreak could occur in the UK if the virus were to be imported and, given the severe welfare and economic consequences of AHS, this would have devastating consequences to the naïve UK equine population.

Using open-access taxonomic and spatial information to create a comprehensive database for the study of mammalian and avian livestock and pet infections.

What are all the species of pathogen that affect our livestock? As 6 out of every 10 human pathogens came from animals, with a good number from livestock and pets, it seems likely that the majority that emerge in the future, and which could threaten or devastate human health, will come from animals. Only 10 years ago, the first comprehensive pathogen list was compiled for humans; we still have no equivalent for animals. Here we describe the creation of a novel pathogen database, and present outputs from the database that demonstrate its value. The ENHanCEd Infectious Diseases database (EID2) is open-access and evidence-based, and it describes the pathogens of humans and animals, their host and vector species, and also their global occurrence. The EID2 systematically collates information on pathogens into a single resource using evidence from the NCBI Taxonomy database, the NCBI Nucleotide database, the NCBI MeSH (Medical Subject Headings) library and PubMed. Information about pathogens is assigned using data-mining of meta-data and semi-automated literature searches. Here we focus on 47 mammalian and avian hosts, including humans and animals commonly used in Europe as food or kept as pets. Currently, the EID2 evidence suggests that: • Within these host species, 793 (30.5%) pathogens were bacteria species, 395 (15.2%) fungi, 705 (27.1%) helminths, 372 (14.3%) protozoa and 332 (12.8%) viruses. • The odds of pathogens being emerging compared to not emerging differed by taxonomic division, and increased when pathogens had greater numbers of host species associated with them, and were zoonotic rather than non-zoonotic. • The odds of pathogens being zoonotic compared to non-zoonotic differed by taxonomic division and also increased when associated with greater host numbers. • The pathogens affecting the greatest number of hosts included: Escherichia coli, Giardia intestinalis, Toxoplasma gondii, Anaplasma phagocytophilum, Cryptosporidium parvum, Rabies virus, Staphylococcus aureus, Neospora caninum and Echinococcus granulosus. • The pathogens of humans and domestic animal hosts are characterised by 4223 interactions between pathogen and host species, with the greatest number found in: humans, sheep/goats, cattle, small mammals, pigs, dogs and equids. • The number of pathogen species varied by European country. The odds of a pathogen being found in Europe compared to the rest of the world differed by taxonomic division, and increased if they were emerging compared to not emerging, or had a larger number of host species associated with them.

Culicoides vectors of bluetongue virus in Chester Zoo.

On four nights in June 2008, light traps were operated for Culicoides biting midges, the vector species for bluetongue virus (BTV), at five sites in Chester Zoo in north-west England. Over 35,000 Culicoides midges, of 25 species, were captured, including high densities inside animal enclosures. Over 94 per cent of all the Culicoides trapped were females of the Obsoletus group, which is implicated as the vector of BTV serotype 8 in northern Europe. The mean catch of this group per trap per night was over 1500, suggesting a potential risk of BTV transmission if the virus is introduced to Chester Zoo in the animals or midges in the summer.

The influence of host number on the attraction of biting midges, Culicoides spp., to light traps.

A preliminary study was undertaken to investigate how the number of sheep below a light-suction trap affects the number of female Culicoides obsoletus Meigen (Diptera: Ceratopogonidae) caught. As the number of sheep increased from zero to three, the number of midges caught increased, but there appeared to be no further increase when six sheep were used. The lack of increase between three and six sheep is attributable to different activity rates on certain nights, perhaps in response to weather, and suggests, therefore, that catches in light traps increase linearly with sheep numbers, at least for small host numbers.

Seroprevalence and spatial distribution of Fasciola hepatica-infected dairy herds in England and Wales.

The seroprevalence of Fasciola hepatica infection in a population of commercial dairy herds in England and Wales was estimated using an ELISA that detected antibodies to F hepatica in bulk tank milk. A total of 3130 milk samples, obtained as convenience samples from two commercial milk-testing laboratories, were tested during the winter of 2006/07. Herds considered to be seropositive were categorised as low positive, medium positive or high positive. A geospatial map was drawn to show the distribution of infected herds and the seroprevalence of exposure at regional level, using the Nomenclature of Units for Territorial Statistics boundaries, and at a finer spatial level defined by postcode area. Overall, 76 per cent (95 per cent confidence interval [CI] 74 to 77 per cent) of herds carried antibodies to F hepatica; the seroprevalence in England was 72 per cent (95 per cent CI 70 to 74 per cent) and in Wales it was 84 per cent (95 per cent CI 82 to 86 per cent). The highest prevalences of exposure were found in north-west England, where more than 47 per cent of herds were in the high positive exposure category.

Evaluation of housing as a means to protect cattle from Culicoides biting midges, the vectors of bluetongue virus.

The housing of animals at night was investigated as a possible means of protecting them from attack by Culicoides biting midges (Diptera: Ceratopogonidae), the vectors of bluetongue. Light-trap catches of Culicoides were compared inside and outside animal housing, in the presence and absence of cattle. A three-replicate, 4 x 4 Latin square design was used at four farms in Bala, north Wales, over 12 nights in May and June 2007, and the experiment repeated in October. In the two studies, respectively, >70 000 and >4500 Culicoides were trapped, of which 93% and 86%, respectively, were of the Culicoides obsoletus group. Across the four farms, in May and June, the presence of cattle increased catches of C. obsoletus by 2.3 times, and outside traps caught 6.5 times more insects than inside traps. Similar patterns were apparent in October, but the difference between inside and outside catches was reduced. Catches were strongly correlated with minimum temperature and maximum wind speed and these two variables explained a large amount of night-to-night variation in catch. Outside catches were reduced, to a greater extent than inside catches, by colder minimum temperatures and higher maximum wind speeds. These conditions occur more frequently in October than in May and June, thereby suppressing outside catches more than inside catches, and reducing the apparent degree of exophily of C. obsoletus in autumn. The results suggest that the risk of animals receiving bites from C. obsoletus is reduced by housing at both times of year and the benefit would be greatest on warm, still nights when outside catches are at their greatest.