Pathogen detection and disease diagnosis in wildlife: challenges and opportunities.

The availability of rapid, highly sensitive and specific molecular and serologic diagnostic assays, such as competitive enzyme-linked immunosorbent assay (cELISA), has expedited the diagnosis of emerging transboundary animal diseases, including bluetongue (BT) and African horse sickness (AHS), and facilitated more thorough characterisation of their epidemiology. The development of assays based on real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect and identify the numerous serotypes of BT virus (BTV) and AHS virus (AHSV) has aided in-depth studies of the epidemiology of BTV infection in California and AHSV infection in South Africa. The subsequent evaluation of pan-serotype, real-time, RT-PCR-positive samples through the use of serotype-specific RT-PCR assays allows the rapid identification of virus serotypes, reducing the need for expensive and time-consuming conventional methods, such as virus isolation and serotype-specific virus neutralisation assays. These molecular assays and cELISA platforms provide tools that have enhanced epidemiologic surveillance strategies and improved our understanding of potentially altered Culicoides midge behaviour when infected with BTV. They have also supported the detection of subclinical AHSV infection of vaccinated horses in South Africa. Moreover, in conjunction with whole genome sequence analysis, these tests have clarified that the mechanism behind recent outbreaks of AHS in the AHS-controlled area of South Africa was the result of the reversion to virulence and/or genome reassortment of live attenuated vaccine viruses. This review focuses on the use of contemporary molecular diagnostic assays in the context of recent epidemiologic studies and explores their advantages over historic virus isolation and serologic techniques.

La disponibilité d'essais diagnostiques moléculaires et sérologiques rapides, hautement sensibles et spécifiques tels que l'épreuve immuno-enzymatique de compétition (ELISAc), a accéléré le diagnostic des maladies animales transfrontalières émergentes, dont la fièvre catarrhale ovine (FCO) et la peste équine, et contribué à dresser un tableau épidémiologique plus complet de ces maladies. Grâce à la mise au point d'essais basés sur l'amplification en chaîne par polymérase en temps réel couplée à une transcription inverse (RT­PCR) qui permettent de détecter et d'identifier les nombreux sérotypes du virus de la fièvre catarrhale du mouton et du virus de la peste équine, des études approfondies ont pu être conduites sur l'épidémiologie de l'infection par le virus de la fièvre catarrhale du mouton en Californie et de l'infection par le virus de la peste équine en Afrique du Sud. L'évaluation postérieure des échantillons positifs à une RT­PCR en temps réel de groupe (détectant le virus quel que soit le sérotype) au moyen de RT­PCR spécifiques de chaque sérotype permet d'identifier rapidement le sérotype causal et de limiter le recours à des méthodes classiques onéreuses et chronophages comme l'isolement viral ou les essais de neutralisation virale spécifiques de chaque sérotype. Les outils fournis par ces essais moléculaires et par les plateformes ELISAc ont renforcé les stratégies de surveillance épidémiologique et permis de mieux connaître les altérations potentielles de comportement chez les tiques Culicoides infectées par le virus de la fièvre catarrhale du mouton. Ils ont également contribué à détecter les cas d'infection asymptomatique par le virus de la peste équine chez des chevaux vaccinés en Afrique du Sud. En outre, associés avec l'analyse de séquences du génome entier, ces tests ont révélé que le mécanisme sous-jacent aux récents foyers de peste équine dans la zone de contrôle en Afrique du Sud correspondait à une réversion vers la virulence et/ou à un réassortiment du génome des souches de vaccin à virus vivant atténué. Les auteurs passent en revue l'utilisation des essais de diagnostic moléculaire de nouvelle génération dans le contexte de récentes études épidémiologiques et cherchent à établir leurs avantages par rapport aux techniques classiques d'isolement viral et de recherche sérologique.

La existencia de ensayos moleculares y serológicos de diagnóstico rápidos y de gran sensibilidad y especificidad, como el ensayo inmunoenzimático de competición (ELISAc), ha acelerado el diagnóstico de enfermedades animales transfronterizas emergentes, como la lengua azul o la peste equina, y facilitado una caracterización más exhaustiva de su epidemiología. La creación de ensayos basados en la reacción en cadena de la polimerasa acoplada a transcripción inversa (RT?PCR) en tiempo real para detectar y caracterizar los numerosos serotipos de los virus de la lengua azul y la peste equina ha ayudado a estudiar a fondo la epidemiología de sendos episodios infecciosos causados por el virus de la lengua azul en California y por el virus de la peste equina en Sudáfrica. El subsiguiente análisis de las muestras positivas a la prueba de RT?PC en tiempo real de cualquier serotipo con empleo de ensayos RT?PCR dirigidos específicamente contra uno u otro serotipo permite identificar rápidamente los serotipos víricos, lo que hace menos necesario el uso de métodos convencionales más caros y largos, como el aislamiento del virus o técnicas de neutralización vírica adaptadas específicamente a un serotipo. Estos dispositivos de ensayo molecular o de ELISAc ponen a nuestra disposición herramientas que potencian las estrategias de vigilancia epidemiológica y ayudan a conocer mejor las eventuales alteraciones del comportamiento de los jejenes Culicoides al ser infectados por el virus de la lengua azul. Estas técnicas han ayudado también a detectar en Sudáfrica casos de infección asintomática por el virus de la peste equina en caballos vacunados. Estas pruebas, además, empleadas en combinación con el análisis de secuencias genómicas completas, han servido para aclarar que el mecanismo subyacente a los recientes brotes de peste equina surgidos en la zona de Sudáfrica donde la enfermedad estaba bajo control fue fruto de la reversión a la virulencia y/o el reordenamiento genómico de virus vacunales atenuados. Los autores, centrándose en el uso de modernos ensayos moleculares de diagnóstico como parte de recientes estudios epidemiológicos, examinan las ventajas que ofrecen en comparación con las tradicionales técnicas serológicas y de aislamiento vírico.

Draft Genome Sequence of a Listeria monocytogenes Isolate of Core Genome Multilocus Sequence Typing Complex Type 2521 from Ready-to-Eat Meat Sausage Related to an Outbreak (Sigma1) in Germany.

We report here the draft genome sequence of a Listeria monocytogenes strain, core genome multilocus sequence typing (cgMLST) complex type 2521 (CT2521), isolated from ready-to-eat meat sausage related to a protracted and supraregional listeriosis outbreak (Sigma1) in Germany from 2014 to 2019.

Biocide-Tolerant Listeria monocytogenes Isolates from German Food Production Plants Do Not Show Cross-Resistance to Clinically Relevant Antibiotics.

Contamination of food during processing is recognized as a main transmission route of Listeria monocytogenes To prevent microbial contamination, biocides are widely applied as disinfectants in food processing plants. However, there are concerns about the development of antimicrobial resistance in foodborne pathogens due to widespread biocide usage. In our study, 93 L. monocytogenes isolates from German food production facilities were (i) tested for biocide and antibiotic susceptibility using broth microdilution assays, (ii) analyzed for links between reduced biocide susceptibility and antibiotic resistance, and (iii) characterized by whole-genome sequencing, including the detection of genes coding for biocide tolerance, antibiotic resistance, and other virulence factors. Fifteen L. monocytogenes isolates were tolerant to benzalkonium chloride (BAC), and genes conferring BAC tolerance were found in 13 of them. Antibiotic resistance was not associated with biocide tolerance. BAC-tolerant isolates were assigned to 6 multilocus sequence type (MLST) clonal complexes, and most of them harbored internalin A pseudogenes with premature stop codons or deletions (n = 9). Our study demonstrated a high genetic diversity among the investigated isolates including genotypes that are frequently involved in human infections. Although in vitro adaptation studies to biocides have raised concerns about increasing cross-resistance to antibiotics, our results do not provide evidence for this phenomenon in field isolates.IMPORTANCE Foodborne pathogens such as L. monocytogenes can persist in food production environments for a long time, causing perennial outbreaks. Hence, bacterial pathogens are able to survive cleaning and disinfection procedures. Accordingly, they may be repeatedly exposed to sublethal concentrations of disinfectants, which might result in bacterial adaptation to these biocides. Furthermore, antibiotic coresistance and cross-resistance are known to evolve under biocide selection pressure in vitro Hence, antimicrobial tolerance seems to play a crucial role in the resilience and persistence of foodborne pathogens in the food chain and might reduce therapeutic options in infectious diseases.

Molecular Survey on Brucellosis in Rodents and Shrews - Natural Reservoirs of Novel Brucella Species in Germany?

Brucellosis is a widespread zoonotic disease introduced from animal reservoirs to humans. In Germany, bovine and ovine/caprine brucellosis were eradicated more than a decade ago and mandatory measures in livestock have been implemented to keep the officially brucellosis-free status. In contrast, surveillance of wildlife is still challenging, and reliable data on the prevalence of brucellae in small mammal populations do not exist. To assess the epidemiology of Brucella spp. in rodents and shrews, a molecular survey was carried out. A total of 537 rodents and shrews were trapped in four federal states located throughout Germany and investigated for the presence of Brucella. Using a two-step molecular assay based on the detection of the Brucella-specific bcsp31 and IS711 sequences in tissue samples, 14.2% (n = 76) of the tested animals were positive. These originated mainly from western and south-western Germany, where preliminary analyses indicate population density-dependent Brucella prevalence in voles (Myodes glareolus) and mice (Apodemus spp.). recA typing revealed a close relationship to a potentially novel Brucella species recently isolated from red foxes (Vulpes vulpes) in Austria. The molecular detection of brucellae in various rodent taxa and for the first time in shrew species shows that these animals may be naturally infected or at least have a history of exposure to Brucella spp.

Isolation and Identification of Brucella melitensis Biovar 3 from Vaccinated Small Ruminants: A Public Health Threat in Kosovo.

In 2011, a human brucellosis case with severe clinical symptoms was reported at the University Clinic for Infectious Diseases in Prishtina, Kosovo. A trace-back investigation was conducted to find the source of human infection. A total of 49 blood samples and 15 corresponding milk samples from sheep and goats raised on the patient's farm were taken for serological and molecular analysis. Serology using RBT and CFT revealed 11 positive animals. Twelve milk samples were PCR positive. A Brucella strain isolated from a goat's milk sample was classified as Brucella melitensis biovar 3, indicating the first ever isolation and report in Kosovo. The use of the Bruce-ladder PCR provided differentiation between the field strain and the vaccine strain. Hence, the accidental transmission of the vaccine strain Rev 1 that was previously used for the vaccination of the farm animals could be excluded. The findings of this study show that brucellosis is still a public health threat in Kosovo despite control measures.

Draft Genome Sequences of Klebsiella oxytoca Isolates Originating from a Highly Contaminated Liquid Hand Soap Product.

In 2013, contaminated liquid soap was detected by routine microbiological monitoring of consumer products through state health authorities. Because of its high load of Klebsiella oxytoca, the liquid soap was notified via the European Union Rapid Alert System for Dangerous Non-Food Products (EU-RAPEX) and recalled. Here, we present two draft genome sequences and a summary of their general features.

First Draft Genome Sequence of a Human Coxiella burnetii Isolate, Originating from the Largest Q Fever Outbreak Ever Reported, the Netherlands, 2007 to 2010.

In 2009, Coxiella burnetii caused a large regional outbreak of Q fever in South Limburg, the Netherlands. Here, we announce the genome draft sequence of a human C. burnetii isolate, strain NL-Limburg, originating from this outbreak, including a brief summary of the genome's general features.

The quest for a true One Health perspective of brucellosis.

One Health is an interdisciplinary collaboration that aims at mitigating risks to human health arising from microorganisms present in non-human animal species, which have the potential to be transmitted and cause disease in humans. Different degrees of scientific collaboration and sectoral integration are needed for different types of zoonotic diseases, depending on the health and associated economic gains that can be expected from a One Health approach. Indeed, mitigating zoonotic risks related to emerging diseases with pandemic potential is different from mitigating risks related to endemic zoonotic diseases like brucellosis. Likewise, management of brucellosis at the wildlife-livestock interface in wildlife conservation areas is in essence different from mitigating transmission of a given Brucella species within its preferential host species, which in turn is different from mitigating the spillover of a given Brucella species to non-preferential host species, humans included. Brucellosis economic models often oversimplify and/or wrongly assess transmission between reservoir hosts and spillover hosts. Moreover,they may not properly value non-market outcomes, such as avoidance of human disease, consumer confidence and conservation biology issues. As a result, uncertainty is such that the economic predictions of these models can be questionable. Therefore, understanding the infection biology of Brucella species is a prerequisite. This paper reviews and highlights important features of the infection biology of Brucella species and the changing epidemiology of brucellosis that need to be integrated into a true One Health perspective of brucellosis.

New developments in the diagnostic procedures for zoonotic brucellosis in humans.

At present, laboratory diagnosis of human brucellosis is based on isolation of the bacteria from clinical samples followed by standard microbiological tube testing, detection of anti-Brucella antibodies using various serological tests, and the use of molecular methods for the detection of Brucella DNA. None of these diagnostic tools can be used on its own to reliably detect the causative agent. Cultures give a low yield and subsequent phenotypic characterisation is time consuming, meaning that the initiation of adequate antibiotic therapy is frequently delayed. Serological tests seem to be more effective but are not internationally standardised. Moreover, antibodies can remain detectable despite successful therapy, cross-reacting antibodies may occur, and variable cut-offs for different levels of endemicity are lacking. Molecular assays may reduce diagnostic delays in clinical laboratories, but diagnostic criteria for active infection have not yet been defined. This article reviews the latest microbiological methods for the diagnosis of human brucellosis and outlines developments for the future.

Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century.

Following the recent discovery of new Brucella strains from different animal species and from the environment, ten Brucella species are nowadays included in the genus Brucella. Although the intracellular trafficking of Brucella is well described, the strategies developed by Brucella to survive and multiply in phagocytic and non-phagocytic cells, particularly to access nutriments during its intracellular journey, are still largely unknown. Metabolism and virulence of Brucella are now considered to be two sides of the same coin. Mechanisms presiding to the colonization of the pregnant uterus in different animal species are not known. Vaccination is the cornerstone of control programs in livestock and although the S19, RB51 (both in cattle) and Rev 1 (in sheep and goats) vaccines have been successfully used worldwide, they have drawbacks and thus the ideal brucellosis vaccine is still very much awaited. There is no vaccine available for pigs and wildlife. Animal brucellosis control strategies differ in the developed and the developing world. Most emphasis is put on eradication and on risk analysis to avoid the re-introduction of Brucella in the developed world. Information related to the prevalence of brucellosis is still scarce in the developing world and control programs are rarely implemented. Since there is no vaccine available for humans, prevention of human brucellosis relies on its control in the animal reservoir. Brucella is also considered to be an agent to be used in bio- and agroterrorism attacks. At the animal/ecosystem/human interface it is critical to reduce opportunities for Brucella to jump host species as already seen in livestock, wildlife and humans. This task is a challenge for the future in terms of veterinary public health, as for wildlife and ecosystem managers and will need a "One Health" approach to be successful.

Development and clinical evaluation of a PCR assay targeting the metalloprotease gene (mprA) of B. pseudomallei.

A PCR assay targeting the metalloprotease gene (mprA) of Burkholderia pseudomallei was developed for the specific detection of this organism in pure cultures and clinical samples. All other closely related organisms including B. mallei the causative agent of glanders, and B. thailandensis tested negative. Burkholderia pseudomallei DNA was successfully amplified from paraffin-embedded lung tissue of a camel with a generalized B. pseudomallei infection. The developed PCR assay can be used as a simple tool for the specific and sensitive detection of B. pseudomallei.

Re-emergence of Francisella tularensis in Germany: fatal tularaemia in a colony of semi-free-living marmosets (Callithrix jacchus).

Francisella tularensis was identified as the cause of a die-off which occurred among a colony of semi-free-living common marmosets (Callithrix jacchus). During the outbreak 5 out of 62 animals died of tularaemia in a research facility located in the district of Goettingen, Germany. All animals had been born at the facility suggesting an endemic infection. A total of five culture isolates were recovered and characterized as F. tularensis holarctica, biovar I. These cultures represent the first isolates obtained in the Federal Republic of Germany for more than 45 years. The outbreak area shows several geographical and ecological characteristics known to favour long-term presence of F. tularensis. Persistence of the pathogen in the remote region along the former German-German border, continuous re-introduction from eastern European countries after destruction of the 'Iron curtain' or introduction through migrating birds are testable hypotheses which could explain the emergence of tularaemia in this particular region.

[Glanders--a comprehensive review]. / Ein Ubersichtsreferat zur Rotzerkrankung.

Since 1990 the number of glanders outbreaks in race, military and pleasure horses in Asia and South America is steadily increasing. Glanders, which is eradicated in Western Europe, Australia and Northern America, is currently considered a re-emerging disease. Consequently, the disease may be introduced into glanders-free regions by subclinical carriers at any time. The causative agent of glanders, Burkholderia (B.) mallei, is highly contagious and leads to chronic disease in horses whereas in donkeys and mules the disease is acute and often fatal. Occurrence of the disease leads to international trading restrictions and infected animals immediately have to be culled and safely disposed off. In humans B. mallei infection results in a severe clinical course, and is fatal without appropriate therapy. Its pathogenicity makes B. mallei a potential biological agent that may be used in bioterroristic attacks. Due to the eradication of glanders in the second half of the last century, veterinarians in western European countries are no longer familiar with its clinical presentation in solipeds. Having these facts in mind, this review describes the epidemiology, clinical signs, pathology and the current eradication strategy of this interesting zoonosis. Pictures of imported endurance horses infected with glanders taken during an eradication campaign in Dubai, United Arab Emirates, in 2004 illustrate most typical clinical findings.

Seroprevalence of anti-Yersinia antibodies in healthy Austrians.

Yersiniosis is caused by Y. enterocolitica and Y. pseudotuberculosis mostly presenting as intestinal infection. The infection is usually acquired from contaminated food. The aim of this study was to determine the seroprevalence of anti-Yersinia antibodies in Austrians. Sera of 750 healthy Austrians from all nine states were tested for anti-Yersinia IgG antibodies using the recomBlot Yersinia Westernblot kit. Overall seroprevalence was 29.7%. Seroprevalence increased significantly with age from 24.7% in the group of the 19 to 24 year olds to 38.5% in the group of persons older than 44 years. The seroprevalence of anti-Yersinia antibodies varied within the states between 18% and 43.5%. The high seroprevalence of anti-Yersinia antibodies in contrast to only approximately 100 reported yersiniosis cases per year points to the fact that the majority of infections is either subclinical or mild.

Seroprevalence of brucellosis, tularemia, and yersiniosis in wild boars (Sus scrofa) from north-eastern Germany.

Brucellosis and tularemia are classical zoonotic diseases transmitted from an animal reservoir to humans. Both, wildlife and domestic animals, contribute to the spreading of these zoonoses. The surveillance of the animal health status is strictly regulated for domestic animals, whereas systematic disease monitoring in wildlife does not exist. The aim of the present study was to provide data on the prevalence of anti-Brucella, anti-Francisella and anti-Yersinia antibodies in wild boars from North-Eastern Germany to assess public health risks. A total of 763 sera of wild boars from Mecklenburg-Western Pomerania hunted in 1995/1996 were tested using a commercially available Brucella suis ELISA, an in-house lipopolysaccharide (LPS)-based Francisella ELISA, and commercially available Western blot kits for the detection of anti-Francisella and anti-Yersinia antibodies. The Yersinia enterocolitica O:9 LPS is able to induce serological cross-reactions indistinguishable from brucellosis due to a similar immunodominant epitope in the Brucella O-polysaccharide. The Yersinia Western blot assay was, therefore, based on five recombinant Yersinia outer proteins which have been proved to be specific for the serodiagnosis of yersiniosis. Anti-Brucella, anti-Francisella and anti-Yersinia antibodies were detected in 22.0%, 3.1%, and 62.6% of the wild boars, respectively. The high seroprevalence of tularemia and brucellosis in wild boars indicates that natural foci of these zoonoses are present in wildlife in Germany. However, the impact of transmission of zoonotic pathogens from wildlife to livestock is unknown. Only careful and systematic monitoring will help to prevent the (re)emergence of these zoonotic diseases in domestic animals and consequently human infection.

Diagnostic procedures in tularaemia with special focus on molecular and immunological techniques.

Tularaemia is a severe bacterial zoonosis caused by the highly infectious agent Francisella tularensis. It is endemic in countries of the northern hemisphere ranging from North America to Europe, Asia and Japan. Very recently, Francisella-like strains causing disease in humans were described from tropical northern Australia. In the last decade, efforts have been made to develop sensitive and specific immunological and molecular techniques for the laboratory diagnosis of tularaemia and also for the definite identification of members of the species F. tularensis and its four subspecies. Screening for the keyword 'Francisella' a Medline search over the last decade was performed and articles describing diagnostic methods for tularaemia and its causative agent were selected. Besides classical microbiological techniques (cultivation, biochemical profiling, susceptibility testing) several new immunological and molecular approaches to identify F. tularensis have been introduced employing highly specific antibodies and various polymerase chain reaction (PCR)-based methods. Whereas direct antigen detection by enzyme-linked immunosorbent assay (ELISA) or immunofluorescence might allow early presumptive diagnosis of tularaemia, these methods--like all PCR techniques--still await further evaluation. Therefore, diagnosis of tularaemia still relies mainly on the demonstration of specific antibodies in the host. ELISA and immunoblot methods started to replace the standard tube or micro-agglutination assays. However, the diagnostic value of antibody detection in the very early clinical phase of tularaemia is limited. Francisella tularensis is regarded as a 'highest priority' biological agent (category 'A' according to the CDC, Atlanta, GA, USA), thus rapid and reliable diagnosis of tularaemia is required not only for a timely onset of therapy, the handling of outbreak investigations but also for the surveillance of endemic foci. Only very recently, evaluated test kits for serological diagnosis of human tularaemia became available, while the introduction of standardized molecular techniques for detection and typing is still missing.

Serodiagnosis of Burkholderia mallei infections in horses: state-of-the-art and perspectives.

Burkholderia mallei causes glanders or farcy in solipeds, a disease that must be reported to the OIE (Office International des Epizooties, Paris, France). The number of reported outbreaks has increased steadily during the last decade. Serodiagnosis is hampered by the considerable number of false-positives and -negatives of the internationally prescribed tests. The major problem leading to low sensitivity and specificity of complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e. crude preparations of whole cells. Future perspectives for the development and evaluation of serological test kits using well-characterized single antigens are discussed in the light of recent molecular research on B. mallei and the closely related saprozoonotic agent B. pseudomallei.

Human brucellosis in a nonendemic country: a report from Germany, 2002 and 2003.

Human brucellosis has become a rare disease in Germany since the eradication of bovine and ovine/caprine brucellosis in this country. Therefore, most physicians are unfamiliar with the illnesses clinical presentation, diagnostic tools, and therapeutic strategies. This retrospective study was carried out to evaluate the epidemiological, clinical, and laboratory features of human brucellosis in Germany in the years 2002 and 2003. Thirty-one bacterial isolates from 30 patients sent to the German national reference laboratory were characterized using the genus-specific bcsp31 real-time PCR, the species-specific AMOS-PCR, and standard microbiological methods for the detection and identification of Brucella spp. The medical records of all patients with bacteriologically confirmed brucellosis were evaluated. All 31 isolates proved to be Brucella (30 Brucella melitensis and 1 Brucella suis). Most of the brucellosis patients were infected in endemic countries while visiting friends and relatives during their summer holidays. One case of laboratory-acquired infection was identified. Brucellosis was transmitted mainly by the consumption of contaminated unpasteurized milk or cheese from goats and sheep. The patients presented primarily with flu-like symptoms, i.e. fever, chills, sweating, headaches, arthralgia, and myalgia. In most cases, however, symptoms and signs of focal complications, e.g. spondylitis, endocarditis, and meningoencephalitis, predominated. The rate of complications was much higher than that in endemic countries, presumably as a result of diagnostic delay due to a low index of suspicion. In summary, physicians in nonendemic countries such as Germany must be aware of brucellosis being a possible cause of fever of unknown origin in immigrants and tourists travelling from endemic countries.

Management in der Behandlung von Patienten nach Einsatz biologischer Agenzien.

The risk of terrorist attacks with weapons of mass destruction like biological agents is increasing. Biological agents can be disseminated as aerosols or by contaminating food and beverages. The multitude of agents and the different pathways of transmission cause very different clinical presentations. Natural infections with potential biological agents in Germany are rare and in most cases imported from endemic areas abroad. It is crucial to include these diseases in the spectrum of differential diagnosis. Local and state health departments have to be notified as early as possible in dubious cases. Public health management can be efficient only, if there is high reporting discipline and all epidemic measures are well coordinated.