Parasitol Res (2009) 105:313–319 DOI 10.1007/s00436-009-1416-y

ORIGINAL PAPER

Bluetongue disease in Germany (2007–2008): monitoring of entomological aspects

Heinz Mehlhorn & Volker Walldorf & Sven Klimpel & Günter Schaub & Ellen Kiel & René Focke & Gabriele Liebisch & Arndt Liebisch & Doreen Werner & Christian Bauer & Henning Clausen & Burkhard Bauer & Martin Geier & Thomas Hörbrand & Hans-Joachim Bätza & Franz J. Conraths & Bernd Hoffmann & Martin Beer

Received: 11 March 2009 /Accepted: 11 March 2009 /Published online: 26 March 2009 # Springer-Verlag 2009

Abstract In the summer of 2006, a bluetongue epidemic cells was formed that covered the monitoring area. As a started in the border area of Belgium, The Netherlands, and rule, one trap was placed into each grid cell. The Germany, spread within 2 years over large areas of Western monitoring program started at the end of March 2007 and and Central Europe, and caused substantial losses in farm lasted until May 2008. It included the catching of ruminants. Especially sheep and cattle were severely by ultraviolet light traps—done each month from days 1 affected, leading to a case–fatality ratio of nearly 40% in until 8, the selection of midges of the obsoletus, sheep (Conraths et al., Emerg Inf Dis 15(3):433–435, Culicoides pulicaris group, and other Culicoides spp., the 2009). The German federal ministry of food, agriculture, testing of midges for bluetongue virus (BTV) by polymerase and consumer protection (BMELV) established a country- chain reaction (PCR), and the daily registration of weather wide monitoring on the occurrence of the vectors of this data at each trap site for the whole monitoring period. The virus, i.e., midges (family ) of the genus following main results were obtained: (1) Members of Culicoides. The monitoring was done on 91 sites, most of the C. obsoletus group were most commonly found in the which were localized in the 150-km restriction zone that traps, reaching often 3/4 of the catches. The African and existed in December 2006. A grid consisting of 45×45 km2 South European vector of BTV—the species Culicoides

: H. Mehlhorn (*) : V. Walldorf : S. Klimpel H. Clausen B. Bauer Department of Parasitology, Heinrich Heine University, Institute for Parasitology and Tropical Düsseldorf 40225, Germany Veterinary Medicine, Free University Berlin, e-mail: [email protected] Berlin 14163, Germany

G. Schaub M. Geier : T. Hörbrand Department of Special Zoology, Fa. Biogents, Regensburg 93053, Germany Ruhr University, Bochum 44801, Germany : E. Kiel R. Focke H.-J. Bätza AG Gewässerökologie und Naturschutz, Bundesministerium für Ernährung Carl von Ossietzky University, Oldenburg 26111, Germany Landwirtschaft und Verbraucherschutz (BMELV), Bonn 53123, Germany G. Liebisch : A. Liebisch Zecklab, Burgwedel 30938, Germany F. J. Conraths Institute of Epidemiology, D. Werner Friedrich-Loeffler-Institut, German Entomological Institute, Wusterhausen 16868, Germany Leibniz-Zentrum für Agrarlandforschung (ZALF), Münchenberg 15374, Germany B. Hoffmann : M. Beer C. Bauer Institute of Diagnostic Virology, Department of Parasitology, Friedrich-Loeffler-Institut, Justus Liebig University, Giessen 53392, Germany Greifswald-Insel Riems 17493, Germany 314 Parasitol Res (2009) 105:313–319 imicola—was never found. (2) Members of the C. obsoletus letters sent to Robert Koch—the founder of modern group were most frequently found infected with BTV microbiology—that this peculiar disease attacking imported besides a few cases in the C. pulicaris group and other ruminants might be the result of the action of a “virus”, species. (3) Members of the C. obsoletus group were also similar to those described by Loeffler and Frosch (1897)in found in winter. Their numbers were reduced, however, and the foot-and-mouth disease. Therefore, bluetongue was also they were caught mostly close to stables. Therefore, a true described for a long time as “pseudo-foot-and-mouth -free period does not exist during the year in Germany. disease”. The recent name “bluetongue disease” refers to (4) The amounts of midges caught daily depended on the the appearance of symptoms in the late stage of lethal cases weather conditions. If it was cold and/or windy, the traps due to cyanosis (blue color of the tongue and of the mucous contained only a few specimens. Since the months from epithelia as signs of the lack of oxygen). January to May 2008 were considerably colder (at all farms) In August 2006, the disease was suddenly found in than their correspondents in 2007, the growing of the Central Europe and started from Belgium, Germany, and population of midges started 2–3 months later in 2008 than The Netherlands, and it is quick and constant in spreading in 2007. (5) The highest populations of midges occurred in (Mehlhorn et al. 2007, 2008a; Wilson and Mellor, 2008; both years (2007 and 2008) during the months September and Hoffmann et al. 2008; Meiswinkel et al. 2007; Saegerman October. This corresponded significantly to the finding of et al. 2008; Toussaint et al. 2007; Conraths et al. 2007, highest numbers of infected midges and to the number of 2009; Hoffmann et al. 2009; Darpel et al. 2007). diseased cattle and sheep during these 2 months. (6) It is After a local, permanent screening of suspected vectors noteworthy that in general, the first virus-positive midges of at two farms in Aachen (North Rhine-Westphalia) in the the species C. obsoletus were found about 1 1/2 months later months August 2006 until January 2007 (Mehlhorn et al. than the first clinical cases had occurred or later than the first 2007), the German federal ministry of food, agriculture, and PCR-proven virus-positive sentinel had been docu- consumer protection (BMELV) installed in the year 2007 a mented. In 2007, the first BTV-positive cattle were detected monitoring system on the abundance of midges and their in May in North Rhine-Westphalia, while the first positive role as vectors of the BTD. The monitoring program was Culicoides specimens were only found in August on the designed according to a “Working document on enhancing same farm. Evaluating these main results of the entomolog- bluetongue monitoring and surveillance in the EU” issued ical monitoring and the fact that many wild ruminants have by the Directorate General for Health and Consumer Affairs also been infected with BTV, it becomes evident that to ensure harmonized monitoring approaches in the bluetongue disease has become endemic in Central Europe, European Union. At that time in Western and Central and that only constant effort including vaccination and Europe, only BTV serotype 8 was found, which had never perhaps also insecticidal protection of cattle and sheep will before been found in Europe, while South Africa harbors keep the economical losses at a reasonable level. The approximately 20 out of a total of 24 known serotypes. following papers (1–10) in this journal will contribute more The 10 papers following this introducing overview in the details obtained from this worldwide unique entomological present volume of the journal (Mehlhorn et al. 2009; monitoring: Bartsch et al. 2009; Bauer et al. 2009; Stephan et Vo rs pr ac h e t a l. 2009; Balczun et al. 2009;Kieletal. al. 2009; Clausen et al. 2009; Hörbrand and Geier 2009; 2009; Kiehl et al. 2009; Bartsch et al. 2009; Clausen et al. Kiehl et al. 2009;Mehlhornetal.2009;Kieletal.2009; 2009; Stephan et al. 2009; Bauer et al. 2009; Hörbrand and Vo rs pr ac h e t a l. 2009; Balczun et al. 2009. Geier 2009) describe the results obtained during this monitoring program, which ran from the end of March 2007 until the end of May 2008. Introduction

Bluetongue disease (BTD) was first described in 1876 in Methods and aims South Africa by an anonymous author in a veterinarian report (Anonymous 1876). It occurred in cattle and sheep Tasks of the project and later became referred to as “fever” and “malarial catarrhal fever of sheep” by Hutcheon (1881, 1902), The German monitoring project had the task to find threatening all imports of ruminants from Europe, e.g., those answers to the following questions: of the “Bures”,who—immigrating from the Netherlands— introduced the name “bekziekte” for this peculiar disease. 1. Has Culicoides imicola, the vector of the bluetongue The Swiss scientist Theiler—famous for his studies on East virus (BTV) in South Africa and South Europe, arrived Coast fever of cattle (theileriosis: Mehlhorn et al. 1992; in Central Europe, and is it involved here in the Dobbelaere and McKeever 2002)—suggested in 1906, in transmission of BTV? Parasitol Res (2009) 105:313–319 315

2. Are the midges of the Culicoides obsoletus group, which had been found as vectors of BTV in Aachen (Western Germany; Mehlhorn et al. 2007), present in the whole Germany in the same quantities (approximately 3/4 of the catches) as in Western Germany? 3. Are there other vectors of BTV than members of the C. obsoletus group? 4. What are the months with the highest rates of occurrence of midges? 5. Is there any midge-free period during the year in Germany? Some groups enlarged their goals and tried to contribute to further questions: 1. Where and when do the midges attack their hosts? 2. Which are the conclusions that can be drawn with respect to protection from bites of vector midges? 3. Are there hints on the pathways of introduction of BTV in Central Europe? Fig. 1 Diagrammatic construction plan of the BG-Sentinel™ midge 4. Are there peculiar regulations needed in future with trap provided by Fa. Biogents (Regensburg). The ultraviolet lamp (see respect to animal transportations, e. g. spraying of Fig. 2) is not shown. are attracted by ultraviolet light during insecticides to avoid transportation of biting insects? night and become sucked into a catch back (dry catches) or into an ethanol filled catch beaker (wet catches) by help of an air stream produced by a fan (detail 12)

Installation and running of the monitoring project catching insects during the night time between dusk and In January 2007, the BMELV invited several research groups dawn, the farmers transferred the catches in fresh 70% with entomological experience to join the virologists and ethanol inside of 100-ml plastic bottles and sent them by epidemiologists of the Friedrich-Loeffler-Institut for a regular mail to one of the above cited institutes, where the common program to monitor the putative vectors of BTV. insects were investigated by means of stereo light micro- During several meetings, the following ten groups agreed to scopes. The midges were separated from the other insects, cooperate: (1) Prof. Dr. H. Mehlhorn (Düsseldorf), as classified as C. obsoletus (group), Culicoides pulicaris coordinator, (2) PD Dr. M. Beer (Riems), (3) Dr. C. Bauer (group), or other Culicoides spp. (Fig. 3) and stored in fresh (Gießen), (4) PD Dr. C. Clausen (Berlin), (5) PD Dr. F. J. 70% ethanol. The female midges—separated into groups of Conraths (Wusterhausen), (6) Dr. G. Geier (Regensburg), (7) fed and unfed individuals—were finally sent each month to Prof. Dr. E. Kiel (Oldenburg), (8) Prof. Dr. A. Liebisch and the German national reference laboratory for bluetongue Dr. G. Liebisch (Burgwedel), (9) Prof. Dr. G. Schaub disease at the FLI (Riems), where groups of 50 female (Bochum), and (10) Dr. D. Werner (Berlin). midges were pooled and examined for BTV serotype To select the sites for monitoring, a grid consisting of 8 genome by real-time polymerase chain reaction (PCR). 45×45 km2 cells was formed that covered the monitoring The obtained data were introduced into a database collect- area. As a rule, one trap was placed into each grid cell. ing the results of all groups and species involved. This Ninety-one sites, normally on farms, were selected with the database was also fed with the data obtained from the help of the local veterinary authorities. Most sites were different weather stations. localized in the 150-km restriction zone that existed in December 2006. By the end of March 2007, one ultraviolet lamp trap (Biogents, Regensburg, Germany; Figs. 1 and 2) Results was (Hobo H8 Pro, Fa. Onset Computer Corp., MA, USA) placed on each selected farm accompanied by a weather The detailed results of the monitoring in the different station, which collected and registered electronically the German regions are presented in the ten papers following relevant data at intervals of 4 h. The farmers were informed this introducing overview in the present volume of the how to handle the traps during the catching period lasting journal (Mehlhorn et al. 2009; Kiehl et al. 2009; Vorsprach from day 1 to 8 of each month. After the eighth day of et al. 2009; Balczun et al. 2009; Kiel et al. 2009; Clausen 316 Parasitol Res (2009) 105:313–319

Fig. 2 Trap in action during night showing UV-light Fig. 3 Life cycle stages of Culicoides species. a Adult female. b Larva hatching from egg. c Larval stage. d Pupa. e Anterior protrusion of pupa Fig. 4 Scanning electron micrograph of the head of a female Culicoides obsoletus. Note the large compound eyes and the short but very strong mouthparts. The antennae show only a few hairs

et al. 2009; Bartsch et al. 2009; Stephan et al. 2009; Bauer relatives came second and reached up to about 20% of et al. 2009; Hörbrand and Geier 2009). However, the the caught midges, while the others (belonging to up to common features can be summarized as follows: 24 other species or taxa) occurred in rather scarce numbers. This was also true for C. dewulfi, which— 1. The African and South European BTV vector C. according to literature—wasexpectedtobemore imicola was never found in Germany during the whole common as it was found in high abundance in some monitoring period. sites in the Netherlands (Saegerman et al., 2008). Only 2. The 91 farms of the monitoring program were situated on a few farms and only during a few catching periods at different heights above sea level and in different do members of the C. pulicaris group occurred in weather regions with considerable differences in the higher numbers than members of the C. obsoletus yearly mean temperatures. The traps were exposed to group. changing winds under different circumstances. Despite 3. As in 2006 (Mehlhorn et al. 2007), members of the these differences, members of the C. obsoletus group C. obsoletus group were found infected with BTV (see Mehlhorn et al. 2009) was most abundant in the 8 since numerous pools were found positive for BTV entire monitoring area, representing in most catches 8 beginning in August 2007 until November 2007. more than 70% of all caught midges. In some catches, Among them, several groups/pools of unfed female even more than 90% of the determined midges specimens of the C. obsoletus group (Fig. 4) contained belonged to the C. obsoletus (group). C. pulicaris and the virus, too. Therefore, it is likely that the main vector Parasitol Res (2009) 105:313–319 317

(s) of BTV 8 in Germany belong to the C. obsoletus used during the establishment and description of the group. However, in addition to pools of midges of the different Culicoides species are often misleading and C. obsoletus group, several pools of with midges of the do not respond to modern criteria. Therefore, several C. pulicaris group were proven to be carriers of BTV species, which look very similar to C. obsoletus or 8 in 2007. However, the number of positive C. pulicaris C. pulicaris, cannot clearly become differentiated. group pools was considerable lower than in midges of Thus, many authors describe groups of C. obsoletus the C. obsoletus group. In the catches from North or C. pulicaris species or name these groups “com- Rhine-Westphalia and Brandenburg (Mehlhorn et al. plexes”. Both are not systemic units. Thus, the true 2009; Clausen et al. 2009), also, unfed females of the C. existence of species has to be tested by molecular pulicaris group contained the virus. These findings biological methods, as was started by several authors underline that members of the C. pulicaris group may (Lit.c.f.Kiehletal.2009). There, it turned out that be suitable as vectors for BTV, but they seem less several species can be united in given species such as important than members of C. obsoletus group. C. obsoletus or C. pulicaris. 4. The catches indicated that there is no midge-free period during the year in the monitoring area, since midges were caught also during the cold months in December until February. This supports the idea that BTV may be Conclusions able to overwinter in midges, and that transmission may occur during the cold season at a very low level. This is The present monitoring showed that midges which are furthermore underlined by the finding that midges able to transmit BTV occurring in the entire monitoring apparently stay inside the stables, since in traps placed areinhighabundance,whilethemainvectorinthe inside stables or at stable doors, many fed females were Mediterranean and Africa, C. imicola, was not found in caught. Germany. This makes it unlikely that BTD was introduced 5. The highest numbers of midges were found in the by northward wandering infected C. imicola. This seems months September and October at most places when also reasonable because BTV 8 has never been detected in also the highest numbers of BTV-infected pools of Southern Europe. In Germany, at least member of the midges were detected. C. obsoletus and the C. pulicaris groups may act as 6. The first BTV-positive pools were not found before vectors of BTV. Midges of these groups overwinter inside August 2007, although the first cases of clinical or PCR- or close to stables. The invasion of BTV may have been positive cattle had been documented already in May stabilized by the infection of wild ruminants which may 2007. This indicates that the sensitivity of the midge serve as reservoir for the virus. These facts make it monitoring is lower than the sensitivity of the clinical necessary to protect susceptible domestic animals from surveillance in ruminants. Moreover, the number of BTV infection or clinical disease. One method is to treat infected midges is rather low at the beginning of the stables and/or the herds with long-lasting insecticides transmission season and increases in the months of July, (Mehlhorn et al. 2008a, b, c, d; Schmahl et al. 2008). August, until November, where it starts to decrease. Alternatively, the animals can be vaccinated against the 7. The method to investigate pools of about 50 female relevant BTV serotypes. For Europe, an inactivated midges for BTV genome might be rather crude since it vaccine against serotype 8 was developed in a very short is not clear how many positive midges are needed until period from 2007 until its first inoculation in May 2008 a pool of 50 midges reacts virus-positive. Thus, it might into sheep and cattle (Table 1). In combination with the be suspected that a larger amount of virus positive existing immunity after natural infection, the mass midges occurs earlier than now seen with the present immunization cattle and sheep led to a considerable method used. reduction of new infections. However, the success of this 8. The microscopic species determination during the present vaccination campaign is endangered by the presence of monitoring was mainly based on the appearance of the BTV serotype 1 in France and the occurrence of serotype wing feathers, where peculiar black dots (formed by fine 6 in The Netherlands and Germany in 2008. Thus, the hair) and white fields (without hair) were combined with a vaccination—although being very effective—has to be characteristic pattern of wing veins. Especially the adapted to new BTV serotypes as was done in other parts disappearance of hair in some specimens (due to the of Europe in the years before (see Table 1). catching and preparation procedures) made it often However, both methods of prophylaxis (vaccination and difficult to a clear species determination. Furthermore, protection against bites) give hope to decrease the other criteria (such as the shape of palps, the shape possible losses due to BTV. Since other virus diseases— of ovary, ootheca, and hair at the feet) that had been including those of humans—are luring at the doors of good 318 Parasitol Res (2009) 105:313–319

old Europe, special attention is needed in times of enormously increasing globalization when animals or troops are transported from overseas back to Europe. Ofcourse,evensuchanintenseandbroadmonitoring project as the present one on BTV vectors left a series of questions without significant or final answers. It remained unsolved, where the involved vector midges have their definitive breeding sites and how long it takes until maturity is reached. Therefore, it remained unclear, 2000

– too, whether the virus is potentially transmitted to the eggs and larvae of the Culicoides species. Furthermore, 1999 experimentshavetobedoneinordertofindoutwhether other blood-sucking or licking are 2006

– mechanical vectors of BTV. In addition, the questions, how many bites of midges and how many viruses are needed to establish a persistent infection inside a vertebrate host, remain unsolved. Thus, ongoing research 2006 2006 2005 – – is urgently needed, which, however, requires significant funding and establishment of positions for scientists, in this for a long time, completely neglected field of medical and veterinary entomology. 2006 2005 –

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