Rainer Oehme

Distribution of Clinically Relevant Borrelia Genospecies in Ticks Assessed by a Novel, Single-Run, Real-Time PCR

ABSTRACT A LightCycler-based PCR protocol was developed which targets the ospA gene for the identification and quantification of the different Borrelia burgdorferi sensu lato species in culture and in ticks, based on the use of a fluorescently labeled probe (HybProbe) and an internally labeled primer. The detection limit of the PCR was 1 to 10 spirochetes. A melting temperature determined from the melting curve of the amplified product immediately after thermal cycling allowed the differentiation of the three different B. burgdorferi sensu lato genospecies (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii) that are clinically relevant in Europe in a single PCR run. This method represents a simplified approach to study the association of different Borrelia species in ticks, the risk of Lyme borreliosis, and the putatively species-specific clinical sequelae. To determine the reliability of the real-time PCR protocol, we studied the prevalence of B. burgdorferi sensu lato infection in Ixodes ricinus ticks. A total of 1,055 ticks were collected by flagging vegetation in five different sites in the region of Konstanz (south Germany) and were examined for the distribution of B. burgdorferi species by real-time PCR. The mean infection rate was 35%. Of 548 adult ticks, 40% were positive, and of 507 nymphs, 30% were positive. The predominant genospecies (with 18% mixed infections) in the examined areas was B. afzelii (53%), followed by B. garinii (18%) and B. burgdorferi sensu stricto (11%); 0.8% of the infecting Borrelia could not be identified.

Molecular Epidemiology of Norovirus in Outbreaks of Gastroenteritis in Southwest Germany from 2001 to 2004

ABSTRACT The identification and molecular epidemiology of norovirus in outbreaks of gastroenteritis were studied during a 3-year period in Germany. Specimens (n = 316) from 159 nonbacterial gastroenteritis outbreaks from March 2001 to June 2004 were analyzed for the presence of noroviruses by reverse transcriptase PCR. Outbreaks were most frequent in elderly peoples homes and care centers (43%), followed by hospitals (24%). Molecular analyses of strains from 148 outbreaks showed that there were up to 12 genotypes involved in the outbreaks. Genogroup II noroviruses were responsible for 95% of the outbreaks. Cocirculation of more than one strain in the same outbreak and cocirculation of genogroup I and II strains in the same place were observed. Genogroup II4 (Grimsby-like) was the most prevalent strain, accounting for 48% and 67% of the outbreaks in 2002 and 2003, respectively. The genogroup IIb (Castell/Suria) genotype was observed in all the years of the study. Epidemiological and molecular data indicated that there was a major shift of the predominant strain that coincided with the appearance of a new variant of genogroup II4 in 2002. By the application of reverse transcriptase PCR, this study has demonstrated the importance and dynamism of noroviruses in Germany.

Spread of ticks and tick-borne diseases in Germany due to global warming

Tick-transmitted diseases like tick-borne encephalitis and Lyme Borreliosis have been well known in Germany for decades. Global climate changes may influence the emergence and reemergence of diseases. Ongoing research now gives an additional focus on other tick-borne pathogens such as Coxiella burnetii, Rickettsia conorii, Anaplasma phagocytophilum and Babesia spp., the causative agents of Q-fever, Mediterranean spotted fever, Anaplasmosis and Babesiosis, respectively. The epidemiology of these pathogens was investigated on ticks as well as on rodents, the main hosts. Therefore adults of Dermacentor spp. (n = 862) and rodents (n = 119) were collected and examined for the existence of C. burnetii and Rickettsia spp. by polymerase chain reaction (PCR). In none of the ticks and rodents C. burnetii could be detected, in contrast to Rickettsia spp. where the infection rate in ticks was about 20%. Over and above that, nymphs and adults of Ixodes ricinus were also collected and investigated by PCR for A. phagocytophilum (n = 5,424), Rickettsia helvetica (n = 1,187) and Babesia spp. (n = 3,113). Thereby infection rates of 1%, 8.9% and 1%, respectively, could be determined. The prevalence in rodents was 5.3% for A. phagocytophilum and 0.8% for Babesia microti. None of the rodents was R. helvetica positive.

Network “Rodent-borne pathogens” in Germany: longitudinal studies on the geographical distribution and prevalence of hantavirus infections

Hantavirus infections are known in Germany since the 1980s. While the overall antibody prevalence against hantaviruses in the general human population was estimated to be about 1–2%, an average of 100–200 clinical cases are recorded annually. In the years 2005 and 2007 in particular, a large increase of the number of human hantavirus infections in Germany was observed. The most affected regions were located in the federal states of Baden-Wuerttemberg, Bavaria, North Rhine Westphalia, and Lower Saxony. In contrast to the well-documented situation in humans, the knowledge of the geographical distribution and frequency of hantavirus infections in their rodent reservoirs as well as any changes thereof was very limited. Hence, the network “Rodent-borne pathogens” was established in Germany allowing synergistic investigations of the rodent population dynamics, the prevalence and evolution of hantaviruses and other rodent-associated pathogens as well as their underlying mechanisms in order to understand their impact on the frequency of human infections. A monitoring of hantaviruses in rodents from endemic regions (Baden-Wuerttemberg, Bavaria, North Rhine Westphalia, Lower Saxony) and regions with a low number of human cases (Mecklenburg Western-Pomerania, Brandenburg, Saxony, Saxony-Anhalt) was initiated. Within outbreak regions, a high prevalence of Puumala virus (PUUV) was detected in bank voles. Initial longitudinal studies in North Rhine Westphalia (city of Cologne), Bavaria (Lower Bavaria), and Lower Saxony (rural region close to Osnabrück) demonstrated a continuing presence of PUUV in the bank vole populations. These longitudinal studies will allow conclusions about the evolution of hantaviruses and other rodent-borne pathogens and changes in their distribution, which can be used for a risk assessment of human infections. This may become very important in order to evaluate changes in the epidemiology of rodent-borne pathogens in the light of expected global climate changes in the future.

Occurrence of different Borrelia burgdorferi sensu lato genospecies including B. afzelii, B. bavariensis, and B. spielmanii in hedgehogs (Erinaceus spp.) in Europe.

In order to determine whether European hedgehogs (Erinaceus europaeus and E. roumanicus) play a role in the epidemiological cycle of Borrelia burgdorferi sensu lato in Central Europe and Great Britain, tissue samples of hedgehogs from Germany (n=211), Austria (n=4), the Czech Republic (n=22), and the U.K. (n=32) were tested for the presence of these tick-borne pathogens. PCR for amplification of the B. burgdorferi s.l.-specific 5S-23S intergenic spacer region as well as the outer surface protein A (ospA) gene were used. B. burgdorferi s.l. DNA was detected in 35 of the 259 E. europaeus and in 2 of 10 E. roumanicus. B. burgdorferi prevalences in E. europaeus ranged from 0% (U.K.) to 37.5% (Czech Republic), for E. roumanicus from 0% (Czech Republic) to 50.0% (Austria). Sequencing revealed the occurrence of 3 different B. burgdorferi genospecies in E. europaeus: B. afzelii was the dominant genospecies, followed by B. bavariensis (previously B. garinii OspA serotype 4) and B. spielmanii, the latter was detected for the first time in Hamburg (Germany). B. afzelii and B. bavariensis were also found in E. roumanicus. Our results suggest that hedgehogs modulate the epidemiology of certain species of the B. burgdorferi s.l. complex, potentially affecting the distribution and abundance of individual B. burgdorferi s.l. genospecies in various habitats. We hypothesise that juvenile or individuals with low immune competence in particular, have a high reservoir potential for the 3 genospecies identified here.

Multiple Synchronous Outbreaks of Puumala Virus, Germany, 2010

To investigate 2,017 cases of hantavirus disease in Germany, we compared 38 new patient-derived Puumala virus RNA sequences identified in 2010 with bank vole–derived small segment RNA sequences. The epidemic process was driven by outbreaks of 6 Puumala virus clades comprising strains of human and vole origin. Each clade corresponded to a different outbreak region.

Prevalence of Coxiella burnetii and Rickettsia spp. in ticks and rodents in southern Germany.

Coxiella burnetii, the causative agent of Q fever, and Rickettsia spp. are bacterial pathogens that can be transmitted by ticks of the genus Dermacentor (i.e., Dermacentor marginatus and D. reticulatus). In Germany, the occurrence of these ticks is currently limited to few areas. However, due to increasing temperatures, these vectors will likely extend their distribution in the future, and C. burnetii and Rickettsia spp. might spread with them. To assess the prospective risk of human infections by these agents, it is important to know their current distribution. We collected 666 adult Dermacentor spp. and 119 rodents, mainly Microtus arvalis, in 3 Q fever endemic areas in southern Germany. Ticks and rodent organ pools were screened by PCR for C. burnetii and Rickettsia spp. No evidence of C. burnetii infections could be found in ticks or rodents, suggesting that these animals do not play an essential role in the epidemiology of Q fever in Germany. Rickettsia raoultii and R. slovaca could be detected in 30.3% and 0.75% of all examined ticks, respectively. In contrast, no rickettsia infections could be found in any rodent samples. Both rickettsia species can cause tick-borne lymphadenopathy (TIBOLA), a usually mild human disease. Because of the possible transmission of these rickettsiae to humans, TIBOLA should be considered in the differential diagnosis of tick-borne diseases. Our data show that a spread of these rickettsiae is possible in Germany and that more studies on the distribution of these agents are necessary.

Presence of an emerging subclone of Francisella tularensis holarctica in Ixodes ricinus ticks from south-western Germany

The zoonotic disease tularaemia is caused by the bacterial pathogen Francisella tularensis. Although the causative agent is known for 100 years, knowledge of its enzootic cycles is still rudimentary. Apart from tabanids and mosquitoes, hard ticks have been described as important vectors and potential reservoirs for F. tularensis. Available data on the incidence of human tularaemia indicate an increase in cases in the federal state of Baden-Wuerttemberg. To determine whether ticks are involved in the reported increase in F. tularensis infections in humans and wildlife in this south-western part of Germany, 916 Ixodes ricinus and 211 adult Dermacentor marginatus and D. reticulatus ticks were collected in two different locations. Screening for the presence of F. tularensis was performed by real-time PCR of the 16S rRNA gene. Of the 95 pools of I. ricinus ticks (representing 916 individual ticks), 8 tick pools (8.4%) were positive in this PCR. 30-bp deletion PCR confirmed that the F. tularensis subspecies holarctica was present. FtM24 VNTR analysis revealed that they belong to the emerging Franco-Iberian subclone group of F. tularensis holarctica. Of the 211 ticks of the genus Dermacentor, 35 randomly chosen DNAs were subjected to 16S rRNA gene screening PCR; 20 of these (57%) gave positive signals. For cluster analysis, the lpnA gene region of all Francisella-positive I. ricinus pools and 6 Dermacentor ticks with a positive reaction in the screening PCR was amplified and sequenced. In the resulting neighbour-joining tree, all Francisella-positive I. ricinus samples clustered with sequences of F. tularensis, whilst all Dermacentor tick samples clustered with FLE (Francisella-like endosymbiont) sequences. This study shows that I. ricinus ticks may serve as vectors and/or reservoirs of F. tularensis in Germany and supports the hypothesis that the state of Baden-Wuerttemberg represents an emerging endemic focus of tularaemia.

Prevalences of tick-borne encephalitis virus and Borrelia burgdorferi sensu lato in Ixodes ricinus populations of the Rhine-Main region, Germany.

Tick-borne encephalitis (TBE) and Lyme borreliosis are the most common tick-borne zooanthroponoses in Germany. The federal risk map for TBE in this country is based on recorded cases of human infection, whereas information on the vector-based prevalence of either pathogen is fragmentary. In this study, a total of 12,497 host-seeking nymphal and adult Ixodes ricinus ticks (Acari: Ixodidae) were collected from March to October 2009 and April to June 2010, in 5 TBE non-risk and 4 TBE risk areas of the Rhine-Main region (Hesse) via flagging. A total of 3615 ticks was examined for infection with Borrelia burgdorferi sensu lato and 9115 ticks were analyzed for TBE virus (TBEV). Pathogens were detected by real-time polymerase chain reaction. Among 3615 questing ticks, 344 (9.5%) were found infected with B. burgdorferi sensu lato. Five Borrelia genospecies were identified by sequencing the OspA gene: B. afzelii (81.3%), B. garinii (14.0%), B. valaisiana (2.7%), B. spielmanii (1.3%), and B. bavariensis (0.7%). TBE infection of ticks differed between areas classified as TBE risk and TBE non-risk areas. While the prevalence of TBEV was between 0 and 0.2% (3 of 3947 ticks) in the TBE risk areas, no TBEV-infected tick was detected from TBE non-risk areas. The results show that B. burgdorferi sensu lato occurred in all 9 examined locations, indicating that Lyme borreliosis is prevalent in the Rhine-Main region, whereas TBEV was detected only in previously classified risk areas.

Detection and characterization of sapoviruses in outbreaks of gastroenteritis in southwest Germany.

BACKGROUND Sapovirus (SV) has been reported from many countries as one of the major causes of gastroenteritis in infants, but its importance for a broader age range is not well appreciated. While previous studies in our regional laboratory and other parts of Germany have identified Norovirus (NV) as a major cause of gastroenteritis in Germany, the prevalence of SV has not been recorded. OBJECTIVES To investigate the prevalence of SV in outbreaks of gastroenteritis in Baden-Württemberg, Germany. STUDY DESIGN Stool specimens from 34 outbreaks of gastroenteritis collected in Baden-Württemberg, Germany in 2002 and 2003, which tested negative for other enteric pathogens were tested for SV by RT-PCR. Positive samples were sequenced and typed using phylogenetic analysis. RESULTS SV was identified in two of 157 (1.3%) specimens. These two viruses belonged to different genotypes of SV and had a nucleotide similarity of only 57%. CONCLUSIONS SV circulates within the study area, but is not an important cause of gastroenteritis in the area.