Peter Kimmig

Pathogens and symbionts in ticks: prevalence of Anaplasma phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rickettsia sp., and Babesia sp. in Southern Germany

Tick-transmitted diseases like tick-borne encephalitis and Lyme borreliosis have been well known in Germany for decades. Ongoing research now gives an additional focus to a broad range of other bacteria and parasites in ticks like Anaplasma phagocytophilum, former Ehrlichia sp., Rickettsia sp. and Babesia sp. Knowledge about the prevalence of these infectious agents in ticks is an important prerequisite for risk assessment of human diseases. Therefore nymphs and adult Ixodes ricinus ticks were collected and examined for Anaplasma phagocytophilum (n = 5424 ticks), Rickettsia sp. (n = 1187), and Babesia sp. (n = 3113). For the detection of Anaplasma phagocytophilum, DNA from the 16S rDNA gene was amplified by nested PCR and hybridized with a DIG-labeled oligonucleotide probe. The examination of Rickettsia sp. was performed by single PCR. A partial sequence of the citrate synthase gene was amplified. As a target for the detection of Babesia sp., DNA from the 18S rDNA gene was amplified, also by single PCR. All positive PCR products were sequenced to control specificity. Anaplasma phagocytophilum was detected by PCR in n = 103 (1.9%) out of 5,424 examined ticks from 11 investigation areas. However, not all positive PCR products hybridized using DIG-labeled oligonucleotide probe. Thus, the result of sequencing indicated that only 1.0% (n = 54) belonged to Anaplasma phagocytophilum and nearly half of these PCR products (0.9%) were identified as Wolbachia sp. Rickettsia sp. in Ixodes ricinus ticks from 3 areas were found in n = 105 (8.9%) out of 1,187 ticks examined (range from 13.3% to 5.6%). Sequencing showed Rickettsia helvetica exclusively. In about 2.6% of Rickettsia-positive ticks, double infection with Anaplasma phagocytophilum was found. Babesia sp. was detected in n= 31 (1.0%) out of 3,113 ticks examined, which originated from 4 different areas. By sequencing, n = 28 (90.0%) were identified as Babesia divergens. Three of all Babesia-positive ticks were identified as harboring Babesia microti. The detection of Anaplasma phagocytophilum, Rickettsia sp. and Babesia sp. demonstrates their possible role as a source of human infection in Germany.

Foci of tick-borne diseases in Southwest Germany

Presently known tick-borne diseases in Germany include Lyme borreliosis, tick-borne encephalitis (TBE-virus, western subtype), Q-fever, babesiosis and presumably ehrlichiosis. Blood samples of 4,368 forestry workers in the State of Baden-Wuerttemberg (B-W), southwestern Germany, were tested for the presence of antibodies against Borrelia burgdorferi sensu lato, TBE-virus and Ehrlichia spp. (genogroup E. phagocytophila). Furthermore 12,327 ticks (Ixodes ricinus) collected in various areas of B-W were analysed by PCR and genotyping for the prevalence of pathogen RNA and DNA. The human seroprevalence rates of antibodies to B. burgdorferi sensu lato ranged from 18% to 52%, for TBE-virus from 0% to 43% and for Ehrlichia spp. from 5% to 16% in various counties of the State. The foci of B. burgdorferi and TBE-virus as indicated by antibody prevalence in humans are only partly overlapping with each other. The highest rates of TBE-virus antibodies are in concordance with available clinical data. However antibody prevalence up to 27% in areas with no reports of clinical cases was found, suggesting that TBE occurs throughout the State of B-W. The prevalence of Ehrlichia spp. antibodies suggests that ehrlichiosis plays a role as a tick-borne disease in Germany, but as long as no clinical data are available, this will remain unclear. Investigations of ticks for TBE-virus (n = 9,189) by nested PCR showed prevalence rates from 0% to 2.3% and for Ehrlichia spp. (n = 1,963) from 2.6% to 3.1%. Examination of ticks (n = 3,138) for the presence of B. burgdorferi sensu lato DNA was performed by PCR and revealed prevalence rates from 13.9% up to 24%. Furthermore 1,106 samples positive for B. burgdorferi sensu lato were used for genotyping. B. afzelii DNA was found in 407 ticks (36.8%), followed by B. garinii (21.9%), B. valaisiana (13.7%), and B. burgdorferi sensu stricto (9.9%). Double infection was found in 6.4% and triple infection in 0.8% of the ticks. 10.5% of the positive samples could not be classified. Prevention of tick-borne diseases has to focus on behavioural intervention to reduce individual tick exposure by proper behaviour in the environment, as a large-scale control of the tick population seems impossible and thus reduction of Lyme borreliosis and TBE through tick control is unlikely. Vaccination against TBE-virus should not only be recommended for high endemic areas but also for persons with a high individual risk.

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.

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.

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.

Diagnosis of acute Q fever with emphasis on enzyme-linked immunosorbent assay and nested polymerase chain reaction regarding the time of serum collection.

A commercially available enzyme-linked immunosorbent assay (ELISA) (Virion/Serion [Wuerzburg, Germany]), an indirect fluorescent antibody test (IFAT) (BIOS/Focus [Cypress, CA]), and a nested polymerase chain reaction (PCR) were explored for diagnosis of acute Q fever in reference to time of serum collection. Serum samples of 22 patients with acute Q fever collected around the fifth day of illness were included. A sensitivity of 30% by ELISA and 80% by IFAT (P = 0.1) was found for the first 5 days of illness and 92% by ELISA and 83% by IFAT during the sixth and eleventh day. PCR revealed a positive result in 8 cases (36%) with 6 cases deriving from the first 5 days of illness. We conclude that ELISA aids especially in the diagnosis of Q fever after 5 days of illness. The benefit of PCR as an additional tool to ELISA was especially evident in the early days of serum sampling.

Being a parasitoid of parasites: host finding in the tick wasp Ixodiphagus hookeri by odours from mammals

The parasitic wasp Ixodiphagus hookeri Howard (Hymenoptera: Encyrtidae) parasitizes larvae and nymphs of a number of tick species worldwide. Ticks themselves are parasitic on vertebrate hosts. To study the specificity and reliability of vertebrate odours used by I. hookeri for host location, we conducted bioassays in a four‐chamber olfactometer. Wasps were arrested by carbon dioxide and by odours from roe deer faeces and odours from hair of roe deer and wild boar. Odours from faeces of cattle, rabbit, and field mouse as well as odours from hair of cattle and field mouse had no effect. Odours from faeces of the host tick species Ixodes ricinus L. (Acari: Ixodidae) were attractive only up to a distance of 1 cm. Thus, I. hookeri reacts to general (carbon dioxide) and specific vertebrate odours from wild boar and deer. Examination of freshly shot specimens demonstrates that deer and wild boars are infested with a sufficient number of tick nymphs to tap the full reproductive potential of an I. hookeri female, which makes cues from these mammal species reliable. These results indicate that I. hookeri locates its hosts using specific and reliable mammal odours and that ticks are parasitized on their vertebrate hosts. The implications of this host‐finding strategy and its benefits for the parasitoid are discussed.

Untersuchungen zur Epidemiologie der FSME in Baden-Württemberg

Seit Beginn der neunziger Jahre haben die FSME-Fälle in Baden-Württemberg um ein Mehrfaches zugenommen. Zur Abklärung der aktuellen epidemiologischen Situation wurden daher im Endemiegebiet Freiburg Zecken auf Befall mit dem FSME-Virus untersucht. Für den Virusnachweis fand eine RT-PCR mit anschließender nested PCR Verwendung. Die Originalmethode von Ramelow et al. wurde hierbei in we-sentlichen Punkten modifiziert: Die Reverse Transkription und die 1. PCR erfolgte mit nur einem Enzym, der rTth DNA-Polymerase (Perkin Elmer); dies führte zu einer Vereinfachung der Technik bei gleichzeitiger Erhöhung der Sensitivität. Eine Steigerung der Spezifität wurde zum einen durch Erhöhen der Annealing Temperatur auf 56°C bzw. 52°C, zum anderen durch Blotten der PCR-Produkte und Hybridisierung mit speziellen Sonden erzielt. Hierbei erwies sich das als apathogen geltende Langat-Virus durchweg als positive Kontrolle geeignet; darüber hinaus konnte mit diesem die Sensitivität quantifiziert und eine geeignete Zecken-Poolgröße ermittelt werden. Mit diesem Verfahren wurden insgesamt 1212 Nymphen und 653 Adulte (345. Weibchen und 299 Männchen) aus dem Endemiegebiet auf FSME untersucht. Bei den Nymphen wurde eine Befallsrate von 0,17%, bei den Adulten von 0,46% ermittelt; die mittlere Durchseuchungsrate lag bei 0,2%. Die Plausibilität dieser Zecken-Befallsrate im Hinblick auf die im Untersuchungsgebiet aufgetretenen FSME-Fälle wird diskutiert. Since the early nineties TBE cases in Baden-Württemberg have several times increased. Therefore ticks from the endemic area of Freiburg have been examined for TBE virus affection in order to clarify the current epidemiological situation. A RT-PCR with following nested PCR was used for virus detection, in which the original method of Ramelow et al. was essentially modified. Both, the reverse transcription and the first PCR were performed by a single enzyme, the rTth-DNA-Polymerase (Perkin Elmer), simplifying the technique and increasing the sensitivity at the same time. A specifity intensification was on the one hand gained by raising the annealing temperature up to 56°C and 52°C respectively, on the other hand by blotting the PCR products and by hybridization with special probes. Regarded as apathogenous, the Langat Virus turned out to be the most suitable item in providing a positive control. Moreover the latter made it possible to quantify the sensivity and to determine an appropriate tick pool size. Altogether 1212 nymphs and 653 adults (345 females and 299 males) from the upper endemic area were examined by this method. Among nymphs the affection rate came to 0,17%, among adults to 0,46%, leading to an average infestation rate of 0,27%. The plausibility of this tick affection ist discussed with regard to the TBE cases occuring in the Freiburg area.

What Else Besides TBE and Borreliosis? Tick-Transmitted Pathogens in Germany and Beyond

Tick-borne diseases are an important problem in European countries. In this chapter, we review the biology and distribution of five tick-transmitted pathogens in Germany and neighbouring countries Austria and Switzerland. We focus on the bacterial pathogens Rickettsia spp., Anaplasma phagocytophilum and Francisella tularensis, the protozoan parasite Babesia spp. as well as on Eyach virus. The diagnosis of these pathogens is difficult, because the majority of the infections result in mild or self-limited diseases. However, all these pathogens may induce severe clinical symptoms; therefore, their importance is not to be underestimated in the diagnosis of tick-borne diseases.