Exp Appl Acarol (2008) 44:307–314 DOI 10.1007/s10493-008-9150-1
Larvae of chigger mites Neotrombicula spp. (Acari: Trombiculidae) exhibited Borrelia but no Anaplasma infections: a Weld study including birds from the Czech Carpathians as hosts of chiggers Ivan Literak · Alexandr A. Stekolnikov · Oldrich Sychra · Lenka Dubska · Veronika Taragelova
Received: 21 January 2008 / Accepted: 28 March 2008 / Published online: 10 April 2008 © Springer Science+Business Media B.V. 2008
Abstract Chigger mites were collected from 1,080 wild birds of 37 species at Certak (Czech Republic), in the western Carpathian Mountains, from 29 July to 24 September 2005. The prevalence of infestation with chigger larvae was 7%. A total of 325 chigger specimens from 10 bird species was identiWed and three chigger species were found: Neotrombicula autumnalis, N. carpathica, and N. inopinata, the latter two species being reported on new hosts. Neotrombicula carpathica is reported in the Czech Republic for the Wrst time. A total of 509 chigger larvae found on 79 host specimens were examined by polymerase chain reaction (PCR) for the presence of Borrelia burgdorferi s.l. DNA (fragments of the rrf (5S)—rrl (23S) intergenic spacer), and Anaplasma phagocytophilum DNA (epank1 gene). A fragment of speciWc Borrelia DNA was ampliWed through PCR in one sample, and the PCR product was further analyzed by reverse line blotting assay, whereby both genospecies of B. garinii and B. valaisiana were proved. This sample pooled Wve chigger larvae collected from one Sylvia atricapilla on 11 August 2005. No A. phagocytophilum DNA was ampliWed. We conclude that larvae of the genus Neotrombicula can be infected with Borrelia genospecies originated from their present or former hosts. Keywords Neotrombicula · Borrelia garinii · Borrelia valaisiana · Anaplasma · Birds · Czech Republic
I. Literak (&) · O. Sychra · L. Dubska Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, 612 42 Brno, Czech Republic e-mail:
[email protected] A. A. Stekolnikov Zoological Institute, Russian Academy of Sciences, Saint Petersburg, Russia V. Taragelova Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia
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Introduction The role of chigger larvae (Acari: Trombiculidae) as vectors of the rickettsia Orientia tsutsugamushi is well known in Asia and other parts of the eastern hemisphere. In Europe, chigger larvae have been suggested as possible vectors of the bacteria Anaplasma phagocytophilum (Fernandez-Soto et al. 2001) and Borrelia burgdorferi sensu lato (Kampen et al. 2004). A total of 20 specimens of unfed Neotrombicula autumnalis larvae, collected on vegetation in a mountainous zone of Spain, were tested for the presence of A. phagocytophilum based on detection of the species speciWc 151-bp segment of the 16S rRNA gene. This test showed at least a 10% infection with A. phagocytophilum (Fernandez-Soto et al. 2001). A further 30 specimens, collected from mice in the urban and periurban area of a small agricultural town in Spain, did not contain the A. phagocytophilum DNA. Fernandez-Soto et al. (2001) speculated that chigger larvae could contribute to the transmission of A. phagocytophilum. They also concluded that, as the infection occurred in unfed larvae of chiggers, N. autumnalis were true carriers of the bacteria, inheriting A. phagocytophilum through a transovarian transmission pathway. Larvae of N. autumnalis were screened for infection with B. burgdorferi s.l. by means of polymerase chain reaction (PCR) in Germany (Kampen et al. 2004), where 1,380 larvae found on vegetation and 634 larvae collected from 100 trapped micromammals were tested. Borrelial DNA was ampliWed from one larva feeding on Crocidura russula. Such a negligible quantity suggested either that the uptake of borrelial spirochetes by feeding chiggers was extremely rare or that ingested spirochetes are rapidly digested. Results of experiments following a Weld study implied a possible transmission of B. garinii from mouse to chigger larva, and a transstadial and transovarial transmission of B. afzelii in their chigger host (Kampen et al. 2004). Nevertheless, the vector competence of Neotrombicula larvae remains unclear. The aims of this study have been to describe the prevalence of chigger larvae in wild birds in the Czech Carpathian Mountains during the postbreeding period and to elucidate whether chigger larvae play a role in the life history of tick-borne bacteria, as suggested in studies by Fernandez-Soto et al. (2001) and Kampen et al. (2004).
Materials and methods Study location, birds examined, collection and identiWcation of chiggers, collection and examination of ticks Ixodes ricinus Field investigations were conducted at Certak (49°34⬘ N, 17°59⬘ E) in the northeastern part of the Czech Republic, Certak is situated in the foothills of the western Beskidy mountain range, representing the westernmost part of the western Carpathian Mountains. Situated 370–400 m above sea level, this is a densely populated area with intensive agriculture and large areas of mixed forest. Birds were captured using mist nets placed in a forest near a stream. Mist nets were also placed along the forest edge adjoining cattle pastures. Birds were mist-netted from 29 July to 24 September 2005, and the nets were checked once every hour. Captured birds were processed and released back into the wild as quickly as possible to minimize disturbance. A total of 1,080 birds of 37 species were examined. Ectoparasites, including chiggers, were collected using tweezers and preserved in 70% ethanol. In the laboratory, about half of the chigger specimens were mounted in FaureBerlese medium and identiWed using a light microscope with phase contrast and an ocular
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micrometer. The monograph by Kudryashova (1998) and taxonomic papers by Stekolnikov (1997, 2001) were used for identiWcation of chiggers. This material is now deposited in the Zoological Institute, Saint Petersburg, Russia. The remaining chigger specimens were examined for presence of speciWc bacterial DNA. Ticks Ixodes ricinus found on a bird, on which chigger larvae positive for Borrelia DNA were found, were also tested individually for the presence of borrelial DNA in the same manner as were the chigger larvae. PCR detection of pathogens Chigger larvae collected from Turdus merula and T. philomelos were tested individually. Twelve and 67 larvae were examined from two specimens of T. merula and nine of T. philomelos, respectively. Chigger larvae from other species were pooled according to the scenario: one bird—one sample. Numbers of samples from diVerent host species, and total numbers of chigger larvae (in parentheses), were as follows: Cyanocita caeruleus 1 (8), Erithacus rubecula 19 (126), Parus major 5 (15), Poecile palustris 1 (1), Prunella modularis 20 (186), Pyrrhula pyrrhula 1 (1), Sylvia atricapilla 13 (57), and Troglodytes troglodytes 8 (33). A total of 509 chigger larvae found on 79 birds of 10 species were examined. Alkaline hydrolysis (Guy and Stanek 1991) was used to extract genomic DNA from individual chiggers, or pools of chiggers. All samples were subjected to a nested PCR targeting a fragment of the rrf (5S)—rrl (23S) intergenic spacer (IGS) of B. burgdorferi s.l. (Kurtenbach et al. 1998), using B. valaisiana and B. garinii DNA as positive controls. The 218–220 bp ampliWcation products were visualized on 1% agarose gels stained with ethidium bromide. The PCR products were further analyzed by the reverse line blot (RLB) assay using DNA probes speciWc to B. burgdorferi s.l., B. burgdorferi s.s., B. garinii, B. afzelii and B. valaisiana, as described previously by Hanincova et al. (2003) and Kurtenbach et al. (1998). As positive controls, amplicons derived from B. burgdorferi s.s., B. garinii, B. afzelii, and B. valaisiana were also blotted. The PCR approach (Walls et al. 2000), amplifying the epank1 gene with primers speciWc for A. phagocytophilum was used to detect A. phagocytophilum DNA. PCR mixtures with a total volume of 25 l contained primers in Wnal concentration of 0.4 M each, 12.5 l of PCR Master Mix (REDTaq® ReadyMix™, Sigma-Aldrich) and 0.5 l of each mite extract in the case of a pooled reaction, and 1 l of DNA extract for a one-to-one reaction. Anaplasma phagocytophilum DNA was used as a positive control, while LA1 and LA6 primers and thermocycling conditions were as described elsewhere (Walls et al. 2000). The 444-bp ampliWcation products were visualized on 1% agarose gels stained with ethidium bromide.
Results Chigger mites Chigger mites were collected from 1,080 wild birds of 37 species. The prevalence of infestation with chigger larvae was 7%. Twelve species of Passeriformes were infested with chigger larvae (Table 1). A total of 325 chigger larvae were identiWed to the species level. Three species of the genus Neotrombicula were found: Neotrombicula autumnalis (n = 65), N. carpathica (n = 178) and N. inopinata (n = 82).
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1/28 18/196 3/69 1/21 18/55 1/9 1/10 11/324 1/16 9/33 3/31 10/53
Cyanocita caeruleus Erithacus rubecula Parus major Poecile palustris Prunella modularis Pyrrhula pyrrhula Sitta europaea Sylvia atricapilla Sylvia borin Troglodytes troglodytes Turdus merula Turdus philomelos
4 9 4 5 33 11 10 3 6 27 10 19
Infestation prevalence (%)
n.d.—chigger mites were not determined to the species level
No. of infested/No. of examined
Host species
6 99 9 n.d. 86 n.d. 1 40 1 33 10 40
No. of chiggers identiWed (n = 325) 0 30 1 – 10 – 0 7 0 4 4 9
Neotrombicula autumnalis (n = 65)
Table 1 Wild birds infested with chigger larvae in the Czech Carpathians collected during the summer of 2005
6 16 2 – 37 – 0 6 0 9 1 5
N. inopinata (n = 82)
0 53 6 – 39 – 1 27 1 20 5 26
N. carpathica (n = 178)
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We did not Wnd chiggers on the following 25 species: Alcedo atthis (1 specimen examined), Crex crex (1), Dendrocopos major (1), Picus canus (1), Aegithalos caudatus (23), Certhia familiaris (18), Lanius collurio (14), Motacilla cinerea (12), Regulus regulus (3), Poecile montanus (13), Emberiza citrinella (13), Fringilla coelebs (8), Coccothraustes coccothraustes (1), Ficedula albicollis (5), F. hypoleuca (23), Muscicapa striata (13), Phoenicurus phoenicurus (4), Acrocephalus palustris (1), Hippolais icterina (2), Locustella naevia (8), Phylloscopus collybita (42), P. sibilatrix (4), P. trochilus (5), Sylvia communis (18), and S. curruca (1). Detection of Borrelia and Anaplasma DNAs A fragment of the rrf (5S)—rrl (23S) IGS of B. burgdorferi s.l. was ampliWed in one chigger sample. An RLB assay proved the presence of both B. garinii and B. valaisiana. This sample pooled Wve chigger larvae collected from Sylvia atricapilla on 11 August 2005. On 8 August 2005 and 15 August 2005, the same bird, identiWed by its ring number, had been caught and examined at the same location. Chigger larvae and ticks I. ricinus were found on this bird during each of three examinations. The results for two chigger larvae and six ticks (four larvae and two nymphs) collected on 8 August 2005 were negative. The results for three tick larvae collected on 11 August 2005 were also negative, as were the results for 12 chigger larvae and 14 ticks (10 larvae and 4 nymphs) collected on 15 August 2005. In no sample, the epank1 gene of A. phagocytophilum DNA was ampliWed.
Discussion Literak et al. (2001) noted that chiggers were often found on birds during their postbreeding migration in the Slovak and Polish Carpathians. They examined 1,354 birds of 59 species and found the prevalence of infestation to be 12%. As with this study, a high prevalence of infestation was found in Turdus philomelos, Prunella modularis, and Troglodytes troglodytes. The larvae of chiggers, therefore, are common parasites in birds in the Czech, as well as the Slovak and Polish, Carpathians in late summer, and especially in birds that feed on the ground. Neotrombicula autumnalis, a well-known agent of trombidiosis, is the main humanattacking chigger mite in Europe and has been recorded on a vast range of mammal and bird hosts. Neotrombicula carpathica has been reported on rodents from Ukraine (Transcarpathia), Moldova (Kudryashova 1998), and Russia (Krasnodar Krai, Karachay-Cherkessia, Kabardino-Balkaria, and North Ossetia-Alania) (Stekolnikov 2001). In this paper, N. carpathica is recorded for the Wrst time in the Czech Republic, and for the Wrst time on bird hosts. Moreover, there is a supposition, based on the analysis of geographic variability in the N. talmiensis species group, that all Wndings of N. talmiensis in Europe (South Russia, Moldova, Ukraine, Czech Republic, Slovakia, Albania, and Bulgaria) be attributed to N. carpathica (Stekolnikov 2002). This question needs further taxonomic investigations. Neotrombicula inopinata is found in the Czech Republic, Great Britain, Austria, Germany, Bulgaria, France, states of former Yugoslavia, Ukraine (Transcarpathia), and Russia (North Ossetia-Alania and Stavropol Krai) (Daniel et al. 1995; Kudryashova 1998), where it is reported from many species of rodents, insectivores, and three bird species. In this paper, it is recorded for the Wrst time on Cyanocita caeruleus, Parus major, Prunella modularis, Sylvia atricapilla, Troglodytes troglodytes, Turdus merula, and Turdus philomelos.
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We detected two Borrelia species DNAs in chigger mite larvae of the genus Neotrombicula collected from Sylvia atricapilla. Borrelia garinii is associated with neuroborreliosis in humans (Wilske et al. 1996). The main reservoirs of B. garinii in the wild in Central Europe are two bird species, Turdus merula and T. philomelos (Taragelova et al. 2008). Moreover, in ticks collected from birds migrating during the postbreeding period in Sweden, B. garinii was the most frequently detected Borrelia species (Comstedt et al. 2006). We believe that the Neotrombicula larvae become infected while sucking the macerated tissue of the host (Coignoul 1976), possibly of the main reservoirs T. merula or T. philomelos, species commonly occurring at Certak. The prevalence of infestation with Neotrombicula spp. in T. merula and T. philomelos were 10% and 19%, respectively, while infestation with Neotrombicula spp. in S. atricapilla was only 3%. Sylvia atricapilla, however, is not an important reservoir of Borrelia spirochetes (Comstedt et al. 2006; Hanincova et al. 2003; Taragelova et al., 2008). It is noteworthy that our Wnding of Borrelia spp. in larvae of Neotrombicula spp. on 11 August 2005 does not correlate with the negative results for larvae of Neotrombicula spp. collected on 8 and 15 August 2005. Further, it does not correlate with the negative results for I. ricinus ticks collected on 8, 11, and 15 August 2005 from the same bird. If the source of infection of Neotrombicula larvae was S. atricapilla specimen the mites had been collected from, we would expect positive results for Neotrombicula spp. larvae each time they were collected and, particularly, positive results for I. ricinus ticks, which are highly susceptible to Borrelia infection. For this reason, we have to consider other sources than S. atricapilla (perhaps T. merula or T. philomelos), as well as transovarial and transstadial transmission of Borrelia genospecies in Neotrombicula spp., as suggested by Kampen et al. (2004). Should chigger larvae be proved to transmit borrelias, they could contribute to maintaining borrelias in autochthonous natural foci. Although there are rare reports on the association of Borrelia valaisiana with neuroborreliosis (Diza et al. 2004, Saito et al. 2007), this Borrelia species is generally considered as non-pathogenic for humans. Borrelia valaisiana is a frequent spirochete in ticks collected from wild birds in Central Europe and Sweden (Comstedt et al. 2006, Taragelova et al. 2008) and, therefore, certain bird species in Europe are likely reservoirs of B. valaisiana. We suggest (just as for B. garinii) that the host (present or former) of Neotrombicula spp. was a reservoir of B. valaisiana, and that Neotrombicula larvae are infected while sucking the macerated tissue of this host. We summarize that larvae of Neotrombicula spp. can be infected with the Borrelia genospecies originating from their host in the same way as larvae and nymphs of the tick I. ricinus. Ixodes ricinus was found to be the main vector of A. phagocytophilum in Europe (Strle 2004). Anaplasma spp. were found also in a number of dermanyssoid mites throughout the world (Reeves et al. 2006). There is little information on the importance of wild birds in the life history of A. phagocytophilum. Wild birds and their ectoparasites (both I. ricinus ticks and Syringophilidae quill mites) were surveyed for infection with A. phagocytophilum in Poland (Skoracki et al. 2006). Amongst the birds captured in this study, 14 individuals, representing four species, hosted quill mites from the family Syringophilidae. Three of 14 mite pools recovered from 14 mite-infested birds harbored A. phagocytophilum DNA (amplifying both the epank1 and p44 genes). The PCR-positive pools originated from one T. merula and two Sturnus vulgaris. The speciWc biology of syringophilid mites, which parasitize exclusively inside the quills of feathers and feed on the host’s subcutaneous Xuids, implies that they must have acquired the pathogen from a bacteremic bird. These results provide the Wrst indirect evidence that at least some passerine hosts are prone to developing
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systemic infections with A. phagocytophilum under natural conditions. Based on our results of Neotrombicula spp. mite examinations, we cannot support this hypothesis. Both bacterial agents Borrelia spp. and A. phagocytophilum were rarely found in chigger mite larvae collected on wild birds. In next Weld studies, an examinations of the birds the positive chiggers had been derived from should be also included in order to characterize a life cycle with chiggers of these agents and their importance for maintaining of Borrelia and Anaplasma foci in nature. Acknowledgements This study was funded through grant No. MSM6215712402 from the Ministry of Education, Youth and Sports of the Czech Republic and Lenka Dubska received funding through grant No. 524-08-P139 from the Czech Science Foundation. We thank Dr. Miroslav Petrovec (Department of Infectious Diseases, University of Ljubljana, Slovenia) for providing A. phagocytophilum DNA, and Dr. Elena Kocianova (Institute of Virology, Bratislava, Slovakia) for a determination of ticks Ixodes ricinus. The authors declare that the study complies with the current laws of the Czech Republic.
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