Feeding ticks are generally spatially distributed in clusters on vertebrate hosts. To test the effect of clustering on transmission of a tick-borne pathogen, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner-infected Ixodes ricinus L. nymphs and uninfected I. ricinus larvae were allowed to feed together in retaining chambers on uninfected AKR/N mice. Engorged infective nymphs dropped off at days 5, 6, and 7, and the 1st infected larvae that fed in the chambers together with the infected nymphs dropped off at day 5. In contrast, ear biopsies and xenodiagnostic larvae placed on the head remained negative during that period. These results suggest that a cofeeding transmission occurred between B. burgdorferi-infected ticks and noninfected ones in the absence of a disseminated infection. Further investigations are being undertaken to determine whether the mechanism responsible for this cofeeding transmission is similar to that described previously with virus-infected tick

Gern, Lise

Rais, Olivier

RERO DOC Digital Library

SHORT COMMUNICATIONEfficient Transmission of Borrelia burgdorferi BetweenCofeeding Ixodes ricinus Ticks (Acari: Ixodidae)LISE GERN AND OLIVIER RAISInstitut de Zoologie, University de Neuchatel, Neuchatel, SwitzerlandJ. Med. Entomol. 33(1): 189-192 (1996)ABSTRACT Feeding ticks are generally spatially distributed in clusters on vertebrate hosts.To test the effect of clustering on transmission of a tick-borne pathogen, Borrelia burgdorferiJohnson, Schmid, Hyde, Steigerwalt & Brenner-infected Ixodes ricinus L. nymphs and unin-fected I. ricinus larvae were allowed to feed together in retaining chambers on uninfectedAKR/N mice. Engorged infective nymphs dropped off at days 5, 6, and 7, and the 1st infectedlarvae that fed in the chambers together with the infected nymphs dropped off at day 5. Incontrast, ear biopsies and xenodiagnostic larvae placed on the head remained negative duringthat period. These results suggest that a cofeeding transmission occurred between B. hurg-dorferi-infected ticks and noninfected ones in the absence of a disseminated infection. Furtherinvestigations are being undertaken to determine whether the mechanism responsible for thiscofeeding transmission is similar to that described previously with virus-infected ticks.KEY WORDS Borrelia burgdorferi, Ixodes ricinus, cofeeding transmission, localized infec-tion, disseminated infectionBorrelia burgdorferi Johnson, Schmid, Hyde,Steigerwalt & Brenner, the agent of Lyme borre-liosis, is a motile extracellular bacterial organismtransmitted to the host by the bites of infectedticks belonging mostly to the Ixodes ricinus L.complex. When an infected tick feeds on an animalhost, B. burgdorferi spirochetes, present in the tickmidgut, migrate through the midgut wall a fewhours after the onset of the blood meal, reach thehemolymph, and invade various tick organs, in-cluding salivary glands, from which they will betransmitted to the host through saliva (Benach etal. 1987, Ribeiro et al. 1987, Zung et al. 1989, Gernet al. 1990). Once infected, this animal will rep-resent an infecting source for other tick individu-als. Skin is not only the site of inoculation of thespirochetes but also an important target organ inLyme borreliosis. In humans, skin is involved inthe early and late stages of the disease. Spirocheteshave been isolated from the skin of a patient asearly as 10 d after a tick bite (Neubert et al. 1986).Likewise, in laboratory animals, Lyme borreliosisspirochetes seem to remain at the site of inocula-tion for ^ l wk after infecting ticks become en-gorged and detached (Shih et al. 1992, 1993).Feeding ticks are generally spatially distributedin clusters on the vertebrate hosts. This clusteringmay have implications on the transmission of tick-borne pathogens. In fact, transmission of patho-gens from infected ticks to uninfected ticksthrough the host skin has been shown previouslyfor intracellular and nonmotile microorganisms.Efficient transmission of Thogoto virus and tick-borne encephalitis virus has been demonstratedfrom infected ticks to uninfected ticks cofeedingin chambers on nonviremic hosts (Jones et al.1987, Alekseev and Chunikhin 1990, Labuda et al.1992). Because spirochetes seem to remain in theinoculation site for a few days after tick detach-ment, we were interested in testing the effect oftick clustering on the transmission of B. burgdor-feri and in investigating the hypothesis of a possi-ble cofeeding transmission between infected anduninfected I. ricinus ticks.Materials and MethodsTicks, Bacteria, and Mice. Ticks used in thisstudy came from a laboratory colony of spirochete-free /. ricinus ticks maintained at the Institut deZoologie (Neuchatel) according to the methods de-scribed by Graf (1978). The spirochetal isolate ofB. burgdorferi (ZS7) used in this study was ob-tained initially from Ulrich Schaible and MarkusSimon (Max-Planck Institut fiir Immunbiologie,Freiburg, Germany). The spirochetes were grownin modified Barbour—Stoenner—Kelly (BSKII) me-dium (Barbour 1984). The ZS7 isolate belongs tothe B. burgdorferi sensu stricto type (Wallich et al.1992). Female mice of the strains AKR/N andBALB/c were bred at the Institut de Zoologie,Neuchatel. Animal care and manipulations were inaccordance with the Swiss Federal Welfare Laws(LPA and OPA). This study was conducted in ac-cordance with the permit issued by the CantonalDepartment of Agriculture.0022-2585/96/0189-0192$02.00/0 © 1996 Entomological Society of America190 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 33, no. 1Table 1. Infection of I. ricinus nymphal ticks fed as larvae on mice together with B. biirgdorferi-infuvlcil nymphsand isolation of spirochetes from mice by culturing ear biopsiesMouse no.1234Chamber12headbiopsy12headbiopsy12headbiopsy12headbiopsyDay 2No. infected/tested2/8 (25)4/10 (40)0/5Negative3/11 (27)1/14 (7)No tickNegative5/9 (56)3/8 (38)0/4Negative3/7 (43)5/13 (39)No tickNegativeDay 5No. infected/tested2/9 (22)2/2 (100)ndNegative10/13 (77)4/7 (57)ndNegative6/8 (75)4/6 (67)0/12Negative0/1No tick0/6NegativeDay of placement of ticks on miceDay 8No. infected/testedNo tickNo tickNo tickNegative3/4 (75)1/2 (50)No tickNegative3/4 (75)No tick0/16NegativendndndndDay 11No. infected/tested9/9 (100)10/10 (100)0/6Negative4/5 (80)1/1 (100)0/9NegativendndndNegativendndndndDay 14No. infected/testedNo tick3/3 (100)0/1Negative3/3 (100)No tickNo tickNegativendndndndndndndndDay 29No. infected/tested1/5 (20)4/5 (80)4/5 (80)PositivendndndndndndndndndndndndPercentage of infected ticks in parentheses. No tick, ticks did not molt; nd, not done.Infection of Ticks. I. ricinus nymphs that hadbeen infected by capillary feeding (Gern et al.1990, Hu et al. 1992) with BSKII medium con-taining 8 X 107 spirochetes per milliliter of strainZS7 as described previously (Gern et al. 1994)were allowed to feed on uninfected BALB/c mice.One month later, these infected mice were infest-ed with uninfected /. ricinus larvae. The derivedinfected nymphs were used in the cofeeding ex-periment. The infection rate of the unfed I. ricinusnymphs was 70% as determined by direct immu-nofluorescent antibody assay.Spirochete Detection in Ticks. Ticks wereevaluated for B. burgdorferi after molting using adirect immunofluorescence antibody test as de-scribed previously (Gern et al. 1991). Briefly, tickswere squashed on slides and treated with a fluo-rescein isothiocyanate-labeled conjugate preparedaccording to Peacock et al. (1971).Cofeeding Experiment. Two retaining cham-bers were glued on the backs of each of 4 unin-fected AKR/N mice as described by Mbow et al.(1994). Each chamber was inspected carefully dai-ly and if necessary glue was added. A collar wasplaced around the neck of each mouse.To determine whether cofeeding transmission ofB. burgdorferi can occur between infected andnoninfected ticks, 7 infected /. ricinus nymphs (in-fection rate 70%) were placed into each chamberon day 0, and a=20 uninfected larvae were addedinto each chamber on days 2 and 5. To control forthe presence of a disseminated infection while in-fected nymphs were feeding, —20 larvae were add-ed freely (that is, not held within chambers) on thehead of each mouse on days 2 and 5 and ear bi-opsies were taken on days 2 and 5, according toSinsky and Piesman (1989). After detachment ofinfected nymphs, the localized versus disseminatedinfection was monitored by adding larvae in thechambers and on the head of the mice and by cul-turing ear biopsies on days 8, 11, 14, and 29.Mice were kept in separate cages over trays ofwater, and engorged ticks that had dropped fromthe head into the water were collected daily.Chambers were examined daily, and the time ofdetachment of nymphs and larvae that had fed inthe chambers and on the head was noted. En-gorged ticks were placed into vials and stored atroom temperature and 95% RH. Ticks were ex-amined for spirochetes after molting using immu-nofluorescence as described above.ResultsOn day 0, four AKR/N mice were infested withinfected /. ricinus nymphs. Engorged infected /.ricinus nymphs dropped off on days 5, 6, and 7after placement into the retaining chambers. Fromeach chamber, 6-7 engorged nymphal ticks werecollected. All nymphs fed in the 2 chambers onmice no. 2 (n = 7 and 6) and no. 4 (n = 7 and 6)dropped off on day 5, nymphal ticks fed on mouseno. 3 dropped off on day 5 (n = 6 and 5) and day6 (n = 1 and 1), and nymphs placed on mouse no.1 dropped off on day 5 (n = 5 and 5), day 6 (n =1; chamber 1), and day 7 (n = 1; chamber 2).Cofeeding transmission from infected nymphsto uninfected I. ricinus larvae was observed forboth retaining chambers placed on each of the 4AKR/N mice (=8/8 chambers positive) (Table 1).The 1st batch of I. ricinus larvae fed in chamberswas shown to be infected by B. burgdorferi (infec-tion rate 7-56%) for all of the 8 chambers (Table1). These larvae had been placed on mice on day2 and fed simultaneously with infected nymphs forat least 3 d. Infection appeared to remain localizedJanuary 1996 GERN AND RAIS: COFEEDING TRANSMISSION OF B. hurgdorferi 191while cofeeding transmission of B. burgdorferi oc-curred between infected nymphs and uninfectedlarvae. This was indicated by the fact that cultureof ear biopsies taken from all mice on day 2 andon day 5 and examination of a total of 27 larvaecollected from the head of 3 of 4 animals on days2 or 5, or both, failed to detect B. burgdorferi.After detachment of the infected nymphs, lo-calized infection was observed for 3 of 3 mice bydetection of spirochetes in larvae placed in thechambers on day 8 (mouse no. 3); on days 8, 11,and 14 (mouse no. 2); and on days 11, 14, and 29(mouse no. 1) (Table 1).The appearance of a disseminated infection wasmonitored by examining larvae collected from thehead of 1 mouse (mouse no. 3) on day 8, 2 mice(mice no. 1 and 2) on day 11, 1 mouse (mouse no.1) on days 14 and 29 (Table 1). Only the batch oflarvae placed on day 29 on mouse 1 was foundpositive. Ear biopsies taken on days 8 and 11 frommice 1, 2, and 3 remained negative, as well as earbiopsies taken on day 14 from mice 1 and 2. Incontrast, an ear biopsy taken on day 29 frommouse no. 1 was positive.DiscussionTick feeding behavior is characterized by thelong duration of the blood meal compared withthat of most other hematophagous arthropods. In-deed, Ixodid ticks feed for days to weeks on theirhosts before they drop off. Consequently, in nat-ural conditions, a great number of ticks may feedat the same time on the same host. Moreover, ticksare often spatially distributed in clusters on theirhosts. This clustering may have some effect on thetransmission of tick-borne pathogens, as previ-ously demonstrated with Thogoto and tick-borneencephalitis viruses (Jones et al. 1987, Alekseevand Chunikhin 1990, Labuda et al. 1992). Thosestudies showed that efficient transmission of theseorganisms can occur between cofeeding tickseven when the hosts do not develop a detectableviremia.The vectors of the Lyme borreliosis agent, B.burgdorferi, are all ixodid ticks that feed for severaldays on their hosts. When B. burgdorferi-infectedticks feed on an animal, the spirochetes are inject-ed into the host skin and may remain at the in-oculation site for a few days after the end of theblood meal (Shih et al. 1992,1993). This promptedus to investigate whetlier cofeeding transmission ofa motile and extracellular microorganism like B.imrgdorferi may occur between infected and un-infected ticks while feeding on a host that had notpreviously been exposed to the infection.In our cofeeding experiment, infected /. ricinuslarvae that had fed simultaneously with infectednymphs in the retaining chambers were collectedfrom all 8 chambers of the 4 AKR/N mice. Thisshows that cofeeding transmission of B. burgdor-feri occurred between infected and noninfectedticks via the host skin in all sites where these tickswere attached. By contrast, no infected larvae werecollected from the head of the animals during thesame period, demonstrating that infection was lo-calized. These results suggest that infection did notimmediately disseminate from the inoculation site.The lack of success in isolating B. burgdorferi fromear biopsies collected on days 2 and 5 was consis-tent with localization of the infection to the skinsite in which infected ticks fed. Our results alsocorroborate previous findings that B. burgdorferiremains in the skin near the inoculation site beforedissemination (Shih et al. 1992, 1993). Localizedinfections were observed for the duration of theobservation period for each mouse (range, 5—29 d).Evidence of a disseminated infection was shownby the detection of B. burgdorferi in an ear biopsyand in 4 of 5 larvae collected from the head ofmouse 1 on day 29. Additional studies are neededto determine the time of development of a dissem-inated infection.Because I. ricinus ticks may be infected by dif-ferent B. burgdorferi species (Hu et al. 1994), co-feeding transmission of various spirochetes mayoccur between I. ricinus ticks infected by differentB. burgdorferi species, allowing exchange of spi-rochetes among ticks, and may partly explain thepresence of different B. burgdorferi phenotypes/genotypes in 1 tick, as observed in adult I. ricinusticks (Leuba-Garcia et al. 1994).Whether B. burgdorferi cofeeding transmissioncan occur between ticks feeding on natural hostspecies remains to be studied. Nevertheless, thedemonstration of cofeeding transmission of B.burgdorferi has important implications for evalu-ating the role, in B. burgdorferi transmission, ofdifferent vertebrate hosts of the tick vector. Mostattempts to determine the ability of a particularvertebrate species to support B. burgdorferi infec-tion (and to transmit the infection to feeding ticks)have been designed to detect disseminated infec-tion. However by means of cofeeding transmission,it is theoretically possible that tick hosts such asdeer may be capable of supporting localized trans-mission even though they may not develop a dis-seminated infection.AcknowledgmentsSpecial thanks to P. F. Humair for his invaluable helpand to Patricia Nuttall for her comments to the manu-script and interesting discussions. We also thank MilanLabuda and Floriane de Marval for discussions, and Mar-kus Simon and Ulrich Schaible for the strain ZS7. Thisresearch was financially supported by the Swiss NationalScience Foundation (No. 32-299964-90), and the FederalOffice for Education and Science (No. 93.0363) as partof the European Union Biomedical and Health Research(Biomed I) Programme (No. BMH-CT93-1183).References CitedAlekseev, A. N., and S. P. Chunikhin. 1990. Ex-change of tickborne encephalitis virus between Ixod-192 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 33, no. 1idae simultaneously feeding on the animals with sub-threshold levels of viraemia. Med. Parazitol. Parazit.Bolezni. 2: 48-50.Harbour, A. G. 1984. Isolation and cultivation of Lymedisease spirochetes. Yale J. Biol. Med. 57: 521-525.Bcnach, J. L., J. L. Coleman, R. A. Skinner, and E.M. Rosier. 1987. Adult Ixodes dammini in rabbits:a hypothesis for the development and transmission ofBorrelia burgdorferi. J. Infect. Dis. 155: 1300-1306.Gem, L., Z. Zhu, and A. Aeschlimann. 1990. Devel-opment of Borrelia burgdorferi in Ixodes ricinus fe-males during blood feeding. Ann. Parasitol. Hum.Comp. 65: 89-93.Gcrn, L., L. N. Toutoungi, C. M. Hu, and A. Aeschli-mann. 1991. Ixodes (Pholeoixodes) hexagonus, anefficient vector of Borrelia burgdorferi in the labora-tory. Med. Vet. Entomol. 5: 431-435.Gern, L., O. Rais, C. Capiau, P. Hauser, Y. Lobet, E.Sinioen, P. Voet, and J. Petre. 1994. Immuniza-tion of mice by recombinant OspA preparations andprotection against Borrelia burgdorferi infection in-duced by Ixodes ricinus tick bites. Immunol. Lett. 39:249-258.Graf, J. F. 1978. Copulation, nutrition et ponte chezIxodes ricinus L. (Ixodoidea: Ixodidae), lere partde.Bull. Soc. Entomol. Suisse 51: 89-97.Hu, C. M., L. Gern, and A. Aeschlimann. 1992.Changes in the protein profiles and antigenicity of dif-ferent Borrelia burgdorferi strains after reintroduc-tion to Ixodes ricinus ticks. Parasitol. Immunol. 14:415-427.Hu, C. M., S. Leuba-Garcia, A. Aeschlimann, M. D.Kramer, and L. Gern. 1994. Comparison in theimmunological properties of Borrelia burgdorferi iso-lates from Ixodes ricinus derived from three endemicareas in Switzerland. Epidemiol. Infect. 112: 533-542.Jones, L. D., C. R. Davies, G. M. Steele, and P. A.Nuttall. 1987. A novel mode of arbovirus transmis-sion involving non-viraemic host. Science (Washing-ton, DC) 237: 775-777.Labuda, M., L. O. Jones, T. Williams, V. Oanielova,and P. A. Nuttall. 1992. Efficient transmission oftick-borne encephalitis virus between co-feeding ticks.J. Med. Entomol. 30: 295-299.Leuba-Garcia, S., M. D. Kramer, R. Wallich, and L.Gern. 1994. Characterization of Borrelia burgdor-feri isolated from different organs of Ixodes ricinusticks collected in nature. Zentralbl. Bakteriol. 280:468-475.Mbow, M. L., M. Christe, B. Rutti, and M. Brossard.1994. Absence of acquired resistance to nymphalIxodes ricinus ticks in BALB/c mice developing cu-taneous reactions. J. Parasitol. 80: 81-87.Neubert, U., H. E. Krampitz, and H. Engl. 1986.Microbiological findings in erythema (chronicum)migrans and related disorders. Zentralbl. Bakteriol.Hyg. Ser. A 263: 237-252.Peacock, M., W. Burgdorfer, and R. S. Ormsbec.1971. Rapid fluorescent-antibody conjugation pro-cedure. Infect. Immun. 3: 355-357.Ribeiro, J.M.C., T. N. Mather, J. Piesman, and A.Spielman. 1987. Dissemination and salivary deliv-ery of Lyme disease spirochetes in vector ticks. J.Med. Entomol. 24: 201-205.Shin, C. M., R. J. Pollack, S. R. Telford III, and A.Spielman. 1992. Delayed dissemination of Lymedisease spirochetes from the site of deposition in theskin of mice. J. Infect. Dis. 166: 827-831.Shih, C. M., S. R. Telford III, R. J. Pollack, and A.Spielman. 1993. Rapid dissemination by the agentof Lyme disease in hosts that permit fulminating in-fection. Infect. Immunol. 61: 2396-2399.Sinsky, R. J., and J. Piesman. 1989. Ear punch bi-opsy method for detection and isolation of Borreliaburgdorferi from rodents. J. Clin. Microbiol. 27:1723-1727.Wallich, R., S. E. Moter, M. D. Kramer, L. Gern, H.Hofman, U. E. Schaible, and M. M. Simon. 1992.Untersuchungen zur genotypischen and phaenotyp-ischen Heterogenitaet von Borrelia burgdorferi, demErreger der Lyme Borreliose, pp. 176-190. In D.Hassler [ed.], Infection Taschenbuch. MMV Medizin,Miinchen.Zung, J. L., S. Lewengrub, M. A. Rudzinska, A. Spiel-man, S. R. Telford III, and J. Piesman. 1989.Fine structural evidence for the penetration of theLyme disease spirochete Borrelia burgdorferi throughthe gut and salivary tissues of Ixodes dammini. Can.J. Zool. 67: 1737-1748.Received for publication 7 March 1995; accepted 28July 1995.

Efficient Transmission of Borrelia burgdorferi Between Cofeeding Ixodes ricinus Ticks (Acari: Ixodidae)

Abstract

Similar works

Full text

Available Versions

RERO DOC Digital Library