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Experimental and AppliedAcarology ISSN 0168-8162Volume 52Number 4 Exp Appl Acarol (2010)52:383-392DOI 10.1007/s10493-010-9375-7

Co-feeding as a route for transmissionof Rickettsia conorii israelensis betweenRhipicephalus sanguineus ticks

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Co-feeding as a route for transmission of Rickettsiaconorii israelensis between Rhipicephalus sanguineusticks

G. Zemtsova • L. F. Killmaster • K. Y. Mumcuoglu • M. L. Levin

Received: 25 May 2010 / Accepted: 10 June 2010 / Published online: 30 June 2010� U.S. Government 2010

Abstract Rickettsia conorii is widely distributed in Europe, Asia, and Africa. The brown dog

tick, Rhipicephalus sanguineus, is the recognized vector of R. conorii. In this study, we assessed

the efficiency of R. conorii israelensis transmission between co-feeding Rh. sanguineus ticks.

Infected Rh. sanguineus adults and uninfected nymphs were fed simultaneously upon either

naıve dogs or a dog previously exposed to this agent. When ticks were placed upon naıve dogs,

92–100% of nymphs acquired the infection and 80–88% of infected engorged nymphs trans-

mitted it transstadially. When ticks were placed upon a seropositive dog, only 8–28.5% of

recipient nymphs became infected. Our results establish the first evidence for efficient natural

transmission of R. conorii israelensis between co-feeding ticks upon both naıve and seropos-

itive dogs. This route of transmission can ensure continuous circulation of R. conorii israelensisin tick vectors even in the absence of naıve reservoir hosts.

Keywords Rickettsia conorii � Rhipicephalus sanguineus � Co-feeding �Transstadial transmission � Infection � Immunity

Introduction

Rickettsia conorii is the infectious agent which causes Mediterranean spotted fever or

Boutonneuse fever in man. It is endemic in many countries of Europe and the Middle East

The findings and conclusions described in this manuscript are those of the authors and do not necessarilyrepresent the views of the Centers for Disease Control and Prevention and the Department of Health andHuman Services.

G. Zemtsova � L. F. Killmaster � M. L. Levin (&)Rickettsial Zoonoses Branch, Mail Stop G-13, National Center for Zoonotic,Vector-Borne and Enteric Diseases, Centers for Disease Control and Prevention,1600 Clifton Road NE, Atlanta, GA 30333, USAe-mail: MLevin@cdc.gov

K. Y. MumcuogluDepartment of Microbiology and Molecular Genetics, The Kuvin Centerfor the Study of Infectious and Tropical Diseases, 91120 Jerusalem, Israel

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that are adjacent to the Mediterranean, Black, and Caspian Seas. Different subspecies of R.conorii, closely related to the reference strain Malish, cause clinically similar though

serotypically distinct diseases (Zhu et al. 2005). R. conorii subspecies caspia is the

causative agent of Astrakhan fever (Tarasevich and Mediannikov 2006). R. conorii sub-

species Indian tick typhus causes Indian tick typhus (Jain et al. 2008). R. conorii sub-

species israelensis, the causative agent of Israeli tick typhus (ISTT), has been described in

Israel, Italy, and Portugal (Mumcuoglu et al. 2002; Blanc and Caminopetros 1932; Tringali

et al. 1986; Peter et al. 1990; Bacellar et al. 1991; Raoult et al. 1992; Psaroulaki et al. 1999;

Bernasconi et al. 2002; Giammanco et al. 2003). Israeli tick typhus is characterized by

fever, headache, and rash after a tick bite (Mumcuoglu et al. 2002; Zhu et al. 2005). In

Europe and in the Mediterranean region, the brown dog tick, Rhipicephalus sanguineus, is

the recognized vector of various strains of R. conorii (Bacellar et al. 1991; Bernasconi et al.

2002; Blanc and Caminopetros 1932; Giammanco et al. 2003; Mumcuoglu et al. 2002;

Peter et al. 1984; Peter et al. 1990; Psaroulaki et al. 1999; Raoult et al. 1992; Tringali et al.

1986). The most common hosts of adult Rh. sanguineus are medium to large-size mammals

such as hedgehogs, foxes, badgers, goats and dogs, while the immature ticks may feed

upon rodents, insectivores and lagomorphs (Mumcuoglu et al. 2002; Peter et al. 1984). A

number of experimental studies have documented transovarial and transstadial transmis-

sion of R. conorii by infected Rh. sanguineus ticks (Blanc and Caminopetros 1932;

Mumcuoglu et al. 2002; Neitz et al. 1941; Socolovschi et al. 2009); however, there are no

data on pathways by which uninfected Rh. sanguineus can acquire the agent.

Co-feeding or non-systemic transmission pathway is the exchange of a pathogen

between infected and uninfected arthropod vectors feeding simultaneously on a host in the

absence of systemic infection (Jones et al. 1987). Three major forms of co-feeding

transmission have been described: transsalival—exchange of pathogen in the saliva of

infected arthropods to uninfected arthropods feeding in close proximity to one another;

distant—transmission of pathogen between arthropods feeding simultaneously at a distance

greater than 1 cm (Alekseev and Chunikhin 1990, 1991); and extended—transmission of

pathogen to uninfected arthropods feeding subsequent to, but in the same location, as

infected arthropods (Randolph et al. 1996). Co-feeding as a mechanism of transmission of

tick-borne pathogens between infected and uninfected ticks may have significant epide-

miological consequences in certain enzootic cycles where systemic infection of vertebrate

hosts may not occur (Randolph et al. 1996). Moreover, this pathway might be realized on

immune animals (Ogden et al. 1997).

This mechanism has been described for the Thogoto virus (Jones et al. 1987, 1990,

1997), the tick-borne encephalitis virus (Alekseev and Chunikhin 1992; Chunikhin 1990;

Labuda et al. 1993a, b), Borrelia burgdorferi (Piesman and Happ 2001; Randolph et al.

1996), Anaplasma phagocytophilum (Levin and Fish 2000), and Rickettsia massiliae(Matsumoto et al. 2005). However, occurrence and efficiency of this transmission mech-

anism vary significantly even among closely related species of pathogens. For example,

Borrelia afzelii is acquired by up to 55% of larvae co-feeding with infected nymphs, while

for B. burgdorferi s.st. the efficiency of such transmission is 0 to 5% (Piesman and Happ

2001; Richter et al. 2002). Similarly, non-systemic transmission of A. marginale did not

occur between infected and uninfected Dermacentor spp. ticks that fed at the same site,

while A. phagocytophilum was acquired by up to 11% of co-feeding Ixodes scapularis(Kocan and de la Fuente 2003; Levin and Fish 2000).

Here, we describe the phenomenon of transmission of R. conorii israelensis between co-

feeding Rh. sanguineus ticks in the absence of detectable host bacteriemia and evaluate its

efficiency in naıve and seropositive hosts.

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Materials and methods

For this study, we used an isolate of R. conorii sub-species israelensis (strain T-487)

originating from Rh. sanguineus ticks collected in Kfar Vitkin, Israel. We chose this

particular isolate because it had shown to be infectious to Rh. sanguineus and to cause only

low mortality in ticks, whereas infection with R. conorii strain Malish was detrimental to

infected ticks (Levin et al. 2009). The agent was grown in Vero E6 cells at 32�C in

antibiotic-free Minimal Essential Medium supplemented with 2% fetal calf serum and

2 mg/ml L-glutamine. Prior to introduction into laboratory dogs and ticks the agent was

purified by Renografin density gradient centrifugation, frozen at -70�C in SPG (with

5MM MgCl2 and 1% Renografin76), and titered by plaque assay on Vero E6 cells, as

previously described (Eremeeva et al. 2003).

Rhipicephalus sanguineus ticks for this study were derived from colonies of North

American and Mediterranean origins maintained in our laboratory as previously described

(Levin et al. 2009; Troughton and Levin 2007). The North American colony originated

from adult ticks collected in Oklahoma, USA in 2001, and the Mediterranean colony

originated from adult ticks collected in Israel in 2005. Specific pathogen free New Zealand

white rabbits (Oryctolagus cuniculus) were used as hosts for feeding all developmental

stages of uninfected ticks. Between feedings, ticks were kept in environmental incubators

at 24 ± 1�C and 90% relative humidity.

Cohorts of infected adult ticks were produced by initial feeding of uninfected larvae

from both colonies upon dogs inoculated intravenously with R. conorii israelensis, and

following feeding of infected nymphs upon a naıve rabbit as previously described (Levin

et al. 2009). The resulting prevalence of infection was 50.0 ± 22.48 and 30.0 ± 20.61% in

adult ticks from North American and Mediterranean colonies, respectively.

For experimental procedures, we used 18-month old cross-breed beagle male dogs.

Ticks were placed inside 7 cm diameter feeding bags attached to a shaven area on the back

of each dog. Infected adults, requiring 9 days for full engorgement, were placed on the

host. Uninfected nymphs feeding for approximately four to 5 days were added 4 days later

in order for both the donor and recipient ticks to complete engorgement simultaneously.

In the first experiment, 30 females and 30 males Rh. sanguineus infected with R. conoriiisraelensis from either North American or Mediterranean colonies (donor ticks) were

placed upon two naıve dogs. Three hundred uninfected nymphs (recipient ticks) from the

corresponding colonies were added into the same feeding bags.

For the second experiment, we used only the natural—sympatric vector-pathogen

combination, i.e. R. conorii israelensis and Rh. sanguineus from our Mediterranean colony.

Infected adult Rh. sanguineus were derived from nymphs infected with R. conorii isra-elensis during the first experiment. This time, ticks were placed upon a dog that had been

exposed to R. conorii israelensis via infected tick bite 5 months earlier. Complete recovery

of the dog from the previous infection was evidenced by repeated negative results of whole

blood PCR, and gradual decline of antibody titer from its peak of C2,048 to 256, at the

time of tick infestation described herein. Twenty of each male and female ticks from the

infected cohort were placed upon the dog on day one. On day five, 200 uninfected recipient

nymphs from the Mediterranean colony were added into the feeding bag already containing

infected adults, and another 200 nymphs were placed into a separate feeding bag attached

at a distance of 10 cm from the first one.

All dogs were observed and monitored for signs of infection twice daily. Blood and

serum samples were collected twice weekly and tested for the presence of rickettsial DNA

and anti- R. conorii antibodies. Engorged ticks were collected and counted daily for the

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duration of infestation. Engorged donor and recipient ticks were tested individually by

PCR for the presence of Rickettsia conorii israelensis DNA. Donor males were collected

and tested immediately after engorgement of all female ticks, and females were tested after

oviposition. Subsamples of engorged nymphs from each feeding bag were tested imme-

diately after detachment from the host. The remaining engorged nymphs were allowed to

molt into the adult stage and the resulting ticks were also tested for presence of R. conoriiisraelensis.

DNA was extracted from ticks and blood samples using IsoQuick nucleic acid

extraction kit (ORCA Research Inc., Bothell, WA) to maximize sensitivity (Schwartz et al.

1997). The presence of rickettsial DNA was detected by PCR using primers RR190-547F

and RR190-701R to amplify a 154-bp fragment of the rompA gene of Rickettsia as

described by Eremeeva et al. (Eremeeva et al. 2003). Water was used as a no-template

negative control. IFA was performed on dog sera, as previously described (Lennette et al.

1995), using FITC labeled goat anti-dog IgG (heavy and light chains) conjugate diluted per

manufacturer’s recommendations (KPL, Inc. Gaithersburg, Maryland, USA). Serum

samples were initially screened at 1/16 and 1/256 dilutions, and positive samples were

titrated to the endpoint in a two-fold dilution series. Serologic data are reported as the

reciprocal of the last dilution showing positive fluorescence.

The statistical analysis was conducted with a chi-square test.

Results

Co-feeding upon naıve dogs

When cohorts of infected adult ticks were placed upon naıve dogs, the host animals did not

exhibit any symptoms of infection (fever, lethargy, anorexia) for the 9-day duration of tick

feeding. Blood PCR was also negative during this period indicating either absence or

undetectable levels of bacteremia. The dogs, however, seroconverted by day 10 post-

infestation demonstrating that they were exposed and susceptible to R. conorii israelensisinfection. Peak antibody titers in both dogs reached C4,096.

A total of 44 North American and 46 Mediterranean adult Rh. sanguineus ticks suc-

cessfully fed to repletion upon the naıve dogs. All these were tested individually by PCR

and 29 and 43 ticks from North American and Mediterranean colonies respectively were

found to contain R. conorii israelensis DNA. The resulting indices of prevalence of

infection 65.9% and 93.5% were significantly higher in engorged adult ticks from either

colony than in corresponding cohorts prior to feeding (P = 0.004 and P \ 0.001)

(Table 1). At the same time, prevalence of infection in engorged Mediterranean

Rh. sanguineus ticks was significantly higher than in ticks of North American origin

(P = 0.001).

Of recipient nymphs co-feeding with infected adults, 198 (66%) North American and

214 (71%) Mediterranean ticks successfully fed to repletion. Among these engorged

nymphs, 100 and 90% of the tested ticks from the North American and Mediterranean

colonies respectively contained R. conorii israelensis DNA (Table 1). Molting success for

ticks from the North American and Mediterranean colonies was 89.9 and 74.7%, respec-

tively. After nymphs molted into adult stage, the prevalence of infection in resulting ticks

remained high—80 and 88% among North American ticks and Mediterranean ticks,

respectively (Table 1). The detected prevalence of infection in resulting adults from the

Mediterranean colony was significantly lower than that in the corresponding cohort of

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engorged nymphs (P = 0.036). On the other hand, prevalence of infection in ticks from the

North American colony decreased only slightly during the molt (P = 0.736). Overall, the

majority of nymphs from either colony were able to transmit R. conorii israelensistransstadially following acquisition of the agent while co-feeding with infected ticks upon

a naıve host.

Co-feeding upon a seropositive dog

When a cohort of adult (Mediterranean) Rh. sanguineus ticks infected with R. conoriiisraelensis was placed upon a dog that had previously been exposed to the same rickettsial

isolate, the dog did not exhibit any symptoms of illness (fever, lethargy, anorexia) for the

9-day duration of tick feeding. Blood PCR also remained negative during this period.

A total of 22 adult Rh. sanguineus fed to repletion upon the seropositive dog. All these

were tested individually by PCR and only 2 (1 male and 1 female) were found to contain

R. conorii israelensis DNA. The resulting prevalence of infection 9.1 ± 12.3% was sig-

nificantly lower in engorged adult ticks than in the same cohort prior to feeding—

88.0 ± 13.0% (P \ 0.001) (Table 2).

Out of 200 nymphs placed in the same feeding bag with adult ticks, 57 successfully fed

to repletion. Of the same number of nymphs placed in a separate bag, 92 survived through

infestation. Only seven (28.0%) of the 25 tested engorged nymphs fed in the same bag with

Table 1 Acquisition of Rickettsia conorii by uninfected Rhipicephalus sanguineus nymphs feedingsimultaneously and in immediate proximity with infected adult ticks upon naıve dogs

Tick origin Prevalence of infection in donor—adult ticks (abs & % ±CI)a

Prevalence of infection in recipient—nymphalticks (abs & % ±CI)a

Prior to feeding After feeding Priortofeeding

After feeding Molted intoadults

North American—Rh. sanguineus

10/20(50.0 ± 22.48%)

29/44(65.9 ± 14.2%)

0/50(0%)

20/20 (100%) 20/25(80.0 ± 16.0%)

Mediterranean—Rh. sanguineus

6/20(30.0 ± 20.61%)

43/46(93.5 ± 7.2%)

0/50(0%)

18/20(90.0 ± 13.5%)

22/25(88.0 ± 13.0%)

a Indices of prevalence of infection are expressed as percentage ±0.95 confidence interval

Table 2 Acquisition of Rickettsia conorii by uninfected Rhipicephalus sanguineus nymphs feedingsimultaneously with infected adult ticks upon a seropositive dog

Prevalence of infection: positive/tested (% ±CI)a

Prior to feeding After feeding Molted into adults

Donor—adult ticks 22/25 (88.0 ± 13.0%) 2/22 (9.1 ± 12.3%) NA

Nymphs fedwith the donors

0/50 (0%) 7/25 (28.0 ± 18.0%) 0/23 (0%)

Nymphs fed 10 cmfrom the donors

0/50 (0%) 2/25 (8.0 ± 10.6%) 1/20 (5.0 ± 9.8%)

Nymphs—total 0/100 (0%) 9/50 (18.0 ± 10.8%) 1/43 (2.3 ± 4.6%)

a Indices of prevalence of infection are expressed as percentage ±0.95 confidence interval

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the infected adults contained rickettsial DNA (Table 2). Prevalence of infection in these

engorged ticks was significantly lower than among recipient nymphs of Mediterranean

origin fed in the previous experiment upon a Rickettsia-naıve dog (P \ 0.001) (Tables 1,

2). Acquisition of infection by recipient nymphs fed in a separate distant bag was even

lower, where only two (8.0%) of the 25 tested engorged ticks contained the rickettsial

DNA by the end of the feeding period. Although the detected difference in the prevalence

of infection between recipient nymphs feeding in the two bags indicates only borderline

statistical significance (P = 0.066), it suggests that the efficiency of R. conorii israelensistransmission among co-feeding ticks diminishes with distance between donors and

recipients. After the recipient nymphs that fed upon the seropositive dog molted into adult

stage, the prevalence of rickettsial infection decreased further, and only one (2.3%) of the

total 43 tested ticks remained positive.

Discussion

The occurrence of non-systemic transmission between infected and uninfected ticks

feeding simultaneously on a host has been described for a number of viral and bacterial

agents (Alekseev and Chunikhin 1992; Chunikhin 1990; Labuda et al. 1993a, b; Randolph

et al. 1996). This pathway is important for continuous circulation of tick-borne pathogens

as it can be realized in situations and environments where competent amplifying reservoir

hosts are scarce or absent. The efficiency of such transmission varies considerably

depending on the nature of the pathogen; species and life stages of the tick vectors

involved; the simultaneity of feeding of donor and recipient ticks; and the density of

infestation. For example, R. massiliae was transmitted from infected Rh. turanicus males to

69% of previously uninfected R. sanguineus females (Matsumoto et al. 2005). Approxi-

mately 33% of I. ricinus larvae acquired B. burgdorferi from infected nymphs if both

stages were placed on a host simultaneously, and up to 61% of recipient ticks became

infected if they were placed at the end of nymphal feeding (Gern and Rais 1996). Con-

versely, in experiments on transmission of B. burgdorferi between co-feeding nymphs and

larvae of I. scapularis, no larvae became infected when low densities of ticks were used,

while the high densities of ticks produced 5% of infected larvae (Piesman and Happ 2001).

Our results demonstrate highly efficient exchange of R. conorii israelensis between

infected and uninfected Rh. sanguineus ticks feeding in close proximity upon a naıve dog.

The agent was successfully transmitted from infected adult ticks to previously uninfected

nymphs while the hosts had neither detectable bacteremia nor clinical symptoms of

infection. The pathogen acquired by nymphs was further efficiently carried transstadially

by the absolute majority of ticks. Cohorts of engorged nymphs from both the North

American and Mediterranean colonies had 100 and 91.7% ticks, respectively positive for

rickettsial DNA. Prevalence of infection among ticks from these cohorts after they molted

into adults was 80 and 88%, respectively. The data impart that, in the absence of host

antibodies, 80 to 95% of individual ticks ingesting R. conorii israelensis during feeding

successfully carried the agent through the molting process. In addition, the prevalence of

infection among cohorts of adult (donor) ticks increased significantly following their

simultaneous feeding upon a Rickettsia-naıve host (Table 1). A significant increase in the

prevalence of R. conorii infection in the donor ticks while feeding upon naive dogs shows

that the agent is successfully transmitted not only from adult to nymphal ticks, but also

between infected and uninfected adults.

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Acquisition of R. conorii israelensis by co-feeding was observed among Rh. sanguineusof both Mediterranean and North American origins. Although the efficiency of co-feeding

transmission between adult ticks from the North American colony appeared significantly

lower than that of the Mediterranean colony, acquisition of R. conorii israelensis by

co-feeding nymphs was almost universal in both colonies.

When R. conorii israelensis -infected and uninfected ticks were co-fed upon a dog with

persisting antibodies against the same agent, the outcome was radically different. Less than

one-third of the recipient nymphs feeding in close proximity with donor ticks acquired the

agent. Efficiency of this transmission farther decreased for ticks feeding further away from

the donors. This indicates negative correlation of the co-feeding transmission efficiency

with the distance between donor and recipient ticks. Furthermore, only a small proportion

of nymphs that ingested the agent while feeding upon a seropositive host managed to

transmit it transstadially. A cohort of engorged nymphs with the overall prevalence of

infection of 18.0% produced a cohort of adult ticks with the prevalence of infection of only

2.3%—an eight-fold decrease (P = 0.015). In other words, merely one out of every eight

nymphs ingesting R. conorii israelensis together with antibody-laden blood carried this

agent through the molt, compared to 80-95% efficiency of transstadial transmission in the

absence of antibodies. This may be due either to ticks ingesting rickettsiae already killed

by the host antibodies, or to continued neutralization of the agent inside molting ticks, or

both.

In addition, the majority of previously infected donor ticks appeared to lose the agent

after feeding upon a seropositive dog, with a ten-fold decrease in the prevalence of

R. conorii israelensis infection from 88% prior to feeding to 9% in the engorged adult ticks

(Table 2). This suggests that host immunoglobulins present in the blood-meal may a play

role in the elimination of the agent from a large proportion of previously infected adult

ticks.

Similar effects had been demonstrated in case of the agent of Lyme disease (Fikrig et al.

1992). However, unlike B. burgdorferi, which resides primarily in the midgut of an unfed

tick and thus is easily accessible to host derived antibodies ingested by a tick during blood

feeding (de Silva et al. 1996; Fikrig et al. 1992), rickettsial agents are not restricted to the

midgut (Baldridge et al. 2007). Nonetheless, host immunoglobulins, including IgG anti-

bodies, ingested by ticks during blood feeding are known to cross the gut wall and retain

their immunological properties in tick hemolymph. This phenomenon has been demon-

strated for several species of ixodid and argasid ticks (Ackerman et al. 1981; Ben-Yakir

1989; Ben-Yakir et al. 1987; Brossard and Rais 1984; Chinzei and Minoura 1987; Fujisaki

et al. 1984; Mbogo et al. 1992). Furthermore, feeding upon immune animals appears to

result in decreased efficiency of nymph-to-adult transstadial transmission of another tick-

borne intracellular pathogen, A. phagocytophilum in ticks (Levin and Fish 2000). Our data

indicate that R. conorii israelensis may also be affected by host immune mechanisms and

that feeding on immune animals decreases the efficiency of transstadial transmission of

R. conorii israelensis as well (Table 2). The mechanisms and locations of the interaction

between host immunoglobulins and an intracellular parasite such as Rickettsia inside a tick

are the subject of an ongoing investigation.

It is noteworthy that after Rh. sanguineus of Mediterranean origin completed their

engorgement upon a seropositive host, rickettsial DNA was detected in a larger proportion

of previously uninfected nymphs than in previously infected adults. This was in contrast to

the high rate of acquisition of the agent by both adult and nymphal ticks from the same

colony while co-feeding upon a naıve seronegative host. This curious difference may be

due to a larger blood volume and thus a larger amount of antibodies ingested by adult ticks

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compared to nymphs, resulting in more thorough elimination of the agent. On the other

hand, adult Rh. sanguineus feed for 8–10 days while nymphs complete their engorgement

in only 4–5 days. It is possible that during short period of nymphal feeding host antibodies

did not have as much time to eliminate the agent as in longer feeding adults. This

hypothesis of time-dependant elimination of R. conorii israelensis in vector ticks by host

antibodies is indirectly supported by the fact that engorged nymphs continued to lose

infection through the molting process resulting in much lower efficiency of transstadial

transmission in ticks fed upon a seropositive host than in those fed upon a seronegative

one.

However, the presence of host antibodies did not completely prevent transmission of

R. conorii israelensis to the next generation of ticks. Co-feeding of uninfected nymphs

side-by-side with infected adults upon a seropositive host still resulted in production of a

new cohort of infected adult Rh. sanguineus albeit with a low prevalence of infection. This

route of transmission may ensure continued circulation of the pathogen among vector ticks

even in the absence of naıve reservoir hosts.

Thus, our study provides the first evidence for efficient natural transmission of

R. conorii israelensis between co-feeding ticks in the absence of detectable bacteremia in

the host. Rh. sanguineus of both Mediterranean and North American origins are capable of

maintaining natural circulation of the agent by this route. Success of R. conorii israelensistransmission via co-feeding inversely depends on the distance between attachment sites of

infected and uninfected ticks on a host. Transmission of R. conorii israelensis via co-

feeding was observed upon both naıve and seropositive dogs. Although the success of both

the co-feeding and transstadial transmission greatly diminished after feeding upon a

seropositive dog, presence of host antibodies did not completely prevent passage of the

agent into new adults.

Further investigation is needed to elucidate the nature of the specific rickettsial antigen

target for the antibody which affects co-feeding transmission of R. conorii israelensis on a

seropositive dog, as well as potential exploitation of this phenomenon to control circulation

of this pathogen and to prevent its transmission to humans.

Acknowledgments We thank Davidia N. Grant and Maria L. Zambrano for their invaluable help withhandling live ticks and robotic equipment. We gratefully acknowledge Dr. Sandor E. Karpathy, Dr. MarinaEremeeva, and Dr. Gregory Dasch for their contributions to this study.

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