Michael L. Levin

Comparison of the reservoir competence of medium-sized mammals and Peromyscus leucopus for Anaplasma phagocytophilum in Connecticut.

In the northeastern United States, Anaplasma phagocytophilum, the agent of human granulocytic ehrlichiosis (HGE), is transmitted by the tick vector Ixodes scapularis. The white-footed mouse Peromyscus leucopus is a competent reservoir for this agent, but the reservoir competence of non-Peromyscus hosts of I. scapularis has not been studied. Here, we report data confirming reservoir competence of medium-sized mammals for A. phagocytophilum. Raccoons, Virginia opossums, gray squirrels, and striped skunks were live-trapped in June-August of 1998-1999 at two locations in Connecticut. Captured animals were kept for several days at the laboratory in wire-mesh cages over water to allow naturally attached ticks to drop off. Samples of blood and serum were taken from each animal prior to its release at the site of capture. Engorged ticks collected from each animal were allowed to molt. Resulting I. scapularis nymphs and adults were tested for the presence of A. phagocytophilum DNA by polymerase chain reaction, as were the blood samples from the animals. A. phagocytophilum DNA was detected in the blood of >10% of the raccoons tested. Raccoons, opossums, squirrels, and skunks produced adult I. scapularis infected with the agent of HGE. Prevalence of infection was the highest in adult ticks fed as nymphs upon raccoons (23%) and the lowest in those fed upon skunks and opossums (5-7%). The agent was present in nymphal I. scapularis fed as larvae upon raccoons and squirrels, but not in ticks fed upon skunks or opossums. We also tested the ability of I. scapularis to transmit A. phagocytophilum to laboratory-reared white-footed mice after acquiring it from medium-sized mammals. Ticks that acquired the agent from raccoons and squirrels successfully transmitted it to mice. Thus, raccoons and gray squirrels are reservoir-competent for the agent of HGE-they become naturally infected, and are capable of transmitting the infection to feeding ticks.

Inability of a Variant Strain of Anaplasma phagocytophilum to Infect Mice

Nymphal Ixodes scapularis ticks were collected from several sites in Rhode Island. Polymerase chain reaction and DNA sequencing were used to determine the presence and prevalence of Anaplasma phagocytophilum human agent (AP-ha) and a genetic variant not associated with human disease (AP-variant 1). The remaining ticks from each cohort were allowed to feed to repletion on either white-footed (Peromyscus leucopus) or DBA/2 (Mus musculus) mice. The engorged ticks and murine blood samples were evaluated for the presence of AP-ha and AP-variant 1. Although a high percentage of the infecting ticks harbored AP-variant 1, only AP-ha was amplified from the murine blood samples. Additional ticks were fed on immunocompromised SCID mice, and, again, only AP-ha was capable of establishing an infection, and only AP-ha could be detected by xenodiagnosis. These data suggest that AP-variant 1 cannot establish an infection in mice, and we propose that AP-variant 1 has an alternative natural reservoir, possibly white-tailed deer.

Vector Competence of the Tick Ixodes ricinus for Transmission of Bartonella birtlesii

Bartonella spp. are facultative intracellular vector-borne bacteria associated with several emerging diseases in humans and animals all over the world. The potential for involvement of ticks in transmission of Bartonella spp. has been heartily debated for many years. However, most of the data supporting bartonellae transmission by ticks come from molecular and serological epidemiological surveys in humans and animals providing only indirect evidences without a direct proof of tick vector competence for transmission of bartonellae. We used a murine model to assess the vector competence of Ixodes ricinus for Bartonella birtlesii. Larval and nymphal I. ricinus were fed on a B. birtlesii-infected mouse. The nymphs successfully transmitted B. birtlesii to naïve mice as bacteria were recovered from both the mouse blood and liver at seven and 16 days after tick bites. The female adults successfully emitted the bacteria into uninfected blood after three or more days of tick attachment, when fed via membrane feeding system. Histochemical staining showed the presence of bacteria in salivary glands and muscle tissues of partially engorged adult ticks, which had molted from the infected nymphs. These results confirm the vector competence of I. ricinus for B. birtlesii and represent the first in vivo demonstration of a Bartonella sp. transmission by ticks. Consequently, bartonelloses should be now included in the differential diagnosis for patients exposed to tick bites.

Life Cycles of Seven Ixodid Tick Species (Acari: Ixodidae) Under Standardized Laboratory Conditions

Abstract Studies of transmission, maintenance, infectivity, virulence, and pathogenicity of tick-borne agents require the use of large numbers of live laboratory-raised ticks. Colonies of Ixodes scapularis Say, Ixodes pacificus Cooley & Kohls, Amblyomma americanum (L.), Dermacentor occidentalis Marx, Dermacentor variabilis (Say), Hemaphysalis leporispalustris (Packard), and Rhipicephalus sanguineus (Latrielle) have been maintained in our laboratory at the Centers for Disease Control and Prevention for five to 18 continuous generations. New Zealand White rabbits (Oryctolagus cuniculus) are used as hosts for all tick species and developmental stages. Between feedings, ticks are stored in environmental incubators at 22–24°C and 90% RH with a day/night photoperiod of 16:8 (L:D) h. The duration of feeding, molting, preoviposition, and periods of postmolting development were recorded. Here, we describe the life cycles of these common North American tick species under standardized laboratory conditions. At 22–24°C, the minimal time needed for each species to complete one life cycle was as follows: I. scapularis, 204–219 d; I. pacificus, 214–229 d; R. sanguineus, 162–177 d; H. leporispalustris, 209–224 d; D. variabilis, 176–191 d; D. occidentalis, 180–195 d; and A. americanum, 192–211 d.

Detection of Rickettsia, Borrelia, and Bartonella in Carios kelleyi (Acari: Argasidae)

Abstract Carios kelleyi (Colley & Kohls 1941), a tick associated with bats and bat habitats, has been reported to feed on humans, but there is little published data regarding the presence of vector-borne pathogens in these ticks. C. kelleyi nymphs and adults were collected from residential and community buildings in Jackson County, Iowa, and tested by polymerase chain reaction for Rickettsia, Borrelia, Bartonella, Coxiella, and Anaplasma. Rickettsia DNA was detected in 28 of 31 live ticks. Sequences of the 17-kDa and rOmpA genes suggest that this agent is a novel spotted fever group Rickettsia. Transstadial and transovarial transmission of this Rickettsia were demonstrated. The flagellin gene of a Borrelia, closely related to B. turicatae, was detected in one of 31 live ticks. The 16S-23S intergenic spacer region of Bartonella henselae also was detected in one of 31 live ticks. Coxiella or A. phagocytophilum DNA were not detected in these ticks.

Systematics and ecology of the brown dog tick, Rhipicephalus sanguineus

Rhipicephalus sanguineus, the brown dog tick, kennel tick, or pan-tropical dog tick, is probably the most widespread ixodid tick, colonising both human and canine dwellings. It is of great significance in both human and veterinary medicine as a vector of several important pathogens and can also cause clinical illness in heavy infestations of dogs. However, there are still significant taxonomic uncertainties, and it is probable that some records of this tick refer to other species. Most past reviews have been broad-ranging or have focused on the pathogens that R. sanguineus transmits, and relatively few have considered its biology in detail or the issues around its true identity. The present review has paid most attention to the taxonomy of R. sanguineus and to the ecological factors that influence tick survival and distribution, particularly in a climate change context. It is evident that more research is required on the systematics of the R. sanguineus species complex, in order to elucidate the epidemiology and prevention of the diseases that it transmits, and to provide the relevant biological data required for development of predictive models of future tick distribution in a climate change scenario.

Isolation and propagation of the Ap-Variant 1 strain of Anaplasma phagocytophilum in a tick cell line.

ABSTRACT The first tissue culture isolates of the unique Anaplasma phagocytophilum strain, Ap-Variant 1, were obtained in the Ixodes scapularis tick-derived cell line ISE6. Two isolates were from goat blood samples: one from a goat infected with I. scapularis ticks from Rhode Island and a second from a goat infected by serial passage of blood from the first infected goat. Eight isolates were made directly from I. scapularis ticks collected from white-tailed deer in Minnesota and represent the first isolations of an Anaplasma species directly from ticks. Each of the 10 isolates had a 16S rRNA gene sequence identical to that previously described for Ap-Variant 1, but differences within the ank gene were found that suggest natural variation. Prevalence of Anaplasma in the Minnesota ticks was 63.9%; 23 of 36 ticks tested by PCR were positive. Six of the tick-derived isolates were obtained from a set of 18 PCR-positive ticks, for a 33.3% isolation success rate. The conservation of host tropism among the Rhode Island and Minnesota isolates of Ap-Variant 1 was examined by use of experimental infections of mice and a goat. A Minnesota tick-derived isolate (MN-61-2) was used to inoculate naïve animals, and this isolate was able to infect a goat but unable to infect each of five mice, confirming that the Minnesota isolates have the same host tropism as Ap-Variant 1 from the northeastern United States. Light and electron microscopy of the Ap-Variant 1 isolate MN-61-2 in ISE6 cells showed cytoplasmic inclusions characteristic of A. phagocytophilum with pleomorphic bacteria in membrane-bound vacuoles and both electron-dense and electron-lucent forms.

Coxiella Symbionts in the Cayenne Tick Amblyomma cajennense

Members of the Coxiella genus are intracellular bacteria that can infect a variety of animals including humans. A symbiotic Coxiella was recently described in Amblyomma americanum ticks in the Northern Hemisphere with no further investigations of other Amblyomma species in other geographic regions. These ixodid ticks represent a group of important vectors for human infectious agents. In the present work, we have demonstrated that symbiotic Coxiella (SCox) are widespread, occurring in South America and infecting 100% of all life stages and eggs of the Cayenne ticks Amblyomma cajennense from Brazil and the USA. Using light microscopy, in situ hybridization, and PCR, we demonstrated SCox in salivary glands, ovaries, and the intestines of A. cajennense. These symbionts are vertically and transtadially transmitted in laboratory reared A. cajennense, and quantitative PCR analyses indicate that SCox are more abundant in adult female ticks, reaching values corresponding to an 11×, 38×, and 200× increase in SCox 16S rRNA gene copy number in unfed females, compared to unfed nymphs, larvae, and eggs, respectively. Phylogenetic analyses showed distinct SCox subpopulations in the USA and Brazil and demonstrated that SCox bacteria do not group with pathogenic Coxiella burnetii.

Co-feeding as a route for transmission of Rickettsia conorii israelensis between Rhipicephalus sanguineus ticks.

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 naïve dogs or a dog previously exposed to this agent. When ticks were placed upon naïve dogs, 92–100% of nymphs acquired the infection and 80–88% of infected engorged nymphs transmitted 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 naïve and seropositive dogs. This route of transmission can ensure continuous circulation of R. conorii israelensis in tick vectors even in the absence of naïve reservoir hosts.

Interference Between the Agents of Lyme Disease and Human Granulocytic Ehrlichiosis in a Natural Reservoir Host

Agents of Lyme disease (Borrelia burgdorferi) and human granulocytic ehrlichiosis (Ehrlichia phagocytophila) are perpetuated in a natural cycle involving the black-legged tick (Ixodes scapularis) and its vertebrate hosts. Using I. scapularis nymphs as the mode of infectious challenge, we studied how infection with one pathogen in white-footed mice (Peromyscus leucopus) affects their ability to acquire the other agent and subsequently to infect larvae, which these agents would do in nature. Two groups of mice were infected with either B. burgdorferi or E. phagocytophila. One week later, B. burgdorferi-infected mice were challenged with E. phagocytophila, and E. phagocytophila-infected mice were challenged with B. burgdorferi. Simultaneously, two control groups of uninfected mice were infected with each agent from the same tick cohorts used on the first groups of mice. Uninfected I. scapularis larvae were fed on all mice for xenodiagnosis at weekly intervals lasting 2 months. For the B. burgdorferi challenge, all control and E. phagocytophila-infected mice acquired B. burgdorferi. However, fewer xenodiagnostic larvae acquired B. burgdorferi from mice with mixed infections compared with mice infected with B. burgdorferi only. For the E. phagocytophila challenge, all five control mice acquired E. phagocytophila, but only two of five mice infected with B. burgdorferi subsequently acquired E. phagocytophila. Consequently, mice with both infections produced fewer xenodiagnostic ticks infected with E. phagocytophila than mice infected with E. phagocytophila only. Thus, a primary infection with either B. burgdorferi or E. phagocytophila in mice inhibited transmission of a second agent, suggesting interference between these two agents.