Amanda D. Loftis

Rickettsial agents in Egyptian ticks collected from domestic animals.

To assess the presence of rickettsial pathogens in ticks from Egypt, we collected ticks from domestic and peridomestic animals between June 2002 and July 2003. DNA extracts from 1019 ticks were tested, using PCR and sequencing, for Anaplasma spp., Bartonella spp., Coxiella burnetii, Ehrlichia spp., and Rickettsia spp. Ticks included: 29 Argas persicus, 10 Hyalomma anatolicum anatolicum, 55 Hyalomma anatolicum excavatum, 174 Hyalomma dromedarii, 2 Hyalomma impeltatum, 3 Hyalomma marginatum rufipes, 55 unidentified nymphal Hyalomma, 625 Rhipicephalus (Boophilus) annulatus, 49 Rhipicephalus sanguineus, and 17 Rhipicephalus turanicus. Ticks were collected predominantly (>80%) from buffalo, cattle, and camels, with smaller numbers from chicken and rabbit sheds, sheep, foxes, a domestic dog, a hedgehog, and a black rat. We detected Anaplasma marginale, Coxiella burnetii, Rickettsia aeschlimannii, and four novel genotypes similar to: “Anaplasma platys,” Ehrlichia canis, Ehrlichia spp. reported from Asian ticks, and a Rickettsiales endosymbiont of Ixodes ricinus.

Borrelia, Coxiella, and Rickettsia in Carios capensis (Acari: Argasidae) from a brown pelican (Pelecanus occidentalis) rookery in South Carolina, USA

Argasid ticks are vectors of viral and bacterial agents of humans and animals. Carios capensis, a tick of seabirds, infests the nests of brown pelicans, Pelecanus occidentalis, and other ground nesting birds along the coast of South Carolina. This tick is associated with pelican nest abandonment and could pose a threat to humans visiting pelican rookeries if visitors are exposed to ticks harboring infectious agents. We collected ticks from a pelican rookery on Deveaux Bank, South Carolina and screened 64 individual ticks, six pools of larvae, and an egg mass for DNA from Bartonella, Borrelia, Coxiella, and Rickettsia by polymerase chain reaction amplification and sequencing. Ticks harbored DNA from “Borrelia lonestari”, a novel Coxiella sp., and three species of Rickettsia, including Rickettsia felis and two undescribed Rickettsia spp. DNA from the Coxiella and two undescribed Rickettsia were detected in unfed larvae that emerged in the laboratory, which implies these agents are transmitted vertically by female ticks. We partially characterize the novel Coxiella by molecular means.

The first report of human illness associated with the Panola Mountain Ehrlichia species: a case report

IntroductionTwo species of Ehrlichia are known to cause human illness. Several other species have been discovered in ticks and animals, and recent reports suggest that some of these Ehrlichia species might be human pathogens. We report here the first association of a recently discovered pathogen, the Panola Mountain Ehrlichia species, with a case of human illness.Case presentationA 31-year-old man from Atlanta, Georgia (GA) in the United States of America (USA) presented with a persistent sore neck of 3 weeks duration following a tick bite. DNA from the Panola Mountain Ehrlichia species, which was recently discovered in a goat in Georgia, was detected in an acute blood sample. Serologic testing was inconclusive. Polymerase chain reaction tests for other tick-borne diseases found in this region were negative. The patient rapidly improved in response to doxycycline therapy.ConclusionDetection of Ehrlichia DNA in an acute blood sample meets the Centers for Disease Control and Prevention laboratory confirmation criteria for ehrlichiosis, and response to doxycycline provides supporting clinical evidence. The Panola Mountain Ehrlichia species, an emerging pathogen transmitted by ticks in the eastern USA, should be considered as a possible cause of tick-borne illness in this region.

Rickettsial pathogens in the tropical rat mite Ornithonyssus bacoti (Acari: Macronyssidae) from Egyptian rats (Rattus spp.)

We collected and tested 616 tropical rat mites (Ornithonyssus bacoti (Hirst)) from rats (Rattus norvegicus (Berkenhout) and R. rattus (Linnaeus)) throughout 14 governorates in Egypt and tested DNA extracts from pools of these mites for Bartonella spp., Coxiella burnetii, and Rickettsia spp. by PCR amplification and sequencing. Three different mite-associated bacterial agents, including one Bartonella and two Rickettsia spp., were detected in eight pools of mites. Further research could demonstrate the vector potential of mites and pathogenicity of these agents to humans or animals.

Geographic distribution and genetic diversity of the Ehrlichia sp. from Panola Mountain in Amblyomma americanum

BackgroundA novel Ehrlichia, closely related to Ehrlichia ruminantium, was recently discovered from Panola Mountain State Park, GA, USA. We conducted a study to determine if this agent was recently introduced into the United States.MethodsWe developed a sensitive PCR assay based on the conserved gltA (citrate synthase) gene and tested DNA samples extracted from 1964 field-collected and 1835 human-biting Amblyomma americanum from 23 eastern states of the USA.ResultsThe novel agent was detected in 36 ticks collected from 10 states between 1998 and 2006. Infected ticks were collected both from vegetation (n = 14, 0.7%) and from humans (n = 22, 1.2%). Fragments of the conserved gltA gene and the variable map1 gene were sequenced from positive samples. Two distinct clades, with 10.5% nucleic acid divergence over the 730 bp map1 sequence, were identified.ConclusionThese data suggest that the Panola Mountain Ehrlichia was not recently introduced to the United States; this agent has an extensive distribution throughout the range of its tick vector, has been present in some locations for several years, and displays genetic variability. Furthermore, people in several states were exposed to this agent through the bite of infected ticks, underscoring the potential public health risk of this emerging ehrlichiosis.

Molecular and Biological Characterization of a Novel Coxiella-like Agent from Carios capensis

Abstract: The genus Coxiella is currently defined by a single monotypic species, Coxiella burnetii. Novel Coxiella spp. have been detected in ticks throughout the world. These bacteria have not been cultured or named, and their evolutionary relationships to C. burnetii are poorly known. A novel Coxiella‐like agent was detected by PCR amplification and sequencing of DNA extracted from 64 pelican ticks, Carios capensis, from Devoux Bank, South Carolina, USA. PCR was used to amplify and characterize genes from the new bacterium. Sequences from some metabolic and housekeeping genes shared a 92–98% similarity to C. burnetii, but other genes such as the IS1111 transposon, com1, and 5S and 16S rRNA genes were not amplified by conventional PCR. Transovarial and transtadial transmission and environmental shedding of the agent were detected by PCR.

Detection of Bacterial Agents in Amblyomma americanum (Acari: Ixodidae) from Georgia, USA, and the use of a Multiplex Assay to Differentiate Ehrlichia chaffeensis and Ehrlichia ewingii

ABSTRACT Amblyomma americanum, the lone star tick, is the most common and most aggressive human biting tick in the Southeastern United States. It is known to transmit the agents of human ehrlichioses, Ehrlichia chaffeensis and Ehrlichia ewingii. In addition, it carries agents of unspecified pathogenicity to humans, including Rickettsia amblyommii, Borrelia lonestari, and the newly emerging Panola Mountain Ehrlichia (PME). Surveillance of these ticks for recognized or emerging pathogens is necessary for assessing the risk of human infection. From 2005 to 2009, we surveyed A. americanum ticks from four locations in the state of Georgia. Ticks (1,183 adults, 2,954 nymphs, and 99 larval batches) were tested using a multiplex real-time polymerase chain reaction (PCR) assay designed to detect and discriminate DNA from Rickettsia spp., E. chaffeensis, and E. ewingii, This assay was capable of detecting as few as 10 gene copies of the aforementioned agents. Ticks were also tested for PME and B. lonestari by nested PCR. The prevalence of infection ranged from 0 to 2.5% for E. chaffeensis, 0 to 3.9% for E. ewingii, 0 to 2.2% for PME, 17 to 83.1% for R. amblyommii, and 0 to 3.1% for B. lonestari. There were 46 (4.1%) individual adults positive for two agents, and two females that were each positive for three agents. Two larval batches were positive for both B. lonestari and R. amblyommii, indicating the potential for transovarial transmission of both agents from a single female. Although infrequent in occurrence, the dynamics of coinfections in individual ticks should be explored further, given the potential implications for differential diagnosis and severity of human illness.

High-throughput molecular testing of ticks using a liquid-handling robot.

Abstract To meet the need for high-throughput sample testing, DNA extraction kits based on the 96-well plate format have been developed for use with blood and tissue samples. These methods have not been applied to DNA extractions from ticks. To meet this need, we developed a high-throughput method for DNA extraction and polymerase chain reaction (PCR) testing of tick samples. A liquid-handling robot was used to extract DNA in a 96-well binding column plate with vacuum manifold. The quantity, purity, and quality of DNA recovered from Ixodes scapularis Say, 1821 nymphs with this method were reproducible and comparable with existing manual DNA extraction techniques. The DNA yield from pools of five nymphal ticks averaged 0.432 ± 0.04 μg (95% CI). The robot also prepared real-time PCR reactions in 96-well plates, directly from the extracted DNA. A modification of the existing P20 tool resulted in accurate pipetting of 1- to 2-μl volumes with a reproducibility of ±0.038 μl when dispensing 1.0 μl. By using this process, 96 samples can be extracted and tested while reducing human labor to ≈30 min.

Population Survey of Egyptian Arthropods for Rickettsial Agents

Abstract:u2002 Between June 2002 and July 2003, 987 fleas, representing four species, and 1019 ticks, representing one argasid and eight ixodid species, were collected from Egyptian animals. These arthropods were tested for rickettsial agents using polymerase chain reaction. DNAs from Anaplasma and Ehrlichia spp. were detected in 13 ticks. Previously undescribed Bartonella spp. were detected in 21 fleas. Coxiella burnetii was detected in two fleas and 20 ticks. Rickettsia typhi was detected in 27 fleas from 10 cities. Spotted fever group rickettsiae were detected in both fleas and ticks and included Rickettsia aeschlimanii and an unnamed Rickettsia sp.

Susceptibility of Mice (Mus musculus) to Repeated Infestation with Amblyomma americanum (Acari: Ixodidae) Ticks

Abstract Laboratory mice, Mus musculus (L.), BALB/c strain, were assessed for their ability to develop resistance to repeated infestation by Amblyomma americanum (L.) ticks. Mice were infested five consecutive times with A. americanum nymphs. No decrease in tick viability was seen after five infestations, suggesting that BALB/c mice do not develop immune-mediated resistance to A. americanum. In contrast, tick viability was significantly reduced in the second infestation of a New Zealand White rabbit, a laboratory animal known to develop resistance to A. americanum.