Manual for maintenance of multi-host ixodid ticks in the laboratory.

Use of laboratory animals as hosts for blood-sucking arthropods remains a time-proven and the most efficient method for establishment and propagation of slowly feeding ixodid ticks, despite introduction of techniques involving artificial feeding on either animal skins or synthetic membranes. New Zealand White rabbits are usually the most accessible and most suitable hosts routinely used for establishment and maintenance of a large variety of multi-host tick species. Here we describe standard procedures for maintaining colonies of multi-host ixodid ticks by feeding all developmental stages (larvae, nymphs, and adults) upon New Zealand White rabbits. When needed, the same procedures can be easily adapted to other species of laboratory or domestic animals from mice to dogs and goats. A summary of our experience in maintaining laboratory colonies of Ixodes scapularis, Ixodes pacificus, Amblyomma americanum, Dermacentor variabilis, Dermacentor occidentalis, Haemaphysalis leporispalustris, and Rhipicephalus sanguineus with descriptions of the complete laboratory life cycles and reliable production of uninfected ticks under standardized conditions has been published by Troughton and Levin (J Med Entomol 44:732-740, 2007). Here we provide step-by-step recommendations for various procedures used in the maintenance of ixodid tick colonies based on over 20 years of experience.

Effects of Rickettsia amblyommatis Infection on the Vector Competence of Amblyomma americanum Ticks for Rickettsia rickettsii.

Although Dermacentor spp. ticks are considered the primary vectors of Rickettsia rickettsii in the United States, other North American tick species are also capable of transmitting the agent, including the lone star tick-Amblyomma americanum. The lone star tick is an aggressive human-biting tick abundant in the South, Central, and Mid-Atlantic United States, which has been shown to be a competent vector of R. rickettsii in laboratory studies. However in nature, A. americanum frequently carry Rickettsia amblyommatis-another member of the spotted fever group-with the prevalence of infection reaching 84% in some populations. It has been postulated that the presence of an endosymbiotic Rickettsia in a significant proportion of a vector population would diminish or even block transmission of pathogenic Rickettsia in ticks from generation to generation due to transovarial interference. We measured the ability of R. amblyommatis-infected A. americanum to acquire R. rickettsii from an infected host with a bloodmeal, and transmit it transstadially, horizontally (to a susceptible host), and vertically to the next generation. Larvae from both the R. amblyommatis-infected and R. amblyommatis-free cohorts acquired R. rickettsii from infected guinea pigs, but the presence of the symbiont diminished the ability of coinfected engorged larvae to transmit R. rickettsii transstadially. Conversely, acquisition of R. rickettsii by cofeeding was unaffected in R. amblyommatis-infected nymphs and adults; prevalence of R. rickettsii in engorged adults reached 97% in both R. amblyommatis-infected and R. amblyommatis-free cohorts. In guinea pigs exposed to dually infected nymphs, R. rickettsii infection was milder than in those fed upon nymphs infected with R. rickettsii only. The frequency of transovarial transmission of R. rickettsii in the R. amblyommatis-infected cohort (31%) appeared lower than that in the R. amblyommatis-free cohort (48%), but the difference was not statistically significant. Larval progenies of dually infected A. americanum females transmitted R. rickettsii to naïve guinea pigs confirming viability of the pathogen. Thus, the vector competence of A. americanum for R. rickettsii was not significantly affected by R. amblyommatis.

Vector competence of Amblyomma americanum (Acari: Ixodidae) for Rickettsia rickettsii.

Rickettsia rickettsii - the etiologic agent of Rocky Mountain spotted fever (RMSF) - is widely spread across the Americas. In the US, Dermacentor spp. ticks are identified as primary vectors of R. rickettsii and Rhipicephalus sanguineus s.l. has been implicated in transmission of this pathogen in several locations in the Southwest. Conversely, ticks of the genus Amblyomma are recognized vectors of RMSF in Central and South America, but not in the US. A. americanum is one of the most aggressive human-biting ticks in the US, whose geographical range overlaps with that of reported RMSF cases. Despite sporadic findings of R. rickettsii DNA in field-collected A. americanum and circumstantial association of this species with human RMSF cases, its vector competence for R. rickettsii has not been appropriately studied. Therefore, we assessed the ability of A. americanum to acquire and transmit two geographically distant isolates of R. rickettsii. The Di-6 isolate of R. rickettsii used in this study originated in Virginia and the AZ-3 isolate originated in Arizona. Under laboratory conditions, A. americanum demonstrated vector competence for both isolates, although the efficiency of acquisition and transovarial transmission was higher for Di-6 than for AZ-3 isolate. Uninfected larvae acquired the pathogen from systemically infected guinea pigs, as well as while feeding side by side with Rickettsia-infected ticks on non-rickettsiemic hosts. Once acquired, R. rickettsii was successfully maintained through the tick molting process and transmitted to susceptible animals during subsequent feedings. Guinea pigs and dogs infested with infected A. americanum developed fever, scrotal edema and dermatitis or macular rash. R. rickettsii DNA was identified in animal blood, skin, and internal organs. The prevalence of infection within tick cohorts gradually increased due to side-by-side feeding of infected and uninfected individuals from 33 to 49% in freshly molted nymphs to 71-98% in engorged females. Moreover, R. rickettsii was transmitted transovarially by approximately 28% and 14% of females infected with Di-6 and AZ-3 isolates, respectively. Hence, A. americanum is capable of acquiring, maintaining and transmitting R. rickettsii isolates originating from two different geographical regions of the US, at least under laboratory conditions. Its role in ecology and epidemiology of RMSF in the US deserves further investigation.