LABORATORY COLONIES OF TICKS Michael L. Levin Lauren Schumacher Medical Entomology Laboratory, Rickettsial Zoonoses Branch Centers for Disease Control and Prevention (CDC) Saravanan Thangamani Insectary Services Division, Galveston National Laboratory University of Texas Medical Branch (UTMB) Table of Contents Foreword Introduction Chapter 1: Maintenance of Multi-Host Ixodid Ticks in the Laboratory 1.1 Equipping and Operating an Insectary 1.2 General Methods for Rearing Ticks 1.3 Tick Culture Chapter 2: Maintenance of Argasid Ticks in the Laboratory Appendix I – Recommended reading on Tick life cycles under laboratory conditions. Appendix II - Vacuum Aspirator Assembly Foreword Methods in Tick Research is part of a comprehensive collection of new rearing and handling protocols for vector species of importance to human health was borne out of the Vector Biology Research Resources Workshop held in June 2015 at the National Institutes of Health with the generous support by BEI Resources. This effort was inspired by the BEI manual, Methods in Anopheles Research, started by Mark Benedict and widely expanded by Paul Howell, which has become the gold standard of mosquito rearing and manipulation protocols. It continues to be the go‐to resource for laboratory‐ based scientists conducting basic research and public health entomologists from malaria endemic countries alike. We would like to thank David Bland, Paul Howell, Michael Levin, Kevin Macaluso, Claudio Meneses, Tobin Rowland, Saravanan Thangamani, and Margaret (Peggy) Wirth, for sharing their techniques and expertise, and for putting together these protocols. These protocols are intended as living, breathing documents with ample room for improvement based on a specific lab's capacity and infrastructure. They are intended as guidelines only, especially with regards to research involving vertebrate animals or biohazards, and arthropod containment, which require institutional approval tailored to individual laboratories. We hope that the community can benefit significantly from the generation of this comprehensive set of new protocols and stimulate new work in vector biology and vector‐borne diseases. Kristin Michel ([email protected], Kansas State University) and Lyric Bartholomay ([email protected], University of Wisconsin‐Madison) To provide feedback on this or any of the vector resources protocols, please send an email to [email protected]. Disclaimer. BEI Resources is funded under contract HHSN272201000027C by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services. The views expressed in this publication neither imply review nor endorsement by HHS, nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government. Introduction Worldwide, ticks are second only to mosquitoes as vectors of human disease. Many modern studies of the virulence of tick-borne pathogens in animals have been performed using pathogens grown and maintained in artificial culture, cell lines, or serial passages of animal blood or tissue homogenates with needle inoculation as the primary mode of infection. However, the dynamics, pathogenesis, and symptoms of infection as well as the subsequent immune response to infection and recovery strongly depend on the route of pathogen introduction into a susceptible host. In natural transmission, tickborne pathogens enter the vertebrate host with tick saliva, which assists establishment of infection by modifying the host immunological and cellular responses at the site of tick attachment. Ticks thus provide a modified environment in which bacteria and parasites can differentiate and proliferate and then migrate to other tissues to ensure successful biological transmission to the next invertebrate host. These tick factors can directly influence the infectivity and virulence of tick-borne agents as well as alter host responses. Consequently, conditions of natural transmission are poorly replicated when artificial methods of proliferation, maintenance, and inoculation into animals are used. Results of such laboratory studies may be difficult to extrapolate and apply to natural infections. The transmission, maintenance, infectivity, virulence, and pathogenicity of tick-borne agents can be studied in the laboratory by using tick bite as the natural mode of infection. Accomplishment of these studies requires maintenance of laboratory colonies of many epidemiologically important hard tick vectors. General setup of a tick laboratory, tick feeding protocols, and environmental requirements necessary for maintenance of ixodid tick colonies had been previously described by Patrick & Hair (1975)1 and Sonenshine (1999)2. Here we provide step-by- step recommendations for various procedures used in maintenance of ixodid tick colonies based on our 20+ years of experience. A list of publications describing laboratory life cycles of various ticks species and specific environmental conditions required for successful establishment and maintenance of laboratory colonies is included below. 1 Patrick, C. D., and J. A. Hair. 1975. Laboratory rearing procedures and equipment for multi-host ticks (Acarina: Ixodidae). J. Med. Entomol. 12: 389-390. 2 Sonenshine, D. E. 1999. Maintenance of ticks in the laboratory, pp. 57-82. In K. Maramorosch and F. Mahmood (eds.), Maintenance of Human, Animal, and Plant Pathogen Vectors. Science Publishers, INC, Enfield, NH, USA. Chapter 1: Maintenance of Multi-Host Ixodid Ticks in the Laboratory 1.1 Equipping and Operating an Insectary Page 1 of 3 Chapter 1: Maintenance of Multi-Host Ixodid Ticks in the Laboratory 1.1 Equipping and Operating an Insectary Michael L. Levin and Lauren Schumacher Medical Entomology Laboratory, Rickettsial Zoonoses Branch Centers for Disease Control and Prevention 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 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 of maintaining colonies of multi-host ixodid ticks by feeding all developmental stages (larvae, nymphs, and adults) upon NZW rabbits. When needed, the same procedures here 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 I. scapularis, I. pacificus, A. americanum, D. variabilis, D. occidentalis, H. leporispalustris, and R. sanguineus with descriptions of the complete laboratory life cycles and reliable production of uninfected ticks under standardized conditions had been published by Troughton and Levin (2007). Biosafety Establishment and maintenance of live colonies of ticks carry substantial biological risk for the personnel both inside and outside the tick-handling facility. This risk is associated with ticks’ obligatory hematophagy, ability to crawl under the protective equipment (PPE) and personal clothing, remain hidden and/or attached to the host as well as survive on or under furniture (e.g. on a counter, in an elevator, on a door handle, on a telephone receiver) for long periods of time. Unlike flying insects, whose escape and dispersal may be impeded by airlocks, ticks can and will crawl through doorways, or hitch a ride on clothing and packaging. In addition, ticks are competent and efficient vectors of multiple pathogenic and obligate or facultative symbiotic microorganisms. While it is possible to test representative samples of ticks in the colony for the presence or absence of known pathogens, there are no guarantees that colonized ticks do not carry some yet unknown pathogenic organisms (e.g. only recently discovered Panola Mountain Ehrlichia, Heartland virus), or that bacteria previously known as Chapter 1: Maintenance of Multi-Host Ixodid Ticks in the Laboratory 1.1 Equipping and Operating an Insectary Page 2 of 3 endosymbionts of ticks are not indeed horizontally transmissible pathogens (e.g. Rickettsia slovaca, R. amblyommii). Therefore, it is imperative that live ticks are always handled in a specially designed and designated laboratory with implemented, strict biosafety procedures – at least ACL level 2 precautions – preventing an escape of hematophagous arthropods. All live ticks, regardless of their origin and/or the goal of a particular study, should be treated and handled as potentially infected. Appropriate PPE must be worn at all times by all personnel entering the designated facility regardless of task(s) performed or its duration. PPE must be inspected for ticks and appropriately removed when exiting the facility – Tick Laboratory PPE must not be allowed outside of the tick-designated area. Personal Protective Equipment Materials Needed: Disposable White Gown or Coveralls Hair cover Disposable Gloves Shoe covers (as needed for animal work) Respirator (as needed for animal work) Face shield or safety glasses (as needed for animal work) Proper personal protective equipment (PPE) should be worn at all times when working in the laboratory. Personal protective equipment required for the lab includes a white gown/ coveralls, a hairnet or a cap (hair must not be touching the gown), and properly