Update on BEI Resources for Parasitology and Arthropod Vector Research

Abstract

BEI Resources has contributed to the advancement of parasitic diseases research for over 16 years. The accessibility of our reference strains and reagents is relevant to the development of new therapeutics and vaccines. Here we provide a resource update with emphasis on the new assets for toxoplasmosis and vector research.

Mission of BEI Resources

BEI Resources was established by the National Institute of Allergy and Infectious Diseases (NIAID) as a comprehensive resource of reagents for the scientific communityⁱ. The primary mission of the program is the acquisition, authentication, preservation, and distribution of a range of unique and quality-assured research materials to aid in the development and evaluation of vaccines, therapeutics, and diagnostics, with a focus on emerging and re-emerging infectionsⁱⁱ.

Resources for Parasitic and Vector-Borne Diseases

Access to reference strains and reagents is critical to the generation of basic research for elucidating aspects of the host-parasite relationship. BEI Resources enables this research by providing community-contributed reference strains of Toxoplasma gondii, kinetoplastids, malaria parasites, and other protozoan pathogens at no cost. Since our previous review, published in 2010 [1], BEI Resources integrated critical NIAID programs, including the Malaria Research and Reference Reagent Resource Center (MR4)ⁱⁱⁱ, the Schistosome Resource Center (SR3)^iv, the Filariasis Research Reagent Resource Center (FR3)^v, and arthropod vectors of disease such as mosquitoes, ticks, triatomines, and sand flies. Over the last decade, materials available for protozoan parasite research have grown to greater than 500 strains representing ten different genera, in addition to antibodies, recombinant proteins, nucleic acids, and expression vectors (Table 1)^vi.

Table 1.

BEI Resources for Protozoan Parasite Research

Group	Species represented	Highlightsa
Apicomplexans	Babesia microti Babesia duncani Cryptosporidium parvum Plasmodium falciparum Plasmodium vivax Plasmodium berghei Plasmodium yoelii Plasmodium chabaudi Toxoplasma gondii	• Reference genome strains • GFP and luciferase expressing strains • Knockout strains • Toxoplasma genetic crosses • In vivo and in vitro strains of Babesia spp. • Antimalarial resistant Plasmodium sp. • DNAs, plasmids, and primers • Monoclonal antibodies
Kinetoplastids	Leishmania spp. Trypanosoma cruzi Trypanosoma brucei brucei Trypanosoma brucei gambiense Trypanosoma brucei rhodesiense	• Reference strains of Leishmania • Reference genome strains of T. cruzi • Reference strains of T. brucei (Lister, TREU927, STIB 247, EATRO, TREU667) • T. brucei rhodesiense strains used in drug-susceptibility studies • β-gal-, GFP-, and luciferase- expressing strains • DNAs • Monoclonal antibodies
Intestinal parasites	Entamoeba histolytica Giardia lamblia	• Reference genome strains • Luciferase–expressing Entamoeba invadens • DNAs
Free-living pathogenic amoebae	Acanthamoeba spp. Balamuthia mandrillaris Naegleria fowleri	• CDC clinical isolates from keratitis and primary amoebic meningoencephalitis cases • DNAs

^aAbbreviations: β-gal, β-galactosidase; CDC, Centers for Disease Control; GFP, green fluorescent protein.

Expansion of Resources for Toxoplasmosis Research

BEI Resources has experienced a substantial increase in toxoplasmosis-related biomaterials during the last decade concomitant with significant advances in Toxoplasma gondii research. The repository is the sole public source of clonal lines derived from genetic crosses between types I and II, types I and III, and types II and III of T. gondii. Studies mapping virulence determinants in these recombinant progeny have shown that polymorphic kinases and pseudokinases secreted from parasite rhoptry organelles have important roles in the phenotypic differences observed between representative strains of the three Toxoplasma types [2].

T. gondii is amenable to genetic manipulation and is frequently used as a model system to study the basic biology of apicomplexan parasites. BEI Resources houses knockout strains deficient in mouse virulence due to the absence of various rhoptry kinases (ROPs) (Table 2). Functions of ROPs include modulation of host immune response genes, STAT proteins, and interferon-γ-inducible p47 GTPases [3-6]. BEI has also acquired transgenic strains that allow the dissection of signaling pathways and conditional silencing of essential genes in Toxoplasma [7-9] (Table 2). Additional resources include reference strains used in whole-genome sequencing (WGS) projects and monoclonal antibodies to parasite surface antigens and proteins of secretory organelles^vi.

Table 2.

Knockout and Transgenic Toxoplasma Strains Available in BEI^a

Strain	BEI no.	Comments
RHΔrop5	NR-49332	• ROP5 knockout in RH background [6] • Deficient in mouse virulence
RHΔrop16	NR-49333	• ROP16 knockout in RH background [5] • Deficient in mouse virulence
RHΔrop16 + rop16	NR-49334	• Complemented ROP16 mutant [5]
RHΔrop18	NR-49442	• ROP18 knockout in RH background [4] • Deficient in mouse virulence
RHΔrop17	NR-51143	• ROP17 knockout in RH background [3] • Deficient in mouse virulence
RHΔrop17Δrop18	NR-51144	• ROP17/ROP18 double knockout in RH background [3] • Deficient in mouse virulence
RH TIR1-3Flag	NR-51145	• Transgenic RH strain expressing the Oryza sativa FLAG-tagged TIR1 auxin receptor [7] • Allows the introduction of transgenes with an auxin-inducible degron (AID) tag • The addition of auxin promotes rapid degradation of AID-tagged proteins in TIR1-3FLAG parasites
RH MIC2-GLuc-C-myc	NR-51148	• Transgenic RH strain expressing the Gaussia luciferase (GLuc) reporter protein fused to C-myc-tagged microneme protein MIC2 [8] • Used for highly sensitive and rapid detection of microneme secretion in a high-throughput format.
RH GCaMP6f	NR-51149	• Transgenic RH strain expressing the fluorescent Ca2+ indicator GCaMP6f [8] • Used to detect Ca2+ fluctuations in real time in response to different stimuli
RHΔku80∷DiCre: T2A:CAT	NR-51627	• Transgenic RH strain that expresses dimerizable Cre recombinase (DiCre) [9] • A chloramphenicol acetyltransferase (CAT) selectable marker is located between the two DiCre subunits to prevent the loss of the recombinase • Stable line that allows the conditional deletion of parasite genes upon rapamycin treatment

RH is a prototypical type I virulent strain. It was originally isolated in 1939 from a 6 year old patient in Cincinnati, Ohio (Sabin AB. Toxoplasmic encephalitis in children. J Am Med Assoc 1941;116:801-807).

^aThe table represents a condensed list of genetically modified strains deposited in the last 4 years. A complete list is available in the BEI Resources online catalog^vi.

Reference Biomaterials for Kinetoplastid Research

Kinetoplastid parasites are transmitted by arthropod vectors, and the diseases they cause are among the 20 neglected tropical diseases listed by the World Health Organization (WHO)^vii. The pursuit of vaccines and chemotherapeutic agents against Trypanosoma cruzi (Chagas' disease), Trypanosoma brucei (African trypanosomiasis), and Leishmania spp. (leishmaniasis) is an active area of research. In this context, mouse models are critical to understanding the mechanisms of disease and the development of vaccines and new therapeutics. BEI Resources provides reference strains of T. brucei (STIB 247, TREU667, Lab 110 EATRO) and T. brucei gambiense (STIB 386) used in mouse models of trypanosomiasis, and T. brucei rhodesiense clinical isolates used in drug-susceptibility studies in vivo [10] (Table 1). For therapeutics research, transgenic parasite lines expressing reporter genes enable high-throughput screening of candidate compounds. BEI Resources houses transgenic T. cruzi and Leishmania that express enzymes that catalyze colorimetric reactions (i.e., lacZ, β-lac), as well as lines engineered with firefly luciferase or the more sensitive red-shifted luciferase gene PpyRE9 [11]. These genetic reporter parasites allow the rapid quantification of growth and streamline the methodology for scoring inhibitor assays using in vitro and in vivo systems. BEI Resources also provides monoclonal antibodies to promastigote and amastigote stages of Leishmania spp. and trypomastigote and amastigote stages of T. cruzi^vi.

In Vivo and In Vitro Cultures of Babesia spp.

Babesia spp. are parasites of red blood cells and the causative agents of babesiosis, an emergent infectious disease in the USA. Primary transmission occurs by ixodid ticks, but the parasites may be passively acquired during pregnancy and blood transfusion [12]. The majority of human cases of babesiosis in the USA are caused by Babesia microti, with a lower incidence from Babesia duncani infection. Babesia divergens is the primary cause of babesiosis in Europe [12]. BEI Resources provides in vivo reference strains of B. microti that have been fully sequenced [13] as well as in vitro lines of B. duncani WA-1 and the B. divergenslike MO1 strain. The availability of in vitro lines for Babesia research enables drug-susceptibility screening in culture and facilitates studies of parasite invasion and metabolism.

Resources for Malaria Research

NIAID’s MR4 was initially funded in 1998 as a central resource for malaria reagents and served as a hub for malaria investigators until integration with BEI Resources in 2010. The reagents include over 150 laboratory standard, genetic recombinant, and newly adapted Plasmodium falciparum parasite lines, as well as antibodies, antigens, and nucleic acids for basic research, diagnostics, vaccine development, and drug screening. Highlights include drug-susceptible and sequenced genomic standards (P. falciparum 3D7), artemisinin- and piperaquine-resistant parasite lines, as well as multiple rodent malaria species (Plasmodium berghei, Plasmodium yoelii, Plasmodium chabaudi, Plasmodium vinckei). Parasites produced in vitro are supplied with drug-susceptibility and msp2 sequence profiles. Methods in Malaria Research, a widely used laboratory manual of numerous malaria protocols, developed in conjunction with EVIMalaR and the Karolinska Institutet, is available as a free download from the BEI Resources website^viii.

Resources for Vector Research

Live anopheline mosquito malaria vectors have been available to investigators sincethe early stages of MR4 and have been integrated into BEI Resources. More than 45 colonies of Anopheles gambiae complex species, Anopheles funestus, Anopheles stephensi, and other malaria vectors have since been complemented with Aedes aegypti strains and Culex spp., including vectors of filarial worms. The Vector Resources program has also been expanded to include eight Ixodid tick species, four sand fly species, and a triatomine, including Ixodes scapularis and Ixodes ricinus tick vectors of babesiosis, Phlebotomus papatasi, sand fly vector of Leishmania major (cutaneous leishmaniasis), Lutzomyia longipalpis, sand fly vector of Leishmania infantum chagasi (infantile visceral leishmaniasis), and Rhodnius prolixus, triatomine vector of T. cruzi (Chagas' disease). Methods protocol manuals for mosquito, tick, and sand fly insectary management and research are available as a free download from the BEI Resources website^ix.

Concluding Remarks

The value of NIAID’s BEI Resources program is exemplified by a user base approaching 10 000 investigators, 20% of which are engaged in parasitology and arthropod vector research. Over 200 parasite and vector-borne disease publications cite reagents obtained from BEI Resources annually. The continued expansion, use, and citation of this program, and the value brought to the investigator of unique, well-characterized strains and reagents is integral to the future support of neglected and emerging diseases of global importance.