Short communication Infectivity of Giardia duodenalis Assemblages A and E for the gerbil and axenisation of duodenal trophozoites Ely Bénéré a , Thomas Geurden b , Lucy Robertson c , Tim Van Assche a , Paul Cos a , Louis Maes a, ⁎ a Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, B-2020 Antwerp, Belgium b Laboratory of Parasitology, Faculty of Veterinary Medicine, University of Ghent, B-9820 Merelbeke, Belgium c Laboratory of Parasitology, Norwegian School of Veterinary Science, 0033 Oslo, Norway abstract article info Article history: Received 24 December 2009 Received in revised form 2 August 2010 Accepted 2 August 2010 Available online 6 August 2010 Keywords: Giardia duodenalis Field isolates Gerbil In vitro axenisation Molecular genotyping Trophozoite Establishing in vitro cultures of Giardia duodenalis trophozoites from faecal cysts remains very difficult due to poor excystation and bacterial contamination. This study investigated an alternative approach starting from duodenal trophozoites of gerbils that were artificially infected with field isolates from humans (Assemblages A and B) and cattle (Assemblage E and mixed E/A). Gerbil infection was successful for Assemblages A (1/1) and B (1/3) from humans, and for E (1/2) and mixed E/A (6/6) from cattle. Despite the fact that some isolates subsequently failed or were difficult to establish in vitro, several Assemblage A and Assemblage E in vitro trophozoite cultures were successful, however, subsequent cloning required adaptation of the standard TYI-S-33 medium whereby different medium supplements were required for promoting growth. Excess of L-cysteine (2 mg/ml) and ascorbic acid (0.2 mg/ml) supported cloning of Assemblage A, while L-glutathione (7.8 mg/ml) was required for Assemblage E. This is a first description of in vitro axenisation of Assemblage E trophozoites from cattle. © 2010 Elsevier Ireland Ltd. All rights reserved. Giardia duodenalis is a ubiquitous flagellate protozoon showing a considerable genetic and biotypic diversity and found in the small intestine of man and a wide variety of animal hosts [1,2]. Two Assemblages (A and B) infect man and several other host species; other strains are host-specific: Assemblage E in hoofed livestock, Assemblages C and D in dogs and Assemblage F in cats [3,4]. Mixed Assemblages A and E infections have been described in cattle with a high degree of genetic polymorphism and different E-subtypes circulating on the same farm or between cattle farms [5,6]. Both parasite (infectivity and virulence) and host (nutritional and immunological status) related factors influence the outcome of infection, but its pathogenesis is not yet fully understood [7,8]. The in-depth study of biological differences between isolates is largely dependent on the availability of in vitro trophozoite cultures, which have already been established for Assemblages A and B [3,9]. To expand to other Assemblages, isolates are usually only available as faecal cysts and starting in vitro cultures prove to be very difficult due to variations in excystation and adaptation, and the ever-present bacterial contamination [10]. Obtaining duodenal trophozoites from artificially infected gerbils has been suggested as a valid alternative [10,11] since this animal species is considered a good experimental host that can be infected with a low dose of cysts and develops a pathogenesis similar to that in the original host [12]. Adequate infection has also been obtained in suckling mice [13] but is far less practical because of their small size, difficult handling and low output of cysts. Important to mention is that several of the abovementioned studies did not genotype the isolates, precluding proper interpreta- tion of Assemblage-specific infectivity for laboratory animals. The aim of this study was twofold: 1) to investigate whether field strains from man and cattle could be successfully established in vitro as trophozoite cultures using the gerbil, and 2) to optimise a protocol for cloning genetically heterogeneous field isolates. Trophozoites were grown and maintained in TYI-S-33 medium as previously described [14]. Faecal cyst samples from human and cattle were suspended in water followed by overnight sedimentation. Additional purification was obtained by flotation on sugar (specific gravity 1.2); thereafter the concentration was determined by staining with fluorescent-labelled monoclonal antibody (Merifluor Cryptospo- ridium/Giardia kit; Meridian Diagnostics Inc., Cincinnati, OH, USA). Cyst isolates were stored at 4 °C in water supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 0.1 μg/ml ampho- tericin B to suppress bacterial and fungal overgrowth. Four to twelve week-old specific pathogen-free (SPF) gerbils (Janvier, St Isle, France) were housed individually and checked for the absence of Giardia by examination of faeces collected over 3 consecutive days. The different isolates were used as fresh as possible, but in some cases were already up to 3 weeks old (Table 1). The gerbils were fasted overnight before oral inoculation with the highest possible dose of cysts [15]. All animal Parasitology International 59 (2010) 634–637 ⁎ Corresponding author. Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium. Tel.: +32 3 265 33 54; fax: +32 3 265 33 26. E-mail address: louis.maes@ua.ac.be (L. Maes). 1383-5769/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2010.08.001 Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint