Downloaded from www.microbiologyresearch.org by IP: 54.166.144.74 On: Thu, 30 Mar 2017 18:15:40 Novel model for the in vivo study of central nervous system infection due to Acanthamoeba spp. (T4 genotype) Parisa Nakhostin Mortazavi, 1 Graham Goldsworthy, 1 Ruth Kirk 2 and Naveed Ahmed Khan 1,3 Correspondence Naveed Ahmed Khan naveed.khan@nottingham.ac.uk 1 School of Biological and Chemical Sciences, Birkbeck, University of London, London, UK 2 School of Life Sciences, Kingston University, Kingston upon Thames, Surrey, UK 3 School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, England, UK Received 28 July 2008 Accepted 22 December 2008 In this study it was shown for what is believed to be the first time that the African migratory locust can be used as a model for the study of Acanthamoeba pathogenesis. Mature adult locusts were injected intra-abdominally with 10 ml suspension of 10 6 Acanthamoeba (a clinical isolate of the T4 genotype) in culture medium, or with the same volume of sterile culture medium. Locusts injected with Acanthamoeba showed significant weight loss and reduced production of faeces compared with control locusts. Furthermore, injection of amoebae killed all of the locusts within 17 days at room temperature, although the speed of kill was temperature and dose dependent. When samples of faecal pellets and various tissues of infected locusts were cultured on non-nutrient agar plates containing bacterial lawns, live amoebae were recovered from haemolymph, flight muscle and fat body samples, but not from faeces. When brains dissected from locusts were incubated with an anti-amoebic drug (100 mM chlorhexidine) to kill extracellular amoebae, and then washed, homogenized and cultured on bacteria-seeded non-nutrient agar plates, only lysates from amoebae-infected locusts were positive for Acanthamoeba. This strongly suggests that amoebae invade the locust brain and, indeed, trophozoites of Acanthamoeba could be identified within the brain in histological sections of brains from infected locusts, but not from uninfected locusts. These findings support the view that locusts can be used as a model for the study of Acanthamoeba pathogenesis in vivo. INTRODUCTION Members of the genus Acanthamoeba are the causative agents of granulomatous encephalitis, a fatal subacute to chronic disease, and amoebic keratitis, a painful sight- threatening infection. Acanthamoeba spp. are also respons- ible for cutaneous lesions and sinusitis in AIDS patients (Schuster, 2002; Marciano-Cabral & Cabral, 2003; Schuster & Visvesvara, 2004; Khan, 2007). Although generally considered a rare infection, Acantha- moeba encephalitis is a major concern in human health, in particular with the increasing populations of AIDS patients as a result of human immunodeficiency virus (HIV) type 1 infection worldwide (estimated by the World Health Organization to be 33 million, as of 2007). The approxi- mate rate of death due to Acanthamoeba encephalitis is estimated to be at least 1.57 per 10 000 HIV/AIDS deaths (Khan, 2006). A distressing aspect is that Acanthamoeba encephalitis is almost always fatal, despite advances in antimicrobial chemotherapy (Khan, 2007). This is due to an incomplete understanding of the pathogenesis and pathophysiology of the disease. Haematogenous spread followed by blood–brain barrier penetration is thought to be the likely route of invasion of the central nervous system (CNS) by amoebae. However, the precise mechanisms associated with Acanthamoeba invasion of the intravascular space, evasion of the host immune defences and traversal of the blood–brain barrier in vivo require investigation. Current studies in vivo have relied on vertebrate models to study Acanthamoeba encephalitis. Although physiologic- ally relevant, mammalian models are expensive, not routinely available in many laboratories, require labour- intensive management and have ethical implications. For these reasons, the use of Locusta migratoria, the African migratory locust, as a model for the study of Acanthamoeba pathogenesis was tested in this study. Locusts are routinely reared in our laboratories and can be handled with ease, as well as injected with large volumes, and a significant Abbreviation: CNS, central nervous system. Journal of Medical Microbiology (2009), 58, 503–508 DOI 10.1099/jmm.0.005462-0 005462 G 2009 SGM Printed in Great Britain 503