Invasion of Erythrocytes by Babesia Bovis
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Invasion of erythrocytes by Babesia bovis Invasion of erythrocytes by Babesia bovis Invasie van erythrocyten door Babesia bovis (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de Rector Magnificus, Prof. Dr. W. H. Gispen ingevolge het besluit van het College voor Promoties in het openbaar te verdedigen op 29 januari 2004 des middags te 14.30 uur Door Fasila Razzia Gaffar Geboren op 19 oktober 1973, te Paramaribo/Suriname Promotor: Prof. Dr. A.W.C.A. Cornelissen Co-promotor: Dr. E. de Vries The research described in this thesis was performed at the Department of Infectious Diseases and Immunology, Division of Parasitology and Tropical Veterinary Medicine, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands and supported by NWO-WOTRO, Netherlands foundation for Advancement of Tropical Research. Printing of this thesis was supported by Intervet Parasitology, Division Parasitology, GlaxoSmithKline and Eijkman Graduate school for Immunology and infectious Diseases. ISBN: 90-393-3594-X Opgedragen aan : Mohamed Junas Gaffar Hakiema & Hamza Kariman Omslag is een kreatie van Afsal Mohammad Aziz Gaffar CONTENTS CHAPTER 1 General Introduction 9 CHAPTER 2 Characterisation of erythrocyte invasion by Babesia bovis 29 merozoites efficiently released from their host cell after high voltage pulsing CHAPTER 3 Babesia bovis merozoites invade human, ovine, equine, 45 porcine and caprine erythrocytes by a sialic acid-dependent mechanism followed by developmental arrest after a single round of cell fission CHAPTER 4 Erythrocyte invasion by Babesia bovis merozoites is 61 inhibited by polyclonal antisera directed against peptides derived from a homologue of Plasmodium falciparum Apical Membrane Antigen 1 (AMA-1) CHAPTER 5 A Babesia bovis merozoite protein with a domain architecture 79 highly similar to the thrombospondin related anonymous protein (TRAP) present in Plasmodium sporozoites CHAPTER 6 A study on the possibilities for obtaining genetic transformation 99 of Babesia bovis CHAPTER 7 An aminoacid substitution in Babesia bovis 113 Dihydrofolate Reductase-Thymidylate Synthase is correlated to cross-resistance against pyrimethamine and WR99210 CHAPTER 8 General Discussion 131 REFERENCES 142 SUMMARY/ SAMENVATTING 157 CURRICULUM VITAE 165 DANKWOORD 167 CHAPTER 1 GENERAL INTRODUCTION “Reciteer in de naam van uw Heer, die geschapen heeft (Qur’an 96:1)” General Introduction INTRODUCTION Members of the genus Babesia cause one of the most common parasitic infections worldwide in wild and domestic animals (98, 140). Economically, bovine babesiosis, caused by B. bovis, B. bigemina, B. major and B. divergens forms a serious problem as it is widely distributed and threatens the health and safety of about 500 million cattle in tropical and subtropical regions of the world (98). Although no Babesia species primarily infect humans, some of the species, like B. divergens, can be transmitted to humans (93). B. bovis infection results in high mortality rates among susceptible cattle, and causes a virulent disease characterized by fever, anemia, anorexia and hypotensive shock syndrome. Parasitized erythrocytes are often sequestered in the capillary beds of the brain and lung, resulting in low peripheral parasitemia, but causing severe pathology like cerebral babesiosis and respiratory distress, which eventually can lead to death. The pathogenicity varies both between and within species but in many cases has a high rate of mortality in untreated animals. THE APICOMPLEXA Members of the phylum Apicomplexa cause parasitic diseases from which the most important disease, malaria, invokes major health problems in human populations in tropical and subtropical countries. Apicomplexan parasites are intracellular protozoan organisms that can invade a specific or even a wide range of different host cells, depending on the species. The characteristic common feature of all members of this phylum is the unique set of organelles localised at the anterior end of their invasive forms that have showed to play a crucial role in the invasion of the host cell (176). These apical organelles are micronemes, rhoptries and dense granules. Besides these organelles they also contain polar rings and some species contain a conoid which lies within the polar rings. Apicomplexans are transmitted to new hosts by different vectors. Malaria parasites for instance, are transmitted by infected mosquitoes while Babesia species are transmitted by infected ticks. 10 Chapter 1 THE GENUS BABESIA Babesia organisms are protozoan parasites that are all transmitted by ticks to their specific vertebrate host. Completion of a life cycle and therefore the maintenance of Babesia spp. is completely dependent on both the tick and the vertebrate host (140). The taxonomic classification of Babesia spp. places them in the phylum Apicomplexa and the order Piroplasmida. Piroplasms often have a pear-shaped appearance inside their host cell. The order of Piroplasmida is divided in Babesiidae and Theileriidae (98, 115). Babesiosis and Theileriosis are both diagnosed largely on clinical signs and symptoms with confirmation by microscopic observation of the parasites with their specific host blood cells and often DNA-based tests, like reverse line blot (82). True Babesia species exclusively invade erythrocytes, whereas Theileria species undergo a pre- erythrocytic cycle in lymphocytes. Examination of stained blood films shows Babesia organisms within red cells, where single and duplicated parasites are found. Babesia species are grouped into the small and large Babesia (98). The invasive parasitic stage of Babesia species inside erythrocytes will grow and differentiate to trophozoites. The diameter of trophozoites divides Babesia species into small and large Babesia. Trophozoites of the small Babesia are 1.0 to 2.5 µm and include B. bovis, B. gibsoni, B. ovis and B. divergens. Trophozoites of large Babesia are 2.5 to 5.0 µm including B. caballi and B. canis. All apicomplexans have a complex life cycle with both asexual and sexual reproduction. Pathogenesis of Babesia species results from the asexual erythrocytic stage, where the parasite undergoes multiple rounds of invasion and replication in the host erythrocytes. The distribution of all the different Babesia species is governed by the geographical distribution of the tick vectors that transmit them. LIFE CYCLE The life cycle of B. bovis takes place in two hosts (Fig. 1). Asexual development takes place in the bovine host and sexual development in the tick vector (140). 11 General Introduction Fig. 1. Life cycle of Babesia spp. in the tick vector and vertebrate host (140) Asexual stage Babesia spp. infected ticks have accumulated mature sporozoites in their salivary glands. When an infected tick takes a blood meal on its host it will pass the content of its salivary gland, including the parasites, into the host. The sporozoite invades a red blood cell and will multiply itself by a single round of binary fission. In contrast to the malaria and Theileria life cycle, where sporozoites invade hepatocytes and white blood cells respectively, B. bovis sporozoites enter the red blood cell. Inside the red blood cell sporozoites differentiate to trophozoites. Babesia multiplies inside the red cell by a single round of binary fission after which the red cell ruptures and daughter parasites, called merozoites, are released into the bloodstream and directly invade a new red blood cell. This cycle is repeated over and over again and is called the merogony stage. During the merogony stage the parasite feeds on haemoglobin and takes up serum nutrients to be able to synthesize amino acids. Nutrients and hemoglobin are taken up by the parasite through pinocytosis at the area around the micropore. Once the parasite is inside the erythrocyte it will start replicating and at the same time will also modify the host cell making it a more suitable place for them. Structural alterations of infected host erythrocytes have been shown to take place. These changes are induced by the parasite and lead to formation of knob-structures (2). Antigenic variation takes place of the proteins (VESA, variant erythrocyte surface antigen) localized on the 12 Chapter 1 erythrocyte membranes (5). Besides formation of merozoites, a few percent of the merozoites invading new erythrocytes differentiate into gametocytes. In apicomplexans like Toxoplasma gondii and Plasmodium spp. male (microgametocytes) and female (macrogametocytes) gametocytes have been found. In Babesia spp. no clear description has been given of gametocytes in the vertebrate host. In ticks that have ingested infected blood several types of parasite with long rays and spikes have been described that were originally called strahlenkörper. One type does not appear to develop further whereas others undergo further multiplication, even after they have emerged from the erythrocyte. These forms may represent micro- and macrogametes although firm evidence is lacking (140). Sexual stage Sexual development takes place in the gut of the tick, after a feeding tick has ingested gametocytes. Gametocytes will develop into gametes when taken up by the tick. Two morphologically different strahlenkörper will fertilize and form a zygote. The zygote is the only parasite stage that is diploid and directly after the first meiotic division the parasite is haploid again. The zygote differentiates into a kinete and can now pass the intestinal wall. Once