Experimental investigation of avian malaria parasites (Plasmodium, Haemosporida): linkage of traditional and molecular data ; Paukščių maliarinių parazitų (Plasmodium, Haemosporida) eksperimentiniai tyrimai: tradicinių ir molekulinių duomenų saitai

Jurga

Sujets

• Avian malaria
• Microscopy
• PCR
• PGR
• Plasmodium
• Zoology

Informations

• Publié par: vilnius_university
• Publié le: 01 janvier 2009
• Nombre de lectures: 47
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

INSTITUTE OF ECOLOGY OF VILNIUS UNIVERSITY
VILNIUS UNIVERSITY










Vaidas Palinauskas




EXPERIMENTAL INVESTIGATION OF AVIAN MALARIA
PARASITES (PLASMODIUM, HAEMOSPORIDA): LINKAGE
OF TRADITIONAL AND MOLECULAR DATA



Doctoral Dissertation
Biomedical Sciences, Zoology (05 B)

















Vilnius, 2009 Dissertation research was carried out at the Institute of Ecology of Vilnius
University in 2005 – 2009

Research Supervisor:
corresponding member of the Lithuanian Academy of Sciences dr. habil.
Gediminas Valki ūnas (Institute of Ecology of Vilnius University, Biomedical
sciences, Zoology – 05 B)

Consultant Supervisor:
prof. dr. Staffan Bensch (Department of Animal Ecology of Lund University,
Sweden, Biomedical sciences, Zoology – 05 B)


2CONTENTS
INTRODUCTION .............................................................................................................. 6
1. LITERATURE REVIEW ............................................................................................. 12
1.1. Brief outline of the life cycle of Plasmodium parasites ......................................... 12
1.2. Brief review of studies of avian malaria parasites ................................................. 14
1.2.1. Investigation of avian malaria parasites using traditional methods ................. 16
1.2.2. Polymerase chain reaction (PCR)-based methods; a comparison with
microscopy in the studies of avian malaria parasites ................................................ 18
1.3. Necessity of development of new approaches and molecular markers for
investigations of avian malaria parasites and closely related haematozoa .................... 20
1.4. Specificity and virulence of Plasmodium species .................................................. 22
1.5. Interaction between parasites and their hosts during simultaneous infections ....... 24
1.6. Brief summary of drug development to treat avian malaria ................................... 25
1.7. Genetic diversity of avian malaria parasites and a potential for their expansion
to new regions .............................................................................................................. 27
2. MATERIALS AND METHODS ................................................................................. 30
2.1. Study site and material .......................................................................................... 30
2.2. Collection of material ............................................................................................ 32
2.2.1. Collection of birds .......................................................................................... 32
2.2.2. Obtaining of bird blood and preparation of the material for microscopic
examination and DNA studies .................................................................................. 34
2.3. Blood smear examination; morphological and morphometric investigation of
parasites ........................................................................................................................ 35
2.4. Extraction of DNA, PCR and sequencing .............................................................. 36
2.5. Dissection of single cells of parasites; extraction of DNA from single cells,
PCR and sequencing ..................................................................................................... 39
2.5.1. Dissection of single cells and extraction of DNA ........................................... 39
2.5.2. Design of primers for small amount of DNA; PCR and sequencing .............. 42
2.6. Design of experiments for infection of birds ......................................................... 43
2.6.1. Experiments for development of molecular identification of
morphospecies of avian malaria parasites ................................................................ 43
2.6.2. Experimental infection of birds with Plasmodium relictum (lineage SGS1) .. 45
32.6.3. Experimental simultaneous infection of birds with Plasmodium relictum
(lineage SGS1) and P. ashfordi (GRW2) ................................................................. 47
2.7. Treatment of experimental avian malaria with Malarone™ and a combination
of Malarone™ and primaquine ..................................................................................... 48
2.8. Statistical analysis ................................................................................................. 50
3. RESULTS AND DISCUSSION ................................................................................... 51
3.1. A comparative analysis of microscopy and PCR-based detection methods for
avian malaria parasites and closely related haematozoa ............................................... 51
3.2. Molecular identification of Plasmodium relictum and Plasmodium
circumflexum; linkage of PCR-based and morphology data and some taxonomic
consideration in identification of Plasmodium spp. ...................................................... 58
3.3. Laser microdissection of single cells for PCR-based studies of Plasmodium
spp. and closely related blood parasites ........................................................................ 68
3.4. Development of Plasmodium relictum (lineage SGS1) infection in
experimentally infected birds ....................................................................................... 72
3.4.1. Specificity of (lineage SGS1) for birds in regard to
their phylogenetic relationships ................................................................................ 73
3.4.2. Prepatent period and dynamic of parasitemia of Plasmodium relictum
(lineage SGS1) in experimentally infected birds ...................................................... 74
3.4.3. The effects of Plasmodium relictum (lineage SGS1) on body mass,
temperature and haematocrit value of birds .............................................................. 78
3.4.4. Plasmodium relictum (lineage SGS1) effects on internal organs of birds ...... 85
3.5. Development of simultaneous infection with Plasmodium relictum (lineage
SGS1) and P. ashfordi (GRW2) in experimentally infected birds ................................ 87
3.5.1. Specificity of Plasmodium ashfordi (lineage GRW2) for experimentally
infected birds ............................................................................................................ 88
3.5.2. Prepatent period and the dynamics of parasitemia of Plasmodium relictum
(lineage SGS1) and P. ashfordi (GRW2) during simultaneous infection in
experimentally infected birds ................................................................................... 89
3.5.3. The effects of Plasmodium relictum (lineage SGS1) and P. ashfordi
(GRW2) simultaneous infection on body mass and haematocrit value of birds ....... 94
3.6. Efficacy of the antimalarial drug Malarone™ and a combination of
Malarone™ and primaquine for treatment of avian malaria ......................................... 97
43.7. Geographic distribution of Plasmodium relictum lineages in the house sparrow
Passer domesticus in Europe ...................................................................................... 101
4. GENERALIZATION ................................................................................................. 105
CONCLUSIONS ............................................................................................................ 110
ACKNOWLEDGEMENTS ........................................................................................... 112
REFERENCES ............................................................................................................... 115
LIST OF PUBLICATIONS ON THE DISSERTATION TOPIC ................................... 134

5INTRODUCTION

Relevance of the study. Malaria parasites (Plasmodium, Plasmodiidae)
parasitise and cause disease, which is well known as malaria, in a wide range of
vertebrate hosts: amphibians, reptiles, birds and mammals including humans
(Valki ūnas, 2005; Martinsen et al., 2008). A shortcoming of recent malaria
research is that the great majority of studies on general malariology deal with a
handful of Plasmodium species infecting humans, some other primates and mice
(Carlton et al., 2002; Sherman, 2005; Pain et al., 2008). Zoological studies,
particularly experimental ones, on malaria parasites infecting reptiles and birds
remain scarce. Moreover, fragmentary information is available about
relationships between bird haemosporidians and their vectors (Valki ūnas, 2005;
Ishtiaq et al., 2008; Kimura et al., 2009). These are obstacles for developing
general models of Plasmodium parasites evolution and for understanding the
evolutionary biology of this large and diverse group of parasites, particularly
from the perspectives of zoology and evolutionary biology.
Avian malaria parasites are responsible for severe diseases in some
domestic and wild birds (Valki ūnas, 2005). These parasites are cosmopolitan in
distribution; they are widespread in Europe, including the Baltic region
(Waldenström et al., 2002; Križanauskien ė e