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Life cycle studies and transmission
mechanisms of myxozoan parasites

Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-
Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades

vorgelegt von

Dennis Marc Kallert

aus Erlangen

Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 3. März 2006

Vorsitzender der
Prüfungskommission: Prof. Dr. D.-P. Häder

Erstberichterstatter: Prof. Dr. W. Haas

Zweitberichterstatter: Prof. Dr. Dr. M. El-Matbouli -Contents-

1. The Phylum Myxozoa Grassé 1970: Common features
2. Current knowledge on myxozoan life cycles8
3. Host invasion by actinospores 10
1. Animals and parasite cultivation
1.1. Fish for experimental infections
1.2. Oligochaetes 12
1.3. Parasites 13
1.4. Animals for substrate isolation 14
2. Experimental infections
2.1. Henneguya nuesslini Schuberg & Schröder 1905
2.2. Myxobolus parviformis sp.n. 16
3. Viability assay 17
4. Discharge experiments 18
4.1. Test substrates
4.2. Experimental set-up 19
4.2.1. Test for mechanical and chemical stimuli
4.2.2. Bulk experiments 20
4.2.3. Frequency dependency 21
2+4.2.4. Ca dependency 22
5. Cinematography of polar filament discharge
6. Sporoplasm emission
7. Mucus fractionation 23
7.1. Heat and incubation
7.2. Ashing 23
7.3. Acetone precipitation 24
- 1 - -Contents-

7.4. Alcian blue precipitation 24
7.5. Fluorescamin derivatisation
7.6. Extraction with activated charcoal 25
7.7. Lipid extraction
7.7.1. Ether-extraction
7.7.2. Lipid isolation 26
7.8. Chemical fractionation
7.8.1. TFMS Deglycosylation
7.8.2. Sialic acid extraction 27
7.9. Enzymatic fractionation 28
7.10. Chromatographic fractionation 29
7.10.1. Ion-exchange
7.10.2. Lichroprep RP 18 30
8. Pure chemicals 30
9. Chemical analyses 32
9.1. Proteins 32
9.2. Amino compounds
9.3. Neutral sugars 32
9.4. Urea 32
9.5. Sialic acids 33
9.6. TLC-separation of lipid classes 33
9.7. Hydrophilic TLC 34
9.8. RP-HPLC 35
9.9. GC/MS 35
9.10. UV-Spectroscopy and fluorescence detection 36
9.12. NMR-spectroscopy 37
10. Chemicals 38
11. Statistical methods 38
1. Life cycle experiments
1.1. Henneguya nuesslini
1.2. Myxobolus parviformis sp. n. 43
2. Viability assay 51
3. Polar filament discharge 51
- 2 - -Contents-

3.1. Visualisation of polar filament discharge 51
3.2. Mechanical and chemical stimuli 53
3.3. Experimental conditions for bulk experiments
2+ 3.4. Ca dependency 54
3.5. Host specificity 55
3.5.1. Myxobolus cerebralis
3.5.2. Henneguya nuesslini 58
3.5.3. Myxobolus parviformis
3.6. Analysis of chemical signals for polar filament discharge 59
3.6.1. Ultrafiltration 59
3.6.2. Lipids 60
3.6.3. Amino compounds 61
3.6.4. Nucleotides 62
3.6.5. Carbohydrates 63
3.6.6. Proteins 68
3.6.7. Stability and small molecular compounds 70
3.6.8. Extraction with activated charcoal 72
3.6.9. Chromatographic fractionation
4. Sporoplasm emission 77
4.1. Emission time course
4.2. Dependence from discharge 78
4.3. Host specificity
4.4. Emission stimuli
5. Analyses 79
5.1. Substrate osmolality 79
5.2. Urea 80
5.3. Lipids 81
5.4. Hydrophilic TLC Analyses 83
5.5. Gradient chromatography 87
5.6. UV-Spectroscopy 89
5.7. HPIC-detection
5.8. Amino acids 91
5.9. Proteins 92
5.10. Carbohydrates 93
5.10.1. Neutral sugars
5.10.2. Sialic acids 94
5.10.3. GC-MS for monosaccharides
5.11. NMR-spectroscopy 95
- 3 - -Contents-

1. Myxozoan lifecycles
2. Host invasion by actinospores 103
3. Host signals for polar filament discharge 107
4. Impacts on myxozoan transmission 113

- 4 - -Abbreviations-


1 J One bond
AA Amino acid
BSA Bovine submaxillary mucin
cAMP Cyclic adenosine monophosphate
cGMP Cyclic guanonophosphate
cIMP Cyclic inosine monophosphate
DO Deuterium oxide2
df Degrees of freedom
GAGs Glycosaminoglycans
GC/MSGas chromatography/massspectroscopy
H & E Haematoxylin/eosin dye
HMQC Hetero multiple quantum coherence
HPIC-IPAD High performance ion exchange chromatography with integrated pulsed
amperometric detection
HPLC High performance liquid chromatography
l Litres
MDD Metal dye detection
MWCO Molecular weight cut-off
NBD-Cl 4-chloro-7-nitrobenzofurazan
NMR Nuclear magnetic resonance
p.e.Post exposure
p.i. Postinfection
PBSPhosphatebuffered saline
PCR Polymerase chain reaction
PI Propidium iodide
RFLP Restriction fragment length polymorphism
SDBS Data base system for organic compounds
SEM Standard error of the mean
SFW Standard fresh water
TAM Triactinomyxon
v/v Volume per volume
w/v Weight per volume
- 5 - - I. Introduction-

I. Introduction

1. The Phylum Myxozoa Grassé 1970: Common features

About 1350 species in 52 genera belong to the Myxozoa, an obligate parasitic
group forming a separate phylum of multicellular metazoan parasites mainly of
teleosts. Invertebrates like oligochaetes, bryozoans and polychaetes serve as
secondary hosts. Despite being well-known as fish parasites, Myxozoa was also
discovered in trematodes (Overstreet 1976, Siau et al. 1981), reptiles (Lom 1990) and
amphibians (McAllister et al. 1995). Developmental stages were found in waterfowl
(Lowenstine et al. 2002), in nervous systems of mammalians (Friedrich et al. 2000)
and myxospores were even detected in human feces (Lebbad and Wilcox 1998,
Moncada et al. 2001). The members of the most abundant genus, Myxobolus
(Myxobolidae), have recently been reviewed by Eiras et al. (2005).
Myxosporea were first described 1841 by Müller (named ‘Psorospermien’) and
Štolc (1899) already noted the metazoan character of these organisms. Nevertheless,
until the second half of the last century, the Myxozoa were commonly assigned to the
protozoans. Due to the morphological variability within species and their highly
reduced body organization, the taxonomy and the phylogenetic position of these
obscure parasites are still subject of numerous controversies (Kent et al. 1994, 2001,
Siddall et al. 1995, Anderson et al. 1998, Canning & Okamura 2004). An outgrowth of
the ongoing phylogenetical work was the development of comprehensive PCR-based
assays suitable for diagnostics.
Although rather few species exert problematic symptoms, some members are
severe pathogens of teleosts. Several species have a significant ecological and
economic impact on freshwater and marine fish populations in Europe and the USA.
Whirling disease, caused by the cosmopolitan parasite Myxobolus cerebralis, is still
considered as one of the most devastating diseases among salmonid populations
(Nehring & Walker 1996, Gilbert and Granath, 2003). It has been responsible for
declines of the wild rainbow trout population in more than 22 northern american states
(Hedrick et al. 1998). Other important pathogens of cultured fish include Tetracapsula
bryosalmonae (proliferative kidney disease of salmonids) and Sphaerospora renicola
(swimbladder inflammation of carp). Myxospores have been shown to resist freezing
and passage through alimentary tracts of poikilothermic animals (e.g. birds, see El-
- 6 - - I. Introduction-

Matbouli and Hoffmann 1991) and are therefore highly adapted to environmental
Morphologically, the myxozoans share some unique features defining phylum
affiliation. The class Malacosporea, probably an ancestral myxozoan group with few
species described that partially resemble different (nemathelminth) characteristics, will
be excluded in this study. Myxosporean specimens are uniformly small sized
(myxospores 10-25 µm, actinospores up to 300µm), thereby showing an immense
biodiversity in shape and morphological variation. Actinospores differ from
myxospores by their triradial symmetry and the softer valves. Developmental stages
lack cilia and cellular fission seems to occur without centrioles. Especially peculiar is
the development including secondary and tertiary cell stages formed by endogeny
during proliferation, a feature that is very rare in animals and otherwise found only in
some protozoans. Most denominating are the polar capsules, specialised cell
organelles reminiscent of cnidarian nemtocysts that are used for attachment to the
host. Though recent findings revealed strong evidence for triploblast bilateral
ancestors of the myxozoan phylum (Schlegel et al., 1996; Anderson et al. 1998;
Okamura et al., 2002), there are arguments for a cnidarian origin of the polar capsules
(discussed in Canning and Okamura, 2004). For example, the polar filaments were
compared to the stinging threads of the parasitic cnidarian Polypodium hydriforme by
Ibragimov (2001).
Figure 1. Schematical structure of a myxozoan triactinomyxon-type actinospore.

- 7 - - I. Introduction-

2. Current knowledge on myxozoan life cycles

About 25 whole myxozoan life cycles have been elucidated today (see Kent et al.
2001). With few exceptions, they include two alternating hosts, an aquatic invertebrate
(oligochaetes or bryozoans) and a vertebrate host, mainly teleost fish (Wolf & Markiw,
1984). Life cycle studies of myxozoans are difficult to conduct, most research has
been carried out with M. cerebralis, which is available through continuous cultivation
(El-Matbouli et al. 1992, Hedrick & El-Matbouli 2002). The complete chronological
development of a myxozoan has only been obtained for M. cerebralis (El-Matbouli &
Hoffmann 1989, El-Matbouli et al. 1995).
Waterborne actinospores (Fig. 1) released from oligochaetes infect the fish via gills
or skin. Amoeboid sporoplasms containing the infective secondary cells leave the
actinospore valve construct and actively penetrate the host integument. In the
respective target tissue, they develop from trophozoits into sporogenic plasmodia.
Coelozoic or histozoic parasitism occurs mostly extracellular, while some species
penetrate host cells for multiplication. Some myxosporeans form extrasporogonic (e.g.
blood-) stages and multiply several times by internal cleavage. Most species tend to
infect specific kinds of organs, tissues or cell types (Molnar 2002, Eszterbauer 2004).
M. cerebralis developmental stages migrate through several tissues along peripheral
nerves to reach the cartilage (El-Matbouli et al. 1995). During growth, somatic and
generative nuclei are found. At the end of the intrapiscine development, myxospores
composed of at least six cells are formed shedding at least two rigid (Myxosporea) or
soft (Malacosporea) protective shell valves. They are formed in pansporoblasts inside
plasmodia or develop in coelozoical or intercellular in pseudoplasmodia.
Although the existing knowledge on transmission is limited, mxospores are thought
to be released from the fish hosts by death of the host (e.g. predation), but there is an
indication that release may also occur from live fish by Myxobolus artus (Ogawa et al.
1992) and most likely some gill-dwelling species. They infect the intestine of
invertebrate hosts by ingestion. Vegetative and sporogonic stages of actinosporeans
are located intercellularly in the intestinal epithelium or in the coelom. Actinospores
released from the oligochaete host inflate to develop their final habitus and initiate a
new development in fish (Fig. 2). The release of myxo- and actinospores is often
seasonal; therefore the whole life cycle may last for 1-2 years although the
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