Biology of parasitic protozoa
IX. Microsporidia (Opisthokonta, Fungi)
Andrea Bardůnek Valigurová
andreav@sci.muni.cz
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5 supergroups = megagroups
Microsporidia
• monophyletic group
• about 1,500 species in 187 genera
• traditionally thought to be a unique phylum
of spore-forming protozoa
• smallest of eukaryotes = size of spores vary
in range 1-40 μm (in medically important
species usually 1-4 µm)
• smallest eukaryotic genomes; 2.5 to 11.6 Mb
in size
• mitochondria highly reduced to mitosomes
• obligate intracellular parasites
• most infect insects, but they are also
responsible for common diseases of
crustaceans and fish
• several species also infect humans
Microsporidia
• spores with inner chitin wall and outer
proteinaceous wall are highly resistant in the
environment  surviving for months to years
• spore germination occurs when
microenvironmental stimuli result in extrusion of
the polar filament (tube)
• polar filament responsible for injecting the
sporoplasm into the host cell (epithelial cells,
macrophages, endothelial cells)
• replication by binary division within a
parasitophorous vacuole
• development of proliferative forms (meronts) that
undergo binary division and differentiate into
sporoblasts and sporonts
https://doi.org/10.1146/annurev.micro.56.012302.160854
https://doi.org/10.1146/annurev.micro.56.012302.160854
Extrusion of polar tube
Microsporidia perfect
parasites
✓ low pathogenicity
✓ metabolically completely dependent on
the host cell
✓ intracellular development during which
they "behave" more like an organelle of
the host cell
✓ host cells react to their presence by
muscle hypertrophies (xenomas in fish)
✓ host cell dies only after spore formation
✓ spores are resistant to environmental
conditions
✓ spread by water and contaminated food
https://doi.org/10.1016/j.pt.2004.04.009
https://doi.org/10.3354/dao02133
Microsporidia in fish
https://doi.org/10.3354/dao02133
Microsporidia in fish - xenomas
Glugea hertwigi infecting Osmerus mordax. Variably sized xenomas in the gastrointestinal tract of infected rainbow
smelt. A) Heavily infected individual with uniformly small xenomas (scale bar = 1 cm). B) Moderate infection with uniformly large
xenomas (scale bar = 1 cm). C) Individual with a mixed infection of small and large xenomas (scale bar = 4 mm).
Xenoma is a growth caused by various protists and fungi, most notably microsporidia. The host cell
undergoes hypertrophy and has many, mostly polyploid, nuclei. This outcome is due to the
microsporidian parasite proliferating inside the host cell. Symbiotic co-existence develops between
host cell and microsporidian and both partners turn into a well-organised xenoparasitic complex.
Different types of xenomas of fish microsporidia
https://folia.paru.cas.cz/artkey/fol-200501-0010_Microsporidian_xenomas_in_fish_seen_in_wider_perspective.php
1) Early stage of Spraguea lophii xenoma;
parasite mass (X) occupies only part of the
host ganglion cell. Bodian. 2) Advanced stage
of S. lophii xenoma in ganglion. Note the
different staining of parasite mass at the
periphery (p) with Nosemoides-type spores
and in the centre (c) with Nosema-type spores.
3) “Cystic” stages preceding formation of huge
xenomas of Ichthyosporidium giganteum.
Compartments contain different stages of
merogonial proliferation. 4) Xenoma of
Tetramicra brevifilum, in a liquid-filled cavity in
host liver parenchyma. 5) Mature xenoma of
Glugea anomala in the body cavity. 6) Xenoma
of Loma branchialis in fish gills. 7) Xenoma of
T. brevifilum in folded-over shape in the host
muscle tissue. 8) Loma acerinae xenoma with
a centrally located host cell nucleus in the
subepithelial connective tissue of intestine. 1-8
= HE. 9-10) Parts of the wall of similar, mature
G. plecoglossi xenomas (X), localised in host
testes (T). Xenoma wall and mature encircling
connective tissue (present in 10) are stained
red. Van Gieson.
Early stages of xenoma development
https://folia.paru.cas.cz/artkey/fol-200501-0010_Microsporidian_xenomas_in_fish_seen_in_wider_perspective.php
Xenomas of Glugea anomala in early stages of
development. 22) Spontaneous infection with G. anomala.
23-25) Early xenomas with hypertrophic branched nuclei
and cylindrical meronts, which predominate in 24 and 25.
26-31) Examples of xenoma transformation due to the onset
of proliferative inflammation of the host. 26) Glugea
plecoglossi infection in ovaries. HE. 27) Proliferation of
granulation tissue in Loma acerinae visualised by Masson’s
trichrome staining. 28) Xenoma of Tetramicra brevifilum
transformed into granuloma in the liver. 29) Granulomatous
lesion at the site of Glugea anomala xenoma in the
glandular part of host stomach wall. 30) Granuloma in fish
ovary replacing G. anomala xenoma. 31) Spraguea lophii
xenoma partly transformed into a granuloma. 32) Overview
of a massive spontaneous infection of G. anomala in fish
intestine. HE.
Encephalitozoon cuniculi
• the most common and important animal pathogen
• natural infections with E. cuniculi in a wide range of
hosts (e.g. rabbits, mice, cats, dogs, foxes, humans)
• important pathogenic agens of pet rabbits with 37-
68% seroprevalence
• transmission - transplacental, ingestion or inhalation
of spores passed in the urine
• most cases of infection are asymptomatic
• infection of brain and kidneys
• clinical signs: liver and kidney failure and
calcification, limb weakness and pressure on the
inner ear can leading to a loss of balance and
hopping in circles, ataxia, head tilt, hind limb
paralysis
• end phase signs: more frequent and stronger
seizure attack, coma and death https://www.youtube.com/watch?v=MER1YAwaO70&t=8s
Microsporidia in mammals
Nosema apis / Nosema ceranae
• causative agent of bee dysentery
• intestinal epithelium of bees
Life cycle of Nosema
Infection begins when a bee ingests Nosema spores, which then germinate inside the midgut of the bee. Parasite enters the
host enterocytes and begins to absorb nutrients  damage to the cell and increased susceptibility to secondary infections.
Nosema grows and multiplies infesting more of the midgut cells and produces spores. Several million spores can be produced
in a single bee worker. Spores either germinate within the bee’s midgut, infecting new cells, or pass through the bee’s digestive
system. Faecal material containing spores can contaminate food and water sources, where they can then be ingested by other
bees. Spores can also be spread to non-infected bees when they clean contaminated combs, or rob contaminated hives and
ingest spores in the process.
✓ hypopharyngeal (brood food) glands of infected nurse bees lose the
ability to produce royal jelly which is fed to honey bee brood
✓ high proportion of eggs laid by the queen of an infected colony may fail to
produce mature larvae
✓ infected queens cease egg-laying and die within a few weeks
✓ young infected nurse bees cease brood rearing turning to guarding and
foraging duties - usually undertaken by older bees
✓ reduction of life expectancy of infected bees - in spring and summer,
infected bees live half as long as non-infected bees
✓ increase of dysentery in adult bees although Nosema is not the prime
cause of dysentery
Effect of Nosema on honey bees
Nosema bombycis
• first described microsporidia
• parasite of silkworm caterpillars, transovarian transmission
• Pasteur recommended rearing caterpillars from clutches of microscopically
inspected females
Nosema locustae
• biocontrol
Vairimorpha necatrix
• butterfly caterpillars
Pleistophora hyphessobryconis
• muscles of neon tetra
Other significant species of microsporidia
Grasshopper control with Nosema locustae baits
Grasshopper control with Nosema locustae baits
Human microsporidiosis
• at least 15 microsporidian species that have been identified as human pathogens
Enterocytozoon bieneusi
Encephalitozoon cuniculi
Encephalitozoon intestinalis
Encephalitozoon hellem
• other genera and species - isolated infections
• opportunistic parasites with worldwide distribution
• mostly in severely immunocompromised patients with AIDS, but also in
immunocompromised not infected with HIV and immunocompetent persons
• diverse clinical manifestations, depending on species and route of infection, while E.
bieneusi-associated diarrhoea is the most common one
• some of them are thermotolerant (causing systemic infections), while some are only
capable of causing eye infections
• disseminated infection can be fatal
• many domestic and wild animals may be naturally infected with various medicallyimportant
microsporidia
Human microsporidiosis
Infective stage is the resistant spore (1) which germinates, rapidly everting its polar tubule which contacts eukaryotic host cell membrane (2). Spore
injects the infective sporoplasm into host cell through the polar tubule (3). Inside the cell, the sporoplasm enters proliferative phase marked by extensive
multiplication via merogony (binary fission or multiple fission), creating meronts (4). Location of this stage within host cell varies by genus; it can occur
either in direct contact with host cell cytosol (Enterocytozoon, Nosema), inside a parasitophorous vacuole of unknown origin (Encephalitozoon), in a
parasite-secreted envelope (Pleistophora, Trachipleistophora), or surrounded by host cell endoplasmic reticulum (Endoreticulatus, Vittaforma) (5).
Following proliferative phase, meronts undergo sporogony in which the thick spore wall and invasion apparatus develop, creating sporonts and
eventually mature spores when all organelles are polarised. When the spores increase in number, completely filling host cell cytoplasm, cell membrane
is disrupted, and spores are released to the surroundings (6). Free mature spores can infect new cells to continue the cycle.
Mature spores of intestinal species may be shed in feces, but the route of transmission is uncertain for many species. Exposure to spores in water or in
soil appears to be a potentially major route. Cases of donor-derived microsporidiosis (Encephalitozoon cuniculi) following bone marrow, kidney, liver,
and heart transplantation have been confirmed.
Proposed transmission of Trachipleistophora hominis in humans
https://doi.org/10.3389/fcimb.2022.924007
Human microsporidiosis
https://doi.org/10.1128/microbiolspec.FUNK-0018-2016
• examined material: stool, urine, BAL, biopsy material, autopsy material
• detection of spores by light microscopy:
✓ chromotrope based staining - Gram, Ziehl-Nielsen
✓ chemofluorescent staining - Calcofluor White M2R, Uvitex
• serological test for antibody detection (IFAT, ELISA)
• transmission electron microscopy
• PCR
Diagnosis of microsporidiosis
Cultivation of microsporidia
Pathology and diagnosis of microsporidiosis
https://doi.org/10.1128/CMR.00010-20
Clinical images from patients with microsporidiosis. A) Encephalitozoon hellem keratoconjunctivitis demonstrating punctate
corneal lesions (white arrows). B) Endoscopy of jejunal mucosa of a patient with gastrointestinal microsporidiosis due to E.
bieneusi demonstrating fusion of the villi. C) Endoscopic retrograde cholangiogram (ERCP) of a patient with HIV infection (AIDS)
and sclerosing cholangitis due to E. bieneusi infection. ERCP demonstrates diffuse dilations of the common bile duct with irregular
walls, plus areas of narrowing and dilation (arrows) of the intrahepatic bile ducts.
Pathology and diagnosis of microsporidiosis
https://doi.org/10.1128/CMR.00010-20
Microsporidia in stool, urine, and other clinical
specimens. A-B) Chromotrope staining of stool
specimens from patients with AIDS and diarrhoea
demonstrating spores of Enterocytozoon bieneusi
measuring 0.7-1 x 1.1-1.6 μm. Spores have a pink
to reddish hue with this stain. Stained spores can
have a safety pin appearance as well. C)
Conjunctival scraping from a patient with E.
hellem keratoconjunctivitis stained with
chemifluorescent brightening agent (staining the
chitin) demonstrating microsporidian spores. D)
Stool specimen from a patient with AIDS and E.
intestinalis infection stained with a quick hot
Gramchromotrope staining demonstrating spores
of 1-1.2 x 2-2.5 μm. Spores have a violet hue with
this stain. E) Chromotrope staining of urine
sediment from a patient with HIV infection who
had a disseminated E. cuniculi infection
demonstrating pink-to red-coloured spores that
are 1.0 to 1.5 by 2.0 to 3.0mm. F) Methylene blueazure
II-fuchsin staining of a touch preparation of
an intestinal biopsy specimen (obtained by
endoscopy) from a patient with intestinal E.
bieneusi infection demonstrating intracellular
spores.
Pathology and diagnosis of microsporidiosis
Biopsy specimens from patients with
microsporidiosis. A) Intestinal biopsy
specimen from patient with Encephalitozoon
intestinalis (arrow) infection. Methylene
blueazure II-fuchsin stain. B) Muscle tissue from
a patient with rheumatoid arthritis treated with
antibody to TNF-a demonstrating Anncaliia
algerae (arrow) myositis. HE. C) Liver biopsy
specimen from an immunodeficient mouse
infected with Encephalitozoon cuniculi (arrows).
Chromotrope stain. D) Renal biopsy revealing
Encephalitozoon hellem (black spores) within
lumen of renal tubule. Steiner stain. E) Brain
tissue from a rabbit with torticollis,
demonstrating a microgranuloma with central
necrosis due to Encephalitozoon cuniculi
infection. No spores are seen in this image. HE.
F) Intestinal villus biopsy specimen from a
patient with AIDS and Enterocytozoon bieneusi
infection (arrow). Methylene blue-azure II-
fuchsin.
https://doi.org/10.1128/CMR.00010-20
Pathology and diagnosis of microsporidiosis
https://doi.org/10.1128/CMR.00010-20
TEM of biopsy specimens from patients with microsporidiosis. A) Duodenal epithelium from a patient with AIDS and
Enterocytozoon bieneusi infection demonstrating proliferating stages (P) and late sporogonial plasmodia (Sp). The arrow points
to a sloughing enterocyte containing mature spores. B) Intestinal biopsy specimen from a patient with AIDS and E. intestinalis
infection demonstrating spores within vacuoles containing a fibrillar matrix. C) E. bieneusi spore demonstrating the characteristic
finding of two rows of three cross sections of the polar tube (arrow). D) Muscle biopsy specimen from a patient with rheumatoid
arthritis on an anti-TNF-a antibody who presented with myositis. Proliferative forms are seen in the biopsy specimen with a
characteristic diplokaryotic nucleus (N). E) Conjunctival biopsy specimen from a patient with keratoconjunctivitis due to E. hellem
demonstrating characteristic spores (arrowheads) with a single row of six cross sections of polar tube.
The overall prevalence of microsporidia in countries worldwide is indicated by colour depth on a world map. The outer and inner pie
charts show the overall infected hosts and hosts infected by different microsporidia in each country, respectively. The number on the
outer and inner pie charts represents the number of positive samples of microsporidia detected in different hosts and pathogens.
Global geographic prevalence of microsporidia
https://doi.org/10.1186/s13071-021-04700-x
Thank you for your attention ☺
Lectures
✓ Introduction: BPP 2022 I
✓ Euglenozoa (Excavata): BPP 2022 II
✓ Fornicata / Preaxostyla / Parabasala (Excavata): BPP 2022 III
✓ Apicomplexa I (SAR): BPP 2022 IV
✓ Apicomplexa II (SAR): BPP 2022 V
✓ Amoebae (Excavata, Amoebozoa): BPP 2022 VI
✓ Ciliophora, Opalinata (SAR): BPP 2022 VII
✓ Pneumocystis (Opisthokonta, Fungi): BPP 2022 VIII
✓ Microsporidia (Opisthokonta, Fungi): BPP 2022 IX
 Myxozoa (Opisthokonta, Animalia): BPP 2022 X