Microsporidia are single-celled, obligate intracellular, eukaryotic parasites which infect a wide range of invertebrate and vertebrate hosts (Canning & Lom 1986; Lom & Dykova 1992; Desportes-Livage 2000 and Shaw & Kent 1999). Amongst their hosts are a range of animal species such as rodents, rabbits, primates (including humans) but the most common hosts are insects and fish.
Microsporidia belong to the phylum Microspora which are characterised by the production of infective spores which possess an elaborate extrusion apparatus (Weiss 2001).
Identification of microsporidia up to the genus level is based on their ultrastructural characteristics and electron microscopy (EM) is considered the gold standard technique for species identification (Lom and Dykova 1992).
Discovery and classification
Microsporidia were discovered in 1857 and over 1200 species are known to date (Shaw & Kent 1999). 156 species infect fish and these fall within 14 genera (Table 1) (reviewed in Lom 2002; Lom & Nilsen 2003).
Originally, Microsporidia were considered to be protozoa but they have been, not far long ago, reclassified as fungi in the general phylum Microsporidia (Keeling & Fast 2002 and Sprague & Becnel 1998).
Possession of a chitin spore wall by microsporidia potentially links them to fungi. They are considered to be true eukaryotes because they have a membrane-bound nucleus, intra-cytoplasmic membrane system and chromosome separation on mitotic spindles. However, they have a less complex and smaller genome than other eukaryotes (Garcia 2002; Keeling 2009).
Microsporidia in Fish
Fish become infected with microsporidia when they ingest spores from other infected fish, that is to say, by direct per oral transmission (Dykova 1995). The parasites then invade the host cell’s cytoplasm and eventually displace the nucleus and other organelles during their course of development. The cytoplasm is reduced to a thin layer around the parasites. The host cell surrounds the parasites with mitochondria too, from which the latter obtains energy. Formation of large hypertrophic cells known as xenomas (Fig. 1) ensues (Lom & Nilsen 2003). These cells can reach sizes of 400 – 500 µm and can be seen grossly as white cysts (Matthews & Matthews 1980 and Ralphs & Matthews 1986).
Xenomas, cyst-like structures, comprise the hypertrophic host cell contents together with Microsporidia at multiple stages of development (Lom & Dykova 2005). A symbiotic relationship develops between the parasite and host, which exhibit physiological integration. This helps the host to confine the parasites in only the infected cells, thereby limiting their spread to other cells and tissues. The parasites are provided with optimal growth conditions reminiscent of a culture tube in addition to protection from host immune attacks, since they are masked with host component (Lom & Dykova 1992).
Figure 1: Xenomas in the body cavity of the golden orfe.
Figure 1: Large hypertrophic cells (xenomas) in the body cavity of the golden orfe. (Picture provided by Dr. R. Kirk, Kingston University London).
Xenomas cause wasting of fish tissue leading to huge losses in catch value as well as reduced growth rates. Some cause serious lesions which result into the demise of infected fish when they destroy the host cells (Lom & Dykova 1992). Microsporidiosis is considered to be a chronic lethal infection by Shaw and Kent 1999.
Dykova and Lom (1978) stated that not all fish Microsporidia species cause xenomas. 9 out of the 14 genera of fish Microsporidia cause xenomas in table (1).
Table 1: Microsporidia genera and their fish hosts.
GlugeaStickleback, pond smelt, flounder, ayu
HeterosporisJapanese eel, Jewel cichlid
KabatanaChinook, Masu, sockeye & pink salmon; rainbow trout
LomaAtlantic cod; haddock; pink, Coho, red, dog, sockeye & Chinook salmon; rainbow trout
MicrofilumLutjanus fulgens (Teleost)
MicrosporidiumCoho, red, dog & Chinook salmon; rainbow trout; nilem; shiped catfish
NucleosporaSalmon, Atlantic halibut
OvipleistophoraGolden shiner, European chub
PleistophoraSalmon, bream, pacific cod, turbot, roach
Table 1. The 14 known genera of Microsporidia that infect fish (reviewed in Lom 2002; Lom & Nilsen 2003), of which 9 (shown in red) cause xenomas in their hosts (reviewed in Lom 2002; Lom & Dykova 2005).
Microsporidia undergo a complicated life cycle which involves merogony or schizogony and sporogony. Merogony is the proliferative stage during which numerous parasites are produced by binary or multiple fission whereas sporogony involves the production of mature spores. Both stages take place inside the host cell (Lom & Dykova 1992).
Initially, the sporoplasm is extruded by breakage and eversion of the polar tube through the thinnest apical part of the spore, with a build up of high pressure inside the spore being the driving force behind this mechanism. The polar tube pierces the host cell and literally injects the sporoplasm into it. This marks the start of development within the host (Lom & Dykova 1992).
Generally Microsporidia are small organisms and the largest developmental stages measure up to 50 µm (Dykova 1995).
The spore (Fig. 2)is the infectious stage of Microsporidia, and the only stage viable outside of the host, due to possession of a tough outer wall. The spore wall is solid, occurs in one piece and consists of a thin outer glycoproteinous exospore and a thick inner chitin endospore. Spores occur in different shapes such as rod-shaped and spherical but oval or ellipsoidal are the most common. They measure between 3 and 10 µm in length and possess an elaborate hatching apparatus. The polaroplast, polar filament and posterior vacuole are the three most important parts of the spore involved in infection (Lom & Dykova 1992; Dykova 1995). The spore has a very intricate morphology (Canning & Lom 1986), but the most conspicuous part is the polar tube which extends obliquely from the anchoring disc at the anterior end to the posterior end of the spore, where it coils beneath the spore wall. A single or diplokaryon nucleus occurs in the sporoplasm between the polaroplast and the posterior vacuole (Dykova 1995).
Figure 2: A generalised diagram of a Microsporidia spore.
Figure 2: Diagram of an oval-shaped spore showing a diprokaryon nucleus, the wall and other components involved in infection such as the polar tube and posterior vacuole (picture obtained from…).
Microsporidia have a huge impact on aquaculture since they cause severe diseases in fish, resulting in a reduction in the growth of fish stocks and productivity in the fish farming sector (Lom & Dykova 1992; Dykova 1995). The economic significance of Microsporidia has therefore promoted extensive research into the pathology that they cause to their hosts (Lom 2002).
This research project was conducted with the aim of investigating the nature and the extent of damage caused by xenomas in fish and the host’s response to infection (microsporidiosis) using histological techniques and light microscopy, as well as identifying the Microsporidia species infecting the golden orfe. Since there is limited research on Microsporidia, this would help provide more information regarding their pathology which could consequently be vital in controlling the impact they have on fish farming
Materials and Methods
Fish (golden orfe) obtained from a pond at Burton Bradstock in Dorset were dissected to remove gill and body cavity tissues infected with Microsporidia. These were fixed in 10% neutral buffered formalin for 48 hours and stored in industrial methylated spirit (IMS). The gill tissue was then decalcified overnight in Osteosoft (Merck, Germany) and again stored in 70% IMS. The tissues were then dehydrated in a series of alcohol of ascending concentrations, embedded in wax and serially sectioned at 5 to 10 µm (appendix 1). Staining with Masson’s tricrhome (appendix 2) was done before examining the sections using light microscopy (phase contrast).
Image analysis technique was used to critically examine the stained sections in order to identify and evaluate the histopathological changes caused by Microsporidia.
Initial macroscopic examination of the fish revealed compression of the organs of the body cavity by the xenomas; cyst-like structures which occur as circular or ovoid lumps (figure 1). Histological examination of the stained serial sections of the body cavity tissue revealed the localisation of the cysts (xenomas) mostly with in the subepithelial connective tissue of the gut (figure 3a). In many sections, the cysts were seen to occur in large numbers and in close proximity with one another (figure 3b). This was an indication that the body cavity tissues, and the fish in general, were heavily infected. The cysts appeared to be intact and were surrounded by a continuous membrane (figures 3b & 4). This showed confinement of Microsporidia parasites within the infected hypertrophied cell.
The xenomas contained large numbers of parasites which appeared to be at different stages of development (figure 4), for example meronts and spores. The merogonial stages of development were seen as whitish round or amorphous masses within the cysts. However, individual meronts could not be discerned (figure 4). The Microsporidia spores, which stained deep red with Masson’s trichrome (Joseph et al. 2006), were evident within the entire xenoma (figure 4). Unfortunately, the internal morphology of the spores could not be examined with light microscopy.
Host cell organelles such as nuclei were not observed, as these were probably displaced by the developing parasites. Secondary xenomas, cysts developing within another cyst, were also seen and these too contained developing parasites (figure 6). This was an indication that the xenomas were developing or mature.
Fibroblasts were observed within the connective tissue surrounding the xenomas (figure 5). These, presumably, played a role in laying down blue-staining collagen fibres around the xenomas as part of the inflammatory response from the host towards the parasites, which is known as a granulomatous response (figure 7).
Unfortunately, no xenomas were found within the gills on histological examination, even though cysts were observed before the tissues were removed from the fish. This could have been due to destruction of these cysts, which were probably young, during surgical removal and/or chemical processing of the tissues. Telangiectasis (gill lamellar dilations) was observed in some slides, on closer examination, but this was not to be mistaken for xenomas.
These observations were also made by other authors such as Abdel-Ghaffar et al. (2011); Gandhi, Locatelli & Feist (1995) and Peyghan et al. (2009)
Microsporidia infect a variety of marine and fresh water fish. This study used the fresh water ornamental fish, the golden orfe (Leuciscus idus), to examine the histopathological effect that Microsporidia have on fish in general. However, there is not much research that has been carried out regarding the histopathological effect that microsporidia have on golden orfe.
The xenomas (cyst-like structures) observed grossly within the body cavity of the fish were generally spherical, as was noted by Matos et al (2003) too. They presumably exerted pressure onto the organs involved in feeding such as the gut, liver and intestines, hence impairing their vital functions (Ralphs & Matthews 1986).
This not only led to morphological modifications but also functional failure due to thickening of the gut wall and eventual occlusion of the lumen. This may be the limiting factor which affects growth in the infected fish and the indirect cause of their death (Dykova 1995).
Occurrence of xenomas in large numbers and close proximity to each other (as seen in figure 3b) was suggestive of a heavy infection, which resulted in the death of the fish. This is in agreement with Lom & Dykova (1992) who stated that Microsporidia provoke severe disease in wild and farmed fish populations causing major losses.
According to the observations made in this study, parasites at different stages of development occurred within the xenomas. Merogonial stages which appeared as white masses and deep red staining spores were seen inside the xenomas which were surrounded by a continuous membrane. The parasites were confined within these cyst-like structures inside infected cells and this limited their spread to other tissues within the host. This mechanism is used by the host to control the infection. However, the parasites use it too as a means of evading recognition and destruction by the host’s immune cells such as macrophages, as stated by Lom & Dykova (1992). The parasites replace the cell organelles and cause hypertrophy of the infected cell, before destroying it. The host cell type is difficult to recognise following transformation into a xenoma (Dykova 1995). The xenoma and its components are morphologically and physiologically integrated to from a separate entity which develops at the expense of the host (Lom & Dykova 2005).
The observation of a granulomatous reaction, a process achieved by fibroblasts laying down collagen fibres around the xenoma wall, is a type of host response towards the parasite and is in agreement with observations made by Lom and Dykova (1992, 2005) and Shaw & Kent (1999).
One of the demerits of this study was that the morphology of the spores was vague and their internal structures could not be examined well enough, due to the low resolving power of the light microscope. This rendered identification of the Microsporidia species infecting the fish studied in this study impossible. Lom & Dykova (1992) stated that identification of Microsporidia was based on the ultrastructural features of the spores and/or the characteristic cell structure of the developmental stages, for example the polar tube. Electron microscopy is the gold-standard method to use in species identification of Microsporidia. In addition to this, Masson’s trichrome stain…
However, this study showed that Microsporidia infection in fish led to hypertrophy of infected cells, with the formation of the xenomas. These observations concur with those of Lom & Dykova (1992); Lom & Nilsen (2003) and other researchers, for example, Abdel-Ghaffar et al. (2011); Gandhi, Locatelli & Feist (1995) and Peyghan et al. (2009). From the commercial point of view, Microsporidia are considered parasites of significant importance by Lom & Dykova (1992) and Dykova (1995) who stated that microsporidia infection reduced the growth of fish stocks which resulted in losses within the fish sector.
Currently, not much research has been done in regard to the histopathological effect that Microsporidia cause to their fish hosts, and other animals in general and therefore a lot more remains to be learnt about these pathogens. Fields that require much more extensive research include the mechanisms by which xenomas are formed and the nature of the xenomas, how the parasites evade the host immune system, invasion of host cells by parasites using their extrusion apparatus, host response towards infection, not to mention but a few. Lom (2002) proposed that a detailed knowledge of fish Microsporidia morphology and their taxonomy would greatly facilitate species determination.
This would help provide vital information that would be used to design and produce drugs in order to reduce the impact that Microsporidia have on agriculture and aquaculture.
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