Chapter One - Enterocytozoon bieneusi of animals—With an ‘Australian twist’

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Abstract

Enterocytozoon bieneusi is a microsporidian microorganism that causes intestinal disease in animals including humans. E. bieneusi is an obligate intracellular pathogen, typically causing severe or chronic diarrhoea, malabsorption and/or wasting. Currently, E. bieneusi is recognised as a fungus, although its exact classification remains contentious. The transmission of E. bieneusi can occur from person to person and/or animals to people. Transmission is usually via the faecal-oral route through E. bieneusi spore-contaminated water, environment or food, or direct contact with infected individuals. Enterocytozoon bieneusi genotypes are usually identified and classified by PCR-based sequencing of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA. To date, ~ 600 distinct genotypes of E. bieneusi have been recorded in ~ 170 species of animals, including various orders of mammals and reptiles as well as insects in > 40 countries. Moreover, E. bieneusi has also been found in recreational water, irrigation water, and treated raw- and waste-waters. Although many studies have been conducted on the epidemiology of E. bieneusi, prevalence surveys of animals and humans are scant in some countries, such as Australia, and transmission routes of individual genotypes and related risk factors are poorly understood. This article/chapter reviews aspects of the taxonomy, biology and epidemiology of E. bieneusi; the diagnosis, treatment and prevention of microsporidiosis; critically appraises the naming system for E. bieneusi genotypes as well as the phylogenetic relationships of these genotypes; provides new insights into the prevalence and genetic composition of E. bieneusi populations in animals in parts of Australia using molecular epidemiological tools; and proposes some areas for future research in the E. bieneusi/microsporidiosis field.

Introduction

Microsporidia are eukaryotic microorganisms represented by 187 genera and > 1300 species (Vávra and Lukeš, 2013). More than 14 species are recorded from humans, of which Enterocytozoon bieneusi is the commonest microsporidian (Anane and Attouchi, 2010; Mathis et al., 2005; Santín-Durán, 2015). E. bieneusi is an obligate intracellular pathogen, usually infecting intestinal epithelial cells and typically causing severe or chronic diarrhoea, malabsorption and/or wasting (Didier and Weiss, 2006; Kotler and Orenstein, 1998). E. bieneusi is presently recognised as a fungus, although the exact classification remains contentious (Adl et al., 2005; Vávra and Lukeš, 2013), and there is some evidence suggesting that the Microsporidia is a phylum within the superphylum Opisthosporida—which is a sister group to the Fungi (Karpov et al., 2014).

The transmission of E. bieneusi can occur from person to person (Gumbo et al., 1999; Leelayoova et al., 2005) and/or animals to people (Cama et al., 2007; Kondova et al., 1998; Tzipori et al., 1997). Transmission is usually via the faecal-oral route through E. bieneusi spore-contaminated water or enviroknment (Ayed et al., 2012; Galván et al., 2013), food (Decraene et al., 2012; Jędrzejewski et al., 2007) or direct contact with infected individuals (Leelayoova et al., 2005; Pagornrat et al., 2009). Interestingly, a study also identified the presence of E. bieneusi in the respiratory tracts of HIV/AIDS patients (Botterel et al., 2002); however, whether air is an actual transmission vehicle for E. bieneusi still requires verification, although Graczyk et al. (2007) did detect E. bieneusi spores in air samples.

Enterocytozoon bieneusi genotypes are usually identified and classified by PCR-based sequencing of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA (Santín-Durán, 2015). To date, ~ 600 distinct genotypes of E. bieneusi have been recorded in ~ 170 species of animals, including various orders of mammals (Artiodactyla, Carnivora, Diprotodontia, Lagomorpha, Perissodactyla, Primates, Rodentia and Struthioniformes), birds (Anseriformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Psittaciformes and Struthioniformes) and reptiles (Squamata) as well as insects (Diptera) in > 40 countries (Supplementary Table 1). Moreover, E. bieneusi has also been found in water samples (recreational water, irrigation water, and treated raw- and waste-waters) (reviewed by Fayer and Santín-Durán, 2014). Thus, the United States Environmental Protection Agency (EPA) listed this microsporidian in its Contaminant Candidate List of Waterborne Organisms (EPA, 1998), and the National Institute of Allergy and Infectious Diseases (NIAID) classified E. bieneusi as a Category B Priority Pathogen (Didier and Weiss, 2006). Although many studies have been conducted on the epidemiology of E. bieneusi (reviewed by Fayer and Santín-Durán, 2014), prevalence surveys of animals and humans are scant in some countries, such as Australia, and transmission routes of individual genotypes and related risk factors are poorly understood.

The purpose of this article/chapter was to: (i) review the current state of knowledge of the taxonomy and biology of E. bieneusi, the pathogenesis of microsporidiosis and aspects of the treatment and prevention of this disease; (ii) summarise available diagnostic and analytical methods, and evaluate advantages and limitations of these techniques; (iii) review the molecular epidemiology of E. bieneusi in animals and humans and risk factors associated with infection and prevalence of E. bieneusi; (iv) improve the naming system of E. bieneusi genotypes and reassess the phylogenetic relationships of these genotypes; (ix) re-examine the genetic make-up of E. bieneusi populations in animals and humans in parts of Australia in a broader context; and (v) make conclusions regarding the epidemiology of E. bieneusi/microsporidiosis and propose future research directions.

Section snippets

Taxonomy

Microsporidia is a diverse phylum of spore-forming microorganisms, first identified in 1857 (Nägeli, 1857). The classification of the Microsporidia has been controversial for decades since its discovery. Microsporidia were classified as schizomycete fungi in 1857, as ‘sporozoan’ protists in 1882 (Balbiani, 1882), and then as a subgroup of the Cnidosporidia within the Sporozoa (now the Apicomplexa) in 1901—a scheme that was followed for > 100 years (Keeling, 2009). Subsequently, in 1983, a new

Life cycle and biology

Microsporidia are obligate intracellular, spore-forming, eukaryotic microorganisms (Stentiford et al., 2016). Generally, their life cycle involves the infectious phase (the spore), and within the host cells there are the proliferative and sporogonic phases (Vávra and Larsson, 2014). The spore is the only life cycle stage that survives in the environment (Vávra and Larsson, 2014).

A typical microsporidian spore (Fig. 1; cf. Stentiford et al., 2016) is bound by both an outer exospore and an

Survival of microsporidians in the environment

Although there has been no research directly on the survival of E. bieneusi spores in the environment, there have been studies on related microsporidians. The spores of microsporidia are very resistant in the environment and can survive for months to years, depending on conditions (reviewed by Fayer, 2015). The impact of environmental factors, such as solar radiation, temperature and humidity, on the viability of microsporidian spores has been studied for some species including Encephalitozoon

Pathogenesis of microsporidiosis due to E. bieneusi

The acquisition of E. bieneusi infections is via the faecal-oral route, either through E. bieneusi spore-contaminated water/food, or direct contact with infected individuals or their faeces (Santín and Fayer, 2009a). Typically, E. bieneusi infection is usually limited to the small intestine (duodenum and jejunum) (Shadduck and Orenstein, 1993), but respiratory tract infections can occur (e.g. Botterel et al., 2002; Graczyk et al., 2007). At a microscopic level, villus atrophy and crypt

Treatment and prevention

Currently, the chemotherapy of microsporidiosis relies on the use of albendazole, fumagillin or nitazoxanide, which appear to be the most effective compounds against microsporidian pathogens. Alternative treatments are under evaluation (Costa and Weiss, 2000; Didier et al., 2004, Didier et al., 2005; Groß, 2003).

Albendazole, a derivative of benzimidazole, is widely used as an antiparasitic drug (Groß, 2003; Horton, 2002). The target of benzimidazole is the β-subunit of tubulin, which is a major

Microsporidian genomics

Insights into microsporidian genomes are critical for understanding their molecular biology and biochemistry, and could, at an applied level, underpin the development of new interventions.

More than 27 microsporidian genomes have been reported to date. The genome of Encephalitozoon cuniculi was the first to be sequenced; it is ~ 2.9 Mb in size (Biderre et al., 1999; Corradi et al., 2010; Dia et al., 2007; Katinka et al., 2001), and the genomes of other taxa range in size from 2.5 to 25 Mb (Keeling

Detection and identification of microsporidia

The methods for the detection of microsporidia have evolved from traditional microscopic diagnosis to serological tests and molecular-based (see 8.1 Microscopic detection, 8.2 Serological/immunological detection techniques, 8.3 Molecular techniques). Traditional methods used include the direct detection/identification of microsporidial stages by transmission electron microscopy (TEM) and/or light microscopy. Serological tests include the enzyme-linked immunosorbent assay (ELISA),

Molecular epidemiology of E. bieneusi

E. bieneusi is widely distributed and has been detected in as many as 42 countries (Supplementary Table 1). This pathogen can infect humans and a broad range of animals, including birds (i.e. Anseriformes, Columbiformes, Falconiformes, Galliformes, Gruiformes, Passeriformes, Psittaciformes and Struthioniformes), mammals (i.e. Artiodactyla, Carnivora, Chiroptera, Diprotodontia, Lagomorpha, Perissodactyla, Primates and Rodentia), Insecta (i.e. Diptera) and Reptilia (i.e. Squamata) (Supplementary

Knowledge gaps regarding E. bieneusi in animals

An appraisal of the literature on E. bieneusi and microsporidiosis research (Section 9) has revealed advances in this field, but it also identified some significant knowledge gaps. Clearly, microsporidiosis is a disease of both animal and human importance worldwide. Epidemiological studies in a number of developed and developing countries around the world have recorded, at varying prevalences, genotypes of E. bieneusi that occur either in humans or in animals other than humans, and some that

Recent insights into the prevalence and genetic make-up of E. bieneusi populations in animals in parts of Australia

There was a clear need for epidemiological studies of E. bieneusi and microsporidiosis in Australia. Based on published literature and in order to enable comparative analyses, we elected to employ nested-PCR-based sequencing of ITS and/or SSU of nuclear ribosomal DNA for the detection and characterisation of genotypes of E. bieneusi in faecal samples from wild and domesticated animals and humans in parts of Australia.

A need for precise naming of E. bieneusi genotypes—Issues and recommendations

Prior to 2005, < 100 E. bieneusi genotypes were recognised (Drosten et al., 2005), such that a well-defined system for naming E. bieneusi genotypes was not essential. However, the number of genotypes has increased to hundreds, which has led to some issues and confusion around the naming of E. bieneusi genotypes, including: (1) Multiple genotype names being linked to the same ITS sequence; (2) SSU and/or LSU sequences being used to define new E. bieneusi genotypes.

In order to address these

Early proposals regarding groupings of E. bieneusi genotypes based on ITS sequence data to identify host affiliations

In order to understand the current state of E. bieneusi genotype groupings, it is useful to have a brief review of its history. When the first few ITS sequences were aligned it was apparent that the number of nucleotide differences present could be useful for defining unique ‘strains’ which could potentially be associated with varying levels of pathogenicity (Rinder et al., 1997). Eventually, as more and more sequences accumulated in GenBank from a broad array of human, agricultural and

A critical re-appraisal of phylogenetic relationships of E. bieneusi genotypes

Here, we offer an alternative grouping-scheme utilising three distinct phylogenetic methods, taking into account statistical support (bootstraps and posterior probabilities), branch lengths, topology and/or historical groupings. We recognise that ITS might be a fallible marker for this purpose, but given that it is presently recognised as the ‘best’ genetic marker for E. bieneusi, we attempt to make sense of the current grouping scheme and make suggestions for an alternative scheme that can be

Need for large-scale temporal/longitudinal and spatial epidemiological studies of E. bieneusi and other enteric pathogens

Many studies of E. bieneusi have been conducted worldwide and near 600 E. bieneusi genotypes have been recorded to date; > 500 of these genotypes appear to show zoonotic potential based on comparative genetic and phylogenetic analyses in peer-reviewed publications (see Supplementary Table 1; Supplementary Fig. 1; Fig. 2, Fig. 3). To date (7th April 2019), 52 E. bieneusi genotypes have been identified in both animals and humans, many genotypes of which have been commonly found in humans and a

Concluding remarks

Recent molecular epidemiological investigations of E. bieneusi from animals and humans from different geographic locations in Australia have revealed that wildlife (deer, marsupials and red foxes), farmed cattle and alpacas, as well as domestic cats and dogs, represent host reservoirs of E. bieneusi potentially capable of infecting humans via water, the environment or direct contact. In water catchments, animals carrying E. bieneusi and water contaminated by E. bieneusi spores may act as

Acknowledgements

Y.Z. was the recipient of scholarships from the Chinese Scholarship Council (CSC) and the University of Melbourne. Research was supported by Melbourne Water Corporation and the Australian Research Council (grant number LP160101299; RBG and A.V.K.). The authors are very grateful to an anonymous reviewer for their very objective and constructive suggestions/comments on the submitted manuscript.

Supplementary materials

Supplementary figures and tables can be accessed via the online version at https://doi.org/10.1016/bs.apar.2020.10.001.

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