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The opportunistic pathogen Encephalitozoon cuniculi in wild living Murinae and Arvicolinae in Central Europe
... Although each strain has a preferential host species, it is known that E. cuniculi has a low host specificity. For example, genotype III ("dog strain") was detected also in wild small rodents [10,11]. The main host of E. hellem are humans, but it has been found also in birds. ...
... In other studies from Central Europe, different tissue samples were used for detection of Encephalitozoon spp. For example, Perec-Matysiak et al. [11] examined by nested PCR spleen and faecal samples of wild small mammals trapped in nature reserves and suburban recreational areas in Slovakia, Poland, and the Czech Republic. They found Encephalitozoon spp. in 15% of animals, which was only slightly higher than the positivity in our study. ...
... Based on molecular techniques, the presence of 4 different genotypes of E. cuniculi (genotypes I-IV) was previously confirmed [8,9]. Genotyping analysis of positive samples from our study showed that 69% (22/32) were E. cuniculi genotype II and 3% (1/32) E. hellem genotype 1 A. E. cuniculi genotype II was also proved in 93% of wild rodents from Slovakia, Poland, and the Czech Republic, while genotypes I and III were proved only in 1.5% and 6% of animals, respectively [11]. In contrast, Sak et al. [17] proved genotype I to be more frequent (58%) compared to genotype II (42%) in wild small mammals from the Czech Republic. ...
Purpose
Parasites of genus Encephalitozoon are well known pathogens of domestic animals however less attention was paid to its spread among wildlife that can play an important role of reservoir of infection. The aim of the study was to conduct molecular detection and genotype characterization of Encephalitozoon spp. in wild small mammals trapped in localities both near to and at a large distance from residential areas.
Methods
In total, 300 wild small mammals (274 Rodentia and 26 Eulipotyphla) were trapped in 41 localities of the Czech Republic and tested by nested PCR for Encephalitozoon spp.
Results
The DNA of Encephalitozoon spp. was proved in tissues (brain or liver) of 11% (32/300) of animals. There was a statistically significant difference ( p < 0.001) in positivity among animal species with the most infected species Micromys minutus (50%, 4/8) and Myodes glareolus (17%, 9/53). There was also statistically significant difference ( p < 0.001) between localities with the higher positivity (29%, 12/42) in localities near to residential areas, compared to localities with a large distance from residential areas (8%, 20/258). Sex and age of wild small mammals did not have effect on their positivity. Genotyping analysis revealed E. cuniculi genotype II in 22 samples and E. hellem genotype 1 A in one sample.
Conclusion
This study brings new information on the molecular characterization of Encephalitozoon spp. isolated from wild small mammals trapped in two different areas (localities in near to residential areas and localities with a large distance from residential areas).
... However, although each strain has a preferential host species, in practice, it is currently known that E. cuniculi has a low host specificity. For example, genotype I was also detected in horses (Wagnerová et al. 2012) and gorillas and chimpanzees (Sak et al. 2011b), genotype II in blue foxes and cats (Benz et al. 2011), genotype III in wild small rodents (Hofmannová et al. 2014;Perec-Matysiak et al. 2019) and pigs (Reetz et al. 2009), and genotype IV in dogs (Nell et al. 2015). In addition, in humans, apart from strain type IV, the other three strains (I, II, and III) were also diagnosed, proving its zoonotic potential (Sak et al. 2011a). ...
... In addition to organs and tissues, nested or real-time PCR has been also carried out in animals and humans using samples from feces Weber et al. 1997;Fournier et al. 2000;Lobo et al. 2003;Sak et al. 2011a;Sak et al. 2011b;Perec-Matysiak et al. 2019), urine Fournier et al. 2000;Baneux and Pognan 2003;Csokai et al. 2009;Hein et al. 2014;Zietek et al. 2014;Abu-Akkada et al. 2015a;Boer et al. 2021), and cerebrospinal fluid (Csokai et al. 2009;Hein et al. 2014;Jeklova et al. 2020), allowing the diagnosis of the disease in clinical cases and the identification of infected animals excreting spores in epidemiological prevalence surveys. The PCR technique in urine (Baneux and Pognan 2003;Csokai et al. 2009) and in cerebrospinal fluid (Csokai et al. 2009;Jeklova et al. 2020) has been associated with low sensitivity, which can lead to false negative results. ...
... Rodents have often been used to study E. cuniculi by allowing, in an experimental context, the evaluation of the parasite dissemination within the host's body (Kotkova et al. 2013;Sak et al. 2017), the possibility of vertical transmission (Kotková et al. 2018), and the therapeutic response to drugs (Kotkova et al. 2013;Lallo et al. 2013;Sak et al. 2017Sak et al. , 2020. Furthermore, it is known that wild rodents are capable of being infected by this agent, functioning as potential "reservoirs" and sources of transmission for predatory species, other domestic animals, and even humans, as described in epidemiological surveys (Hersteinsson et al. 1993;Meredith et al. 2015;Perec-Matysiak et al. 2019) and case reports (Hofmannová et al. 2014;Kitz et al. 2018). For example, the overall prevalence of E. cuniculi in wild rodents was 5.31% in the UK (Meredith et al. 2015) and 15% in Central Europe (Perec-Matysiak et al. 2019), according to epidemiological data obtained through serological and molecular methods, respectively. ...
Encephalitozoon cuniculi is a microsporidian parasite mostly associated with its natural host, the rabbit (Oryctolagus cuniculus). However, other animals can be infected, like other mammals, birds, and even humans. Although it usually causes subclinical infection, it can also lead to encephalitozoonosis, a clinical disease characterized by neurological, ocular, and/or renal signs that can be even fatal, especially in immunocompromised individuals. Therefore, this multidisciplinary review contributes with updated information about the E. cuniculi, deepening in its molecular and genetic characterization, its mechanisms of infection and transmission, and its prevalence among different species and geographic locations, in a One Health perspective. Recent information about the diagnostic and therapeutic approach in the main host species and the prophylaxis and infection control measures currently suggested are also discussed.
... In both provinces, only genotype TURK1B of E. hellem was found (Table 3). In this study, the sequences of 48 positive samples isolated from diarrheic pigs with genotype III of E. cuniculi were 100% identical to the sequence (KX189630) from Apodemus agrarius, a striped field mouse in a study from Poland [36]. At the same time, four samples with genotype III sequences were identical and had the highest degree of similarity (99.5-98.97%) ...
... A genotype III sequence found in one diarrheic pig sample in this study had 99.65% homology to a genotype III sequence (KJ577583) isolate from Lagurus lagurus (steppe lemming) in the Czech Republic [37] (Table 4, Figure S1(B)). In addition, eight positive E. cuniculi genotype II samples showed complete sequence identity with genotype II (accession No. KX189632) from yellow-necked mouse [Apodemus flavicollis] in Poland [36]. One sample of E. cuniculi genotype II had 98.92% homology with isolate from yellow-necked mouse (accession No. KX189632) ( Table 4, Figure S1(A)). ...
Background
Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp., and Giardia duodenalis (G. intestinalis) are enteric pathogens that cause diarrhea in pigs. This study aimed to determine the prevalence of these enteric parasites and their coinfection with E. bieneusi in diarrheic pigs in Southwest China (Chongqing and Sichuan) using nested polymerase chain reaction (nPCR) based methods.
Results
A total of 514 fecal samples were collected from diarrheic pigs from 14 pig farms in Chongqing (five farms) and Sichuan (nine farms) Provinces. The prevalence of Encephalitozoon spp., Cryptosporidium spp. and G. duodenalis was 16.14% (83/514), 0% (0/514), and 8.95% (46/514), respectively. Nested PCR revealed 305 mono-infections of E. bieneusi, six of E. cuniculi, two of E. hellem, and nine of G. duodenalis and 106 concurrent infections of E. bieneusi with the other enteric pathogens. No infections of E. intestinalis and Cryptosporidium species were detected. The highest coinfection was detected between E. bieneusi and E. cuniculi (10.5%, 54/514), followed by E. bieneusi and G. duodenalis (5.8%, 30/514) and E. bieneusi and E. hellem (2.9%, 15/514). E. bieneusi was the most frequently detected enteric pathogen, followed by E. cuniculi, G. duodenalis and E. hellem. There was a significant age-related difference in the prevalence of E. cuniculi in fattening pigs (χ² = 15.266, df = 3, P = 0.002) and G. duodenalis in suckling pigs (χ² = 11.92, df = 3, P = 0.008) compared with the other age groups. Sequence analysis of the ITS region of Encephalitozoon species showed two genotypes (II and III) for E. cuniculi and one (TURK1B) for E. hellem. Only G. duodenalis assemblage A was identified in all nested PCR-positive samples. E. bieneusi was found more often than other enteric pathogens.
Conclusions
This study showed that E. bieneusi, Encephalitozoon spp. [E. cuniculi and E. hellem] and G. duodenalis were common enteric parasites in diarrheic pigs in Chongqing and Sichuan Provinces. In case of both mono-infection and coinfection, E. bieneusi was the most common enteric pathogen in diarrheic pigs. Thus, it may be a significant cause of diarrhea in pigs. Precautions should be taken to prevent the spread of these enteric parasites.
... Rodents may also be a reservoir of E. cuniculi for foxes, minks, and cats. Research involving wild rodents in Poland, Czechia, and Slovakia has showed a 15% prevalence in these animals [14][15][16]. E. cuniculi antibodies were found in 0% to 26% of the population of cats, and in 30% of the population of dogs [17][18][19][20][21]. ...
Encephalitozoonosis is a disease caused by E. cuniculi. It is diagnosed primarily in rabbits but is less frequently so in other animal species. E. cuniculi is classified among Microsporidia—fungi frequently found in the environment, that are resistant to numerous external factors. Apart from rabbits, rodents form the next group of animals most exposed to infection with these pathogens. The objective of the study was to analyze the prevalence of E. cuniculi infection in guinea pigs with different clinical disorders. The study included 67 animals with E. cuniculi infection confirmed via real-time PCR. The infected animals most frequently exhibited nervous and urinary system symptoms, as well as issues with vision organs, while several animals were also recorded as having problems with the respiratory system and thyroid gland dysfunction. The study shows that encephalitozoonosis constitutes a significant problem in rodents kept as domestic animals, which in turn may be a source of infection for humans.
... 2,3 While E. cuniculi genotype I has rarely been recorded in asymptomatic birds, rodents, rabbits, dogs, horses, boars, buffalos, great apes, and seldom in humans [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] and E. cuniculi genotype III has been identified only in lemmings, rabbits, birds, swine, dogs, blue foxes, snow leopard, tamarins and squirrel monkeys, and also in humans, causing a variety of pathologies ranging from asymptomatic to fatal infections, 4,[19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] Encephalitozoon cuniculi genotype II has been found in many different species of hosts causing a wide spectrum of pathogenesis. [35][36][37][38][39][40] The recently described genotype IV has been detected in cats, dogs, marmots, and in immunosuppressed humans, 3,41,42 causing disseminated encephalitozoonosis in the latter species. However, contrary to the first three genotypes, genotype IV is not available for in vitro culture and thus for laboratory experiments. ...
Background
Microsporidia of the genus Encephalitozoon are usually associated with severe infections in immunodeficient hosts while, in immunocompetent ones, microsporidiosis produces minimal clinically apparent disease. Despite their microscopic size, microsporidia are capable of causing systemic infection within a few days. However, the mechanisms by which microsporidia reach target tissues during acute infection remain unclear. Out of four genotypes of Encephalitozoon cuniculi, only three are available for experimental studies, with E. cuniculi genotype II being the best characterized.
Methods
In the present study, we tested the association between inflammation induction in immunocompetent and immunodeficient mice and the presence of spores of E. cuniculi genotypes I and III in selected organs using molecular methods and compared the results with previously published data on E. cuniculi genotype II.
Results
We reported the positive connection between inflammation induction and the significant increase of E. cuniculi genotypes I and III occurrence in inflammatory foci in both immunocompetent BALB/c and immunodeficient severe combined immunodeficient (SCID) mice in the acute phase of infection. The induction of inflammation resulted in increased concentration of E. cuniculi of both genotypes in the site of inflammation, as previously reported for E. cuniculi genotype II. Moreover, our study extended the spectrum of differences among E. cuniculi genotypes by the variations in dispersal rate within host bodies after experimentally induced inflammation.
Conclusion
The results imply possible involvement of immune cells serving as vehicles transporting E. cuniculi towards inflammation foci. The elucidation of possible connection with pro-inflammatory immune responses represents an important challenge with implications for human health and the development of therapeutic strategies.
... Kitz et al. [43] described a case of encephalitozoonosis in a group of captive Barbary striped grass mice (Lemniscomys barbarus) in a zoo collection. Perec-Matysiak et al. [44] reported the presence of this microsporidium in wild living rodents from Poland, the Czech Republic and Slovakia, identifying as the most frequent species E. cuniculi genotype II (92.5%), followed by E. cuniculi genotypes I (1.5%) and III (6.0%). ...
The involvement of animals for therapeutic purposes has very ancient roots. To date, it is clear that animal-assisted interventions (AAIs), in addition to ensuring the replacement of missing or deficient affects, improves psychophysiological parameters connected to human health. However, AAI could potentially present risks related to the transmission of infectious agents from animals to humans. Among these microorganisms, E. cuniculi is a microspore which induces pathological effects (fever, headache, nausea, vomiting, diarrhea, breathlessness, respiratory symptoms, and weakness) in both humans and animals. Consequently, an accurate and fast diagnosis of E. cuniculi infection, as well as the identification of new diagnostic approaches, is of fundamental importance. This literature review was carried out to provide an extensive and comprehensive analysis of the most recent diagnostic techniques to prevent and care for E. cuniculi-associated risks in the AAI field.
... The initial database search yielded a total of 1695 records (Fig. 1); finally, 22 papers were eligible for final meta-analysis after duplicate/non-eligible remove [21,22,[26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. Among the 22 eligible studies, six studies had more than one data (Table 1). ...
Background
Microsporidiosis as a zoonotic disease has caused serious health problems in high-risk groups, including immunosuppressed individuals. Among the potential animal reservoirs of microsporidia, rodents play a key role due to close-contact with humans and their dispersion in different environments. Therefore, this systematic review and meta-analysis aimed to assess the global status and genetic diversity of microsporidia infection in different rodents.Methods
The standard protocol of preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines were followed. Scopus, PubMed, Web of Science, and Google Scholar were searched from 1 January 2000 to 15 April 2021. All peer-reviewed original research articles describing the molecular prevalence of microsporidia infection in rodents were included. Inclusion and exclusion criteria were applied. The point estimates and 95% confidence intervals were calculated using a random-effects model. The variance between studies (heterogeneity) were quantified by I2 index.ResultsOf 1695 retrieved studies, 22 articles (including 34 datasets) were included for final meta-analysis. The pooled global molecular prevalence (95% CI) of microsporidia infection in rodents was 14.2% (95% CI 10.9–18.3%). The highest prevalence of microsporidia was found in Apodemus spp. 27.3% (95% CI 15–44.5%). Enterocytozoon bieneusi was the most common pathogen (26/34; 76.47% studies) according to PCR-based methods, and the genotype D as the highest reported genotype (15 studies).Conclusions
The findings of the study showed a relatively high prevalence of microsporidia infection in rodents as a potential animal reservoir for infecting human. Given the relatively high incidence of microsporidiosis, designing strategies for control, and prevention of microsporidia infection in rodents should be recommended.
... While E. cuniculi genotype II has been found in many different species of hosts inducing causing wide spectrum of pathogenesis (Weber et al., 1994;Mertens et al., 1997;Didier et al., 1998;Didier 2005;Perec-Matysiak et al.,2019), the other genotypes have been reported less frequently. Encephalitozoon cuniculi genotype I has been recorded only rarely in asymptomatic birds (Kašičková et al., 2009), dogs (Engelhardt et al., 2017), great apes (Sak et al., 2011b, rodents (Sak et al. 2011a;Tsukada et al., 2013), horses (Wagnerová et al., 2012;Laatamna et al., 2015), rabbits (Kimura et al., 2013;Deng et al., 2020), boars (Němejc et al., 2014), buffalos and seldom in humans (Ditrich et al., 2011;Halánová et al., 2013;Tavalla et al., 2017); whereas E. cunciculi genotype III has been identified in lemmings, rabbits, birds, swine, dogs, blue foxes, snow leopards, tamarins and squirrel monkeys, and also in humans, causing a variety of pathologies ranging from asymptomatic to lethal infections (Botha et al., 1986;Cutlip and Beall, 1989;van Dellen et al., 1989;Didier et al., 1994;Guscetti et al., 2003;Reetz et al., 2004Reetz et al., , 2009Mathis et al., 2005;Asakura et al., 2006;Juan-Sallés et al., 2006;Kašičková et al., 2009;Snowden et al., 2009;Valenčáková et al., 2011;Hocevar et al., 2014;Hofmannová et al., 2014;Meng et al., 2014;Scurrell et al., 2015;12. ...
Out of three genotypes of Encephalitozoon cuniculi (I–III) available for experimental studies, E. cuniculi genotype I remains the less characterized.
This study describes for the first time individual phases of microsporidiosis caused by E. cuniculi genotype I and efficacy of albendazole treatment in immunocompetent BALB/c and C57Bl/6 mice and immunodeficient SCID, CD4-/- and CD8-/- mice using molecular detection and quantification methods.
We demonstrate asymptomatic infection despite an intense dissemination of microsporidia into most organs within the first weeks post infection, followed by a chronic infection characterized by significant microsporidia persistence in immunocompetent, CD4-/- and CD8-/- mice and a lethal outcome for SCID mice. Albendazole application led to loss E. cuniculi genotype I infection in immunocompetent mouse strains, decreased spore burden by half in CD4-/- and CD8-/- mice, and prolongation of survival of SCID mice.
These results showed Encephalitozoon cuniculi genotype I infection extend and albendazole sensitivity was comparable to E. cuniculi genotype II, but the infection onset speed and mortality rate was similar to E. cuniculi genotype III. These imply that differences in the course of infection and the response to treatment depend not only on immunological status of the host, but also on the genotype causing the infection.
Purpose
This study aims to inform about the clinical image, diagnostic possibilities, and treatment options for cats and dogs diagnosed with ocular encephalitozoonosis.
Materials and Methods
Medical records of 7 dogs and 75 cats with ocular encephalitozoonosis presented at the Clinical Unit of Ophthalmology of the University of Veterinary Medicine Vienna between 08/2006 and 02/2022 have been compiled. Diagnosis was based on the presence of cataracts and positive antibody titer against Encephalitozoon cuniculi . Patient history, details on the characteristics of the cataracts, level of antibody titers, and the treatment regimen were summarized, as are the results of histological stainings of surgical samples to determine the presence of Encephalitozoon cuniculi , as well as PCR analyses to identify its sub‐strains.
Results
Our analysis of the clinical data shows that focal cortical anterior cataracts often associated with anterior uveitis and lesions in the fundus are the most prevalent manifestation of ocular encephalitozoonosis in cats and dogs.
Conclusion
Serological testing and PCR analysis of lens material are the most important diagnostic tools. Timely phacoemulsification with adjuvant anti‐inflammatory and antiparasitic medication is the most promising therapy for Encephalitozoon cuniculi‐induced cataracts.
Case series summary
Three domestic shorthair cats from California presented to veterinary ophthalmologists with immature cataracts. Other presenting clinical signs included corneal edema, anisocoria, anterior uveitis, elevated intraocular pressure, blepharospasm and/or lethargy. All patients were immunocompromised due to concurrent diseases and/or immunomodulatory drugs. Diagnostics included serial comprehensive ophthalmic examinations with tonometry, ocular ultrasound, electroretinogram and testing for other causes of feline uveitis. Testing for Encephalitozoon cuniculi included serology, histopathology and/or PCR of aqueous humor, lens material or paraffin-embedded whole eye. Treatments included antiparasitic medication, anti-inflammatory medication and supportive care in all three cases. Surgical treatment included enucleation (one case), bilateral phacoemulsification and unilateral intraocular lens placement (one case) and bilateral phacoemulsification with bilateral endolaser ciliary body ablation and bilateral intraocular lens implantation (one case). Both cats for which serologic testing for E cuniculi was performed were positive (1:64–1:4096). In all cats, diagnosis of intraocular E cuniculi was based on at least one of the following: lens histopathology or PCR of aqueous humor, lens material or paraffin-embedded ocular tissue. The clinical visual outcome was best in the patient undergoing phacoemulsification at the earliest stage of the cataract.
Relevance and novel information
Encephalitozoon cuniculi should be considered as a differential cause of cataracts and uveitis in cats in California, the rest of the USA and likely worldwide.
Microsporidia are pathogenic organism related to fungi. They cause infections in a wide variety of mammals as well as in avian, amphibian, and reptilian hosts. Many microsporidia species play an important role in the development of serious diseases that have significant implications in human and veterinary medicine. While microsporidia were originally considered to be opportunistic pathogens in humans, it is now understood that infections also occur in immune competent humans. Encephalitozoon cuniculi, Encephalitozoon intestinalis, and Enterocytozoon bieneusi are primarily mammalian pathogens. However, many other species of microsporidia that have some other primary host that is not a mammal have been reported to cause sporadic mammalian infections. Experimental models and observations in natural infections have demonstrated that microsporidia can cause a latent infection in mammalian hosts. This chapter reviews the published studies on mammalian microsporidiosis and the data on chronic infections due to these enigmatic pathogens.
Background
Rickettsiae are obligate intracellular alpha-proteobacteria. They are transmitted via arthropod vectors, which transmit the bacteria between animals and occasionally to humans. So far, much research has been conducted to indicate reservoir hosts for these microorganisms, but our knowledge is still non-exhaustive. Therefore, this survey was undertaken to investigate the presence of Rickettsia spp. in wild-living small rodents from south-western Poland.
Results
In total, 337 samples (193 from spleen and 144 from blood) obtained from 193 wild-living rodents: Apodemus agrarius, Apodemus flavicollis, and Myodes glareolus were tested by qPCR for Rickettsia spp. based on a fragment of gltA gene. The prevalence of infection was 17.6% (34/193). Subsequently, the positive samples were analysed by conventional PCR targeting the gltA gene fragment. The genus Rickettsia was confirmed by sequence analysis in four samples from the blood. In two blood samples from A. agrarius, the identified pathogen was Rickettsia helvetica. The Rickettsia obtained from A. flavicollis was assigned to Rickettsia felis-like organisms group. One isolate from A. agrarius could be determined only to the genus level.
Conclusions
This study shows the presence of Rickettsia DNA in tissues of wild-living rodents, suggesting some potential role of these animals in temporarily maintaining and spreading the bacteria in enzootic cycles.
Within the microsporidian genus Encephalitozoon, three species, Encephalitozoon cuniculi, Encephalitozoon hellem and Encephalitozoon intestinalis have been described. Several orders of the Class Aves (Passeriformes, Psittaciformes, Apodiformes, Ciconiiformis, Gruiformes, Columbiformes, Suliformes, Podicipediformes, Anseriformes, Struthioniformes, Falconiformes) and of the Class Mammalia (Rodentia, Lagomorpha, Primates, Artyodactyla, Soricomorpha, Chiroptera, Carnivora) can become infected. Especially E. cuniculi has a very broad host range while E. hellem is mainly distributed amongst birds. E. intestinalis has so far been detected only sporadically in wild animals. Although genotyping allows the identification of strains with a certain host preference, recent studies have demonstrated that they have no strict host specificity. Accordingly, humans can become infected with any of the four strains of E. cuniculi as well as with E. hellem or E. intestinalis, the latter being the most common. Especially, but not exclusively, immunocompromised people are at risk. Environmental contamination with as well as direct transmission of Encephalitozoon is therefore highly relevant for public health. Moreover, endangered species might be threatened by the spread of pathogens into their habitats. In captivity, clinically overt and often fatal disease seems to occur frequently. In conclusion, Encephalitozoon appears to be common in wild warm-blooded animals and these hosts may present important reservoirs for environmental contamination and maintenance of the pathogens. Similar to domestic animals, asymptomatic infections seem to occur frequently but in captive wild animals severe disease has also been reported. Detailed investigations into the epidemiology and clinical relevance of these microsporidia will permit a full appraisal of their role as pathogens.
Increased risk of zoonotic transmission of the potential human pathogenic species Enterocytozoon bieneusi, Encephalitozoon intestinalis and Encephalitozoon cuniculi was detected in wild immunocompetent mice (Mus musculus musculus; n=280). Analysis was conducted with the use of PMP1/PMP2 primers and SYBR Green RT-PCR. Using Real Time PCR and comparing the sequences with sequences in the GenBank, E. bieneusi was detected in 3 samples (1.07 %), E. cuniculi in 1 sample (0.35 %) and E. intestinalis in 1 sample (0.35 %). The results of this report document the low host specificity of detected microsporidia species, and imply the importance of synanthropic rodents as a potential source of human microsporidial infection.
Microsporidia were identified in stool specimens by histochemistry and PCR of 30 (18.9%) of 159 HIV-infected patients presenting
to the S. P. Botkin Memorial Clinical Hospital of Infectious Diseases, St. Petersburg, Russia. The higher prevalence of Encephalitozoon intestinalis, in 21 (12.8%) patients, than of Enterocytozoon bieneusi, in 2 patients (1.2%), was unexpected. Encephalitozoon cuniculi was detected in three patients: one with strain I and two with strain II. Encephalitozoon hellem was detected in one patient, and two patients were identified as being infected by Microsporidium species. One patient was infected with both E. intestinalis and E. cuniculi. In two patients, the microsporidian species were not identifiable. No statistically significant differences in gender, age,
and stage of AIDS were observed between the microsporidian-positive and -negative HIV-infected patients. HIV-infected patients
diagnosed with microsporidian infection, however, were significantly more likely to exhibit ≤100 CD4+ T cells/μl blood (20/30 patients [67%]; odds ratio [OR], 3.150; 95% confidence interval [CI95], 1.280 to 7.750; P = 0.0116) and weight loss of >10% of the baseline (19/30 patients [63%]; odds ratio, 2.995; CI95, 1.100 to 8.158; P = 0.0352) than HIV-infected patients not diagnosed with microsporidian infection. In summary, this is the first report describing
the diagnosis of microsporidian infection of HIV-infected patients in Russia and the first detection of E. cuniculi strain II in a human.
Knowledge about the protozoan parasite fauna in voles (Arvicolinae) in Austria is rather limited, although some of these pathogens play an important role in human medicine and cause zoonoses (e.g., Toxoplasma gondii and Encephalitozoon cuniculi). Others are of relevance in veterinary medicine and have a negative economic impact (e.g., Neospora caninum). Two hundred sixty-eight common voles (Microtus arvalis) and 86 water voles (Arvicola terrestris) from the most western Austrian province, Vorarlberg, were analyzed with PCR techniques for infections with T. gondii, N. caninum, and E. cuniculi. Brain tissues of two common voles (0.7%) and of four water voles (4.7%) tested positive for T. gondii. Furthermore, analysis of four common voles (1.5%) and two water voles (2.3%) generated positive findings for N. caninum, and brain tissues of 16 common voles (6%) and six water voles (7%) tested positive for E. cuniculi. Accordingly, this study not only demonstrates the autochthonous existence of the zoonotic parasites T. gondii and E. cuniculi in voles in Vorarlberg, it also provides the first evidence of an occurrence of N. caninum in animals of the subfamily Arvicolinae, and it is an additional contribution to investigations of the sylvatic cycle of N. caninum.
Disseminated microsporidiosis is a life-threatening opportunistic infection. Here, we report about a previously undescribed
genovar of Encephalitozoon cuniculi causing disseminated infection in a non-HIV-infected renal transplant recipient. Disseminated microsporidiosis must be considered
in the differential diagnosis of chronic fever in renal allograft recipients, even those without urinary symptoms.
Antibodies to Encephalitozoon cuniculi were found in wild arctic foxes (Alopex lagopus), feral mink (Mustela vison), wood mice (Apodemus sylvaticus) and house mice (Mus musculus) in Iceland. Animals with antibodies were found throughout the country. No lesions attributable to encephalitozoonosis were found in adult animals necropsied. However, one arctic fox cub with a neurological disorder had pathological and serological evidence of encephalitozoonosis.
The ribosomal DNA internal transcribed spacer (ITS) region of a recently cultured human Encephalitozoon cuniculi isolate was analyzed by gene amplification and DNA sequencing. Restriction endonuclease digestion (FokI) and double-stranded DNA heteroduplex mobility shift analysis were performed to determine their utility for strain differentiation. The human E. cuniculi isolate was identical to E. cuniculi III, which had been isolated only from domestic dogs until now. The patient providing the isolate owned a pet dog, but no microsporidia were detected in the pet's urine.
Encephalitozoon cuniculi has a wide host range among mammals, but whether it represents a homogeneous species is a subject of controversy. We have isolated, cultivated (in human MRC-5 cells) and, for the first time, characterized by immunological and molecular biological methods four isolates of E. cuniculi from Norwegian blue foxes with a history of encephalitozoonosis. The isolates were compared with nine isolates from domestic rabbits from Switzerland. Two E. cuniculi subtypes were identified according to their host species. A 5'-GTTT-3' tetranucleotide repeat was present twice in the rDNA intergenic spacer in all isolates from foxes as opposed to three times in all isolates from rabbits. Furthermore, random amplified polymorphic DNA analysis showed one polymorphic band among the subtypes, and Western-blot analysis using serum from an infected fox discriminated between the two subtypes on the basis of their banding patterns in the ranges of 31-33 and 38-40 kDa. The 5'-GTTT-3' tetranucleotide repeat is a valuable genetic marker for these two subtypes of E. cuniculi and will be of use in continued studies on the molecular epidemiology of this parasite.
Encephalitozoon intestinalis (Septata intestinalis) is the second most prevalent microsporidian species infecting humans, but it has not been described in other animal species.
This investigation examined 10 domestic animal stool samples (8 mammalian, 2 avian) containing spores detected by anti-Encephalitozoon monoclonal antibody immunofluorescence (FA). The presence of E. intestinalis but not Encephalitozoon hellem or Encephalitozoon cuniculi was confirmed in 6 of 8 mammalian stool samples by species-specific FA and polymerase chain reaction. Clusters of spores
inside epithelial cells were observed in feces of five mammals (donkey, dog, pig, cow, and goat) using “quick-hot” Gram-chromotrope
stain. None of the 10 samples reacted with anti-E. hellem or anti-E. cuniculi sera, nor were they amplified with species-specific primers for E. hellem and E. cuniculi. To our knowledge, this is the first identification of E. intestinalis in animals other than humans. The data shown herein suggest the possibility that E. intestinalis infection may be zoonotic in origin.
Microsporidia are increasingly recognized as causing opportunistic infections in immunocompromised individuals. Encephalitozoon cuniculi is probably the most studied mammalian microsporidian that infects insects and mammals, including man. In this study, 8 E. cuniculi isolates were compared and were found to fall into 3 strains. Strain type I includes the rabbit type isolate, as well as isolates from an additional rabbit, a dwarf rabbit, and a mouse. Strain type II includes 2 murine isolates and strain type III includes 2 isolates obtained from domestic dogs. By SDS-PAGE, the 3 strains differ primarily in the molecular weight range of 54-59 kDa where strain type I displays an apparent broad singlet at 57 kDa, strain type II displays an apparent doublet at 54 and 58 kDa, and strain type III displays an apparent broad band at 59 kDa. Antigenic differences were detected in the molecular weight regions of 54-58 kDa as well as 28-40 kDa by Western blot immunodetection using murine antisera raised against E. cuniculi, Encephalitozoon hellem, and the Encephalitozoon-like Septata intestinalis. Polymerase chain reaction (PCR) products containing only small subunit rDNA sequences from the different E. cuniculi isolates formed homoduplexes whereas PCR products containing intergenic rRNA gene sequences formed heteroduplexes in mobility shift analyses. Fok I digestion of the PCR products containing the intergenic rRNA gene region resulted in unique restriction fragment length polymorphism patterns, and DNA sequencing demonstrated that in the intergenic spacer region, the sequence 5'-GTTT-3' was repeated 3 times in strain type I, twice in strain type II, and 4 times in strain type III. This study indicates that there exist at least 3 E. cuniculi strains which may become important in the epidemiology of human E. cuniculi infections. Furthermore, as additional E. cuniculi isolates are characterized, these strains will be named or reclassified once the criteria for taxonomy and phylogenetic tree construction for microsporidia become better defined.
Microsporidia are single-celled, obligate intracellular parasites that were recently reclassified from protozoa to fungi. Microsporidia are considered a cause of emerging and opportunistic infections in humans, and species infecting humans also infect a wide range of animals, raising the concern for zoonotic transmission. Persistent or self-limiting diarrhea are the most common symptoms associated with microsporidiosis in immune-deficient or immune-competent individuals, respectively. Microsporidian spores appear to be relatively resistant under environmental conditions, and species of microsporidia infecting humans and animals have been identified in water sources, raising concern about water-borne transmission. Sensitive and specific immunomagnetic bead separation and PCR-based methods are being developed and applied for detecting microsporidia in infected hosts and water sources for generating more reliable prevalence data. The most effective drugs for treating microsporidiosis in humans currently include albendazole, which is effective against the Encephalitozoon species but not against Enterocytozoon bieneusi, and fumagillin, which has broader anti-microsporidia activity but is toxic in mammals, suggesting a need to identify better drugs. Strategies to capture and disinfect microsporidia in water are being developed and include filtration, coagulation, chlorination, gamma-irradiation, and ozonation.
Microsporidia are long-known parasitic organisms of almost every animal group, including invertebrates and vertebrates. Microsporidia emerged as important opportunistic pathogens in humans when AIDS became pandemic and, more recently, have also increasingly been detected in otherwise immunocompromised patients, including organ transplant recipients, and in immunocompetent persons with corneal infection or diarrhea. Two species causing rare infections in humans, Encephalitozoon cuniculi and Brachiola vesicularum, had previously been described from animal hosts (vertebrates and insects, respectively). However, several new microsporidial species, including Enterocytozoon bieneusi, the most prevalent human microsporidian causing human immunodeficiency virus-associated diarrhea, have been discovered in humans, raising the question of their natural origin. Vertebrate hosts are now identified for all four major microsporidial species infecting humans (E. bieneusi and the three Encephalitozoon spp.), implying a zoonotic nature of these parasites. Molecular studies have identified phenotypic and/or genetic variability within these species, indicating that they are not uniform, and have allowed the question of their zoonotic potential to be addressed. The focus of this review is the zoonotic potential of the various microsporidia and a brief update on other microsporidia which have no known host or an invertebrate host and which cause rare infections in humans.
Disseminated microsporidiosis was diagnosed in an adult female Egyptian fruit bat that died unexpectedly in a zoo. Gross findings, which were minimal, included poor body condition, bilateral renomegaly, and mottling of the liver. Histopathological lesions, which were particularly pronounced in the urogenital tract and liver, consisted primarily of inflammation associated with intracytoplasmic microsporidian spores. Polymerase chain reaction -based methods were used to establish the identity of the microsporidian as Encephalitozoon hellem. E. hellem is an emerging cause of human and avian disease, manifested mainly as opportunistic infection in immunosuppressed patients. This report describes the first documented case of E. hellem in a non-human mammalian species.
Disseminated encephalitozoonosis was diagnosed in 2 sibling, juvenile, cotton-top tamarins (Saguinus oedipus) and 3 sibling, neonatal, emperor tamarins (S. imperator) by use of histologic examination, histochemical analysis, electron microscopy, and polymerase chain reaction (PCR) analysis with nucleotide sequencing. All tamarins were captive born at zoos in North America and died with no premonitory signs of disease. The main pathologic findings were myocarditis (4/5), hepatitis (3/5), interstitial pneumonia (3/5), skeletal myositis (3/5), meningoencephalitis (2/5), adrenalitis (2/5), tubulointerstitial nephritis (1/5), myelitis (1/5), sympathetic ganglioneuritis (1/5), and retinitis (1/5). Central nervous system lesions were the most prominent findings in cotton-top tamarins. The inflammation was predominantly lymphocytic and suppurative in cotton-top tamarins, whereas emperor tamarins had granulomatous or lymphoplasmacytic lesions. Intralesional periodic acid-Schiff-, gram-, or acid-fast (or all 3)-positive, oval-to-elliptical shaped organisms were found in 1 cotton-top and the 3 emperor tamarins. By electron microscopy, these organisms were consistent with microsporidia of the genus Encephalitozoon. E. cuniculi genotype III was detected by PCR analysis and sequencing in paraffin-embedded brain, lung, and bone marrow specimens from the cotton-top tamarins. Although PCR results were negative for one of the emperor tamarins, their dam was seropositive for E. cuniculi by ELISA and Western blot immunodetection. These findings and recent reports of encephalitozoonosis in tamarins in Europe suggest that E. cuniculi infection may be an emerging disease in callitrichids, causing high neonatal and juvenile mortality in some colonies. The death of 2 less than 1-day-old emperor tamarins from a seropositive dam supports the likelihood of vertical transmission in some of the cases reported here.
Diversity of Enterocytozoon bieneusi genotypes in wild small rodent populations still remains incomplete and only few molecular studies have been conducted among these hosts. Therefore, the aim of this study was to determine whether small rodents, i.e., Apodemus agrarius, Apodemus flavicollis, Mus musculus and Myodes glareolus act as hosts of E. bieneusi and can play an important role in spore spreading in the environment of south-western Poland. Molecular analyses were conducted to determine pathogen genotypes. A total of 191 fecal and 251 spleen samples collected from 311 rodent individuals were examined for the occurrence of E. bieneusi by PCR amplifying ITS gene. The overall prevalence of E. bieneusi in rodent samples was 38.9%. The nucleotide sequences of ITS region of E. bieneusi revealed the presence a total of 12 genotypes with two being already known, i.e., D and gorilla 1 genotypes. The remaining ten are novel genotypes (WR1-WR10) which segregated into three groups in a neighbor joining phylogeny. This study reports for the first time E. bieneusi occurrence in wild living rodents in Poland and shows extensive genetic diversity within E. bieneusi isolates of rodent origin.
Encephalitozoon is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts. In this study, we used nested PCR to investigate the presence of Encephalitozoon infection in Rodentia and Soricomorpha in Japan. We attempted to amplify and sequence Encephalitozoon-specific DNA from brain and viscera samples of 180 animals collected between 2008 and 2010. Forty-three samples (23.9%) from the orders Rodentia and Soricomorpha were positive for Encephalitozoon. This study is the first report of Encephalitozoon infection in Rodentia and Soricomorpha in Japan, and our findings suggest that these hosts may play a role in the spread of microsporidian spores in the environment.
Encephalitozoon cuniculi is an obligate intracellular microsporidian that is the causal agent of encephalitozoonosis, an important and emerging disease in both humans and animals. Little is known about its occurrence in wildlife. In this study, serum samples from 793 wild rodents [178 bank voles (BV), 312 field voles (FV) and 303 wood mice (WM)], 96 foxes and 27 domestic cats from three study areas in the UK were tested for the presence of antibodies to E. cuniculi using a direct agglutination test (DAT). Seroprevalence in the wild rodents ranged from 1.00% to 10.67% depending on species (overall 5.31%) and was significantly higher in foxes [49.50% (50/96)]. None of the 27 cats sampled were found to be seropositive. This is the first report of seroprevalence to E. cuniculi in BV, FV, WM, foxes and cats in the UK and provides some evidence that foxes could act as sentinels for the presence of E. cuniculi in rodents. The study demonstrates that wildlife species could be significant reservoirs of infection for both domestic animals and humans.
Microsporidia have emerged as causes of opportunistic infections associated with diarrhea and wasting in AIDS patients. This review describes recent reports of microsporidiosis in HIV-infected individuals and the growing awareness of microsporidiosis in non-HIV-infected populations.
Microsporidia were only rarely recognized as causes of disease in humans until the AIDS pandemic. Implementation of combination antiretroviral therapy (cART) to curtail HIV replication and restore immune status drastically reduced the occurrence of opportunistic infections, including those due to microsporidia, in HIV-infected individuals. In developing countries where cART is not always accessible, microsporidiosis continues to be problematic. Improvement of diagnostic methods over the previous 25 years led to identification of several new species of microsporidia, many of which disseminate from enteric to systemic sites of infection and contribute to some unexpected lesions. Among non-HIV-infected but immune-suppressed individuals, microsporidia have infected organ transplant recipients, children, the elderly, and patients with malignant disease and diabetes. In otherwise healthy immune-competent HIV seronegative populations, self-limiting diarrhea occurred in travelers and as a result of a foodborne outbreak associated with contaminated cucumbers. Keratitis due to microsporidiosis has become problematic and a recent longitudinal evaluation demonstrated that non-HIV-infected individuals seropositive for microsporidia who had no clinical signs continued to intermittently shed organisms in feces and urine.
Greater awareness and implementation of better diagnostic methods are demonstrating that microsporidia contribute to a wide range of clinical syndromes in HIV-infected and non-HIV-infected people. As such, microsporidia should be considered in differential diagnoses if no other cause can be defined.
Microsporidia are recognized as a major aetiological agent in chronic diarrhoea of immunocompromised patients. Their detection by light microscopy is hampered by the small size of the spores. A simple and rapid DNA extraction method has been developed for the detection of microsporidian DNA by PCR directly from stool specimens. It can be performed at room temperature in a 1.5-ml microcentrifuge tube format in less than 1 hour. The subsequent nested polymerase chain reaction permits the detection of 3-100 spores in a 0.1-g stool sample. The amplification products can be verified and the species Enterocytozoon bieneusi, Encephalitozoon cuniculi and Encephalitozoon (Septata) intestinalis distinguished by a simple restriction endonuclease digest.
Microsporidia are small, single-celled, obligately intracellular parasites that have caused significant agricultural losses and interference with biomedical research. Interest in the microsporidia is growing, as these organisms are recognized as agents of opportunistic infections in persons with AIDS and in organ transplant recipients. Microsporidiosis is also being recognized in children and travelers, and furthermore, concern exists about the potential of zoonotic and waterborne transmission of microsporidia to humans. This article reviews the basic biology and epidemiology of microsporidiosis in mammals.
The microsporidian species Encephalitozoon cuniculi can infect a wide variety of mammals including man. It is a common parasite in rabbits and several sporadic infections in laboratory rats have been described. Based on molecular data three E. cuniculi strains have been identified. Here we describe the first in vitro propagation of E. cuniculi, which was isolated from a free-ranging rat (Rattus norvegicus). The rat was one of three seropositive animals among 23 rats captured in the city of Zurich. The new isolate was further characterised as strain II ("mouse"-strain) based on the rDNA internal transcribed spacer sequence. Western blot analysis of this isolate revealed slight differences to other available strain II isolates originating from laboratory mice and farmed blue foxes. The new isolate caused disseminated infection in liver and lung upon oral inoculation of Brown Norway (BN) rats and was transmitted to sentinel rats. This rat-adapted isolate will be valuable to study the pathogenesis of Encephalitozoon infections in the rat model.
Microsporidian spores have been detected by Chromotrope 2R and calcofluor stains in fecal samples of three free-ranging human-habituated mountain gorillas in Uganda and in two people who share gorilla habitats. All spore isolates have been identified by PCR with species-specific primers and fluorescent in situ hybridization with a species-specific oligonucleotide probe to be Encephalitozoon intestinalis. Sequencing analyses of the full length SSUrRNA amplified from all spore isolates were identical with Enc. intestinalis SSUrRNA GenBank SIU09929. Sequences generated from a fragment containing the internal transcribed spacer of these isolates were identical to GenBank sequence Y11611, i.e., Enc. intestinalis of anthroponotic origin. A single pathogen genotype in two genetically distant but geographically united host groups indicates anthropozoonotic transmission of Enc. intestinalis. It is highly unlikely that these two identical Enc. intestinalis genotypes were acquired independently by gorillas and people; it is much more probable that one group initiated infection of the other.
The farmed blue fox (Alopex lagopus) is particularly susceptible to congenital infections of the microsporidian species Encephalitozoon cuniculi. This report is based on an outbreak of the disease in Finland with high mortality. Five pups (four males and one female) with prolonged disease were examined. The pups had moderate pathological alterations in the kidneys and mild lesions were found in the brains, hearts, salivary and prostatic glands. Diagnosis of E. cuniculi infection was made from serological tests (ELISA, CIA, IFAT), and by in vitro isolation of the parasite from the brain of all five pups investigated. The identity was confirmed by molecular means as E. cuniculi strain II ('mouse strain'). Novel histopathological lesions not described as yet in fox encephalitozoonosis are presented. These include cerebral infarction and necrotizing inflammation of the renal pelvis. The sources and mechanisms of spreading of E. cuniculi to blue foxes are discussed.
To determine how long waterborne spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis could survive at environmental temperatures, culture-derived spores were stored in water at 10, 15, 20, 25, and 30 C and tested for infectivity in monolayer cultures of Madin Darby bovine kidney (MDBK) cells. At 10 C, spores of E. intestinalis were still infective after 12 mo, whereas those of E. hellem and E. cuniculi were infective for 9 and 3 mo, respectively. At 15 C, spores of the same species remained infective for 10, 6, and 2 mo, and at 20 C, for 7, 5, and 1 mo, respectively. At 25 C, spores of E. intestinalis and E. hellem were infective for 3 mo, but those of E. cuniculi were infective for only 3 wk. At 30 C, the former 2 species were infective for 3 wk and 1 mo, respectively, and the latter species for only 1 wk. These findings indicate that spores of different species of Encephalitozoon differ in their longevity and temperature tolerance, but at temperatures from 10 to 30 C, all 3 have the potential to remain infective in the environment long enough to become widely dispersed.
In the present paper some aspects of the biology and various factors influencing the potential for environmental contamination with protozoan parasites infective stages implicated in water and foodborne diseases are described. The major protozoan species that affect humans are Entamoeba histolytica, Acanthamoeba sp., Neagleria sp. Giardia intestinalis, Cryptosporidium parvum, Cyclospora cayetanensis, Toxoplasma gondii, Isospora/Sarcocystis sp. Encephalitozoom intestinals and Enterocytozoon bieneuisi. These parasites exist in the environment as oocyst, cysts or spores, which are the transmissive stages in many environmental conditions, e.g. water, soil, food as well as being infective stages to subsequent generation of hosts. Global concern with parasitic contamination of our environment must influence development of better detection methods and of evaluation and risk assessment of these infections. In this paper, the biology, waterborne and foodborne transmission, as well as methods for detection and control of Cryptosporidium parvum, Giardia intestinals and Toxoplasma gondii will be described.
Microsporidia have emerged as causes of infectious diseases in AIDS patients, organ transplant recipients, children, travelers, contact lens wearers, and the elderly. These organisms are small single-celled, obligate intracellular parasites that were considered to be early eukaryotic protozoa but were recently reclassified with the fungi. Of the 14 species of microsporidia currently known to infect humans, Enterocytozoon bieneusi and Encephalitozoon intestinalis are the most common causes of human infections and are associated with diarrhea and systemic disease. Species of microsporidia infecting humans have been identified in water sources as well as in wild, domestic, and food-producing farm animals, raising concerns for waterborne, foodborne, and zoonotic transmission. Current therapies for microsporidiosis include albendazole which is a benzimidazole that inhibits microtubule assembly and is effective against several microsporidia, including the Encephalitozoon species, but is less effective against E. bieneusi. Fumagillin, an antibiotic and anti-angiogenic compound produced by Aspergillus fumigatus, is more broadly effective against Encephalitozoon spp. and Enterocytozoon bieneusi but is toxic when administered systemically to mammals. Gene target studies have focused on methionine aminopeptidase 2 (MetAP2) for characterizing the mechanism of action and for identifying more effective, less toxic fumagillin-related drugs. Polyamine analogues have shown promise in demonstrating anti-microsporidial activity in culture and in animal models, and a gene encoding topoisomerase IV was identified in Vittaforma corneae, raising prospects for studies on fluoroquinolone efficacy against microsporidia.
Thoracic fluid (pleural fluid and clotted blood) from 206 foxes were examined for antibodies to Toxoplasma gondii and 220 thoracic fluid samples were tested for Neospora caninum antibodies using indirect immunofluorescent antibody tests (IFAT). A total of 115 (56%) and six (3%) foxes had antibodies to T. gondii and N. caninum, respectively. The brains from 148 foxes were examined for histological lesions and pathological changes suggestive of parasitic encephalitis were observed in 33 (22%). Two thirds of these foxes had antibodies to T. gondii and one fox had antibodies to both T. gondii and N. caninum. PCR assays carried out on DNA extracted from the 33 brains with histological lesions were negative for N. caninum but one of the brains was positive for T. gondii. Microsporidian DNA was also amplified from the brains of two of these foxes. Sequencing these amplicons revealed 100% homology with Encephalitozoon (Septata) intestinalis in one fox and Encephalitozoon cuniculi in the second fox. This is the first report of Encephalitozoon infections in wildlife in Ireland.
Epidemiology of microsporidia in human infections
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More than a rabbit´s tale -Encephalitozoon spp. in wild mammals and birds
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