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Encephalitozoon cuniculi: Implementation of a new fluorimetric method for the detection of anti-microsporidia antibodies
Experimental Parasitology 126 (2010) 267–269
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Research Brief
Encephalitozoon cuniculi: Implementation of a new fluorimetric method for the detection of anti-microsporidia antibodies Soledad Fenoy a, Amaranta A. Pérez-Fernánz b, Marta Rodero b, Carmen del Águila a, Carmen Cuéllar b,* a b
Laboratorio de Parasitología, Facultad de Farmacia, Universidad CEU San Pablo, Montepríncipe, Boadilla del Monte, 28668 Madrid, Spain Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
a r t i c l e
i n f o
Article history: Received 28 December 2009 Received in revised form 23 April 2010 Accepted 27 April 2010 Available online 21 May 2010 Keywords: Fluorimetric assay Encephalitozoon cuniculi Immunodiagnostic methods Microsporidia
a b s t r a c t A new fluorimetric method for the diagnosis of microsporidia was compared to the indirect immunofluorescence (IIF) method. Plates were coated with Encephalitozoon cuniculi spores, sera were incubated and an anti-human IgG FITC-conjugate was added. Finally, the plates were read using a fluorimeter. The results obtained were compared using the IIF technique confirming the positive sera with Fluorescence Index (FI) values of 3.75 and 5.24 in the fluorimetric method. Sera with FI values of 2.03 and 2.35 had borderline results when the IIF technique was used. The present results confirm the usefulness of fluorimetric methods in the diagnosis of human microsporidia, both in cases of the absence of immunodeficiency as well as in epidemiological studies. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction The number of genera implicated in human microsporidiosis has increased at the same rate as the improvements in diagnostic techniques, and interest in this group of parasites has grown accordingly. The most common diagnostic method is the modified trichrome stain (Weber et al., 1992). This technique does not differentiate among different species and needs to be performed by trained personnel and confirmation of the diagnosis must be carried out by indirect immunofluorescence assay (IIF) or PCR analysis (da Silva et al., 1997, 1996; del Aguila et al., 1997; Gainzarain et al., 1998; Matos et al., 2002; Visvesvara et al., 1994). Both techniques also have certain drawbacks, such as the human factor for IIF, or the need for specialized laboratories for PCR. For these reasons, knowledge of the true prevalence of microsporidia is complex and laborious. In order to find a rapid, simple and reliable alternative to investigate the degree of contact with microsporidia, we have introduced a new fluorimetric diagnostic method.
2. Materials and methods Eighty-eight sera from a population of normal unpaid-volunteer blood donors from the ‘‘Cruz Roja” hospital of Madrid were selected. Consent was obtained from each participant in the study. Hyperimmune rabbit serum developed against Encephalitozoon cuniculi spores and normal rabbit serum were used as controls. * Corresponding author. E-mail address: [email protected] (C. Cuéllar). 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.04.025
E. cuniculi (ECLD) was cultured on E6 monolayers and spores were Percoll purified before use (Visvesvara et al., 1995). Plates (Costar, Corning, NY, USA) were sensitized overnight at 4 °C by the addition of 106 spores of E. cuniculi per well, dried, and fixed with methanol:acetone (1:1). After washing with 0.05% PBS– Tween 20 (PBS–Tween), wells were blocked by the addition of 200 ll per well of 0.1% BSA in PBS, for 1 h at 37 °C. After washing, 100 ll of duplicate dilutions of sera at 1/200 in PBS–Tween, containing 0.1% BSA were added and incubated at 37 °C for 2 h. Once the plates were washed, 100 ll per well of a goat anti-human IgG FITC-conjugate (Sigma–Aldrich) at 1/64 dilution in PBS–Tween, 0.1% BSA, was added and incubated for 1 h at 37 °C. After washing, the plates were read in a fluorimeter (TECAN Infinite F200, Switzerland). Simultaneously, an assay without spores was provided as a control. Results were expressed as fluorescence indexes (FI) by the ratio between the arbitrary fluorescence units (AFU) resulting from the test serum and the AFU from the non-specific reaction with the BSA used in the blocking. In the first step, the assay conditions were selected using a rabbit hyperimmune serum as the positive control (data not shown). For the IIF test, the slides were coated with 5000 E. cuniculi spores per well and serum samples were diluted in PBS (1/64 and 1/128). After the addition of the FITC-conjugate, the slides were read using a fluorescent microscope. 3. Results The mean FI ± SD from the 88 human sera tested using the new fluorimetric analysis (FA) was 1.73 ± 0.58 (95% confidence
268
S. Fenoy et al. / Experimental Parasitology 126 (2010) 267–269
7.5
4. Discussion
FI
5.0
2.5
0.0
A
B
C
sera
D
E
Fig. 1. Fluorescence indexes (FI) of the 88 human sera tested by the new fluorimetric assay using E. cuniculi spores. A, all the sera; B, sera with FI P 3 (Group I); C, sera with FI between 2–2.9 (Group II); D, sera with FI between 1.5–1.9 (Group III); E, sera with FI 6 1.5 (Group IV). Each point represents a single serum.
interval 1.61–1.85), range 0.76–5.24. The results obtained with sera after FA were divided into four groups according to their FI as follows: Group I: sera with FI P 3 (two sera); Group II: FI values between 2.9–2 (17 sera); Group III: FI values between 1.9 and 1.5 (31 sera) and Group IV: FI values below 1.5 (38 sera) (Fig. 1). We observed that means were significantly different by one-way analysis of variance and after analyzing the results using the Tukey’s multiple comparison test significant differences were observed among all the groups (p < 0.001). Positive control used in the FA method showed FI values over 3. Serum from normal rabbit used as negative control showed FI less than 1.5. To confirm the results, all the sera studied were tested by IIF, confirming the positivity of the two sera from Group I which presented FI values of 3.75 and 5.24 (Fig. 2). Two of the 14 sera from the Group II, with FI values of 2.03 and 2.35, had borderline results when the IIF technique was used. Sera from Groups III and IV with values between 1.9 and less than 1.5 showed no fluorescence. Positive control used in the IF method showed strong fluorescence. Serum from normal rabbit used as negative control showed no fluorescence.
There is little knowledge of actual exposure to microsporidia in the general population and may be due to the diagnostic reference methods used, trichrome stain (Weber et al., 1992) or PCR methods (da Silva et al., 1997, 1996; Visvesvara et al., 1994) which are laborious and expensive. These methods have been applied mainly in feces samples to establish an actual infection, since the majority of human microsporidia produce diarrhea; however, these methods are not the most suitable alternative in epidemiological studies where a high number of samples are studied. ELISA and IIF methods have been the most used assays in these studies. In spite of their specificity, both methods have the inconvenience of the need for previous preparation of a soluble antigen (ELISA), and trained personnel (IIF). This new FA method allows us to use of spores from several microspora to identify the etiological agent using serum samples in a simple manner. As mentioned above, sera were divided into four groups according to their FI values: P3, 2.9–2, 1.9–1.5 and <1.5. The comparison to IIF technique showed that only sera with FI P 3 were positive, representing 2.27% of the 88 studied sera. These results are in accordance with those obtained by Omura et al. (2007) who detected a similar seroprevalence (1.05%) in healthy individuals in Japan using anti-polar tube IgG. The same authors obtained a significant increasing in seroprevalence (36%) using anti-polar tube IgM. Conversely, Halánová et al. (2003) found, 5.7% of seropositivity by IIF in Slovak Republic. To our knowledge our results are the first study on seroprevalence using spores of E. cuniculi in Spain. However, it is important to highlight the fact that, although E. cuniculi spores have been used to detect anti-microsporidia antibodies, the method cannot discriminate between different spores of microsporidia since human polyclonal sera were used. The only other similar study in Spain was carried out by del Aguila et al. (2001) but with E. intestinalis, where 2.7% of positive sera were obtained in a group of 406 blood donors from Madrid. The present results confirm the usefulness of fluorimetric methods in the diagnosis of human microsporidia, both in cases of the absence of immunodeficiency as well as in epidemiological studies. On the other hand, due to the zoonotic potential of E. cuniculi and other microsporidia, this method may be used to detect and control infections in rabbits and other pets capable of transmitting their diseases to human beings. Recent works by Santaniello et al. (2009) confirm that rabbit is the main reservoir of E. cuniculi with epidemiological relevance and immediate public health
Fig. 2. Positive indirect immunofluorescence test (IIF) of the two human sera with Fluorescence indexes (FI) P 3 (Group I) by the new E. cuniculi fluorimetric assay. Panel A, sera with FI = 3.75. Panel B, sera with FI = 5.24.
S. Fenoy et al. / Experimental Parasitology 126 (2010) 267–269
importance because its presence has been demonstrated not only in neurologically diseased rabbits but also in healthy and other diseased rabbits (Igarashi et al., 2008). Acknowledgment We are grateful to Brian Crilly for his help in the preparation of the manuscript. References da Silva, A.J., Schwartz, D.A., Visvesvara, G.S., de Moura, H., Slemenda, S.B., Pieniazek, N.J., 1996. Sensitive PCR diagnosis of Infections by Enterocytozoon bieneusi (microsporidia) using primers based on the region coding for smallsubunit rRNA. Journal of Clinical Microbiology 34, 986–987. da Silva, A.J., Bornay-Llinares, F.J., del Aguila, A., Moura, H., Peralta, J.M., Sobottka, I., Schwartz, D.A., Visvesvara, G.S., Slemenda, S.B., Pieniazek, N.J., 1997. Diagnosis of Enterocytozoon bieneusi (microsporidia) infections by polymerase chain reaction in stool samples using primers based on the region coding for smallsubunit ribosomal RNA. Archives of Pathology and Laboratory Medicine 121, 874–879. del Aguila, C., Rueda, C., De la Camara, C., Fenoy, S., 2001. Seroprevalence of antiEncephalitozoon antibodies in Spanish immunocompetent subjects. Journal of Eukaryotic Microbiology Supplement 75S, 78S. del Aguila, C., Lopez-Velez, R., Fenoy, S., Turrientes, C., Cobo, J., Navajas, R., Visvesvara, G.S., Croppo, G.P., Da Silva, A.J., Pieniazek, N.J., 1997. Identification of Enterocytozoon bieneusi spores in respiratory samples from an AIDS patient with a 2-year history of intestinal microsporidiosis. Journal of Clinical Microbiology 35, 1862–1866. Gainzarain, J.C., Canut, A., Lozano, M., Labora, A., Carreras, F., Fenoy, S., Navajas, R., Pieniazek, N.J., da Silva, A.J., del Aguila, C., 1998. Detection of Enterocytozoon
269
bieneusi in two human immunodeficiency virus-negative patients with chronic diarrhea by polymerase chain reaction in duodenal biopsy specimens and review. Clinical and Infectious Diseases 27, 394–398. Halanova, M., Cislakova, L., Valencakova, A., Balent, P., Adam, J., Travnicek, M., 2003. Serological screening of occurrence of antibodies to Encephalitozoon cuniculi in humans and animals in Eastern Slovakia. Annals of Agricultural and Environmental Medicine 10, 117–120. Igarashi, M., Oohashi, E., Dautu, G., Ueno, A., Kariya, T., Furuya, K., 2008. High seroprevalence of Encephalitozoon cuniculi in pet rabbits in Japan. Journal of Veterinary Medicine Sciences 70, 1301–1304. Matos, O., Lobo, M.L., Goncalves, L., Antunes, F., 2002. Diagnostic use of 3 techniques for identification of microsporidian spores among AIDS patients in Portugal. Scandinavian Journal of Infectious Diseases 34, 591–593. Omura, M., Furuya, K., Kudo, S., Sugiura, W., Azuma, H., 2007. Detecting immunoglobulin M antibodies against microsporidian Encephalitozoon cuniculi polar tubes in sera from healthy and human immunodeficiency virus-infected persons in Japan. Clinical and Vaccine Immunology 14, 168–172. Santaniello, A., Dipineto, L., Rinaldi, L., Menna, L.F., Cringoli, G., Fioretti, A., 2009. Serological survey of Encephalitozoon cuniculi in farm rabbits in Italy. Research in Veterinary Sciences 87, 67–69. Visvesvara, G.S., da Silva, A.J., Croppo, G.P., Pieniazek, N.J., Leitch, G.J., Ferguson, D., de Moura, H., Wallace, S., Slemenda, S.B., Tyrrell, I., 1995. In vitro culture and serologic and molecular identification of Septata intestinalis isolated from urine of a patient with AIDS. Journal of Clinical Microbiology 33, 930–936. Visvesvara, G.S., Leitch, G.J., da Silva, A.J., Croppo, G.P., Moura, H., Wallace, S., Slemenda, S.B., Schwartz, D.A., Moss, D., Bryan, R.T., 1994. Polyclonal and monoclonal antibody and PCR-amplified small-subunit rRNA identification of a microsporidian, Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection. Journal of Clinical Microbiology 32, 2760– 2768. Weber, R., Bryan, R.T., Owen, R.L., Wilcox, C.M., Gorelkin, L., Visvesvara, G.S., 1992. Improved light-microscopical detection of microsporidia spores in stool and duodenal aspirates. The Enteric Opportunistic Infections Working Group. New England Journal of Medicine 326, 161–166.
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Research Brief
Encephalitozoon cuniculi: Implementation of a new fluorimetric method for the detection of anti-microsporidia antibodies Soledad Fenoy a, Amaranta A. Pérez-Fernánz b, Marta Rodero b, Carmen del Águila a, Carmen Cuéllar b,* a b
Laboratorio de Parasitología, Facultad de Farmacia, Universidad CEU San Pablo, Montepríncipe, Boadilla del Monte, 28668 Madrid, Spain Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
a r t i c l e
i n f o
Article history: Received 28 December 2009 Received in revised form 23 April 2010 Accepted 27 April 2010 Available online 21 May 2010 Keywords: Fluorimetric assay Encephalitozoon cuniculi Immunodiagnostic methods Microsporidia
a b s t r a c t A new fluorimetric method for the diagnosis of microsporidia was compared to the indirect immunofluorescence (IIF) method. Plates were coated with Encephalitozoon cuniculi spores, sera were incubated and an anti-human IgG FITC-conjugate was added. Finally, the plates were read using a fluorimeter. The results obtained were compared using the IIF technique confirming the positive sera with Fluorescence Index (FI) values of 3.75 and 5.24 in the fluorimetric method. Sera with FI values of 2.03 and 2.35 had borderline results when the IIF technique was used. The present results confirm the usefulness of fluorimetric methods in the diagnosis of human microsporidia, both in cases of the absence of immunodeficiency as well as in epidemiological studies. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction The number of genera implicated in human microsporidiosis has increased at the same rate as the improvements in diagnostic techniques, and interest in this group of parasites has grown accordingly. The most common diagnostic method is the modified trichrome stain (Weber et al., 1992). This technique does not differentiate among different species and needs to be performed by trained personnel and confirmation of the diagnosis must be carried out by indirect immunofluorescence assay (IIF) or PCR analysis (da Silva et al., 1997, 1996; del Aguila et al., 1997; Gainzarain et al., 1998; Matos et al., 2002; Visvesvara et al., 1994). Both techniques also have certain drawbacks, such as the human factor for IIF, or the need for specialized laboratories for PCR. For these reasons, knowledge of the true prevalence of microsporidia is complex and laborious. In order to find a rapid, simple and reliable alternative to investigate the degree of contact with microsporidia, we have introduced a new fluorimetric diagnostic method.
2. Materials and methods Eighty-eight sera from a population of normal unpaid-volunteer blood donors from the ‘‘Cruz Roja” hospital of Madrid were selected. Consent was obtained from each participant in the study. Hyperimmune rabbit serum developed against Encephalitozoon cuniculi spores and normal rabbit serum were used as controls. * Corresponding author. E-mail address: [email protected] (C. Cuéllar). 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.04.025
E. cuniculi (ECLD) was cultured on E6 monolayers and spores were Percoll purified before use (Visvesvara et al., 1995). Plates (Costar, Corning, NY, USA) were sensitized overnight at 4 °C by the addition of 106 spores of E. cuniculi per well, dried, and fixed with methanol:acetone (1:1). After washing with 0.05% PBS– Tween 20 (PBS–Tween), wells were blocked by the addition of 200 ll per well of 0.1% BSA in PBS, for 1 h at 37 °C. After washing, 100 ll of duplicate dilutions of sera at 1/200 in PBS–Tween, containing 0.1% BSA were added and incubated at 37 °C for 2 h. Once the plates were washed, 100 ll per well of a goat anti-human IgG FITC-conjugate (Sigma–Aldrich) at 1/64 dilution in PBS–Tween, 0.1% BSA, was added and incubated for 1 h at 37 °C. After washing, the plates were read in a fluorimeter (TECAN Infinite F200, Switzerland). Simultaneously, an assay without spores was provided as a control. Results were expressed as fluorescence indexes (FI) by the ratio between the arbitrary fluorescence units (AFU) resulting from the test serum and the AFU from the non-specific reaction with the BSA used in the blocking. In the first step, the assay conditions were selected using a rabbit hyperimmune serum as the positive control (data not shown). For the IIF test, the slides were coated with 5000 E. cuniculi spores per well and serum samples were diluted in PBS (1/64 and 1/128). After the addition of the FITC-conjugate, the slides were read using a fluorescent microscope. 3. Results The mean FI ± SD from the 88 human sera tested using the new fluorimetric analysis (FA) was 1.73 ± 0.58 (95% confidence
268
S. Fenoy et al. / Experimental Parasitology 126 (2010) 267–269
7.5
4. Discussion
FI
5.0
2.5
0.0
A
B
C
sera
D
E
Fig. 1. Fluorescence indexes (FI) of the 88 human sera tested by the new fluorimetric assay using E. cuniculi spores. A, all the sera; B, sera with FI P 3 (Group I); C, sera with FI between 2–2.9 (Group II); D, sera with FI between 1.5–1.9 (Group III); E, sera with FI 6 1.5 (Group IV). Each point represents a single serum.
interval 1.61–1.85), range 0.76–5.24. The results obtained with sera after FA were divided into four groups according to their FI as follows: Group I: sera with FI P 3 (two sera); Group II: FI values between 2.9–2 (17 sera); Group III: FI values between 1.9 and 1.5 (31 sera) and Group IV: FI values below 1.5 (38 sera) (Fig. 1). We observed that means were significantly different by one-way analysis of variance and after analyzing the results using the Tukey’s multiple comparison test significant differences were observed among all the groups (p < 0.001). Positive control used in the FA method showed FI values over 3. Serum from normal rabbit used as negative control showed FI less than 1.5. To confirm the results, all the sera studied were tested by IIF, confirming the positivity of the two sera from Group I which presented FI values of 3.75 and 5.24 (Fig. 2). Two of the 14 sera from the Group II, with FI values of 2.03 and 2.35, had borderline results when the IIF technique was used. Sera from Groups III and IV with values between 1.9 and less than 1.5 showed no fluorescence. Positive control used in the IF method showed strong fluorescence. Serum from normal rabbit used as negative control showed no fluorescence.
There is little knowledge of actual exposure to microsporidia in the general population and may be due to the diagnostic reference methods used, trichrome stain (Weber et al., 1992) or PCR methods (da Silva et al., 1997, 1996; Visvesvara et al., 1994) which are laborious and expensive. These methods have been applied mainly in feces samples to establish an actual infection, since the majority of human microsporidia produce diarrhea; however, these methods are not the most suitable alternative in epidemiological studies where a high number of samples are studied. ELISA and IIF methods have been the most used assays in these studies. In spite of their specificity, both methods have the inconvenience of the need for previous preparation of a soluble antigen (ELISA), and trained personnel (IIF). This new FA method allows us to use of spores from several microspora to identify the etiological agent using serum samples in a simple manner. As mentioned above, sera were divided into four groups according to their FI values: P3, 2.9–2, 1.9–1.5 and <1.5. The comparison to IIF technique showed that only sera with FI P 3 were positive, representing 2.27% of the 88 studied sera. These results are in accordance with those obtained by Omura et al. (2007) who detected a similar seroprevalence (1.05%) in healthy individuals in Japan using anti-polar tube IgG. The same authors obtained a significant increasing in seroprevalence (36%) using anti-polar tube IgM. Conversely, Halánová et al. (2003) found, 5.7% of seropositivity by IIF in Slovak Republic. To our knowledge our results are the first study on seroprevalence using spores of E. cuniculi in Spain. However, it is important to highlight the fact that, although E. cuniculi spores have been used to detect anti-microsporidia antibodies, the method cannot discriminate between different spores of microsporidia since human polyclonal sera were used. The only other similar study in Spain was carried out by del Aguila et al. (2001) but with E. intestinalis, where 2.7% of positive sera were obtained in a group of 406 blood donors from Madrid. The present results confirm the usefulness of fluorimetric methods in the diagnosis of human microsporidia, both in cases of the absence of immunodeficiency as well as in epidemiological studies. On the other hand, due to the zoonotic potential of E. cuniculi and other microsporidia, this method may be used to detect and control infections in rabbits and other pets capable of transmitting their diseases to human beings. Recent works by Santaniello et al. (2009) confirm that rabbit is the main reservoir of E. cuniculi with epidemiological relevance and immediate public health
Fig. 2. Positive indirect immunofluorescence test (IIF) of the two human sera with Fluorescence indexes (FI) P 3 (Group I) by the new E. cuniculi fluorimetric assay. Panel A, sera with FI = 3.75. Panel B, sera with FI = 5.24.
S. Fenoy et al. / Experimental Parasitology 126 (2010) 267–269
importance because its presence has been demonstrated not only in neurologically diseased rabbits but also in healthy and other diseased rabbits (Igarashi et al., 2008). Acknowledgment We are grateful to Brian Crilly for his help in the preparation of the manuscript. References da Silva, A.J., Schwartz, D.A., Visvesvara, G.S., de Moura, H., Slemenda, S.B., Pieniazek, N.J., 1996. Sensitive PCR diagnosis of Infections by Enterocytozoon bieneusi (microsporidia) using primers based on the region coding for smallsubunit rRNA. Journal of Clinical Microbiology 34, 986–987. da Silva, A.J., Bornay-Llinares, F.J., del Aguila, A., Moura, H., Peralta, J.M., Sobottka, I., Schwartz, D.A., Visvesvara, G.S., Slemenda, S.B., Pieniazek, N.J., 1997. Diagnosis of Enterocytozoon bieneusi (microsporidia) infections by polymerase chain reaction in stool samples using primers based on the region coding for smallsubunit ribosomal RNA. Archives of Pathology and Laboratory Medicine 121, 874–879. del Aguila, C., Rueda, C., De la Camara, C., Fenoy, S., 2001. Seroprevalence of antiEncephalitozoon antibodies in Spanish immunocompetent subjects. Journal of Eukaryotic Microbiology Supplement 75S, 78S. del Aguila, C., Lopez-Velez, R., Fenoy, S., Turrientes, C., Cobo, J., Navajas, R., Visvesvara, G.S., Croppo, G.P., Da Silva, A.J., Pieniazek, N.J., 1997. Identification of Enterocytozoon bieneusi spores in respiratory samples from an AIDS patient with a 2-year history of intestinal microsporidiosis. Journal of Clinical Microbiology 35, 1862–1866. Gainzarain, J.C., Canut, A., Lozano, M., Labora, A., Carreras, F., Fenoy, S., Navajas, R., Pieniazek, N.J., da Silva, A.J., del Aguila, C., 1998. Detection of Enterocytozoon
269
bieneusi in two human immunodeficiency virus-negative patients with chronic diarrhea by polymerase chain reaction in duodenal biopsy specimens and review. Clinical and Infectious Diseases 27, 394–398. Halanova, M., Cislakova, L., Valencakova, A., Balent, P., Adam, J., Travnicek, M., 2003. Serological screening of occurrence of antibodies to Encephalitozoon cuniculi in humans and animals in Eastern Slovakia. Annals of Agricultural and Environmental Medicine 10, 117–120. Igarashi, M., Oohashi, E., Dautu, G., Ueno, A., Kariya, T., Furuya, K., 2008. High seroprevalence of Encephalitozoon cuniculi in pet rabbits in Japan. Journal of Veterinary Medicine Sciences 70, 1301–1304. Matos, O., Lobo, M.L., Goncalves, L., Antunes, F., 2002. Diagnostic use of 3 techniques for identification of microsporidian spores among AIDS patients in Portugal. Scandinavian Journal of Infectious Diseases 34, 591–593. Omura, M., Furuya, K., Kudo, S., Sugiura, W., Azuma, H., 2007. Detecting immunoglobulin M antibodies against microsporidian Encephalitozoon cuniculi polar tubes in sera from healthy and human immunodeficiency virus-infected persons in Japan. Clinical and Vaccine Immunology 14, 168–172. Santaniello, A., Dipineto, L., Rinaldi, L., Menna, L.F., Cringoli, G., Fioretti, A., 2009. Serological survey of Encephalitozoon cuniculi in farm rabbits in Italy. Research in Veterinary Sciences 87, 67–69. Visvesvara, G.S., da Silva, A.J., Croppo, G.P., Pieniazek, N.J., Leitch, G.J., Ferguson, D., de Moura, H., Wallace, S., Slemenda, S.B., Tyrrell, I., 1995. In vitro culture and serologic and molecular identification of Septata intestinalis isolated from urine of a patient with AIDS. Journal of Clinical Microbiology 33, 930–936. Visvesvara, G.S., Leitch, G.J., da Silva, A.J., Croppo, G.P., Moura, H., Wallace, S., Slemenda, S.B., Schwartz, D.A., Moss, D., Bryan, R.T., 1994. Polyclonal and monoclonal antibody and PCR-amplified small-subunit rRNA identification of a microsporidian, Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection. Journal of Clinical Microbiology 32, 2760– 2768. Weber, R., Bryan, R.T., Owen, R.L., Wilcox, C.M., Gorelkin, L., Visvesvara, G.S., 1992. Improved light-microscopical detection of microsporidia spores in stool and duodenal aspirates. The Enteric Opportunistic Infections Working Group. New England Journal of Medicine 326, 161–166.