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Microsporidia in household dogs and cats in Iran; a zoonotic concern
Veterinary Parasitology 185 (2012) 121–123
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Microsporidia in household dogs and cats in Iran; a zoonotic concern Sh. Jamshidi a,∗ , A. Shojaee Tabrizi b , M. Bahrami c , H. Momtaz c a b c
Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Shiraz, Shiraz, Iran Department of Microbiology, Faculty of Veterinary Medicine, Islamic Azad University, Shahrekord Branch, Sharekord, Iran
a r t i c l e
i n f o
Article history: Received 12 February 2011 Received in revised form 27 September 2011 Accepted 3 October 2011 Keywords: Microsporidia Dog Cat Iran
a b s t r a c t Microsporidia in dogs and cats is primarily caused by the obligate, intracellular parasite Encephalitozoon cuniculi, which is a member of the phylum Microsporidia. The aim of the current study is the detection of this parasite in stool samples of small animals of Iran, by polymerase chain reaction. Microsporidia spp. was found in 31% (31/100) of dogs (E. cuniculi (18/100), Encephalitozoon bieneusi (8/100) and Encephalitozoon intestinalis (5/100)), and 7.5% (3/40) of the specimens obtained from cats were infected with E. bieneusi. Sequencing of PCR products confirmed these results. In conclusion, Microsporidia infection seems to be fairly common in pet animals of Iran, especially in dogs. This finding could indicate the importance of pet animals as zoonotic reservoirs of microsporidial human infections. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Microsporidia are an exceptionally diverse group of organisms comprising more than 1200 species classified into approximately 100 genera that were recently reclassified from protozoa to fungi. Their life cycle includes stages resulting in characteristically small (1–4 m), environmentally resistant, infecting spores (Fayer, 2004). These unicellular, obligate intracellular eukaryotes have long been known to be causative agents of economically important diseases in every major animal group, especially insects, fish and mammals and they emerged as important opportunistic pathogens when AIDS became pandemic (Wittner, 1999). The clinical course of microsporidiosis depends on the immune status of the host and the site of infection. Clinically silent chronic infections usually develop in immunologically competent hosts that are either naturally or experimentally infected with Encephalitozoon cuniculi. Immune-compromised hosts infected with microsporidia are most likely to develop disseminated disease that often contributes to death (Snowden and Shadduck, 1999). E. cuniculi causes significant disease in
carnivores such as domestic dogs, blue fox (Alopex lagopus) and mink. Interestingly, these animals have developed hyperimmune responses resulting in immune complex formation and renal disease (Shadduck and Orenstein, 1993; Didier et al., 1998; Snowden and Shadduck, 1999). In humans, Encephalitozoon bieneusi infections remain localized to the small intestine, leading to persistent diarrhea. Species of Encephalitozoon, Trachipleistophora and Pleistophora often disseminate to cause sinusitis, keratoconjunctivitis, hepatitis, myositis, peritonitis, nephritis, encephalitis or pneumonia in immune-deficient individuals (Didier et al., 2004). Many species of microsporidia found to infect humans also infect a wide range of animals supporting the likelihood that zoonotic transmission occurs. Thus, the question of whether pet animals are the source of human infections is reasonable. In this study, PCR-based and microscopic methods were used to detect microsporidial spores in stool samples of pet dogs and cats having close contact with humans in Isfahan (Iran). 2. Materials and methods 2.1. Animals
∗ Corresponding author. Tel.: +98 21 6111 7193; fax: +98 21 6933222. E-mail address: [email protected] (Sh. Jamshidi). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.10.002
This study has been approved by the Iranian laboratory animal ethics framework under the supervision
122
Sh. Jamshidi et al. / Veterinary Parasitology 185 (2012) 121–123
of the Iranian Society for the Prevention of Cruelty to Animals. For this study, 100 stool samples from dogs (mean age = 4 years) and 40 samples from cats (mean age = 1.5 years) were collected from asymptomatic healthy animals which were referred to the Isfahan small animal clinic during a 6-month period in 2008. Samples were transferred to the diagnostic laboratory of Shahrekord Azad University and stored at 4 ◦ C until further analysis. 2.2. DNA extraction and PCR assays DNA was extracted from the fecal samples using instructions recommended by Laird et al. (1991). Fivehundred microliters of lysis buffer (EDTA 0.5 M, 100 mM Tris–HCl, SDS 10%, NACL 15 M, Proteinase K 20 mg/ml) was added to 500 l of each stool sample and incubated at 55 ◦ C for 2–3 h. Subsequently, 100 l phenol, 98 l chloroform and 4 l isoamyl alcohol were added to 200 l of lysed solution and after 30–60 s, vortexed at 4000 rpm for 4 min. The supernatant was collected and the same volume of pure ethanol was added and centrifugation was done at 4000 rpm for 5 min. After washing the DNA with 75% ethanol, the samples were centrifuged at 7000 rpm for 5 min, the supernatant was discarded and the DNA was dried. One hundred microliters of distilled water was then added to the DNA and stored at −20 ◦ C.
Table 1 Results of PCR on stool samples of household dogs and cats.
Dog (N = 100) Cat (N = 40)
E. cuniculi
E. bieneusi
E. intestinalis
18 (58%) 0
8 (25.8%) 3 (7.5%)
5 (16.1%) 0
2.4. Light microscopy For identification of microsporidia, fecal smears were stained with the Weber’s chromotrope as described (Weber et al., 1992). At least 50 fields of each stained smear were examined by light microscope under oil immersion at a magnification of 1000× before being considered negative. 3. Results PCR amplification: Out of 100 stool samples of dogs, 31cases were positive and a diagnostic band of 549 bp (E. cuniculi) was seen in 18 samples, 607 bp (E. bieneusi) in 8 samples and 520 bp (E. intestinalis) in 5 samples. All of the positive results belong to over one-year-old dogs. DNA of E. bieneusi (607 bp) was amplified in stool samples from 3/40 (7.5%) cats (Table 1). Light microscopy: With Weber’s chromotropebased stain, all PCR-positive samples showed spores of microsporidia that stained pinkish red (Fig. 1) and no spore was detected in the PCR-negative samples. 4. Discussion
2.3. PCR Microsporidial SSU-rRNA coding regions were amplified using the following species-specific primers: EBIEF1/EBIER1 for E. bieneusi (Da Silva et al., 1996), SINTF/SINTR for Encephalitozoon intestinalis (Da Silva et al., 1997), ECUNF/ECUNR for E. cuniculi and EHELF/EHELR for Encephalitozoon hellem (Visvesvara et al., 1994). All polymerase chain reactions (PCR) were performed in an Eppendorf thermocycler (Mastercycler gradient, Eppendorf GA, Hamburg, Germany) in a final volume of 50 L containing 1 g of extracted DNA, 5 L of 10× PCR buffer (Fermentas, Lithuania), 0.2 mM of dNTP, 2 mmol/L MgCl2 , 2 mol/L of each primer and 1 U of Taq DNA polymerase (Fermentas, Lithuania). Conditions for PCR reactions were: denaturing at 94 ◦ C for 50 s, annealing at 58 ◦ C for 50 s and extension at 72 ◦ C for 45 s. In all cases, 34 cycles were completed. Twenty microliters of each amplification underwent gel electrophoresis [1.5% (w/v) agarose gel with 0.3% ethidium bromide in 10% Tris–borate EDTA buffer (TBE)] and was visualized under UV transilluminator; the size of the expected fragments was compared to a 100-bp reference marker (Fermentas Inc., Glen Burnie, MD, USA). Since the positive control sample was not used in our molecular study, to confirm or reject the PCR results, the PCR products of the primary positive samples were purified using a PCR product purification kit (Roche Applied Science, Germany) and sent to the Macrogen Co. (South Korea) for sequencing.
Microsporidia are considered a cause of emerging and opportunistic infections in human, and species infecting humans also infect a wide range of animals, raising the concern for zoonotic transmission (Didier et al., 2004). However, the exact source of microsporidial human infection and routes of transmission remain unclear. Spores could be distributed into the environment via fecal, urine and respiratory excretions of infected animals and persons,
Fig. 1. Pinkish red spores of Microsporidia from a stool sample of a dog stained with Weber’s chromotrope (magnification 1000×). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Sh. Jamshidi et al. / Veterinary Parasitology 185 (2012) 121–123
which could all be possible sources of infection (Lores et al., 2002). Microsporidial infections can lead to severe diseases such as chronic diarrhea and significant weight loss in humans as well as animals (Snowden and Shadduck, 1999), however, it seems that many animals can carry these organisms without obvious clinical manifestations. These clinically silent carriers seem to be an even greater risk to public health than a sick animal. In the present study, for the first time, both molecular and microscopic methods were used to detect microsporidial spores in fecal samples of healthy pet cats and dogs in Iran, as a potential reservoir of human infections. Thirty-one percent of stool samples obtained from 100 household dogs were positive with PCR, which were confirmed by microscopic evaluations as well. This rate of infection should be believed significantly high, especially when considering that immuno-compromised persons and/or children may have close contact with them. The rate of infection in cats (7.5%) was lower than dogs, but it is still high and it seems that both dogs and cats have a serious role as reservoirs of infection to humans and are important in the epidemiology of microsporidiosis. E. cuniculi was detected in more than half of our positive samples (58%). Direct evidence of zoonotic transmission of microsporidiosis was reported in a child who seroconverted after being exposed to a litter of puppies infected with E. cuniculi (McInnes and Stewart, 1991). As previously mentioned, this species usually causes significant disease in domestic dogs (Shadduck and Orenstein, 1993; Didier et al., 1998; Snowden and Shadduck, 1999), but interestingly, none of our studied group had clinical signs of disease. This evidence again shows that a clinically normal dog can harbor even fully pathogenic strains and act only as a shedder. In almost 26% (8/31) of the positive specimens, E. bieneusi was found, which in 16% (5/31) E. intestinalis was found. It is not clear whether their presence in the fecal samples is the result of an active infection or not, but it obviously demonstrates that these animals can shed them into the environment, suggesting that indirect zoonotic transmission of microsporidia between animals and humans could occur through contaminated food, water, or aerosols.
123
Finally, the role of animals in the transmission of microsporidial human infections requires further study. However, our results show that small animals, especially dogs, even when they are apparently normal could be shedders of Microsporidia via their feces, and owners, especially those who are old or have a child and/or an immunodeficient patient, should be informed with practical suggestions designed to reduce this low risk. References Da Silva, A.J., Slemenda, S.B., Visvesvara, G.S., Schwartz, D.A., Wilcox, C.M., Wallace, S., Pieniazek, N.J., 1997. Detection of Septata intestinalis (microsporidia) Cali et al., 1993 using polymerase chain reaction primers targeting the small subunit ribosomal RNA coding region. Mol. Diagn. 2, 47–52. 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 small-subunit rRNA. J. Clin. Microbiol. 34 (4), 986–987. Didier, E.S., Stovall, M.E., Green, L.C., Brindley, P.J., Sestak, K., Didier, P.J., 2004. Epidemiology of microsporidiosis: sources and modes of transmission. Vet. Parasitol. 126, 145–166. Didier, E.S., Snowden, K.F., Shadduck, J.A., 1998. Biology of microsporidian species infecting mammals. Adv. Parasitol. 40, 283–320. Fayer, R., 2004. Infectivity of microsporidia spores stored in seawater at environmental temperatures. J. Parasitol. 90, 654–657. Laird, P.W., Zijderveld, A., Linders, K., Rudnicki, M.A., Jaenisch, R., Berns, A., 1991. Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 19 (15), 4293. Lores, B., del Aguila, C., Arias, C., 2002. Enterocytozoon bieneusi (microsporidia) in faecal samples from domestic animals from Galicia, Spain. Mem. Inst. Oswaldo Cruz 97, 941–945. McInnes, E.F., Stewart, C.G., 1991. The pathology of subclinical infection of Encephalitozoon cuniculi in canine dams producing pups with overt encephalitozoonosis. J. S. Afr. Vet. Assoc. 62, 51–54. Shadduck, J.A., Orenstein, J.M., 1993. Comparative pathology of microsporidiosis. Arch. Pathol. Lab. Med. 117, 1215–1219. Snowden, K.F., Shadduck, J.A., 1999. Microsporidia of higher vertebrates. In: Wittner, M., Weiss, L. (Eds.), The Microsporidia and Microsporidiosis. American Society of Microbiology, Washington, DC, pp. 393–419. 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., et al., 1994. Polyclonal and monoclonal antibody and PCR-amplified smallsubunit rRNA identification of a microsporidian, Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection. J. Clin. Microbiol. 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. N. Engl. J. Med. 326, 161–166. Wittner, M., 1999. Historic perspective on the microsporidia: expanding horizons. In: Wittner, M., Weiss, L. (Eds.), The Microsporidia and Microsporidiosis. American Society of Microbiology, Washington, DC, pp. 1–6.
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Microsporidia in household dogs and cats in Iran; a zoonotic concern Sh. Jamshidi a,∗ , A. Shojaee Tabrizi b , M. Bahrami c , H. Momtaz c a b c
Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Shiraz, Shiraz, Iran Department of Microbiology, Faculty of Veterinary Medicine, Islamic Azad University, Shahrekord Branch, Sharekord, Iran
a r t i c l e
i n f o
Article history: Received 12 February 2011 Received in revised form 27 September 2011 Accepted 3 October 2011 Keywords: Microsporidia Dog Cat Iran
a b s t r a c t Microsporidia in dogs and cats is primarily caused by the obligate, intracellular parasite Encephalitozoon cuniculi, which is a member of the phylum Microsporidia. The aim of the current study is the detection of this parasite in stool samples of small animals of Iran, by polymerase chain reaction. Microsporidia spp. was found in 31% (31/100) of dogs (E. cuniculi (18/100), Encephalitozoon bieneusi (8/100) and Encephalitozoon intestinalis (5/100)), and 7.5% (3/40) of the specimens obtained from cats were infected with E. bieneusi. Sequencing of PCR products confirmed these results. In conclusion, Microsporidia infection seems to be fairly common in pet animals of Iran, especially in dogs. This finding could indicate the importance of pet animals as zoonotic reservoirs of microsporidial human infections. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Microsporidia are an exceptionally diverse group of organisms comprising more than 1200 species classified into approximately 100 genera that were recently reclassified from protozoa to fungi. Their life cycle includes stages resulting in characteristically small (1–4 m), environmentally resistant, infecting spores (Fayer, 2004). These unicellular, obligate intracellular eukaryotes have long been known to be causative agents of economically important diseases in every major animal group, especially insects, fish and mammals and they emerged as important opportunistic pathogens when AIDS became pandemic (Wittner, 1999). The clinical course of microsporidiosis depends on the immune status of the host and the site of infection. Clinically silent chronic infections usually develop in immunologically competent hosts that are either naturally or experimentally infected with Encephalitozoon cuniculi. Immune-compromised hosts infected with microsporidia are most likely to develop disseminated disease that often contributes to death (Snowden and Shadduck, 1999). E. cuniculi causes significant disease in
carnivores such as domestic dogs, blue fox (Alopex lagopus) and mink. Interestingly, these animals have developed hyperimmune responses resulting in immune complex formation and renal disease (Shadduck and Orenstein, 1993; Didier et al., 1998; Snowden and Shadduck, 1999). In humans, Encephalitozoon bieneusi infections remain localized to the small intestine, leading to persistent diarrhea. Species of Encephalitozoon, Trachipleistophora and Pleistophora often disseminate to cause sinusitis, keratoconjunctivitis, hepatitis, myositis, peritonitis, nephritis, encephalitis or pneumonia in immune-deficient individuals (Didier et al., 2004). Many species of microsporidia found to infect humans also infect a wide range of animals supporting the likelihood that zoonotic transmission occurs. Thus, the question of whether pet animals are the source of human infections is reasonable. In this study, PCR-based and microscopic methods were used to detect microsporidial spores in stool samples of pet dogs and cats having close contact with humans in Isfahan (Iran). 2. Materials and methods 2.1. Animals
∗ Corresponding author. Tel.: +98 21 6111 7193; fax: +98 21 6933222. E-mail address: [email protected] (Sh. Jamshidi). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.10.002
This study has been approved by the Iranian laboratory animal ethics framework under the supervision
122
Sh. Jamshidi et al. / Veterinary Parasitology 185 (2012) 121–123
of the Iranian Society for the Prevention of Cruelty to Animals. For this study, 100 stool samples from dogs (mean age = 4 years) and 40 samples from cats (mean age = 1.5 years) were collected from asymptomatic healthy animals which were referred to the Isfahan small animal clinic during a 6-month period in 2008. Samples were transferred to the diagnostic laboratory of Shahrekord Azad University and stored at 4 ◦ C until further analysis. 2.2. DNA extraction and PCR assays DNA was extracted from the fecal samples using instructions recommended by Laird et al. (1991). Fivehundred microliters of lysis buffer (EDTA 0.5 M, 100 mM Tris–HCl, SDS 10%, NACL 15 M, Proteinase K 20 mg/ml) was added to 500 l of each stool sample and incubated at 55 ◦ C for 2–3 h. Subsequently, 100 l phenol, 98 l chloroform and 4 l isoamyl alcohol were added to 200 l of lysed solution and after 30–60 s, vortexed at 4000 rpm for 4 min. The supernatant was collected and the same volume of pure ethanol was added and centrifugation was done at 4000 rpm for 5 min. After washing the DNA with 75% ethanol, the samples were centrifuged at 7000 rpm for 5 min, the supernatant was discarded and the DNA was dried. One hundred microliters of distilled water was then added to the DNA and stored at −20 ◦ C.
Table 1 Results of PCR on stool samples of household dogs and cats.
Dog (N = 100) Cat (N = 40)
E. cuniculi
E. bieneusi
E. intestinalis
18 (58%) 0
8 (25.8%) 3 (7.5%)
5 (16.1%) 0
2.4. Light microscopy For identification of microsporidia, fecal smears were stained with the Weber’s chromotrope as described (Weber et al., 1992). At least 50 fields of each stained smear were examined by light microscope under oil immersion at a magnification of 1000× before being considered negative. 3. Results PCR amplification: Out of 100 stool samples of dogs, 31cases were positive and a diagnostic band of 549 bp (E. cuniculi) was seen in 18 samples, 607 bp (E. bieneusi) in 8 samples and 520 bp (E. intestinalis) in 5 samples. All of the positive results belong to over one-year-old dogs. DNA of E. bieneusi (607 bp) was amplified in stool samples from 3/40 (7.5%) cats (Table 1). Light microscopy: With Weber’s chromotropebased stain, all PCR-positive samples showed spores of microsporidia that stained pinkish red (Fig. 1) and no spore was detected in the PCR-negative samples. 4. Discussion
2.3. PCR Microsporidial SSU-rRNA coding regions were amplified using the following species-specific primers: EBIEF1/EBIER1 for E. bieneusi (Da Silva et al., 1996), SINTF/SINTR for Encephalitozoon intestinalis (Da Silva et al., 1997), ECUNF/ECUNR for E. cuniculi and EHELF/EHELR for Encephalitozoon hellem (Visvesvara et al., 1994). All polymerase chain reactions (PCR) were performed in an Eppendorf thermocycler (Mastercycler gradient, Eppendorf GA, Hamburg, Germany) in a final volume of 50 L containing 1 g of extracted DNA, 5 L of 10× PCR buffer (Fermentas, Lithuania), 0.2 mM of dNTP, 2 mmol/L MgCl2 , 2 mol/L of each primer and 1 U of Taq DNA polymerase (Fermentas, Lithuania). Conditions for PCR reactions were: denaturing at 94 ◦ C for 50 s, annealing at 58 ◦ C for 50 s and extension at 72 ◦ C for 45 s. In all cases, 34 cycles were completed. Twenty microliters of each amplification underwent gel electrophoresis [1.5% (w/v) agarose gel with 0.3% ethidium bromide in 10% Tris–borate EDTA buffer (TBE)] and was visualized under UV transilluminator; the size of the expected fragments was compared to a 100-bp reference marker (Fermentas Inc., Glen Burnie, MD, USA). Since the positive control sample was not used in our molecular study, to confirm or reject the PCR results, the PCR products of the primary positive samples were purified using a PCR product purification kit (Roche Applied Science, Germany) and sent to the Macrogen Co. (South Korea) for sequencing.
Microsporidia are considered a cause of emerging and opportunistic infections in human, and species infecting humans also infect a wide range of animals, raising the concern for zoonotic transmission (Didier et al., 2004). However, the exact source of microsporidial human infection and routes of transmission remain unclear. Spores could be distributed into the environment via fecal, urine and respiratory excretions of infected animals and persons,
Fig. 1. Pinkish red spores of Microsporidia from a stool sample of a dog stained with Weber’s chromotrope (magnification 1000×). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Sh. Jamshidi et al. / Veterinary Parasitology 185 (2012) 121–123
which could all be possible sources of infection (Lores et al., 2002). Microsporidial infections can lead to severe diseases such as chronic diarrhea and significant weight loss in humans as well as animals (Snowden and Shadduck, 1999), however, it seems that many animals can carry these organisms without obvious clinical manifestations. These clinically silent carriers seem to be an even greater risk to public health than a sick animal. In the present study, for the first time, both molecular and microscopic methods were used to detect microsporidial spores in fecal samples of healthy pet cats and dogs in Iran, as a potential reservoir of human infections. Thirty-one percent of stool samples obtained from 100 household dogs were positive with PCR, which were confirmed by microscopic evaluations as well. This rate of infection should be believed significantly high, especially when considering that immuno-compromised persons and/or children may have close contact with them. The rate of infection in cats (7.5%) was lower than dogs, but it is still high and it seems that both dogs and cats have a serious role as reservoirs of infection to humans and are important in the epidemiology of microsporidiosis. E. cuniculi was detected in more than half of our positive samples (58%). Direct evidence of zoonotic transmission of microsporidiosis was reported in a child who seroconverted after being exposed to a litter of puppies infected with E. cuniculi (McInnes and Stewart, 1991). As previously mentioned, this species usually causes significant disease in domestic dogs (Shadduck and Orenstein, 1993; Didier et al., 1998; Snowden and Shadduck, 1999), but interestingly, none of our studied group had clinical signs of disease. This evidence again shows that a clinically normal dog can harbor even fully pathogenic strains and act only as a shedder. In almost 26% (8/31) of the positive specimens, E. bieneusi was found, which in 16% (5/31) E. intestinalis was found. It is not clear whether their presence in the fecal samples is the result of an active infection or not, but it obviously demonstrates that these animals can shed them into the environment, suggesting that indirect zoonotic transmission of microsporidia between animals and humans could occur through contaminated food, water, or aerosols.
123
Finally, the role of animals in the transmission of microsporidial human infections requires further study. However, our results show that small animals, especially dogs, even when they are apparently normal could be shedders of Microsporidia via their feces, and owners, especially those who are old or have a child and/or an immunodeficient patient, should be informed with practical suggestions designed to reduce this low risk. References Da Silva, A.J., Slemenda, S.B., Visvesvara, G.S., Schwartz, D.A., Wilcox, C.M., Wallace, S., Pieniazek, N.J., 1997. Detection of Septata intestinalis (microsporidia) Cali et al., 1993 using polymerase chain reaction primers targeting the small subunit ribosomal RNA coding region. Mol. Diagn. 2, 47–52. 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 small-subunit rRNA. J. Clin. Microbiol. 34 (4), 986–987. Didier, E.S., Stovall, M.E., Green, L.C., Brindley, P.J., Sestak, K., Didier, P.J., 2004. Epidemiology of microsporidiosis: sources and modes of transmission. Vet. Parasitol. 126, 145–166. Didier, E.S., Snowden, K.F., Shadduck, J.A., 1998. Biology of microsporidian species infecting mammals. Adv. Parasitol. 40, 283–320. Fayer, R., 2004. Infectivity of microsporidia spores stored in seawater at environmental temperatures. J. Parasitol. 90, 654–657. Laird, P.W., Zijderveld, A., Linders, K., Rudnicki, M.A., Jaenisch, R., Berns, A., 1991. Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 19 (15), 4293. Lores, B., del Aguila, C., Arias, C., 2002. Enterocytozoon bieneusi (microsporidia) in faecal samples from domestic animals from Galicia, Spain. Mem. Inst. Oswaldo Cruz 97, 941–945. McInnes, E.F., Stewart, C.G., 1991. The pathology of subclinical infection of Encephalitozoon cuniculi in canine dams producing pups with overt encephalitozoonosis. J. S. Afr. Vet. Assoc. 62, 51–54. Shadduck, J.A., Orenstein, J.M., 1993. Comparative pathology of microsporidiosis. Arch. Pathol. Lab. Med. 117, 1215–1219. Snowden, K.F., Shadduck, J.A., 1999. Microsporidia of higher vertebrates. In: Wittner, M., Weiss, L. (Eds.), The Microsporidia and Microsporidiosis. American Society of Microbiology, Washington, DC, pp. 393–419. 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., et al., 1994. Polyclonal and monoclonal antibody and PCR-amplified smallsubunit rRNA identification of a microsporidian, Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection. J. Clin. Microbiol. 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. N. Engl. J. Med. 326, 161–166. Wittner, M., 1999. Historic perspective on the microsporidia: expanding horizons. In: Wittner, M., Weiss, L. (Eds.), The Microsporidia and Microsporidiosis. American Society of Microbiology, Washington, DC, pp. 1–6.